Death regulator Nedd2-like caspase: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - Death regulator Nedd2-like caspase

Synonyms - Dronc

Cytological map position - 67C4--5

Function - caspase

Keywords - programmed cell death, apoptosis

Symbol - Nc

FlyBase ID: FBgn0026404

Genetic map position -

Classification - p20 and p10 domains, Caspase (ICE-like protease)

Cellular location - cytoplasmic



Precomputed BLAST | Entrez Gene

Recent literature
Orme, M. H., et al. (2016). The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles. Nat Commun 7: 10972. PubMed ID: 26960254
Summary:
Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. This study found that the Drosophila unconventional myosin Crinkled (Ck) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. The data indicate that CK acts as substrate adaptor, recruiting Shaggy46/GSK3-β to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, these results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.

Lee, T. V., Kamber Kaya, H. E., Simin, R., Baehrecke, E. H. and Bergmann, A. (2016). The initiator caspase Dronc is subject of enhanced autophagy upon proteasome impairment in Drosophila. Cell Death Differ [Epub ahead of print]. PubMed ID: 27104928
Summary:
A major function of ubiquitylation is to deliver target proteins to the proteasome for degradation. In the apoptotic pathway in Drosophila, the inhibitor of apoptosis protein 1 (Diap1) regulates the activity of the initiator caspase Dronc (death regulator Nedd2-like caspase; caspase-9 ortholog) by ubiquitylation, supposedly targeting Dronc for degradation by the proteasome. Using a genetic approach, this study showed that Dronc protein fails to accumulate in epithelial cells with impaired proteasome function suggesting that it is not degraded by the proteasome, contrary to the expectation. Similarly, decreased autophagy, an alternative catabolic pathway, does not result in increased Dronc protein levels. However, combined impairment of the proteasome and autophagy triggers accumulation of Dronc protein levels suggesting that autophagy compensates for the loss of the proteasome with respect to Dronc turnover. Consistently, it was shown that loss of the proteasome enhances endogenous autophagy in epithelial cells. It is proposed that enhanced autophagy degrades Dronc if proteasome function is impaired.
Koerver, L., Melzer, J., Roca, E. A., Teichert, D., Glatter, T., Arama, E. and Broemer, M. (2016). The de-ubiquitylating enzyme DUBA is essential for spermatogenesis in Drosophila. Cell Death Differ[Epub ahead of print]. PubMed ID: 27518434
Summary:
De-ubiquitylating enzymes (DUBs) reverse protein ubiquitylation and thereby control essential cellular functions. A screen for a DUB that counteracts caspase ubiquitylation to regulate cell survival identified the Drosophila ovarian tumour-type DUB DUBA (CG6091). DUBA physically interact with the initiator caspase Death regulator Nedd2-like caspase (Dronc) and de-ubiquitylates it, thereby contributing to efficient inhibitor of apoptosis-antagonist-induced apoptosis in the fly eye. Searching also for non-apoptotic functions of DUBA, Duba-null mutants were found to be male sterile and display defects in spermatid individualisation, a process that depends on non-apoptotic caspase activity. Spermatids of DUBA-deficient flies showed reduced caspase activity and lack critical structures of the individualisation process. Biochemical characterisation revealed an obligate activation step of DUBA by phosphorylation. With genetic rescue experiments it was demonstrated that DUBA phosphorylation and catalytic activity are crucial in vivo for DUBA function in spermatogenesis. These results demonstrate for the first time the importance of de-ubiquitylation for fly spermatogenesis.
Kamber Kaya, H.E., Ditzel, M., Meier, P. and Bergmann, A. (2017). An inhibitory mono-ubiquitylation of the Drosophila initiator caspase Dronc functions in both apoptotic and non-apoptotic pathways. PLoS Genet 13: e1006438. PubMed ID: 28207763
Summary:
Apoptosis is an evolutionary conserved cell death mechanism, which requires activation of initiator and effector caspases. The Drosophila initiator caspase Dronc, the ortholog of mammalian Caspase-2 and Caspase-9, has an N-terminal CARD domain that recruits Dronc into the apoptosome for activation. In addition to its role in apoptosis, Dronc also has non-apoptotic functions such as compensatory proliferation. One mechanism to control the activation of Dronc is ubiquitylation. However, the mechanistic details of ubiquitylation of Dronc are less clear. For example, monomeric inactive Dronc is subject to non-degradative ubiquitylation in living cells, while ubiquitylation of active apoptosome-bound Dronc triggers its proteolytic degradation in apoptotic cells. This study examined the role of non-degradative ubiquitylation of Dronc in living cells in vivo, i.e. in the context of a multi-cellular organism. In vivo data suggest that in living cells Dronc is mono-ubiquitylated on Lys78 (K78) in its CARD domain. This ubiquitylation prevents activation of Dronc in the apoptosome and protects cells from apoptosis. Furthermore, K78 ubiquitylation plays an inhibitory role for non-apoptotic functions of Dronc. Further, not all of the non-apoptotic functions of Dronc require its catalytic activity. In conclusion, data demonstrate a mechanism whereby Dronc's apoptotic and non-apoptotic activities can be kept silenced in a non-degradative manner through a single ubiquitylation event in living cells.

Jo, J., Im, S. H., Babcock, D. T., Iyer, S. C., Gunawan, F., Cox, D. N. and Galko, M. J. (2017). Drosophila caspase activity is required independently of apoptosis to produce active TNF/Eiger during nociceptive sensitization. Cell Death Dis 8(5): e2786. PubMed ID: 28492538
Summary:
Tumor necrosis factor (TNF) signaling is required for inflammatory nociceptive (pain) sensitization in Drosophila and vertebrates. Nociceptive sensitization in Drosophila larvae following UV-induced tissue damage is accompanied by epidermal apoptosis and requires epidermal-derived TNF/Eiger and the initiator caspase, Dronc. In this study, apoptotic cell death and thermal nociceptive sensitization are genetically and procedurally separable in a Drosophila model of UV-induced nociceptive sensitization. Activation of epidermal Dronc induces TNF-dependent but effector caspase-independent nociceptive sensitization in the absence of UV. In addition, knockdown of Dronc attenuated nociceptive sensitization induced by full-length TNF/Eiger but not by a constitutively soluble form. UV irradiation induced TNF production in both in vitro and in vivo, but TNF secretion into hemolymph was not sufficient to induce thermal nociceptive sensitization. Downstream mediators of TNF-induced sensitization included two TNF receptor-associated factors, a p38 kinase, and the transcription factor nuclear factor kappa B. Finally, sensory neuron-specific microarray analysis revealed downstream TNF target genes induced during thermal nociceptive sensitization. One of these, enhancer of zeste (E(z)), functions downstream of TNF during thermal nociceptive sensitization. These findings suggest that an initiator caspase is involved in TNF processing/secretion during nociceptive sensitization, and that TNF activation leads to a specific downstream signaling cascade and gene transcription required for sensitization. These findings have implications for both the evolution of inflammatory caspase function following tissue damage signals and the action of TNF during sensitization in vertebrates.
Hara, Y., Sudo, T., Togane, Y., Akagawa, H. and Tsujimura, H. (2018). Cell death in neural precursor cells and neurons before neurite formation prevents the emergence of abnormal neural structures in the Drosophila optic lobe. Dev Biol. PubMed ID: 29447906
Summary:
Programmed cell death is a conserved strategy for neural development both in vertebrates and invertebrates and is recognized at various developmental stages in the brain from neurogenesis to adulthood. This study investigated the effect of inhibition of cell death on optic lobe development. Cell death was shown to occur in neural precursor cells and neurons before neurite formation and functions to prevent various developmental abnormalities. When neuronal cell death was inhibited by an effector caspase inhibitor, p35, multiple abnormal neuropil structures arose during optic lobe development-e.g., enlarged or fused neuropils, misrouted neurons and abnormal neurite lumps. Inhibition of cell death also induced morphogenetic defects in the lamina and medulla development. These defects were reproduced in the mutant of an initiator caspase, dronc. If cell death was a mechanism for removing the abnormal neuropil structures, it would also be expected to be observed in mutants defective for corpse clearance. However, they were not observed in these mutants. When dead cell-membranes were visualized with Apoliner, they were observed only in cortices and not in neuropils. These results suggest that the cell death occurs before mature neurite formation. Moreover, it was found that inhibition of cell death induced ectopic neuroepithelial cells, neuroblasts and ganglion mother cells in late pupal stages, at sites where the outer and inner proliferation centers were located at earlier developmental stages. Caspase-3 activation was observed in the neuroepithelial cells and neuroblasts in the proliferation centers. These results indicate that cell death is required for elimination of the precursor cells composing the proliferation centers. This study substantiates an essential role of early neural cell death for ensuring normal development of the central nervous system.
Baena-Lopez, L. A., Arthurton, L., Bischoff, M., Vincent, J. P., Alexandre, C. and McGregor, R. (2018). Novel initiator caspase reporters uncover unknown features of caspase-activating cells. Development. PubMed ID: 30413561
Summary:
The caspase-mediated regulation of many cellular processes, including apoptosis, justifies the substantial interest in understanding all of the biological features of these enzymes. To complement functional assays, it is critical to identify caspase-activating cells in live tissues. This work describes novel initiator caspase-reporters that, for the first time, provide direct information concerning the initial steps of the caspase activation cascade in Drosophila tissues. A caspase-sensor has capitalized on the rapid subcellular localization change of a fluorescent marker to uncover novel cellular apoptotic events. These refer to the actin-mediated positioning of the nucleus before cell delamination. The other construct has benefited from a caspase-induced nuclear translocation of a QF transcription factor. This feature enables the genetic manipulation of caspase-activating cells, whilst showing the spatio-temporal patterns of initiator caspase activity. Collectively, these sensors offer experimental opportunities not available by using previous reporters and therefore they have been proven useful to illuminate unknown aspects of caspase-dependent processes in apoptotic and non-apoptotic cellular scenarios.
BIOLOGICAL OVERVIEW

Drosophila Nedd2-like caspase, referred to here and in the literature as Dronc (Drosophila Nedd2-like caspase, not to be confused with Death related ced-3/Nedd2-like protein), is a caspase recruitment domain-containing Drosophila caspase that is expressed in a temporally and spatially restricted fashion during development. Dronc is the only fly caspase known to be regulated by the hormone ecdysone. Ectopic expression of dronc in the developing fly eye leads to increased cell death and an ablated eye phenotype that can be suppressed by halving the dosage of the genes in the H99 complex (reaper, hid, and grim) and enhanced by mutations in diap1 (thread). The dronc eye ablation phenotype can be suppressed by coexpression of the baculoviral caspase inhibitor p35. Dronc also interacts, both genetically and biochemically, with the CED-4/Apaf-1 fly homolog, Ark. Furthermore, extracts made from Ark homozygous mutant flies have reduced ability to process Dronc, showing that Ark is required for Dronc processing. Using the RNA interference technique, it has been shown that loss of Dronc function in early Drosophila embryos results in a dramatic decrease in cell death, indicating that Dronc is important for programmed cell death during embryogenesis. These results suggest that Dronc is a key caspase mediating programmed cell death in Drosophila (Quinn, 2000).

Caspases are cysteine proteases that act as central effectors of programmed cell death. These proteases are synthesized as precursor molecules that are processed in cells undergoing apoptosis to generate two subunits that fold into a tetrameric active enzyme conformation. In mammals, 14 caspases have been described thus far, which can be grouped into two classes based upon the length of their prodomain. Caspases containing a long prodomain, such as caspase-2, -8, -9, and -10, appear to be activated first by a proximity-induced autoprocessing mechanism involving clustering of procaspase molecules, often assisted by specific adaptor molecules. These caspases contain specific protein-protein interaction domains in the prodomain region that mediate their interaction with their respective adaptors or mediate dimerization of procaspase molecules. For example, caspase-2 and caspase-9 contain caspase recruitment domains (CARDs) in their prodomain, whereas caspase-8 and caspase-10 contain two copies of death effector domains (DEDs). The CARD in caspase-2 is required for homodimerization, whereas the CARD in caspase-9 interacts with the mammalian CED-4-like adaptor Apaf-1 (Drosophila homolog, Apaf-1-related-killer). Cytochrome c- and dATP-dependent oligomerization of Apaf-1, followed by interaction of oligomerized Apaf-1 with procaspase-9, results in proximity-induced activation of caspase-9. One of the DEDs in caspase-8 interacts with the DED in the adaptor FADD, a molecule that helps recruit procaspase-8 to activated death receptors of the tumor necrosis factor receptor family. Again, this adaptor-mediated recruitment of the procaspase molecules is believed to be sufficient for caspase activation. Once the caspases containing long prodomains are activated, they are believed to activate downstream caspases that lack specific protein-protein interaction domains, thereby initiating a cascade of caspase activation (Quinn, 2000 and references therein).

In Drosophila, three proteins, Reaper, Hid, and Grim, play critical roles in apoptosis. These proteins act upstream of caspase activation and appear to be required to counteract the caspase inhibitors Diap1 and Diap2, which are Drosophila homologs of the baculovirus inhibitor of apoptosis, IAP. In Drosophila, specific mutations in diap1 (thread) show increased cell death in the embryo, and Diap1 has been shown to bind to and inhibit the activity of Drosophila effector caspases. The Drosophila Apaf-1-related-killer (Ark) and a proapoptotic CED-9/Bcl-2 homolog, Debcl/Drob-1/dBorg-1, have been described. There are seven known caspases in Drosophila, including five published ones (Dcp-1, Dredd, Drice, Dronc, and Decay, and two unpublished ones (Damm and Strica; GenBankTM accession numbers AF240763 and AF242734, respectively). Dredd and Dronc contain long prodomains carrying DEDs and a CARD, respectively, suggesting that these two caspases may act as upstream caspases. Strica also contains a long prodomain, but it lacks any CARD/DED sequences. However, Dcp-1, Drice, Decay, and Damm lack long prodomains and are thus similar to downstream effector caspases in mammals. A dcp-1 mutation results in larval lethality and melanotic tumors. Additionally, dcp-1 mutants show a defect in transfer of nurse cell cytoplasmic contents to developing oocytes, suggesting that dcp-1 may also be required for Drosophila oogenesis. Because no mutants for other Drosophila caspases are currently available, their precise functions remain unknown. However, a number of indirect observations point to a role for other Drosophila caspases in apoptosis in vivo. For example, dredd mRNA accumulates in embryonic cells undergoing programmed cell death; in nurse cells in the ovary at a time that coincides with nurse cell death, dronc mRNA, although widely expressed during development, is up-regulated by ecdysone in larval salivary glands and midgut before histolysis of these tissues. jAntibody depletion experiments suggest that Drice is required for apoptotic activity in the S2 Drosophila cell line. Furthermore, a deficiency uncovering dredd dominantly suppresses the ablated eye phenotype due to ectopic expression of rpr, hid, or grim, and a mini-gene of dredd reverses this suppression, showing that dredd is important for PCD in vivo (Quinn, 2000 and references therein).

Overexpression of Dronc induces cell death in mammalian cells (Dorstyn, 1999). In two recent studies (Meier, 2000 and Hawkins, 2000), it has been shown that ectopic expression of dronc promotes apoptosis in the developing Drosophila eye that can be suppressed by co-expression of diap1. Furthermore, in yeast, Diap1 inhibits Dronc activity, and this inhibition is abrogated by co-expression of Hid or Grim (Hawkins, 2000). Diap1 binds to the prodomain of Dronc, and, consistent with this, expression of a truncated version of Dronc lacking the prodomain results in a more severely ablated eye phenotype that can not be rescued by co-expression of Diap1 (Meier, 2000). These studies also show that a mutated version of dronc (containing a mutation in the caspase active site) acts in a dominant negative manner to suppress rpr- and hid-induced cell death (Meier, 2000). Likewise, a deficiency removing the dronc gene is able to dominantly suppress rpr- and hid-induced cell death in the eye (Meier, 2000), showing that Dronc mediates cell death by Rpr and Hid. In addition, these studies showed that Dronc associates with the effector caspase Drice and is able to process Drice to the active form (Meier, 2000 and Hawkins, 2000). Ectopic expression of dronc at various developmental stages results in apoptosis. The dronc eye ablation phenotype can also be blocked by co-expression of the baculoviral caspase inhibitor p35. Confirming these genetic interactions, Dronc can form complexes with Diap1, Grim, Ark, and P35; however, the interaction between Dronc and Grim or P35 is indirect. Because a direct binding between Dronc and P35 could not be demonstrated in vitro, it is likely that P35 interacts with and inhibits a downstream caspase rather than Dronc itself. One possible candidate is Drice, which has been shown to interact with both P35 (19) and Dronc (Meier, 2000), or Dcp-1, which can cleave P35. Ark is required for Dronc activation because Dronc is poorly processed in extracts from Ark homozygous mutant flies. Because specific dronc mutants are currently unavailable, the technique of RNA interference (RNAi) was used to ablate dronc mRNA in embryos. Dronc is shown to be essential for cell death during embryogenesis and these results suggest a central function for Dronc in the cell death effector machinery in Drosophila (Quinn, 2000 and references therein).

Based on homology and the ability of Dronc to form a complex with Ark, Dronc is expected to be a functional homolog of CED-3/caspase-9. Therefore, Dronc is expected to be downstream of Ark and the proteins of the H99 complex, which are known to induce PCD by activating caspases. Consistent with this, a dronc deficiency or expression of the dominant negative dronc mutant is able to suppress the ablated eye phenotype of GMR-hid and GMR-rpr (Meier, 2000; Hawkins, 2000 and Quinn, 2000). Because overexpression of upstream caspases generally results in autoactivation, ectopic expression of dronc was expected to be epistatic to (downstream of) the H99 genes and Ark. However, halving the dosage of the H99 genes or Ark suppresses the GMR-dronc eye phenotype, suggesting that the H99 genes and Ark are rate-limiting for dronc function (Quinn, 2000). This may be explained by the possibility that Dronc, when overexpressed as a zymogen, is not able to self-activate very efficiently and may therefore be dependent on the dosage of upstream activating genes. Another possibility is that the suppression of GMR-dronc by halving the dosage of the H99 genes may be a result of a feedback amplification loop between active caspases and Rpr, Hid, and Grim (Quinn, 2000 and references therein).

Consistent with the genetic interaction, Dronc forms a complex with the H99 gene product Grim when co-expressed in cells. However, this interaction is not direct and may occur through Diap1, which can bind to Dronc and to Grim (Meier, 2000; Hawkins, 2000 and Quinn, 2000). The significance of the in vivo interaction between Grim and Dronc is unclear and requires further investigation. As a CED-3/caspase-9 homolog, activation of Dronc is expected to require Ark, and, consistent with this, Dronc and Ark form a complex in SL2 cells. A complex of Ark and Dronc has also been observed in 293T cells that results in generation of the cleaved, active form of Dronc. Ark mutant extracts are defective in their ability to generate the cleaved active form of Dronc and have lower levels of active caspases. These results, together with genetic data, suggest that Dronc is likely to be a functional homolog of CED-3/caspase-9 because it is activated by Ark, the CED-4/Apaf-1 homolog. Furthermore, the amino-terminal region of Ark containing the CARD and CED-4 homology domain is sufficient for Dronc binding (Quinn, 2000).

The Drosophila apoptosis inhibitor Diap1 inhibits the activity of Drice and Dcp-1 and is antagonized by Rpr, Hid, or Grim. The data showing a dose-dependent enhancement of GMR-Dronc by diap1 mutations and that Dronc and Diap1 form a complex, are consistent with Diap1 also acting as an inhibitor of Dronc. Diap1 and Dronc interact genetically and biochemically and the prodomain of Dronc is required for the Diap1 interaction (Meier, 2000). Expression of diap1 or diap2 (to a lesser extent) is able to suppress the GMR-dronc phenotype, indicating that Diap2 as well as Diap1 can prevent Dronc-mediated cell killing. However, because a diap2 deficiency does not show a dominant enhancement of GMR-dronc, and Diap2 does not form a complex with Dronc, it is likely that the suppression of GMR-Dronc by GMR-Diap2 is indirect, perhaps via the inhibition of downstream caspases. The genetic and biochemical observations for a role for Diap1, but not Diap2, in suppressing Dronc function are consistent with previous studies showing that diap1 and diap2 function differently in inhibiting cell death. Halving the dosage of diap1, but not diap2, enhances rpr-, hid-, or grim-induced cell death, whereas overexpression of diap1 or diap2 can inhibit rpr- or hid-induced death, but only overexpression of diap1 can inhibit grim-induced cell death. Additional studies are required to further explore the precise roles of Diap1 and Diap2 in the Drosophila cell death pathway (Quinn, 2000).

In summary, Dronc is essential for cell death in early embryos and ectopic expression of Dronc can induce cell death in flies. Furthermore, Dronc is likely to be a functional homolog of CED-3/caspase-9 in flies. The data showing genetic and physical interactions between Dronc and P35, Grim, Ark, and Diap1 provide a framework for further investigation of the PCD pathway in flies (Quinn, 2000).

Extracellular reactive oxygen species drive apoptosis-induced proliferation via Drosophila macrophages

Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. This study shows that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. It is proposed that in an immortalized ('undead') model of AiP, in which the activity of the effector caspases is blocked, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer (Fogarty, 2016).

The role of ROSs as a regulated form of redox signaling in damage detection and damage response is becoming increasingly clear. This study has shown that in Drosophila, extracellular ROSs generated by the NADPH oxidase Duox drive compensatory proliferation and overgrowth following hid-induced activation of the initiator caspase Dronc in developing epithelial tissues. At least one consequence of ROS production is the activation of hemocytes at undead epithelial disc tissue. Furthermore, the work implies that extracellular ROS and hemocytes are part of the feedback amplification loop between Hid, Dronc, and JNK that occurs during stress-induced apoptosis. Finally, hemocytes release the TNF ligand Eiger, which promotes JNK activation in epithelial disc cells (Fogarty, 2016).

This work helps to understand why JNK activation occurs mostly in apoptotic/undead cells but occasionally also in neighboring surviving cells. Because the data indicate that hemocytes trigger JNK activation in epithelial cells, the location of hemocytes on the imaginal discs determines which epithelial cells receive the signal for JNK activation. Nevertheless, the possibility is not excluded that there is also an autonomous manner of Dronc-induced JNK activation in undead/apoptotic cells (Fogarty, 2016).

In the context of apoptosis, hemocytes engulf and degrade dying cells. However, there is no evidence that hemocytes have this role in the undead AiP model. No Caspase-3 (CC3) material is observed in hemocytes attached to undead tissue. Therefore, the role of hemocytes in driving proliferation is less clear and likely context dependent. In Drosophila embryos, hemocytes are required for epidermal wound healing, but this is a nonproliferative process. With respect to tumor models in Drosophila, much of the research to date has focused on the tumor-suppressing role of hemocytes and the innate immune response. However, a few reports have implicated hemocytes as tumor promoters in a neoplastic tumor model. Consistently, in the undead model of AiP, this study found that hemocytes have an overgrowth- and tumor-promoting role. Therefore, the state of the damaged tissue and the signals produced by the epithelium may have differential effects on hemocyte response (Fogarty, 2016).

In a recent study, ROSs were found to be required for tissue repair of wing imaginal discs in a regenerative (p35-independent) model of AiP, consistent with the current work. Although a role of hemocytes was not investigated in this study, it should be noted that p35-independent AiP models do not cause overgrowth, whereas undead ones such as the ey>hid-p35 AiP model do. It is therefore possible that ROSs in p35-independent AiP models are necessary for tissue repair independent of hemocytes, whereas ROSs in conjunction with ROS-activated hemocytes in undead models mediate the overgrowth of the affected tissue. Future work will clarify the overgrowth-promoting function of hemocytes. These considerations are reminiscent of mammalian systems, where many solid tumors are known to host alternatively activated (M2) tumor-associated macrophages, which promote tumor growth and are associated with a poor prognosis (Fogarty, 2016).

Because tumors are considered 'wounds that do not heal', the undead model of AiP is seen as a tool to probe the dynamic interactions and intercellular signaling events that occur in the chronic wound microenvironment. Future studies will investigate the specific mechanisms of hemocyte-induced growth and the tumor-promoting role of inflammation in Drosophila as well as roles of additional tissue types, such as the fat body, on modulating tumorous growth (Fogarty, 2016).

Plasma membrane localization of apoptotic caspases for non-apoptotic functions

Caspases are best characterized for their function in apoptosis. However, they also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), where caspases release mitogens for compensatory proliferation independently of their apoptotic role. This study reports that the unconventional myosin, Myo1D, which is known for its involvement in left/right development, is an important mediator of AiP in Drosophila. Mechanistically, Myo1D translocates the initiator caspase Dronc to the basal side of the plasma membrane of epithelial cells where Dronc promotes the activation of the NADPH-oxidase Duox for reactive oxygen species generation and AiP in a non-apoptotic manner. It is proposed that the basal side of the plasma membrane constitutes a non-apoptotic compartment for caspases. Finally, Myo1D promotes tumor growth and invasiveness of the neoplastic scrib Ras(V12) model. Together, these studies have identified a new function of Myo1D for AiP and tumorigenesis and reveal a mechanism by which cells sequester apoptotic caspases in a non-apoptotic compartment at the plasma membrane (Amcheslavsky, 2018).

Under stress conditions, when a large number of cells are dying, there is a need for compensatory proliferation to replace the lost cells with new cells. Work using several model organisms has shown that, under these conditions, apoptotic cells can release mitogenic signals that induce proliferation of surviving cells for the replacement of dying cells. Because apoptotic cells are actively triggering this type of compensatory proliferation, this process has been termed apoptosis-induced proliferation (AiP) (Amcheslavsky, 2018).

Caspases are Cys proteases that are the main effectors of apoptosis. They are produced as inactive zymogens with a prodomain and after processing a large and small subunit. There are initiator and effector caspases. Initiator caspases carry protein/protein interacting motifs in their prodomains, which mediate their incorporation into large multimeric protein complexes. For example, the mammalian initiator caspase-9 is recruited into the Apaf-1 apoptosome, while its Drosophila ortholog Dronc forms the apoptosome with the Apaf-1 homolog Dark. Effector caspases such as mammalian caspase-3, or Drosophila DrICE and Dcp-1, are proteolytically processed by activated initiator caspases and mediate the apoptotic process (Amcheslavsky, 2018).

In addition to apoptosis, caspases are also mediating AiP. They trigger the release of Wnt, bone morphogenetic protein (BMP)/transforming growth factor β (TGF-β), epidermal growth factor (EGF), and Hedgehog mitogens for AiP. This has been best studied for the Drosophila initiator caspase Dronc using the 'undead' AiP model in which apoptotic signaling is induced by expression of upstream cell death factors such as hid, but the execution of apoptosis is blocked by co-expression of the effector caspase inhibitor p35, thus rendering cells in an undead condition. Because P35 inhibits apoptosis, but not Dronc, Dronc can still mediate non-apoptotic functions such as AiP. When hid and p35 are co-expressed using the ey-Gal4 driver (ey > hid,p35), which is expressed in epithelial cells of eye imaginal discs, Dronc continuously signals for AiP and triggers hyper-proliferation. Consequently, the discs are enlarged and the resulting heads of the adult flies are overgrown . In genetic screens, screening was carried out for suppressors of the overgrowth phenotype of undead (ey > hid,p35) adult heads to identify genes and mechanisms involved in AiP (Amcheslavsky, 2018).

Mechanistically, this study showed that, in undead cells, Dronc stimulates the NADPH-oxidase Duox for the production of extracellular reactive oxygen species (eROS). eROS recruit and activate hemocytes, Drosophila immune cells similar to macrophages, to the undead imaginal disc. In turn, hemocytes release the tumor necrosis factor-like ligand Eiger, which induces JNK activity in epithelial disc cells. JNK promotes the expression of the apoptotic genes reaper and hid, which initiate a positive feedback loop to maintain undead signaling (Fogarty, 2016). In addition, it induces the release of the mitogens Wingless (Wg), a Wnt-like gene in Drosophila, decapentaplegic, a BMP/TGF-β homolog, and Spitz, an EGF ligand, which all promote AiP (Amcheslavsky, 2018).

In addition to undead AiP, there is also 'genuine' AiP, during which dying cells complete the apoptotic process, and the response of the affected tissue to replace the dying cells is examined. In contrast to undead AiP, genuine AiP does not promote overgrowth. Therefore, although most genes identified in undead AiP also have important roles in genuine AiP, there must be differences between the two AiP models. In any case, genuine AiP is used as a model of tissue regeneration, while the hyper-proliferation of undead AiP serves as a tumorigenic model (Amcheslavsky, 2018).

Class I unconventional myosins are conserved actin-based motor proteins, composed of the N-terminal head (motor) region with an ATP binding motif (including P-, switch1-, and switch2 loops) and an actin-binding domain, a neck region characterized by two to three IQ motifs, and a C-terminal tail domain that interacts with phospholipids at membranes. Mammals have eight class I myosins, Drosophila has three, Myosin 1D (Myo1D, also known as Myo31DF), MyoIC (Myo61F), and Myo95E. While Myo1D and Myo1C are involved in left/right (L/R) development of visceral organs, the function of Myo95E is unknown (Amcheslavsky, 2018).

Although Drosophila is a bilateral organism, certain visceral organs such as the gut and the coiling of the spermiducts around the gut, which occurs in a morphogenetic movement termed male terminalia rotation, display L/R asymmetry. In Myo1D mutants, the chirality of these asymmetric organs and movements are reversed. For example, the male terminalia rotation during pupal development, which, in wild-type, occurs for 360° in clockwise (dextral) orientation, proceeds in Myo1D mutants sinistrally, defining Myo1D as dextral determinant. Myo1D engages the actin cytoskeleton and adherens junctions for this movement (Amcheslavsky, 2018).

Overexpression of Myo1C antagonizes the dextral activity of Myo1D by displacing it from adherens junctions. However, the loss-of-function phenotype of Myo1C did not confirm this antagonizing function. Instead, while Myo1C single mutants do not display any L/R defect, the Myo1C Myo1D double mutant has a stronger sinistral male terminalia phenotype than Myo1D mutants indicating that Myo1C has a partially redundant dextral activity with Myo1D (Amcheslavsky, 2018).

It has long been known that genes in the apoptosis pathway, such as hid, dronc, and drICE, are also involved in male terminalia rotation in Drosophila. Indeed, localized apoptotic activity is required for this L/R process. How Myo1D and the apoptosis pathway interact for male terminalia rotation is not very well understood. Interestingly, mutants of the JNK signaling pathway or overexpression of puckered, an inhibitor of JNK activity, also display defects in male terminalia rotation (Amcheslavsky, 2018).

This study reports that Myo1D is an essential component of AiP in the undead model. Genetic inactivation of Myo1D strongly suppresses ey > hid,p35-induced overgrowth of the head capsule, while overexpression of Myo1D enhances it. Myo1D promotes the generation of ROS by Duox for AiP signaling. Further mechanistic analysis reveals that Myo1D is required for membrane localization of Dronc, specifically to the basal side of the plasma membrane of undead epithelial disc and salivary gland cells. Here, Dronc exerts a non-apoptotic function resulting in Duox activation. It is proposed that the basal side of the plasma membrane constitutes a non-apoptotic compartment that allows non-apoptotic processes of Dronc and potentially other caspases to occur. Therefore, in addition to the dextral activity of Myo1D, this study identified a second function of Myo1D for the control of apoptosis-induced proliferation (Amcheslavsky, 2018).

Mechanistically, it was found that Myo1D is involved in the localization of the initiator caspase Dronc to the basal side of the plasma membrane of undead DP disc and SG cells. Myo1D interacts with Dronc, suggesting that it may directly translocate Dronc to the plasma membrane. However, Myo1D does not appear to be a cleavage target of the caspase Dronc (Amcheslavsky, 2018).

The observed localization of Dronc to the basal side of the plasma membrane in undead DP cells is critical for the mechanism of AiP. Undead cells attract hemocytes to the discs in a Dronc- and Duox-dependent manner. However, that occurs at the basal side of DP cells of imaginal discs because the basal side is exposed to the hemolymph that contains circulating hemocytes, while the apical side faces the lumen between the DP and the PM. Consistently, there is also an enrichment of Duox at the basal side of the plasma membrane. Therefore, in order to be able to activate Duox for ROS generation and hemocyte activation, Dronc needs to be specifically present at the basal side of the plasma membrane (Amcheslavsky, 2018).

It has long been known that caspases, including Dronc, have non-apoptotic functions in addition to their well characterized role in apoptosis. This paper reveals one mechanism by which cells may activate a caspase (Dronc) without the detrimental consequences of apoptosis. The sequestration of Dronc to the basal side of the plasma membrane in a Myo1D-dependent manner and the low abundance of Dronc's apoptotic partner Dark at the plasma membrane may ensure localized and controlled apoptosome activity which is sufficient for AiP, but not for killing cells. Alternatively, apoptotic substrates needed for the execution of apoptosis may not be present at the plasma membrane or in insufficient amount to pass the apoptotic threshold (Amcheslavsky, 2018).

While this study addressed the role of membrane localization of Dronc under undead conditions, recently membrane-localized Dronc was shown in SGs under normal conditions, which explains the membrane localization of Dronc at control SGs. Here, membrane-localized Dronc is required for F-actin cytoskeleton dismantling at the end of larval development in a non-apoptotic manner. In addition to the plasma membrane, the outer mitochondrial membrane has been shown to provide a non-apoptotic platform for caspase activation, in this case during sperm maturation. Therefore, membranes in general may provide a local environment for non-apoptotic caspase activities (Amcheslavsky, 2018).

The membrane localization of Dronc in SGs is mediated by Tango7, which has previously been implicated in spermatid maturation. As mentioned above, membrane-localized Dronc is required for dismantling of the cortical F-actin cytoskeleton in SGs of late larvae. However, while Tango7 RNAi blocks actin dismantling, Myo1D RNAi does not , suggesting that the roles of Tango7 and Myo1D for membrane localization of Dronc are different from each other. That also explains why in undead SGs the membrane localization of Dronc strongly increases in a Myo1D-dependent manner. Unfortunately, it was not possible to test if Tango7 is involved in AiP. Tango7 RNAi in eye imaginal discs results in complete loss of the disc. Tango7 encodes the homolog of eukaryotic translation initiation factor 3m (eIF3m), suggesting that it may also have an important requirement for protein translation, explaining the loss of the eye disc by Tango7 RNAi (Amcheslavsky, 2018).

In addition to Myo1D and Tango7, there is at least one other factor, Crinkled (Ck), which directs Dronc to non-apoptotic functions. Ck bridges the interaction between Dronc and the kinase Shaggy/glycogen synthase kinase beta (GSK-β), resulting in the selective activation of Shaggy/GSK-β, which then promotes non-apoptotic activities such as the specification of scutellar bristles, border cell migration, and correct branching of the aristae. Interestingly, Ck encodes another unconventional myosin, a member of the class VII myosin family, potentially suggesting that other myosins may also direct non-apoptotic functions to caspases (Amcheslavsky, 2018).

Myo1D and the apoptotic machinery have been linked to male terminalia rotation, an L/R process during pupal development. Indeed, apoptosis is required for Myo1D-dependent male terminalia rotation. It is unknown how Myo1D interacts with the apoptotic machinery to direct this L/R movement. In future studies, it will be interesting to examine if the Myo1D-dependent mechanism identified here for AiP also applies to male terminalia rotation or whether a separate mechanism exists in this context (Amcheslavsky, 2018).

Myo1D not only localizes Dronc to the plasma membrane, it also stabilizes it. Dronc is activated in undead cells, and activated Dronc is subject of increased protein degradation. Thus, Myo1D prevents degradation of Dronc by changing its subcellular localization to the plasma membrane (Amcheslavsky, 2018).

Myo1D has a very strong requirement for AiP in the undead model, and a requirement in the scrib-/-RasV12 tumorigenesis model, yet it does not appear to play any significant role in genuine AiP. In fact, Myo1D is the first gene identified that is essential for the hyper-proliferation of undead AiP, but not required for the regeneration of genuine AiP. The mechanism revealed in this paper provides an explanation for this behavior. During genuine AiP, cells are allowed to undergo apoptosis, which requires cytosolic Dronc activity. Although ROS are generated during genuine AiP, the origin of these ROS has not been determined and may not require the plasma membrane-localized Duox. Therefore, a key difference between genuine AiP and undead AiP, and potentially between other regenerative versus tumorigenic models, may be the altered localization of Dronc to a non-apoptotic compartment at the plasma membrane, and a shift from balanced apoptosis and proliferation to dominant proliferation. The next big question will be to examine what exactly is prompting Myo1D to drive this re-localization of Dronc under sustained undead conditions, but not under the limited regenerative conditions of the genuine AiP models, and whether that answer provides any insight into the cancer versus wound healing models (Amcheslavsky, 2018).

In conclusion, in addition to its role in L/R development, this study identified a second function of Myo1D for AiP and tumorigenesis. The basal side of the plasma membrane was identified as a non-apoptotic environment for caspase function. In future work, it will be important to identify the mechanisms by which Dronc mediates its non-apoptotic functions at the plasma membrane for AiP and other cellular processes that require membrane localization of Dronc and other caspases (Amcheslavsky, 2018).


REGULATION
Promoter

Dronc is an apical Drosophila caspase essential for programmed cell death during fly development. During metamorphosis, dronc gene expression is regulated by the steroid hormone ecdysone, which also regulates the levels of a number of other critical cell death proteins. As dronc protein levels are important in determining caspase activation and initiation of cell death, the regulation of the dronc promoter was analyzed using transgenic flies expressing a LacZ reporter gene under the control of the dronc promoter. These results indicate that dronc expression is highly dynamic during Drosophila development, and is controlled both spatially and temporally. While a 2.3 kb dronc promoter region contains most of the information required for correct gene expression, a 1.1 kb promoter region is expressed in some tissues and not others. During larval-pupal metamorphosis, two ecdysone-induced transcription factors, Broad-Complex and E93, are required for correct dronc expression. These data suggest that the dronc promoter is regulated in a highly complex manner, and provides an ideal system to explore the temporal and spatial regulation of gene expression driven by nuclear hormone receptors (Daish, 2003).

Experiments outlined in this paper demonstrate that 2.3 kb of the dronc promoter is largely sufficient for temporal expression (compared to endogenous dronc) throughout development. Previous experiments have shown that dronc is predominantly expressed in the larval and prepupal salivary glands and midgut, and larval brain lobes. 2.3 kb of the dronc promoter contains all necessary elements for correct spatial regulation of dronc expression in these tissues (Daish, 2003).

In order to identify transcription factors responsible for both temporal and spatial regulation of dronc and ecdysone-mediated PCD, it is of vital importance to elucidate the regions of the promoter essential for dronc expression in different tissues. In addition, it would be of interest to determine if there is a single promoter region controlling the spatial expression profile of dronc, or if different promoter regions are required in different tissues. LacZ transgenic reporter experiments reveal that the 2.3 kb promoter is the minimal requirement for correct expression in brain lobes and salivary glands. Furthermore, the region between 1.1 and 2.3 kb contains transcription factor-binding sites essential for expression in these tissues. This region also seems to harbor a repressor element important to keep dronc levels low during periods when ecdysone titers are low. Surprisingly, regulation of dronc transcription is markedly different in the midgut. The region between 1.1 and 2.3 kb is not important for transcription in this tissue, because 1.1 kb of the promoter is sufficient for expression. These results clearly demonstrate that distinct regions of the promoter are required for expression in different tissues, and implies that different transcription factors regulate dronc expression in a tissue-dependent manner (Daish, 2003).

The two ecdysone-induced transcription factors BR-C and E93 are essential for dronc expression in salivary glands. In the midgut, however, only E93 seems to be important. The results of dronc promoter-LacZ transgenic expression in flies deficient in BR-C and E93 are consistent with recent findings. LacZ expression driven by the 2.8 kb promoter is severely impaired in salivary glands of BR-C (rbp5 and npr) or E93 mutants, whereas expression is impaired only in the midgut of E93 mutant background animals. This further supports the idea that the mechanisms governing dronc regulation are tissue specific. The key questions arising from these experiments are: why does the BR-C Z1 isoform (rbp5 mutant) regulate dronc in the salivary glands and not in the midgut? What factors are binding to the 1.1-2.3 kb region of the promoter in salivary glands, and why are they not as important in the midgut? Previous results show that either BR-C Z1- or BR-C Z1-regulated proteins bind to the dronc proximal promoter and control its expression. Transactivation of the 2.8 kb promoter by BR-C Z1, however, was only seen in specific cell types. Given that BR-C Z1 is also expressed in the midgut, this implies that it may be acting through cofactors which are not expressed in the midgut, yet are specifically recruited to the dronc promoter. Alternatively, BR-C Z1 induces the expression of another factor which binds to the promoter, and this factor is absent in the midgut (Daish, 2003).

Since the proximal promoter alone (0.54 kb) is not sufficient for expression in the salivary gland, it is believed that BR-C Z1 (or a Z1-regulated protein) is cooperating with other transcription factors binding upstream (1.1-2.3 kb), that are essential for salivary gland expression. It has been shown that E93 acts through the first 600 bp of the dronc promoter by transactivation studies; however, no direct binding of E93 to the dronc (or any other) promoter has been shown so far. Additionally, a preliminary analysis indicates the presence of an EcR/Usp-binding site between 1.1 and 2.3 kb of the dronc promoter, and in vitro experiments show that this element may be important in regulating dronc expression. Since the proximal promoter (0.54 kb) alone is not sufficient for expression, cooperation of BR-C and E93 with EcR/Usp and other unknown factors may be important for temporal and spatial regulation of dronc expression during development. Identification of these factors will be important for fully understanding dronc transcription during development (Daish, 2003).

Overall, this study has established the minimal dronc promoter requirement for spatial and temporal expression to be within the 2.3 kb region upstream of the dronc gene. This region is important for both BR-C- and E93-mediated transcription in salivary glands and E93 transcription in the midgut. Importantly, the 1.1-2.3 kb promoter region harbors elements important for salivary gland expression and a putative repressor element. The 0.54-1.1 kb promoter region is important for expression in the midgut. These regions will form the basis of future experiments designed to identify factors necessary for the regulation of dronc expression during PCD (Daish, 2003).

Regulation of Dronc expression by ecdysone

The steroid hormone ecdysone has been shown to mediate apoptosis of larval tissues during pupariation. To investigate whether ecdysone also induces expression of dronc, the levels of dronc mRNA were examined after the addition of ecdysone to second instar larval midgut and salivary gland tissues, which normally show only low levels of dronc mRNA. After 1 hr exposure to ecdysone there was a several-fold increase in dronc mRNA levels in the early second instar larval midgut tissue, indicating that ecdysone can induce dronc expression in the midgut. However, salivary glands from early second instar larvae do not show dronc induction after ecdysone treatment. Because salivary glands normally undergo apoptosis later than midgut tissues, it is possible that the failure of ecdysone to induce dronc in salivary glands is the result of the absence of a developmentally controlled factor required for ecdysone-induced gene expression. For this reason, whether ecdysone could induce dronc expression in salivary glands at a later developmental stage was examined. Salivary glands from late second instar larvae, which normally only express very low levels of dronc, were found to strongly express dronc 1 hr after ecdysone treatment. Thus, ecdysone induces dronc expression in both midgut and salivary gland tissues (Dorstyn, 1999).

Characterization of Dronc enzymatic activity

To check whether DRONC is indeed a caspase, recombinant DRONC was generated in E. coli and its proteolytic activity was assessed on synthetic fluorogenic peptide substrates. Expression of both the full-length DRONC precursor or truncated DRONC lacking the putative prodomain (residues 1-113) generated enzyme that showed low-level activity on a caspase-3 substrate DEVD-afc. However, DRONC activity on the caspase-2 pentapeptide substrate VDVAD-amc was 5-fold higher than on DEVD-afc, suggesting that, similar to caspase-2, the minimum substrate requirement for DRONC includes a P5 residue. Under identical conditions, recombinant DRONC and caspase-2 show approximately similar activities on VDVAD and DEVD substrates. No significant cleavage by DRONC of caspase-1 substrate YVAD-afc was observed (Dorstyn, 1999).

Since DRONC interacts with drICE, the ability of active DRONC to cleave drICE CdeltaA, lamin DmO , the DNA fragmentation factor DREP-1 and the baculovirus caspase inhibitor p35 was assayed. Both DRONC and drICE cleave drICE CdeltaA, lamin DmO and DREP-1. The cleavage products generated by DRONC and drICE are clearly different, indicating that DRONC and drICE each cleave lamin DmO and DREP-1 at different sites. Unlike drICE, however, DRONC is unable to cleave p35. Together, these results indicate that dronc encodes a catalytically active protease and that its unique active site PFCRG pentapeptide confers upon it a different substrate specificity from classical caspases such as drICE that share the QAC(R/Q/G)(G/E) active site pentapeptide consensus (Meier, 2000).

Many caspases induce apoptosis when expressed in mammalian cells. It was therefore asked whether pro-DRONC, DeltaN DRONC or the catalytically inactive mutant of DeltaN DRONC (DeltaN DRONC CdeltaA) kill Rat-1 fibroblasts. Expression of DeltaN DRONC, which lacks its pro-domain, is very effective at inducing cell death, as is expression of either of the positive controls, caspase-8 and the Fas pathway adaptor FADD. However, in complete contrast, expression of full-length DRONC exerts no lethal effect. DRONC therefore resembles caspases-4 and -5 , both of which kill mammalian cells only when expressed without their respective pro-domains. As in S.pombe, the catalytically inactive DeltaN DRONC CdeltaA mutant has no effect on Rat-1 cell viability, consistent with a requirement for the caspase activity of DRONC to induce Rat-1 cell death. The lack of toxicity of full-length DRONC in Rat-1 cells is in stark contrast to the situation in S.pombe in which both pro-DRONC and DeltaN DRONC are toxic and undergo autocatalytic activation. One possible explanation for this discrepancy is that mammalian cells contain cellular factors that suppress pro-DRONC activation by binding its pro-domain. If true, deletion of the pro-domain in DeltaN DRONC would then render the caspase no longer inhibitable by such putative factors, resulting in the spontaneous activation of DeltaN DRONC and consequent cell death. Cell line-specific variations in levels of such putative inhibitory factors might explain why the efficacy with which pro-DRONC induces cell death is variable amongst different cell types. In this context, it is noteworthy that although pro-DRONC does not induce cell death in Rat-1 cells, it is lethal to NIH 3T3 cells (Meier, 2000).

Some caspases autocatalytically cleave and activate themselves. To determine if and where DRONC cleaves itself, a COOH-terminal His6-tagged version of DRONC was expressed and purified from E. coli. The purified protein consisted of two major bands, presumably consisting of the processed large and small subunits. This protein is active as a caspase. To determine the site of cleavage between the large and small subunits, Edman degradation amino-terminal sequencing was performed on the smaller band. The NH2-terminal sequence that was determined occurs COOH-terminal to the sequence TQTE352, suggesting that DRONC cleaves itself following a glutamate rather than an aspartate. To test this hypothesis DRONC TQTE352 was directly mutated to TQTA352. 35S-Labeled wild type DRONC and DRONC TQTAE352A were generated by in vitro translation and incubated with bacterially produced DRONC or DCP-1. DRONC cleaved itself to generate a product corresponding in size to the prodomain and large subunit. This band is not seen when DRONC TQTAE352A is the substrate, consistent with the hypothesis that DRONC processes itself following TQTE352. DCP-1 and drICE cleave DRONC at several sites. This cleavage is unaffected by the presence of the TQTA352 mutation, suggesting that these caspases cleave elsewhere in DRONC, perhaps in the DRONC prodomain. To explore the possibility of cleavage within the DRONC prodomain a form of DRONC, DRONCpD4A, was generated in which the P1 aspartates of four potential caspase target sites within the prodomain, DEKD66, ESVD110, DESD113, and DIVD135, were changed to alanine. 35S-labeled in vitro translated DRONCpD4A was still processed by wild type DRONC, but not by DCP-1. Similar results were obtained with cleavage by drICE. Thus DCP-1 and drICE can process DRONC within the prodomain, but not at the large-small subunit boundary. If this processing occurs in vivo it may serve as a point of regulation of DRONC function (Hawkins, 2000).

Positional scanning synthetic combinatorial libraries (PS-SCL) have been a useful tool to determine cleavage site specificities of other caspases. The PS-SCL is composed of three separate sublibraries of 8,000 compounds each. In each sublibrary, one position is defined with one of 20 amino acids (excluding cysteine), while the remaining two positions contain a mixture of amino acids present in approximately equimolar concentrations. Analysis of the three sublibraries (20 samples each) affords a complete understanding of the amino acid preferences in the P2, P3, and P4 positions. This approach was used to characterize DRONC's preferences for given amino acids at each of these positions. The positional scanning synthetic combinatorial libraries available all contain aspartate at the P1 position. While DRONC cleaves itself after glutamate, it is also able to cleave protein substrates after aspartate. Thus it was reasoned that the existing aspartate-based libraries would yield useful information about DRONCs cleavage specificity. DRONC shows a strong preference for Thr, Ile, or Val at the P2 position. A wider spectrum of amino acids was tolerated at the P3 and P4 positions. This analysis suggests that TATD constitutes an optimal DRONC P1 aspartate tetrapeptide cleavage site (Hawkins, 2000).

The results of the PS-SCL analysis are supported by experiments in which DRONC activity was tested directly with a number of commonly used tetrapeptide activity substrates. DRONC shows highest levels of activity with the tetrapeptides VEID-AMC and IETD-AMC, and somewhat lower levels of activity with DEVD-AMC. However, little if any activity is seen with WEHD-AMC or YVAD-AMC, which are predicted to be poor substrates. DRONC has higher levels of activity with the pentapeptide GIETD-AMC than with the tetrapeptide IETD-AMC. This suggests that a P5 residue is important for optimal DRONC activity (Hawkins, 2000).

To further characterize DRONCs cleavage preferences assays were carried out in which the cleavage activities of DRONC and DCP-1 were measured for two different peptide substrates: Ac-TQTE-AFC and Ac-DEVD-AFC. Ac-TQTE-AFC is derived from the known DRONC autoprocessing site and is also predicted to correspond to a good DRONC cleavage site based on the results obtained from PS-SCL analysis. Ac-DEVD-AFC is a tetrapeptide substrate for caspases generally grouped together as effectors of apoptosis (group II caspases). DRONCs activity is low in absolute terms compared with DCP-1. However, DRONC shows a clear cleavage preference for the Ac-TQTE-AFC substrate over Ac-DEVD-AFC. As expected, DCP-1, which has a common variant of the standard caspase active site pentapeptide (QACQG), has a strong preference for the tetrapeptide substrate with a P1 aspartate, Ac-DEVD-AFC (Hawkins, 2000).

Despite the fact that DRONC shows relatively low levels of activity with tetrapeptide substrates containing a P1 aspartate, DRONC is efficiently inhibited by the broad range tripeptide caspase inhibitor carbobenzoxy-VAD-fluoromethyl ketone (z-VAD-fmk) (Hawkins, 2000).

It was of interest to determine DRONC's P1 specificity with respect to aspartate and glutamate. To do this a second tetrapeptide substrate, Ac-TQTD-AFC, was synthesized that differs from Ac-TQTE-AFC only by the P1 residue. These substrates were used to measure DRONCs activity. DRONC shows only a slight preference for cleavage of tetrapeptide substrates with a P1 aspartate over those with a P1 glutamate. DRONC is, however, a particularly poor catalyst of tetrapeptide hydrolysis. The calculated activity values for DRONC are roughly 40-180-fold lower than those described for caspase-9, which itself is a very inefficient enzyme in isolation as compared with most other caspases. This may reflect the fact that DRONC has an intrinsically low turnover rate or that optimal in vitro assay conditions have not been identified. However, DRONC activity may also be regulated allosterically through interactions with the Drosophila homolog of Apaf-1, HAC-1, and DARK in a manner similar to that of mammalian caspase-9 by Apaf-1. Alternatively, since DRONC shows similar levels of activity to DCP-1 on the protein substrate drICE, optimal DRONC cleavage may require additional sequences surrounding the target site (Hawkins, 2000).

If DRONC is an apical cell death caspase, likely substrates include other Drosophila caspases. drICE is a good candidate to be such a target since immunodepletion experiments show that drICE is required for rpr-dependent apoptotic events in cell extracts, and genetic interactions suggest that DRONC contributes to rpr-, hid-, and grim-dependent cell death. 35S-labeled in vitro translated drICE was generated and it was incubated with bacterially produced DRONC or DCP-1. drICE was efficiently cleaved by DRONC and DCP-1. The generation of a band corresponding in size to that of the mature large subunit Ala29-Asp230 was observed. Several other cleavage products were also generated. These correspond to full-length drICE lacking the prodomain, Ala29-Val339 and a fragment comprising the prodomain and large subunit processed at the COOH terminus of the large-small subunit linker region, 1-Asp230. To show that DRONC and DCP-1 process drICE at TETD230, the proposed natural drICE cleavage site between large and small subunits, this site was changed to TETA230. DRONC and DCP-1 do not cleave 35S-labeled in vitro translated drICE TETA230 between the large and small subunit, implying that they both cleave drICE at TETD230. These results, taken together with the observed site of DRONC autoprocessing in bacteria and in vitro , and the results of tetrapeptide cleavage experiments, argue that DRONC cleaves following glutamate as well as aspartate. DRONC efficiently processesdrICE at QTETD230. However, it processes DCP-1 very poorly at the equivalent site in the large-small subunit linker, VTETD215. These results are consistent with the possibility that the optimal DRONC peptide substrate is a pentapeptide (Hawkins, 2000).

The drICE TETA230 mutant is still cleaved by DCP-1 at one position, perhaps at the prodomain-large subunit boundary. To explore this possibility the P1 aspartate of the proposed prodomain-large subunit boundary caspase target site, DHTD28, was altered to alanine, generating drICED28A. DRONC and DCP-1 both process drICED28A to generate a fragment corresponding in size to the prodomain and large subunit processed at Asp230. A slightly smaller band, probably corresponding to the prodomain and the large subunit processed at the NH2 terminus of the large-small subunit linker, 1-Asp217, was also produced. However, no bands corresponding in size to full-length drICE lacking the prodomain or the fully processed large subunit were observed. These observations demonstrate that DCP-1 processes drICE in the prodomain as well as at TETD230 (Hawkins, 2000).

Addition of DRONC to in vitro translated drICE results in production of a mature drICE large subunit lacking prodomain sequences, but DRONC is unable to process drICE TETA230 within the prodomain. These observations suggested that drICE cleaved by DRONC at TETD230 is autocatalytically removing its own prodomain. To test this possibility DRONC and DCP-1 were incubated with an in vitro translated version of drICE, drICEC211A, in which the active site cysteine was changed to alanine. This caspase should remain inactive following cleavage at TETD230. DRONC cleavage of drICEC211A results in the appearance of only a single band corresponding to the prodomain and large subunit. This observation suggests that drICE autocatalytically removes its own prodomain following cleavage between the large and small subunits. Mature drICEC211A large subunit was generated in the presence of DCP-1. This further supports the argument that DCP-1 cleaves drICE in the prodomain as well as at the large-small subunit boundary (Hawkins, 2000).

What purpose could be served by DRONC having an altered cleavage specificity? One possibility is simply that DRONC has unique targets other than itself, and that a different target site preference is required for cleavage of these substrates. DRONC's novel cleavage site specificity, in conjunction with the sequence of the linker between the large and small subunits, may also provide a mechanism for limiting DRONC's ability to become activated by other caspase cascades. DCP-1 or drICE do not process DRONC to any significant extent at the large-small subunit boundary. This is not surprising because there are only two aspartates in the linker region between the large and small subunits, DEYD324 and KWPD348. Based on positional scanning synthetic combinatorial library analysis of tetrapeptide substrates, these sequences are predicted to be very poor substrates for all known mammalian caspases and DCP-1. The possibility that processing of DRONC by unknown proteases occurs at these sites in vivo cannot be ruled out. However, because DRONC is able to process itself in the linker region at TQTE352, but other tested caspases are not, it seems reasonable that DRONC's altered cleavage specificity, coupled with the lack of good target sites for other caspases in the large-small subunit linker region, may serve at least in part to make activation of DRONC more strictly DRONC-dependent. This may provide a mechanism for limiting cross-talk between other caspase cascades and pathways activated by DRONC (Hawkins, 2000).

Regulation of Dronc induction of apoptosis

Although loss of the inhibitor of apoptosis (IAP) protein DIAP1 has been shown to result in caspase activation and spontaneous cell death in Drosophila cells and embryos, the point at which DIAP1 normally functions to inhibit caspase activation is unknown. Depletion of the DIAP1 protein in Drosophila S2 cells or the Sf-IAP protein in Spodoptera frugiperda Sf21 cells by RNA interference (RNAi) or cycloheximide treatment results in rapid and widespread caspase-dependent apoptosis. Co-silencing of dronc< or dark largely suppresses this apoptosis, indicating that DIAP1 is normally required to inhibit an activity dependent on these proteins. Silencing of dronc also inhibits Ice processing following stimulation of apoptosis, demonstrating that DRONC functions as an apical caspase in S2 cells. Silencing of diap1 or treatment with UV light induces DRONC processing, which occurs in two steps. The first step appears to occur continuously even in the absence of an apoptotic signal and to be dependent on DARK, because full-length DRONC accumulates when dark is silenced in non-apoptotic cells. In addition, treatment with the proteasome inhibitor MG132 results in accumulation of this initially processed form of DRONC, but not full-length DRONC, in non-apoptotic cells. The second step in DRONC processing is observed only in apoptotic cells. These results indicate that the initial step in DRONC processing occurs continuously via a DARK-dependent mechanism in Drosophila cells and that DIAP1 is required to prevent excess accumulation of this first form of processed DRONC, presumably through its ability to act as a ubiquitin-protein ligase (Muro, 2002).

Dronc induction of apoptosis

A number of caspases have been shown to induce apoptosis when overexpressed. DRONC was transiently co-expressed with ß-galactosidase in NIH 3T3 cells. At 48 hr posttransfection approximately 60% of the ß-galactosidase positive cells had undergone apoptosis. DRONC-induced cell death was almost completely abolished by coexpression of baculovirus P35 and inhibited to a lesser extent by MIHA, OpIAP, and Bcl-2. CrmA, an inhibitor of caspase-1, is least effective in inhibiting DRONC-induced apoptosis. A substitution mutation of the catalytic Cys-318 to Gly completely abolishes the cell-killing activity of DRONC, suggesting that cysteine protease activity is responsible for the apoptotic function of DRONC. The localization of DRONC protein was examined in transfected cells by using DRONC-GFP fusion constructs. Fusion of GFP to the carboxyl terminal of DRONC does not affect its cell-killing activity. At 24 hr, when most of the transfected cells appeared morphologically normal, DRONC was mostly localized in the cytoplasmic fraction of cells. In some cells, DRONC protein appeared to be concentrated asymmetrically near the cellular nucleus, possibly associated with some subcellular structures. Staining of transfected cells with mitochondrial markers suggests that DRONC does not localize to mitochondria. At 48 hr after transfection, DRONC-GFP protein is uniformly distributed in apoptotic cells (Dorstyn, 1999).

To determine whether ectopic expression of DRONC can induce cell death in D.melanogaster, the GAL4/UAS system was used to express various forms of DRONC in the developing Drosophila compound eye. Independent transgenic Drosophila lines were generated carrying pro-dronc, DeltaN dronc, pro-dronc CdeltaA, DeltaN dronc CdeltaA or dronc-card (the pro-domain of DRONC on its own) under the control of GAL4-upstream activating sequences (UAS). These flies were then crossed with Drosophila strains expressing GAL4 under the control of the glass multimer reporter in differentiating photoreceptors and pigment cells posterior to the morphogenetic furrow in the eye imaginal disc. The DRONC-induced phenotypes that were observed were of variable severity, depending on the insertion line used, presumably because of insertion site-specific effects on the transgene expression level. Accordingly, one representative weak UAS-pro-dronc (pro-droncW) and one representative strong UAS-pro-dronc line (pro-droncS) were selected for further characterization, along with one UAS-DeltaN dronc line (Meier, 2000).

Pro-droncW flies carrying one copy of the transgene exhibit a 'spotted eye' phenotype when crossed with GMR-gal4 flies: although pro-droncW flies are white+, and should therefore have red eyes, their eyes appeared white with occasional red spots. Such eyes have an essentially normal external morphology and size, in contrast to eyes expressing Rpr under the control of GMR, which are severely reduced in size. By comparison, pro-droncS and DeltaN dronc transgenic flies exhibit dramatically 'roughened eyes' that are severely reduced in size. Scanning electron microscopy (SEM) analysis of pro-droncS and DeltaN dronc eyes confirms that surface morphology is severely distorted, erupted and rough. As with pro-droncW flies, eyes from pro-droncS and DeltaN dronc flies are white, not red. This consequence of DRONC expression in eyes is particularly intriguing given that expression of Rpr dramatically reduces eye size yet has no effect on eye color. The phenotypes induced by DRONC expression are a consequence of DRONC caspase activity since overexpression of catalytically inactive CdeltaA mutants of DRONC exerts no detectable effect on eye development (Meier, 2000).

To investigate in detail the consequences of DRONC expression on the survival of photoreceptor and pigment cells underlying the eye surface, transverse sections of adult transgenic eyes were examined. Surprisingly, even in the pro-droncW flies, no normal cellular structures of either pigment or photoreceptor cells were visible: only remnants of pigment cells and vacuole-like structures remained. These remnant pigment cells, containing the red pigment pteridine, were responsible for the red 'spots' observed in the pro-droncW fly eyes. It was therefore concluded that GMR-driven DRONC expression kills both pigment and photoreceptor cells (Meier, 2000).

One possibility is that the ablation of internal eye structures seen in dronc transgenic flies may result from excess cell death in the developing eye disc. Third instar larval eye discs were examined for the appearance of apoptotic cells using acridine orange, which stains apoptotic cells. Compared with controls, third larval instar eye discs expressing DeltaN DRONC exhibit dramatic and super-numerary apoptosis posterior to the morphogenetic furrow. In contrast, no such sign of excessive apoptosis is evident in eye discs from third instar larvae expressing full-length pro-droncW. However, during later development (60 h after puparium formation), eye discs of pro-droncW pupae exhibit a dramatic increase in numbers of apoptotic cells. It is presumably this very late activation of apoptosis, essentially after the eye lens structure has formed, which gives the eyes of pro-droncW flies their characteristic morphology wherein the eyes show an essentially normal outer structure with internal ablation. In contrast, the devastating 'small eye' phenotype seen in pro-droncS, DeltaN dronc or GMR-rpr transgenic flies is consistent with the observed induction of cell death much earlier during larval eye development (Meier, 2000).

The pro-domain-less DeltaN DRONC generates a consistently more severe eye ablation phenotype than does pro-DRONC. Indeed, all DeltaN dronc transgenic lines die when crossed with GMR-gal4 and maintained at 25°C, although viability of some of these lines can be sustained by crossing them to a weak GMR-gal4 driver line and maintaining them at 18°C. The lethality is most likely not to be a trivial result of misexpression of GMR-gal4 in tissues other than the developing eye but, rather, to be due to the inability of DeltaN DRONC flies to open the pupae case with their heads because of extreme head malformation. As a consequence, such flies die trapped in their pupae cases. In confirmation of this, it was found that flies with severely deformed and black eyes can indeed be rescued by manually opening the puparium at the end of their development (Meier, 2000).

To examine the function of Dronc in a whole animal, transgenic flies were generated containing Dronc tagged with GFP or the inactive dronc mutant, droncC318G, also tagged with GFP, under the control of the yeast UAS(GAL4) in pUAST. Expression of these constructs was then achieved by crossing flies to various GAL4 drivers. To show that the constructs were expressed, UAS-dronc and UAS-droncC318G flies were crossed to flies containing the GMR-GAL4 driver, allowing expression in the posterior region of third instar larval eye imaginal discs. Eye imaginal discs from these third instar larvae stained specifically in the posterior region with antiGFP and antiDronc antibodies, demonstrating that high levels of specific expression were achieved and that the antiDronc antibody was specifically detecting Dronc protein. To determine whether ectopic overexpression of dronc could induce cell death, acridine orange staining of these eye imaginal discs was carried out to detect dying cells. Expression of the droncC318G construct has little effect on the normal pattern of dying cells in the eye imaginal disc, whereas wild type dronc expression results in a massive induction of cell death in the posterior part of the eye disc. Expression of dronc during embryogenesis or in different tissues during larval development using the heat shock-inducible hsp70-GAL4 driver also results in ectopic cell death (Quinn, 2000).

To examine the phenotypic consequence of expression of dronc in the eye disc, progeny of the cross of GMR-GAL4 to the UAS-dronc construct were allowed to develop into adults. Many died as pupae, which has been observed previously and attributed to the poor ability of the adults to break through the pupal case. The few adults from this cross that survive exhibit severely ablated eyes. By contrast, no death during the pupal stage was observed with flies from the cross of GMR-GAL4 to UAS- droncC318G, and adult flies showed normal eyes. Thus, the expression of dronc results in an almost complete ablation of the eye, similar to that obtained with expression of the apoptosis inducers rpr, hid, or grim from the GMR enhancer (Quinn, 2000).

Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).

Analysis of the Drosophila genome sequence reveals a single predicted transcript that encodes a type II membrane protein with structural similarities to members of the TNF superfamily. This protein is referred to as Eiger, in memory of the numerous mountaineers that have been killed by the Eiger Nordwand, the 'wall of death.' The Eiger protein contains a cytoplasmic domain, a transmembrane region between amino acid residues 36 and 62, and an extracellular domain of 353 amino acids. The C-terminal TNF homology domain (THD) of Eiger shows comparable homology to several human TNF family members (20%–25% identity). In situ hybridization has revealed a weak expression in imaginal discs with a pronounced pattern in the eye (Moreno, 2002).

Like a subset of human TNF ligands, Eiger can induce caspase-dependent apoptosis. Targeted expression of Eiger in the eyes and wings of Drosophila causes a severe ablation of these organs, and Eiger-expressing cell clones are rapidly eliminated. Both of these effects can be suppressed by coexpression of the pan-caspase inhibitor p35 (Moreno, 2002).

Caspase-8 is the key initiator caspase of death ligand-induced apoptosis in mammals. Upon stimulation by TNF, the adaptor protein FADD recruits and aggregates several molecules of procaspase-8 that mutually cleave and activate each other. Due to the involvement of an extracellular ligand, this pathway has been referred to as the 'extrinsic death pathway'. DREDD is the Drosophila caspase most similar to caspase-8 and has been shown to physically interact with Drosophila FADD. Surprisingly, complete removal of DREDD function fails to block Eiger-induced apoptosis, indicating that Eiger triggers cell death by a DREDD/caspase-8-independent pathway (Moreno, 2002).

The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).

Genetic interaction of dronc with apoptosis genes

To examine genetic interactions between Dronc and other apoptotic pathway genes, two UAS-dronc transgenic lines (#23 and #80) were chosen that result in relatively low lethality when crossed to GMR-GAL4 and a recombinant second chromosome was generated for each of these transgenes with GMR-GAL4. When GMR-GAL4 UAS-dronc#80 was crossed to wild type w1118 flies at 25°C, adult flies that exhibited slightly rough and mottled eyes were observed. A similar phenotype has been observed in previous studies and has been shown to be due to ablation of the pigment and photoreceptor cells. Similar results were observed for GMR-GAL4, UAS-dronc#23. This phenotype became more severe when expression of dronc via GMR-GAL4 was increased by raising the temperature to 29°C. Because this eye phenotype can be modified by increasing the expression of dronc, it provided a dosage-sensitive system for examining genetic interactions between dronc and other genes of the apoptosis pathway. To test this further, whether co-expression of the baculovirus caspase inhibitor P35 from the GMR enhancer was able to suppress the eye phenotype of GMR-dronc at 29°C was examined. Co-expression of GMR-p35 dramatically improves the eye ablation phenotype of GMR-dronc. Thus, in this system, Dronc is sensitive to P35 in the Drosophila eye (Quinn, 2000).

Whether Dronc is able to induce cell death in the hemocyte-derived SL2 cells was also examined. Surprisingly, transfection of these cells with full-length Dronc only resulted in 25% cell death. Because previous studies have shown that Diap1 binds to the prodomain of Dronc and may inhibit Dronc function, a truncated version of Dronc lacking the prodomain (MPD-Dronc) was transfected into SL2 cells. This resulted in a significant increase in cell death (50%). Because previous studies had failed to observe an effect of the caspase inhibitor P35 on dronc-induced cell death, a test was performed to see whether co-transfection of P35 could suppress MPD-Dronc-induced cell death. In contrast to previous results, P35 was able to significantly suppress MPD-Dronc-induced cell death in SL2 cells. This result is consistent with a previous observation showing that P35 inhibits Dronc-induced cell death in a mammalian overexpression system. However, it should be noted that co-expression of P35 does not rescue MPD-Dronc-induced cell death as well as Diap1, z-VAD-fluoromethylketone, or the dominant negative Dronc mutant, DroncC318G, although rescue is significantly better than that observed with Diap2 (Quinn, 2000).

Whether the GMR-dronc eye phenotype is sensitive to halving the dosage of the various Drosophila apoptosis-regulatory genes was tested. To assess whether the GMR-dronc eye phenotype is sensitive to the dosage of the H99 genes (reaper, hid, and grim), GMR-dronc flies were crossed to a deficiency removing the H99 genes, Df(3L)H99, at 29°C. The H99 deficiency dominantly suppressed the GMR-dronc eye phenotype. Thus, the cell death-inducing activity of dronc is sensitive to the dosage of the H99 genes. Furthermore, halving the dosage of dronc using a deficiency modifies the ablated eye phenotype of GMR-hid and GMR-rpr, suggesting that dronc is downstream of hid and rpr. To determine whether there was a genetic interaction with dronc and dark, whether decreasing the dosage of dark modified the eye phenotype of GMR-dronc at 29°C was examined. Three different P-element alleles of dark (darkCD4, darkCD8, and darkl(2)k11502) show suppression of the GMR-dronc eye phenotype, indicating that Dark plays a role in promoting Dronc-induced cell death in the eye. Halving the dosage of diap1 using deficiencies or the specific allele thread5 dominantly enhances the GMR-dronc eye phenotype at 25°C . In addition, these diap1 mutations dominantly enhance the lethality associated with GMR-dronc, resulting in at least 10-fold lower numbers of GMR-dronc/+; Df(diap1)/+ adult flies than expected. In contrast, a deficiency removing diap2 showed no effect on the GMR-dronc phenotype, and no lethal effects were observed. Thus diap1, but not a deficiency removing diap2, shows a dosage-sensitive interaction with dronc. By contrast, ectopic expression of diap1 or diap2 from the GMR promoter shows suppression of the GMR-dronc ablated eye phenotype, although GMR-diap2 results in much weaker suppression than GMR-diap1. Thus, both Diap1 and Diap2 are capable of directly or indirectly blocking Dronc-mediated cell death (Quinn, 2000).

Mutations that remove DRONC are not available. Therefore, to examine a possible role for DRONC as a cell death effector a form of DRONC, DRONCC318S, was generated in which the active site cysteine was altered to serine. Expression of similar forms of other caspases results in a suppression of caspase activity and caspase-dependent cell death. This may occur as a result of interaction of DRONCC318S with the Drosophila homolog of the caspase-activating protein Apaf-1, thus preventing the Drosophila Apaf-1 from binding to wild type DRONC and promoting its activation in a manner similar to that described for mammalian Apaf-1 and caspase-9. Transgenic Drosophila were generated in which DRONCC318S was expressed under the control of a promoter, known as GMR, that drives transgene expression specifically in the developing fly eye. The eyes of these flies, known as GMR-DRONCC318S flies, appear similar to those of wild type flies. To assay the ability of DRONCC318S to block cell death, GMR-DRONCC318S flies were crossed to flies overexpressing rpr (GMR-rpr), hid (GMR-hid), or grim (GMR-grim) under the control of the same promoter. GMR-driven expression of rpr, hid, or grim results in a small eye phenotype due to activation of caspase-dependent cell death. However, flies coexpressing GMR-DRONCC318S and one of the cell death activators showed a dramatic suppression of the small eye phenotype, indicating that cell death had been suppressed. The possibility cannot be ruled out that this suppression is a result of DRONCC318S forming nonproductive interactions with the Drosophila Apaf-1 that block its ability to activate other long prodomain caspases such as DCP-2/DREDD. However, these possibilities notwithstanding, these results suggest that DRONC activity is important for bringing about rpr-, hid-, and grim-dependent cell death (Hawkins, 2000).

Flies were generated that expressed full-length wild type DRONC under GMR control. While phenotypes displayed by individuals within a line were similar, different lines displayed eyes with various degrees of eye disruption, presumably owing to genomic position effects on the expression level of the transgene. By manipulating the number of copies of the GMR-DRONC transgene in animals, a phenotypic series was inferred in which low levels of DRONC expression (GMR-DRONCW flies) resulted in no outward phenotype, while higher levels of expression (GMR-DRONCM flies) resulted in cell death late in retinal development. These flies had eyes that were normal in size and shape, but that were largely white due to a loss of retinal pigment. Tangential sections through the eyes of GMR-DRONCM flies showed that all retinal cells, including photoreceptors, were missing. Increasing DRONC expression levels still further (GMR-DRONCS flies) resulted in flies with small eyes, similar to those seen in animals overexpressing rpr, hid, or grim. These observations show that DRONC expression in the eye induces cell death in a dose-dependent manner. Consistent with this interpretation, third instar eye imaginal discs from animals expressing GMR-DRONCS show high levels of staining with the vital dye acridine orange, which is taken up and retained by dying cells (Hawkins, 2000).

DIAP1, a Drosophila member of the IAP family of caspase inhibitors, suppresses rpr-, hid-, and grim-dependent cell death in the fly. It was reasoned that if expression of DRONC was activating the same pathway, then the GMR-DRONC eye phenotype might be sensitive to the levels of DIAP1. To test this hypothesis the amount of DIAP1 in the eye was decreased by crossing a strong loss-of-function DIAP1 point mutant, thread 5 (th5), to GMR-DRONCM flies. th5 heterozygotes are phenotypically wild type. However, flies that are heterozygous for th5, and that express GMR-DRONCM, show an enhancement of the GMR-DRONC-dependent small eye phenotype. In contrast, small eyed GMR-DRONCS flies that overexpress DIAP1 because they carry a GMR-DIAP1 transgene show a strong suppression of the small eye and pigment loss phenotypes. These observations, are consistent with the idea that DRONC activity is negatively regulated by DIAP1. However, they do not exclude the possibility that DIAP1's effects on the DRONC overexpression phenotypes are due, at least in part, to DIAP1's ability to suppress the activity of caspases such as drICE, that are activated by DRONC (Hawkins, 2000).

Genetic and biochemical evidence suggests that one mechanism by which RPR, HID, and GRIM promote apoptosis is by blocking DIAP1's ability to inhibit caspase activation or activity, thereby promoting caspase-dependent cell death. To determine if DRONCs activity could be regulated in a similar manner tests were performed to see whether RPR, HID, or GRIM could interfere with DIAP1-dependent inhibition of DRONC-dependent yeast cell death. Yeast were generated in which DRONC was expressed under GAL1 control and DIAP1 was expressed under the control of the copper-inducible CUP1 promoter. A third GAL1 vector was then introduced that was either empty or that expressed RPR, HID, or GRIM. Cells expressing GAL1-DRONC and empty vectors died when plated on medium containing galactose and 100 µM copper, but cells expressing GAL1-DRONC and CUP1-DIAP1 survived. Coexpression of GAL1-RPR had no effect on the survival of yeast expressing GAL1-DRONC and CUP1-DIAP1. However, coexpression of GAL1-HID or GAL1-GRIM completely blocked the survival of these cells. Thus, while these experiments do not exclude the possibility that HID and GRIM might alter DRONC activity directly, they are consistent with other observations arguing that these proteins mediate their effects on caspase activity, and thus presumably caspase-dependent yeast cell killing, by virtue of their interactions with DIAP1 (Watkins, 2000).

Reaper, Hid, and Grim are three Drosophila cell death activators that each contain a conserved NH2 -terminal Reaper-Hid-Grim (RHG) motif. The importance of the RHG motifs in Reaper and Grim have been examined for their different abilities to activate cell death during development. Analysis of chimeric R/Grim and G/Reaper proteins indicates that the Reaper and Grim RHG motifs are functionally distinct and help to determine specific cell death activation properties. A truncated GrimC protein lacking the RHG motif retains an ability to induce cell death, and unlike Grim, R/Grim, or G/Reaper, its actions are not efficiently blocked by the cell death inhibitors Diap1, Diap2, p35, or a dominant/negative Dronc caspase. Finally, a second region of sequence similarity was identified in Reaper, Hid, and Grim, that may be important for shared RHG motif-independent activities (Wing, 2001).

Do Reaper, Hid, and Grim share RHG-independent functions? Both truncated ReaperC and GrimC proteins induce cell death in developing tissues, indicating that regions outside the RHG motif also have death-inducing activities. Surprisingly, it was found that cell death induced by GrimC or ReaperC is only partially repressed by p35, suggesting a distinct mode of action compared with native Reaper or Grim. Similar to Reaper, Hid and Grim, GrimC does apparently act through Dronc, since GrimC-induced death is partially suppressed by a dominant/negative DroncC318S protein. However, the persistence of some eye cell death in the presence of DroncC318S indicates that GrimC and ReaperC also act through alternate pathways. Perhaps GrimC acts through pro-apoptotic Drosophila Bcl-2 orthologs that may induce cell death which is not blocked by p35. Another interesting possibilty is that GrimC might act via a Drosophila ortholog of Scythe, a Xenopus cell death regulator that binds Reaper, Hid, and Grim independently of the RHG motif (Wing, 2001 and references therein).

Role of Cytochrome C in caspase activation in Drosophila cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. Cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and the initiator caspase Dronc and effector caspase Ice are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, Debcl, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular Dronc and Ice appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, Dronc is recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals (Dorstyn, 2002).

In mammalian cell extracts, addition of cytochrome c and dATP results in the formation of an ~700-kD complex, commonly known as an apoptosome. Studies using purified components have demonstrated that the apoptosome, consisting of Apaf-1, cytochrome c, and procaspase-9, is necessary for caspase-9 activation. Since formation of an apoptosome in Drosophila has not been demonstrated and because cytochrome c is not released from mitochondria during apoptosis, whether a cytochrome c-dependent apoptosome containing Dronc is formed in Drosophila cells was tested. Cell extracts prepared from BG2 cells were fractionated by gel filtration chromatography and individual fractions were analyzed by immunoblotting using specific antibodies. In cell extracts kept at 4°C, the majority of Dronc was eluted in its monomeric form (50 kD) in fractions 20-22. Extracts that were incubated at 27°C with or without cytochrome c and dATP showed a shift of some of the Dronc protein to fractions 3-5, which correspond to a molecular mass of >670 kD. The shift in the absence of added cytochrome c may suggest that endogenous cytochrome c present in cell extracts could be sufficient to allow the formation of the large complex containing Dronc. Similar results have been seen using mammalian cell extracts. Drosophila cells grow at 27°C, however, when the cell extracts are incubated at 37°C, the majority of the Dronc is recruited to the >700-kD complex and there is increased processing of proDronc and proIce. The reason for this is not clear, however recombinant Dronc and Ice and extracts prepared from apoptotic BG2 cells show considerably more caspase activities at 37°C than at 27°C (Dorstyn, 2002).

Does the large complex contains Ice? In cell extracts incubated at 4°C, the majority of the Ice precursor remains in its monomeric form, although some appears to be dimeric. Incubation of cell extracts at 27°C or 37°C, with or without cytochrome c/dATP, results in the recruitment of a fraction of Ice to the high molecular mass complex. Interestingly, in extracts incubated at 37°C, most of the Ice in the high molecular mass complex is processed, whereas most of the monomeric Ice is in the precursor form. These results suggest the formation of an apoptosome containing Dronc and Ice in Drosophila cell extracts (Dorstyn, 2002).

To further explore the role of cytochrome c in the formation of the Dronc-containing complex, cytochrome c was immunodepleted from S100 fractions. These fractions were then subjected to gel filtration experiments. When incubated at 27°C, a small fraction of Dronc is found in the high molecular mass complex. Addition of cytochrome c and dATP causes a significant increase in the recruitment of Dronc to the >700-kD complex. Immunoblotting the fractions with the cytochrome c antibody shows that incubation of S100 at 27°C results in the recruitment of a significant proportion of cytochrome c to the >700-kD complex. Interestingly, only dimeric (26 kD) cytochrome c is detected in the >700-kD complex. These results suggest that cytochrome c and dATP, at least in part, are responsible for the formation of the complex (Dorstyn, 2002).

In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Silencing expression of either or both DC3 and DC4 has no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. Loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 fail to activate caspases in Drosophila cell lysates, but, remarkably, induce caspase activation in extracts from human cells. Overall, these results argue that DARK-mediated DRONC activation occurs independently of cytochrome c (Dorstyn, 2004).

The data clearly show that neither of the two cytochrome c species in Drosophila are required for caspase activation or apoptosis. Previous studies reported that a P-element insertion in the dc3 gene (bln1) results in loss of DRICE activity in testis (Arama, 2003). However, a recent report indicates that the bln1 P-element insertion also disrupts a number of other genes (Huh, 2004), thus questioning whether DC3 is responsible for DRICE activity. Additionally, DRICE activation during spermatogenesis appears to be independent of DARK and DRONC (Huh, 2004). If DC3 is required for caspase activation in Drosophila, a loss of function mutation in dc3 should lead to severe developmental defects and lethality. Furthermore, although a tissue-specific function has been suggested for DC3, it is unlikely that DC3 functions only during spermatogenesis, given its ubiquitous expression. Although disruption of the dc4 gene is embryonic lethal, DC4 cannot induce caspase activation and apoptosis in Drosophila cells (Dorstyn, 2004).

The question remains: how does DARK mediates DRONC activation? One possibility is that other factors can substitute for cytochrome c function during apoptosis. Alternatively, removal of DIAP1 from DRONC may be sufficient to allow an interaction with DARK and activation. Given that transcription plays a major role in developmental PCD in Drosophila, changes in the concentration of DIAP1, DRONC, and DARK proteins could facilitate caspase activation in the fly. These studies, combined with published work, demonstrate that Drosophila and mammalian cytochrome c proteins are functionally similar since they can both mediate respiration and Apaf-1 activation in mammalian cell lysates. Therefore, the requirement for cytochrome c in caspase activation in mammals is likely to have evolved late in evolution (Dorstyn, 2004).

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) results in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), are significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, this study demonstrates that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Liu, 2009).

The status quo action of JH has been well documented in several insect orders, particularly in Coleoptera, Orthoptera and Lepidoptera, in which JH treatment causes supernumerary larval molting and JH deficiency triggers precocious metamorphosis. However, as JH does not cause supernumerary larval molting in flies, evidence for the status quo action of JH in Drosophila has remained elusive. From past studies and from the experimental data presented in this study, it is concluded that the status quo hypothesis does indeed apply to JH action in Drosophila. First, although JH application during the final larval instar or during the prepupal stage has little effect on the differentiation of adult head and thoracic epidermis in Drosophila, it does prevent normal adult differentiation of the abdominal epidermis. After JH treatment, a second pupal, rather than an adult, abdominal cuticle is formed in Diptera. Second, JH or a JH agonist applied to Drosophila at the onset of metamorphosis results in lethality during pupal-adult metamorphosis. Similarly, global overexpression of jhamt (Juvenile hormone acid methyl transferase) results in severe defects during the pupal-adult transition and eventually death (Niwa, 2008). Third, CA ablation leading to JH deficiency causes precocious and enhanced fat body PCD. Fourth, JH deficiency results in pupal lethality and delayed larval development, although JH deficiency is not sufficient to cause precocious metamorphosis. The composite data demonstrate that JH in Drosophila does have status quo actions on the abdominal epidermis during pupal-adult metamorphosis and on the fat body during larval-pupal metamorphosis. It is concluded that the status quo action of JH in Drosophila is functionally important, but more subtle than that in Coleoptera, Orthoptera and Lepidoptera. However, it is not clear whether JH is essential for embryonic and earlier larval development because the CA cells are not completely ablated in the JH-deficient animals until the early-wandering (EW) stage. To address this question, it would be necessary to generate a mutant (i.e., of jhamt) that interrupts JH but not the farnesyl pyrophosphate biosynthesis pathway (Liu, 2009).

The insect fat body is analogous to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. In response to 20E pulses, Drosophila larval organs undergo a developmental remodeling process during metamorphosis. Blocking the 20E signal specifically in the fat body during the larval-pupal transition (Lsp2>; UAS-EcRDN) prevented the fat body from undergoing PCD and cell dissociation (Liu, 2009).

The experimental data in this paper demonstrates that JH prevents caspase-dependent PCD in the fat body during the larval-pupal transition in Drosophila. First, JH deficiency in Aug21>, UAS-grim resulted in the fat body undergoing precocious and enhanced PCD and cell dissociation. Aug21> is a GAL4 driver that specifically targets gene expression to the CA. Precocious and enhanced apoptosis appeared as early as L3D1 in the JH-deficient animals. Methoprene application on L3D1 was able to rescue ~40% of the pupae to adults, but it failed to rescue post-EW. Second, 2D-DIGE/MS and qPCR analyses indicated that the fat body in the JH-deficient animals has multiple developmental defects. The upregulation of the caspase genes Dronc and Drice should account for the PCD in the fat body, as overexpression of Dronc in the fat body causes PCD, cell dissociation, and thus lethality. Overexpression of Dronc or Drice in cells and tissues is sufficient to cause caspase-dependent PCD. Third, the 20E-triggered transcriptional cascade in the fat body was downregulated in the JH-deficient animals, indicating that JH does not suppress the 20E-triggered transcriptional cascade in preventing caspase-dependent PCD in the fat body (Liu, 2009).

The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of EcR. One might expect that perfect timing, titer and receptor response of JH and 20E are required to ensure accurate PCD in a tissue- and stage-specific manner during Drosophila metamorphosis. In the JH-deficient animals, the upregulation of Dronc and Drice resulted in precocious and enhanced PCD, such that the JH-deficient animals are committed to die during the larval-pupal transition. This hypothesis was strengthened by overexpression of Dronc specifically in the fat body, which caused larval lethality. Taken together, it is concluded that JH antagonizes 20E-induced caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Liu, 2009).

Based on the phenotypes and gene expression profiles in the four fly lines used, it is concluded that JH counteracts MET and GCE to prevent caspase-dependent PCD. First, the Met-overexpressing animals died during larval life, with precocious and enhanced PCD and cell dissociation in the fat body. Dramatic upregulation of Dronc and Drice was observed when Met was specifically overexpressed in the fat body and this upregulation was significantly decreased by methoprene application demonstrating that JH is epistatic to MET and GCE. Moreover, the Dronc-overexpressing animals exhibited similar phenotypes to the Met-overexpressing animals. Second, in the fat body of the JH-deficient animals, PCD and the expression of Dronc and Drice were upregulated but not as significantly as in the Met-overexpressing animals. This might explain why the JH-deficient animals did not die until early pupal life. Third, both the global JH-overexpressing animals and the Met/gce-deficient animals died during the pupal-adult transition. In these animals, Dronc and Drice were downregulated and caspase-dependent PCD was decreased in the fat body, implying that these animals died from a lack of caspase-dependent PCD. Weak mutants of Dronc and Drice mutants die during pupal life, showing that caspase-dependent PCD is essential for Drosophila metamorphosis. In addition, it was also observed that methoprene application at the onset of metamorphosis results in delayed fat body remodeling (Liu, 2009).

In the future, it will be crucial to elucidate the detailed molecular mechanism of how JH counteracts MET and GCE to prevent caspase-dependent PCD. In Drosophila S2 cells, the transcriptional activity of MET is dependent on the JH concentration and both MET-MET and MET-GCE interactions can be greatly diminished by JH. The bHLH-PAS transcription factors typically function as hetero- or homodimers. If MET/GCE is the Juvenile Hormone Receptor (JHR), the transcriptional activities of the dimerized MET/GCE and the JH-MET/GCE complex should differ. In other words, the dimerized MET/GCE should induce transcription of Dronc and Drice and, in turn, JH binding to form the JH-MET/GCE complex should reduce this induction. Although there are no examples in the literature in which a receptor, without ligand, acts as a transcriptional activator and the transcriptional activity of the receptor is diminished when the ligand is bound, it could be speculated that the JHR is a unique hormone receptor and perhaps that is the reason why it has yet to be isolated and characterized. Unfortunately, the experiments described here were conducted in Drosophila S2 cells, where the possibility of an endogenous JHR could not be eliminated. Although MET/GCE is definitely a key component in the JH signal transduction pathway, whether MET/GCE is the bona fide JHR remains conjecture (Liu, 2009).

It is very likely that MET cross-talks with EcR-USP via a large molecular complex. One can hypothesize that MET promotes 20E action in the absence of JH and suppresses 20E action in the presence of JH, a model which is favored. Drosophila FKBP39 (FK506-BP1) could be a key component in this complex because it physically interacts with MET, EcR and USP, and binds the D. melanogaster JH response element 1. Moreover, Drosophila FKBP39 inhibits 20E-induced autophagy (Juhász., 2007). Further analysis of the complex will be crucial to precisely define the molecular mechanism of cross-talk between the action of JH and 20E (Liu, 2009).

In summary, it is concluded that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila. The Drosophila fat body has provided an excellent model for studying the long-standing question of JH signal transduction. To finally settle the question of the bona fide JHR and to understand the precisely defined molecular mechanism of JH action requires further research at a variety of levels in several species of insects that can be genetically manipulated, such as Drosophila, Bombyx and Tribolium (Liu, 2009).

Caspase signalling in the absence of apoptosis drives Jnk-dependent invasion

Tumours evolve several mechanisms to evade apoptosis, yet many resected carcinomas show significantly elevated caspase activity. Moreover, caspase activity is positively correlated with tumour aggression and adverse patient outcome. These observations indicate that caspases might have a functional role in promoting tumour invasion and metastasis. Using a Drosophila model of invasion, this study shows that precise effector caspase activity drives cell invasion without initiating apoptosis. Affected cells express the matrix metalloprotinase Mmp1 and invade by activating Jnk. These results link Jnk and effector caspase signalling during the invasive process and suggest that tumours under apoptotic stresses from treatment, immune surveillance or intrinsic signals might be induced further along the metastatic cascade (Rudrapatna, 2013).

Overall, these results indicate that effector caspase activity below levels sufficient to direct cell death might be optimal for migration of transformed cells. This signalling promotes migration through Jnk, consistent with previous studies showing that Jnk lies downstream of Dronc. Caspase activation of Jnk frequently leads to compensatory proliferation, a homeostatic programme of cell replacement after apoptosis. Compensatory proliferation studies of 'undead cells' have come to opposite conclusions concerning the role of Drice. The current work is consistent with the mammalian literature placing the JNK pathway as a caspase target (Rudrapatna, 2013).

Effector caspases are active in tumours in situ and are associated with metastasis; the current results indicate that cells with moderate caspase activity that are protected from apoptosis are prone to migration. In this view, therapeutic interventions proposed to increase tumour apoptosis might paradoxically exacerbate malignancy, as has been previously suggested. Tumour inflammation has also been suggested to promote metastasis and might do so via stimulation of the extrinsic apoptosis pathway. Tumour cells commonly contain high levels of XIAP, which blocks caspases' active site in a manner similar to P35. This might provide an important mechanism directing tumours to metastasize, though the experiments emphasize the importance of precise caspase activity. A better understanding of caspases' role in tumour progression might enhance ability to predict a tumour's progression and the impact of treatments designed to promote the apoptosis process (Rudrapatna, 2013).

Protein Interactions

The physical interaction between DRONC and drICE was assessed by testing for the ability of the two proteins to co-immunoprecipitate from cell extracts. FLAG-tagged, full-length, catalytically inactive DRONC (pro-DRONC CdeltaA, 1-451) was co-expressed in 293T cells together with Myc-tagged catalytically inactive pro-drICE CdeltaA (1-339), DeltaN drICE CdeltaA (29-339) or Bcl-10. The mammalian protein Bcl-10 that contains an N-terminal CARD was used as the control in the co-immunoprecipitation experiments. Pro-DRONC specifically co-immunoprecipitates both pro-drICE and DeltaN drICE, but not Bcl-10, indicating that DRONC and drICE form a stable complex in cell extracts (Meier, 2000).

The observed difference between the pro-apoptotic activity of pro-DRONC and pro-domain-lacking DeltaN DRONC in Drosophila and mammalian cells raises the possibility that spontaneous activation of pro-DRONC is suppressed through interaction of its pro-domain with some putative cellular inhibitor. To identify such an inhibitor, Drosophila proteins were sought that interact specifically with the DRONC pro-domain in a yeast two-hybrid assay using a 0-24 h Drosophila embryonic cDNA library. From 1 × 106 yeast transformants, 56 DRONC-interacting clones were recovered, of which 17 encoded DIAP1. The second BIR domain of DIAP1 is necessary and sufficient for the interaction with the pro-domain of DRONC. This is particularly intriguing since the BIR2 region of DIAP1 is also known to interact physically with, and block the pro-apoptotic activity of, Rpr, Grim and Hid (Meier, 2000).

To verify the observed interaction between DIAP1 and DRONC, co-immunoprecipitation experiments were performed on cellular extracts obtained from 293T cells. FLAG-tagged pro-DRONC CdeltaA, DeltaN DRONC CdeltaA and DRONC-CARD (the pro-domain of DRONC on its own) were each tested for interaction with Myc-tagged DIAP1 deletion mutants (BIR1/2, 1-341; BIR1, 1-146; and BIR2, 177-341). As expected, full-length DRONC and the isolated pro-domain of DRONC (DRONC-CARD) both co-immunoprecipitate with BIR1/2 and BIR2 but not with BIR1, consistent with yeast two-hybrid data showing that the BIR2 domain of DIAP1 is required for the interaction with DRONC. Somewhat surprisingly, however, DeltaN DRONC lacking the pro-domain also co-immunoprecipitated with DIAP1, although to a far lesser extent than full-length DRONC or DRONC-CARD. The BIR2 region of DIAP1 is required for this interaction between DeltaN DRONC and DIAP1, since DeltaN DRONC forms stable complexes only with BIR1/2 and BIR2 and not with BIR1. Taken together, these results indicate that DIAP1 physically interacts with unprocessed pro-caspase DRONC and that the BIR2 region of DIAP1 is able to bind both the pro-domain and the core region of DRONC (Meier, 2000).

Several lines of evidence suggest that pro-DRONC activation is negatively regulated via its pro-domain. This is best illustrated by the biology underlying the relatively weak eye phenotype in flies expressing full length pro-droncW: (1) most UAS-pro-dronc lines are viable when crossed to a strong GMR-gal4 line and kept at 25°C: in contrast, virtually all GMR-driven DeltaN dronc transgenic lines tested die under such conditions; (2) most pro-dronc lines exhibit essentially normal outer eye structure, whereas rare surviving DeltaN dronc transgenic flies never display this 'weak' eye phenotype and have severely deformed eyes; (3) ectopic expression of pro-DRONC induces no significant increase of cell death in the eye discs of third instar larvae, whereas excessive cell death is evident posterior to the morphogenetic furrow in the eye discs of third instar larvae expressing DeltaN DRONC; (4) ectopic expression of DRONC in mammalian Rat-1 cells induces apoptosis only when its pro-domain had been removed, suggesting the existence of an innate inhibitor of DRONC activation acting through the DRONC-CARD domain. All of these observations implicate the DRONC pro-domain in repressing activation of the caspase and suggest that DRONC activation is kept in abeyance in metazoan cells through the action of some CARD-binding innate inhibitor. In contrast, studies of DRONC in S.pombe unambiguously show that isolated pro-DRONC is, by itself, perfectly capable of undergoing catalytic autoprocessing resulting in its activation. Indeed, in yeast, pro-DRONC proved more toxic than DeltaN DRONC, suggesting that the presence of the pro-domain may actually enhance DRONC activation in the absence of other modulating influences (Meier, 2000).

In insect cells, a candidate for such an innate DRONC repressor is the inhibitor of apoptosis, DIAP1, which interacts with the DRONC pro-domain: co-expression of DIAP1 completely reverts the eye ablation phenotype of pro-droncW flies, whereas the eye ablation phenotype induced by DeltaN DRONC is largely unaffected. If endogenous DIAP1, or an analog, were not expressed in the Drosophila eye until very late in its development, this would provide the requisite mechanism for holding the activity of DRONC in abeyance until very late, so generating the 'spotted eye' phenotype observed. Indeed, heterozygosity at the diap1 locus greatly enhances the eye phenotype induced by pro-DRONC overexpression, indicating that endogenous DIAP1 negatively regulates DRONC activation in vivo. This is analogous to the way in which c-IAP1, c-IAP2 and XIAP bind to, and inhibit activation of, the pro-form of the apical caspase-9 in mammalian cells. The notion that it is DIAP1, in particular, that most likely fulfils the role of in vivo suppressor of DRONC is reinforced by studies in yeast that show that while DIAP1 both interacts with, and protects from the lethal effects of, pro-DRONC, Drosophila DIAP2 and the mammalian IAP homologs MIHA, MIHB, MIHC, MIHD and XIAP offer no such protection (Meier, 2000).

Currently, very little is known about how IAPs suppress apoptosis, although the most convincing biological evidence for the ability of IAPs to regulate cell death comes from genetic studies in D.melanogaster. Deletion of the chromosomal region encoding DIAP1 enhances cell death induced by ectopic expression of Rpr, and genetic loss of DIAP1 function leads to early and widespread apoptosis, indicating that DIAP1 is essential for survival of many cell types. Furthermore, overexpression of DIAP1 suppresses cell death induced by either Rpr, Grim or Hid through direct interaction between these various pro-apoptotic proteins and the second BIR domain of DIAP1, the same BIR domain that is sufficient for its interaction with pro-DRONC. It is noteworthy that it is also the second BIR repeat of the mammalian IAP family members c-IAP1, c-IAP2 and XIAP that appears sufficient for their anti-apoptotic activity (Meier, 2000 and references therein).

The finding that DIAP1 directly binds to and inhibits cell death caused by ectopic expression of DRONC, as well as by Rpr, Grim and Hid, underscores the key role played by DIAP1 in the regulation of apoptosis in D.melanogaster and raises the possibility that Rpr, Hid or Grim may exert some, or all, of their pro-apoptotic action through displacement of DIAP1 from the pro-domain of DRONC, so allowing activation of the caspase and consequent cell death. The isolation of DIAP1 mutants that display greatly reduced binding for Rpr, Hid and Grim and significantly suppress Rpr, Hid and Grim cell killing strongly supports this idea. According to this model, IAPs function as 'guardians' of the apoptotic machinery: these guardians act to suppress the chance of spontaneous activation of the intrinsic cell death machinery by neutralizing pro-apoptotic caspases, so establishing a buffered threshold that must be either exceeded or neutralized in order to initiate the destruction of a cell (Meier, 2000).

The biochemical interaction of Dronc with various apoptosis regulators was examined by co-precipitation methods. When 293T cells were transfected with Dronc-GFP and Diap1-Myc or Diap2-Myc, it was found that Diap1, but not Diap2, was present in the Dronc immunoprecipitated complex, showing that Dronc and Diap1 can interact. Dronc can form a complex with Diap1, but not with Diap2, in SL2 cells. The H99 proteins, Rpr, Hid, and Grim, were examined after transfecting 293T cells with Dronc-GFP and with Rpr-FLAG, Hid-FLAG, or Grim-FLAG constructs. Proteins associating with antiGFP-immunoprecipitated Dronc were immunoblotted with the antiFLAG antibody. Dronc co-immunoprecipitates with Grim, but not with Rpr or Hid, suggesting that Dronc forms a complex with Grim. Whether Dronc can form a complex with P35 was examined by transfecting SL2 cells with Dronc tagged with the HA and 6xHis epitopes and with HA-tagged P35. P35 is present in the Dronc complex, indicating that these proteins can associate (Quinn, 2000).

To determine whether the interactions of Dronc with Grim or P35 are direct, whether these proteins can interact in vitro was tested. Whereas Grim can bind to Diap1 in vitro, Dronc is unable to bind to either Grim or P35. Thus, under the conditions where Grim and Diap1 can interact, Dronc and P35 or Grim do not interact, suggesting that the interaction observed between Dronc and Grim or P35 in cells is indirect. Furthermore, the addition of Diap1 allows Grim to be co-immunoprecipitated with Dronc, suggesting that the complex formed in vivo between Dronc and Grim may be mediated by Diap1 (Quinn, 2000).

The amino-terminal region of Apaf-1-related-killer (Ark) containing the CARD and CED-4/Apaf-1 homology domains but lacking the WD40 repeats has been shown to bind to Dredd and Drice. Furthermore, in mammalian tissue culture cells, Dark has been shown to bind to Dronc. To investigate the region of Dark required for the interaction of Dark with Dronc, SL2 cells were transfected with a construct containing a Myc-tagged Dark amino-terminal region [Ark(1-411)], containing the CARD and CED-4/Apaf-1 homology domains, alone or with a FLAG-tagged Dronc construct. Dark-Myc was immunoprecipitated with antiMyc antibodies; pelleted complexes, and cell lysates were analyzed by immunoblotting with antiFLAG antibodies. Dronc-FLAG protein (50% of the protein present in the lysate) was detected in the antiMyc-Arc immunoprecipitate. Thus, the amino-terminal region of Dark containing the CARD domain and the CED-4/Apaf-1 homology region is sufficient for Dronc association in vivo (Quinn, 2000).

To test the requirement of Ark for Dronc activation, extracts prepared from darkCD8 homozygous flies were examined for caspase activity and for their ability to cleave Dronc in vitro. Ark mutant flies have reduced caspase activity compared with wild type for several caspase substrates. VEID is the preferred substrate for Dronc, although VDVAD is also cleaved well by Dronc. DEVD is a caspase-3 substrate that is cleaved poorly by Dronc but preferred by the downstream caspases Dcp1, Decay, and Drice. Thus, Ark mutant extracts contain lower cleavage activity toward both preferred Dronc substrates and preferred downstream caspase substrates. Lower caspase activity has also been observed in extracts from Ark mutant embryos. Furthermore, Ark mutant extracts showed considerably reduced ability to cleave Dronc to its active form, showing that Ark is important for Dronc processing. Because darkCD8 (and darkCD4) are hypomorphic mutants, and it is not known whether they are completely null because a deficiency of the Ark region is not available, the residual Dronc processing observed may be due to residual Ark activity or to an alternative mechanism (Quinn, 2000).

Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival in Drosophila and appear to neutralize the cell death machinery by binding to and ubiquitylating pro-apoptotic caspases. Cell death is triggered when 'Reaper-like' proteins bind to IAPs and liberate caspases from IAPs. The thioredoxin peroxidase Jafrac2 has been identified as an IAP-interacting protein in Drosophila cells that harbors a conserved N-terminal IAP-binding motif. In healthy cells, Jafrac2 resides in the endoplasmic reticulum but is rapidly released into the cytosol following induction of apoptosis. Mature Jafrac2 interacts genetically and biochemically with DIAP1 and promotes cell death in tissue culture cells and the Drosophila developing eye. In common with Rpr, Jafrac2-mediated cell death is contingent on DIAP1 binding because mutations that abolish the Jafrac2-DIAP1 interaction suppress the eye phenotype caused by Jafrac2 expression. Jafrac2 displaces Dronc from DIAP1 by competing with Dronc for the binding of DIAP1, consistent with the idea that Jafrac2 triggers cell death by liberating Dronc from DIAP1-mediated inhibition (Tenev, 2002).

Jafrac2 was recovered as a DIAP1-interacting protein in the cell using the tandem affinity purification (TAP) system. Like Rpr, Grim, Hid, Sickle, Smac/DIABLO and HtrA2/Omi, Jafrac2 bears a conserved N-terminal IAP-binding motif (IBM) essential for IAP interaction. Jafrac2 is synthesized as a precursor protein with an N-terminal signal peptide that targets it to the ER. Upon import into the ER, the signal peptide of Jafrac2 is cleaved off, thereby exposing the IAP interacting domain that allows this mature Jafrac2 isoform to interact with DIAP1, DIAP2 and XIAP (Tenev, 2002).

In living cells Jafrac2 is compartmentalized and sequestered in the ER away from IAPs, where it exists exclusively in the processed from. This is evident because mature Jafrac2, like cytochrome c, which is compartmentalized in mitochondria, remains associated with the membrane fraction in healthy cells. Following stimulation of apoptosis by UV irradiation or ER stress-inducing agents, mature Jafrac2 is released from the membrane fraction and is present in the cytosol where it can interact with DIAP1 and DIAP2. Because the pro-apoptotic, IAP-interacting form of Jafrac2 is released only upon cell death insult, the major regulatory step for Jafrac2 appears to be its release from the ER lumen. The release of Jafrac2 from the ER of UV-irradiated cells occurs early in UV-mediated apoptosis. This is evident because Jafrac2 expression becomes diffuse in otherwise morphologically normal cells within 3-4 h following UV exposure. In similar experiments, the mitochondrial release of cytochrome c, Smac/DIABLO and HtrA2/Omi that occurs, early in apoptosis, also became apparent within 3-4 h following UV treatment. Thus, Jafrac2 resembles Smac/DIABLO and HtrA2/Omi that are similarly compartmentalized in healthy cells and that promote caspase activation after their release from mitochondria following the cell death trigger. Furthermore, analogous to Smac/DIABLO and HtrA2/Omi, Jafrac2 also requires N-terminal processing to generate its pro-apoptotic form. Hence, Jafrac2, Smac/DIABLO and HtrA2/Omi all undergo a maturation process through cleaving off their signal peptide following import into their respective organelles. This organelle-specific maturation ensures that newly synthesized Jafrac2, Smac/DIABLO and HtrA2/Omi will not promote apoptosis prior to their sequestration into organelles (Tenev, 2002).

In common with Rpr, Grim and Hid, Jafrac2 interacts genetically and biochemically with DIAP1 and is able to promote cell death. In the Drosophila eye and tissue culture cells, mature Jafrac2, like Rpr, efficiently induces cell death in a DIAP1-binding dependent manner. Recent studies have suggested that Rpr and Grim antagonize the anti-apoptotic activity of IAPs by two distinct mechanisms -- (1) by a mechanism that requires DIAP1 binding, Rpr promotes DIAP1 self ubiquitylation and proteasomal degradation and (2) Rpr and Grim were also found to repress global protein translation by a mechanism that does not rely on IAP binding. The Ub fusion technique has been used to examine whether Jafrac2 and Rpr possess apoptosis-promoting activities that are independent of IAP binding. In vivo Rpr and Jafrac2 promote cell death exclusively in an IAP-binding dependent manner because mutations that impair the binding between DIAP1 and Rpr or Jafrac2 completely abolish their ability to induce cell death in the developing eye and tissue culture cells. Thus, Rpr and Jafrac2 that fail to bind to DIAP1 also fail to induce cell death. Mutations in endogenous diap1, which greatly impair the binding of DIAP1 to Rpr or Jafrac2, suppress Rpr and Jafrac2-mediated cell killing. Together, these data argue that in common with Rpr, mature Jafrac2 promotes cell death, and this activity is contingent upon their binding to DIAP1 (Tenev, 2002).

The interaction between Jafrac2 and the DIAP1 BIR2 domain is indispensable for its pro-apoptotic function. Interestingly, Jafrac2 and Dronc share a common binding site in the BIR2 domain that is distinct from the site of interaction between the DIAP1 BIR2 domain and Rpr and Hid. The th4 mutation of DIAP1's BIR2 domain greatly diminishes binding to Jafrac2 and Dronc, whereas the same mutation does not affect its binding to Rpr and Hid. In addition, the th23-4 DIAP1 mutation that greatly impairs the binding of DIAP1 to Rpr and Hid does not affect the DIAP1-Jafrac2 interaction. Consistent with the biochemical data, flies carrying the th4 mutation, which abolishes Jafrac2 binding, display strongly suppressed Jafrac2-induced eye ablation but enhanced Rpr-induced cell death in the eye (Tenev, 2002).

Several lines of evidence show that the IBM of Jafrac2 is essential for IAP binding and induction of apoptosis. Mutations that delete or obstruct the N-terminus of mature Jafrac2 abrogate the ability of Jafrac2 to bind to DIAP1 and trigger cell death. The view that Jafrac2 harbors a bona fide IAP-binding motif is strongly supported by crystal structure analyses that have identified Ala1 of IBMs as the critical residue to anchor this motif to the BIR surface of IAPs. In addition to the requirement of Ala1, there is a strong preference for Pro3. In accordance with other IBMs, the putative IBM of mature Jafrac2 bears Ala1 and Pro3. Furthermore, the IBM of Rpr is functionally interchangeable with the IBM of Jafrac2. A chimeric Rpr mutant (AKP-Rpr) in which the IBM of Rpr was replaced with the IBM of Jafrac2, displayed the same phenotype and cell death promoting efficacy as wild-type Rpr (AVA-Rpr) in both the Drosophila developing eye and tissue culture cells. Together, these results reveal that whereas Jafrac2 and Rpr share a common IAP-binding motif, they also have some distinct DIAP1-binding requirements that presumably give these interactions their specificity (Tenev, 2002).

Physical interaction between DIAP1 and caspases is essential to regulate apoptosis in vivo because embryos with a homozygous mutation that abolishes Dronc binding die early during embryogenesis due to widespread apoptosis. Unrestrained cell death caused by loss of DIAP1 function requires the Drosophila Apaf-1 homolog DARK because a mutation in dark rescues DIAP1-dependent defects. Thus, loss of DIAP1 function allows DARK-dependent caspase activation. Although activation of downstream, effector caspases is required for normal cell death, the activation of initiator caspases, such as Dronc, is rate limiting for the activation of this cascade. The observed unrestrained cell death caused by loss of DIAP1 function is likely to be triggered by the initiator caspase Dronc because DIAP1 normally suppresses Dronc activation, which in turn is mediated by DARK. In line with the current model on caspase activation, it is argued that the DIAP1-mediated inhibition of Dronc is the key regulatory step in controlling cell death. This view is supported by the observation that flies with diap1 mutations that either abolish binding or ubiquitylation of Dronc completely fail to suppress Dronc- mediated cell death in vivo. Thus, DIAP1 suppresses Dronc activation by binding to and targeting Dronc for ubiquitylation. However, when Rpr-like molecules displace DIAP1 from Dronc, Dronc is recruited into a 700 kDa size apoptosome protein complex that results in Dronc activation. Consequently, cell death is triggered when Dronc is liberated from DIAP1. Thus, the key event in regulating the caspase cascade appears to be inhibition of Dronc by DIAP1 (Tenev, 2002).

Several lines of evidence support the notion that Jafrac2 promotes cell death by interfering with the Dronc-DIAP1 interaction, thereby displacing and liberating Dronc from DIAP1. (1) Jafrac2 and Dronc bind to the same site of the BIR2 domain of DIAP1, since the BIR2 th4 mutation of DIAP1 equally abolished Dronc and Jafrac2 binding. In contrast, Rpr and Hid binding to the th4 DIAP1 mutant remains unaffected. (2) Jafrac2 competes with Dronc for the binding of DIAP1, and Jafrac2 possesses a significantly higher DIAP1-binding affinity compared with that of Dronc to DIAP1, as would be expected of a protein that displaces Dronc from DIAP1. (3) Ectopic expression of Jafrac2 in the developing Drosophila eye causes a phenotype that is highly reminiscent of the phenotype observed in flies ectopically expressing Dronc. (4) Heterozygosity at the dronc locus rescued the eye-ablation phenotype induced by Jafrac2, indicates that apoptotic signal transduction initiated by Jafrac2 is mediated through Dronc. Taken together, these results indicate that Jafrac2 promotes cell death by liberating Dronc from the anti-apoptotic activity of DIAP1 (Tenev, 2002).

The observation that Jafrac2, like the apoptotic inducers Rpr, Grim and Hid, induces apoptosis through binding to DIAP1 places Jafrac2 in a potentially pivotal position to regulate apoptosis. The findings are consistent with a model whereby Jafrac2 promotes apoptosis by displacing DIAP1 from Dronc, so allowing activation of the caspase cascade and consequent cell death. The idea is favored whereby Jafrac2 function is additive to, but independent of, Rpr. The early release of Jafrac2 from the ER of UV-irradiated cells is consistent with the view that Jafrac2 is involved in the initiation of apoptosis. Thus, Jafrac2 is released from the ER at a time when other early apoptotic events occur, such as the mitochondrial release of cytochrome c, Smac/DIABLO and HtrA2/Omi in mammalian cells. Once released, Jafrac2 interacts with DIAP1 and thereby liberates Dronc, which in turn is activated by DARK. In line with the notion that Jafrac2 functions in a complementary but distinct cell death pathway to Rpr, Grim and Hid, it is found that a chromosomal deletion that includes the jafrac2 locus does not suppress the eye phenotypes caused by ectopic expression of Rpr, Grim and Hid. However, it is possible that Jafrac2 may also be part of a positive feedback mechanism, which cooperates with Rpr-like proteins to promote apoptosis in response to cellular damage. These two alternatives cannot be distinguished because no jafrac2 mutant flies are available and Jafrac2 is refractory to the effect of dsRNA interference (Tenev, 2002).

The data are consistent with the idea that Jafrac2, with its thioredoxin peroxidase activity and IAP-binding ability, contains two distinct functions. In healthy cells, Jafrac2 may fulfil a 'housekeeping' role through its peroxidase activity by protecting the cell from oxidative damage. Consistent with this view, members of the peroxiredoxin protein family play an important role in protecting cells against oxidative damage by scavenging intracellularly generated reactive oxygen species, such as H2O2. However, upon UV irradiation, mature Jafrac2 is released from the ER and competes with Dronc for the binding of DIAP1 that is independent of its peroxidase activity. Consequently, Jafrac2 liberates Dronc from DIAP1 inhibition and allows activation of the proteolytic caspase cascade, resulting in cell death (Tenev, 2002).

Members of the IAP family block activation of the intrinsic cell death machinery by binding to and neutralizing the activity of pro-apoptotic caspases. In Drosophila melanogaster, the pro-apoptotic proteins Reaper Rpr, Grim and Hid all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. In vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, the RING finger of DIAP1 promotes the ubiquitination of both itself and of Dronc. Disruption of the DIAP1 RING finger does not inhibit its binding to Rpr, Hid or Dronc, but completely abrogates ubiquitination of Dronc. These data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination (Wilson, 2002).

DRONC is involved in ROS-induced apoptosis caused by impairment of the selD/sps1 homolog in Drosophila

The cellular antioxidant defense systems neutralize the cytotoxic by-products referred to as reactive oxygen species (ROS). Among them, selenoproteins have important antioxidant and detoxification functions. The interference in selenoprotein biosynthesis results in accumulation of ROS and consequently in a toxic intracellular environment. The resulting ROS imbalance can trigger apoptosis to eliminate the deleterious cells. In Drosophila, a null mutation in the selD gene (homologous to the human selenophosphate synthetase type 1) causes an impairment of selenoprotein biosynthesis, a ROS burst and lethality. This mutation (known as selDptuf) can serve as a tool to understand the link between ROS accumulation and cell death. To this aim, the mechanism by which selDptuf mutant cells become apoptotic was analyzed in Drosophila imaginal discs. The apoptotic effect of selDptuf does not require the activity of the Ras/MAPK-dependent proapoptotic gene hid, but results in stabilization of the tumor suppressor protein p53 and transcription of the Drosophila pro-apoptotic gene reaper (rpr). Genetic evidence supports the idea that the initiator caspase DRONC is activated and that the effector caspase DRICE is processed to commit selDptuf mutant cells to death. Moreover, the ectopic expression of the inhibitor of apoptosis DIAP1 rescues the cellular viability of selDptuf mutant cells. These observations indicate that selDptuf ROS-induced apoptosis in Drosophila is mainly driven by the caspase-dependent p53/Rpr pathway (Morey, 2003).

Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination

The inhibitor of apoptosis protein DIAP1 inhibits Dronc-dependent cell death by ubiquitinating Dronc. The pro-death proteins Reaper, Hid and Grim (RHG) promote apoptosis by antagonizing DIAP1 function. This study reports the structural basis of Dronc recognition by DIAP1 as well as a novel mechanism by which the RHG proteins remove DIAP1-mediated downregulation of Dronc. Biochemical and structural analyses revealed that the second BIR (BIR2) domain of DIAP1 recognizes a 12-residue sequence in Dronc. This recognition is essential for DIAP1 binding to Dronc, and for targeting Dronc for ubiquitination. Notably, the Dronc-binding surface on BIR2 coincides with that required for binding to the N termini of the RHG proteins, which competitively eliminate DIAP1-mediated ubiquitination of Dronc. These observations reveal the molecular mechanisms of how DIAP1 recognizes Dronc, and more importantly, how the RHG proteins remove DIAP1-mediated ubiquitination of Dronc (Chai, 2003).

Drosophila caspase transduces Shaggy/GSK-3beta kinase activity in neural precursor development

Caspases are well known for their role in the execution of apoptotic programs, in which they cleave specific target proteins, leading to the elimination of cells, and for their role in cytokine maturation. In this study, a novel substrate was identified, that, through cleavage by caspases, can regulate Drosophila neural precursor development. Shaggy (Sgg)46 protein, an isoform encoded by the sgg gene and essential for the negative regulation of Wingless signaling, is cleaved by the Dark-dependent caspase. This cleavage converts it to an active kinase, which contributes to the formation of neural precursor [sensory organ precursor (SOP)] cells. This evidence suggests that caspase regulation of the wingless pathway is not associated with apoptotic cell death. These results imply a novel role for caspases in modulating cell signaling pathways through substrate cleavage in neural precursor development (Kanuka, 2005; full text of article).

Previous genetic studies of sgg mutant flies showed the interesting observation that some phenotypes of sgg mutants can be rescued by the expression of sgg10 or sgg39 (the other sgg isoform similar to sgg10), but not sgg46, suggesting that Sgg46 might be an inactive form. The Sgg10 kinase phosphorylates the Arm protein and induces its degradation. Various forms of Sgg protein were tested for this activity. Expression of Sgg10 induces Arm phosphorylation and degradation in a kinase-dependent manner. In contrast, full-length Sgg46 did not produce the same effects on the Arm protein. Interestingly, expression of a putative cleaved form of Sgg46, containing the kinase domain (myc-Sgg46 DeltaN235 and myc-Sgg46 DeltaN300), led to Arm phosphorylation and degradation in a manner similar to that of Sgg10. These results suggest that full-length Sgg46 is an inactive form that can be converted into an active kinase via caspase-dependent cleavage (Kanuka, 2005).

Whether these findings would be applicable to macrochaete and SOP cell development in vivo was tested by using transgenic flies expressing Sgg proteins. The ectopic expression of Sgg10 by sca-GAL4 caused the loss of macrochaetes and SOP cells. No apoptotic cells in the myc-Sgg10 protein-expressing region of the wing disc could be detected, indicating that this disappearance did not result from the death of SOP cells. Consistent with the immunoblotting results, full-length Sgg46 did not influence macrochaete and SOP cell formation, whereas the cleaved form of Sgg46 (Sgg46 DeltaN300) worked in a manner similar to that of Sgg10. After crossing sca-GAL4+UAS-DRONC DN to UAS-sgg10, most F1 progeny showed a clear loss of macrochaetes in the scutellum, indicating that Sgg kinase activation might be downstream of caspases. These observations suggest that the processing of Sgg46 by caspases leads to the formation of an active kinase that can negatively regulate SOP cell development (Kanuka, 2005).

Finally, whether Sgg46 contributes significantly to macrochaete and SOP cell formation in vivo was investigated. The ectopic expression of Sgg46 D235G/D300G by sca-GAL4 significantly induced extra macrochaetes and SOP cells. Since Sgg46 D235G/D300G could not be cleaved by caspases, this noncleaved Sgg46 might act as dominant-negative form against endogenous Sgg function. Furthermore, an ectopic knockdown of Sgg protein expression by dsRNA-expressing constructs revealed that the specific reduction of the Sgg46 protein induced extra macrochaetes. However, inhibition of Sgg46 is less effective at producing extra macrochaetes than inhibiting Dark or DRONC, suggesting that modulation of Sgg kinase activity may not be the only mechanism contributing to SOP formation. It still remains to be examined whether or not Sgg46 is actually cleaved and converted into an active form in proneural clusters, and will require further examination in vivo. Based on the findings that loss of Sgg function or inhibition of caspase activity resulted in extra macrochaetes mainly in the scutellum of the adult notum (pSC and aSC), where Wingless is highly expressed, and that caspases are activated in scabrous-expressing cluster, it could be considered that scabrous-expressing SOP cells that will produce pSC and aSC macrochaetes are located in specific region, where precise formation of each set of macrochaetes might require both (1) Wg expression (to increase bristle) and (2) caspase activation (to decrease bristle). Thus, it appears that Dark-dependent caspase signaling mediates the total Sgg kinase activity by processing Sgg46 into an active form, thereby negatively regulating Wingless-sensitive macrochaete development (Kanuka, 2005).

Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease

Although essential in mammals, in flies the importance of mitochondrial outer membrane permeabilization for apoptosis remains highly controversial. This study demonstrates that Drosophila Omi (dOmi; FlyBase term: HtrA2), a fly homologue of the serine protease Omi/HtrA2, is a developmentally regulated mitochondrial intermembrane space protein that undergoes processive cleavage, in situ, to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the proapoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. In summary, this study demonstratesin flies that mitochondrial permeabilization not only occurs during apoptosis but also results in the release of a bona fide proapoptotic protein (Challa, 2007).

The role of mitochondria in fly apoptosis remains highly controversial, due in large part to disagreement over whether mitochondria undergo losses in Δψm (mitochondrial membrane potential) and MOMP (mitochondrial outer membrane permeabilization) following stress. Moreover, although mitochondrial release of cytochrome c in mammalian cells initiates formation of the Apaf-1 apoptosome complex and activation of caspases, there is disagreement over the importance of cytochrome c for promoting cell death in flies. The cytochrome c debate notwithstanding, there are additional mitochondrial proteins in mammals that play a role in promoting apoptosis, including the dual IAP antagonist and serine protease, Omi/HtrA2 (Hegde, 2001; Martins, 2001; Suzuki, 2001; Verhagen, 2001). In these studies, attempts were made to determine if the Drosophila homologue of Omi might likewise participate in cell death. It was found that dOmi was highly homologous to hOmi, particularly within the serine protease domain, and that its expression was developmentally regulated. dOmi was imported into fly mitochondria and processed in situ, resulting in the removal of its mitochondrial targeting sequence (MTS) and exposure of two distinct IAP binding motif (IBMs). The mature forms of dOmi were then released into the cytoplasm following stress, through both caspase-dependent and -independent processes. However, once in the cytosol, dOmi induced cell death in S2 cells and in the developing fly eye, primarily through proteolytic degradation of DIAP1 and likely other substrates (Challa, 2007).

Indeed, catalytically inactive Δ79-dOmiS266A and Δ92-dOmiS266A failed to induce significant apoptosis, which was somewhat surprising, given that both forms of dOmi selectively bound to the BIR2 domain in DIAP1 and displaced the initiator caspase DRONC. In particular, the affinity of Δ79-dOmi for BIR2 was lower than that observed for Rpr-IBM, but was slightly higher than that observed for mature Smac with XIAP-BIR3. So why did dOmi require its proteolytic activity to induce cell death, rather than inducing rapid IBM-dependent apoptosis? Notably, unlike other fly IAP antagonists, which exhibit partial preference for either the BIR1 or BIR2 domains, dOmi completely failed to bind the BIR1 domain in DIAP1 and did not displace the active effector caspase DrICE. Thus, it is possible that the continued inhibition of DrICE by DIAP1 was sufficient to inhibit cell death. There is precedence for such a scenario in mammals, since it has been shown that XIAP mutants that fail to bind and inhibit caspase-9 can still prevent apoptosis through inhibition of caspase-3 alone (Challa, 2007).

One of the primary differences between fly and mammalian IAP antagonists relates to their abilities to independently induce apoptosis. Indeed, Rpr, Hid, and Grim induce robust cell death in both cultured cells and, whereas overexpression of mature Smac in the cytoplasm of mammalian cells generally fails to induce apoptosis in the absence of an accompanying prodeath stimulus. A potential explanation for these results may involve their relative capacities to induce RING-dependent autoubiquitinylation upon binding to IAPs. Indeed, while many IAP antagonists in the fly induce DIAP1 autoubiquitinylation, Smac appears to suppress XIAP autoubiquitinylation. In these studies, dOmi failed to induce or suppress DIAP1 autoubiquitinylation upon binding to its BIR2 domain. Thus, in the absence of dOmi's proteolytic activity, DIAP1 may again be free to maintain its inhibition of DrICE via its BIR1 domain. By contrast, given that DIAP1 can protect cells by targeting active DRONC for proteosomal degradation, it is also plausible that DIAP1 might regulate cell death, in part by, promoting the turnover of dOmi. It has been reported that the DIAP1 binding mutant, DRONC (F118E), induces significantly more cell death than wild-type DRONC, when expressed in the developing fly eye, and correspondingly, this study found that Δ92-dOmi consistently produced a more severe phenotype than Δ79-dOmi, in accordance with their relative affinities for DIAP1 (Challa, 2007).

Others have reconciled such differences between the mammalian and fly IAP antagonists by arguing that, in contrast to the Apaf-1·caspase-9 apoptosome complex, the DARK·DRONC apoptosome complex is constitutively active. Consequently, DIAP1 is required to continuously ubiquitinylate DRONC and mediate its turnover in order to prevent cell death. In this model, Rpr, Hid, or Grim need only displace this active DRONC, in order to promote the activation of effector caspases and induce apoptosis. However, recent studies suggest that, at least for Rpr and Grim, the C-terminus of these IAP antagonists play important roles in promoting both mitochondrial injury and/or inhibition of protein translation. These alternative functions for Rpr and Grim may be necessary to first initiate caspase activation, after which the IBMs serve to displace these active caspases from DIAP1. Therefore, it could be that binding of dOmi to DIAP1-BIR2 per se does not induce apoptosis, because in the absence of another stimulus, there may be very little active DRONC to displace. In any event, regardless of whether dOmi induces cell killing solely through its proteolytic activity, or functions as a pure IAP antagonist in certain contexts, these studies suggest that mitochondria may play a far more important role in apoptosis in the fly than previously thought (Challa, 2007).

The role of the RING-finger protein Elfless in Drosophila spermatogenesis and apoptosis

elfless (CG15150, FBgn0032660) maps to polytene region 36DE 5' (left) of reduced ocelli/Pray for Elves (PFE) on chromosome 2L and is predicted to encode a 187 amino acid RING finger E3 ubiquitin ligase that is putatively involved in programmed cell death (PCD, e.g., apoptosis). Several experimental approaches were used to characterize CG15150/elfless and test whether defects in this gene underlie the male sterile phenotype associated with overlapping chromosomal deficiencies of region 36DE. elfless expression is greatly enhanced in the testes and the expression pattern of UAS-elfless-EGFP driven by elfless-Gal4 is restricted to the tail cyst cell nuclei of the testes. Despite this, elfless transgenes failed to rescue the male sterile phenotype in Df/Df flies. Furthermore, null alleles of elfless, generated either by imprecise excision of an upstream P-element or by FLP-FRT deletion between two flanking piggyBac elements, are fertile. In a gain-of-function setting in the eye, it was found that elfless genetically interacts with key members of the apoptotic pathway including the initiator caspase Dronc and the ubiquitin conjugating enzyme UbcD1. DIAP1, but not UbcD1, protein levels are increased in heads of flies expressing Elfless-EGFP in the eye, and in testes of flies expressing elfless-Gal4 driven Elfless-EGFP. Based on these findings, it is speculated that Elfless may regulate tail cyst cell degradation to provide an advantageous, though not essential, function in the testis (Caldwell, 2009).

Emerging data are now elucidating the roles of key members of the apoptosome, including the caspases Dronc and Drice, in spermatid individualization in Drosophila. The molecules are thought to be inhibited by dBruce, a ubiquitin-conjugating enzyme with a BIR domain. In effect, dBruce may be acting in the spermatid cysts in a manner similar to another documented BIR-domain protein, DIAP1. This study examined the role of the E3 ubiquitin ligase, Elfless, and shows that, consistent with its prediction as a RING finger protein, Elfless interacts with key members of the apoptotic pathway (Caldwell, 2009).

It is proposed that Elfless acts to directly or indirectly regulate UbcD1 activity in the apoptotic pathway. w; GMR-Gal4; UAS-elfless in an otherwise wild type background produces pigment cell defects reminiscent of those of w; GMR-Gal4; UAS-Dronc. As previously shown, w; GMR-Gal4; UAS-Dronc in a ubcD1 heterozygous mutant background produces an eye phenotype slightly worse than w; GMR-Gal4; UAS-Dronc alone which suggests that Dronc may also be a target of UbcD1. Finally, inhibition of apoptosis through Dronc is effectively lost in w; GMR-Gal4; UAS-Dronc in a diap1/+ background; the eye defects exhibited in these lines are severe and flies die as pharate adults. w; GMR-Gal4; UAS-elfless flies in a ubcD1/+ mutant background similarly die as pharate adults and the eye phenotype is significantly worse than that of w; GMR-Gal4; UAS-elfless alone; pigmentation is absent in these flies and the size of the eye is reduced due to decreased UbcD1-mediated inhibition of Dronc and DIAP1. These data suggested that Elfless may be regulating UbcD1 activity (Caldwell, 2009).

While Elfless and UbcD1 had been shown to interact by yeast-two hybrid, it has not been possible to confirm a direct association between Elfless and either DIAP1 or UbcD1 by co-immunoprecipitation experiments. Nevertheless, it is clear from Western blot analysis that mis-expression of Elfless-EGFP in the eye or testes does not significantly change the level of UbcD1 protein but does increase DIAP1 protein levels. Thus, if Elfless is downregulating UbcD1 activity, it seems to be doing so without changing UbcD1 levels. Since DIAP1 auto-ubiquitination is UbcD1-dependent, Elfless-mediated downregulation of UbcD1 activity is consistent with reduced DIAP1 auto-ubiquitination and degradation, resulting in higher DIAP1. While on the one hand this would increase the anti-apoptotic activity of DIAP1, UbcD1 downregulation on the other hand would also increase the pro-apoptotic activity of Dronc, effectively circumventing DIAP1, and producing the somewhat mild eye phenotype evident in w; GMR-Gal4; UAS-elfless in an otherwise wild type background. Consistent with this model, in ubcD1 heterozygotes the eye phenotype is severely worsened and lethality is evident, while in Dronc heterozygotes, the eye phenotype is improved (Caldwell, 2009).

Although this study clearly demonstrates a genetic interaction of elfless with Dronc and ubcD1 in PCD and it is proposed that mis-expressed Elfless in the eye negatively regulates UbcD1 activity, no essential role in fertility could be ascribed to this locus, despite the promising role of this molecule in sperm development based on expression profiling and the Df/Df male sterility phenotype. Furthermore, an elfless transgene is not sufficient to rescue the male sterility associated with these deficiencies in polytene region 36DE. Despite the lack of a sterility phenotype when the RING finger protein encoded by elfless is deleted from the genome, the fact that elfless is functionally retained in the genome by selection indicates that elfless performs an important and advantageous, albeit redundant, function in the testes. Thus, while in laboratory vials, males are able to produce normal numbers of offspring, subtle functional differences can be quite significant in wild populations. What advantage could Elfess provide in the tail cyst cell? One possibility is that mobilization of Elfless in the nuclei of these cells facilitates tail cyst cell degeneration for more efficient resorption; sperm release from the cyst can still take place, but there may be a physiological burden on the testis during the life-time of the fly. The fact that Elfless is in the nucleus may suggest that it targets gene expression at this transitional stage of tail cyst cells. Full activation of apoptosis by Elfless in tail cyst cells is unlikely, as Elfless is nuclear, and no TUNEL staining in wildtype testes appears in the late stage cysts. Nevertheless, apoptosis signaling can be quite different in varying developmental contexts, and there is precedent for enlisting branches of the apoptotic pathway in different developmental processes. For example, Caspase-3 is activated in the cystic bulge of the developing spermatocyst, while the correct balance of DIAP1 levels is important for cellular mobilization of border cells and other cells during oogenesis. More incisive future approaches will be needed to discern the roles that Elfless and other subtle modulators play in mating and evolution (Caldwell, 2009).

Dronc caspase exerts a non-apoptotic function to restrain phospho-Numb-induced ectopic neuroblast formation in Drosophila

Drosophila neuroblasts have served as a model to understand how the balance of stem cell self-renewal versus differentiation is achieved. Drosophila Numb protein regulates this process through its preferential segregation into the differentiating daughter cell. How Numb restricts the proliferation and self-renewal potentials of the recipient cell remains enigmatic. This study shows that phosphorylation at conserved sites regulates the tumor suppressor activity of Numb. Enforced expression of a phospho-mimetic form of Numb (Numb-TS4D) or genetic manipulation that boosts phospho-Numb levels, attenuates endogenous Numb activity and causes ectopic neuroblast formation (ENF). This effect on neuroblast homeostasis occurs only in the type II neuroblast lineage, which generates intermediate neural progenitors (INPs). INPs undergo a maturation process and multiple rounds of asymmetric division to produce GMCs and differentiated progenies. This study identified Dronc caspase as a novel binding partner of Numb, and demonstrates that overexpression of Dronc suppresses the effects of Numb-TS4D in a non-apoptotic and possibly non-catalytic manner. Reduction of Dronc activity facilitates ENF induced by phospho-Numb. These findings uncover a molecular mechanism that regulates Numb activity and suggest a novel role for Dronc caspase in regulating neural stem cell homeostasis (Ouyang, 2011).

Proper balance of the self-renewal versus differentiation of stem cells is crucial for tissue homeostasis. Disruption of this process could contribute to tumorigenesis. Numb has been identified as a key player that limits the proliferation potential of neuroblasts and INPs. This study elucidates the mechanisms of Numb action in this process and uncover a novel mechanism by which Numb activity is regulated at the post-translational level. The results suggest a model in which phosphorylation of Numb at conserved sites within its functionally important PTB domain impairs its association with the caspase Dronc and attenuates its tumor suppressor activity in type II neuroblasts (Ouyang, 2011).

As a defining feature of Numb protein is its asymmetric localization in stem cells and progenitors, previous studies of Numb have been focused on the control of its asymmetric localization. A number of factors have been identified to regulate Numb localization, including its binding partner Pon and kinases such as aPKC, Aurora A and Polo. This presents evidence that phosphorylation of Numb at the putative Polo sites primarily affect Numb activity in negatively regulating Notch signaling through promoting the endocytosis of Spdo. Although not all the identified Polo phosphorylation sites in Numb perfectly match the optimal consensus sequence initially defined for Polo, the Polo consensus sequence being defined is evolving, and specific characterized phosphorylation sites in other Polo substrates actually do not conform to the above consensus sequences. A common feature appears to be negatively charged residues surrounding the S/T residues; all five sites identified in Numb have this feature. Moreover, evidence is provided that the sites that were identified are responsive to phosphorylation controlled by Polo and PP2A. More importantly, phosphorylation of Numb at these sites has a significant effect on NSC homeostasis (Ouyang, 2011).

Polo kinase was shown to also control Numb asymmetric localization by phosphorylating Pon, an adaptor protein for Numb. Loss of Numb asymmetry in polo mutants contributes to ENF. The increased neuroblasts in polo mutants largely occur in the type I lineage. This study demonstrates that overexpression of Polo impairs Numb activity and leads to ENF in type II lineage. In this situation, Pon is presumably also phosphorylated by Polo. However, its positive effect on Numb asymmetric localization is likely to be overridden by impairment of Numb activity by Polo. This underlines the importance of Numb activity regulation in vivo and further indicates that Polo kinase acts on diverse targets to control neuroblast homeostasis. This study shows that phosphorylation of Numb by Polo is probably antagonized by PP2A action in type II lineage, which presumably serves to fine-tune Numb activity through dephosphorylation. Interestingly, the relationships between Polo and PP2A in type I lineage is different from that in type II lineage. In type I neuroblasts, overexpression of Polo can rescue PP2A loss-of-function phenotype, consistent with Polo being positively regulated by PP2A at the transcription level. Elucidation of the mechanisms mediating these differential effects will help lead to an understanding of the distinct behaviors of neuroblasts in these two lineages (Ouyang, 2011).

Deregulation of Numb phosphorylation contributes to loss of Numb activity and eventually leads to unrestrained ENF. Given the conservation of the phospho-sites identified in this study and the potential role of Numb in tumor suppression in mammals, mutations in Numb itself or in some kinases/phosphatases that affect Numb phosphorylation under pathophysiological conditions could contribute to cancer in humans. In lung and breast cancer tissues, Polo expression is upregulated. It is possible that under such pathophysiological conditions, Numb becomes hyperphosphorylated and consequently loses its antagonistic effect on Notch signaling, which could have detrimental consequences on tissue homeostasis. The phosphorylation sites of Numb identified in this study are conserved in mammals. It would be interesting to test in the future whether this phospho-epitope could be detected in human tumor samples (Ouyang, 2011).

This study demonstrated that ENF induced by phospho-Numb occurs specifically in type II lineages, consistent with Numb primarily acting in type II lineage to restrict the proliferation of INPs. It is conceivable that Numb is also phosphorylated by Polo kinase in type I lineage. However, certain unidentified factors might block the effect of phospho-Numb on type I neuroblasts. It is also possible that type I and type II lineages might employ different molecular mechanisms to control their stem cell self-renewal and differentiation, considering their different origin and modes of neurogenesis. Consistent with this notion, the Numb/Notch pathway has been suggested to be dispensable in the type I lineage (Ouyang, 2011).

The prominent brain tumor phenotype induced by Numb-TS4D provides an excellent system with which to identify novel molecules involved in controlling NSC homeostasis. This study shows that Dronc, a newly identified binding partner of Numb, is involved in regulating neuroblast homeostasis. Overexpression of Dronc is sufficient to attenuate Numb-TS4D-induced ENF without promoting neuroblast apoptosis. At the mechanistic level, this study shows that Dronc appears to act upstream of Notch to regulate Numb function, apparently in a process that does not strictly depend on its catalytic activity. Importantly, reduction of dronc function results in neuroblasts being more susceptible to the effect of phospho-Numb on neuroblast homeostasis. In addition, Dronc RNAi is able to further increase ectopic neuroblasts in numbS52F mutant, indicating that Dronc-Numb interaction is normally involved in regulating neuroblast homeostasis. Accumulating evidence suggests that caspases, in addition to their pro-apoptotic functions, also participate in other developmental process without inducing cell death. For example, Dronc has been implicated in a non-autonomous role in compensatory proliferation. It would be interesting to examine in the future whether Dronc transduces a signal from the neighboring niche cells via cell-cell interaction to establish neuroblast homeostatic control. It is also worth noting that mice deficient for caspase 2 (Casp2), which is closely related to Dronc in Drosophila, develop normally as their wild-type siblings; however, the fibroblasts from Casp2 null animals are easily transformed when challenged with oncogenic insults (Ho, 2009). The downstream effectors mediating this effect are not known. It would therefore be interesting to test whether the Numb/Dronc pathway identified here is generally involved in stem cell and cancer biology (Ouyang, 2011).

Structural mechanisms of DIAP1 auto-inhibition and DIAP1-mediated inhibition of drICE

The Drosophila inhibitor of apoptosis protein DIAP1 exists in an auto-inhibited conformation, unable to suppress the effector caspase drICE. Auto-inhibition is disabled by caspase-mediated cleavage of DIAP1 after Asp20. The cleaved DIAP1 binds to mature drICE, inhibits its protease activity, and, presumably, also targets drICE for ubiquitylation. DIAP1-mediated suppression of drICE is effectively antagonized by the pro-apoptotic proteins Reaper, Hid, and Grim (RHG). Despite rigorous effort, the molecular mechanisms behind these observations are enigmatic. This study reports a 2.4 Å crystal structure of uncleaved DIAP1-BIR1, which reveals how the amino-terminal sequences recognize a conserved surface groove in BIR1 to achieve auto-inhibition, and a 3.5 Å crystal structure of active drICE bound to cleaved DIAP1-BIR1, which provides a structural explanation to DIAP1-mediated inhibition of drICE. These structures and associated biochemical analyses, together with published reports, define the molecular determinants that govern the interplay among DIAP1, drICE and the RHG proteins (Li, 2011).

The structural and biochemical information presented in this study gives rise to a model on the interplay of Dronc, drICE, DIAP1, and the RHG proteins. During homeostasis, DIAP1 targets the Dronc zymogen for ubiquitylation and presumably proteasome-mediated degradation. This regulation depends on the interaction between a peptide fragment of Dronc and the conserved groove on DIAP1-BIR2. DIAP1 exists in an auto-inhibited conformation. Caspase-mediated cleavage of DIAP1 after Asp20 disables auto-inhibition, allowing the resulting DIAP1 fragment to bind to and inhibit active drICE. During apoptosis, the RHG proteins use their N-terminal peptides to compete with drICE and Dronc for binding to the conserved peptide-binding grooves on the BIR1 and BIR2 domains, respectively. Such competition results in the release of drICE and Dronc from DIAP1. The freed Dronc zymogen is activated by the Dark apoptosome and the mature Dronc cleaves and activates drICE. In this regard, drICE, DIAP1 and the RHG proteins together provide a fail-safe mechanism to ensure appropriate drICE activation only under bona fide apoptotic conditions (Li, 2011).

The underpinning of this regulatory network is competition among multiple protein-protein interactions mediated by the conserved grooves of the BIR1 and BIR2 domains of DIAP1. Auto-inhibition of DIAP1-BIR1 is achieved by occupation of this groove by its own N-terminal sequence ASVV. The free peptide ASVV does not stably associate with BIR1; the covalent linkage facilitates the binding by increasing the local concentration of ASVV. This binding arrangement allows disabling of auto-inhibition upon cleavage of DIAP1 after Asp20. Inhibition of drICE by BIR1 requires occupation of this groove by the N-terminal sequences ALGS of drICE. Similar to ASVV, the free ALGS peptide exhibited no detectable binding to the BIR1 fragment. Three weak interfaces between BIR1 and drICE cooperate to yield a stable hetero-tetramer with a KD of approximately 1-2 microM. Removal of drICE inhibition by BIR1 depends on the interactions between RHG and the peptide-binding groove, with KD values of 0.12-0.76 microM. Endowing RHG with the strongest interactions ensures an apoptotic phenotype once the RHG proteins are activated in cells. It is acknowledged that the proposed model may be simplistic, as the network of protein-protein interactions and regulation is likely to be more complex in vivo. Nevertheless, the biophysical underpinnings described in this model are likely to have a role in various stages of apoptosis regulation (Li, 2011).

An IAP-binding motif, though not ostensibly abbreviated as IBM, was originally defined to contain four contiguous amino acids that resemble the Smac tetrapeptide AVPI. This structurally defined motif, with binding affinities of 0.1-1 microM, has stringent requirement for the first (P1), third (P3) and fourth (P4) amino acids. The P1 residue must be Ala, which binds to a small hydrophobic pocket on one end of the conserved groove on BIR domain. The P4 residue must be hydrophobic, preferably bulky, to occupy a greasy pocket on the other end of the groove. Deletion of P1 or P4 in a tetrapeptide results in abrogation of stable interaction with the BIR domain. The P3 residue is either Pro or Ala. Pro as P3, with its unique backbone configuration, optimizes simultaneous binding by both P1 and P4 residues for DIAP1-BIR2 or XIAP-BIR3. Ala as P3 can be better accommodated by DIAP1-BIR1 and XIAP-BIR2. In recent studies1, IBM was redefined to contain three contiguous amino acids, with P3 no longer restricted to Pro or Ala. Such tripeptides, ALG/AKG for drICE/Dcp1, or their longer variants, ALGS/AKGC for drICE/Dcp1, do not meet the structural criteria for IBM. Importantly, these peptides in isolation do not form a stable complex with any BIR domain. The definition of such motifs as IBMs insinuates the incorrect assumption that such free peptide motifs may stably interact with the BIR domain. This assumption, in turn, has engendered ample confusion in data interpretation in recent years (Li, 2011).

An IBM at the N-terminus of the caspase-9 small subunit recognizes a conserved surface groove on XIAP-BIR3; this interaction locks caspase-9 in the inhibited state. During apoptosis, Smac/Diablo uses a similar tetrapeptide motif to occupy the BIR3 groove, hence releasing caspase-9 and relieving XIAP-mediated inhibition. Caspase-3 or -7 is inhibited by an 18-residue peptide segment preceding the BIR2 domain of XIAP. Because both caspase-3 and drICE are inhibited directly at the active sites by XIAP and DIAP1, respectively, the overall appearance of the two BIR-caspase complexes is similar. It should be noted, however, that the essential interactions and key features are quite different. For example, caspase-3 or -7 can be inhibited by an isolated 18-residue peptide fused to GST; but the intact BIR1 domain of DIAP1 is absolutely required for drICE inhibition. The orientation of the BIR domain relative to the caspase is different by approximately 90 degrees between caspase-3/BIR2 and drICE/BIR1. Importantly, the peptide-binding groove of XIAP-BIR2 does not have an apparent role in the inhibition of caspase-3 and -7 (Li, 2011).

Using purified, recombinant proteins, this study showed that the BIR1 domain of DIAP1 only forms a stable complex with active drICE following caspase-mediated cleavage of DIAP1 after Asp20. The uncleaved DIAP1-BIR1 exhibited very weak binding to drICE. These observations contrast the report that both uncleaved and cleaved DIAP1-BIR1 bound to drICE similarly using coimmunoprecipitation. The cleaved DIAP1, but not the uncleaved DIAP1, potently inhibits the proteolytic activity of drICE towards both peptide and protein substrates. These observations unambiguously demonstrate that drICE sequestered by cleaved DIAP1 remains catalytically inactive. In fact, the conclusion that DIAP1-sequestered drICE was catalytically active, contradicted the biochemical observation that no protease activity was detectable towards peptide or protein substrate21. It is not uncommon for a substrate to be converted into a protease inhibitor upon cleavage, as exemplified by the pan-caspase inhibitor p35 (Li, 2011).

The key question is not whether BIR1 inhibits drICE, but why BIR1-sequestered drICE continues to exhibit proteolytic activity towards the uncleaved DIAP1. A time course analysis of DIAP1 cleavage shows that the protease activity of drICE was slowed down considerably over time, as the concentration of inhibitor -- cleaved DIAP1 -- increased. The level of drICE activity at the 15-minute time point was at least 6-fold higher than that at 90-minute point. This observation again illustrates that the cleaved DIAP1 is a bona fide inhibitor of drICE (Li, 2011).

Some of the contrasting claims about the regulation of drICE by DIAP1 might be attributable to the limitations of the investigative methods. Biochemical and biophysical investigations, employing homogeneous, recombinant proteins, usually provide mechanistic answers to questions that pertain to protein-protein interactions and enzyme activities. The caveat, however, is whether such observations are biologically relevant, and if yes, to what extent these findings are important. By contrast, investigation by cellular biochemistry, exemplified by coimmunoprecipitation, provides important clues to molecular mechanisms. For both approaches, caution must be exercised for the interpretation of results. Notably, in some cases, the contrasting claims can be reconciled by a complex system. For example, despite structural data demonstrating that the auto-inhibition of DIAP1 involves the binding of its N-terminal sequences to the BIR1 domain, it remains theoretically possible that additional interactions between the N- and C-terminal domains of DIAP1 may contribute to its auto-inhibition1. The best example is Apaf-1, whose auto-inhibition entails two elements: one by the C-terminal WD40 repeats and the other within the N-terminal half. Binding to cytochrome c relieves the auto-inhibition by the WD40 repeats31, and exchange of ADP for ATP defeats the auto-inhibition imposed by intra-domain interactions within the N-terminal-half (Li, 2011).

Structure of the Drosophila apoptosome at 6.9 &aRing; resolution

The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. This study shows that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. The 3D structure of a double ring was determined at approximately 6.9 &ARing; resolution, and a model was created of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has 'lost' a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution (Yuan, 2011).

Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila

Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. This study reports the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome (Pang, 2015).

This study presents the cryo-EM structures of the Dark apoptosome and the multimeric Dronc-Dark complex at overall resolutions of 4.0 and 4.1 Å, respectively. Notably, the EM density in the central region of the structures exhibits considerably higher resolutions, which allow assignment of specific side chains and atomic interactions. Because the overall domain organization of Dark is identical to that of Apaf-1, the structures reveal for the first time conserved atomic features of an apoptosome from a higher organism. The observed structural features of the Dark apoptosome, most of which are likely preserved in the Apaf-1 apoptosome, reveal the underpinnings of initiator caspase activation. Supporting this analysis, structure of the Dark protomer can be very well aligned with that of the activated Apaf-1 protomer from the Apaf-1 apoptosom (Pang, 2015).

This study presents the cryo-EM structures of the Dark apoptosome and the multimeric Dronc-Dark complex at overall resolutions of 4.0 and 4.1 Å, respectively. Notably, the EM density in the central region of the structures exhibits considerably higher resolutions, which allow assignment of specific side chains and atomic interactions. Because the overall domain organization of Dark is identical to that of Apaf-1, the structures reveal for the first time conserved atomic features of an apoptosome from a higher organism. The observed structural features of the Dark apoptosome, most of which are likely preserved in the Apaf-1 apoptosome, reveal the underpinnings of initiator caspase activation. Supporting this analysis, structure of the Dark protomer can be very well aligned with that of the activated Apaf-1 protomer from the Apaf-1 apoptosome (Pang, 2015).


DEVELOPMENTAL BIOLOGY
Promoter

Dronc is an apical Drosophila caspase essential for programmed cell death during fly development. During metamorphosis, dronc gene expression is regulated by the steroid hormone ecdysone, which also regulates the levels of a number of other critical cell death proteins. As dronc protein levels are important in determining caspase activation and initiation of cell death, the regulation of the dronc promoter was analyzed using transgenic flies expressing a LacZ reporter gene under the control of the dronc promoter. These results indicate that dronc expression is highly dynamic during Drosophila development, and is controlled both spatially and temporally. While a 2.3 kb dronc promoter region contains most of the information required for correct gene expression, a 1.1 kb promoter region is expressed in some tissues and not others. During larval-pupal metamorphosis, two ecdysone-induced transcription factors, Broad-Complex and E93, are required for correct dronc expression. These data suggest that the dronc promoter is regulated in a highly complex manner, and provides an ideal system to explore the temporal and spatial regulation of gene expression driven by nuclear hormone receptors (Daish, 2003).

Experiments outlined in this paper demonstrate that 2.3 kb of the dronc promoter is largely sufficient for temporal expression (compared to endogenous dronc) throughout development. Previous experiments have shown that dronc is predominantly expressed in the larval and prepupal salivary glands and midgut, and larval brain lobes. 2.3 kb of the dronc promoter contains all necessary elements for correct spatial regulation of dronc expression in these tissues (Daish, 2003).

In order to identify transcription factors responsible for both temporal and spatial regulation of dronc and ecdysone-mediated PCD, it is of vital importance to elucidate the regions of the promoter essential for dronc expression in different tissues. In addition, it would be of interest to determine if there is a single promoter region controlling the spatial expression profile of dronc, or if different promoter regions are required in different tissues. LacZ transgenic reporter experiments reveal that the 2.3 kb promoter is the minimal requirement for correct expression in brain lobes and salivary glands. Furthermore, the region between 1.1 and 2.3 kb contains transcription factor-binding sites essential for expression in these tissues. This region also seems to harbor a repressor element important to keep dronc levels low during periods when ecdysone titers are low. Surprisingly, regulation of dronc transcription is markedly different in the midgut. The region between 1.1 and 2.3 kb is not important for transcription in this tissue, because 1.1 kb of the promoter is sufficient for expression. These results clearly demonstrate that distinct regions of the promoter are required for expression in different tissues, and implies that different transcription factors regulate dronc expression in a tissue-dependent manner (Daish, 2003).

The two ecdysone-induced transcription factors BR-C and E93 are essential for dronc expression in salivary glands. In the midgut, however, only E93 seems to be important. The results of dronc promoter-LacZ transgenic expression in flies deficient in BR-C and E93 are consistent with recent findings. LacZ expression driven by the 2.8 kb promoter is severely impaired in salivary glands of BR-C (rbp5 and npr) or E93 mutants, whereas expression is impaired only in the midgut of E93 mutant background animals. This further supports the idea that the mechanisms governing dronc regulation are tissue specific. The key questions arising from these experiments are: why does the BR-C Z1 isoform (rbp5 mutant) regulate dronc in the salivary glands and not in the midgut? What factors are binding to the 1.1-2.3 kb region of the promoter in salivary glands, and why are they not as important in the midgut? Previous results show that either BR-C Z1- or BR-C Z1-regulated proteins bind to the dronc proximal promoter and control its expression. Transactivation of the 2.8 kb promoter by BR-C Z1, however, was only seen in specific cell types. Given that BR-C Z1 is also expressed in the midgut, this implies that it may be acting through cofactors which are not expressed in the midgut, yet are specifically recruited to the dronc promoter. Alternatively, BR-C Z1 induces the expression of another factor which binds to the promoter, and this factor is absent in the midgut (Daish, 2003).

Since the proximal promoter alone (0.54 kb) is not sufficient for expression in the salivary gland, it is believed that BR-C Z1 (or a Z1-regulated protein) is cooperating with other transcription factors binding upstream (1.1-2.3 kb), that are essential for salivary gland expression. It has been shown that E93 acts through the first 600 bp of the dronc promoter by transactivation studies; however, no direct binding of E93 to the dronc (or any other) promoter has been shown so far. Additionally, a preliminary analysis indicates the presence of an EcR/Usp-binding site between 1.1 and 2.3 kb of the dronc promoter, and in vitro experiments show that this element may be important in regulating dronc expression. Since the proximal promoter (0.54 kb) alone is not sufficient for expression, cooperation of BR-C and E93 with EcR/Usp and other unknown factors may be important for temporal and spatial regulation of dronc expression during development. Identification of these factors will be important for fully understanding dronc transcription during development (Daish, 2003).

Overall, this study has established the minimal dronc promoter requirement for spatial and temporal expression to be within the 2.3 kb region upstream of the dronc gene. This region is important for both BR-C- and E93-mediated transcription in salivary glands and E93 transcription in the midgut. Importantly, the 1.1-2.3 kb promoter region harbors elements important for salivary gland expression and a putative repressor element. The 0.54-1.1 kb promoter region is important for expression in the midgut. These regions will form the basis of future experiments designed to identify factors necessary for the regulation of dronc expression during PCD (Daish, 2003).

Regulation of Dronc expression by ecdysone

The steroid hormone ecdysone has been shown to mediate apoptosis of larval tissues during pupariation. To investigate whether ecdysone also induces expression of dronc, the levels of dronc mRNA were examined after the addition of ecdysone to second instar larval midgut and salivary gland tissues, which normally show only low levels of dronc mRNA. After 1 hr exposure to ecdysone there was a several-fold increase in dronc mRNA levels in the early second instar larval midgut tissue, indicating that ecdysone can induce dronc expression in the midgut. However, salivary glands from early second instar larvae do not show dronc induction after ecdysone treatment. Because salivary glands normally undergo apoptosis later than midgut tissues, it is possible that the failure of ecdysone to induce dronc in salivary glands is the result of the absence of a developmentally controlled factor required for ecdysone-induced gene expression. For this reason, whether ecdysone could induce dronc expression in salivary glands at a later developmental stage was examined. Salivary glands from late second instar larvae, which normally only express very low levels of dronc, were found to strongly express dronc 1 hr after ecdysone treatment. Thus, ecdysone induces dronc expression in both midgut and salivary gland tissues (Dorstyn, 1999).

Characterization of Dronc enzymatic activity

To check whether DRONC is indeed a caspase, recombinant DRONC was generated in E. coli and its proteolytic activity was assessed on synthetic fluorogenic peptide substrates. Expression of both the full-length DRONC precursor or truncated DRONC lacking the putative prodomain (residues 1-113) generated enzyme that showed low-level activity on a caspase-3 substrate DEVD-afc. However, DRONC activity on the caspase-2 pentapeptide substrate VDVAD-amc was 5-fold higher than on DEVD-afc, suggesting that, similar to caspase-2, the minimum substrate requirement for DRONC includes a P5 residue. Under identical conditions, recombinant DRONC and caspase-2 show approximately similar activities on VDVAD and DEVD substrates. No significant cleavage by DRONC of caspase-1 substrate YVAD-afc was observed (Dorstyn, 1999).

Since DRONC interacts with drICE, the ability of active DRONC to cleave drICE CdeltaA, lamin DmO , the DNA fragmentation factor DREP-1 and the baculovirus caspase inhibitor p35 was assayed. Both DRONC and drICE cleave drICE CdeltaA, lamin DmO and DREP-1. The cleavage products generated by DRONC and drICE are clearly different, indicating that DRONC and drICE each cleave lamin DmO and DREP-1 at different sites. Unlike drICE, however, DRONC is unable to cleave p35. Together, these results indicate that dronc encodes a catalytically active protease and that its unique active site PFCRG pentapeptide confers upon it a different substrate specificity from classical caspases such as drICE that share the QAC(R/Q/G)(G/E) active site pentapeptide consensus (Meier, 2000).

Many caspases induce apoptosis when expressed in mammalian cells. It was therefore asked whether pro-DRONC, DeltaN DRONC or the catalytically inactive mutant of DeltaN DRONC (DeltaN DRONC CdeltaA) kill Rat-1 fibroblasts. Expression of DeltaN DRONC, which lacks its pro-domain, is very effective at inducing cell death, as is expression of either of the positive controls, caspase-8 and the Fas pathway adaptor FADD. However, in complete contrast, expression of full-length DRONC exerts no lethal effect. DRONC therefore resembles caspases-4 and -5 , both of which kill mammalian cells only when expressed without their respective pro-domains. As in S.pombe, the catalytically inactive DeltaN DRONC CdeltaA mutant has no effect on Rat-1 cell viability, consistent with a requirement for the caspase activity of DRONC to induce Rat-1 cell death. The lack of toxicity of full-length DRONC in Rat-1 cells is in stark contrast to the situation in S.pombe in which both pro-DRONC and DeltaN DRONC are toxic and undergo autocatalytic activation. One possible explanation for this discrepancy is that mammalian cells contain cellular factors that suppress pro-DRONC activation by binding its pro-domain. If true, deletion of the pro-domain in DeltaN DRONC would then render the caspase no longer inhibitable by such putative factors, resulting in the spontaneous activation of DeltaN DRONC and consequent cell death. Cell line-specific variations in levels of such putative inhibitory factors might explain why the efficacy with which pro-DRONC induces cell death is variable amongst different cell types. In this context, it is noteworthy that although pro-DRONC does not induce cell death in Rat-1 cells, it is lethal to NIH 3T3 cells (Meier, 2000).

Some caspases autocatalytically cleave and activate themselves. To determine if and where DRONC cleaves itself, a COOH-terminal His6-tagged version of DRONC was expressed and purified from E. coli. The purified protein consisted of two major bands, presumably consisting of the processed large and small subunits. This protein is active as a caspase. To determine the site of cleavage between the large and small subunits, Edman degradation amino-terminal sequencing was performed on the smaller band. The NH2-terminal sequence that was determined occurs COOH-terminal to the sequence TQTE352, suggesting that DRONC cleaves itself following a glutamate rather than an aspartate. To test this hypothesis DRONC TQTE352 was directly mutated to TQTA352. 35S-Labeled wild type DRONC and DRONC TQTAE352A were generated by in vitro translation and incubated with bacterially produced DRONC or DCP-1. DRONC cleaved itself to generate a product corresponding in size to the prodomain and large subunit. This band is not seen when DRONC TQTAE352A is the substrate, consistent with the hypothesis that DRONC processes itself following TQTE352. DCP-1 and drICE cleave DRONC at several sites. This cleavage is unaffected by the presence of the TQTA352 mutation, suggesting that these caspases cleave elsewhere in DRONC, perhaps in the DRONC prodomain. To explore the possibility of cleavage within the DRONC prodomain a form of DRONC, DRONCpD4A, was generated in which the P1 aspartates of four potential caspase target sites within the prodomain, DEKD66, ESVD110, DESD113, and DIVD135, were changed to alanine. 35S-labeled in vitro translated DRONCpD4A was still processed by wild type DRONC, but not by DCP-1. Similar results were obtained with cleavage by drICE. Thus DCP-1 and drICE can process DRONC within the prodomain, but not at the large-small subunit boundary. If this processing occurs in vivo it may serve as a point of regulation of DRONC function (Hawkins, 2000).

Positional scanning synthetic combinatorial libraries (PS-SCL) have been a useful tool to determine cleavage site specificities of other caspases. The PS-SCL is composed of three separate sublibraries of 8,000 compounds each. In each sublibrary, one position is defined with one of 20 amino acids (excluding cysteine), while the remaining two positions contain a mixture of amino acids present in approximately equimolar concentrations. Analysis of the three sublibraries (20 samples each) affords a complete understanding of the amino acid preferences in the P2, P3, and P4 positions. This approach was used to characterize DRONC's preferences for given amino acids at each of these positions. The positional scanning synthetic combinatorial libraries available all contain aspartate at the P1 position. While DRONC cleaves itself after glutamate, it is also able to cleave protein substrates after aspartate. Thus it was reasoned that the existing aspartate-based libraries would yield useful information about DRONCs cleavage specificity. DRONC shows a strong preference for Thr, Ile, or Val at the P2 position. A wider spectrum of amino acids was tolerated at the P3 and P4 positions. This analysis suggests that TATD constitutes an optimal DRONC P1 aspartate tetrapeptide cleavage site (Hawkins, 2000).

The results of the PS-SCL analysis are supported by experiments in which DRONC activity was tested directly with a number of commonly used tetrapeptide activity substrates. DRONC shows highest levels of activity with the tetrapeptides VEID-AMC and IETD-AMC, and somewhat lower levels of activity with DEVD-AMC. However, little if any activity is seen with WEHD-AMC or YVAD-AMC, which are predicted to be poor substrates. DRONC has higher levels of activity with the pentapeptide GIETD-AMC than with the tetrapeptide IETD-AMC. This suggests that a P5 residue is important for optimal DRONC activity (Hawkins, 2000).

To further characterize DRONCs cleavage preferences assays were carried out in which the cleavage activities of DRONC and DCP-1 were measured for two different peptide substrates: Ac-TQTE-AFC and Ac-DEVD-AFC. Ac-TQTE-AFC is derived from the known DRONC autoprocessing site and is also predicted to correspond to a good DRONC cleavage site based on the results obtained from PS-SCL analysis. Ac-DEVD-AFC is a tetrapeptide substrate for caspases generally grouped together as effectors of apoptosis (group II caspases). DRONCs activity is low in absolute terms compared with DCP-1. However, DRONC shows a clear cleavage preference for the Ac-TQTE-AFC substrate over Ac-DEVD-AFC. As expected, DCP-1, which has a common variant of the standard caspase active site pentapeptide (QACQG), has a strong preference for the tetrapeptide substrate with a P1 aspartate, Ac-DEVD-AFC (Hawkins, 2000).

Despite the fact that DRONC shows relatively low levels of activity with tetrapeptide substrates containing a P1 aspartate, DRONC is efficiently inhibited by the broad range tripeptide caspase inhibitor carbobenzoxy-VAD-fluoromethyl ketone (z-VAD-fmk) (Hawkins, 2000).

It was of interest to determine DRONC's P1 specificity with respect to aspartate and glutamate. To do this a second tetrapeptide substrate, Ac-TQTD-AFC, was synthesized that differs from Ac-TQTE-AFC only by the P1 residue. These substrates were used to measure DRONCs activity. DRONC shows only a slight preference for cleavage of tetrapeptide substrates with a P1 aspartate over those with a P1 glutamate. DRONC is, however, a particularly poor catalyst of tetrapeptide hydrolysis. The calculated activity values for DRONC are roughly 40-180-fold lower than those described for caspase-9, which itself is a very inefficient enzyme in isolation as compared with most other caspases. This may reflect the fact that DRONC has an intrinsically low turnover rate or that optimal in vitro assay conditions have not been identified. However, DRONC activity may also be regulated allosterically through interactions with the Drosophila homolog of Apaf-1, HAC-1, and DARK in a manner similar to that of mammalian caspase-9 by Apaf-1. Alternatively, since DRONC shows similar levels of activity to DCP-1 on the protein substrate drICE, optimal DRONC cleavage may require additional sequences surrounding the target site (Hawkins, 2000).

If DRONC is an apical cell death caspase, likely substrates include other Drosophila caspases. drICE is a good candidate to be such a target since immunodepletion experiments show that drICE is required for rpr-dependent apoptotic events in cell extracts, and genetic interactions suggest that DRONC contributes to rpr-, hid-, and grim-dependent cell death. 35S-labeled in vitro translated drICE was generated and it was incubated with bacterially produced DRONC or DCP-1. drICE was efficiently cleaved by DRONC and DCP-1. The generation of a band corresponding in size to that of the mature large subunit Ala29-Asp230 was observed. Several other cleavage products were also generated. These correspond to full-length drICE lacking the prodomain, Ala29-Val339 and a fragment comprising the prodomain and large subunit processed at the COOH terminus of the large-small subunit linker region, 1-Asp230. To show that DRONC and DCP-1 process drICE at TETD230, the proposed natural drICE cleavage site between large and small subunits, this site was changed to TETA230. DRONC and DCP-1 do not cleave 35S-labeled in vitro translated drICE TETA230 between the large and small subunit, implying that they both cleave drICE at TETD230. These results, taken together with the observed site of DRONC autoprocessing in bacteria and in vitro , and the results of tetrapeptide cleavage experiments, argue that DRONC cleaves following glutamate as well as aspartate. DRONC efficiently processesdrICE at QTETD230. However, it processes DCP-1 very poorly at the equivalent site in the large-small subunit linker, VTETD215. These results are consistent with the possibility that the optimal DRONC peptide substrate is a pentapeptide (Hawkins, 2000).

The drICE TETA230 mutant is still cleaved by DCP-1 at one position, perhaps at the prodomain-large subunit boundary. To explore this possibility the P1 aspartate of the proposed prodomain-large subunit boundary caspase target site, DHTD28, was altered to alanine, generating drICED28A. DRONC and DCP-1 both process drICED28A to generate a fragment corresponding in size to the prodomain and large subunit processed at Asp230. A slightly smaller band, probably corresponding to the prodomain and the large subunit processed at the NH2 terminus of the large-small subunit linker, 1-Asp217, was also produced. However, no bands corresponding in size to full-length drICE lacking the prodomain or the fully processed large subunit were observed. These observations demonstrate that DCP-1 processes drICE in the prodomain as well as at TETD230 (Hawkins, 2000).

Addition of DRONC to in vitro translated drICE results in production of a mature drICE large subunit lacking prodomain sequences, but DRONC is unable to process drICE TETA230 within the prodomain. These observations suggested that drICE cleaved by DRONC at TETD230 is autocatalytically removing its own prodomain. To test this possibility DRONC and DCP-1 were incubated with an in vitro translated version of drICE, drICEC211A, in which the active site cysteine was changed to alanine. This caspase should remain inactive following cleavage at TETD230. DRONC cleavage of drICEC211A results in the appearance of only a single band corresponding to the prodomain and large subunit. This observation suggests that drICE autocatalytically removes its own prodomain following cleavage between the large and small subunits. Mature drICEC211A large subunit was generated in the presence of DCP-1. This further supports the argument that DCP-1 cleaves drICE in the prodomain as well as at the large-small subunit boundary (Hawkins, 2000).

What purpose could be served by DRONC having an altered cleavage specificity? One possibility is simply that DRONC has unique targets other than itself, and that a different target site preference is required for cleavage of these substrates. DRONC's novel cleavage site specificity, in conjunction with the sequence of the linker between the large and small subunits, may also provide a mechanism for limiting DRONC's ability to become activated by other caspase cascades. DCP-1 or drICE do not process DRONC to any significant extent at the large-small subunit boundary. This is not surprising because there are only two aspartates in the linker region between the large and small subunits, DEYD324 and KWPD348. Based on positional scanning synthetic combinatorial library analysis of tetrapeptide substrates, these sequences are predicted to be very poor substrates for all known mammalian caspases and DCP-1. The possibility that processing of DRONC by unknown proteases occurs at these sites in vivo cannot be ruled out. However, because DRONC is able to process itself in the linker region at TQTE352, but other tested caspases are not, it seems reasonable that DRONC's altered cleavage specificity, coupled with the lack of good target sites for other caspases in the large-small subunit linker region, may serve at least in part to make activation of DRONC more strictly DRONC-dependent. This may provide a mechanism for limiting cross-talk between other caspase cascades and pathways activated by DRONC (Hawkins, 2000).

Regulation of Dronc induction of apoptosis

Although loss of the inhibitor of apoptosis (IAP) protein DIAP1 has been shown to result in caspase activation and spontaneous cell death in Drosophila cells and embryos, the point at which DIAP1 normally functions to inhibit caspase activation is unknown. Depletion of the DIAP1 protein in Drosophila S2 cells or the Sf-IAP protein in Spodoptera frugiperda Sf21 cells by RNA interference (RNAi) or cycloheximide treatment results in rapid and widespread caspase-dependent apoptosis. Co-silencing of dronc< or dark largely suppresses this apoptosis, indicating that DIAP1 is normally required to inhibit an activity dependent on these proteins. Silencing of dronc also inhibits Ice processing following stimulation of apoptosis, demonstrating that DRONC functions as an apical caspase in S2 cells. Silencing of diap1 or treatment with UV light induces DRONC processing, which occurs in two steps. The first step appears to occur continuously even in the absence of an apoptotic signal and to be dependent on DARK, because full-length DRONC accumulates when dark is silenced in non-apoptotic cells. In addition, treatment with the proteasome inhibitor MG132 results in accumulation of this initially processed form of DRONC, but not full-length DRONC, in non-apoptotic cells. The second step in DRONC processing is observed only in apoptotic cells. These results indicate that the initial step in DRONC processing occurs continuously via a DARK-dependent mechanism in Drosophila cells and that DIAP1 is required to prevent excess accumulation of this first form of processed DRONC, presumably through its ability to act as a ubiquitin-protein ligase (Muro, 2002).

Dronc induction of apoptosis

A number of caspases have been shown to induce apoptosis when overexpressed. DRONC was transiently co-expressed with ß-galactosidase in NIH 3T3 cells. At 48 hr posttransfection approximately 60% of the ß-galactosidase positive cells had undergone apoptosis. DRONC-induced cell death was almost completely abolished by coexpression of baculovirus P35 and inhibited to a lesser extent by MIHA, OpIAP, and Bcl-2. CrmA, an inhibitor of caspase-1, is least effective in inhibiting DRONC-induced apoptosis. A substitution mutation of the catalytic Cys-318 to Gly completely abolishes the cell-killing activity of DRONC, suggesting that cysteine protease activity is responsible for the apoptotic function of DRONC. The localization of DRONC protein was examined in transfected cells by using DRONC-GFP fusion constructs. Fusion of GFP to the carboxyl terminal of DRONC does not affect its cell-killing activity. At 24 hr, when most of the transfected cells appeared morphologically normal, DRONC was mostly localized in the cytoplasmic fraction of cells. In some cells, DRONC protein appeared to be concentrated asymmetrically near the cellular nucleus, possibly associated with some subcellular structures. Staining of transfected cells with mitochondrial markers suggests that DRONC does not localize to mitochondria. At 48 hr after transfection, DRONC-GFP protein is uniformly distributed in apoptotic cells (Dorstyn, 1999).

To determine whether ectopic expression of DRONC can induce cell death in D.melanogaster, the GAL4/UAS system was used to express various forms of DRONC in the developing Drosophila compound eye. Independent transgenic Drosophila lines were generated carrying pro-dronc, DeltaN dronc, pro-dronc CdeltaA, DeltaN dronc CdeltaA or dronc-card (the pro-domain of DRONC on its own) under the control of GAL4-upstream activating sequences (UAS). These flies were then crossed with Drosophila strains expressing GAL4 under the control of the glass multimer reporter in differentiating photoreceptors and pigment cells posterior to the morphogenetic furrow in the eye imaginal disc. The DRONC-induced phenotypes that were observed were of variable severity, depending on the insertion line used, presumably because of insertion site-specific effects on the transgene expression level. Accordingly, one representative weak UAS-pro-dronc (pro-droncW) and one representative strong UAS-pro-dronc line (pro-droncS) were selected for further characterization, along with one UAS-DeltaN dronc line (Meier, 2000).

Pro-droncW flies carrying one copy of the transgene exhibit a 'spotted eye' phenotype when crossed with GMR-gal4 flies: although pro-droncW flies are white+, and should therefore have red eyes, their eyes appeared white with occasional red spots. Such eyes have an essentially normal external morphology and size, in contrast to eyes expressing Rpr under the control of GMR, which are severely reduced in size. By comparison, pro-droncS and DeltaN dronc transgenic flies exhibit dramatically 'roughened eyes' that are severely reduced in size. Scanning electron microscopy (SEM) analysis of pro-droncS and DeltaN dronc eyes confirms that surface morphology is severely distorted, erupted and rough. As with pro-droncW flies, eyes from pro-droncS and DeltaN dronc flies are white, not red. This consequence of DRONC expression in eyes is particularly intriguing given that expression of Rpr dramatically reduces eye size yet has no effect on eye color. The phenotypes induced by DRONC expression are a consequence of DRONC caspase activity since overexpression of catalytically inactive CdeltaA mutants of DRONC exerts no detectable effect on eye development (Meier, 2000).

To investigate in detail the consequences of DRONC expression on the survival of photoreceptor and pigment cells underlying the eye surface, transverse sections of adult transgenic eyes were examined. Surprisingly, even in the pro-droncW flies, no normal cellular structures of either pigment or photoreceptor cells were visible: only remnants of pigment cells and vacuole-like structures remained. These remnant pigment cells, containing the red pigment pteridine, were responsible for the red 'spots' observed in the pro-droncW fly eyes. It was therefore concluded that GMR-driven DRONC expression kills both pigment and photoreceptor cells (Meier, 2000).

One possibility is that the ablation of internal eye structures seen in dronc transgenic flies may result from excess cell death in the developing eye disc. Third instar larval eye discs were examined for the appearance of apoptotic cells using acridine orange, which stains apoptotic cells. Compared with controls, third larval instar eye discs expressing DeltaN DRONC exhibit dramatic and super-numerary apoptosis posterior to the morphogenetic furrow. In contrast, no such sign of excessive apoptosis is evident in eye discs from third instar larvae expressing full-length pro-droncW. However, during later development (60 h after puparium formation), eye discs of pro-droncW pupae exhibit a dramatic increase in numbers of apoptotic cells. It is presumably this very late activation of apoptosis, essentially after the eye lens structure has formed, which gives the eyes of pro-droncW flies their characteristic morphology wherein the eyes show an essentially normal outer structure with internal ablation. In contrast, the devastating 'small eye' phenotype seen in pro-droncS, DeltaN dronc or GMR-rpr transgenic flies is consistent with the observed induction of cell death much earlier during larval eye development (Meier, 2000).

The pro-domain-less DeltaN DRONC generates a consistently more severe eye ablation phenotype than does pro-DRONC. Indeed, all DeltaN dronc transgenic lines die when crossed with GMR-gal4 and maintained at 25°C, although viability of some of these lines can be sustained by crossing them to a weak GMR-gal4 driver line and maintaining them at 18°C. The lethality is most likely not to be a trivial result of misexpression of GMR-gal4 in tissues other than the developing eye but, rather, to be due to the inability of DeltaN DRONC flies to open the pupae case with their heads because of extreme head malformation. As a consequence, such flies die trapped in their pupae cases. In confirmation of this, it was found that flies with severely deformed and black eyes can indeed be rescued by manually opening the puparium at the end of their development (Meier, 2000).

To examine the function of Dronc in a whole animal, transgenic flies were generated containing Dronc tagged with GFP or the inactive dronc mutant, droncC318G, also tagged with GFP, under the control of the yeast UAS(GAL4) in pUAST. Expression of these constructs was then achieved by crossing flies to various GAL4 drivers. To show that the constructs were expressed, UAS-dronc and UAS-droncC318G flies were crossed to flies containing the GMR-GAL4 driver, allowing expression in the posterior region of third instar larval eye imaginal discs. Eye imaginal discs from these third instar larvae stained specifically in the posterior region with antiGFP and antiDronc antibodies, demonstrating that high levels of specific expression were achieved and that the antiDronc antibody was specifically detecting Dronc protein. To determine whether ectopic overexpression of dronc could induce cell death, acridine orange staining of these eye imaginal discs was carried out to detect dying cells. Expression of the droncC318G construct has little effect on the normal pattern of dying cells in the eye imaginal disc, whereas wild type dronc expression results in a massive induction of cell death in the posterior part of the eye disc. Expression of dronc during embryogenesis or in different tissues during larval development using the heat shock-inducible hsp70-GAL4 driver also results in ectopic cell death (Quinn, 2000).

To examine the phenotypic consequence of expression of dronc in the eye disc, progeny of the cross of GMR-GAL4 to the UAS-dronc construct were allowed to develop into adults. Many died as pupae, which has been observed previously and attributed to the poor ability of the adults to break through the pupal case. The few adults from this cross that survive exhibit severely ablated eyes. By contrast, no death during the pupal stage was observed with flies from the cross of GMR-GAL4 to UAS- droncC318G, and adult flies showed normal eyes. Thus, the expression of dronc results in an almost complete ablation of the eye, similar to that obtained with expression of the apoptosis inducers rpr, hid, or grim from the GMR enhancer (Quinn, 2000).

Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).

Analysis of the Drosophila genome sequence reveals a single predicted transcript that encodes a type II membrane protein with structural similarities to members of the TNF superfamily. This protein is referred to as Eiger, in memory of the numerous mountaineers that have been killed by the Eiger Nordwand, the 'wall of death.' The Eiger protein contains a cytoplasmic domain, a transmembrane region between amino acid residues 36 and 62, and an extracellular domain of 353 amino acids. The C-terminal TNF homology domain (THD) of Eiger shows comparable homology to several human TNF family members (20%–25% identity). In situ hybridization has revealed a weak expression in imaginal discs with a pronounced pattern in the eye (Moreno, 2002).

Like a subset of human TNF ligands, Eiger can induce caspase-dependent apoptosis. Targeted expression of Eiger in the eyes and wings of Drosophila causes a severe ablation of these organs, and Eiger-expressing cell clones are rapidly eliminated. Both of these effects can be suppressed by coexpression of the pan-caspase inhibitor p35 (Moreno, 2002).

Caspase-8 is the key initiator caspase of death ligand-induced apoptosis in mammals. Upon stimulation by TNF, the adaptor protein FADD recruits and aggregates several molecules of procaspase-8 that mutually cleave and activate each other. Due to the involvement of an extracellular ligand, this pathway has been referred to as the 'extrinsic death pathway'. DREDD is the Drosophila caspase most similar to caspase-8 and has been shown to physically interact with Drosophila FADD. Surprisingly, complete removal of DREDD function fails to block Eiger-induced apoptosis, indicating that Eiger triggers cell death by a DREDD/caspase-8-independent pathway (Moreno, 2002).

The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).

Genetic interaction of dronc with apoptosis genes

To examine genetic interactions between Dronc and other apoptotic pathway genes, two UAS-dronc transgenic lines (#23 and #80) were chosen that result in relatively low lethality when crossed to GMR-GAL4 and a recombinant second chromosome was generated for each of these transgenes with GMR-GAL4. When GMR-GAL4 UAS-dronc#80 was crossed to wild type w1118 flies at 25°C, adult flies that exhibited slightly rough and mottled eyes were observed. A similar phenotype has been observed in previous studies and has been shown to be due to ablation of the pigment and photoreceptor cells. Similar results were observed for GMR-GAL4, UAS-dronc#23. This phenotype became more severe when expression of dronc via GMR-GAL4 was increased by raising the temperature to 29°C. Because this eye phenotype can be modified by increasing the expression of dronc, it provided a dosage-sensitive system for examining genetic interactions between dronc and other genes of the apoptosis pathway. To test this further, whether co-expression of the baculovirus caspase inhibitor P35 from the GMR enhancer was able to suppress the eye phenotype of GMR-dronc at 29°C was examined. Co-expression of GMR-p35 dramatically improves the eye ablation phenotype of GMR-dronc. Thus, in this system, Dronc is sensitive to P35 in the Drosophila eye (Quinn, 2000).

Whether Dronc is able to induce cell death in the hemocyte-derived SL2 cells was also examined. Surprisingly, transfection of these cells with full-length Dronc only resulted in 25% cell death. Because previous studies have shown that Diap1 binds to the prodomain of Dronc and may inhibit Dronc function, a truncated version of Dronc lacking the prodomain (MPD-Dronc) was transfected into SL2 cells. This resulted in a significant increase in cell death (50%). Because previous studies had failed to observe an effect of the caspase inhibitor P35 on dronc-induced cell death, a test was performed to see whether co-transfection of P35 could suppress MPD-Dronc-induced cell death. In contrast to previous results, P35 was able to significantly suppress MPD-Dronc-induced cell death in SL2 cells. This result is consistent with a previous observation showing that P35 inhibits Dronc-induced cell death in a mammalian overexpression system. However, it should be noted that co-expression of P35 does not rescue MPD-Dronc-induced cell death as well as Diap1, z-VAD-fluoromethylketone, or the dominant negative Dronc mutant, DroncC318G, although rescue is significantly better than that observed with Diap2 (Quinn, 2000).

Whether the GMR-dronc eye phenotype is sensitive to halving the dosage of the various Drosophila apoptosis-regulatory genes was tested. To assess whether the GMR-dronc eye phenotype is sensitive to the dosage of the H99 genes (reaper, hid, and grim), GMR-dronc flies were crossed to a deficiency removing the H99 genes, Df(3L)H99, at 29°C. The H99 deficiency dominantly suppressed the GMR-dronc eye phenotype. Thus, the cell death-inducing activity of dronc is sensitive to the dosage of the H99 genes. Furthermore, halving the dosage of dronc using a deficiency modifies the ablated eye phenotype of GMR-hid and GMR-rpr, suggesting that dronc is downstream of hid and rpr. To determine whether there was a genetic interaction with dronc and dark, whether decreasing the dosage of dark modified the eye phenotype of GMR-dronc at 29°C was examined. Three different P-element alleles of dark (darkCD4, darkCD8, and darkl(2)k11502) show suppression of the GMR-dronc eye phenotype, indicating that Dark plays a role in promoting Dronc-induced cell death in the eye. Halving the dosage of diap1 using deficiencies or the specific allele thread5 dominantly enhances the GMR-dronc eye phenotype at 25°C . In addition, these diap1 mutations dominantly enhance the lethality associated with GMR-dronc, resulting in at least 10-fold lower numbers of GMR-dronc/+; Df(diap1)/+ adult flies than expected. In contrast, a deficiency removing diap2 showed no effect on the GMR-dronc phenotype, and no lethal effects were observed. Thus diap1, but not a deficiency removing diap2, shows a dosage-sensitive interaction with dronc. By contrast, ectopic expression of diap1 or diap2 from the GMR promoter shows suppression of the GMR-dronc ablated eye phenotype, although GMR-diap2 results in much weaker suppression than GMR-diap1. Thus, both Diap1 and Diap2 are capable of directly or indirectly blocking Dronc-mediated cell death (Quinn, 2000).

Mutations that remove DRONC are not available. Therefore, to examine a possible role for DRONC as a cell death effector a form of DRONC, DRONCC318S, was generated in which the active site cysteine was altered to serine. Expression of similar forms of other caspases results in a suppression of caspase activity and caspase-dependent cell death. This may occur as a result of interaction of DRONCC318S with the Drosophila homolog of the caspase-activating protein Apaf-1, thus preventing the Drosophila Apaf-1 from binding to wild type DRONC and promoting its activation in a manner similar to that described for mammalian Apaf-1 and caspase-9. Transgenic Drosophila were generated in which DRONCC318S was expressed under the control of a promoter, known as GMR, that drives transgene expression specifically in the developing fly eye. The eyes of these flies, known as GMR-DRONCC318S flies, appear similar to those of wild type flies. To assay the ability of DRONCC318S to block cell death, GMR-DRONCC318S flies were crossed to flies overexpressing rpr (GMR-rpr), hid (GMR-hid), or grim (GMR-grim) under the control of the same promoter. GMR-driven expression of rpr, hid, or grim results in a small eye phenotype due to activation of caspase-dependent cell death. However, flies coexpressing GMR-DRONCC318S and one of the cell death activators showed a dramatic suppression of the small eye phenotype, indicating that cell death had been suppressed. The possibility cannot be ruled out that this suppression is a result of DRONCC318S forming nonproductive interactions with the Drosophila Apaf-1 that block its ability to activate other long prodomain caspases such as DCP-2/DREDD. However, these possibilities notwithstanding, these results suggest that DRONC activity is important for bringing about rpr-, hid-, and grim-dependent cell death (Hawkins, 2000).

Flies were generated that expressed full-length wild type DRONC under GMR control. While phenotypes displayed by individuals within a line were similar, different lines displayed eyes with various degrees of eye disruption, presumably owing to genomic position effects on the expression level of the transgene. By manipulating the number of copies of the GMR-DRONC transgene in animals, a phenotypic series was inferred in which low levels of DRONC expression (GMR-DRONCW flies) resulted in no outward phenotype, while higher levels of expression (GMR-DRONCM flies) resulted in cell death late in retinal development. These flies had eyes that were normal in size and shape, but that were largely white due to a loss of retinal pigment. Tangential sections through the eyes of GMR-DRONCM flies showed that all retinal cells, including photoreceptors, were missing. Increasing DRONC expression levels still further (GMR-DRONCS flies) resulted in flies with small eyes, similar to those seen in animals overexpressing rpr, hid, or grim. These observations show that DRONC expression in the eye induces cell death in a dose-dependent manner. Consistent with this interpretation, third instar eye imaginal discs from animals expressing GMR-DRONCS show high levels of staining with the vital dye acridine orange, which is taken up and retained by dying cells (Hawkins, 2000).

DIAP1, a Drosophila member of the IAP family of caspase inhibitors, suppresses rpr-, hid-, and grim-dependent cell death in the fly. It was reasoned that if expression of DRONC was activating the same pathway, then the GMR-DRONC eye phenotype might be sensitive to the levels of DIAP1. To test this hypothesis the amount of DIAP1 in the eye was decreased by crossing a strong loss-of-function DIAP1 point mutant, thread 5 (th5), to GMR-DRONCM flies. th5 heterozygotes are phenotypically wild type. However, flies that are heterozygous for th5, and that express GMR-DRONCM, show an enhancement of the GMR-DRONC-dependent small eye phenotype. In contrast, small eyed GMR-DRONCS flies that overexpress DIAP1 because they carry a GMR-DIAP1 transgene show a strong suppression of the small eye and pigment loss phenotypes. These observations, are consistent with the idea that DRONC activity is negatively regulated by DIAP1. However, they do not exclude the possibility that DIAP1's effects on the DRONC overexpression phenotypes are due, at least in part, to DIAP1's ability to suppress the activity of caspases such as drICE, that are activated by DRONC (Hawkins, 2000).

Genetic and biochemical evidence suggests that one mechanism by which RPR, HID, and GRIM promote apoptosis is by blocking DIAP1's ability to inhibit caspase activation or activity, thereby promoting caspase-dependent cell death. To determine if DRONCs activity could be regulated in a similar manner tests were performed to see whether RPR, HID, or GRIM could interfere with DIAP1-dependent inhibition of DRONC-dependent yeast cell death. Yeast were generated in which DRONC was expressed under GAL1 control and DIAP1 was expressed under the control of the copper-inducible CUP1 promoter. A third GAL1 vector was then introduced that was either empty or that expressed RPR, HID, or GRIM. Cells expressing GAL1-DRONC and empty vectors died when plated on medium containing galactose and 100 µM copper, but cells expressing GAL1-DRONC and CUP1-DIAP1 survived. Coexpression of GAL1-RPR had no effect on the survival of yeast expressing GAL1-DRONC and CUP1-DIAP1. However, coexpression of GAL1-HID or GAL1-GRIM completely blocked the survival of these cells. Thus, while these experiments do not exclude the possibility that HID and GRIM might alter DRONC activity directly, they are consistent with other observations arguing that these proteins mediate their effects on caspase activity, and thus presumably caspase-dependent yeast cell killing, by virtue of their interactions with DIAP1 (Watkins, 2000).

Reaper, Hid, and Grim are three Drosophila cell death activators that each contain a conserved NH2 -terminal Reaper-Hid-Grim (RHG) motif. The importance of the RHG motifs in Reaper and Grim have been examined for their different abilities to activate cell death during development. Analysis of chimeric R/Grim and G/Reaper proteins indicates that the Reaper and Grim RHG motifs are functionally distinct and help to determine specific cell death activation properties. A truncated GrimC protein lacking the RHG motif retains an ability to induce cell death, and unlike Grim, R/Grim, or G/Reaper, its actions are not efficiently blocked by the cell death inhibitors Diap1, Diap2, p35, or a dominant/negative Dronc caspase. Finally, a second region of sequence similarity was identified in Reaper, Hid, and Grim, that may be important for shared RHG motif-independent activities (Wing, 2001).

Do Reaper, Hid, and Grim share RHG-independent functions? Both truncated ReaperC and GrimC proteins induce cell death in developing tissues, indicating that regions outside the RHG motif also have death-inducing activities. Surprisingly, it was found that cell death induced by GrimC or ReaperC is only partially repressed by p35, suggesting a distinct mode of action compared with native Reaper or Grim. Similar to Reaper, Hid and Grim, GrimC does apparently act through Dronc, since GrimC-induced death is partially suppressed by a dominant/negative DroncC318S protein. However, the persistence of some eye cell death in the presence of DroncC318S indicates that GrimC and ReaperC also act through alternate pathways. Perhaps GrimC acts through pro-apoptotic Drosophila Bcl-2 orthologs that may induce cell death which is not blocked by p35. Another interesting possibilty is that GrimC might act via a Drosophila ortholog of Scythe, a Xenopus cell death regulator that binds Reaper, Hid, and Grim independently of the RHG motif (Wing, 2001 and references therein).

Role of Cytochrome C in caspase activation in Drosophila cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. Cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and the initiator caspase Dronc and effector caspase Ice are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, Debcl, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular Dronc and Ice appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, Dronc is recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals (Dorstyn, 2002).

In mammalian cell extracts, addition of cytochrome c and dATP results in the formation of an ~700-kD complex, commonly known as an apoptosome. Studies using purified components have demonstrated that the apoptosome, consisting of Apaf-1, cytochrome c, and procaspase-9, is necessary for caspase-9 activation. Since formation of an apoptosome in Drosophila has not been demonstrated and because cytochrome c is not released from mitochondria during apoptosis, whether a cytochrome c-dependent apoptosome containing Dronc is formed in Drosophila cells was tested. Cell extracts prepared from BG2 cells were fractionated by gel filtration chromatography and individual fractions were analyzed by immunoblotting using specific antibodies. In cell extracts kept at 4°C, the majority of Dronc was eluted in its monomeric form (50 kD) in fractions 20-22. Extracts that were incubated at 27°C with or without cytochrome c and dATP showed a shift of some of the Dronc protein to fractions 3-5, which correspond to a molecular mass of >670 kD. The shift in the absence of added cytochrome c may suggest that endogenous cytochrome c present in cell extracts could be sufficient to allow the formation of the large complex containing Dronc. Similar results have been seen using mammalian cell extracts. Drosophila cells grow at 27°C, however, when the cell extracts are incubated at 37°C, the majority of the Dronc is recruited to the >700-kD complex and there is increased processing of proDronc and proIce. The reason for this is not clear, however recombinant Dronc and Ice and extracts prepared from apoptotic BG2 cells show considerably more caspase activities at 37°C than at 27°C (Dorstyn, 2002).

Does the large complex contains Ice? In cell extracts incubated at 4°C, the majority of the Ice precursor remains in its monomeric form, although some appears to be dimeric. Incubation of cell extracts at 27°C or 37°C, with or without cytochrome c/dATP, results in the recruitment of a fraction of Ice to the high molecular mass complex. Interestingly, in extracts incubated at 37°C, most of the Ice in the high molecular mass complex is processed, whereas most of the monomeric Ice is in the precursor form. These results suggest the formation of an apoptosome containing Dronc and Ice in Drosophila cell extracts (Dorstyn, 2002).

To further explore the role of cytochrome c in the formation of the Dronc-containing complex, cytochrome c was immunodepleted from S100 fractions. These fractions were then subjected to gel filtration experiments. When incubated at 27°C, a small fraction of Dronc is found in the high molecular mass complex. Addition of cytochrome c and dATP causes a significant increase in the recruitment of Dronc to the >700-kD complex. Immunoblotting the fractions with the cytochrome c antibody shows that incubation of S100 at 27°C results in the recruitment of a significant proportion of cytochrome c to the >700-kD complex. Interestingly, only dimeric (26 kD) cytochrome c is detected in the >700-kD complex. These results suggest that cytochrome c and dATP, at least in part, are responsible for the formation of the complex (Dorstyn, 2002).

In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Silencing expression of either or both DC3 and DC4 has no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. Loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 fail to activate caspases in Drosophila cell lysates, but, remarkably, induce caspase activation in extracts from human cells. Overall, these results argue that DARK-mediated DRONC activation occurs independently of cytochrome c (Dorstyn, 2004).

The data clearly show that neither of the two cytochrome c species in Drosophila are required for caspase activation or apoptosis. Previous studies reported that a P-element insertion in the dc3 gene (bln1) results in loss of DRICE activity in testis (Arama, 2003). However, a recent report indicates that the bln1 P-element insertion also disrupts a number of other genes (Huh, 2004), thus questioning whether DC3 is responsible for DRICE activity. Additionally, DRICE activation during spermatogenesis appears to be independent of DARK and DRONC (Huh, 2004). If DC3 is required for caspase activation in Drosophila, a loss of function mutation in dc3 should lead to severe developmental defects and lethality. Furthermore, although a tissue-specific function has been suggested for DC3, it is unlikely that DC3 functions only during spermatogenesis, given its ubiquitous expression. Although disruption of the dc4 gene is embryonic lethal, DC4 cannot induce caspase activation and apoptosis in Drosophila cells (Dorstyn, 2004).

The question remains: how does DARK mediates DRONC activation? One possibility is that other factors can substitute for cytochrome c function during apoptosis. Alternatively, removal of DIAP1 from DRONC may be sufficient to allow an interaction with DARK and activation. Given that transcription plays a major role in developmental PCD in Drosophila, changes in the concentration of DIAP1, DRONC, and DARK proteins could facilitate caspase activation in the fly. These studies, combined with published work, demonstrate that Drosophila and mammalian cytochrome c proteins are functionally similar since they can both mediate respiration and Apaf-1 activation in mammalian cell lysates. Therefore, the requirement for cytochrome c in caspase activation in mammals is likely to have evolved late in evolution (Dorstyn, 2004).

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) results in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), are significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, this study demonstrates that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Liu, 2009).

The status quo action of JH has been well documented in several insect orders, particularly in Coleoptera, Orthoptera and Lepidoptera, in which JH treatment causes supernumerary larval molting and JH deficiency triggers precocious metamorphosis. However, as JH does not cause supernumerary larval molting in flies, evidence for the status quo action of JH in Drosophila has remained elusive. From past studies and from the experimental data presented in this study, it is concluded that the status quo hypothesis does indeed apply to JH action in Drosophila. First, although JH application during the final larval instar or during the prepupal stage has little effect on the differentiation of adult head and thoracic epidermis in Drosophila, it does prevent normal adult differentiation of the abdominal epidermis. After JH treatment, a second pupal, rather than an adult, abdominal cuticle is formed in Diptera. Second, JH or a JH agonist applied to Drosophila at the onset of metamorphosis results in lethality during pupal-adult metamorphosis. Similarly, global overexpression of jhamt (Juvenile hormone acid methyl transferase) results in severe defects during the pupal-adult transition and eventually death (Niwa, 2008). Third, CA ablation leading to JH deficiency causes precocious and enhanced fat body PCD. Fourth, JH deficiency results in pupal lethality and delayed larval development, although JH deficiency is not sufficient to cause precocious metamorphosis. The composite data demonstrate that JH in Drosophila does have status quo actions on the abdominal epidermis during pupal-adult metamorphosis and on the fat body during larval-pupal metamorphosis. It is concluded that the status quo action of JH in Drosophila is functionally important, but more subtle than that in Coleoptera, Orthoptera and Lepidoptera. However, it is not clear whether JH is essential for embryonic and earlier larval development because the CA cells are not completely ablated in the JH-deficient animals until the early-wandering (EW) stage. To address this question, it would be necessary to generate a mutant (i.e., of jhamt) that interrupts JH but not the farnesyl pyrophosphate biosynthesis pathway (Liu, 2009).

The insect fat body is analogous to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. In response to 20E pulses, Drosophila larval organs undergo a developmental remodeling process during metamorphosis. Blocking the 20E signal specifically in the fat body during the larval-pupal transition (Lsp2>; UAS-EcRDN) prevented the fat body from undergoing PCD and cell dissociation (Liu, 2009).

The experimental data in this paper demonstrates that JH prevents caspase-dependent PCD in the fat body during the larval-pupal transition in Drosophila. First, JH deficiency in Aug21>, UAS-grim resulted in the fat body undergoing precocious and enhanced PCD and cell dissociation. Aug21> is a GAL4 driver that specifically targets gene expression to the CA. Precocious and enhanced apoptosis appeared as early as L3D1 in the JH-deficient animals. Methoprene application on L3D1 was able to rescue ~40% of the pupae to adults, but it failed to rescue post-EW. Second, 2D-DIGE/MS and qPCR analyses indicated that the fat body in the JH-deficient animals has multiple developmental defects. The upregulation of the caspase genes Dronc and Drice should account for the PCD in the fat body, as overexpression of Dronc in the fat body causes PCD, cell dissociation, and thus lethality. Overexpression of Dronc or Drice in cells and tissues is sufficient to cause caspase-dependent PCD. Third, the 20E-triggered transcriptional cascade in the fat body was downregulated in the JH-deficient animals, indicating that JH does not suppress the 20E-triggered transcriptional cascade in preventing caspase-dependent PCD in the fat body (Liu, 2009).

The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of EcR. One might expect that perfect timing, titer and receptor response of JH and 20E are required to ensure accurate PCD in a tissue- and stage-specific manner during Drosophila metamorphosis. In the JH-deficient animals, the upregulation of Dronc and Drice resulted in precocious and enhanced PCD, such that the JH-deficient animals are committed to die during the larval-pupal transition. This hypothesis was strengthened by overexpression of Dronc specifically in the fat body, which caused larval lethality. Taken together, it is concluded that JH antagonizes 20E-induced caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Liu, 2009).

Based on the phenotypes and gene expression profiles in the four fly lines used, it is concluded that JH counteracts MET and GCE to prevent caspase-dependent PCD. First, the Met-overexpressing animals died during larval life, with precocious and enhanced PCD and cell dissociation in the fat body. Dramatic upregulation of Dronc and Drice was observed when Met was specifically overexpressed in the fat body and this upregulation was significantly decreased by methoprene application demonstrating that JH is epistatic to MET and GCE. Moreover, the Dronc-overexpressing animals exhibited similar phenotypes to the Met-overexpressing animals. Second, in the fat body of the JH-deficient animals, PCD and the expression of Dronc and Drice were upregulated but not as significantly as in the Met-overexpressing animals. This might explain why the JH-deficient animals did not die until early pupal life. Third, both the global JH-overexpressing animals and the Met/gce-deficient animals died during the pupal-adult transition. In these animals, Dronc and Drice were downregulated and caspase-dependent PCD was decreased in the fat body, implying that these animals died from a lack of caspase-dependent PCD. Weak mutants of Dronc and Drice mutants die during pupal life, showing that caspase-dependent PCD is essential for Drosophila metamorphosis. In addition, it was also observed that methoprene application at the onset of metamorphosis results in delayed fat body remodeling (Liu, 2009).

In the future, it will be crucial to elucidate the detailed molecular mechanism of how JH counteracts MET and GCE to prevent caspase-dependent PCD. In Drosophila S2 cells, the transcriptional activity of MET is dependent on the JH concentration and both MET-MET and MET-GCE interactions can be greatly diminished by JH. The bHLH-PAS transcription factors typically function as hetero- or homodimers. If MET/GCE is the Juvenile Hormone Receptor (JHR), the transcriptional activities of the dimerized MET/GCE and the JH-MET/GCE complex should differ. In other words, the dimerized MET/GCE should induce transcription of Dronc and Drice and, in turn, JH binding to form the JH-MET/GCE complex should reduce this induction. Although there are no examples in the literature in which a receptor, without ligand, acts as a transcriptional activator and the transcriptional activity of the receptor is diminished when the ligand is bound, it could be speculated that the JHR is a unique hormone receptor and perhaps that is the reason why it has yet to be isolated and characterized. Unfortunately, the experiments described here were conducted in Drosophila S2 cells, where the possibility of an endogenous JHR could not be eliminated. Although MET/GCE is definitely a key component in the JH signal transduction pathway, whether MET/GCE is the bona fide JHR remains conjecture (Liu, 2009).

It is very likely that MET cross-talks with EcR-USP via a large molecular complex. One can hypothesize that MET promotes 20E action in the absence of JH and suppresses 20E action in the presence of JH, a model which is favored. Drosophila FKBP39 (FK506-BP1) could be a key component in this complex because it physically interacts with MET, EcR and USP, and binds the D. melanogaster JH response element 1. Moreover, Drosophila FKBP39 inhibits 20E-induced autophagy (Juhász., 2007). Further analysis of the complex will be crucial to precisely define the molecular mechanism of cross-talk between the action of JH and 20E (Liu, 2009).

In summary, it is concluded that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila. The Drosophila fat body has provided an excellent model for studying the long-standing question of JH signal transduction. To finally settle the question of the bona fide JHR and to understand the precisely defined molecular mechanism of JH action requires further research at a variety of levels in several species of insects that can be genetically manipulated, such as Drosophila, Bombyx and Tribolium (Liu, 2009).

Caspase signalling in the absence of apoptosis drives Jnk-dependent invasion

Tumours evolve several mechanisms to evade apoptosis, yet many resected carcinomas show significantly elevated caspase activity. Moreover, caspase activity is positively correlated with tumour aggression and adverse patient outcome. These observations indicate that caspases might have a functional role in promoting tumour invasion and metastasis. Using a Drosophila model of invasion, this study shows that precise effector caspase activity drives cell invasion without initiating apoptosis. Affected cells express the matrix metalloprotinase Mmp1 and invade by activating Jnk. These results link Jnk and effector caspase signalling during the invasive process and suggest that tumours under apoptotic stresses from treatment, immune surveillance or intrinsic signals might be induced further along the metastatic cascade (Rudrapatna, 2013).

Overall, these results indicate that effector caspase activity below levels sufficient to direct cell death might be optimal for migration of transformed cells. This signalling promotes migration through Jnk, consistent with previous studies showing that Jnk lies downstream of Dronc. Caspase activation of Jnk frequently leads to compensatory proliferation, a homeostatic programme of cell replacement after apoptosis. Compensatory proliferation studies of 'undead cells' have come to opposite conclusions concerning the role of Drice. The current work is consistent with the mammalian literature placing the JNK pathway as a caspase target (Rudrapatna, 2013).

Effector caspases are active in tumours in situ and are associated with metastasis; the current results indicate that cells with moderate caspase activity that are protected from apoptosis are prone to migration. In this view, therapeutic interventions proposed to increase tumour apoptosis might paradoxically exacerbate malignancy, as has been previously suggested. Tumour inflammation has also been suggested to promote metastasis and might do so via stimulation of the extrinsic apoptosis pathway. Tumour cells commonly contain high levels of XIAP, which blocks caspases' active site in a manner similar to P35. This might provide an important mechanism directing tumours to metastasize, though the experiments emphasize the importance of precise caspase activity. A better understanding of caspases' role in tumour progression might enhance ability to predict a tumour's progression and the impact of treatments designed to promote the apoptosis process (Rudrapatna, 2013).

Protein Interactions

The physical interaction between DRONC and drICE was assessed by testing for the ability of the two proteins to co-immunoprecipitate from cell extracts. FLAG-tagged, full-length, catalytically inactive DRONC (pro-DRONC CdeltaA, 1-451) was co-expressed in 293T cells together with Myc-tagged catalytically inactive pro-drICE CdeltaA (1-339), DeltaN drICE CdeltaA (29-339) or Bcl-10. The mammalian protein Bcl-10 that contains an N-terminal CARD was used as the control in the co-immunoprecipitation experiments. Pro-DRONC specifically co-immunoprecipitates both pro-drICE and DeltaN drICE, but not Bcl-10, indicating that DRONC and drICE form a stable complex in cell extracts (Meier, 2000).

The observed difference between the pro-apoptotic activity of pro-DRONC and pro-domain-lacking DeltaN DRONC in Drosophila and mammalian cells raises the possibility that spontaneous activation of pro-DRONC is suppressed through interaction of its pro-domain with some putative cellular inhibitor. To identify such an inhibitor, Drosophila proteins were sought that interact specifically with the DRONC pro-domain in a yeast two-hybrid assay using a 0-24 h Drosophila embryonic cDNA library. From 1 × 106 yeast transformants, 56 DRONC-interacting clones were recovered, of which 17 encoded DIAP1. The second BIR domain of DIAP1 is necessary and sufficient for the interaction with the pro-domain of DRONC. This is particularly intriguing since the BIR2 region of DIAP1 is also known to interact physically with, and block the pro-apoptotic activity of, Rpr, Grim and Hid (Meier, 2000).

To verify the observed interaction between DIAP1 and DRONC, co-immunoprecipitation experiments were performed on cellular extracts obtained from 293T cells. FLAG-tagged pro-DRONC CdeltaA, DeltaN DRONC CdeltaA and DRONC-CARD (the pro-domain of DRONC on its own) were each tested for interaction with Myc-tagged DIAP1 deletion mutants (BIR1/2, 1-341; BIR1, 1-146; and BIR2, 177-341). As expected, full-length DRONC and the isolated pro-domain of DRONC (DRONC-CARD) both co-immunoprecipitate with BIR1/2 and BIR2 but not with BIR1, consistent with yeast two-hybrid data showing that the BIR2 domain of DIAP1 is required for the interaction with DRONC. Somewhat surprisingly, however, DeltaN DRONC lacking the pro-domain also co-immunoprecipitated with DIAP1, although to a far lesser extent than full-length DRONC or DRONC-CARD. The BIR2 region of DIAP1 is required for this interaction between DeltaN DRONC and DIAP1, since DeltaN DRONC forms stable complexes only with BIR1/2 and BIR2 and not with BIR1. Taken together, these results indicate that DIAP1 physically interacts with unprocessed pro-caspase DRONC and that the BIR2 region of DIAP1 is able to bind both the pro-domain and the core region of DRONC (Meier, 2000).

Several lines of evidence suggest that pro-DRONC activation is negatively regulated via its pro-domain. This is best illustrated by the biology underlying the relatively weak eye phenotype in flies expressing full length pro-droncW: (1) most UAS-pro-dronc lines are viable when crossed to a strong GMR-gal4 line and kept at 25°C: in contrast, virtually all GMR-driven DeltaN dronc transgenic lines tested die under such conditions; (2) most pro-dronc lines exhibit essentially normal outer eye structure, whereas rare surviving DeltaN dronc transgenic flies never display this 'weak' eye phenotype and have severely deformed eyes; (3) ectopic expression of pro-DRONC induces no significant increase of cell death in the eye discs of third instar larvae, whereas excessive cell death is evident posterior to the morphogenetic furrow in the eye discs of third instar larvae expressing DeltaN DRONC; (4) ectopic expression of DRONC in mammalian Rat-1 cells induces apoptosis only when its pro-domain had been removed, suggesting the existence of an innate inhibitor of DRONC activation acting through the DRONC-CARD domain. All of these observations implicate the DRONC pro-domain in repressing activation of the caspase and suggest that DRONC activation is kept in abeyance in metazoan cells through the action of some CARD-binding innate inhibitor. In contrast, studies of DRONC in S.pombe unambiguously show that isolated pro-DRONC is, by itself, perfectly capable of undergoing catalytic autoprocessing resulting in its activation. Indeed, in yeast, pro-DRONC proved more toxic than DeltaN DRONC, suggesting that the presence of the pro-domain may actually enhance DRONC activation in the absence of other modulating influences (Meier, 2000).

In insect cells, a candidate for such an innate DRONC repressor is the inhibitor of apoptosis, DIAP1, which interacts with the DRONC pro-domain: co-expression of DIAP1 completely reverts the eye ablation phenotype of pro-droncW flies, whereas the eye ablation phenotype induced by DeltaN DRONC is largely unaffected. If endogenous DIAP1, or an analog, were not expressed in the Drosophila eye until very late in its development, this would provide the requisite mechanism for holding the activity of DRONC in abeyance until very late, so generating the 'spotted eye' phenotype observed. Indeed, heterozygosity at the diap1 locus greatly enhances the eye phenotype induced by pro-DRONC overexpression, indicating that endogenous DIAP1 negatively regulates DRONC activation in vivo. This is analogous to the way in which c-IAP1, c-IAP2 and XIAP bind to, and inhibit activation of, the pro-form of the apical caspase-9 in mammalian cells. The notion that it is DIAP1, in particular, that most likely fulfils the role of in vivo suppressor of DRONC is reinforced by studies in yeast that show that while DIAP1 both interacts with, and protects from the lethal effects of, pro-DRONC, Drosophila DIAP2 and the mammalian IAP homologs MIHA, MIHB, MIHC, MIHD and XIAP offer no such protection (Meier, 2000).

Currently, very little is known about how IAPs suppress apoptosis, although the most convincing biological evidence for the ability of IAPs to regulate cell death comes from genetic studies in D.melanogaster. Deletion of the chromosomal region encoding DIAP1 enhances cell death induced by ectopic expression of Rpr, and genetic loss of DIAP1 function leads to early and widespread apoptosis, indicating that DIAP1 is essential for survival of many cell types. Furthermore, overexpression of DIAP1 suppresses cell death induced by either Rpr, Grim or Hid through direct interaction between these various pro-apoptotic proteins and the second BIR domain of DIAP1, the same BIR domain that is sufficient for its interaction with pro-DRONC. It is noteworthy that it is also the second BIR repeat of the mammalian IAP family members c-IAP1, c-IAP2 and XIAP that appears sufficient for their anti-apoptotic activity (Meier, 2000 and references therein).

The finding that DIAP1 directly binds to and inhibits cell death caused by ectopic expression of DRONC, as well as by Rpr, Grim and Hid, underscores the key role played by DIAP1 in the regulation of apoptosis in D.melanogaster and raises the possibility that Rpr, Hid or Grim may exert some, or all, of their pro-apoptotic action through displacement of DIAP1 from the pro-domain of DRONC, so allowing activation of the caspase and consequent cell death. The isolation of DIAP1 mutants that display greatly reduced binding for Rpr, Hid and Grim and significantly suppress Rpr, Hid and Grim cell killing strongly supports this idea. According to this model, IAPs function as 'guardians' of the apoptotic machinery: these guardians act to suppress the chance of spontaneous activation of the intrinsic cell death machinery by neutralizing pro-apoptotic caspases, so establishing a buffered threshold that must be either exceeded or neutralized in order to initiate the destruction of a cell (Meier, 2000).

The biochemical interaction of Dronc with various apoptosis regulators was examined by co-precipitation methods. When 293T cells were transfected with Dronc-GFP and Diap1-Myc or Diap2-Myc, it was found that Diap1, but not Diap2, was present in the Dronc immunoprecipitated complex, showing that Dronc and Diap1 can interact. Dronc can form a complex with Diap1, but not with Diap2, in SL2 cells. The H99 proteins, Rpr, Hid, and Grim, were examined after transfecting 293T cells with Dronc-GFP and with Rpr-FLAG, Hid-FLAG, or Grim-FLAG constructs. Proteins associating with antiGFP-immunoprecipitated Dronc were immunoblotted with the antiFLAG antibody. Dronc co-immunoprecipitates with Grim, but not with Rpr or Hid, suggesting that Dronc forms a complex with Grim. Whether Dronc can form a complex with P35 was examined by transfecting SL2 cells with Dronc tagged with the HA and 6xHis epitopes and with HA-tagged P35. P35 is present in the Dronc complex, indicating that these proteins can associate (Quinn, 2000).

To determine whether the interactions of Dronc with Grim or P35 are direct, whether these proteins can interact in vitro was tested. Whereas Grim can bind to Diap1 in vitro, Dronc is unable to bind to either Grim or P35. Thus, under the conditions where Grim and Diap1 can interact, Dronc and P35 or Grim do not interact, suggesting that the interaction observed between Dronc and Grim or P35 in cells is indirect. Furthermore, the addition of Diap1 allows Grim to be co-immunoprecipitated with Dronc, suggesting that the complex formed in vivo between Dronc and Grim may be mediated by Diap1 (Quinn, 2000).

The amino-terminal region of Apaf-1-related-killer (Ark) containing the CARD and CED-4/Apaf-1 homology domains but lacking the WD40 repeats has been shown to bind to Dredd and Drice. Furthermore, in mammalian tissue culture cells, Dark has been shown to bind to Dronc. To investigate the region of Dark required for the interaction of Dark with Dronc, SL2 cells were transfected with a construct containing a Myc-tagged Dark amino-terminal region [Ark(1-411)], containing the CARD and CED-4/Apaf-1 homology domains, alone or with a FLAG-tagged Dronc construct. Dark-Myc was immunoprecipitated with antiMyc antibodies; pelleted complexes, and cell lysates were analyzed by immunoblotting with antiFLAG antibodies. Dronc-FLAG protein (50% of the protein present in the lysate) was detected in the antiMyc-Arc immunoprecipitate. Thus, the amino-terminal region of Dark containing the CARD domain and the CED-4/Apaf-1 homology region is sufficient for Dronc association in vivo (Quinn, 2000).

To test the requirement of Ark for Dronc activation, extracts prepared from darkCD8 homozygous flies were examined for caspase activity and for their ability to cleave Dronc in vitro. Ark mutant flies have reduced caspase activity compared with wild type for several caspase substrates. VEID is the preferred substrate for Dronc, although VDVAD is also cleaved well by Dronc. DEVD is a caspase-3 substrate that is cleaved poorly by Dronc but preferred by the downstream caspases Dcp1, Decay, and Drice. Thus, Ark mutant extracts contain lower cleavage activity toward both preferred Dronc substrates and preferred downstream caspase substrates. Lower caspase activity has also been observed in extracts from Ark mutant embryos. Furthermore, Ark mutant extracts showed considerably reduced ability to cleave Dronc to its active form, showing that Ark is important for Dronc processing. Because darkCD8 (and darkCD4) are hypomorphic mutants, and it is not known whether they are completely null because a deficiency of the Ark region is not available, the residual Dronc processing observed may be due to residual Ark activity or to an alternative mechanism (Quinn, 2000).

Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival in Drosophila and appear to neutralize the cell death machinery by binding to and ubiquitylating pro-apoptotic caspases. Cell death is triggered when 'Reaper-like' proteins bind to IAPs and liberate caspases from IAPs. The thioredoxin peroxidase Jafrac2 has been identified as an IAP-interacting protein in Drosophila cells that harbors a conserved N-terminal IAP-binding motif. In healthy cells, Jafrac2 resides in the endoplasmic reticulum but is rapidly released into the cytosol following induction of apoptosis. Mature Jafrac2 interacts genetically and biochemically with DIAP1 and promotes cell death in tissue culture cells and the Drosophila developing eye. In common with Rpr, Jafrac2-mediated cell death is contingent on DIAP1 binding because mutations that abolish the Jafrac2-DIAP1 interaction suppress the eye phenotype caused by Jafrac2 expression. Jafrac2 displaces Dronc from DIAP1 by competing with Dronc for the binding of DIAP1, consistent with the idea that Jafrac2 triggers cell death by liberating Dronc from DIAP1-mediated inhibition (Tenev, 2002).

Jafrac2 was recovered as a DIAP1-interacting protein in the cell using the tandem affinity purification (TAP) system. Like Rpr, Grim, Hid, Sickle, Smac/DIABLO and HtrA2/Omi, Jafrac2 bears a conserved N-terminal IAP-binding motif (IBM) essential for IAP interaction. Jafrac2 is synthesized as a precursor protein with an N-terminal signal peptide that targets it to the ER. Upon import into the ER, the signal peptide of Jafrac2 is cleaved off, thereby exposing the IAP interacting domain that allows this mature Jafrac2 isoform to interact with DIAP1, DIAP2 and XIAP (Tenev, 2002).

In living cells Jafrac2 is compartmentalized and sequestered in the ER away from IAPs, where it exists exclusively in the processed from. This is evident because mature Jafrac2, like cytochrome c, which is compartmentalized in mitochondria, remains associated with the membrane fraction in healthy cells. Following stimulation of apoptosis by UV irradiation or ER stress-inducing agents, mature Jafrac2 is released from the membrane fraction and is present in the cytosol where it can interact with DIAP1 and DIAP2. Because the pro-apoptotic, IAP-interacting form of Jafrac2 is released only upon cell death insult, the major regulatory step for Jafrac2 appears to be its release from the ER lumen. The release of Jafrac2 from the ER of UV-irradiated cells occurs early in UV-mediated apoptosis. This is evident because Jafrac2 expression becomes diffuse in otherwise morphologically normal cells within 3-4 h following UV exposure. In similar experiments, the mitochondrial release of cytochrome c, Smac/DIABLO and HtrA2/Omi that occurs, early in apoptosis, also became apparent within 3-4 h following UV treatment. Thus, Jafrac2 resembles Smac/DIABLO and HtrA2/Omi that are similarly compartmentalized in healthy cells and that promote caspase activation after their release from mitochondria following the cell death trigger. Furthermore, analogous to Smac/DIABLO and HtrA2/Omi, Jafrac2 also requires N-terminal processing to generate its pro-apoptotic form. Hence, Jafrac2, Smac/DIABLO and HtrA2/Omi all undergo a maturation process through cleaving off their signal peptide following import into their respective organelles. This organelle-specific maturation ensures that newly synthesized Jafrac2, Smac/DIABLO and HtrA2/Omi will not promote apoptosis prior to their sequestration into organelles (Tenev, 2002).

In common with Rpr, Grim and Hid, Jafrac2 interacts genetically and biochemically with DIAP1 and is able to promote cell death. In the Drosophila eye and tissue culture cells, mature Jafrac2, like Rpr, efficiently induces cell death in a DIAP1-binding dependent manner. Recent studies have suggested that Rpr and Grim antagonize the anti-apoptotic activity of IAPs by two distinct mechanisms -- (1) by a mechanism that requires DIAP1 binding, Rpr promotes DIAP1 self ubiquitylation and proteasomal degradation and (2) Rpr and Grim were also found to repress global protein translation by a mechanism that does not rely on IAP binding. The Ub fusion technique has been used to examine whether Jafrac2 and Rpr possess apoptosis-promoting activities that are independent of IAP binding. In vivo Rpr and Jafrac2 promote cell death exclusively in an IAP-binding dependent manner because mutations that impair the binding between DIAP1 and Rpr or Jafrac2 completely abolish their ability to induce cell death in the developing eye and tissue culture cells. Thus, Rpr and Jafrac2 that fail to bind to DIAP1 also fail to induce cell death. Mutations in endogenous diap1, which greatly impair the binding of DIAP1 to Rpr or Jafrac2, suppress Rpr and Jafrac2-mediated cell killing. Together, these data argue that in common with Rpr, mature Jafrac2 promotes cell death, and this activity is contingent upon their binding to DIAP1 (Tenev, 2002).

The interaction between Jafrac2 and the DIAP1 BIR2 domain is indispensable for its pro-apoptotic function. Interestingly, Jafrac2 and Dronc share a common binding site in the BIR2 domain that is distinct from the site of interaction between the DIAP1 BIR2 domain and Rpr and Hid. The th4 mutation of DIAP1's BIR2 domain greatly diminishes binding to Jafrac2 and Dronc, whereas the same mutation does not affect its binding to Rpr and Hid. In addition, the th23-4 DIAP1 mutation that greatly impairs the binding of DIAP1 to Rpr and Hid does not affect the DIAP1-Jafrac2 interaction. Consistent with the biochemical data, flies carrying the th4 mutation, which abolishes Jafrac2 binding, display strongly suppressed Jafrac2-induced eye ablation but enhanced Rpr-induced cell death in the eye (Tenev, 2002).

Several lines of evidence show that the IBM of Jafrac2 is essential for IAP binding and induction of apoptosis. Mutations that delete or obstruct the N-terminus of mature Jafrac2 abrogate the ability of Jafrac2 to bind to DIAP1 and trigger cell death. The view that Jafrac2 harbors a bona fide IAP-binding motif is strongly supported by crystal structure analyses that have identified Ala1 of IBMs as the critical residue to anchor this motif to the BIR surface of IAPs. In addition to the requirement of Ala1, there is a strong preference for Pro3. In accordance with other IBMs, the putative IBM of mature Jafrac2 bears Ala1 and Pro3. Furthermore, the IBM of Rpr is functionally interchangeable with the IBM of Jafrac2. A chimeric Rpr mutant (AKP-Rpr) in which the IBM of Rpr was replaced with the IBM of Jafrac2, displayed the same phenotype and cell death promoting efficacy as wild-type Rpr (AVA-Rpr) in both the Drosophila developing eye and tissue culture cells. Together, these results reveal that whereas Jafrac2 and Rpr share a common IAP-binding motif, they also have some distinct DIAP1-binding requirements that presumably give these interactions their specificity (Tenev, 2002).

Physical interaction between DIAP1 and caspases is essential to regulate apoptosis in vivo because embryos with a homozygous mutation that abolishes Dronc binding die early during embryogenesis due to widespread apoptosis. Unrestrained cell death caused by loss of DIAP1 function requires the Drosophila Apaf-1 homolog DARK because a mutation in dark rescues DIAP1-dependent defects. Thus, loss of DIAP1 function allows DARK-dependent caspase activation. Although activation of downstream, effector caspases is required for normal cell death, the activation of initiator caspases, such as Dronc, is rate limiting for the activation of this cascade. The observed unrestrained cell death caused by loss of DIAP1 function is likely to be triggered by the initiator caspase Dronc because DIAP1 normally suppresses Dronc activation, which in turn is mediated by DARK. In line with the current model on caspase activation, it is argued that the DIAP1-mediated inhibition of Dronc is the key regulatory step in controlling cell death. This view is supported by the observation that flies with diap1 mutations that either abolish binding or ubiquitylation of Dronc completely fail to suppress Dronc- mediated cell death in vivo. Thus, DIAP1 suppresses Dronc activation by binding to and targeting Dronc for ubiquitylation. However, when Rpr-like molecules displace DIAP1 from Dronc, Dronc is recruited into a 700 kDa size apoptosome protein complex that results in Dronc activation. Consequently, cell death is triggered when Dronc is liberated from DIAP1. Thus, the key event in regulating the caspase cascade appears to be inhibition of Dronc by DIAP1 (Tenev, 2002).

Several lines of evidence support the notion that Jafrac2 promotes cell death by interfering with the Dronc-DIAP1 interaction, thereby displacing and liberating Dronc from DIAP1. (1) Jafrac2 and Dronc bind to the same site of the BIR2 domain of DIAP1, since the BIR2 th4 mutation of DIAP1 equally abolished Dronc and Jafrac2 binding. In contrast, Rpr and Hid binding to the th4 DIAP1 mutant remains unaffected. (2) Jafrac2 competes with Dronc for the binding of DIAP1, and Jafrac2 possesses a significantly higher DIAP1-binding affinity compared with that of Dronc to DIAP1, as would be expected of a protein that displaces Dronc from DIAP1. (3) Ectopic expression of Jafrac2 in the developing Drosophila eye causes a phenotype that is highly reminiscent of the phenotype observed in flies ectopically expressing Dronc. (4) Heterozygosity at the dronc locus rescued the eye-ablation phenotype induced by Jafrac2, indicates that apoptotic signal transduction initiated by Jafrac2 is mediated through Dronc. Taken together, these results indicate that Jafrac2 promotes cell death by liberating Dronc from the anti-apoptotic activity of DIAP1 (Tenev, 2002).

The observation that Jafrac2, like the apoptotic inducers Rpr, Grim and Hid, induces apoptosis through binding to DIAP1 places Jafrac2 in a potentially pivotal position to regulate apoptosis. The findings are consistent with a model whereby Jafrac2 promotes apoptosis by displacing DIAP1 from Dronc, so allowing activation of the caspase cascade and consequent cell death. The idea is favored whereby Jafrac2 function is additive to, but independent of, Rpr. The early release of Jafrac2 from the ER of UV-irradiated cells is consistent with the view that Jafrac2 is involved in the initiation of apoptosis. Thus, Jafrac2 is released from the ER at a time when other early apoptotic events occur, such as the mitochondrial release of cytochrome c, Smac/DIABLO and HtrA2/Omi in mammalian cells. Once released, Jafrac2 interacts with DIAP1 and thereby liberates Dronc, which in turn is activated by DARK. In line with the notion that Jafrac2 functions in a complementary but distinct cell death pathway to Rpr, Grim and Hid, it is found that a chromosomal deletion that includes the jafrac2 locus does not suppress the eye phenotypes caused by ectopic expression of Rpr, Grim and Hid. However, it is possible that Jafrac2 may also be part of a positive feedback mechanism, which cooperates with Rpr-like proteins to promote apoptosis in response to cellular damage. These two alternatives cannot be distinguished because no jafrac2 mutant flies are available and Jafrac2 is refractory to the effect of dsRNA interference (Tenev, 2002).

The data are consistent with the idea that Jafrac2, with its thioredoxin peroxidase activity and IAP-binding ability, contains two distinct functions. In healthy cells, Jafrac2 may fulfil a 'housekeeping' role through its peroxidase activity by protecting the cell from oxidative damage. Consistent with this view, members of the peroxiredoxin protein family play an important role in protecting cells against oxidative damage by scavenging intracellularly generated reactive oxygen species, such as H2O2. However, upon UV irradiation, mature Jafrac2 is released from the ER and competes with Dronc for the binding of DIAP1 that is independent of its peroxidase activity. Consequently, Jafrac2 liberates Dronc from DIAP1 inhibition and allows activation of the proteolytic caspase cascade, resulting in cell death (Tenev, 2002).

Members of the IAP family block activation of the intrinsic cell death machinery by binding to and neutralizing the activity of pro-apoptotic caspases. In Drosophila melanogaster, the pro-apoptotic proteins Reaper Rpr, Grim and Hid all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. In vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, the RING finger of DIAP1 promotes the ubiquitination of both itself and of Dronc. Disruption of the DIAP1 RING finger does not inhibit its binding to Rpr, Hid or Dronc, but completely abrogates ubiquitination of Dronc. These data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination (Wilson, 2002).

DRONC is involved in ROS-induced apoptosis caused by impairment of the selD/sps1 homolog in Drosophila

The cellular antioxidant defense systems neutralize the cytotoxic by-products referred to as reactive oxygen species (ROS). Among them, selenoproteins have important antioxidant and detoxification functions. The interference in selenoprotein biosynthesis results in accumulation of ROS and consequently in a toxic intracellular environment. The resulting ROS imbalance can trigger apoptosis to eliminate the deleterious cells. In Drosophila, a null mutation in the selD gene (homologous to the human selenophosphate synthetase type 1) causes an impairment of selenoprotein biosynthesis, a ROS burst and lethality. This mutation (known as selDptuf) can serve as a tool to understand the link between ROS accumulation and cell death. To this aim, the mechanism by which selDptuf mutant cells become apoptotic was analyzed in Drosophila imaginal discs. The apoptotic effect of selDptuf does not require the activity of the Ras/MAPK-dependent proapoptotic gene hid, but results in stabilization of the tumor suppressor protein p53 and transcription of the Drosophila pro-apoptotic gene reaper (rpr). Genetic evidence supports the idea that the initiator caspase DRONC is activated and that the effector caspase DRICE is processed to commit selDptuf mutant cells to death. Moreover, the ectopic expression of the inhibitor of apoptosis DIAP1 rescues the cellular viability of selDptuf mutant cells. These observations indicate that selDptuf ROS-induced apoptosis in Drosophila is mainly driven by the caspase-dependent p53/Rpr pathway (Morey, 2003).

Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination

The inhibitor of apoptosis protein DIAP1 inhibits Dronc-dependent cell death by ubiquitinating Dronc. The pro-death proteins Reaper, Hid and Grim (RHG) promote apoptosis by antagonizing DIAP1 function. This study reports the structural basis of Dronc recognition by DIAP1 as well as a novel mechanism by which the RHG proteins remove DIAP1-mediated downregulation of Dronc. Biochemical and structural analyses revealed that the second BIR (BIR2) domain of DIAP1 recognizes a 12-residue sequence in Dronc. This recognition is essential for DIAP1 binding to Dronc, and for targeting Dronc for ubiquitination. Notably, the Dronc-binding surface on BIR2 coincides with that required for binding to the N termini of the RHG proteins, which competitively eliminate DIAP1-mediated ubiquitination of Dronc. These observations reveal the molecular mechanisms of how DIAP1 recognizes Dronc, and more importantly, how the RHG proteins remove DIAP1-mediated ubiquitination of Dronc (Chai, 2003).

Drosophila caspase transduces Shaggy/GSK-3beta kinase activity in neural precursor development

Caspases are well known for their role in the execution of apoptotic programs, in which they cleave specific target proteins, leading to the elimination of cells, and for their role in cytokine maturation. In this study, a novel substrate was identified, that, through cleavage by caspases, can regulate Drosophila neural precursor development. Shaggy (Sgg)46 protein, an isoform encoded by the sgg gene and essential for the negative regulation of Wingless signaling, is cleaved by the Dark-dependent caspase. This cleavage converts it to an active kinase, which contributes to the formation of neural precursor [sensory organ precursor (SOP)] cells. This evidence suggests that caspase regulation of the wingless pathway is not associated with apoptotic cell death. These results imply a novel role for caspases in modulating cell signaling pathways through substrate cleavage in neural precursor development (Kanuka, 2005; full text of article).

Previous genetic studies of sgg mutant flies showed the interesting observation that some phenotypes of sgg mutants can be rescued by the expression of sgg10 or sgg39 (the other sgg isoform similar to sgg10), but not sgg46, suggesting that Sgg46 might be an inactive form. The Sgg10 kinase phosphorylates the Arm protein and induces its degradation. Various forms of Sgg protein were tested for this activity. Expression of Sgg10 induces Arm phosphorylation and degradation in a kinase-dependent manner. In contrast, full-length Sgg46 did not produce the same effects on the Arm protein. Interestingly, expression of a putative cleaved form of Sgg46, containing the kinase domain (myc-Sgg46 DeltaN235 and myc-Sgg46 DeltaN300), led to Arm phosphorylation and degradation in a manner similar to that of Sgg10. These results suggest that full-length Sgg46 is an inactive form that can be converted into an active kinase via caspase-dependent cleavage (Kanuka, 2005).

Whether these findings would be applicable to macrochaete and SOP cell development in vivo was tested by using transgenic flies expressing Sgg proteins. The ectopic expression of Sgg10 by sca-GAL4 caused the loss of macrochaetes and SOP cells. No apoptotic cells in the myc-Sgg10 protein-expressing region of the wing disc could be detected, indicating that this disappearance did not result from the death of SOP cells. Consistent with the immunoblotting results, full-length Sgg46 did not influence macrochaete and SOP cell formation, whereas the cleaved form of Sgg46 (Sgg46 DeltaN300) worked in a manner similar to that of Sgg10. After crossing sca-GAL4+UAS-DRONC DN to UAS-sgg10, most F1 progeny showed a clear loss of macrochaetes in the scutellum, indicating that Sgg kinase activation might be downstream of caspases. These observations suggest that the processing of Sgg46 by caspases leads to the formation of an active kinase that can negatively regulate SOP cell development (Kanuka, 2005).

Finally, whether Sgg46 contributes significantly to macrochaete and SOP cell formation in vivo was investigated. The ectopic expression of Sgg46 D235G/D300G by sca-GAL4 significantly induced extra macrochaetes and SOP cells. Since Sgg46 D235G/D300G could not be cleaved by caspases, this noncleaved Sgg46 might act as dominant-negative form against endogenous Sgg function. Furthermore, an ectopic knockdown of Sgg protein expression by dsRNA-expressing constructs revealed that the specific reduction of the Sgg46 protein induced extra macrochaetes. However, inhibition of Sgg46 is less effective at producing extra macrochaetes than inhibiting Dark or DRONC, suggesting that modulation of Sgg kinase activity may not be the only mechanism contributing to SOP formation. It still remains to be examined whether or not Sgg46 is actually cleaved and converted into an active form in proneural clusters, and will require further examination in vivo. Based on the findings that loss of Sgg function or inhibition of caspase activity resulted in extra macrochaetes mainly in the scutellum of the adult notum (pSC and aSC), where Wingless is highly expressed, and that caspases are activated in scabrous-expressing cluster, it could be considered that scabrous-expressing SOP cells that will produce pSC and aSC macrochaetes are located in specific region, where precise formation of each set of macrochaetes might require both (1) Wg expression (to increase bristle) and (2) caspase activation (to decrease bristle). Thus, it appears that Dark-dependent caspase signaling mediates the total Sgg kinase activity by processing Sgg46 into an active form, thereby negatively regulating Wingless-sensitive macrochaete development (Kanuka, 2005).

Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease

Although essential in mammals, in flies the importance of mitochondrial outer membrane permeabilization for apoptosis remains highly controversial. This study demonstrates that Drosophila Omi (dOmi; FlyBase term: HtrA2), a fly homologue of the serine protease Omi/HtrA2, is a developmentally regulated mitochondrial intermembrane space protein that undergoes processive cleavage, in situ, to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the proapoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. In summary, this study demonstratesin flies that mitochondrial permeabilization not only occurs during apoptosis but also results in the release of a bona fide proapoptotic protein (Challa, 2007).

The role of mitochondria in fly apoptosis remains highly controversial, due in large part to disagreement over whether mitochondria undergo losses in Δψm (mitochondrial membrane potential) and MOMP (mitochondrial outer membrane permeabilization) following stress. Moreover, although mitochondrial release of cytochrome c in mammalian cells initiates formation of the Apaf-1 apoptosome complex and activation of caspases, there is disagreement over the importance of cytochrome c for promoting cell death in flies. The cytochrome c debate notwithstanding, there are additional mitochondrial proteins in mammals that play a role in promoting apoptosis, including the dual IAP antagonist and serine protease, Omi/HtrA2 (Hegde, 2001; Martins, 2001; Suzuki, 2001; Verhagen, 2001). In these studies, attempts were made to determine if the Drosophila homologue of Omi might likewise participate in cell death. It was found that dOmi was highly homologous to hOmi, particularly within the serine protease domain, and that its expression was developmentally regulated. dOmi was imported into fly mitochondria and processed in situ, resulting in the removal of its mitochondrial targeting sequence (MTS) and exposure of two distinct IAP binding motif (IBMs). The mature forms of dOmi were then released into the cytoplasm following stress, through both caspase-dependent and -independent processes. However, once in the cytosol, dOmi induced cell death in S2 cells and in the developing fly eye, primarily through proteolytic degradation of DIAP1 and likely other substrates (Challa, 2007).

Indeed, catalytically inactive Δ79-dOmiS266A and Δ92-dOmiS266A failed to induce significant apoptosis, which was somewhat surprising, given that both forms of dOmi selectively bound to the BIR2 domain in DIAP1 and displaced the initiator caspase DRONC. In particular, the affinity of Δ79-dOmi for BIR2 was lower than that observed for Rpr-IBM, but was slightly higher than that observed for mature Smac with XIAP-BIR3. So why did dOmi require its proteolytic activity to induce cell death, rather than inducing rapid IBM-dependent apoptosis? Notably, unlike other fly IAP antagonists, which exhibit partial preference for either the BIR1 or BIR2 domains, dOmi completely failed to bind the BIR1 domain in DIAP1 and did not displace the active effector caspase DrICE. Thus, it is possible that the continued inhibition of DrICE by DIAP1 was sufficient to inhibit cell death. There is precedence for such a scenario in mammals, since it has been shown that XIAP mutants that fail to bind and inhibit caspase-9 can still prevent apoptosis through inhibition of caspase-3 alone (Challa, 2007).

One of the primary differences between fly and mammalian IAP antagonists relates to their abilities to independently induce apoptosis. Indeed, Rpr, Hid, and Grim induce robust cell death in both cultured cells and, whereas overexpression of mature Smac in the cytoplasm of mammalian cells generally fails to induce apoptosis in the absence of an accompanying prodeath stimulus. A potential explanation for these results may involve their relative capacities to induce RING-dependent autoubiquitinylation upon binding to IAPs. Indeed, while many IAP antagonists in the fly induce DIAP1 autoubiquitinylation, Smac appears to suppress XIAP autoubiquitinylation. In these studies, dOmi failed to induce or suppress DIAP1 autoubiquitinylation upon binding to its BIR2 domain. Thus, in the absence of dOmi's proteolytic activity, DIAP1 may again be free to maintain its inhibition of DrICE via its BIR1 domain. By contrast, given that DIAP1 can protect cells by targeting active DRONC for proteosomal degradation, it is also plausible that DIAP1 might regulate cell death, in part by, promoting the turnover of dOmi. It has been reported that the DIAP1 binding mutant, DRONC (F118E), induces significantly more cell death than wild-type DRONC, when expressed in the developing fly eye, and correspondingly, this study found that Δ92-dOmi consistently produced a more severe phenotype than Δ79-dOmi, in accordance with their relative affinities for DIAP1 (Challa, 2007).

Others have reconciled such differences between the mammalian and fly IAP antagonists by arguing that, in contrast to the Apaf-1·caspase-9 apoptosome complex, the DARK·DRONC apoptosome complex is constitutively active. Consequently, DIAP1 is required to continuously ubiquitinylate DRONC and mediate its turnover in order to prevent cell death. In this model, Rpr, Hid, or Grim need only displace this active DRONC, in order to promote the activation of effector caspases and induce apoptosis. However, recent studies suggest that, at least for Rpr and Grim, the C-terminus of these IAP antagonists play important roles in promoting both mitochondrial injury and/or inhibition of protein translation. These alternative functions for Rpr and Grim may be necessary to first initiate caspase activation, after which the IBMs serve to displace these active caspases from DIAP1. Therefore, it could be that binding of dOmi to DIAP1-BIR2 per se does not induce apoptosis, because in the absence of another stimulus, there may be very little active DRONC to displace. In any event, regardless of whether dOmi induces cell killing solely through its proteolytic activity, or functions as a pure IAP antagonist in certain contexts, these studies suggest that mitochondria may play a far more important role in apoptosis in the fly than previously thought (Challa, 2007).

The role of the RING-finger protein Elfless in Drosophila spermatogenesis and apoptosis

elfless (CG15150, FBgn0032660) maps to polytene region 36DE 5' (left) of reduced ocelli/Pray for Elves (PFE) on chromosome 2L and is predicted to encode a 187 amino acid RING finger E3 ubiquitin ligase that is putatively involved in programmed cell death (PCD, e.g., apoptosis). Several experimental approaches were used to characterize CG15150/elfless and test whether defects in this gene underlie the male sterile phenotype associated with overlapping chromosomal deficiencies of region 36DE. elfless expression is greatly enhanced in the testes and the expression pattern of UAS-elfless-EGFP driven by elfless-Gal4 is restricted to the tail cyst cell nuclei of the testes. Despite this, elfless transgenes failed to rescue the male sterile phenotype in Df/Df flies. Furthermore, null alleles of elfless, generated either by imprecise excision of an upstream P-element or by FLP-FRT deletion between two flanking piggyBac elements, are fertile. In a gain-of-function setting in the eye, it was found that elfless genetically interacts with key members of the apoptotic pathway including the initiator caspase Dronc and the ubiquitin conjugating enzyme UbcD1. DIAP1, but not UbcD1, protein levels are increased in heads of flies expressing Elfless-EGFP in the eye, and in testes of flies expressing elfless-Gal4 driven Elfless-EGFP. Based on these findings, it is speculated that Elfless may regulate tail cyst cell degradation to provide an advantageous, though not essential, function in the testis (Caldwell, 2009).

Emerging data are now elucidating the roles of key members of the apoptosome, including the caspases Dronc and Drice, in spermatid individualization in Drosophila. The molecules are thought to be inhibited by dBruce, a ubiquitin-conjugating enzyme with a BIR domain. In effect, dBruce may be acting in the spermatid cysts in a manner similar to another documented BIR-domain protein, DIAP1. This study examined the role of the E3 ubiquitin ligase, Elfless, and shows that, consistent with its prediction as a RING finger protein, Elfless interacts with key members of the apoptotic pathway (Caldwell, 2009).

It is proposed that Elfless acts to directly or indirectly regulate UbcD1 activity in the apoptotic pathway. w; GMR-Gal4; UAS-elfless in an otherwise wild type background produces pigment cell defects reminiscent of those of w; GMR-Gal4; UAS-Dronc. As previously shown, w; GMR-Gal4; UAS-Dronc in a ubcD1 heterozygous mutant background produces an eye phenotype slightly worse than w; GMR-Gal4; UAS-Dronc alone which suggests that Dronc may also be a target of UbcD1. Finally, inhibition of apoptosis through Dronc is effectively lost in w; GMR-Gal4; UAS-Dronc in a diap1/+ background; the eye defects exhibited in these lines are severe and flies die as pharate adults. w; GMR-Gal4; UAS-elfless flies in a ubcD1/+ mutant background similarly die as pharate adults and the eye phenotype is significantly worse than that of w; GMR-Gal4; UAS-elfless alone; pigmentation is absent in these flies and the size of the eye is reduced due to decreased UbcD1-mediated inhibition of Dronc and DIAP1. These data suggested that Elfless may be regulating UbcD1 activity (Caldwell, 2009).

While Elfless and UbcD1 had been shown to interact by yeast-two hybrid, it has not been possible to confirm a direct association between Elfless and either DIAP1 or UbcD1 by co-immunoprecipitation experiments. Nevertheless, it is clear from Western blot analysis that mis-expression of Elfless-EGFP in the eye or testes does not significantly change the level of UbcD1 protein but does increase DIAP1 protein levels. Thus, if Elfless is downregulating UbcD1 activity, it seems to be doing so without changing UbcD1 levels. Since DIAP1 auto-ubiquitination is UbcD1-dependent, Elfless-mediated downregulation of UbcD1 activity is consistent with reduced DIAP1 auto-ubiquitination and degradation, resulting in higher DIAP1. While on the one hand this would increase the anti-apoptotic activity of DIAP1, UbcD1 downregulation on the other hand would also increase the pro-apoptotic activity of Dronc, effectively circumventing DIAP1, and producing the somewhat mild eye phenotype evident in w; GMR-Gal4; UAS-elfless in an otherwise wild type background. Consistent with this model, in ubcD1 heterozygotes the eye phenotype is severely worsened and lethality is evident, while in Dronc heterozygotes, the eye phenotype is improved (Caldwell, 2009).

Although this study clearly demonstrates a genetic interaction of elfless with Dronc and ubcD1 in PCD and it is proposed that mis-expressed Elfless in the eye negatively regulates UbcD1 activity, no essential role in fertility could be ascribed to this locus, despite the promising role of this molecule in sperm development based on expression profiling and the Df/Df male sterility phenotype. Furthermore, an elfless transgene is not sufficient to rescue the male sterility associated with these deficiencies in polytene region 36DE. Despite the lack of a sterility phenotype when the RING finger protein encoded by elfless is deleted from the genome, the fact that elfless is functionally retained in the genome by selection indicates that elfless performs an important and advantageous, albeit redundant, function in the testes. Thus, while in laboratory vials, males are able to produce normal numbers of offspring, subtle functional differences can be quite significant in wild populations. What advantage could Elfess provide in the tail cyst cell? One possibility is that mobilization of Elfless in the nuclei of these cells facilitates tail cyst cell degeneration for more efficient resorption; sperm release from the cyst can still take place, but there may be a physiological burden on the testis during the life-time of the fly. The fact that Elfless is in the nucleus may suggest that it targets gene expression at this transitional stage of tail cyst cells. Full activation of apoptosis by Elfless in tail cyst cells is unlikely, as Elfless is nuclear, and no TUNEL staining in wildtype testes appears in the late stage cysts. Nevertheless, apoptosis signaling can be quite different in varying developmental contexts, and there is precedent for enlisting branches of the apoptotic pathway in different developmental processes. For example, Caspase-3 is activated in the cystic bulge of the developing spermatocyst, while the correct balance of DIAP1 levels is important for cellular mobilization of border cells and other cells during oogenesis. More incisive future approaches will be needed to discern the roles that Elfless and other subtle modulators play in mating and evolution (Caldwell, 2009).

Dronc caspase exerts a non-apoptotic function to restrain phospho-Numb-induced ectopic neuroblast formation in Drosophila

Drosophila neuroblasts have served as a model to understand how the balance of stem cell self-renewal versus differentiation is achieved. Drosophila Numb protein regulates this process through its preferential segregation into the differentiating daughter cell. How Numb restricts the proliferation and self-renewal potentials of the recipient cell remains enigmatic. This study shows that phosphorylation at conserved sites regulates the tumor suppressor activity of Numb. Enforced expression of a phospho-mimetic form of Numb (Numb-TS4D) or genetic manipulation that boosts phospho-Numb levels, attenuates endogenous Numb activity and causes ectopic neuroblast formation (ENF). This effect on neuroblast homeostasis occurs only in the type II neuroblast lineage, which generates intermediate neural progenitors (INPs). INPs undergo a maturation process and multiple rounds of asymmetric division to produce GMCs and differentiated progenies. This study identified Dronc caspase as a novel binding partner of Numb, and demonstrates that overexpression of Dronc suppresses the effects of Numb-TS4D in a non-apoptotic and possibly non-catalytic manner. Reduction of Dronc activity facilitates ENF induced by phospho-Numb. These findings uncover a molecular mechanism that regulates Numb activity and suggest a novel role for Dronc caspase in regulating neural stem cell homeostasis (Ouyang, 2011).

Proper balance of the self-renewal versus differentiation of stem cells is crucial for tissue homeostasis. Disruption of this process could contribute to tumorigenesis. Numb has been identified as a key player that limits the proliferation potential of neuroblasts and INPs. This study elucidates the mechanisms of Numb action in this process and uncover a novel mechanism by which Numb activity is regulated at the post-translational level. The results suggest a model in which phosphorylation of Numb at conserved sites within its functionally important PTB domain impairs its association with the caspase Dronc and attenuates its tumor suppressor activity in type II neuroblasts (Ouyang, 2011).

As a defining feature of Numb protein is its asymmetric localization in stem cells and progenitors, previous studies of Numb have been focused on the control of its asymmetric localization. A number of factors have been identified to regulate Numb localization, including its binding partner Pon and kinases such as aPKC, Aurora A and Polo. This presents evidence that phosphorylation of Numb at the putative Polo sites primarily affect Numb activity in negatively regulating Notch signaling through promoting the endocytosis of Spdo. Although not all the identified Polo phosphorylation sites in Numb perfectly match the optimal consensus sequence initially defined for Polo, the Polo consensus sequence being defined is evolving, and specific characterized phosphorylation sites in other Polo substrates actually do not conform to the above consensus sequences. A common feature appears to be negatively charged residues surrounding the S/T residues; all five sites identified in Numb have this feature. Moreover, evidence is provided that the sites that were identified are responsive to phosphorylation controlled by Polo and PP2A. More importantly, phosphorylation of Numb at these sites has a significant effect on NSC homeostasis (Ouyang, 2011).

Polo kinase was shown to also control Numb asymmetric localization by phosphorylating Pon, an adaptor protein for Numb. Loss of Numb asymmetry in polo mutants contributes to ENF. The increased neuroblasts in polo mutants largely occur in the type I lineage. This study demonstrates that overexpression of Polo impairs Numb activity and leads to ENF in type II lineage. In this situation, Pon is presumably also phosphorylated by Polo. However, its positive effect on Numb asymmetric localization is likely to be overridden by impairment of Numb activity by Polo. This underlines the importance of Numb activity regulation in vivo and further indicates that Polo kinase acts on diverse targets to control neuroblast homeostasis. This study shows that phosphorylation of Numb by Polo is probably antagonized by PP2A action in type II lineage, which presumably serves to fine-tune Numb activity through dephosphorylation. Interestingly, the relationships between Polo and PP2A in type I lineage is different from that in type II lineage. In type I neuroblasts, overexpression of Polo can rescue PP2A loss-of-function phenotype, consistent with Polo being positively regulated by PP2A at the transcription level. Elucidation of the mechanisms mediating these differential effects will help lead to an understanding of the distinct behaviors of neuroblasts in these two lineages (Ouyang, 2011).

Deregulation of Numb phosphorylation contributes to loss of Numb activity and eventually leads to unrestrained ENF. Given the conservation of the phospho-sites identified in this study and the potential role of Numb in tumor suppression in mammals, mutations in Numb itself or in some kinases/phosphatases that affect Numb phosphorylation under pathophysiological conditions could contribute to cancer in humans. In lung and breast cancer tissues, Polo expression is upregulated. It is possible that under such pathophysiological conditions, Numb becomes hyperphosphorylated and consequently loses its antagonistic effect on Notch signaling, which could have detrimental consequences on tissue homeostasis. The phosphorylation sites of Numb identified in this study are conserved in mammals. It would be interesting to test in the future whether this phospho-epitope could be detected in human tumor samples (Ouyang, 2011).

This study demonstrated that ENF induced by phospho-Numb occurs specifically in type II lineages, consistent with Numb primarily acting in type II lineage to restrict the proliferation of INPs. It is conceivable that Numb is also phosphorylated by Polo kinase in type I lineage. However, certain unidentified factors might block the effect of phospho-Numb on type I neuroblasts. It is also possible that type I and type II lineages might employ different molecular mechanisms to control their stem cell self-renewal and differentiation, considering their different origin and modes of neurogenesis. Consistent with this notion, the Numb/Notch pathway has been suggested to be dispensable in the type I lineage (Ouyang, 2011).

The prominent brain tumor phenotype induced by Numb-TS4D provides an excellent system with which to identify novel molecules involved in controlling NSC homeostasis. This study shows that Dronc, a newly identified binding partner of Numb, is involved in regulating neuroblast homeostasis. Overexpression of Dronc is sufficient to attenuate Numb-TS4D-induced ENF without promoting neuroblast apoptosis. At the mechanistic level, this study shows that Dronc appears to act upstream of Notch to regulate Numb function, apparently in a process that does not strictly depend on its catalytic activity. Importantly, reduction of dronc function results in neuroblasts being more susceptible to the effect of phospho-Numb on neuroblast homeostasis. In addition, Dronc RNAi is able to further increase ectopic neuroblasts in numbS52F mutant, indicating that Dronc-Numb interaction is normally involved in regulating neuroblast homeostasis. Accumulating evidence suggests that caspases, in addition to their pro-apoptotic functions, also participate in other developmental process without inducing cell death. For example, Dronc has been implicated in a non-autonomous role in compensatory proliferation. It would be interesting to examine in the future whether Dronc transduces a signal from the neighboring niche cells via cell-cell interaction to establish neuroblast homeostatic control. It is also worth noting that mice deficient for caspase 2 (Casp2), which is closely related to Dronc in Drosophila, develop normally as their wild-type siblings; however, the fibroblasts from Casp2 null animals are easily transformed when challenged with oncogenic insults (Ho, 2009). The downstream effectors mediating this effect are not known. It would therefore be interesting to test whether the Numb/Dronc pathway identified here is generally involved in stem cell and cancer biology (Ouyang, 2011).

Structural mechanisms of DIAP1 auto-inhibition and DIAP1-mediated inhibition of drICE

The Drosophila inhibitor of apoptosis protein DIAP1 exists in an auto-inhibited conformation, unable to suppress the effector caspase drICE. Auto-inhibition is disabled by caspase-mediated cleavage of DIAP1 after Asp20. The cleaved DIAP1 binds to mature drICE, inhibits its protease activity, and, presumably, also targets drICE for ubiquitylation. DIAP1-mediated suppression of drICE is effectively antagonized by the pro-apoptotic proteins Reaper, Hid, and Grim (RHG). Despite rigorous effort, the molecular mechanisms behind these observations are enigmatic. This study reports a 2.4 Å crystal structure of uncleaved DIAP1-BIR1, which reveals how the amino-terminal sequences recognize a conserved surface groove in BIR1 to achieve auto-inhibition, and a 3.5 Å crystal structure of active drICE bound to cleaved DIAP1-BIR1, which provides a structural explanation to DIAP1-mediated inhibition of drICE. These structures and associated biochemical analyses, together with published reports, define the molecular determinants that govern the interplay among DIAP1, drICE and the RHG proteins (Li, 2011).

The structural and biochemical information presented in this study gives rise to a model on the interplay of Dronc, drICE, DIAP1, and the RHG proteins. During homeostasis, DIAP1 targets the Dronc zymogen for ubiquitylation and presumably proteasome-mediated degradation. This regulation depends on the interaction between a peptide fragment of Dronc and the conserved groove on DIAP1-BIR2. DIAP1 exists in an auto-inhibited conformation. Caspase-mediated cleavage of DIAP1 after Asp20 disables auto-inhibition, allowing the resulting DIAP1 fragment to bind to and inhibit active drICE. During apoptosis, the RHG proteins use their N-terminal peptides to compete with drICE and Dronc for binding to the conserved peptide-binding grooves on the BIR1 and BIR2 domains, respectively. Such competition results in the release of drICE and Dronc from DIAP1. The freed Dronc zymogen is activated by the Dark apoptosome and the mature Dronc cleaves and activates drICE. In this regard, drICE, DIAP1 and the RHG proteins together provide a fail-safe mechanism to ensure appropriate drICE activation only under bona fide apoptotic conditions (Li, 2011).

The underpinning of this regulatory network is competition among multiple protein-protein interactions mediated by the conserved grooves of the BIR1 and BIR2 domains of DIAP1. Auto-inhibition of DIAP1-BIR1 is achieved by occupation of this groove by its own N-terminal sequence ASVV. The free peptide ASVV does not stably associate with BIR1; the covalent linkage facilitates the binding by increasing the local concentration of ASVV. This binding arrangement allows disabling of auto-inhibition upon cleavage of DIAP1 after Asp20. Inhibition of drICE by BIR1 requires occupation of this groove by the N-terminal sequences ALGS of drICE. Similar to ASVV, the free ALGS peptide exhibited no detectable binding to the BIR1 fragment. Three weak interfaces between BIR1 and drICE cooperate to yield a stable hetero-tetramer with a KD of approximately 1-2 microM. Removal of drICE inhibition by BIR1 depends on the interactions between RHG and the peptide-binding groove, with KD values of 0.12-0.76 microM. Endowing RHG with the strongest interactions ensures an apoptotic phenotype once the RHG proteins are activated in cells. It is acknowledged that the proposed model may be simplistic, as the network of protein-protein interactions and regulation is likely to be more complex in vivo. Nevertheless, the biophysical underpinnings described in this model are likely to have a role in various stages of apoptosis regulation (Li, 2011).

An IAP-binding motif, though not ostensibly abbreviated as IBM, was originally defined to contain four contiguous amino acids that resemble the Smac tetrapeptide AVPI. This structurally defined motif, with binding affinities of 0.1-1 microM, has stringent requirement for the first (P1), third (P3) and fourth (P4) amino acids. The P1 residue must be Ala, which binds to a small hydrophobic pocket on one end of the conserved groove on BIR domain. The P4 residue must be hydrophobic, preferably bulky, to occupy a greasy pocket on the other end of the groove. Deletion of P1 or P4 in a tetrapeptide results in abrogation of stable interaction with the BIR domain. The P3 residue is either Pro or Ala. Pro as P3, with its unique backbone configuration, optimizes simultaneous binding by both P1 and P4 residues for DIAP1-BIR2 or XIAP-BIR3. Ala as P3 can be better accommodated by DIAP1-BIR1 and XIAP-BIR2. In recent studies1, IBM was redefined to contain three contiguous amino acids, with P3 no longer restricted to Pro or Ala. Such tripeptides, ALG/AKG for drICE/Dcp1, or their longer variants, ALGS/AKGC for drICE/Dcp1, do not meet the structural criteria for IBM. Importantly, these peptides in isolation do not form a stable complex with any BIR domain. The definition of such motifs as IBMs insinuates the incorrect assumption that such free peptide motifs may stably interact with the BIR domain. This assumption, in turn, has engendered ample confusion in data interpretation in recent years (Li, 2011).

An IBM at the N-terminus of the caspase-9 small subunit recognizes a conserved surface groove on XIAP-BIR3; this interaction locks caspase-9 in the inhibited state. During apoptosis, Smac/Diablo uses a similar tetrapeptide motif to occupy the BIR3 groove, hence releasing caspase-9 and relieving XIAP-mediated inhibition. Caspase-3 or -7 is inhibited by an 18-residue peptide segment preceding the BIR2 domain of XIAP. Because both caspase-3 and drICE are inhibited directly at the active sites by XIAP and DIAP1, respectively, the overall appearance of the two BIR-caspase complexes is similar. It should be noted, however, that the essential interactions and key features are quite different. For example, caspase-3 or -7 can be inhibited by an isolated 18-residue peptide fused to GST; but the intact BIR1 domain of DIAP1 is absolutely required for drICE inhibition. The orientation of the BIR domain relative to the caspase is different by approximately 90 degrees between caspase-3/BIR2 and drICE/BIR1. Importantly, the peptide-binding groove of XIAP-BIR2 does not have an apparent role in the inhibition of caspase-3 and -7 (Li, 2011).

Using purified, recombinant proteins, this study showed that the BIR1 domain of DIAP1 only forms a stable complex with active drICE following caspase-mediated cleavage of DIAP1 after Asp20. The uncleaved DIAP1-BIR1 exhibited very weak binding to drICE. These observations contrast the report that both uncleaved and cleaved DIAP1-BIR1 bound to drICE similarly using coimmunoprecipitation. The cleaved DIAP1, but not the uncleaved DIAP1, potently inhibits the proteolytic activity of drICE towards both peptide and protein substrates. These observations unambiguously demonstrate that drICE sequestered by cleaved DIAP1 remains catalytically inactive. In fact, the conclusion that DIAP1-sequestered drICE was catalytically active, contradicted the biochemical observation that no protease activity was detectable towards peptide or protein substrate21. It is not uncommon for a substrate to be converted into a protease inhibitor upon cleavage, as exemplified by the pan-caspase inhibitor p35 (Li, 2011).

The key question is not whether BIR1 inhibits drICE, but why BIR1-sequestered drICE continues to exhibit proteolytic activity towards the uncleaved DIAP1. A time course analysis of DIAP1 cleavage shows that the protease activity of drICE was slowed down considerably over time, as the concentration of inhibitor -- cleaved DIAP1 -- increased. The level of drICE activity at the 15-minute time point was at least 6-fold higher than that at 90-minute point. This observation again illustrates that the cleaved DIAP1 is a bona fide inhibitor of drICE (Li, 2011).

Some of the contrasting claims about the regulation of drICE by DIAP1 might be attributable to the limitations of the investigative methods. Biochemical and biophysical investigations, employing homogeneous, recombinant proteins, usually provide mechanistic answers to questions that pertain to protein-protein interactions and enzyme activities. The caveat, however, is whether such observations are biologically relevant, and if yes, to what extent these findings are important. By contrast, investigation by cellular biochemistry, exemplified by coimmunoprecipitation, provides important clues to molecular mechanisms. For both approaches, caution must be exercised for the interpretation of results. Notably, in some cases, the contrasting claims can be reconciled by a complex system. For example, despite structural data demonstrating that the auto-inhibition of DIAP1 involves the binding of its N-terminal sequences to the BIR1 domain, it remains theoretically possible that additional interactions between the N- and C-terminal domains of DIAP1 may contribute to its auto-inhibition1. The best example is Apaf-1, whose auto-inhibition entails two elements: one by the C-terminal WD40 repeats and the other within the N-terminal half. Binding to cytochrome c relieves the auto-inhibition by the WD40 repeats31, and exchange of ADP for ATP defeats the auto-inhibition imposed by intra-domain interactions within the N-terminal-half (Li, 2011).

Structure of the Drosophila apoptosome at 6.9 &aRing; resolution

The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. This study shows that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. The 3D structure of a double ring was determined at approximately 6.9 &ARing; resolution, and a model was created of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has 'lost' a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution (Yuan, 2011).

Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila

Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. This study reports the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome (Pang, 2015).

This study presents the cryo-EM structures of the Dark apoptosome and the multimeric Dronc-Dark complex at overall resolutions of 4.0 and 4.1 Å, respectively. Notably, the EM density in the central region of the structures exhibits considerably higher resolutions, which allow assignment of specific side chains and atomic interactions. Because the overall domain organization of Dark is identical to that of Apaf-1, the structures reveal for the first time conserved atomic features of an apoptosome from a higher organism. The observed structural features of the Dark apoptosome, most of which are likely preserved in the Apaf-1 apoptosome, reveal the underpinnings of initiator caspase activation. Supporting this analysis, structure of the Dark protomer can be very well aligned with that of the activated Apaf-1 protomer from the Apaf-1 apoptosom (Pang, 2015).

This study presents the cryo-EM structures of the Dark apoptosome and the multimeric Dronc-Dark complex at overall resolutions of 4.0 and 4.1 Å, respectively. Notably, the EM density in the central region of the structures exhibits considerably higher resolutions, which allow assignment of specific side chains and atomic interactions. Because the overall domain organization of Dark is identical to that of Apaf-1, the structures reveal for the first time conserved atomic features of an apoptosome from a higher organism. The observed structural features of the Dark apoptosome, most of which are likely preserved in the Apaf-1 apoptosome, reveal the underpinnings of initiator caspase activation. Supporting this analysis, structure of the Dark protomer can be very well aligned with that of the activated Apaf-1 protomer from the Apaf-1 apoptosome (Pang, 2015).


EFFECTS OF MUTATION

Effects of Mutation or Deletion

Because specific mutations in dronc are currently not available, the technique of RNAi was used to ablate dronc gene function during embryogenesis. RNAi, a technique developed in Caenorhabditis elegans, has recently been successfully used in Drosophila and mammalian cells to specifically ablate gene function. Dronc double-stranded mRNA was injected into precellularized embryos, and samples were aged until stage 13. Embryos were analyzed by Dronc antibody staining to assess the efficiency of the Dronc protein ablation and by TUNEL assays to reveal apoptotic cells. In addition, embryos were also stained with neural differentiation marker monoclonal antibody 22C10 to reveal whether ablation of dronc affects neural development. At stage 13, uninjected embryos show Dronc expression throughout the embryo and a large number of TUNEL-positive cells. In contrast, in stage 13 dronc RNAi embryos, Dronc protein is undetectable, and very few TUNEL-positive cells are observed. Buffer-injected control embryos show no decrease in cell death, but rather more cells are TUNEL-positive. At least 400 dronc RNAi-injected embryos were examined, and the results are consistent for all embryos. Although dronc RNAi-injected embryos fail to hatch, examination of embryonic structures using Nomarski optics shows no apparent gross structural defects. Furthermore, staining with neural marker 22C10 shows that neural differentiation is normal. These results show that dronc is essential for induction of cell death during embryogenesis. Because Dronc shares very limited (<25%) nucleotide sequence homology with all Drosophila caspases, dronc RNAi is unlikely to affect the function of other caspases (Quinn, 2000).

Does DRONC acts in an Rpr-dependent or an Rpr-independent death pathway? To investigate this question more carefully, whether Rpr-induced cell death is sensitive to dronc gene dosage was examined. Because no single gene mutations in dronc are currently available, mutant flies were used with a larger chromosomal deletion that includes the dronc locus [Df(3L)AC1]. Df(3L)AC1 was crossed to GMR-rpr flies and it was found that flies carrying Df(3L)AC1 show a significant suppression of the Rpr eye phenotype. Furthermore, Df(3L)AC1 also suppresses Hid-mediated cell killing in the eye. To investigate further whether this observed suppression is due specifically to loss of dronc, whether the expression of dominant-negative DRONC mutants (pro-DRONC CdeltaA and DRONC-CARD) also suppresses the Rpr eye phenotype was assessed. Pro-DRONC CdeltaA strongly suppresses Rpr cell killing, and, surprisingly, the pro-domain of DRONC on its own (DRONC-CARD) completely rescues the Rpr eye phenotype. These results, in which DRONC function is ablated either by the Df(3L)AC1 deletion or by the action of dominant-negative DRONC, are consistent with the notion that DRONC is a rate-limiting caspase in the Rpr and Hid death pathway (Meier, 2000).

Studies have indicated that Rob-1 (aka Debcl) can induce apoptosis by a caspase independent mechanism. Although Rob-1 strongly stimulates caspase activation when overexpressed in S2 cells, its ability to kill cells is barely antagonized by the caspase-related apoptosis inhibitors p35 and DIAP2, or by an active-site mutant of a Drosophila apical caspase, DRONC (Nedd2-like caspase). Furthermore, in transgenic flies, rough-eye phenotype caused by overexpression of Rob-1 is not completely suppressed by coexpression of p35. Similarly, previous studies on the proapoptotic Bax, Bak, and Mtd suggest that they all induce apoptosis in the presence of broad caspase inhibitors. Moreover, both Bax and Bak can induce mitochondrial dysfunction and also kill yeast, which lack endogenous caspases. Both Rpr- and Hid-induced cell death are blocked by coexpression of baculovirus p35. In contrast, overexpression of p35 shows no effect on Rob-1-induced apoptosis. Higher levels of p35 expression exhibit only a slight protective effect against Rob-1-induced cell killing. Similarly, DIAP2, which inhibits both Rpr- and Hid-induced apoptosis, or an active-site mutant of the CARD (caspase recruitment domain)-containing Drosophila caspase, DRONC, shows little effect on Rob-1-induced apoptosis, suggesting that the Rob-1-stimulated cell-death pathway is downstream or independent of DIAP2 and DRONC. Thus, overexpression of Rob-1 induces apoptosis, probably through a caspase-independent pathway partly distinct from that used by other known Drosophila killer proteins (Rpr, Hid, and Grim). Rob-1 overexpressed in human embryonic kidney 293T cells also exhibits a proapoptotic activity that is only slightly inhibited by p35. Thus, Rob-1, like mammalian Bax, Bak, and Mtd, may activate cell death by inducing both caspase-dependent and -independent pathways. The latter pathways probably can be antagonized by an as-yet-unidentified antiapoptotic member of the Drosophila Bcl-2 family. In the nematode C. elegans, in which two Bcl-2/CED-9 family members, CED-9 and EGL-1, have been identified, all cell deaths occur in a caspase-dependent manner. The presence of a Bax-like protein, Rob-1, in Drosophila might indicate the acquisition of a caspase-independent cell death pathway through evolution (Igaki, 2000).

Among the seven caspases encoded in the fly genome, only dronc contains a caspase recruitment domain. To assess the function of this gene in development, a null mutation in dronc was produced. Animals lacking zygotic dronc are defective for programmed cell death (PCD) and arrest as early pupae. These mutants present a range of defects, including extensive hyperplasia of hematopoietic tissues, supernumerary neuronal cells, and head involution failure. dronc genetically interacts with the Ced4/Apaf1 counterpart, Dark, and adult structures lacking dronc are disrupted for fine patterning. Furthermore, in diverse models of metabolic injury, dronc cells are completely insensitive to induction of cell killing. These findings establish dronc as an essential regulator of cell number in development and illustrate broad requirements for this apical caspase in adaptive responses during stress-induced apoptosis (Chew, 2004).

Dronc and compensatory cell proliferation

In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Apoptotic cells are shown to express the secretory factors Wingless and Decapentaplegic. When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation is observed. Significantly, Wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, evidence is provided that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation (Ryoo, 2004).

To investigate how the inhibition of diap1 may lead to mitogen expression, attention was focused on Dronc and the Jun N-terminal Kinase (JNK) pathway. Dronc has been implicated in compensatory proliferation, and its activity can be inhibited by the expression of droncDN. In addition, the JNK signaling pathway was considered as a candidate, since its activity is known to correlate with many forms of stress-provoked apoptosis, including disruption of morphogens, cell competition, and rpr expression. In Drosophila, the JNK pathway can be effectively blocked by the expression of puckered (puc), which encodes a phosphatase that negatively regulates JNK (Ryoo, 2004).

To induce patches of undead cells, wing imaginal discs were generated with mosaic clones expressing hid and p35. 48 hr after induction, these imaginal discs contained hid-expressing clones that autonomously induced wg. Using this experimental setup, it was asked whether additional expression of either droncDN or puc would block wg induction in undead cells. When droncDN was coexpressed, a subset of the hid-expressing population was still able to induce wg. In contrast, when puc was coexpressed, wg induction by hid was almost completely blocked. These results provide evidence that the JNK pathway is required for wg induction under these conditions but fail to uncover a similar requirement for Dronc (Ryoo, 2004).

To independently investigate the role of puc and droncDN in compensatory proliferation, the size of wing discs harboring undead cells was measured and they were compared with those of the sibling controls. Under the experimental conditions, wing discs harboring hid- and p35-expressing clones were on average 53% larger than their sibling controls. Coexpression of puc within these undead clones significantly limited growth, resulting in only a small increase in wing disc size that was not statistically significant. In contrast, coexpression of droncDN did not limit growth. Wing size measurements also correlated with the degree of wg induction. The larger size of discs harboring hid- and p35-expressing cells is not due simply to extra cell survival: (1) these undead cells are derived from the normal lineage; (2) the size of wing discs expressing hid, p35, and puc serves as a control. In this case, although a large number of undead cells were generated, no significant increase in disc size was observed, in stark contrast to the discs expressing hid and p35 only. It is concluded that the JNK pathway is required for the nonautonomous growth promoting activity of the undead cells (Ryoo, 2004).

To confirm a role of puc in imaginal disc growth, rpr and p35 werecoexpressed in wild-type and puc−/+ imaginal discs. Like hid, rpr is a DIAP1 antagonist, but with a weaker cell killing activity when overexpressed in imaginal disc cells. In a puc+/+ background, a small amount of ectopic wg expression was observed, indicative of rpr's weaker DIAP1 inhibiting activity. In contrast, ectopic wg expression was strongly enhanced in puc−/+ discs. Because the puc allele used, pucE69, also acts as a lacZ reporter, JNK pathway induction could be monitored simultaneously. wg induction in undead cells correlates very well with puc-lacZ expression, with a stronger induction at the center of the wing pouch. These results further support the role of JNK in the induction of wg (Ryoo, 2004).

Next to be tested was whether the reduction of puc had an effect on apoptosis-induced cell proliferation. Whereas puc−/+ discs expressing only p35 had BrdU incorporation similar to wild-type discs, coexpression of rpr and p35 in puc−/+ led to a significant increase in BrdU incorporation. Also, the size of these discs were on average 41% larger than those coexpressing rpr and p35 in a puc+/+ background. Taken together, these results show that diap1 inhibition leads to JNK activation and that JNK activity promotes wg induction and cell proliferation (Ryoo, 2004).

To directly test if JNK signaling can activate wg and dpp expression, hepCA, a constitutively active form of hemipterous (hep), the Drosophila JNK kinase was conditionally expressed. Expression of hepCA causes induction of wg-lacZ within 22 hr and to a lesser extent also dpp-lacZ. These ß-gal-expressing cells shifted basally and were apoptotic as assayed by anti-active caspase-3 antibody labeling. Hid protein levels were also elevated in these cells. Significantly, since p35 was not use to block apoptosis in this experiment, this demonstrates that wg and dpp can be induced not only in undead cells, but also in 'real' apoptotic cells (Ryoo, 2004).

This study provides evidence that the central apoptotic regulators can control the activity of mitogenic pathways. In particular, inhibition of DIAP1, either via expression of Reaper and Hid or by mutational inactivation, leads to the induction of the putative mitogens wg and dpp. When apoptosis was initiated through DIAP1 inhibition but cells were kept alive by blocking caspases, the resulting 'undead cells' exhibited strong mitogenic activity and stimulated tissue overgrowth. Inhibiting wg signaling with a conditional TCFDN blocked cell proliferation in imaginal discs, indicating that wg has an essential mitogenic function. Finally, evidence was provided that the JNK pathway mediates mitogen expression and imaginal disc overgrowth in response to rpr and hid. Based on these results, it is proposed that apoptotic cells actively signal to induce compensatory proliferation. DIAP1 inhibits both caspases as well as dTRAF1. According to this model, when DIAP1 is inhibited in response to cellular injury, the JNK pathway is activated and wg/dpp are induced in apoptotic cells. Secretion of these factors stimulates growth of proliferation-competent neighboring cells and leads to compensatory proliferation (Ryoo, 2004).

This study provides clear genetic evidence that diap1 is involved in compensatory proliferation. Overall, similar results were obtained with hypomorphic diap1 alleles (diap122-8s, diap133-1s), a null allele (diap1th5), and inactivation of diap1 by expression of Reaper and Hid. However, whereas expression of p35 effectively blocked apoptosis of diap122-8s/22-8s cells and in response to Reaper/Hid, it only partially suppressed the death of diap1th5/th5 cells. Consequently, the generation of undead cells was less efficient with the diap1th5 mutation. Moreover, these results suggest that the JNK pathway transduces the signal to activate mitogen expression and cell proliferation. Since IAPs have been shown to ubiquitylate TRAFs in both mammals and Drosophila and since no evidence was found for Dronc in growth promotion, it is attractive to speculate that JNK is regulated through direct DIAP1/TRAF1 interaction (Ryoo, 2004).

An important unresolved question is why compensatory proliferation is seen only in response to cellular injury, but not during normal developmental apoptosis. In particular, inactivation of DIAP1 by Reaper, Hid, and Grim is restricted not only to injury-provoked apoptosis, but also underlies most developmental cell deaths. One possible explanation is that activation of the JNK pathway is key to mitogenic signaling of apoptotic cells. Consistent with this idea, the JNK pathway is activated in response to tissue stress and injury, but not during developmental apoptosis. Furthermore, this study shows that JNK signaling can induce the expression of wg/dpp and nonautonomous cell proliferation. Therefore, it is possible that robust JNK activation and compensatory proliferation require the combined input of stress and apoptotic signals (Ryoo, 2004).

DRONC is required for specific developmental cell death pathways and stress-induced apoptosis

Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. This study describes the generation of a specific mutant of the Drosophila gene encoding Dronc, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and these studies show that Dronc is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, Dronc is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. These results indicate that Dronc is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of Dronc (Daish, 2004).

A Drosophila P element line, KG02994, was obtained with an insertion 113 bp upstream of the 5' UTR of the dronc gene. Analysis of KG02994 shows that this line is a hypomorphic allele of dronc. The KG02994 insertion was used to generate P element excision mutants in the attempt to delete dronc. Three potential dronc mutant lines were identified as containing a deletion. One line, named droncZ, was selected for further studies of DRONC function. The breakpoints were confirmed by sequencing as being 1926 bp upstream of the dronc transcription start site and within the intron of dronc, 890 bp downstream from the ATG. Inspection of the Drosophila genome sequence indicates that a gene of unknown function, CG6685, is within this deleted region. This confounds any conclusions that could be drawn from the observed larval lethal phenotype of the droncZ deletion line regarding Dronc function (Daish, 2004).

The dronc deletion mutants (droncZ) arrest at the late larval stage with melanotic tumors and developmental defects in larval tissues. CG66854 animals fail to form a normal puparium following gut clearance and arrest as partially contracted larvae with melanotic tumors. All droncd5 animals pupate but arrest prior to the pharate adult stage (Daish, 2004).

It was necessary to assess, in a droncZ background, the ability of the respective complementation transgenes to emulate the endogenous expression profiles and restore viability when combined in the same animal. The CG66854/droncd5; droncZ/droncZ animals survived to adult with an eclosion rate of 32.67% of expected. The remaining noneclosed animals develop to an advanced pharate adult stage. These results demonstrate that the discreet phenotypes observed for droncd5 and CG66854 are attributed primarily to the gene product of the nonfunctional gene. Since a detailed expression profile of the CG6685 transcript in development is not known, it is not possible to demonstrate definitively that its correct expression has been restored in droncd5 animals. However, it is believed that the phenotype and lethality observed for droncd5 animals can be primarily attributed to the absence of Dronc and not disruption to the CG6685 expression profile for the following reasons: (1) each of the transgenes is associated with discreet developmental defect profiles and survival boundaries, (2) combining the two complementation transgenes in the same animal significantly rescues droncZ lethality along with the individual lethalities of droncd5 and CG66854; (3) Dronc is present in CG66854 animals; (4) droncd5 animals have CG6685 transcriptional activity, and (5) CG6685 transcript is barely detectable in larval midguts and absent from salivary glands prior to their destruction (Daish, 2004).

To assess the role of dronc in development, the survival and developmental progression of these mutant and complementation lines were analyzed. No significant lethality was found during embryonic stages, as measured by hatching frequency, in any of the mutant or complementation lines. The survival rate of KG02994 homozygotes to the 3L stage was decreased (70% of expected, and eclosion was delayed compared to their sibling heterozygotes. While droncZ and CG66854 animals had a reduced survival frequency to 3L, this was not observed for droncd5 animals. This suggests that the increased larval lethality associated with droncZ and CG66854 is due to the loss of CG6685 gene function and not dronc. Additionally, reduced developmental delay in droncd5 and KG02994 animals than in droncZ and CG66854 animals was observed, with the majority reaching the late 3L stage 1.5 days after wild-type (wt) animals (Daish, 2004).
To investigate the possible causes of lethality in droncd5, the morphology of various larval organs was examined and acridine orange (AO) staining was used to investigate cell death. Although larger in some animals, no obvious morphological defect in droncd5 larval brain lobes was apparent. However, there was significant reduction in AO staining in droncd5 brain lobes compared to wt, which showed many AO-positive cells. Larval eye discs also displayed a dramatic decrease in cell death compared to wt. It is therefore concluded that the loss of Dronc results in a decrease in cell death in the larval brain lobes and eye discs (Daish, 2004).

dronc is transcriptionally upregulated in salivary glands following the late prepupal ecdysone pulse triggering gland histolysis. Larval salivary gland cell death is delayed in KG02994 animals. To further asses the role of Dronc in salivary gland PCD, droncd5 and wt animals were staged to 20 and 30 hr relative to puparium formation (RPF) and sections analyzed by light microscopy. droncd5 animals contained persistent salivary glands with an overall appearance similar to prehistolysed controls including an intact lumen surrounded by nonrounded cells. Adult structures form in droncd5 animals as in the control, indicating continuing pupal development. Progression of autophagy judged by vacuolar dynamics prior to histolysis (13 hr RPF) shows wt salivary gland cells containing variable sized eosin-positive vacuoles while droncd5 cells primarily contain larger vacuoles typical of earlier developmental stages. Ultrastructural analysis of droncd5 salivary glands at 14 hr RPF showed a lack of membrane bound autophagic bodies observed in wt salivary glands, suggesting a lack of characteristic autophagy in the droncd5 salivary glands (Daish, 2004).

No TUNEL-positive nuclei were observed in droncd5 salivary glands at the time when all wt gland cells were TUNEL positive. Hoechst staining of persistent glands showed that nuclear integrity is maintained in droncd5 salivary glands 30 hr RPF. If a dronc-dependent mechanism contributes to salivary gland histolysis, droncd5 glands should be deficient in caspase activity. Consistent with this, caspase activity is reduced in droncd5 salivary glands at 14 hr RPF relative to wt. An increase in active DRICE-like immunostaining was seen in wt salivary glands at 14 hr RPF. However, active DRICE-like immunostaining in droncd5 salivary glands at 14 hr RPF was significantly lower, even though the levels of total DRICE protein in droncd5 and wt were comparable. These results indicate that effector caspase activation in salivary glands is Dronc dependent (Daish, 2004).

To assess whether the genetic regulatory hierarchy upstream of dronc is intact in droncd5 animals, cell death gene expression was analyzed in droncd5 salivary glands. A key ecdysone-regulated transcription factor E93 (which is required for maximal dronc transcription) is upregulated in droncd5 salivary glands at a time similar to wt. hid transcript was also present, indicating that the ecdysone-regulated PCD hierarchy is intact in droncd5 salivary glands (Daish, 2004).

dronc is upregulated following the late 3L ecdysone pulse, which triggers midgut histolysis. However, in contrast to salivary glands, droncd5 midgut histolysis is initiated normally. The fact that DNA fragmentation and caspase activity precedes the time of maximal dronc transcription suggests that aspects of apoptosis are initiated in the midgut in the absence of this apical caspase (Daish, 2004).

Dronc has been shown to function in the Rpr, Hid, and Grim cell death pathway. Since droncd5 is a null mutant and Dronc acts downstream of Rpr, Hid, and Grim, suppression of the Rpr-, Hid-, and Grim-induced phenotypes was expected in a genetic interaction cross with droncd5. As expected, the Rpr-, Hid-, and Grim-induced eye phenotypes are suppressed in the droncd5 heterozygous background, thus confirming that Dronc is required in Rpr-, Grim-, and Hid-mediated death pathways (Daish, 2004).

To investigate the role of Dronc in DNA damage-induced PCD, droncd5 and wt 3L animals were irradiated with 8 Gy γ and the effect on apoptosis and caspase activity was analyzed. Following irradiation, wt larvae showed significant increases in eye disc cell death as observed by AO staining. No similar increase in AO staining was observed in droncd5 eye discs following irradiation. Whole-animal lysates from the same experiment showed an increase in caspase activity. Consistent with the lack of AO staining following irradiation, no significant increase in VDVAD or VEID cleavage was observed for droncd5. Although DEVD showed a marginal increase in activity, it was less than half the control increase (Daish, 2004).

It is concluded that although caspase function is been implicated in Drosophila cell death pathways, most of the previous studies have relied on overexpression of wild-type or dominant-negative transgenes. These studies, while informative, do not provide conclusive evidence for caspase function in developmental PCD. This paper describes the analyses of a dronc mutant alleles in Drosophila. The mutant is a specific point mutation in the dronc gene replacing the deleted allele. The upstream gene CG6685 is essential for development, and deletion of dronc may have effects on the regulation of CG6685 transcription by removal of essential promoter elements. Therefore, the dronc mutant described in this study is an effective way of generating a specific gene disruption if employing a P element excision strategy (Daish, 2004).

While some of the phenotypes in dronc mutants are consistent with previous observations, a number of unexpected findings were uncovered in this study. These include: (1) Dronc plays an essential role in development; (2) despite the fact that Dronc is the only CARD-containing caspase in Drosophila, there are Dronc-independent caspase activation and cell death pathways, (3) Dronc is dispensible for midgut histolysis even though it is highly upregulated by ecdysone in this tissue prior to its histolysis, and (4) evidence that Dronc may be required for some aspects of autophagy is provided. As Dronc has also been recently implicated in some nonapoptotic events, the availability of specific dronc mutants now makes it possible to further explore alternative roles of this apical caspase in diverse developmental events (Daish, 2004).

The two Drosophila cytochrome C proteins can function in both respiration and caspase activation: Analyses of ark and dronc mutants demonstrate that both genes are required for spermatid individualization and that their phenotypes resemble cyt-c-d mutant spermatids and expression of the caspase inhibitor p35 in the testes

Cytochrome C has two apparently separable cellular functions: respiration and caspase activation during apoptosis. While a role of the mitochondria and cytochrome C in the assembly of the apoptosome and caspase activation has been established for mammalian cells, the existence of a comparable function for cytochrome C in invertebrates remains controversial. Drosophila possesses two cytochrome c genes, Cytochrome c proximal and Cytochrome c distal. cyt-c-d is required for caspase activation in an apoptosis-like process during spermatid differentiation, whereas cyt-c-p is required for respiration in the soma. However, both cytochrome C proteins can function interchangeably in respiration and caspase activation, and the difference in their genetic requirements can be attributed to differential expression in the soma and testes. Furthermore, orthologues of the apoptosome components, Ark (Apaf-1) and Dronc (caspase-9), are also required for the proper removal of bulk cytoplasm during spermatogenesis. Finally, several mutants that block caspase activation during spermatogenesis were isolated in a genetic screen, including mutants with defects in spermatid mitochondrial organization. These observations establish a role for the mitochondria in caspase activation during spermatogenesis (Arama, 2006).

In order to identify genes required for caspase activation during spermatid differentiation in Drosophila, attempts were made to identify mutants that lacked activated caspase-3 staining, as detected using CM1 antibody, which detects the active form of the effector casepase drICE. For this purpose, an existing collection was screened of more than 1000 male-sterile mutant lines defective in spermatid individualization that were previously identified among a collection of about 6000 viable mutants. Dissected testes from each line were stained with CM1: 33 lines were identified that were CM1-negative. However, the vast majority of male-sterile lines remained CM1-positive, even though many displayed severe defects in spermatid individualization. Therefore, caspase activation at the onset of spermatid individualization appears to be independent of other aspects of sperm differentiation, such as the assembly of the individualization complex or its movement. One of the mutants, line Z2-1091, failed to complement the sterility of bln1, a P-element insertion in cyt-c-d, and was CM1-negative as a homozygote, in trans to a small deletion removing the cyt-c-d locus [Df(2L)Exel6039], or in trans to the cyt-c-dbln1 allele. In contrast, Z2-1091 complemented the lethality of K13905, a P-element insertion in cyt-c-p, and K13905 complemented the sterility of Z2-1091. Genomic sequence analyses of the transcription units of both cyt-c-d and cyt-c-p in Z2-1091 flies revealed a point mutation of TGG to TGA at codon 62 in cyt-c-d, causing a change of Trp62 into a stop codon that results in a truncation of almost half of the protein. Henceforth this allele will be referred to as cyt-c-dZ2-1091. Given the molecular nature of cyt-c-dZ2-1091, it is very unlikely that this allele affects the function of genes adjacent to cyt-c-d (Arama, 2006).

Effector caspases, such as drICE, can display DEVD cleaving activity. Therefore, it was asked whether wild-type adult testes also contain DEVDase activity, and whether this activity is affected in cyt-c-d mutant testes. Lysates of wild-type testes indeed display detectable levels of DEVDase activity, which were significantly reduced upon treatment with the potent DEVDase inhibitor Z-VAD.fmk. Importantly, this activity was highly reduced in cyt-c-dZ2-1091 mutant testes. These results provide independent evidence for effector caspase activity in wild-type sperm, and they support a role of cytochrome C-d in caspase activation in this system (Arama, 2006).

In mammals, mitochondria are important for the regulation of apoptosis, and it has been shown that they can release several proapoptotic proteins into the cytosol in response to apoptotic stimuli. The best-studied case is the release of cytochrome C, an essential component of the respiratory chain. Cytosolic cytochrome C can bind to and activate Apaf-1, which in turn leads to the activation of caspase-9. However, no comparable role of mitochondrial factors for caspase activation has yet been established in invertebrates. The elimination of cytoplasm during terminal differentiation of spermatids in Drosophila involves an apoptosis-like process that requires caspase activity; a P-element insertion (bln1) in one of the two Drosophila cytochrome c genes, cyt-c-d, has been shown to be associated with male-sterility and loss of effector caspase activation during spermatid individualization. This study demonstrates that the defects in caspase activation and spermatid individualization of bln1 mutant males can be rescued by transgenic expression of the ORF of cyt-c-d. Furthermore, from screening more than a thousand male-sterile lines with defects in sperm individualization for defects in active-caspase (CM1) staining, a nonsense point mutation was identified in cyt-c-d, that recapitulates all the phenotypes observed for bln1. Taken together, these results unequivocally demonstrate that cyt-c-d is necessary for effector caspase activation and sperm terminal differentiation in Drosophila (Arama, 2006).

Two decades ago, the mouse cytochrome c gene was used as a probe for screening a Drosophila genomic library and a fragment was isolated that carried two distinct cytochrome c genes. Northern blot analyses indicated high levels of cyt-c-p expression, while cyt-c-d was reported to be expressed at much lower levels in all stages of development. However, neither the exon/intron organization nor the boundaries of the 5' and 3' UTRs of these genes were determined at the time. As a result, the original Northern analyses were performed with a probe corresponding to the untranscribed genomic region between the two cytochrome c genes that was not suitable to properly assess the size and distribution of cytochrome c transcripts. Unfortunately, this has caused considerable confusion in the field from the start, as even the original report noted that the size of the observed cyt-c-d transcript differed more than two-fold from the predicted size. More recently, relying on the incorrect assumption that cyt-c-d is ubiquitously expressed in the fly, it has been suggested that a loss-of-function mutation in cyt-c-d should lead to severe developmental defects and lethality rather than merely male sterility. However, using a specific cyt-c-d 3' UTR probe reveals a transcript of the predicted size that is absent in cyt-c-dbln1 mutants. Furthermore, the RT-PCR and immunofluorescence analyses presented in this study indicate that cyt-c-d is mainly expressed in the male germ line and is completely absent during embryonic and larval development, while cyt-c-p is expressed in the soma during all stages of development. In light of these findings, it is not surprising that loss-of-function mutations in cyt-c-d cause male sterility, whereas cyt-c-p mutations lead to embryonic lethality. RT-PCR results suggest that cyt-c-p is also expressed in the testis, although to a much lower extent than cyt-c-d. This expression is attributed primarily to the somatic cells of the testis, since no cytochrome C protein is detected in cyt-c-dbln1 elongating spermatids, while cyt-c-p RNA is expressed in cyt-c-dbln1 mutant flies. However, the very low cyt-c-d expression detected in the soma of adult females leaves room for the possibility that cyt-c-d might function in caspase activation in some somatic cells as well (Arama, 2006).

In mammalian cells, release of cytochrome C into the cytosol in response to proapoptotic stimuli can be readily demonstrated. However, previous attempts to detect a similar phenomenon in Drosophila have been unsuccessful. In contrast, apoptotic stimuli can lead to increased cytochrome C immuno-reactivity. A possible limitation is that all these studies were conducted using mammalian antibodies with questionable specificity and sensitivity, and only in a small number of cell types and paradigms. Using an antibody that was raised against Drosophila cytochrome C-d, an increase in a 'grainy signal' was detected upon the onset of individualization, with the highest staining observed in the vicinity of the individualization comple (IC). Since it is highly unlikely that additional cytochrome C-d is being transcribed and imported to the mitochondria at this late stage, the explanation is favored that a conformational change or an exposure of a hidden epitope causes the increase in the intensity of the signal. The activation of Dronc, the Drosophila caspase-9 orthologue, also occurs in association with the IC and depends on the presence of the Drosophila Apaf-1 orthologue, Ark. Moreover, the proapoptotic Hid protein is localized in a similar fashion. What are these structures then, which accumulate apoptotic factors in the vicinity of the IC? One plausible suggestion from the literature is that these structures correspond to 'mitochondrial whorls', which result from the extrusion of material from the minor mitochondrial derivative and constitute the leading component of the IC. These 'whorls' can be labeled using a testes-specific mitochondrial-expressed GFP line. Using this GFP marker, it was found that cytochrome C-d is indeed closely associated with mitochondrial whorls. Therefore, it is possible that an active apoptosome forms in the vicinity of the IC in response to dramatic changes in the mitochondrial architecture that occur at this stage of spermatid differentiation. Similarly, studying the response of Drosophila flight muscle cells to oxygen stress, have recently reported that the cristae within individual mitochondria become locally rearranged in a pattern that they termed a 'swirl'. This process was associated with widespread apoptotic cell death in the flight muscle, which was correlated with a conformational change of cytochrome C manifested by the display of an otherwise hidden epitope. Collectively, these observations suggest that apoptosome-like complexes composed of cytochrome C-d, Ark, and Dronc might be associated with unique mitochondrial swirl-like structures. Consistent with this idea, it was found that the long isoform of Ark that contains the WD40 repeats, the target for cytochrome C binding to mammalian Apaf-1, is the major form detectably expressed in testes (Arama, 2006).

The fact that cytochrome C-d immunoreactivity increases in the vicinity of the IC suggests that the extensive mitochondrial organizations preceding individualization may be partially required for caspase activation. Consistent with this idea, several mutants, such as plnZ2-0516, which display defects in Nebenkern differentiation and caspase activation. However, not all mitochondrial differentiation events are required for caspase activation. For example, CM1 staining is seen in fuzzy onions, a mutant defective in the mitochondrial fusion event that generates the Nebenkern. In contrast, analysis of the pln mutant indicates that proper elongation of the Nebenkern is essential for caspase activation. Therefore, characterization of other mitochondrial mutants may shed light on the connection between mitochondrial organization and caspase activation during sperm differentiation (Arama, 2006).

What are the mechanisms by which cytochrome C-d activates caspases during late spermatogenesis? In vertebrate cells, following its release into the cytosol, cytochrome C binds to the WD40 domain of the adaptor molecule Apaf-1, which in turn multimerizes and recruits the initiator caspase, caspase-9 via interaction of their CARD domains. This complex, known as the apoptosome, further cleaves and activates effector caspases like caspase-3. Although this model has become the prevailing dogma in the field, the phenotype of mice mutant for a Cyt c with drastically reduced apoptogenic function ('KA allele') suggests that the mechanisms for caspase activation may be more complex than what was previously thought. In particular, this study suggests that cytochrome C-independent mechanisms for the activation of Apaf-1 and caspase-9 exist, as well as cytochrome C-dependent but Apaf-1-independent mechanisms for apoptosis. These analyses of ark (Apaf-1) and dronc (caspase-9) loss-of-function mutants demonstrate that both genes are required for spermatid individualization, and that their phenotypes, in particular their failure to properly remove the spermatid cytoplasm into the WB, resemble cyt-c-d mutant spermatids and expression of the caspase inhibitor p35 in the testes. However, some caspase-3-like activity could still be detected in these mutant testes. This may suggest that either the ark and dronc alleles are not null, or that cytochrome C-d also functions in an apoptosome-independent pathway to promote caspase-3 activation. Therefore, the regulation of caspase activation and apoptosis may be more similar between insects and mammals than has been previously appreciated. Further genetic analysis of this pathway in Drosophila may provide general insights into diverse mechanisms of apoptosis activation (Arama, 2006).

Previous observations raised the possibility that the two distinct cytochrome c genes may have evolved to serve distinct functions in respiration and caspase regulation. In order to address this hypothesis, it was asked whether expression of one protein might rescue mutations in the other cytochrome c gene. Surprisingly, it was found that transgenic expression of the cyt-c-p ORF in germ cells rescues caspase activation, spermatid individualization, and sterility of cyt-c-d-/- flies. Therefore, the ability to activate caspases is not restricted to the cytochrome C-d protein, and it is possible that cytochrome C-p functions in apoptosis in at least some somatic cells (Arama, 2006).

Although cyt-c-d is almost exclusively expressed in the male germ cells, ectopic expression of this protein in the soma can rescue the respiration defect and lethality of cyt-c-p-/- mutant flies, demonstrating that cytochrome C-d can function in energy metabolism. This raises the question whether the lack of caspase activation could be due to reduced ATP-levels. Although this is a formal possibility, this explanation is considered very unlikely since mutant spermatids complete many other energy-intensive cellular processes. These include the extensive transformation from round spermatids to 1.8 mm long elongated spermatids, a process that involves extensive remodeling and movement of actin filaments, generation of the axonemal tail, mitochondrial reorganization, plasma/axonemal membranes reorganization, and nuclear condensation and elongation. Since all of these processes can occur in the absence of cytochrome C-d, there is no overt shortage of ATP in cyt-c-d mutants. It is therefore considered very unlikely that ATP has become limiting in these mutant cells. Since earlier stage spermatids express cytochrome C-p, sufficient ATP seems to persist to late developmental stages. In mammalian cells, cellular ATP concentration is sufficiently high (around 2 mM) to keep cultured cell alive for several days upon ATP synthase inhibition. Furthermore, cells in which cytochrome c expression is decreased by RNAi still undergo apoptosis in response to various stimuli. Likewise, it appears that cytochrome C is not essential for the function of mature murine sperm, since mice deficient for the testis specific form of cytochrome C, Cyt cT, are fertile. Taken together, all these observations argue strongly against the possibility that ATP levels in cyt-c-d-/- mutant spermatids would be insufficient for caspase activation (Arama, 2006).

In conclusion, the results presented in this study definitively demonstrate that cytochrome C-d is essential for caspase activation and spermatid individualization. Both cytochrome C proteins of Drosophila are, at least to some extent, functionally interchangeable. The results also indicate that cytochrome C can promote caspase activation in the absence of a functional apoptosome. Given the powerful genetic techniques available, late spermatogenesis of Drosophila promises to be a powerful system to identify novel pathways for mitochondrial regulation of caspase activation (Arama, 2006).

The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death; The primary function of ARK is to facilitate DRONC activation

The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, it has been demonstrated that ARK is essential for most programmed cell death (PCD) during Drosophila development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal is severely delayed in ark mutants. However, PCD occurs normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in Drosophila (Mills, 2006).

ark alleles were obtained in a screen conducted using mitotic recombination for mutations that appear in an increased relative representation of mutant over wild-type (WT) tissue. In these mutants, the mutant clones were larger than the corresponding WT twin spots. The screen of the right arm of chromosome 2 identified mutations in the hippo locus. Four alleles of ark were also obtained from the same screen; these alleles were all lethal at the pupal stage of development as homozygotes or in trans to each other. Sequencing revealed point mutations or deletions in the coding sequence of the ark gene in each of the mutant chromosomes. ark1 had a G to A mutation, resulting in the truncation of the protein after residue 206; ark2 had a C to T mutation, causing protein truncation after residue 660, and ark3 had a deletion after residue 592, generating a frameshift mutation, whereas ark4 possessed a T to G mutation, causing protein truncation after residue 1,357. The mutation in ark1 is predicted to affect both of the reported alternately spliced transcripts of the ark gene. Because all ark mutants were lethal at a similar stage, only ark1 and ark2 were analyzed in these studies (Mills, 2006).

Similar to Apaf-1, ARK consists of a CARD, a nucleotide-binding NB-ARC domain, and multiple WD40 repeats. ark1 mutation truncates the protein in the NB-ARC (CED-4 domain), whereas ark2 leads to a protein lacking most of the WD40 repeats. Both mutants are lethal and have very similar phenotypes, suggesting that they are strong loss-of-function alleles. The phenotypes also indicate that both the NB-ARC and the WD40 domains are essential for ARK function. Unlike the published hypomorphs, all homozygous ark1 and ark2 animals die as pupae. Despite the similar overall phenotypes for ark1 and ark2 alleles, development of ark1 mutants to pupation was significantly delayed when compared with WT or ark2 alleles, suggesting that ark1 may be a stronger allele than ark2. Consequently, the survival of ark1-null animals to early pupae stage was lower than that of the heterozygotes. Although larvae and pupae from both ark mutants appear grossly normal externally, some larval tissues derived from late third instar animals show hyperplasia. For example the larval central nervous system (CNS) was enlarged in both ark mutants. This was particularly evident in the ventral ganglion that appeared to be elongated and contained longer nerve fibers. In ~40% of ark1 and most of the ark2 animals, the wing discs were enlarged. In a small number of both mutants, the eye discs were also enlarged (Mills, 2006).

dronc mutant embryos contain extra cells, and the removal of maternal dronc abolishes most cell death during embryogenesis. dronc-deficient embryos also show an enlargement of the CNS, which is presumably caused by reduced PCD. By staining embryos with anti-embryonic lethal abnormal visual protein (ELAV) antibody to visualize neurons in the CNS and peripheral nervous system, extra neurons were found in chordotonal cell clusters in ark mutant embryos. There were up to three extra cells per cluster in most ark mutant embryos analyzed. Staining of embryos with BP102 antibody, which recognizes CNS axons, showed gross abnormalities in many mutant animals, with ark2 animals often showing more dramatic features. Stronger staining of CNS axons was consistently observed in ark mutant embryos compared with WT animals, which could result from more densely packed axons. In many mutant animals, the ventral nerve cord appeared to be improperly compacted and the spacing between longitudinal axonal tracts was enlarged. This could be attributable to additional cells in the mutants caused by reduced PCD (Mills, 2006).

Since ark mutants essentially phenocopy the loss-of-dronc function, the data argue that these proteins act in a common pathway. Previous experiments using RNA interference have shown that ARK is required for DRONC activation. These results suggest that the primary function of ARK is to facilitate DRONC activation. The observation that metamorphic midgut cell death occurs normally, whereas salivary gland PCD is significantly delayed, suggests that the midgut may provide a model system for studying novel caspase activation and cell death pathways that are independent of the evolutionarily conserved canonical pathway (Mills, 2006).

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases; the initiator caspase DRONC may lack executioner functions

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, since its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. This study further dissects the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. The induction of apoptosis is dependent in each case on expression of ARK and DRONC and surviving cells continue to proliferate. A difference was noted in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these have dramatic effects to sustain cell survival following depletion of DIAP1, but have only minor effects following cellular stress. These results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted (Kiessling, 2006).

This study further dissected the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. The induction of apoptosis is dependent on expression of the APAF-1 homolog ARK, and the initiator caspase, DRONC. Knock-down of either ARK or DRONC led not only to short term cell survival, as is also observed in mammalian cells lacking APAF-1 or caspase-9, but also to long term survival, seen as cellular accumulation as the cells continued to proliferate. This is in striking contrast to observations in mammalian cells lacking APAF-1 or caspase-9, where cells ultimately succumb to 'caspase-independent cell death' and do not proliferate. This difference is most easily explained by the difference in mitochondrial involvement: in mammals, MOMP is associated with the release of potentially toxic factors, such as AIF, endoG, Omi, and others, and with an eventual loss of mitochondrial function, any of which can contribute to death, even when downstream caspase activation is blocked or defective. The apparent absence of MOMP in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted (Kiessling, 2006).

Studies in ARK mutants clearly demonstrate that ARK is required for cell death in vivo, since these mutants display developmental defects, including an enlarged nervous system, and resist death induced by transgenic expression of Grim. Furthermore, genetic studies revealed an epistatic relationship between ARK and DIAP1 by demonstrating that loss of ARK reverses catastrophic defects seen in DIAP1 mutants and rescues developing tissues that would otherwise die from DAIP1 inactivation. The function of ARK is required for hyperactivation of caspases which occurs in the absence of DIAP-1. One might argue that the current findings are therefore merely confirmatory. However, it should be noted that profound developmental defects are observed in mice lacking APAF-1, caspase-9, or caspase-3, which are nevertheless dispensable for stress or oncogene-induced cell death in MEFs and lymphocytes from these mice in vitro and cells of the interdigital web in vitro or in vivo. In fact, there is currently no evidence that a cell capable of proliferation can do so following MOMP, and alternative explanations of developmental defects in these knockout mice (other than survival and proliferation following MOMP) have been offered (Kiessling, 2006).

A rapid loss of the DIAP1 is observed in the S2 cells, when treated with various stressors. The full length DIAP1 protein disappears rapidly and a smaller, 27 kDa fragment accumulates over time. Interestingly, the broad spectrum caspase inhibitor zVAD-fmk does not suppress the degradation of DIAP1, but the 27 kDa cleavage product could not be detected when caspase activation was inhibited. The differences between DIAP1 degradation with or without caspase activity could be explained by the notion that the degradation of DIAP1 after treatment with apoptosis-inducing stimuli is mediated by a combination of cleavage by caspases and proteasomal degradation. Thus, the continued degradation of DIAP1 in the presence of activated caspases produces the 27 kDa fragment. It has been recently reported that caspase-dependent cleavage of DIAP1 is required for DIAP1 loss in an early stage of apoptosis and that cleavage of DIAP1 is required for degradation. Similarly, it was observed that if caspases are inhibited following apoptosis induction, DIAP1 levels remain unaltered for a number of hours, however, the inhibition of caspases does not block DIAP1 degradation at longer times (Kiessling, 2006).

While a requirement for ARK and DRONC was observed under all of the pro-apoptotic conditions in S2 cells, a difference was noted in the effects of knock-down of the executioner caspases DCP-1 and DRICE. Knock-down of either or both of these has dramatic effects to sustain cell survival following knock-down of DIAP1, but has only minor effects following cellular stress (Kiessling, 2006).

Several possible explanations were envisioned for this difference. One possibility is that knock-down of DIAP1 leads to caspase activation uniquely through permitting DRONC function to activate DCP-1 and DRICE, while stressors somehow engage other caspase activation pathways (and other caspases). This, however, is inconsistent with the observation that stress-induced apoptosis is clearly dependent on ARK and DRONC. Alternatively, it may be that stress-induced death also involves inhibition of other IAPs, such as DIAP-2 and dBRUCE, which may have a wider spectrum of effects to engage additional caspases not affected by DIAP1 alone. Previously, however, it was noted that knock-down of DIAP-2 does not trigger apoptosis, but greatly enhances susceptibility to death induced by stressors such as were used in this study. This argues that DIAP-2 function, at least, continues following such stress (such that its knock-down has an effect) and thus it is less likely to be an important explanation for the current effects (Kiessling, 2006).

The final possibility is perhaps the most interesting. The knock-down of DIAP1 leads to death, presumably through permitting low levels of ongoing (and otherwise repressed) caspase activation to function and any subsequent effect may depend on amplification, as active executioner caspases cleave and activate others. Therefore, knock-down of even one caspase in the cell may dampen this amplification so that cells survive. In contrast, the induction of apoptosis by stress may involve not only blockade of DIAP1 function (through the N-termini of Reaper, Hid, Grim and Sickle) but also another signal that amplifies caspase activation upstream of ARK. Such an upstream effect has been suggested by studies of the so-called 'GH3' region in these proteins, that appears to be required for death in Drosophila cells and can function to promote the mitochondrial pathway in vertebrate systems. Reaper, Hid, Grim, and probably Sickle are necessary for stress-induced apoptosis in Drosophila, and therefore their effects are likely to depend on ARK and ARK-DRONC interactions. Nevertheless, this line of reasoning suggests that they function not only to de-repress caspases (though blocking DIAP1), but also to do something else to bypass full dependence on DCP-1 and DRICE, perhaps by amplifying caspase activation at the level of the ARK-DRONC interaction. While speculative, this possibility is intriguing, and suggests that the induction of apoptosis in Drosophila may prove to be more complex than simple models indicate (Kiessling, 2006).

IKK epsilon regulates F actin assembly and interacts with Drosophila IAP1 in cellular morphogenesis

Differentiated cells assume complex shapes through polarized cell migration and growth. These processes require the restricted organization of the actin cytoskeleton at limited subcellular regions. IKKε is a member of the IκB kinase family, and its developmental role has not been clear. Drosophila IKKε localizes to the ruffling membrane of cultured cells and is required for F actin turnover at the cell margin. In IKKε mutants, tracheal terminal cells, bristles, and arista laterals, which require accurate F actin assembly for their polarized elongation, all exhibit aberrantly branched morphology. These phenotypes are sensitive to a change in the dosage of Drosophila inhibitor of apoptosis protein 1 (DIAP1) and the caspase DRONC without apparent change in cell viability. In contrast to this, hyperactivation of IKKε destabilizes F actin-based structures. Expression of a dominant-negative form of IKKε increases the amount of DIAP1. The results suggest that at the physiological level, IKKε acts as a negative regulator of F actin assembly and maintains the fidelity of polarized elongation during cell morphogenesis. This IKKε function involves the negative regulation of the nonapoptotic activity of DIAP1 (Oshima, 2006).

A collective form of cell death requires Homeodomain interacting protein kinase

Post-eclosion elimination of the Drosophila wing epithelium was examined in vivo where collective 'suicide waves' promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. This phenotype wase leveraged in mosaic animals to discover relevant genes. homeodomain interacting protein kinase (HIPK) was shown to be required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers (Link, 2007).

Elimination of cells by programmed cell death (PCD) is a universal feature of development and aging. In both vertebrates and invertebrates, dying cells often progress through a stereotyped set of transformations referred to as apoptosis. In this form of PCD the nucleus condenses, and the collapsing cell corpse fragments into 'apoptotic bodies' that are engulfed by specialized phagocytes or neighboring cells. Apoptosis requires autonomous genetic functions within the dying cell, and extrinsic cues that elicit apoptosis have been investigated in numerous experimental models. Other forms of death are also thought to contribute during development and differ from apoptosis with respect to cellular morphology, mechanism, or mode of activation. These may include necrosis, characterized by swelling of the plasma membrane, or autophagic cell death, which is linked to extensive vacuolization in the cytoplasm. These forms of cell death can be caspase dependent or independent and may or may not be under deliberate genetic control (Link, 2007).

Two conserved protein families comprise central elements of the apoptotic machinery. Orthologous proteins represented by Ced4 in the nematode, Apaf1 in mammals, and Drosophila Ark (Dark) function as activating adaptors for CARD-containing apical caspases. During apoptosis, Ced4/Apaf1/Dark adaptors associate with pro-caspase partners (Ced3, Caspase 9, and Dronc) in a multimeric complex referred to as the 'apoptosome'. This complex is regulated by Bcl2 proteins, but apparently through a diverse group of mechanisms (Link, 2007).

Components of the Drosophila apoptosome have been genetically examined. dark and dronc are recessive, lethal genes. Both exert global functions during PCD and in stress-induced apoptosis. However, their roles in apoptosis are not absolute because rare cell deaths occurred in embryos lacking maternal and zygotic product of either gene. Elimination of dronc in the wing causes a unique, age-dependent phenotype associated with late-onset blemishing throughout the wing blade (Chew, 2004). This study shows that this progressive phenotype is characteristic for wing epithelia that lack apoptogenic functions and is caused by defects in a communal form of PCD where epithelial cells are collectively and rapidly eliminated. These findings were leveraged to discover additional genes required for PCD, and a limited set of loci, many of which were previously unknown to function in cell death, were recovered. This study establish that homeodomain interacting protein kinase (HIPK) is essential for coordinated death in the wing epithelium and, consistent with PCD functions in earlier developmental stages, regulates proper cell number in diverse tissue types (Link, 2007).

Wings mosaic for dronc- tissue exhibit normal morphology at eclosion but develop progressive, melanized blemishes with age (Chew, 2004). Similar methods were applied to determine whether lesions in other apoptogenic genes present a similar phenotype. After eclosion, wings mosaic for Df(H99), a deletion removing the apoptotic activators reaper (rpr), grim, and hid, were morphologically normal at eclosion, but over 3-7 d, melanized blemishes appeared at random throughout the wing (Link, 2007).

Likewise, homozygous driceDelta1 adult 'escapers' deficient for the effector caspase Drice also presented normal wings at eclosion but developed blemishes with age. Wings mosaic for dark82, a null allele of dark, were indistinguishable from wild-type (WT) at eclosion, but within 4 d developed wing blemishes. These late-onset blemishes became markedly more severe as animals aged. Similar yet less severe wing blemishes occurred in adults homozygous for darkCD4, a hypomorphic allele of dark. Together, these observations establish that late-onset progressive blemishing in mosaic wings is a characteristic phenotype shared among mutants in canonical PCD pathways (Link, 2007).

In the wing of newly eclosed adults, PCD removes the epithelium that forms the dorsal and ventral cuticles. To determine whether the cause of the blemish phenotype might trace to defective death in the wing epithelium, this tissue was examined in dark mutants. For these studies, wings of darkCD4 adults were prepared for light and electron microscopy. Histological analyses at the light level showed that on the first day of eclosion, the dorsal and ventral cuticles of WT animals became tightly merged with no intervening tissue evident between these layers. However, even 14 d after eclosion, cells and cell remnants remained situated between the dorsal and ventral cuticles in dark mutants. This 'undead' tissue was most easily visualized in lateral sections through melanized blemishes. Further examination of the persisting epithelium at the EM level showed evidence of intact cells soon after eclosion and ectopic cellular material 24 h after eclosion (Link, 2007).

To directly examine the death of wing epithelial cells in vivo, a transgenic nuclear DsRed reporter was used that driven by vestigial-Gal4 (vg:DsRed), allowing visualization of the fate of these cells soon after eclosion. Observations with this pan-epithelial marker in the wing confirmed earlier studies. Within 1 h of eclosion, intact epithelial cells are clearly present and regularly patterned throughout the wing. 1-2 h later (2-3 h after eclosion), the entire intervein epithelium disappears, manifested here by the abrupt loss of DsRed throughout the wing blade. Live, real-time imaging of the wing in newly eclosed adults revealed unexpected features associated with elimination of the intervein epithelium. Epithelial cells, labeled by nuclear fluorescence, were arranged in a regular, predictable pattern throughout the wing. Then, consistent with nuclear breakdown, fluorescence became redistributed throughout the cell followed by indications of blebbing and the appearance of fragmenting cells. Occasionally, weak fluorescence enclosed in cell corpses condensed to bright punctate bodies. This series of apoptogenic changes spread extremely rapidly throughout the epithelium, appearing here as a collective wave initiating from the peripheral edge and moving across the wing blade (Link, 2007).

Within just 4 min, virtually all nuclei (~450 cells) within a space of ~114 mm2 converted from viable to apoptotic morphology. The process involved tight coordination at the group level because the likelihood of a single cell apoptosing was clearly linked to similar behaviors by nearest neighboring cells over short time frames. Also, the direction and size of the cell death wave may not be fixed in every region of the wing, but centrally located cell groups were generally eliminated earlier (Link, 2007).

Unlike conventional examples of PCD in development, no indication was found that overt engulfment of apoptotic corpses occurred at the site of death. Instead, DsRed-labeled cell remnants were passively swept en masse toward the nearest wing vein where, apparently under hydrostatic pressure, cell debris streamed proximally toward the body through the wing or along the wing vein. Together, these observations describe a communal form of PCD that rapidly eliminates the wing epithelium through coordinated group behavior (Link, 2007).

The vg:DsRed reporter was used to track the fate of mosaic wing epithelia where mutant clones were induced. In sharp contrast to WT wings, abnormally persisting cells could be readily detected as patches of DsRed in the nuclei of epithelial cells in mosaic tissues. For example, wings mosaic for dronc- clones retained extensive patches of persisting DsRed-labeled cells. Here, cells and nuclei were readily detected 4 d after eclosion, and even at 11 d post-eclosion, extensive evidence of cell debris was seen (not depicted). Wings mosaic for the H99 deletion gave identical results. Likewise, adults mutated for dark exhibited persisting cells throughout the wing blade. Consistent with this, rare driceDelta1 escapers also showed evidence of persisting cells after eclosion. These observations link failures in PCD to progressive melanized wing blemishes, raising the possibility that other apoptogenic mutants might also produce this phenotype (Link, 2007).

Unlike previously described wing defects, which are congenital and evident at eclosion, the age-dependent phenotype described in in this study is characteristic of mutations in genes that function in canonical PCD pathways. Moreover, when the dosage of dronc was reduced by half in darkCD4 adults or if WT Dmp53 was removed from these same animals, melanized blemishes became far more severe. These genetic interactions are highly specific because wing defects were never observed in Dmp53- homozygotes or in dronc51 heterozygotes. Numerous other mutants showed no such effects in combination with a dark hypomorph. It was reasoned that, if genetically eliminated, additional regulators and effectors in PCD pathways should phenocopy wings mosaic for dark- or dronc- tissue. A collection of preexisting transposon mutants was screened to capture insertions that exhibit normal wings at eclosion but develop melanized blemishes with age. This strategy exploits the FLP/FRT system together with wing-specific drivers to interrogate animals bearing wing genotypes mosaic for clones of P element-derived lethal mutations. Progeny with mosaic wings were examined for late-onset wing blemishes at 1, 7, and 14 d post-eclosion. Over 1,000 lethal insertions were screened, representing 356 2nd chromosome mutations and 707 3rd chromosome mutations (Link, 2007).

The majority of insertions (87%) produced no visible defects as wing mosaics. 13% of insertions tested produced abnormalities, and these were scored for the phenotypic categories. Congenital defects including notched, blistered, or wrinkled wings occurred alone or occasionally as compound phenotypes. The candidate strains that developed wing blemishing were further subdivided based on phenotypic severity. Insertions in class A developed pronounced blemishes within a week of eclosion, whereas those in class B developed relatively light-colored patches between 1 and 2 wk after eclosion. Mutant lines exhibiting class A phenotypes were rare (~2%). All members of this class lacked blemishes at eclosion and displayed progressive blemishing occasionally associated with fragile and sometimes broken wings. A new allele of dark (l(2)SH0173) was recovered in this class, providing reassuring validation for the screening strategy. Some members among these classes exhibited congenital notches or blisters, but congenital blemishes present at eclosion were not found (Link, 2007).

Inverse PCR was applied to map or confirm insertion sites of many class A and B strains. In addition to darkl(3)SH0173, several mutations associated with genes previously implicated in PCD were isolated. For example, l(2)SH2275 contains an insertion 2 kb upstream of mir-14, a microRNA capable of modulating Rpr-induced cell death. Likewise, l(3)S048915 maps to the first intron of DIAP1 and may represent a hypermorphic allele at this locus. l(3)S055409 maps near misshapen, a gene implicated in cell killing triggered by Rpr or Eiger, the fly counterpart of TNF. Several insertions map in or near transcriptional or translational regulators that might alter the expression of cell death genes. For example, grunge (l(3)S146907), an Atrophin-like protein, functions as a transcriptional repressor, while belle (l(3)S097074) belongs to the DEAD-box family of proteins often implicated in translational regulation and RNA processing. A portion of the class A and B hits were also directly examined for defective PCD by applying the vg:DsRed reporter in mosaic wings. Of the 29 strains tested, 14 showed obvious evidence for persisting cells in the wing epithelium (Link, 2007).

Mutants identified that exhibit both blemishing and persisting cells are likely candidates for PCD genes. One strain, l(3)S134313, produced severe late-onset blemishing and a persisting cell phenotype. After mapping this insertion to the first intron of the HIPK, null alleles at this locus were produced (Link, 2007).

Two FRT-containing P element insertions flanking the coding region of HIPK were used to generate a novel deletion. PCR verified recombination between P elements, and 8 deletion strains were recovered. These validated alleles eliminate exons 4-12, removing over 92% of coding sequence in the predicted HIPK open reading frame. Deletions at the HIPK locus were uniformly lethal before the 3rd instar stage. However, zygotic HIPK is not essential to complete embryogenesis because ~70% of HIPK homozygotes hatch to 1st instar larvae. HIPKD1 was recombined on the FRT79 chromosome to generate adult wings mosaic for this allele, and like the original insertion, these animals also developed robust progressive blemishes and a persisting cell phenotype. Both phenotypes were more severe than the original P insertion, suggesting that the l(3)S134313 allele is hypomorphic for HIPK. These findings link loss of HIPK function to the query phenotypes, establishing that the action of HIPK is essential for post-eclosion PCD in the wing epithelium (Link, 2007).

Using general stains (acridine orange) or TUNEL methods, embryonic PCD was not overtly disturbed in HIPK mutants. To investigate the possibility of more subtle or specific phenotypes, the nervous system was examined using antibodies that label specific populations of neurons affected by the H99 deletion. Using anti-Kruppel antibody, it was confirmed that stage 14-15 WT embryos contained 9-12 Kruppel-positive cells in the Bolwig's Organ. However, a portion of animals lacking maternal HIPK contained as many as 15 cells per organ at a penetrance comparable to H99 animals, which are completely cell death defective. Neurons expressing dHb9, a homeodomain protein marking a subset of cells that persist in cell death-defective H99 embryos, was examined (Rogulja-Ortmann, 2007). In germline clones, distinct classes of dHb9 staining patterns emerged. A subset of animals exhibited extreme patterning defects. Other animals displayed a striking increase in dHb9-positive cell numbers when compared with WT embryos of the parental strain. These data establish that HIPK fundamentally regulates cell numbers in the nervous system, and because the same subpopulation of cells are affected by the H99 mutation, they implicate HIPK as a more general regulator of PCD (Link, 2007).

The pupal eye undergoes reorganization involving cell death of interommatidial cells after pupation. To determine if HIPK regulates cell death in the retina, whole eye clones were generated and the anti-Dlg (discs large) antibody was used to outline cell borders in dissected pupal eyes after pupation. Extra interommatidial cells were frequently retained in whole eye HIPK- clones. This phenotype is overtly similar to animals lacking the apical caspase Dronc and consistent with an essential role in retinal PCD (Link, 2007).

Elimination of the wing epithelium in newly eclosed adults is predictable, easily visualized, and experimentally tractable. The major histomorphologic events involve cell death, delamination, and clearance of corpses and cell remnants. Recent studies established that post-eclosion PCD is under hormonal control and involves the cAMP/PKA pathway (Kimura, 2004). While dying cells in the adult wing present apoptotic features (e.g., sensitivity to p35 and TUNEL positive), elimination of the epithelium is distinct from classical apoptosis in several important respects. (1) Unlike most in vivo models, overt engulfment of cell corpses does not occur at the site of death. Instead, dead or dying cells and their remnants are washed into the thoracic cavity via streaming of material along and through wing veins. (2) Extensive vacuolization is seen in ultrastructural analyses, which could indicate elevated autophagic activity. (3) Widespread and near synchronous death that occurs in this context defines an abrupt group behavior. The process affects dramatic change at the tissue level, causing wholesale loss of intervein cells and coordinated elimination of the entire layer of epithelium. Rather than die independently, these cells die communally, as if responding to coordinated signals propagated throughout the entire epithelium, perhaps involving intercellular gap junctions. This group behavior contrasts with canonical in vivo models where a single cell, surrounded by viable neighbors, sporadically initiates apoptosis (Link, 2007).

It has been proposed that an epithelial-to-mesenchymal transition (EMT) accounts for the removal of epithelial cells after eclosion (Kiger, 2007). Although the results do not exclude EMT associated changes in the newly eclosed wing epithelium, compelling lines of evidence establish that post-eclosion loss of the wing epithelium occurs by PCD in situ -- before cells are removed from the wing. First, before elimination, wing epithelial cells label prominently with TUNEL. Second, every mutation in canonical PCD genes so far tested failed to effectively eliminate the wing epithelium, and at least two of these were recovered in the screen described in this paper. Third, elimination of the wing epithelium was reversed by induction of p35, a broad-spectrum caspase inhibitor. Fourth, using time-lapse microscopy, condensing or pycnotic nuclei, followed by the rapid removal of all cell debris in time frames was detected that was not consistent with active migration. Instead, removal of cell remnants occurred by a passive streaming process, involving perhaps hydrostatic flow of the hemolymph (Link, 2007).

This study sampled over one fifth of all lethal genes and nearly 10% of all genes in the fly genome for the progressive blemish phenotype, a reliable indicator of PCD failure in the wing epithelium. Nearly half of the mutants that produced melanized wing blemishing also displayed a cell death-defective phenotype when examined with the vg:DsRed reporter. The precise link between these defects is unclear, but a likely explanation suggests that as the surrounding cuticle fuses, persisting cells, now deprived for nutrients and oxygen, become necrotic and may initiate melanization. Mutants could arrest at upstream steps, involving the specification or execution of PCD, or they might affect proper clearance of cell corpses from the epithelium. New alleles were recovered of dark (l(2)SH0173) and a likely hypermorph of thread (l(3)S048915), which provides reassuring validation of this prediction (Link, 2007).

By leveraging this distinct phenotype, novel cell death genes were captured, including the Drosophila orthologue of HIPK. Though first identified as an NK homeodomain binding partner, this gene is an essential regulator of PCD and cell numbers in diverse tissue contexts. Of the four mammalian HIPK genes, HIPK2, the predicted ortholog of Drosophila HIPK, has been placed in the p53 stress-response apoptotic pathway (D'Orazi, 2002; Hofmann, 2002; Di Stefano, 2004; Di Stefano, 2005), but whether the Drosophila counterpart similarly impacts this network is not yet known (Link, 2007).

A genome-wide RNAi screen reveals multiple regulators of caspase activation

A genome-wide RNA interference screen was performed to systematically identify regulators of apoptosis induced by DNA damage in Drosophila cells. Forty-seven double- stranded RNAs were identified that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. These data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death (Yi, 2007).

The genes that are specifically involved in caspase-dependent cell death were classified. Substantial induction of caspase activity was observed 8 h after treatment with a topoisomerase II inhibitor, doxorubicin (dox), to induce dose-dependent cell death. Any RNAi suppressing this activity implicates the target gene in early regulation of caspase activation. In addition to dcp-1 RNAi, knockdown of dronc and jra (the Drosophila homolog of c-Jun) significantly suppressed caspase-3/7-like activity in the presence of dox, whereas the negative control, RNAi against calpain A, a calcium-dependent cysteine protease, did not affect this pathway (Yi, 2007).

This analysis was expanded to all of the genes identified in the initial RNAi screen and 20 dsRNAs were discovered that suppressed caspase activation induced by DNA damage. Interestingly, 12 of these genes were found to be epistatic to diap1 (Yi, 2007).

diap1 epistatic analysis was performed to further categorize the genes. DIAP1, the fly orthologue of the mammalian inhibitors of apoptosis proteins, is a direct inhibitor of caspases, and deficiency in DIAP1 leads to rapid caspase activation and apoptosis in vivo. Thus, apoptosis induced by the loss of DIAP1 presents an alternative apoptotic assay independent of DNA damage. Silencing of genes that regulate activation of the core apoptotic machinery may provide protection against apoptosis induced by both DNA damage and the loss of DIAP1. RNAi against dcp-1 partially suppressed cell death induced by the depletion of DIAP1 in Kc cells. Also, dronc RNAi potently protected cells against apoptosis induced by deficiency in DIAP1. Altogether, 32 of the genes confirmed from the primary screen provided significant protection against cell death induced by the silencing of DIAP1 (Yi, 2007).

Interestingly, 12 dsRNAs suppressed caspase-3/7-like activity after dox treatment and protected against cell death induced by diap1 RNAi, suggesting that these genes are required for apoptosis induced by multiple stimuli. To confirm that these genes are necessary for the full activation of caspases, it was determined whether these dsRNAs could suppress spontaneous caspase activity induced by diap1 RNAi. Maximal induction of caspase activity by diap1 RNAi was observed after 24 h, and this effect was completely suppressed by dsRNA against dcp-1. Importantly, ablating 10/12 dsRNAs resulted in the significant suppression of caspase activity compared with diap1 RNAi only (Yi, 2007).

In addition to dronc RNAi, dsRNAs targeting chn and dARD1 provided the strongest suppression of spontaneous caspase activity. Consistent with the observation that RNAi against chn protects against DNA damage-induced cell death, the mammalian orthologue neuron-restrictive silencer factor (NRSF)/RE1-silencing transcription factor (REST) was recently identified as a candidate tumor suppressor in epithelial cells (Westbrook, 2005). Previous work indicates that Chn and NRSF/REST function as a transcriptional repressor of neuronal-specific genes (Chong, 1995; Schoenherr, 1995; Tsuda, 2006), suggesting that cellular differentiation may render cells refractory to caspase activation and apoptosis. Also, several metabolic genes, CG31674, CG14740, and CG12170, were identified that may be involved in the general regulation of caspase activation. It has been demonstrated that NADPH produced by the pentose phosphate pathway regulates the activation of caspase-2 in nutrient-deprived Xenopus laevis oocytes. Together with these results, these observations provide further evidence for an intimate link between the regulation of metabolism and induction of apoptosis (Yi, 2007).

To further explore the significance of these findings, whether silencing the mammalian orthologues of the fly genes identified from the RNAi screen confers protection against dox-induced cell death was investigated in mammalian cells. A set of mammalian orthologues was selected that are believed to be nonredundant. The list includes the orthologues of dMiro, which functions as a Rho-like GTPase; dARD1, which functions as an N-acetyltransferase; CG12170, which functions as a fatty acid synthase; and Chn, which functions as a transcriptional repressor (RHOT1, hARD1, OXSM, and REST, respectively; FlyBase). In addition, Plk3, a mammalian orthologue of Polo, was tested since dsRNA targeting polo potently protected against dox treatment (Yi, 2007).

The ability of siRNAs targeting a gene of interest to protect against DNA damage was tested in HeLa cells. As a positive control, cells were transfected with siRNAs targeting Bax or Bak, two central regulators of mammalian cell death. Indeed, silencing of Bax or Bak resulted in significant protection against dox- induced cell death. It was observed that plk3 RNAi provided partial protection against dox treatment, which is consistent with previous studies implicating Plk3 in stress-induced apoptosis. Interestingly, the knockdown of hARD1 dramatically enhanced cell survival in the presence of dox to levels similar to that of Bak. This protective effect was also evident at the morphological level. In cells transfected with a nontargeting control siRNA, dox treatment resulted in typical apoptotic morphology, including cell rounding and membrane blebbing. In direct contrast, cells transfected with siRNAs against hARD1 maintained a normal and healthy morphology and continued to proliferate in the presence of dox (Yi, 2007).

To examine whether the protection provided by siRNAs targeting hARD1 and plk3 is associated with the suppression of caspase activation, caspase activity was measured in these cells treated with dox. RNAi against plk3 provided partial suppression of caspase activity, again supporting the observed protection phenotype. Interestingly, the depletion of REST resulted in some suppression of caspase activity in the presence of dox even though the protection against cell death was not statistically significant. Consistent with the viability assay, complete suppression of caspase-3/7 activity was observed in cells transfected with hARD1 siRNA. These results indicate that hARD1 is required for caspase-dependent cell death induced by DNA damage. Furthermore, all four siRNAs targeting hARD1 were individually capable of providing robust protection against cell death, strongly suggesting that these siRNAs target hARD1 specifically (Yi, 2007).

Because the silencing of hARD1 dramatically suppressed activation of the downstream caspases, whether activation of the upstream caspases in response to dox treatment is also perturbed was also examined. Remarkably, hARD1 RNAi inhibited the cleavage of caspase-2 and -9 in cells treated with dox, whereas caspase cleavage was readily detected in control cells. Thus, it is proposed that hARD1 regulates the signal transduction pathway apical to the apoptotic machinery in the DNA damage response itself or the activation of upstream caspases (Yi, 2007).

Consistent with the results of the caspase-3/7 assay, silencing of hARD1 completely inhibited the appearance of activated caspase-3 induced by dox. This assay was used for a hARD1 complementation experiment to demonstrate the proapoptotic role of hARD1 in response to DNA damage. A new siRNA pool was used, targeting the 5' untranslated region of hARD1 (5'si); this treatment inhibited caspase-3 cleavage induced by dox treatment. Furthermore, caspase-3 cleavage was observed in reconstituted hARD1 knockdown cells. Because six out of six siRNAs against hARD1 provided strong protection against DNA damage-induced apoptosis and complementation of hARD1-sensitized cells to caspase activation, it is concluded that the functional role of ARD1 for dox-induced apoptosis is evolutionally conserved from Drosophila to mammals (Yi, 2007).

In summary, this study used an unbiased RNAi screening platform in Drosophila cells to identify genes involved in promoting DNA damage-induced apoptosis. Forty-seven dsRNAs were isolated that suppress cell death induced by dox. These genes encode for known apoptotic regulators such as Dronc, the Drosophila orthologue of the known proapoptotic transcriptional factor c-Jun, and an ecdysone-regulated protein, Eip63F-1, thereby validating the primary screen. Furthermore, this study implicates a large class of metabolic genes that were previously not suspected to have a role in modulating caspase activation and apoptosis, such as genes involved in fatty acid biosynthesis (CG11798), amino acid/carbohydrate metabolism (CG31674), citrate metabolism (CG14740), complex carbohydrate metabolism (CG10725), and ribosome biosynthesis (CG6712). These results support the proposal that the cellular metabolic status regulates the threshold for activation of apoptosis and thus plays a critical role in the decision of a cell to live or die (Yi, 2007).

Of particular interest is the identification of ARD1. Evidence is presented that RNAi against ARD1 provides protection against cell death and leads to the suppression of caspase activation induced by DNA damage in fly cells and HeLa cells. Furthermore, deficiency in dARD1 renders fly cells resistant to the spontaneous caspase activity and cell death associated with loss of Diap1. Importantly, substantial evidence is provided that hARD1 is required for caspase activation in the presence of DNA damage in mammalian cells. Cleavage of initiator and executioner caspases are suppressed in hARD1 RNAi cells treated with dox, suggesting that hARD1 functions further upstream of caspase activation, and the complementation of hARD1 knockdown cells restores caspase-3 cleavage. These data indicate that ARD1 is necessary for DNA damage-induced apoptosis in flies and mammals (Yi, 2007).

ARD1 functions in a complex with N-acetyltransferase to catalyze the acetylation of the Nα-terminal residue of newly synthesized polypeptides and has been implicated in the regulation of heterochromatin, DNA repair, and the maintenance of genomic stability in yeast. These studies suggest that ARD1 may be involved in regulating an early step in response to DNA damage. It is anticipated that future studies will focus on determining whether ARD1 functions in similar processes in mammals. The diversity of genes identified in this screen illustrates the complex cellular integration of survival and death signals through multiple pathways (Yi, 2007).

Persephone/Spatzle pathogen sensors mediate the activation of Toll receptor signaling in response to endogenous danger signals in apoptosis-deficient Drosophila

Apoptosis is an evolutionarily conserved mechanism that removes damaged or unwanted cells, effectively maintaining cellular homeostasis. It has long been suggested that a deficiency in this type of naturally occurring cell death could potentially lead to necrosis, resulting in the release of endogenous immunogenic molecules such as DAMPs (damage-associated molecular patterns) and a non-infectious inflammatory response. However, the details about how danger signals from apoptosis-deficient cells are detected and translated to an immune response are largely unknown. This study found that Drosophila mutants deficient for Dronc, the key initiator caspase required for apoptosis, produced the active form of the endogenous Toll ligand Spatzle (Spz). It is speculated that, as a system for sensing potential DAMPs in the hemolymph, the dronc mutants constitutively activate a proteolytic cascade that leads to Spz proteolytic processing. It was demonstrated that Toll signaling activation required the action of Persephone, a CLIP-domain serine protease that usually reacts to microbial proteolytic activities. These findings show that the Persephone proteolytic cascade plays a crucial role in mediating DAMP-induced systemic responses in apoptosis-deficient Drosophila mutants (Ming, 2014).

Proteomic survey reveals altered energetic patterns and metabolic failure prior to retinal degeneration

Inherited mutations that lead to misfolding of the visual pigment rhodopsin (Rho) are a prominent cause of photoreceptor neuron (PN) degeneration and blindness. How Rho proteotoxic stress progressively impairs PN viability remains unknown. To identify the pathways that mediate Rho toxicity in PNs, a comprehensive proteomic profiling of retinas was performed from Drosophila transgenics expressing Rh1P37H, the equivalent of mammalian RhoP23H, the most common Rho mutation linked to blindness in humans. Profiling of young Rh1P37H retinas revealed a coordinated upregulation of energy-producing pathways and attenuation of energy-consuming pathways involving target of rapamycin (TOR) signaling, which was reversed in older retinas at the onset of PN degeneration. The relevance of these metabolic changes to PN survival was probed by using a combination of pharmacological and genetic approaches. Chronic suppression of TOR signaling, using the inhibitor rapamycin, strongly mitigated PN degeneration, indicating that TOR signaling activation by chronic Rh1P37H proteotoxic stress is deleterious for PNs. Genetic inactivation of the endoplasmic reticulum stress-induced JNK/TRAF1 axis as well as the APAF-1/caspase-9 axis, activated by damaged mitochondria, dramatically suppressed Rh1P37H-induced PN degeneration, identifying the mitochondria as novel mediators of Rh1P37H toxicity. It is thus proposed that chronic Rh1P37H proteotoxic stress distorts the energetic profile of PNs leading to metabolic imbalance, mitochondrial failure, and PN degeneration and therapies normalizing metabolic function might be used to alleviate Rh1P37H toxicity in the retina. This study offers a glimpse into the intricate higher order interactions that underlie PN dysfunction and provides a useful resource for identifying other molecular networks that mediate Rho toxicity in PNs (Griciuc, 2014).

Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila

Apoptosis has essential roles in a variety of cellular and developmental processes. Although the pathway is well studied, how the activities of individual components in the pathway are regulated is less understood. In Drosophila, a key component in apoptosis is Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is required to prevent caspase activation. This study demonstrates that Drosophila CG42593 (ubr3), encoding the homolog of mammalian UBR3, has an essential role in regulating the apoptosis pathway. Loss of ubr3 activity causes caspase-dependent apoptosis in Drosophila eye and wing discs. Genetic epistasis analyses show that the apoptosis induced by loss of ubr3 can be suppressed by loss of initiator caspase Drosophila Nedd2-like caspase (Dronc), or by ectopic expression of the apoptosis inhibitor p35, but cannot be rescued by overexpression of DIAP1. Importantly, the activity of Ubr3 in the apoptosis pathway is not dependent on its Ring-domain, which is required for its E3 ligase activity. Furthermore, through the UBR-box domain, Ubr3 physically interacts with the neo-epitope of DIAP1 that is exposed after caspase-mediated cleavage. This interaction promotes the recruitment and ubiquitination of substrate caspases by DIAP1. Together, these data indicate that Ubr3 interacts with DIAP1 and positively regulates DIAP1 activity, possibly by maintaining its active conformation in the apoptosis pathway (Huang, 2014).

Apoptotic mechanisms during competition of ribosomal protein mutant cells: roles of the initiator caspases Dronc and Dream/Strica

Heterozygosity for mutations in ribosomal protein genes frequently leads to a dominant phenotype of retarded growth and small adult bristles in Drosophila (the Minute phenotype). Cells with Minute genotypes are subject to cell competition, characterized by their selective apoptosis and removal in mosaic tissues that contain wild-type cells. Competitive apoptosis was found to depend on the pro-apoptotic reaper, grim and head involution defective genes but was independent of p53. Rp/+ cells were protected by anti-apoptotic baculovirus p35 expression but lacked the usual hallmarks of 'undead' cells. They lacked Dronc activity, and neither expression of dominant-negative Dronc nor dronc knockdown by dsRNA prevented competitive apoptosis, which also continued in dronc null mutant cells or in the absence of the initiator caspases dredd and dream/strica. Only simultaneous knockdown of dronc and dream/strica by dsRNA was sufficient to protect Rp/+ cells from competition. By contrast, Rp/Rp cells were also protected by baculovirus p35, but Rp/Rp death was dronc-dependent, and undead Rp/Rp cells exhibited typical dronc-dependent expression of Wingless. Independence of p53 and unusual dependence on Dream/Strica distinguish competitive cell death from noncompetitive apoptosis of Rp/Rp cells and from many other examples of cell death (Kohn, 2015).

Rp/+ cells in competition with wild-type cells undergo programmed cell death. Regardless of whether Rp/+ clones are in a wild-type background, or wild-type clones are in a Rp/+ background, cell death occurs that can be prevented by the caspase inhibitors baculovirus p35 and DIAP1, or by removing the pro-apoptotic genes grim, reaper and hid. This study shows that competitive cell death involves less Dronc activity than other forms of apoptosis, and can be initiated by Dream/Strica (Kohn, 2015).

Unexpectedly, this study found that Rp/Rp cells die through apoptosis, and therefore can survive and divide in the presence of baculovirus p35. In these experiments, cells homozygous for M(3)95A, a mutation thought to correspond to RpS3 were protected by p35, Dronc-DN or dsRNA for dronc. The findings are not specific to this mutation: p35 protected clones homozygous for the M(3)i55 mutation corresponding to RpS17 (Kohn, 2015).

It is doubtful that cells divide in the complete absence of essential ribosomal proteins. Even if these alleles are null, presumably each clone of Rp/Rp cells begins as a recombinant cell that inherits ribosomes from the Rp/+ mother cell. The results suggest that as ribosome numbers diminish with time and growth, apoptosis is triggered before growth becomes impossible, and Rp/Rp cells can undergo a few more divisions if they are protected from apoptosis. Apoptosis of Rp/Rp cells provides a useful contrast with competitive death of Rp/+ cells within the same preparations (Kohn, 2015).

When p35 protects cells from effector caspases, persistent Dronc activity marks the ‘undead’ cells. Undead Rp/Rp cells were labeled by CM1 and expressed Wg. They lost these undead markers when Dronc-DN was expressed along with p35, confirming Dronc activity. By contrast, Rp/+ cells protected by p35 were unlabeled by CM1 antibodies or for Wg. Therefore, competitive death of Rp/+ cells seems to involve little Dronc activity. Competitive death is initiated either not by Dronc, or by Dronc activity at low levels. The Dronc independence of competitive apoptosis precluded using undead cells to map competitive cell death signals, which has been possible for developmental apoptosis (Kohn, 2015).

The different levels of Dronc activity in Rp/+ and Rp/Rp cells mirrored different dronc requirements. Expression of Dronc-DN protected Rp/Rp cells, albeit less effectively than baculovirus p35, as did dsRNA for dronc. Dronc-DN may be less effective than p35 because Dronc-DN does not inhibit Dronc activity completely. Alternatively, if Rp/Rp cells are subject to competition with Rp/+ cells in addition to cell-autonomous cell death, Rp/Rp cells that were protected from apoptosis by Dronc-DN would still be subject to Dronc-independent competition with neighboring Rp/+ cells, and for this reason would survive less well than Rp/Rp cells protected by p35 (Kohn, 2015).

By contrast, Rp/+ clones were eliminated in the absence of dronc or in the presence of Dronc-DN or dsRNA for dronc. Dronc-DN and dsRNA for dronc may retain some activity, and clones of cells homozygous for dronc null alleles may inherit Dronc protein or RNA, but as these reagents effectively blocked dronc-dependent processes in Rp/Rp cells, competitive death of Rp/+ cells must require less Dronc activity than is normal in other forms of apoptosis, such as that of Rp/Rp cells (Kohn, 2015).

The remaining initiator caspases Dredd and Dream/strica were also dispensable for competitive cell death. Effector caspases, which are clearly both activated and required, must either be activated by multiple initiator caspases redundantly, or by novel mechanisms. It proved possible to express dsRNA for both dronc and dream in competing cells, and this blocked competitive cell death almost completely, indicating that Dronc and Dream/Strica acted redundantly to initiate competitive cell death. It was notable that dsRNA for dronc alone led to some reduction in cell death, although it was not significant statistically in this study. This may indicate that dream does not substitute completely for dronc. However, reduced cell death of Rp/+ cells even in Rp/+ imaginal discs that lack wild-type clones indicates that Rp/+ cells were subject to a background of dronc-dependent cell death not due to cell competition. Alone, dsRNA for dream/strica had no effect. Overall, these data indicate that dronc and dream could each initiate competitive cell death, rendering competitive cell death less dependent on dronc than most apoptosis in Drosophila (Kohn, 2015).

Another group concluded that competitive death of RpS17/+ cells is dronc-dependent, and that p35 expression during cell competition leads to undead Rp/+ cells. Their study, however, did not differentiate the apoptosis of Rp/Rp cells from Rp/+ cells, or note the presence of undead Rp/Rp in the presence of p35.In addition, the M(3)i55 chromosome that is mutant for RpS17 may exhibit a higher background of dronc-dependent, noncompetitive Rp/+ cell death than the M(3)95A chromosome used in this study. It is important to distinguish these dronc-dependent death mechanisms from cell competition (Kohn, 2015).

Dronc is commonly activated in the apoptosome by expression of the pro-apoptotic genes rpr, grim or hid, which lead to the degradation of anti-apoptotic DIAP1 protein. Indeed, rpr and hid transcription is elevated in Rp/+ cells, and deleting rpr, grim and hid prevented cell competition. These data fall short of proof that elevated rpr, grim or hid expression initiate competitive cell death. Transcriptional reporters for hid and rpr were elevated in all Rp/+ cells, most of which were not apoptotic. As Rp/+ cells do not transcribe higher levels of DIAP1 elevated rpr or hid may sensitize Rp/+ cells to cell death without being the specific trigger. Elevating DIAP1 after rpr, grim and hid deletion might desensitize Rp/+ cells, rather than block a specific pathway. An interesting possibility is that Rp/+ cells elevate a nonautonomous survival factor. Such a factor would be present at lower levels near to wild-type cells, potentially providing a trigger for competitive cell death at clone boundaries. Such a scenario remains hypothetical until the factor is identified (Kohn, 2015).

Transcription of rpr and hid in response to stress or γ-irradiation depends on the IRER enhancer. Unlike some other examples of apoptosis, competitive cell death did not depend on this enhancer, ruling out at least one mechanism by which changes in pro-apoptotic gene expression levels could lead to cell competition. The IRER enhancer, as well as the transcription ofhid and rpr, are transcriptional targets of p53, and p53 is activated by Rp mutations in vertebrates. Cell competition continued in the absence of p53, however. In addition, Rp/+ cells did not express ectopic Wg in the presence of p35 , which would be expected if p53 was activated. (Kohn, 2015).

It is significant that competitive cell death in Drosophila continued in the absence of p53 for two reasons. First, it indicated that cell competition was not mediated by the vertebrate nucleolar stress pathway, whereby certain ribosomal proteins stabilize p53 and trigger death of hematopoietic stem cells. Accordingly, the MDM2 ubiquitin ligase that is targeted by vertebrate ribosomal proteins is not encoded in the Drosophila genome. It remains possible that other mechanisms of nucleolar stress occur in Drosophila. Secondly, it has been suggested that cell competition could be a mechanism to remove aneuploid cells. As there is p53-independent removal of aneuploid cells, the findings are consistent with cell competition as the mechanism (Kohn, 2015).

It could be proposed that expression levels determine the contributions of Dronc and Dream/strica to cell death. In this view, Dream/strica levels are inadequate to initiate developmental cell death, but higher levels of Dream/strica proteins in Rp/+ cells become sufficient to initiate competitive apoptosis in the absence of Dronc. There is evidence against this simple model, however, because the lack of undead Rp/+ cells in the presence of baculovirus p35 indicated that Dronc activity was unusually low in competitive apoptosis. In addition, apoptosis of Rp/Rp cells, as well as noncompetitive apoptosis of Rp/+ cells, were both dronc-dependent but independent of dream/strica. The fact that Rp/+ cells are capable of dronc-dependent and dronc/dream-dependent cell death processes is difficult to explain unless competitive cell death specifically activates Dream/strica in addition to Dronc (Kohn, 2015).

Little is yet known concerning the molecular mechanisms of Dream/strica activation. Dream/strica differs from other initiator caspases in that its extensive prodomain is serine/threonine-rich and lacks a caspase recruitment domain or death effector domain, suggesting that it could be activated by a distinct mechanism. The protein interacts with both DIAP1 and DIAP2, and some studies suggest that Dream/strica accelerates aspects of dronc-dependent apoptosis. Dream/strica and Dronc are also required redundantly for nurse cell death during oogenesis and the programmed elimination of certain peptidergic neurons from the CNS during pupariation. Further characterization of mechanisms of Dream/Strica activation may be revealing concerning the induction of competitive apoptosis by interactions between wild-type and Rp/+ cells (Kohn, 2015).


EVOLUTIONARY HOMOLOGS

Domain structure and alternative forms of Caspase-9

A novel isoform of rat caspase-9 has been identified in which the C terminus of full-length caspase-9 is replaced with an alternative peptide sequence. Casp-9-CTD (where CTD is carboxyl-terminal divergent) is expressed in multiple tissues, with the relative highest expression observed in ovary and heart. Casp-9-CTD is found primarily in the cytoplasm and is not detected in the nucleus. Structural predictions suggest that in contrast to full-length caspase-9, casp-9-CTD is not able to be processed. This model is supported by reduced protease activity of casp-9-CTD preparations in vitro and by the lack of detectable processing of casp-9-CTD proenzyme or the induction of cell death following transfection into cells. Both neuronal and non-neuronal cell types transfected with casp-9-CTD are resistant to death evoked by trophic factor deprivation or DNA damage. In addition, cytosolic lysates prepared from cells permanently expressing exogenous casp-9-CTD are resistant to caspase induction by cytochrome c in reconstitution assays. Taken together, these observations indicate that casp-9-CTD acts as a dominant-negative variant. Its expression in various tissues indicates a physiological role in regulating cell death (Angelastro, 2001).

Mechanism of activation of apical caspases

Apoptosis is orchestrated by the concerted action of caspases, activated in a minimal two-step proteolytic cascade. Existing data suggests that apical caspases are activated by adaptor-mediated clustering of inactive zymogens. However, the mechanism by which apical caspases achieve catalytic competence in their recruitment/activation complexes remains unresolved. Proximity-induced activation of apical caspases is attributable to dimerization. Internal proteolysis does not activate these apical caspases but is a secondary event resulting in partial stabilization of activated dimers. Activation of caspases-8 and -9 occurs by dimerization that is fully recapitulated in vitro by kosmotropes, salts with the ability to stabilize the structure of proteins. Further, single amino acid substitutions at the dimer interface abrogate the activity of caspases-8 and -9 introduced into recipient mammalian cells. A unified caspase activation hypothesis is proposed whereby apical caspases are activated by dimerization of monomeric zymogens (Boatright, 2003).

In the death receptor induced apoptotic pathway, caspase-8 autocatalytically cleaves itself at specific cleavage sites. To better understand the regulatory mechanisms behind caspase-8 activation, active wild-type caspase-8 (wtC8) and an uncleavable form of procaspase-8 (uncleavable C8) were compared. wtC8 predominantly exists as a monomer and dimerizes in a concentration and inhibitor binding-dependent fashion. The KD for dimeric wtC8 is ~50 microM and decreases when inhibitor binds. Uncleavable C8 is mainly monomeric, but a small amount that dimerizes is as active as wtC8. Inhibitor binding does not favor dimerization but induces active site rearrangements in uncleavable C8. These findings suggest that dimerization is the crucial factor for caspase-8 activation (Donepudi, 2003).

Interaction of Caspase-9 with inhibitors of apoptosis

X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP. XIAP associates with the active caspase-9-Apaf-1 holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp315. Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevent caspase-9 association with XIAP and its concomitant inhibition. The N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs. Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. These studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis (Srinivasula, 2001).

MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. After UV radiation, MIHA prevents apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functions after release of cytochrome c and DIABLO from the mitochondria and is able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO binds to MIHA and disrupts MIHA's association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis (Ekert, 2001).

The inhibitor of apoptosis (IAP) proteins potently inhibit the catalytic activity of caspases. While profound insight into the inhibition of the effector caspases has been gained in recent years, the mechanism of how the initiator caspase-9 is regulated by IAPs remains enigmatic. This paper reports the crystal structure of caspase-9 in an inhibitory complex with the third baculoviral IAP repeat (BIR3) of XIAP at 2.4 Å resolution. The structure reveals that the BIR3 domain forms a heterodimer with a caspase-9 monomer. Strikingly, the surface of caspase-9 that interacts with BIR3 also mediates its homodimerization. Monomeric caspase-9 is catalytically inactive due to the absence of a supporting sequence element that could be provided by homodimerization. Thus, XIAP sequesters caspase-9 in a monomeric state, which serves to prevent catalytic activity. These studies, in conjunction with other observations, define a unified mechanism for the activation of all caspases (Shiozaki, 2003).

Caspase-9 mutation

The hypothesis that Caspase 9 (Casp9) is a critical upstream activator of caspases has been tested through gene targeting in mice. The majority of Casp9 knockout mice die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis during brain development. Casp9 deletion prevents activation of Casp3 in embryonic brains in vivo, and Casp9-deficient thymocytes show resistance to a subset of apoptotic stimuli, including absence of Casp3-like cleavage and delayed DNA fragmentation. Moreover, the cytochrome c-mediated cleavage of Casp3 is absent in the cytosolic extracts of Casp9-deficient cells but is restored after addition of in vitro-translated Casp9. Together, these results indicate that Casp9 is a critical upstream activator of the caspase cascade in vivo (Kuida, 1998).

Mutation of Caspase 9 (Casp9) results in embryonic lethality and defective brain development associated with decreased apoptosis. Casp9-/- embryonic stem cells and embryonic fibroblasts are resistant to several apoptotic stimuli, including UV and gamma irradiation. Casp9-/- thymocytes are also resistant to dexamethasone- and gamma irradiation-induced apoptosis, but are surprisingly sensitive to apoptosis induced by UV irradiation or anti-CD95. Resistance to apoptosis is accompanied by retention of the mitochondrial membrane potential in mutant cells. In addition, cytochrome c is translocated to the cytosol of Casp9-/- ES cells upon UV stimulation, suggesting that Casp9 acts downstream of cytochrome c. Caspase processing is inhibited in Casp9-/- ES cells but not in thymocytes or splenocytes. Comparison of the requirement for Casp9 and Casp3 in different apoptotic settings indicates the existence of at least four different apoptotic pathways in mammalian cells (Hakem, 1998).

Programmed cell death is critical for normal nervous system development and is regulated by Bcl-2 and Caspase family members. Targeted disruption of bcl-x(L), an antiapoptotic bcl-2 gene family member, causes massive death of immature neurons in the developing nervous system whereas disruption of caspase-9, a proapoptotic caspase gene family member, leads to decreased neuronal apoptosis and neurodevelopmental abnormalities. To determine whether Bcl-X(L) and Caspase-9 interact in an obligate pathway of neuronal apoptosis, bcl-x/caspase-9 double homozygous mutants were generated. The increased apoptosis of immature neurons observed in Bcl-X(L)-deficient embryos is completely prevented by concomitant Caspase-9 deficiency. In contrast, bcl-x(-/-)/caspase-9(-/-) embryonic mice exhibit an expanded ventricular zone and neuronal malformations identical to those observed in mice lacking only Caspase-9. These results indicate both epistatic and independent actions of Bcl-X(L) and Caspase-9 in neuronal programmed cell death. To examine Bcl-2 and Caspase family-dependent apoptotic pathways in telencephalic neurons, the effects of cytosine arabinoside (AraC), a known neuronal apoptosis inducer, were examined on wild-type, Bcl-X(L)-, Bax-, Caspase-9-, Caspase-3-, and p53-deficient telencephalic neurons in vitro. AraC causes extensive apoptosis of wild-type and Bcl-X(L)-deficient neurons. p53- and Bax-deficient neurons show marked protection from AraC-induced death, whereas Caspase-9- and Caspase-3-deficient neurons show minimal or no protection, respectively. These findings contrast with a previous investigation of AraC-induced apoptosis of telencephalic neural precursor cells in which death was completely blocked by p53 or Caspase-9 deficiency but not Bax deficiency. In total, these results indicate a transition from Caspase-9- to Bax- and Bcl-X(L)-mediated neuronal apoptosis (Zaidi, 2001).

Dysregulation of apoptosis contributes to the pathogenesis of many human diseases. As effectors of the apoptotic machinery, caspases are considered potential therapeutic targets. Using an established in vivo model of Fas-mediated apoptosis, it has been demonstrated that elimination of certain caspases is compensated in vivo by the activation of other caspases. Hepatocyte apoptosis and mouse death induced by the Fas agonistic antibody Jo2 requires proapoptotic Bcl-2 family member Bid and uses a Bid-mediated mitochondrial pathway of caspase activation; deficiency in caspases essential for this pathway, caspase-9 or caspase-3, unexpectedly result in rapid activation of alternate caspases after injection of Jo2, and therefore fail to protect mice against Jo2 toxicity. Moreover, both ultraviolet and gamma irradiation, two established inducers of the mitochondrial caspase-activation pathway, also elicit compensatory activation of caspases in cultured caspase-3(-/-) hepatocytes, indicating that the compensatory caspase activation is mediated through the mitochondria. These findings provide direct experimental evidence for compensatory pathways of caspase activation. This issue should therefore be considered in developing caspase inhibitors for therapeutic applications (Zheng, 2000).

Signaling upstream of Caspase-9: Apaf-1 and cytochrome

Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. In turn, activated caspase-9 cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminishes caspase-3 activation. Mutation of the active site of caspase-9 attenuates the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade triggered by cytochrome c and dATP. Several caspases, including caspase-9, have long prodomains at their NH2 termini. This domain has been proposed to function as a caspase recruitment domain (CARD), allowing proteins with such domains to interact with each other. Although Apaf-1 is not a caspase, its NH2-terminal region contains a CARD, suggesting that Apaf-1 may recruit caspase-9 through their respective CARDs. The data suggest that, within the context of full-length Apaf-1, the CARD is not accessible for caspase-9 binding. Cytochrome c and dATP may induce a conformational change in Apaf-1 that exposes its CARD (Li, 1997).

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. A mammalian homolog of CED-4, Apaf-1, can associate with several death proteases in mammalian cells, including caspase-4, caspase-8, caspase-9, and nematode CED-3. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhances the killing activity of caspase-9, which requires the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promotes the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, has been shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL is mediated through both domains of Apaf-1: the CED-4-like domain and the C-terminal domain, which contains WD-40 repeats. Expression of Bcl-XL inhibits the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibits Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL fails to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans (Hu, 1998).

The reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9 is reported. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1.cytochrome c complex. Such a complex can be isolated using gel filtration chromatography and is by itself sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1 to 1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3 (Zou, 1999).

Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large (on the order of approximately 1.4 MDa). dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M(r) values of approximately 1.4 MDa and approximately 700 kDa. Kinetic analysis demonstrates that in vitro the approximately 700-kDa complex is produced more rapidly than the approximately 1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide or N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the complex predominantely formed is the approximately 700-kDa Apaf-1 containing apoptosome complex. This complex processes effector caspases. Thus, the approximately 700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis (Cain, 2000).

Apoptotic response downstream of Caspase-9

The exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homolog, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and three others (caspases-1, -4, and -5) fail to be activated under the same conditions. In vitro association assays confirm that caspase-9 selectively binds to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 do not. Depletion of caspase-9 from cell extracts abrogates cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enables an ordering of the sequence of caspase activation events downstream of caspase-9 and reveals the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9 (Slee, 1999).

In contrast to the autoprocessing of caspase-9, little is known about the biological significance of caspase-9 processing by caspase-3 via a feedback loop in vivo. Antisera were prepared against mouse caspase-9 cleavage sites so that only the activated form of mouse caspase-9 was recognized. Using these antisera and caspase-9- and caspase-3-deficient mouse embryonic fibroblasts, it has been demonstrated that mouse caspase-9 is initially autoprocessed at D(353) and D(368) at low levels during staurosporine-induced apoptosis, whereupon the D(368) and D(168) sites are preferentially processed over D(353) by activated caspase-3 as part of a feedback amplification loop. Ac-DEVD-MCA (caspase-3-like) and Ac-LEHD-MCA (caspase-9-like) cleavage activities clearly show that caspase-9 autoprocessing is necessary for the activation of caspase-3, whereas full activation of caspase-3 and caspase-9 is achieved only through the feedback amplification loop. This feedback amplification loop also plays a predominant role during programmed cell death of dorsal root ganglia neurons at mouse embryonic day 11.5 (Fujita, 2001).

During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome has been examined. A multi-step process is proposed, whereby catalytically active processed or unprocessed caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3. XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex (Bratton, 2001).

Apoptotic protease-activating factor-1 (Apaf-1), a key regulator of the mitochondrial apoptosis pathway, consists of three functional regions: (1) an N-terminal caspase recruitment domain (CARD) that can bind to procaspase-9; (2) a CED-4-like region enabling self-oligomerization, and (3) a regulatory C terminus with WD-40 repeats masking the CARD and CED-4 region. During apoptosis, cytochrome c and dATP can relieve the inhibitory action of the WD-40 repeats and thus enable the oligomerization of Apaf-1 and the subsequent recruitment and activation of procaspase-9. Different apoptotic stimuli induce the caspase-mediated cleavage of Apaf-1 into an 84-kDa fragment. The same Apaf-1 fragment is obtained in vitro by incubation of cell lysates with either cytochrome c/dATP or caspase-3 but not with caspase-6 or caspase-8. Apaf-1 is cleaved at the N terminus, leading to the removal of its CARD H1 helix. An additional cleavage site is located within the WD-40 repeats and enables the oligomerization of p84 into an approximately 440-kDa Apaf-1 multimer even in the absence of cytochrome c. Due to the partial loss of its CARD, the p84 multimer is devoid of caspase-9 or other caspase activity. Thus, these data indicate that Apaf-1 cleavage causes the release of caspases from the apoptosome in the course of apoptosis (Lauber, 2001).

Caspase-9 function in normal development and tissue damage

Caspase-9, an initiator caspase, and caspase-3, an effector caspase, have been suggested to mediate the terminal stages of neuronal apoptosis, but little is known about their activation in vivo. Temporal and spatial aspects of caspase-9 and -3 activation have been examined in olfactory receptor neurons (ORNs) undergoing apoptosis after target removal in vivo. After removal of the olfactory bulb, enhanced expression of procaspase-9 and -3 is observed in ORNs, followed by activation initially at the level of the lesion, then in axons, and only later in the ORN soma. The amyloid precursor-like protein-2 (APLP2) is a caspase substrate that is cleaved in an identical spatiotemporal pattern, suggesting its cleavage is the result of retrograde propagation of a pro-apoptotic signal in a caudorostral wave from the synapse through the axon to the ORN cell body. A null mutation in caspase-3 causes a change in axonal patterning indicative of an overall developmental expansion of the ORN population, and mature ORNs of caspase-3 knock-outs do not undergo caspase-dependent terminal dUTP nick end labeling-positive apoptosis after olfactory bulb removal. These results demonstrate that ORNs require caspase-3 activation to undergo normal developmental and mature target-deprived apoptosis. In addition, an axonal site of action for caspase-3 and -9 has been demonstrated; regulation and activation of caspase-3 and -9 leading to apoptosis is a highly ordered process that occurs initially at the presynaptic level and only later at the cell body after deafferentation (Cowan, 2001).

Caspase-9 is an upstream caspase that can become active in response to cellular damage, including deprivation of growth factors and exposure to oxidative stress in vitro. Little is known, however, about how activation of caspase-9 is temporally and spatially regulated in vivo, e.g., during development. Vimentin has been demonstrated to be the first example of a caspase-9 substrate that is not a downstream procaspase. Immunohistochemical analysis, using a specific antibody against the vimentin fragments generated by caspase-9, shows that caspase-9 cleaves vimentin in apoptotic cells in the embryonic nervous system and the interdigital regions. This result is consistent with observations that gene knockouts of caspase-9 and its activator, Apaf-1, result in developmental defects in these tissues. These results show that the specific antibody is useful for in situ detection of caspase-9 activation in programmed cell death (Nakanishi, 2001).

Sympathetic neurons have the potential to activate two alternative caspase-dependent pathways either of which is capable of mediating death induced by NGF deprivation; these neurons have the potential to switch from one pathway to the other. The presence of these two alternative pathways to trophic factor deprivation-induced death may have implications for ensuring the correct development of the nervous system. In wild-type neurons, a caspase-2-dependent pathway is required for death, and a caspase-9-dependent pathway appears to be suppressed by endogenous inhibitors of apoptosis proteins (IAPs). In contrast, for caspase-2-null neurons, death is dependent on the caspase-9 pathway. The mechanism underlying the shift is the result of a threefold compensatory elevation of caspase-9 expression and a doubling of levels of direct IAP binding protein with low pI/(DIABLO)/second mitochondria-derived activator of caspase (Smac), an IAP inhibitor. These findings resolve seemingly discrepant findings regarding the roles of various caspases after NGF deprivation and raise a cautionary note regarding the interpretation of findings with caspase-null animals. The choice of the death-mediating caspase pathway in the sympathetic neurons is thus dependent on the regulated relative expression of components of the pathways including those of caspases, IAPs, and IAP inhibitors (Troy, 2001).

Mammalian caspases are a family of cysteine proteases that plays a critical role in apoptosis. Caspase-2 processing has been analyzed in human cell lines containing defined mutations in caspase-3 and caspase-9. Caspase-2 processing, during cell death induced by UV irradiation, depends both on caspase-9 and caspase-3 activity, while, during TNF-alpha-dependent apoptosis, capase-2 processing is independent of caspase-9 but still requires caspase-3. In vitro procaspase-2 is the preferred caspase cleaved by caspase-3, while caspase-7 cleaves procaspase-2 with reduced efficiency. Caspase-2-mediated apoptosis requires caspase-9, and cells co-expressing caspase-2 and a dominant negative form of caspase-9 are impaired in activating a normal apoptotic response and release cytochrome c into the cytoplasm. These findings suggest a role played by caspase-2 as a regulator of the mitochondrial integrity and open questions on the mechanisms responsible for its activation during cell death (Paroni, 2001).

Cell death and Huntington's Disease

Huntington's Disease is an inherited neurodegenerative disease that affects the medium spiny neurons in the striatum. The disease is caused by the expansion of a polyglutamine sequence in the N terminus of Huntingtin (Htt), a widely expressed protein. Htt is an antiapoptotic protein in striatal cells and acts by preventing caspase-3 activity. Htt overexpression in other CNS-derived cells can protect them from more than 20 days exposure to fatal stimuli. In particular, cytochrome c continues to be released from mitochondria into the cytosol of cells that overexpress normal Htt. However, procaspase-9 is not processed, indicating that wild-type Htt (wtHtt) acts downstream of cytochrome c release. These data show that Htt inhibits neuronal cell death by interfering with the activity of the apoptosome complex (Rigamonti, 2001).


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Search PubMed for articles about Drosophila Dronc

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