org Ice

Ice


REGULATION

Signaling upstream of Ice

The proapoptotic genes reaper (rpr), grim, and head involution defective (hid) are required for virtually all embryonic apoptosis. The proteins encoded by these genes share a short region of homology at their amino termini. The Drosophila IAP homolog Thread/Diap1 (Th/Diap1) negatively regulates apoptosis during development. It has been proposed that Rpr, Grim, and Hid induce apoptosis by binding and inactivating TH/Diap1. The region of homology between the three proapoptotic proteins has been proposed to bind to the conserved BIR2 domain of TH/Diap1. An analysis of loss-of-function and gain-of-function alleles of th indicates that additional domains of Th/Diap1 are necessary to allow th to inhibit death induced by Rpr, Grim, and Hid. In addition, analysis of loss-of-function mutations demonstrates that th is necessary to block apoptosis very early in embryonic development. This may reflect a requirement to block maternally provided Rpr and Hid, or it may indicate another function of the Th/Diap1 protein (Lisi, 2000).

Several mechanisms of action have been suggested for the antiapoptotic properties of the IAP family of proteins. Among these are the binding of the Drosophila IAPs to the proapoptotic proteins Rpr, Grim, and Hid. This interaction has been demonstrated in overexpression systems, and has been proposed to involve the homologous amino-terminal 14 amino acid sequences of the apoptosis initiators with the second BIR domain of the IAPs. The data presented here suggest that this is an oversimplification. Another mechanism that has been proposed for IAP antiapoptotic activity is the direct binding and inhibition of caspases. Th/Diap1 binds to the Drosophila caspases Ice and DCP-1 and functions to inhibit their ability to induce apoptosis. Here again, this binding activity appears to rest within BIR2 (Lisi, 2000).

These physical interactions support a simple model of IAP action. In this model, IAPs act within viable cells to inhibit caspase function. The action of Rpr, Hid, and Grim interferes with the ability of IAPs to inhibit caspases, thus inducing apoptosis. On the basis of the model, the LOF mutations identified in this study would be predicted to interfere with the ability of the Th/Diap1 protein to inhibit caspase function. This is likely to be true for th109.07, which lacks most of the protein, as well as for th5 and th4, which affect conserved residues in BIR2. BIR2 is sufficient to inhibit apoptosis induced by the active form of the Drosophila caspase Ice. The th9 mutation in BIR1 suggests that this BIR is also important for the full function in caspase inhibition. Alternatively, this change in BIR1 might have long-range effects on BIR2 structure or on protein stability (Lisi, 2000).

salvador (sav) restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis. Expression of hid or reaper (rpr) in the eye imaginal disc results in activation of the effector caspase Drice. An antibody that recognizes the cleaved (activated) form of Drice was used to stain eye discs expressing GMR-hid or GMR-rpr. In wild-type cells, Drice is activated by GMR-hid or GMR-rpr. However, in clones of sav tissue, Drice activation by either GMR-hid or GMR-rpr is almost completely blocked. These experiments indicate that sav blocks activation of Drice by both rpr and hid (Tapon, 2002).

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).

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron degenerative disorder, caused by the loss of telomeric copy of the survival motor neuron gene (SMN1). To better understand how motor neurons are targeted in SMA patients, it is important to study the role of SMN protein in cell death. In this report, RNA interference (RNAi) has been used to study the loss-of-function of SMN in Drosophila S2 cells. A 601 base-pair double-stranded RNA (dsRNA) of Drosophila Smn (dSMN) was used for silencing the dSMN. The data indicate that dSMN RNAi results in more than 90% reduction of both RNA and protein. Further analysis of S2 cells by cell death ELISA and flow cytometry assays revealed that reduction of dSMN expression significantly increases apoptosis. The cell death mediated by SMN depletion is caspase-dependent and specifically due to the activation of the endogenous caspases, DRONC and Ice. Significantly, the effect of dSMN RNAi is reversed by a peptide caspase inhibitor, zVAD-fmk. These results suggest that dSMN is involved in signal pathways of apoptotic cell death in Drosophila. Hence, the model system of reduced SMN expression by RNAi in Drosophila could be exploited for identification of therapeutic targets (Ilangovan, 2003).

Ice is a potential target of microRNA Mir-14

MicroRNAs (miRNAs) are small regulatory RNAs that are between 21 and 25 nucleotides in length and repress gene function through interactions with target mRNAs. The genomes of metazoans encode on the order of several hundred miRNAs, but the processes they regulate have been defined for only a few cases. New inhibitors of apoptotic cell death were sought by testing existing collections of P element insertion lines for their ability to enhance a small-eye phenotype associated with eye-specific expression of the Drosophila cell death activator Reaper. The Drosophila miRNA mir-14 has been identified as a cell death suppressor. Loss of mir-14 enhances Reaper-dependent cell death, whereas ectopic expression suppresses cell death induced by multiple stimuli. Animals lacking mir-14 are viable. However, they are stress sensitive and have a reduced lifespan. mir-14 mutants have elevated levels of the apoptotic effector caspase Ice, suggesting one potential site of action. Mir-14 also regulates fat metabolism. Deletion of mir-14 results in animals with increased levels of triacylglycerol and diacylglycerol, whereas increases in mir-14 copy number have the converse effect (Xu, 2003).

The two C. elegans miRNAs with known functions, lin-4 and let-7, are thought to regulate development by binding to the 3'untranslated region of target transcripts and thereby repressing the translation of their products. In these examples, the analysis of genetic interactions provides important clues as to the identity of targets. In the absence of this sort of information, it is difficult to predict miRNA targets in animals. This is because base pairing between the mature miRNA and its target is imperfect and the rules that govern which base pair interactions are important are unknown. Potential Mir-14 binding sites were sought in a number of apoptotic regulators, including Dronc, Rpr, Hid, and Grim. Potential target sites were identified in the transcripts of several genes, including Ice, Dcp-1, Scythe, SkpA, and Grim (however, the Grim target is present in the 3'UTR, which was absent in the GMR-Grim transgene). Of these, Ice, an apoptotic effector caspase, is of particular interest. Ice is required for at least some cell deaths and is activated by Dronc, which promotes cell death induced by Rpr, Hid, and Grim. Ice levels in adults were measured by using an anti-Ice antibody. Ice is elevated in mir-14Δ1 flies as compared to the wild-type, and this increase is suppressed in the presence of two copies of the mir-14-containing 3.4 kb genomic DNA fragment. Whereas these observations alone do not prove that Ice is a direct target of Mir-14, they do suggest that Ice is regulated, either directly or indirectly, by Mir-14 levels (Xu, 2003).

Caspase activity is regulated by scylla and charybde, homologues of the human apoptotic gene RTP801

Robotic methods and the whole-genome sequence of Drosophila melanogaster were used to facilitate a large-scale expression screen for spatially restricted transcripts in Drosophila embryos. In this screen, scylla (scyl) and charybde (chrb), which code for dorsal transcripts in early Drosophila embryos and are homologous to the human apoptotic gene RTP801, were identified. In Drosophila, both gene products are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation. Gene disruption studies revealed the functional redundancy of scyl and chrb, as well as their requirement for embryonic head involution. From the perspective of functional genomics, these studies demonstrate that global surveys of gene expression can complement traditional genetic screening methods for the identification of genes essential for development: beginning from their spatio-temporal expression profiles and extending to their downstream placement relative to dpp and zen, these studies reveal roles for the scyl and chrb gene products as links between patterning and cell death (Scuderi, 2006).

Based upon the observations that: (1) simultaneous loss of scyl and chrb function leads to a hid-analogous, cell death defective phenotype and (2) scyl and chrb are homologous to the mammalian apoptotic gene RTP801, it was postulated that the scyl and chrb gene products have pro-apoptotic functions in the embryonic Drosophila head. Two lines of experimentation were employed to test this hypothesis. (1) hid expression was examined in scyl chrb double mutant embryos in situ. The scyl and chrb gene products do not function as transcriptional modulators of hid since hid transcription is unaffected in scyl chrb double mutant embryos. (2) A Caspase-3 activity assay was employed to monitor apoptosis in wild-type and scyl chrb double mutant embryos. Activated Caspase-3 has been used previously to specifically label apoptotic cells in Drosophila. Anti-Caspase-3 staining mirrors cell death patterns previously defined by acridine orange and TUNNEL assays in the Drosophila embryo and pupal retina. In this study, dying cells expressing activated Caspase-3 were evident in the head and the nervous system of 95% of embryos derived from matings of Df(3L)vin4/twi:GFP heterozygotes 0-8 h AEL (n = 278). When GFP screening was used to enrich for similarly staged mutant embryos, it was noted that Caspase-3 activity was greatly diminished in mid-stage scyl chrb double mutants. By 8 AEL, 75% of the mutant-enriched population was caspase-negative, in contrast to the unselected population in which only 8% of the embryos were found to be caspase-negative. No gross differences in Caspase-3 activity were found prior to the onset of germ band retraction and head involution. Since cleaved Caspase-3 is a key executioner (and hence marker) of apoptosis, these data support the hypothesis that Scylla and Charybde have pro-apoptotic roles in Drosophila head involution. More generally, Scylla and Charybde likely function as essential death activators in Drosophila since Caspase-3 activation in scyl chrb double mutants is disrupted in the nervous system as well as in the head. The scylla and charybde gene products are not, however, sufficient for cell death since (1) immunostains reveal wild-type patterns of Caspase-3 activation in embryos derived from dl mutant mothers and in which expression of scylla and charybde is greatly expanded and (2) neither scyl nor chrb (alone or in combination) can mimic hid-induced apoptosis in cultured Cos or Hela cells (Scuderi, 2006).

Several lines of evidence indicate that Scylla and Charybde function in the Hid-mediated cell death pathway. (1) A previous phenotypic analysis of scyl chrb mutants revealed their essential roles in regulating cell death in the developing Drosophila eye. Loss-of-function studies have similarly revealed a requirement for Hid in modulating cell death events in early and late stages of Drosophila eye development. (2) In this study, which relied upon deficiencies and RNAi methodologies to generate scyl chrb null double mutants, an earlier developmental requirement for the scyl and chrb gene products was documented. scyl chrb double mutants suffer an embryonic lethality that is associated with defects in the morphogenetic process of head involution. Drosophila homozygous for loss-of-function hid alleles similarly suffer an embryonic lethality and exhibit signature defects in head involution. (3) Molecular characterization of the embryonic lethality in scyl chrb double mutants revealed that Caspase-3 activation is disrupted not only in the morphogenetically aberrant head, but in the CNS as well. In Drosophila, Hid induces apoptosis in midline glia cells failing to activate the EGFR signaling cascade. Together, the significant homologies of scyl and chrb to the mammalian RTP801 gene product that functions as an apoptotic factor in mammalian cell culture systems, as well as the scyl chrb embryonic and eye phenotype studies establish redundant roles for scyl and chrb in Hid-mediated cell death in both embryonic and post-embryonic stages of the Drosophila life cycle (Scuderi, 2006).

Each of the three cell death proteins, hid, rpr and grim, has been implicated in apoptotic events defining segmental boundaries and/or neuronal fates in the CNS, albeit in different paradigms. In the CNS, specificity in neuronal apoptosis is achieved via differential expression of the BX-C Hox gene abd-A, which prevents neuronal apoptosis in posterior segments. Viewed from this perspective, the finding that the Zen and BX-C Drosophila Hox gene products regulate transcription of the scyl and chrb pro-apoptotic genes (and thereby potentially sculpt head and segment boundaries during development) is reminiscent of the Deformed Drosophila Hox protein functioning as a transcriptional activator of the rpr cell death gene. Together, these studies strengthen the idea that Hox-gene-dependent induction of cell death is a general phenomenon in Drosophila (Scuderi, 2006).

Intriguingly, the pro- and anti-apoptotic roles of the Zen and BX-C Homeobox transcription factors in Drosophila embryogenesis correspond to their activation and repression effects on scyl and chrb gene expression. In this regard, scyl, chrb and cell death are activated by Zen in dorsal domains of the developing embryo, whereas ventrally scyl, chrb and cell death are repressed by one or more of BX-C gene products. Hence, in addition to the pro-apoptotic role of Zen, there is evidence for an anti-apoptotic role for the BX-C gene product(s) and in flies as in mouse related transcription factors function in context-specific fashion (Scuderi, 2006).

As a final point, both TGF-β and BMP mammalian members of the TGF-β cytokine superfamily have been documented to induce cell death in numerous developmental contexts. Along these same lines, previous reports in Drosophila have suggested a link between Dpp and cell death but have stopped short of designating this link as direct. Based on molecular and genetic evidence, it is suggested that the Drosophila pro-apoptotic scyl and chrb gene products serve as direct links between Dpp/Zen-mediated patterning and differentiation, in this case, cell death. Thus, in Drosophila as in vertebrates, cytokines of the TGF-β superfamily control both cell death and cell proliferation within the contexts of their cellular environments (Scuderi, 2006).

Given the importance of cell death regulation in development and disease, it is likely that there are several mechanisms by which cell death can be regulated, and, in like fashion, several nodes where independent regulatory pathways may in specific contexts converge. With respect to members of the RTP801 family of apoptotic factors, evidence points to at least two triggers of regulation: cell death can be a pathologic response to stresses such as hypoxia (as is the case for mammalian RTP801) or cell death can be a developmental response to a spatially and temporally restricted cell signaling pathway, such as the Dpp/TGF-β cytokine-mediated signaling pathway (as is the case for Drosophila Scylla and Charybde). Within the context of pathway convergence nodes, it is particularly notable that several reports document cross-talk between the HIF-1 and TGF-β pathways in regulating gene expression and cell death, and thus it is possible that the RTP801/Scylla/Charybde death effectors represent a point of convergence between these two death activating pathways. Consistent with this model is the demonstration that scyl and chrb are hypoxia-inducible in Drosophila (Reiling, 2004). Viewed from this perspective, the genetically defined roles of Scylla and Charybde as pro-apoptotic effectors establish a clear basis for future genetic and biochemical characterization of the mechanism by which activation of cell death programs might occur via Dpp/TGF-β-mediated signaling (Scuderi, 2006).

Distinct mechanisms of apoptosis-induced compensatory proliferation in proliferating and differentiating tissues in the Drosophila eye

In multicellular organisms, apoptotic cells induce compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. This study shows that a second mechanism of apoptosis-induced compensatory proliferation exists. This mechanism is dependent on effector caspases which trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Furthermore, whereas Dpp and Wg signaling is preferentially employed in apoptotic proliferating tissues, Hh signaling is activated in differentiating eye tissues. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell-cycle reentry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent nonapoptotic function (Fan, 2008).

In developing wing discs in which apoptosis was induced by expression of the pro-apoptotic gene hid, loss of the caspase inhibitor DIAP1, or by X-ray treatment, the accumulation of two major mitogens, Dpp and Wg, has been observed in dying cells. Key for this finding is the simultaneous expression of the caspase inhibitor P35. Under these conditions, the dying cells were kept alive ('undead'), allowing accumulation of Dpp and Wg. This accumulation appears to be dependent on the initiator caspase Dronc, because it cannot be blocked by expression of P35 which inhibits effector caspases but not Dronc. In addition, the Drosophila homolog of the tumor suppressor p53, Dp53, has been implicated downstream of Dronc for compensatory proliferation. Notably, these studies on mechanisms of compensatory proliferation were carried out in developing larval wing imaginal discs in Drosophila. Cells in wing discs proliferate extensively during larval stages, and the majority of these cells does not differentiate before they reach pupal development. Hence, the mechanisms of compensatory proliferation have so far only been investigated in situations where most cells are proliferating. Interestingly, apoptosis-induced compensatory proliferation in differentiating eye tissue of third-instar larvae. However, it is unclear whether this form of compensatory proliferation is controlled by a similar mechanism as reported for larval proliferating wing discs (Fan, 2008).

This study revealed that there are at least two distinct mechanisms that promote compensatory proliferation in response to apoptotic activity. The general difference between these two mechanisms lies in the developmental context of the tissue in which compensatory proliferation occurs. In proliferating wing and eye tissues, compensatory proliferation induced by extensive apoptosis is dependent on Dronc and Dp53, which induce Dpp and Wg expression. In contrast, in differentiating eye tissue, apoptosis induces compensatory proliferation through a novel mechanism requiring the effector caspases DrICE and Dcp-1, which induce Hh signaling in a nonapoptotic function (Fan, 2008).

When cells stop proliferating and become committed to adopt cell fate, dramatic changes in gene expression are occurring. Given these changes in developmental plasticity, it is not surprising that distinct mechanisms of apoptosis-induced compensatory proliferation are employed in proliferating versus differentiating tissues. However, it should be noted that the proliferating capacity of differentiating tissues is rather restricted. In GMR-hid eye discs, although hid is expressed in all cells posterior to the MF, compensatory proliferation occurs only in cells that are still undifferentiated. Yet, even though they are undifferentiated they have withdrawn from the cell cycle and, under normal developmental conditions (i.e., without GMR-hid), they would soon be recruited to adopt cell fate. However, the apoptotic environment causing increased Hh signaling appears to be able to trigger reentry of these cells into the cell cycle (Fradkin, 2008).

Interestingly, the Hh signal is specifically increased in photoreceptor neurons requiring a nonapoptotic activity of effector caspases. Hh signaling can then nonautonomously induce proliferation of undifferentiated cells at the basal side of the eye disc. However, overexpression of Hh posterior to the MF in wild-type eye discs alone is not sufficient to induce a comparable wave of compensatory proliferation as in GMR-hid eye discs. This suggests that cell-cycle reentry requires activation of additional factors/pathways stimulated in apoptotic cells (Fradkin, 2008).

Although hid can stimulate increased Hh expression in photoreceptor neurons throughout the posterior half of the eye disc, compensatory proliferation is restricted to a certain distance (six to ten ommatidial columns) from the MF. This corresponds to approximately 6-15 hr of developmental time, and might be the time required for cell-cycle reentry. Similarly, when mammalian cells that have exited the cell cycle are stimulated to reenter the cell cycle, they need about 8 hr to do this. The reason for this delay is unknown. Studying compensatory proliferation in GMR-hid eye discs might provide a genetic model to address this interesting problem (Fradkin, 2008).

It is not clear whether this novel effector caspase-, Hh-dependent pathway of compensatory proliferation also applies to other, or even all, differentiating tissues. However, what this study shows is that there are at least two distinct mechanisms of apoptosis-induced compensatory proliferation. It is also possible that other mechanisms of compensatory proliferation in different developmental contexts are going to be uncovered in the future. Interestingly, in developing larval wing discs, P35-dependent compensatory proliferation has been implicated in cell competition. This suggests that, even in tissue with the same developmental potential, compensatory proliferation can occur with distinct mechanisms (Fradkin, 2008).

How cells sense different developmental contexts and operate distinct proliferating mechanisms in response to apoptotic stress is unknown. Specifically, where is the specificity and selectivity for distinct caspases coming from in tissues of different developmental potential? What are the mechanisms engaged by these caspases to trigger secretion of either Dpp and Wg or Hh? These are questions which need to be addressed in the future (Fan, 2008).

This study has several implications for tumorigenesis. First, many tumors develop when quiescent cells reenter the cell cycle. The mechanisms for cell-cycle reentry are largely unknown. Second, evasion from apoptosis is a hallmark of cancer. Many tumor cells are induced to undergo apoptosis. However, they do not die, because they downregulate essential components of the apoptotic pathway such as Apaf-1 and caspases. Thus, these undead tumor cells might secrete mitogens which might induce compensatory proliferation similar to the Drosophila case. In this way, undead cells might contribute to the growth of the tumor. A similar argument can be made for chemotherapy, which in many cases attempts to activate the apoptotic program in a tumor cell. If the death of the tumor cell is blocked, or slow, mitogens might be produced and the tumor growth could be even more severe. This is very obvious in the apoptotic wing or anterior eye discs in Drosophila when apoptosis is blocked by P35. Under these conditions, overgrown wing and eye tissues are observed. Thus, evasion of apoptosis might directly contribute to tumor growth. Finally, although increased Hh signaling can lead to various cancers, how Hh induces cellular proliferation and tissue overgrowth is not well understood. Mutations in Patched1, a negative regulator of sonic Hh, frequently give rise to human tumors. The exact cause is unknown. These data imply that Hh signaling might be involved in cell-cycle reentry allowing cells to resume proliferation (Fan, 2008).

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).

Heat shock protein-70 (Hsp-70) suppresses paraquat-induced neurodegeneration by inhibiting JNK and Caspase-3 activation in Drosophila model of Parkinson's disease

Parkinson's disease (PD) is one of the most common neurodegenerative disorders with limited clinical interventions. A number of epidemiological as well as case-control studies have revealed an association between pesticide exposure, especially of paraquat (PQ) and occurrence of PD. Hsp70, a molecular chaperone by function, has been shown as one of the modulators of neurological disorders. However, paucity of information regarding the protective role of Hsp70 on PQ-induced PD like symptoms led to a hypothesis that modulation of hsp70 expression in the dopaminergic neurons would improve the health of these cells. Advantage was taken of Drosophila, which is a well-established model for neurological research and also possesses genetic tools for easy manipulation of gene expression with limited ethical concern. Over-expression of hsp70 was found to reduce PQ-induced oxidative stress along with JNK and caspase-3 mediated dopaminergic neuronal cell death in the exposed organism. Further, anti-apoptotic effect of hsp70 was shown to confer better homeostasis in the dopaminergic neurons of the PQ-exposed organism, as evidenced by their improved locomotor performance and survival. The study has merit in the context of human concern since protection of dopaminergic neurons in PQ-exposed organism was observed by over-expressing a human homologue of hsp70, HSPA1L, in these cells. The effect was parallel to that observed with Drosophila hsp70. These findings reflect the potential therapeutic applicability of hsp70 against PQ-induced PD like symptoms in an organism (Shukla, 2014).

Drosophila CK2 promotes Wts to suppress Yki activity for growth control

Drosophila Hippo signaling regulates Wts activity to phosphorylate and inhibit Yki in order to control tissue growth. CK2 is widely expressed and involved in a variety of signaling pathways. This study reports that Drosophila CK2 promotes Wts activity to phosphorylate and inhibit Yki activity, which is independent of Hpo induced Wts promotion. In vivo, CK2 overexpression suppresses hpo mutant induced Ex upregulation and overgrowth phenotype while it cannot affect wts mutant. Consistent with this, knockdown of CK2 upregulates Hpo pathway target expression. It was also found that Drosophila CK2 is essential for tissue growth as a cell death inhibitor as knockdown of CK2 in developing discs induces severe growth defect as well as caspase3 (Drice) signal. Taken together, these results uncover a dual role of CK2: while its major role is promoting cell survive, it may potentially be a growth inhibitor as well (Hu, 2014).

Characterization of Ice enzymic properties

To address whether Ice might be involved in the apoptotic pathway in Drosophila, the effects of its overexpression were analyzed in the S2 Drosophila cell line. A full-length Ice ORF was cloned under the control of a metallothionein promoter and stably transfected into S2 cells. While overexpression of Ice has no direct effect on the cells, it significantly sensitizes S2 cells to apoptosis. A population of S2 cells overexpressing Ice died at a significantly increased rate when induced to die by either cycloheximide or etoposide treatment. The observation that overexpression of Ice does not induce apoptosis in the absence of any apoptotic stimulus is consistent with a model in which caspases are principally post-translationally regulated and this led to an investigation of the role of processing in Ice regulation and activation (Fraser, 1997a).

One of the critical processing events during caspase activation by proteolysis is the removal of an N-terminal prodomain. Given the tight regulation of the activity of full-length Ice in S2 cells even after overexpression, the effects were characterized of expression of an N-terminally truncated form of Ice. An ORF corresponding to amino acids 81-339 of Ice was constructed, cloned under control of a metallothionein promoter and stably transfected into the S2 cells. Induction of expression of this ORF (Ice-N) rapidly results in apoptosis of S2 cells including characteristic blebbing of the cells, chromatin condensation and DNA degradation. Overexpression of a catalytically inactive mutant Ice-N (C211A) has no effect on S2 cells. The cell death induced by overexpression of Ice-N is completely blocked by the caspase inhibitors zVAD.fmk and BocAsp.fmk, consistent with the activity of Ice as a caspase (Fraser, 1997a).

Apoptosis in S2 cells induced by overexpression of rpr, cycloheximide treatment or etoposide treatment is blocked by caspase inhibitors. S2 cells express Ice and it was asked whether Ice is proteolytically processed during apoptosis in these cells as would be expected for a critical caspase in the apoptotic pathway. A rpr ORF was placed under the control of a metallothionein promoter and stably transfected into S2 cells. Following rpr induction, S2 cells undergo apoptosis and endogenous Ice is processed giving rise to both p21 and p12 subunits. Ice processing is first detectable at time points where very little cell death can be seen in the cell pool. Analogous results were obtained for etoposide- and cycloheximide-induced apoptosis (Fraser, 1997a).

Many caspases are known to autoprocess when overexpressed in Escherichia coli and use was made of this to purify mature processed Ice. Ice-N was N-terminally His-tagged and this fusion protein was produced under the control of the trc promoter in E.coli. The resulting protein was purified and found to contain three species when analysed by SDS-PAGE: a p30 form (His-Ice-N), a p21 (His-large subunit) and a small subunit of p12. The protein was >90% pure by SDS-PAGE. Immunoblotting confirmed that the His tag was as expected on the p30 and p21 bands (Fraser, 1997a).

The ability of this purified protease to cleave p35 and lamin DmO was assayed. The baculovirus p35 protein inhibits all cell death in vivo in Drosophila and has been shown to act as a caspase inhibitor by acting as a substrate. If Ice is part of the basal machinery in Drosophila, it should therefore cleave p35. The Drosophila lamin B homolog, lamin DmO, is cleaved during S2 cell apoptosis and therefore represents a potential effector substrate for Ice. 35S-labelled p35 or DmO proteins were incubated with the purified Ice enzyme. p35 is an excellent substrate for Ice, being cleaved to completion within 1 h at 37°C. While the cleavage of DmO is less efficient, the fragments obtained comigrate exactly with the DmO fragments in apoptotic S2 cells. Ice activity in both reactions is completely inhibited by the addition of 5 mM iodoacetamide but not by a variety of other protease inhibitors (TLCK, TPCK, aprotinin and PMSF), confirming that Ice is a cysteine protease (Fraser, 1997a).

The role was examined of Ice in in vitro apoptosis of the Drosophila cell line S2. Sytoplasmic lysates made from S2 cells undergoing apoptosis induced by either reaper (rpr) expression or cycloheximide treatment contain a caspase activity with DEVD specificity which can cleave p35, lamin DmO, Ice and DCP-1 in vitro, and which can trigger chromatin condensation in isolated nuclei. Using antibodies specific to Ice, it has been shown that immunodepletion of Ice from these lysates is sufficient to remove most measurable in vitro apoptotic activity, and that re-addition of exogenous Ice to such immunodepleted lysates restores apoptotic activity. It is concluded that, at least in S2 cells, Ice can be the sole caspase effector of apoptosis (Fraser, 1997b).

Protein Interactions

Many members of the inhibitor of apoptosis (IAP) family inhibit cell death. Existing data suggest at least two mechanisms of action: (1) Drosophila IAPs (D-IAP1 and D-IAP2) and a baculovirus-derived IAP, Op-IAP, physically interact with and inhibit the anti-apoptotic activity of Reaper, HID, and Grim (three genetically defined inducers of apoptosis in Drosophila), and (2) human IAPs (c-IAP1, c-IAP2, and X-IAP) interact with a number of different proteins including specific members of the caspase family of cysteine proteases that are crucial in the execution of cell death. An examination was carried out to see if insect-active IAPs could inhibit apoptosis in insect SF-21 cells induced by selected caspases, Drosophila Ice, Sf-caspase-1, and mammalian caspase-3. D-IAP1 inhibits apoptosis induced by the active forms of all three caspases tested and physically interacts with the active, but not the proform of Ice. MIHA, the mouse homolog of X-IAP and an effective inhibitor of caspase-3, also interacts with and blocks apoptosis induced by active Ice but is relatively ineffective in blocking Sf-caspase-1. Op-IAP and D-IAP2 are unable to effectively inhibit any of the active caspases tested and fail to interact with Ice. The Drosophila IAPs and Op-IAP, but not MIHA, block HID-initiated activation of pro-Ice. It is concluded that D-IAP1 is capable of inhibiting the activation of Ice as well as inhibiting apoptosis induced by the active form of Ice. In contrast, D-IAP2 and Op-IAP are more limited in their inhibitory targets and may be limited to inhibiting the activation of caspases (Kaiser, 1998).

The physical interaction between DRONC and Ice 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-Ice CdeltaA (1-339), DeltaN Ice 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-Ice and DeltaN Ice, but not Bcl-10, indicating that DRONC and Ice form a stable complex in cell extracts (Meier, 2000).

Since DRONC interacts with Ice, the ability of active DRONC to cleave Ice CdeltaA, lamin DmO, the DNA fragmentation factor DREP-1 and the baculovirus caspase inhibitor p35 was assayed. Both DRONC and Ice cleave Ice CdeltaA, lamin DmO and DREP-1. The cleavage products generated by DRONC and Ice are clearly different, indicating that DRONC and Ice each cleave lamin DmO and DREP-1 at different sites. Unlike Ice, 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 Ice that share the QAC(R/Q/G)(G/E) active site pentapeptide consensus (Meier, 2000).

Activation of Ice is Ark dependent

Apaf-1-related-killer (Ark) encodes a Drosophila homolog of mammalian Apaf-1 and Caenorhabditis elegans CED-4, cell-death proteins. Like Apaf-1, but in contrast to CED-4, Ark contains a carboxy-terminal WD-repeat domain necessary for interactions with the mitochondrial protein cytochrome c. Ark selectively associates with caspases. To examine the role of Ark isoforms in the induction of cell death, the effect of Ark overexpression in the Drosophila S2 cell line was analyzed. Transient expression of Dapaf-1S (the short isoform of Ark), like C. elegans CED-4, markedly reduces cell viability in S2 cells. However, neither Dapaf-1L (the long isoform of Ark) nor human Apaf-1 could induce cell death. dapaf-1S was transfected with the antiapoptotic genes C. elegans ced-9, baculovirus caspase inhibitor p35, bcl-xL, or diap2. The overexpression of CED-9 and p35 most effectively prevents Dapaf-1S-induced cell death, and Bcl-xL inhibits apoptosis moderately, suggesting that Dapaf-1S activates endogenous caspase(s) in Drosophila. Transfection of diap2 can block reaper-induced cell death in S2 cells, but not Dapaf-1S-induced cell death, indicating that the Dapaf-1S-induced cell death pathway is downstream or independent of DIAP2. Next, either dapaf-1L or dapaf-1S was transfected together with Ice, which encodes a typical DEVDase. The Ice-induced cell death is greatly enhanced by the overexpression of Dapaf-1L. A synergistic effect of Dapaf-1S and Ice coexpression was not observed. In summary, these results show that the each isoform of Ark has distinct cell death-inducing activity in Drosophila cells (Kanuka, 1999).

Caspase activities were measured in S2 cells expressing Dapaf-1L or Dapaf-1S using two different substrates, which distinguish caspase-1-like proteases (Ac-YVAD-MCA) from caspase-3-like proteases (Ac-DEVD-MCA). High DEVD- but not YVAD-cleaving activities were observed in the cytoplasmic lysates from dapaf-1S-transfected S2 cells, which is consistent with the cell-killing activity of Dapaf-1S. Based on the observation that Dapaf-1L and Dapaf-1S activate distinct types of caspases, the caspase activities present in these lysates were measured using various caspase-specific substrates. This experiment also revealed that Dapaf-1L and Dapaf-1S activate different members of the caspase family (Kanuka, 1999).

To determine the function of these novel YVADase-type caspases activated by Dapaf-1L, it was first hypothesized that these YVADase activities are required for Dapaf-1L-induced Ice activation. When dapaf-1L and Ice were cotransfected into S2 cells, Dapaf-1L dramatically enhanced the DEVDase activities, including that of Ice. In this case, the addition of a YVADase inhibitor (Ac-YVAD-CMK) into the culture medium effectively blocked the Dapaf-1L-induced DEVDase activation. Since Ac-YVAD-CMK could not inhibit Dapaf-1S function against Ice activation, it was concluded that the novel YVADase activities were required for the sequential activation of Ice and other DEVDases in S2 cells. The Drosophila YVADase activities already had been reported in Drosophila S2 cell lysates overexpressing the cytoplasmic region of Fas protein (Kondo, 1997), and several ESTs (expressed sequence tags) were found that encode Drosophila YVADase-like proteins; thus, candidates for Dapaf-1L-activated YVADases may exist and participate in cell death cascades in Drosophila (Kanuka, 1999).

Do Dapaf-1L and Dapaf-1S interact with Ice, a Drosophila DEVDase-type caspase, by forming a physical complex, in a manner similar to that reported for CED-4 and proCED-3, or Apaf-1 and procaspase-9? Ice is essential for the apoptosis in Drosophila S2 cells induced by etoposide, cycloheximide, and the overexpression of rpr or C. elegans ced-4. CED-4 has been shown to activate Ice and bind to it directly (Kanuka, 1999a). Immunoprecipitation analysis reveals that overexpressed Dapaf-1S, but not Dapaf-1L, specifically binds to Ice. Dapaf-1L also interacts with Dronc, a long CARD-containing Drosophila caspase in 293 T cells. Interestingly, in this case, the processed form of DRONC was detected in Dapaf-1L-expressing lysates. These data suggest that the distinct caspase-binding affinities of Dapaf-1L and Dapaf-1S are responsible for the activation of different members of the caspase family. The presence of Apaf-1 isoform (Apaf-1S), which lacks WDRs produced by alternative splicing, may suggest the distinct activation mechanisms of caspases by Apaf-1, and Apaf-1S may be also present in mammals (Kanuka, 1999).

A loss-of-function mutant in the Ark gene was obtained. Ark is located to the cytological position 53F on the right arm of chromosome II. One preexisting lethal P element insertion, l(2)k11502 (P1041) was found in the first noncoding exon of Ark. After removal of the background lethal mutation, this P element mutant may behave as a putative null allele of Ark. It is referred it as the dapaf-1K1 (dpfK1) allele. The expression of Ark mRNA could not be detected in dpfK1 homozygous embryos and larvae by in situ hybridization and RT-PCR. The homozygotes for the dpfK1 mutation were approximately 25% semilethal in pupal stage, and the outer morphology of larvae and adult flies appeared to be normal, except for adult dorsal bristles (Kanuka, 1999).

A significant decrease in apoptotic cells stained by TUNEL was observed in developing embryos lacking maternal and zygotic Ark function. During early- and mid-germ band shortening (stages 11-12), cell death is normally seen in the dorsal region of the head and beneath the developing epithelium of the gnathal segments. Remarkably, the TUNEL-positive signals under the epithelium had disappeared in dpfK1/dpfK1 embryos. If Ark is required for caspase activation that leads to completion of the cell death pathway in the embryo, the embryonic caspase activity should be decreased in the dpfK1 mutant. To test this possibility, caspase activities were measured in the lysates of mixed embryos at 6-18 hr after egg laying (AEL) using various caspase-specific substrates. Large amounts of DEVDase and DQTDase activities were observed in developing embryos that contained many dead cells. In the dpfK1/dpfK1 embryos, these caspase activities were markedly decreased to half compared with their original levels. Whereas the increases of DEVDase activity in wild-type and dpfK1/dpfK1 embryos were observed in the initial stage of embryogenesis, at 3 hr AEL, DEVDase activity was continuously increased in wild type, but not in dpfK1/dpfK1 embryos. These results indicate that Ark is required for caspase activation in embryonic cell death (Kanuka, 1999).

Next it was determined whether cyt c activates caspases in the Drosophila embryo in a Dapaf-1-dependent manner. Although the addition of cyt c and dATP into cytosols of S2 cells could not evoke any caspase activities, a prominent caspase activation (DEVDase) was observed in lysates from wild-type embryos in a cyt c and dATP-dependent manner. The cyt c/dATP-induced caspase activation is not entirely observed in the lysates prepared from dpfK1/dpfK1 homozygous embryos and is effectively blocked by an ATPase inhibitor (FSBA; 5'-p-fluorosulfonylbenzoyl adenosine), which is known to inhibit the function of Apaf-1/CED-4-like molecules. These data strongly suggest that Dapaf-1L/cyt c complex actually contributes to the caspase activation in the embryo (Kanuka, 1999).

Genetic studies of cell death in Drosophila have led to the identification of three apoptotic activators: rpr, head involution defective (hid), and grim. The deletion of all three genes blocks apoptosis in the Drosophila embryo, and overexpression of any one of them is sufficient to kill cells that would normally live. The products of these genes appear to activate one or more caspases, because cell killing by rpr, hid, and grim is blocked by the caspase inhibitor p35. If Ark actually acts as a caspase activator, like Apaf-1/CED-4 in adult flies, the downstream pathways of one or more of these three gene products should depend on Ark function to activate caspases. dpfK1/dpfK1 flies were crossed to the GMR-rpr and GMR-hid strains to examine whether or not there are any genetic interactions. Compared with GMR-rpr adult flies in a wild-type background, GMR-rpr flies homozygous for dpfK1 show significantly improved eye morphology, but no obvious influence on hid-activated cell killing was observed in this case. Ice and Dredd appear to be activated downstream of Rpr in Drosophila S2 cells, and Ice is an essential caspase in rpr-induced cell death, consistent with observations that Dapaf-1L and Dapaf-1S activate Ice in S2 cells. These results suggest that Ark is involved in the rpr-induced cell death pathway, and the contribution of Ark against hid-induced cell death may not be as high as that of rpr (Kanuka, 1999).

The genetic evidence that Ark interacts with rpr and the observation that Dapaf-1L contains WDRs strongly imply that cyt c might act as an initiator for Dapaf-1-mediated caspase activation. Overexpression of rpr and treatment with staurosporine or cycloheximide causes rapid caspase activation and increase of cyt c in digitonin-extracted lysates of Drosophila S2 cells. In S2 cells, immunoprecipitation experiments reveal that released cyt c by rpr directly interacts with Dapaf-1L, but Dapaf-1S, which lacks WDRs, binds to cyt c weakly. These observations suggest that one candidate for the internal signaling molecule between Rpr and Ark could be cyt c, and the target of cyt c would be Dapaf-1L, a structural homolog of mammalian Apaf-1 (Kanuka, 1999).

To reveal the physiological roles of specific caspase-activating cascades initiated by Dapaf-1, morphological defects were sought in the nervous system of the dpfK1 homozygous larvae and adults. At the third-instar larval stage, the brain hemispheres of the dpfK1 mutant are larger than those of the wild-type and contain a markedly decreased number of apoptotic cells. Because the number of cells stained by the antibody against a neural marker (Prospero) actually increases in larval brain in dpfK1/dpfK1, Dapaf-1-dependent cell death might be required for the regulation of the number of neural cells in developing brain (Kanuka, 1999).

The extra sensory organ on the notum is one of the typical structures of the Drosophila peripheral nervous system (PNS). Four large bristles (macrochaetes) are always observed on the wild-type scutellum. However, extra bristles often appear on the scutellum of dpfK1/dpfK1 flies (48%, n = 54). These ectopic bristles may be induced by defects in caspase activation in the developing scutellum because overexpression of a caspase inhibitor P35 using the GAL4/UAS system also induces a similar phenotype. Expression of P35 in the scutellum with sca-GAL4, dpp-GAL4, and ptc-GAL4 results in the formation of ectopic bristles similar to those observed in the dpfK1 mutants. These observations suggest that Dapaf-1-dependent caspase activation may play roles for control of the sensory organ numbers (Kanuka, 1999).

The normal ommatidium in the Drosophila eye consists of photoreceptor cells, pigment cells, and cone cells. The exact numbers of these cells are strictly regulated by extracellular and intracellular mechanisms, including apoptotic cell death. In the eyes of the dpfK1 homozygous adults, abnormal ommatidia with one extra photoreceptor cell are frequently observed. The existence of these extra photoreceptor cells is not caused by the mislocation of R7/R8 cells. In addition, morphology of the pigment cell layer is disorganized compared with the regular pattern of the wild type, and the extra pigment cells are often observed in pupal retina of dpfK1 mutant. Since the numbers of pigment cells are regulated by apoptotic cell deaths, blockade of these cell deaths by caspase inhibitor P35 causes survival of extra pigment cells. These data suggest that the control of the number of photoreceptor cells and the pigment cells might depend on Ark function (Kanuka, 1999).

These findings suggest that the two different caspase activation mechanisms seen in nematodes are both present in Drosophila. The function of Dapaf-1S, fulfilling one of these mechanisms, is to bind to Ice and to activate DEVDase (Ice), resembling the action of CED-4 in C. elegans by which proCED-3 is processed into its mature form (Chinnaiyan, 1997). Dapaf-1L, fulfilling the second of these mechanisms, acts like mammalian Apaf-1, by activating YVADase first, then activating DEVDase (Li, 1997). The mechanism underlying the Dapaf-1L-induced YVADase activation is very similar to that in mammals, which is based on the observation that the inhibition of caspase-1-like protease by the YVAD inhibitor blocks the subsequent activation of caspase-3 in apoptosis induced by Fas antigen, and the observation that Apaf-1 activates procaspase-9, resulting in the subsequent activation of caspase-3 (Li, 1997). These facts lead to an interesting hypothesis that CED-4 acquired WDRs at its C terminus through evolution, which enabled a more advanced regulation of programmed cell death. The WDR of Apaf-1 interacts with cyt c derived from mitochondria in the presence of apoptotic stimuli, and this binding is one of the triggers for Apaf-1-induced caspase activation (Hu, 1998). Cyt c directly interacts with Dapaf-1L and Ark is required for cyt c-dependent caspase activation in lysates from developing embryos. It is possible that cyt c displayed on the surface of mitochondria might activate Dapaf-1. Rpr-induced cyt c release is accelerated by the Rpr-binding protein Scythe in the Xenopus cell-free system. A scythe-like molecule might play a role in Rpr-induced cyt c release in Drosophila. Thus, this finding suggests that the mechanism is evolutionarily conserved by which WDR-containing Apaf-1-like molecules, such as Dapaf-1L, are required for cyt c-dependent caspase activation (Kanuka, 1999).

Alternative splicing of Bcl-x, caspase-2, and CED-4 has been reported. In all cases, splicing isoforms show the opposite functions of their parental product. In addition to these findings, it was found that activation of distinct caspases can be regulated by alternative splicing of Dapaf-1. Dapaf-1L seems to be a latent form because it binds to cyt c. However, Dapaf-1S can work as an active form without cyt c and activate distinct caspase from Dapaf-1L/cyt c complex when it is expressed. Thus, at least two caspase activation mechanisms (one is cyt c dependent, another is by alternative splicing) are present in Drosophila. Since Apaf-1S is also found in the mouse, this type of regulatory mechanism may be also conserved through evolution (Kanuka, 1999).

Programmed cell death via caspase activation is essential for normal development in various species. In C. elegans, CED-4 is thought to be the only molecule responsible for activating CED-3. However, the higher multicellular organisms have acquired more sophisticated caspase-processing procedures in response to various apoptotic demands. In Drosophila, three distinct molecules, Rpr, Hid, and Grim, activate endogenous caspases. The Drosophila caspase activator, Dapaf-1, is shown in this study to be involved in rpr-induced cell death cascades. At the same time, the in vivo function of Ark indicates the existence of complicated caspase activation systems in Drosophila. The Drosophila cell death inducers Rpr and Hid exhibit their functions through caspase activation, but no genetic interaction could be seen between Ark and hid-induced cell death in the compound eye, suggesting that Ark may not contribute so much to caspase activation mechanisms evoked by hid. Although Ark is involved in the execution of the cell death program induced by the overexpression of rpr, the GMR-rpr phenotype could not be completely rescued in a dpfK1 homozygous background. There are two possible interpretions of this result. One is that the allele is not null and therefore, some Rpr-dependent death can still occur. Another interpretation is that the Ark allele is entirely null, and Rpr functions through multiple pathways, one Dapaf-1-dependent and one independent. The latter scenario is preferred. Since no transcripts of Ark in Ark mutant embryo could be detected, and embryonic lysates from dpfK1 homozygous mutant do not respond to cyt c, the Ark allele seems to be null. Since Drosophila cyt c that may be released by rpr binds to Dapaf-1L, Ark could contribute to the cyt c-dependent caspase activation that occurs downstream of Rpr. Multiple caspase activation mechanisms are also suggested by the observation that lysates from the dpfK1 homozygous mutant have half the amounts of activated caspase. These data imply that approximately half of the caspase activity in the embryo is dependent on the Ark functions. However, the rest of the caspases are likely to be activated by another mechanism. Although knockout mice lacking Apaf-1, caspase-3, or caspase-9 exhibit several severe defects in early embryonic development, these phenotypes are observed only in certain tissues and organs, suggesting that Ark and mammalian Apaf-1 participate in caspase activation partially in vivo. The roles of these distinct machineries for caspase activation remain to be elucidated, but because in some cases the expression of both Hid and Rpr is required to kill specific cells or tissues in Drosophila, cumulative caspase activation is probably necessary to induce cell death in some situations (Kanuka, 1999).

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).

Ice 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).

DIAP2 functions as a mechanism-based regulator of drICE that contributes to the caspase activity threshold in living cells

In addition to their well-known function in apoptosis, caspases are also important in several nonapoptotic processes. How caspase activity is restrained and shut down under such nonapoptotic conditions remains unknown. This study shows that Drosophila inhibitor of apoptosis protein 2 (DIAP2) controls the level of caspase activity in living cells. Animals that lack DIAP2 have higher levels of drICE activity. Although diap2-deficient cells remain viable, they are sensitized to apoptosis following treatment with sublethal doses of x-ray irradiation. DIAP2 was found to regulate the effector caspase drICE through a mechanism that resembles the one of the caspase inhibitor p35. As for p35, cleavage of DIAP2 is required for caspase inhibition. The data suggest that DIAP2 forms a covalent adduct with the catalytic machinery of drICE. In addition, DIAP2 also requires a functional RING finger domain to block cell death and target drICE for ubiquitylation. Because DIAP2 efficiently interacts with drICE, these data suggest that DIAP2 controls drICE in its apoptotic and nonapoptotic roles (Ribeiro, 2007).

Caspases are best known for their role in executing apoptosis, however, they also play important signaling roles in nonapoptotic processes, such as regulation of actin dynamics, innate immunity, and cell proliferation, differentiation, and survival. Under such conditions, caspases are activated without killing the cell. Given the irreversible nature of caspase-mediated proteolysis, caspases' activation and activity is subject to complex regulation. After zymogen activation, caspase activity can be controlled by various cellular and viral inhibitors. Interestingly, the viral inhibitors CrmA and p35 neutralize caspases through a fundamentally different mechanism than cellular inhibitors such as the inhibitor of apoptosis (IAP) proteins. CrmA and p35 function as 'suicide substrates,' whereby cleavage of CrmA and p35 is required to block the caspase. Initially, CrmA and p35 are cleaved through a mechanism that resembles substrate hydrolysis. However, during the reaction, the protease's catalytic machinery is trapped via covalent linkage. This strategy is referred to as 'mechanism-based' inhibition because it relies on the caspase's catalytic property. So far, cellular-derived mechanism-based caspase inhibitors have not been identified (Ribeiro, 2007).

IAPs are defined by the presence of one to three copies of the baculovirus IAP repeat (BIR) domain, which functions as a protein interaction module. In addition, certain but not all IAPs also carry a C-terminal RING finger domain, which provides these molecules with E3 ubiquitin-protein ligase activity. In Drosophila, DIAP1-mediated inhibition of caspases is indispensable for cell survival. Mutations that abrogate the physical association of DIAP1 with the effector caspases drICE or DCP-1 or the initiator caspase Dronc cause spontaneous caspase activation and cell death (Ribeiro, 2007).

The involvement of the second Drosophila IAP (DIAP2) in regulating caspases is less clear. Overexpression studies suggest that DIAP2 can suppress cell death induced by the IAP antagonists reaper (Rpr) and head involution defective (Hid) and suppress apoptosis induced by diap1-RNAi. Because diap1-RNAi-mediated death is independent of IAP antagonists, this suggests that DIAP2 can neutralize caspases. Of the seven D. melanogaster caspases, DIAP2 binds only to the effector caspase drICE and the atypical caspase Strica. In contrast, DIAP1 exhibits much broader caspase selectivity because it inhibits Dronc, drICE, and DCP-1. Based on the domain architecture and sequence alignments, DIAP2 is the closest homologue to mammalian IAPs. In contrast, DIAP1 is more related to IAPs from insect viruses. Like the mammalian x-linked IAP (XIAP), DIAP2 carries three BIR domains and a C-terminal RING finger. XIAP blocks apoptosis by acting as a potent enzymatic inhibitor of caspase-3, -7, and -9. Residues located immediately upstream of XIAP's BIR2 domain directly bind to the active site pockets of caspase-3 and -7, thereby obstructing substrate entry. Importantly, XIAP is not cleaved by caspases because it binds to the catalytic pockets of effector caspases in a reverse orientation. Thus, XIAP relies on allosteric mechanisms to block effector caspase activity. XIAP's ability to suppress effector caspases depends on the side chain of Asp148 and, to a lesser extent, Val146. It is predicted that Asp148 must be conserved in all IAPs that neutralize effector caspases. Consistently, Asp148 is conserved in cIAP1 and 2. Intriguingly, DIAP2 also shows homology to XIAP's Val146 and Asp148, as it carries equivalent residues at Val98 and Asp100. In contrast, these residues are absent in DIAP1 (Ribeiro, 2007).

Of all the IAPs, XIAP appears to be the only IAP that potently inhibits caspases in vitro. Other IAPs, such as cIAP1 and 2, are poor inhibitors of caspases under these conditions. cIAP1 and 2 may rely on their E3 ligase activity to block caspases in vivo, making them caspase regulators rather than inhibitors. Although DIAP1 is a relatively good inhibitor of D. melanogaster caspases in vitro, DIAP1-caspase physical association alone is insufficient to neutralize caspases in the fly. In addition to binding, DIAP1 uses two independent mechanisms to regulate caspases. One relies on the E3 ubiquitin protein ligase activity of DIAP1's own RING finger, whereas the other, the 'N-end' rule, functions independently of this domain. The RING finger of DIAP1 is required to target the proform of the initiator caspase Dronc for ubiquitylation and inactivation. In contrast, active effector caspases seem to be neutralized through a mechanism that involves both the RING finger domain as well as the N-end rule degradation machinery that is recruited by DIAP1 (Ribeiro, 2007 and references therein).

In Drosophila, developmentally regulated apoptosis is induced by the IAP antagonists Rpr, Grim, and Hid. Embryos lacking rpr, grim, and hid are virtually devoid of apoptosis and die at the end of embryogenesis with accumulation of supernumeral cells. The current model suggests that Rpr, Grim, and Hid induce apoptosis by binding to the BIR domains of DIAP1, thereby liberating caspases from IAP inhibition. Furthermore, IAP antagonists also deplete DIAP1 protein levels by promoting its degradation and cause mitochondrial permeability. Although Rpr, Grim, and Hid efficiently antagonize DIAP1, they also associate with DIAP2. This implies that DIAP2 contributes to the overall antiapoptotic threshold of a cell, a view that is supported by the notion that RNAi-mediated knockdown of DIAP2 sensitizes cultured cells to stress-induced death. However, analyses using diap2 mutant flies have failed to expose any involvement of DIAP2 in regulating programmed cell death. Instead, they have shown that DIAP2 is required for NF kappaB activation during the innate immune response in D. melanogaster (Ribeiro, 2007 and references therein).

This study used the fluorescence resonance energy transfer (FRET)-based caspase-3 indicator SCAT3 to examine DIAP2's role in regulating caspase activity in vivo. Using diap2 mutant animals, it was found that these animals harbor significantly increased levels of drICE activity. Consistent with higher levels of active caspases, diap2 mutant cells are sensitized to apoptosis after exposure to sublethal doses of x-ray irradiation. Furthermore, DIAP2 tightly associates with the effector caspase drICE and DIAP2-mediated drICE inhibition requires DIAP2 cleavage at Asp100. The data are consistent with the notion that after cleavage, the caspase forms a covalent adduct with DIAP2, which results in the stabilization of the DIAP2-caspase complex. This mode of caspase binding is similar to that of p35 or CrmA. However, in addition to the mechanism-based trapping, DIAP2 also requires a functional RING finger domain to neutralize drICE-mediated cell death. Consistently, it was found that DIAP2 robustly ubiquitylates drICE in vivo (Ribeiro, 2007).

The data suggest that DIAP2 is a caspase regulator that contributes to the caspase activity threshold. Several lines of evidence support this view. First, loss of diap2 causes an increase in basal levels of caspase activity in vivo. Consistent with this increase in basal levels of caspase activity, diap2 mutant cells are sensitized to sublethal doses of x-ray irradiation, and, in tissue culture cells, depletion of DIAP2 by RNAi sensitizes cells to treatment with chemotherapeutic drugs. Second, DIAP2 overexpression in the developing fly eye efficiently suppresses cell death triggered by diap1 RNAi. Importantly, diap1 RNAi causes spontaneous and unrestrained caspase activation and cell death that occurs independently of the action of IAP antagonists. The efficiency with which DIAP2 rescues the diap1 RNAi phenotype is highly reminiscent of that of p35. Both DIAP2 and p35 rescue the diap1 RNAi eye size and pigmentation to an apparent normal morphology but fail to restore the formation of bristles. The notion that DIAP2 expression phenocopies the expression of p35, in the absence of any involvement of IAP antagonists, suggests that DIAP2 can function as a direct caspase inhibitor for a p35-sensitive caspase. Third, DIAP2 physically interacts with the effector caspase drICE and suppresses drICE-mediated cell death. Fourth, endogenous DIAP2 is readily cleaved by caspases, which indicates that DIAP2 encounters caspases in vivo. Moreover, after induction of apoptosis, the timing and extent of DIAP2 cleavage is highly reminiscent to that of DIAP1, which suggests that cleavage of DIAP2 is a relatively early event during apoptosis. Finally, IAP antagonists bind to DIAP1 and 2 with similar efficiencies. Consistent with the view that the caspase inhibitory activity of both IAPs is antagonized by IAP antagonists, it was found that Hid blocks DIAP1 and DIAP2 from binding to drICE. Together, these data support a model in which DIAP1 and 2 coordinately control drICE, thereby blocking the amplification of the caspase cascade. Accordingly, DIAP2 contributes to the apoptotic threshold by regulating drICE. However, DIAP1 controls initiation of apoptosis, as it is the sole IAP that regulates the initiator caspase Dronc and, with it, the formation of the Dronc/Dark apoptosome. Ultimately, programmed cell death is induced when IAP antagonists such as Hid synchronously bind to DIAP1 and 2, allowing apoptosome formation and unhindered amplification of the caspase cascade (Ribeiro, 2007).

Although DIAP2 seems to be a bona fide regulator of drICE, its loss does not cause spontaneous apoptosis. This is most likely caused by its epistatic position in the caspase cascade and its restricted specificity for caspases (see DIAP2's epistatic position in the caspase cascade). Because DIAP2 exclusively regulates drICE and does not bind other caspases, its loss merely causes an increase in caspase activity, presumably because of a small amount of uninhibited active drICE. Under such conditions, drICE activation remains regulated because it requires proteolytic input from Dronc, which, in turn, is under the control of DIAP1. Although loss of DIAP2 results in sublethal levels of active caspases, RNAi-mediated depletion of DIAP1 causes strong, apoptosome-driven activation of downstream effector caspases, which leads to the amplification of the proteolytic signal and death of the cell. Under diap1 RNAi conditions, levels of endogenous DIAP2 seem to be insufficient to suppress the amount of active caspases generated. But, if the levels of DIAP2 are increased, it efficiently blocks diap1 RNAi-mediated cell death. However, when programmed cell death is triggered through the activation of IAP antagonists, both IAPs are targeted equally, thereby releasing the inhibition of the cell death machinery. Thus, controlled activation of caspases and cell survival are ensured by both DIAP1 and 2 (Ribeiro, 2007).

The notion that diap2 mutant animals have increased levels of caspase activity indicates that cells can generate and tolerate a certain amount of activated caspases without undergoing apoptosis. This is consistent with the notion that caspases fulfill important signaling functions independent of executing cell death. For instance, DmIKKepsilon activation was recently found to reduce DIAP1 protein levels, thereby allowing activation of sublethal levels of Dronc, which, in turn, is required for the proper development of sensory organ precursor cells. Thus, modulating caspase activity in a selective, transient, and possibly spatially restricted manner is likely to be a widespread phenomenon that allows caspases to take part in signaling functions without jeopardizing cell viability. Concerted reduction of DIAP1 and 2 protein levels, or their controlled and selective inhibition, may therefore allow controlled activation of caspases and caspase-mediated signaling. The cellular concentration of IAPs would thereby set a cell type-specific threshold for caspase activation and activity. In DIAP2 mutants, however, cells are left in an unbalanced, sensitized state as they accumulate higher than normal levels of active caspases. Accordingly, diap2 mutant third instar larvae are sensitized to apoptosis when subjected to sublethal doses of x-ray irradiation. This sensitized state is only visualized by exposing diap2 mutant animals to sublethal insults. When strong proapoptotic stimuli are used, such as 40 Gy of x-ray irradiation (Huh, 2007) or overexpression of Rpr, Hid, and Grim, diap2 mutant animals display the same cell death phenotypes as WT counterparts (Huh, 2007). Under these strong proapoptotic conditions, IAP antagonists neutralize both IAPs, causing full activation and amplification of the proteolytic caspase cascade. Thus, the contribution of the sublethal amount of active caspases in diap2 mutants seems to be masked by the higher concentration of active caspases generated under such apoptotic conditions (Ribeiro, 2007).

This molecular characterization has uncovered an unexpected mechanism through which DIAP2 restrains the target enzyme drICE. Surprisingly, DIAP2 acts as a pseudosubstrate that, after cleavage, seems to trap the active caspase via a covalent linkage between DIAP2 and the catalytic machinery of drICE. Mutation of the DIAP2 caspase cleavage site abrogates its ability to bind drICE and suppress drICE-mediated cell death. It is unusual for proteins that regulate enzymes to use a mechanism-based strategy, and most types avoid the catalytic machinery by simply blocking the substrate cleft in a lock and key strategy. The cleavage of DIAP2 distinguishes it from all natural inhibitors with the exception of p35, CrmA, serpins, and α-macroglobulins. Serpins and p35 both require cleavage to function as protease inhibitors. p35 arrests proteolysis at the thioacyl intermediate stage, which results in a covalent adduct between p35 and the caspase. In particular, the cleavage site residue Asp87 of p35 links up with the catalytic cysteine of the caspase. Thus, cleaved p35 locks the catalytic machinery of the caspase in a nonproductive inactive configuration. The exquisite sensitivity of the DIAP2-drICE complex to strong nucleophiles such as NH2OH or DTT is diagnostic of a stable thioester adduct between DIAP2 and drICE. Nevertheless, it remains possible that this complex could form because of a disulfide bridge between DIAP2 and the catalytic thiol of drICE. However, the requirement of DIAP2's Asp100 for drICE binding suggests that Asp100 is involved in forming a thioester covalent adduct with the caspase active site Cys. Ultimately, crystal structure or mass spectrometric analysis will be required to identify the chemical nature of this complex. However, the relatively harsh conditions required for sample preparations for mass spectrometric analysis combined with the labile nature of thiol esters will make it difficult to identify a fragment containing a covalent linkage between DIAP2 and drICE. Collectively, the data suggest that the DIAP2-drICE complex is stabilized by a covalent linkage (Ribeiro, 2007).

Despite the notion that p35 and DIAP2 both occupy and produce a covalent linkage with the active site of caspases, the modes of interaction and mechanism of inhibition are very different. Unlike p35, DIAP2 requires additional domains for caspase binding and inhibition. First, DIAP2 needs to associate with drICE through a bimodular interaction, whereby the BIR3 domain binds to the IBM of drICE and the 97-Ser-Val-Val-Asp-100 region of DIAP2 occupies the catalytic pocket of the caspase. Each motif on its own is insufficient for caspase binding as mutation of either the BIR3 or cleavage site interferes with complex formation. Second, physical interaction with drICE is not sufficient to block caspase activity. In addition, DIAP2 requires a functional RING finger domain to regulate drICE-mediated cell death. This is evident because a DIAP2 RING finger mutant fails to suppress cell death induced by RNAi-mediated depletion of DIAP1. Moreover, DIAP2 fails to suppress drICE in vitro. In this respect, DIAP2 functions as mechanism-based regulator (relying on the enzyme's catalytic property to trap it) rather than inhibitor. After capture, DIAP2 targets drICE for ubiquitylation (Ribeiro, 2007).

An important question is why DIAP2 does not function as an inhibitor like p35 if it also establishes a covalent linkage with the catalytic active site cysteine of the caspase. One possibility is that DIAP2 and p35 differ in their ability to protect the highly labile thioester bond from hydrolysis. This view is supported by the notion that the DIAP2-drICE interaction is significantly more sensitive to nucleophile attack than p35-drICE. Thus, in the situation of DIAP2, the thioester linkage with drICE may simply help to stabilize the caspase interaction, whereas in p35-drICE, it is used to irreversibly inhibit the protease. The more labile nature of the DIAP2-drICE complex may allow regulation, which cannot occur with p35. Moreover, p35 inhibits all effector caspases, whereas DIAP2 only regulates drICE. The observation that the nontarget caspase DCP-1 can also cleave DIAP2 but is not inhibited raises the possibility that DCP-1 controls the level of functional DIAP2 through proteolytic inactivation. Examples of such regulation are manifold and include the proteolytic inactivation of serpins by nontarget proteases. Because DIAP2 tightly interacts with drICE, it is likely to control its apoptotic and nonapoptotic functions (Ribeiro, 2007).

Activation of sterol regulatory element-binding protein by the caspase Drice in Drosophila larvae

During larval development in Drosophila, transcriptional activation of target genes by sterol regulatory element-binding protein (dSREBP) is essential for survival. In all cases studied to date, activation of SREBPs requires sequential proteolysis of the membrane-bound precursor by site-1 protease S1PS1P) and site-2 protease (S2P). Cleavage by S2P, within the first membrane-spanning helix of SREBP, releases the transcription factor. In contrast to flies lacking dSREBP, flies lacking dS2P are viable. The Drosophila effector caspase Drice cleaves dSREBP, and cleavage requires an Asp residue at position 386, in the cytoplasmic juxtamembrane stalk. The initiator caspase Dronc does not cleave dSREBP, but animals lacking dS2P require both drice and dronc to complete development. They do not require Dcp1, although this effector caspase also can cleave dSREBP in vitro. Cleavage of dSREBP by Drice releases the amino-terminal transcription factor domain of dSREBP to travel to the nucleus where it mediates the increased transcription of target genes needed for lipid synthesis and uptake. Drice-dependent activation of dSREBP explains why flies lacking dS2P are viable, and flies lacking dSREBP itself are not (Amarneh, 2009).

Cleavage of SREBP by caspases was first reported in 1995 (Pai, 1995; Wang, 1995; Wang, 1996), but the significance of those observations has remained unclear. Both in transfected S2 cells and in vitro, with purified enzyme, Drice cleaves dSREBP, and this cleavage requires an Asp residue at position 386 in the juxtamembrane stalk. Other Asp residues in this region are not required for cleavage. As caspases typically cleave following an Asp residue, the parsimonious interpretation of these data is that Drice cleaves dSREBP following Asp-386 (Amarneh, 2009).

Cleavage of dSREBP by Drice releases the amino terminus. The present data demonstrate that dS2P mutants require drice for survival. Animals doubly mutant for dS2P and drice phenocopy dSREBP null mutants; they survive extremely poorly on unsupplemented medium and show greatly improved survival on medium supplementedwith fatty acids. Conversely, larvae whose only source for dSREBP is a dSREBP transgene, P{dSREBPg(D386A)}, which harbors a substitution of Asp-386 for Ala at the putative cleavage site, are viable in the presence of wild type dS2P. If these transgenic animals lack dS2P, the mutant transgenic larvae die at the end of second instar. Just as observed for the double mutant dS2P, drice larvae, the P{dSREBPg(D386A)} transgenic larvae are substantially rescued by dietary supplementation with fatty acids. The extent of rescue is similar to the rescue of dSREBP189 homozygotes in the same experiment, confirming that lethality results from deficits in fatty acid metabolism (Amarneh, 2009).

The results with the dS2P1/dS2P2; dronc51/dronc51 double mutants are very similar to the results of experiments with driceδ1, even though purified Dronc cannot cleave dSREBP directly. This likely reflects the requirement for the initiator caspase Dronc to cleave the effector caspase Drice, such that in the absence of Dronc, Drice is not activated and cannot cleave dSREBP. Cleavage of Drice by Dronc, for example, is required for activation of Drice during apoptosis. These data suggest that Drice, activated by Dronc, cleaves dSREBP in larvae lacking dS2P (Amarneh, 2009).

Little apoptosis is observed in Drosophila between embryogenesis and pupariation. Cleavage of dSREBP by Drice during larval growth therefore does not appear to be related to apoptosis. Even in the absence of substantial apoptosis, however, mRNAs for Drice and Dronc are detected at low levels in larvae. Genetic data indicate that at least some of that message is translated to yield enzyme that is active during larval life when apoptosis is not observed. Constitutive activation of effector caspases by the apoptosome in the absence of apoptosis is seen elsewhere during Drosophila growth and development. For example, in embryos, there is a basal level of effector caspase activity several hours before the onset of programmed cell death. Caspase activity is also detected in hemocytes from 3rd instar larvae that are likewise not undergoing apoptosis (Amarneh, 2009).

Does caspase cleavage of dSREBP play a role in normal larval physiology or is this phenomenon seen only when dS2P is absent? In larvae lacking dS2P, activation of dSREBP is readily detected in the fat body. The cleavage of dSREBP by Drice in the larval fat body could be a fortuitous consequence of the presence of a caspase site in the juxtamembrane stalk and the coincidental expression of Drice in the larval fat body. Alternatively, caspase cleavage may represent an important and pervasive physiologically relevant means of activating SREBP independently of the previously described machinery (Scap, S1P, and S2P). The present data do not allow firm distinction between these possibilities but an inference may be drawn from sequence data (Amarneh, 2009).

The site of caspase cleavage in SREBP-1 is conserved among its vertebrate homologues, and the site of caspase cleavage in SREBP-2, which is not homologous to the caspase site in SREBP-1, is likewise highly conserved among vertebrate SREBP-2s. Similarly, the caspase site Asp in dSREBP is well conserved among Drosophila SREBPs as is the preceding Thr residue. In the case of mammalian SREBP-1 and -2, and now for dSREBP, the seputative sites have been validated experimentally. The differing positions of these confirmed caspase sites indicate that the absolute position of caspase cleavage within the juxtamembrane stalk is not crucial, whereas the presence of a caspase cleavage site is. Cleavage almost anywhere within this region may offer suitable means of releasing active SREBPand thus bypassing the normal processing machinery (Amarneh, 2009).

Cleavage of dSREBP is usually tightly controlled by end product feedback regulation. If SREBP in the ER membrane is the substrate for caspase cleavage, this would bypass feedback regulation, which relies of the control of ER-to-Golgi transport of SREBP (Amarneh, 2009).

Under what circumstances might a cell or organism need to bypass end product-mediated feedback suppression of the transcription of the genes of lipid synthesis? It may be desirable during periods of rapid membrane synthesis, such as fetal development in mammals. Rapid deposition of large stores of lipid may be another such a case. The mass of the Drosophila larvae increases roughly 200-fold between the time it emerges from the egg and the onset of pupariation about 5 days later. The majority of this increase in mass results from the storage of lipid in the fat body, which is needed to fuel metamorphosis. End product-mediated suppression of the transcription of the genes of lipid synthesis may be incompatible with the need for continued high levels of lipid accumulation and synthesis in the presence of large amounts of lipid already stored (Amarneh, 2009).

In dS2P mutant larvae, transcript abundance of dSREBP target genes is much greater than in larvae lacking dSREBP itself, and activation of dSREBP is readily detected in the fat body. This activation permits the survival of the mutant animals. However, in the complete absence of dS2P, the mutant offspring of mutant mothers survive only half as well as their heterozygous siblings. Their reduced survival results from a deficit in lipid metabolism; they survive at nearly the expected rate on medium supplemented with fatty acids. Therefore, cleavage of dSREBP by Drice is not fully redundant with the usual processing mechanism. Instead, it may serve to augment dSREBP activation in specific tissues to support the rapid deposition of lipid stores during larval life (Amarneh, 2009).

Bicaudal is a conserved substrate for Drosophila and mammalian caspases and is essential for cell survival

Members of the caspase family of cysteine proteases coordinate cell death through restricted proteolysis of diverse protein substrates and play a conserved role in apoptosis from nematodes to man. However, while numerous substrates for the mammalian cell death-associated caspases have now been described, few caspase substrates have been identified in other organisms. This study used a proteomics-based approach to identify proteins that are cleaved by caspases during apoptosis in Drosophila D-Mel2 cells, a subline of the Schneider S2 cell line. This approach identified multiple novel substrates for the fly caspases and revealed that bicaudal/betaNAC is a conserved substrate for Drosophila and mammalian caspases. RNAi-mediated silencing of bicaudal expression in Drosophila D-Mel2 cells resulted in a block to proliferation, followed by spontaneous apoptosis. Similarly, silencing of expression of the mammalian bicaudal homologue, betaNAC, in HeLa, HEK293T, MCF-7 and MRC5 cells also resulted in spontaneous apoptosis. These data suggest that bicaudal/betaNAC is essential for cell survival and is a conserved target of caspases from flies to man (Creagh, 2009).

At present, three substrates for Drosophila caspases, DIAP1, Lamin DmO, and the Drosophila Apaf-1 homologue, ARK, have been identified. A proteomics-based screen resulted in the identification of 14 proteins that underwent caspase-dependent alterations to their relative mobilities on two-dimensional (2D) SDS-PAGE gels. Two of these substrates were cloned and confirmed that they are efficiently cleaved by the Drosophila caspases DrICE and DCP-1. Bicaudal and its human homologue, βNAC, appear to be essential for cell proliferation and cell survival as RNAi-mediated silencing of the expression of these proteins resulted in a block to cell division, followed by spontaneous apoptosis. This suggests that, as well as targeting substrate proteins that contribute to the ordered destruction of the cell, caspases also inactivate key proteins such as βNAC that are essential for cell survival (Creagh, 2009).

Interestingly, βNAC has also been implicated as a negative regulator of programmed cell death in the nematode C. elegans and ablation of this gene product results in massive and unscheduled apoptosis in developing worm embryos. NAC functions to bind short nascent polypeptides as they emerge from the ribosome. The latter event prevents inappropriate interactions with cellular proteins and non-specific binding by the signal recognition particle and consequent targeting to the ER. NAC also prevents the targeting of non-translating ribosomes to the ER. This fundamental role of NAC is reflected in the catastrophic phenotype of null mutations affecting the βNAC-coding sequence gene in a range of species. Loss of βNAC in developing mice leads to post-implantation lethality and mutation of Drosophila bicaudal promotes developmental arrest, which is associated with duplication of the posterior embryonic regions in the place of the anterior embryonic segments (Markesich, 2000). RNAi-mediated silencing of the C. elegans βNAC homologue, ICD-1, also results in developmental arrest associated with massive cell death (Bloss, 2000). Thus, the disablement of βNAC function through caspase-dependent proteolysis may contribute substantially to cellular demise (Creagh, 2009).

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).


Ice: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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