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).
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).
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).
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).
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).
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).
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).
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).
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