To determine if dOmi could induce cell death in the developing fly eye, we generated transgenic flies expressing wild-type 79-dOmi (GMR-gal4;UAS-domi79wt7B), 92-dOmi (GMR-gal4;UAS-domi92wt5A), or their catalytically inactive S266A mutants (GMR-gal4;UAS-dOmi79S266A4A and GMR-gal4;UAS-dOmi92S266A42A). Interestingly, when compared with control flies, expression of 79-dOmi and 92-dOmi resulted in phenotypes ranging from organismal lethality at pupal stages to a rough eye (Figure 5B and C). The effects of 92-dOmi were consistently much stronger than 79-dOmi (Figure 5C), but as previously observed in S2 cells, expression of the catalytically inactive S266A dOmi mutants did not result in any phenotype (Figure 5B). In contrast to the effects of Z-VAD-fmk in S2 cells, expression of the baculoviral caspase inhibitor p35 did not inhibit cell death induced by 79-dOmi or 92-dOmi (Figure 5B, data not shown). GMR-driven expression of dOmi in the fly eye however occurred over a 5–6 day period, beginning with photoreceptor differentiation in the third larval instar and continuing throughout pupal development, whereas the effects of dOmi expression in S2 cells were examined after 1–2 days. Thus, dOmi could promote caspase-dependent apoptosis via its serine protease activity, but in the long term did not require caspase activity in order to induce cell death.

The IBMs in dOmi interact selectively with the BIR2 domain in DIAP1 and displace the initiator caspase DRONC

Rpr, Hid, Grim, Sickle, and Jafrac2 all reportedly induce apoptosis in the fly, by interacting with and displacing the effector caspase DrICE from the BIR1 domain in DIAP1, and/or the initiator caspase DRONC from the BIR2 domain (Chai et al, 2003; Zachariou et al, 2003; Yan et al, 2004). Therefore, since dOmi clearly bound to DIAP1 in an IBM-dependent manner (Figure 3B), it was surprising that this interaction alone failed to induce significant amounts of apoptosis in S2 cells or in the developing fly eye (Figure 5A, S266A versus Vec Ctrl, Figure 5B). In order to resolve this dilemma, we sought to further characterize dOmi's interaction with DIAP1, as well as its role in promoting caspase activation. We began by expressing various DIAP1 truncation mutants as GST fusion proteins and subsequently performed pulldown assays using naïve S2 cell lysates (Figure 6A and B). Importantly, the BIR2 domain in DIAP1 was found to be essential for binding both processed forms of endogenous dOmi, whereas neither the BIR1 nor the RING domains were required (Figure 6B).

Figure 6.

Mature dOmi does not bind to the BIR1 domain in DIAP1 or displace the effector caspase DrICE. (A, B) GST-DIAP1 and truncation mutants were expressed in bacteria and purified to homogeneity. The proteins (500 nM) were then captured using GSH–Sepharose beads and incubated (3 h at 4°C) with naïve S2 cell lysates (100 g), in a final volume of 300 l. The bead complexes were subsequently isolated, separated by SDS–PAGE, and immunoblotted using a rabbit anti-dOmi polyclonal antibody. (C, D) Recombinant DrICE (175 nM) was preincubated with or without GST-BIR1 (inhibitor, 3.5 M) for 30 min at 25°C. Human PARP (substrate, 5.75 M) was then added alone, or in combination with recombinant 79-dOmi, 92-dOmi, or the IBM truncation mutants (AIIQ, ASKM) (4.5 M), and were further incubated for 60 min at 25°C. The 79-IBM peptide and the positive control, Rpr-IBM peptide (5 g), were also tested in separate incubations. All protein complexes were separated by SDS–PAGE, and the gels stained with Coomassie Blue. (E) Structural model of 79-IBM bound to DIAP1-BIR1.

View full figure (190 KB)

Given that dOmi failed to bind BIR1, we predicted that it would be unable to antagonize BIR1-dependent inhibition of DrICE. To provide definitive evidence, we incubated recombinant DrICE with its substrate PARP, either alone or in the presence of GST-BIR1. At its approximate IC₅₀, GST-BIR1 inhibited DrICE-mediated cleavage of PARP by 50% (Figure 6C and D, lanes 1–3). As expected, this inhibition was readily overcome by a Rpr peptide, matching its N-terminal IBM (Rpr-IBM; AVAFYIPD), but not by a control peptide (MKSDFYFQ) (Figure 6D, lanes 4 and 6). More importantly, however, neither recombinant 79-dOmi, 92-dOmi, nor their IBM truncation mutants (AIIQ or ASKM), promoted DrICE-dependent cleavage of PARP (Figure 6C, lanes 4–7). Moreover, unlike Rpr-IBM, the IBM peptide of 79-dOmi (AIIQREDL) also failed to antagonize BIR1-dependent inhibition of DrICE (Figure 6D, lanes 4 and 5). To determine why dOmi failed to displace DrICE, we modeled the 79-IBM into the BIR1 binding pocket of DIAP1, using the previously solved crystal structure for BIR1 bound to Rpr-IBM (PDB code 1SDZ; Yan et al, 2004). As shown in Figure 6E, Arg5 in dOmi appeared to sterically clash with Glu86 in the bottom of the BIR1 pocket, thus preventing 79-dOmi from forming a stable complex with BIR1.

Since mature dOmi bound to the BIR2 domain in DIAP1 (Figure 6B), we predicted that dOmi might displace the initiator caspase DRONC from the BIR2 binding pocket. We therefore incubated GST-BIR2-RING with an N-terminal fragment of DRONC (1–139) and observed the formation of a BIR2-RINGDRONC complex (Figure 7A, lanes 1 and 10), consistent with a previous report (Chai et al, 2003). As expected, addition of 79-dOmi or 92-dOmi to the incubation mixture resulted in a concentration-dependent displacement of DRONC from the complex (Figure 7A, lanes 2–5 and 11–14), with 79-dOmi displaying a higher affinity for BIR2-RING compared to 92-dOmi (K_d0.27 M versus 1.18 M) (Figure 7C). By contrast, neither of the IBM truncation mutants (AIIQ or ASKM) bound to BIR2-RING or displaced DRONC (Figure 7A, lanes 6–9 and 15–18). In additional experiments, the Rpr-IBM peptide also displaced DRONC from the BIR2 binding pocket (Figure 7B), with an affinity similar to that reported for the Hid-IBM (K_d0.036 M versus 0.041 M) (Figure 7C) (Wu et al, 2001). Thus, in our assays, Rpr was 7-fold more potent than 79-dOmi at displacing DRONC (Figure 7C). Nevertheless, the affinity of 79-dOmi for DIAP1-BIR2 was 3-fold higher than that reported for DRONC (Figure 7C) (Chai et al, 2003). Moreover, by comparison, the affinity of 79-dOmi for DIAP1-BIR2 was higher than that reported for Smac with XIAP-BIR3 (Liu et al, 2000), which is compelling, given that DRONC and caspase-9 exhibit virtually identical binding affinities for their respective IAPs (Figure 7C).

Figure 7.

Mature dOmi binds selectively to the BIR2 domain in DIAP1 and displaces the initiator caspase DRONC. (A, B) GST-BIR2-RING (3 M) was incubated with an N-terminal fragment of DRONC (6 M), in the absence or presence of the Rpr-IBM peptide (0–4 M), recombinant 79-dOmi, 92-dOmi, or their corresponding IBM mutants (AIIQ, ASKM) (2–16 M). The dOmi proteins also contained an active-site mutation (S266A), to ensure that dOmi's proteolytic activity did not interfere with the displacement of DRONC. Displacement curves were plotted to determine the EC₅₀ values for each of the Rpr-IBM peptide and dOmi proteins. All protein complexes were then separated by SDS–PAGE, and the gels stained with Coomassie Blue. (C) Comparison of the dissociation constants for specific fly and mammalian IAP antagonists and initiator caspases with their respective IAPs. (D) Structural model of 79-IBM bound to DIAP1-BIR2.

View full figure (283 KB)

The reasons for the selectivity of 79-dOmi for BIR2 over BIR1 were subsequently revealed through modeling studies, using the solved crystal structure of BIR2 bound to Hid-IBM (PDB code 1JD6) (Wu et al, 2001) (Figure 7D). Indeed, the steric clash observed between Arg5 in 79-dOmi and Glu86 in BIR1 (Figure 6D) did not exist in the BIR2 model, as Glu86 is replaced by a glycine in the analogous position (Gly269) (Figure 7D). Arg5 appeared to exhibit some electro-repulsion with Arg260 and Arg262 in BIR2, and thus may account for the reduced affinity of 79-dOmi for BIR2 compared to Rpr and Hid (Figure 7C and D). Collectively, the biochemical and structural data indicate that 79-dOmi can selectively displace DRONC from the BIR2 domain in DIAP1. However, this interaction is insufficient, on its own, to induce significant levels of cell death, perhaps because the BIR1 domain retains its ability to inhibit the effector caspase, DrICE. Indeed, we have previously shown in human cells that the linker-BIR2 domain in XIAP can inhibit the effector caspase-3 and prevent cell death, even when mutations in its BIR3 domain prevent inhibition of the initiator caspase-9 (Bratton et al, 2002).

dOmi alleviates DIAP1 inhibition of caspases by proteolytically degrading DIAP1

Although wild-type dOmi clearly induced cell death in both S2 cells and the developing fly eye via its serine protease activity, it remained unclear precisely how this led to caspase activation. hOmi proteolytically degrades certain IAPs in mammalian cells, including cIAP1, cIAP2, and Bruce/Apollon (Jin et al, 2003; Yang et al, 2003), raising the possibility that dOmi might indirectly increase caspase activity, at least in part, by degrading DIAP1. We therefore examined the effects of dOmi on the expression levels of DIAP1 in S2 cells. As shown in Figure 8A, DIAP1 was largely absent from cells when coexpressed with wild-type 79-dOmi, 92-dOmi, or the IBM mutants (lanes 2, 4, 5, and 7), whereas DIAP1 was readily detected in cells coexpressing the catalytically inactive S266A mutants (lanes 3 and 6). Thus, dOmi's proteolytic activity was responsible for mediating the loss in DIAP1, independent of its IBMs. We next incubated recombinant dOmi with DIAP1 (immunoprecipitated from transfected S2 cells) and found that both 79-dOmi and 92-dOmi directly degraded DIAP1 in a concentration-dependent manner (Figure 8B). However, it was difficult to visualize many of the DIAP1 fragments, due to proteolytic removal of the HA tag. Therefore, we repeated our in vitro cleavage assay by incubating recombinant dOmi with GST-DIAP1 that was first purified and then biotinylated. Under these conditions, dOmi once again proteolytically processed DIAP1 into numerous fragments that were readily visualized by blotting with streptavidin-HRP (Figure 8C).

Figure 8.

dOmi proteolytically degrades DIAP1. (A) S2 cells were cotransfected with pIE1-HA-DIAP1, along with pRmHa3-dOmi (79-dOmi, 92-dOmi), catalytically inactive mutants of dOmi (79-dOmi^S266A, 92-dOmi^S266A), or IBM mutants of dOmi (79-dOmi^GGIQ, 92-dOmi^GGKM). Following the addition of CuSO4 (0.7 mM) to induce expression of dOmi and its mutants, whole-cell lysates were immunoblotted for DIAP1 and dOmi expression levels. (B) HA-DIAP1 was expressed in S2 cells and immunoprecipitated using an anti-HA antibody (262K, Cell Signaling). The immunoprecipitates were then incubated with wild-type 79-dOmi or 92-dOmi (50–200 nM) for 2 h at 37°C and subsequently immunoblotted for HA-DIAP1. (C) Biotinylated GST-DIAP1 (250 ng) was incubated with recombinant 79-dOmi or 92-dOmi (50–200 nM) for 2 h at 37°C, in a total volume of 30 l, and subsequently blotted with streptavidin–HRP.

View full figure (76 KB)

As previously noted, a number of recent studies have suggested that other IAP antagonists in the fly may stimulate DIAP1 autoubiquitinylation and target DIAP1 for destruction by the 26S proteasome (Hays et al, 2002; Holley et al, 2002; Ryoo et al, 2002; Wing et al, 2002; Yoo et al, 2002). dOmi, on the other hand, did not appear to induce DIAP1 autoubiquitinylation, since neither 79-dOmi^S266A nor 92-dOmi^S266A induced a loss in DIAP1, when coexpressed in S2 cells (Figure 8A, lanes 1, 3, and 6). Furthermore, in subsequent in vitro assays using fly embryo lysates, neither recombinant dOmi, nor the 79-IBM peptide, enhanced (or suppressed) the basal level of DIAP1 autoubiquitinylation (data not shown). Thus, dOmi promoted caspase activity and cell death, at least in part by ridding the cell of DIAP1. However, dOmi accomplished this feat, not by stimulating DIAP1 autoubiquitinylation, but rather by directly degrading DIAP1.

The role of mitochondria in fly apoptosis remains highly controversial, due in large part to disagreement over whether mitochondria undergo losses in m and MOMP following stress (Kanuka et al, 1999; Zimmermann et al, 2002; Dorstyn et al, 2004; Senoo-Matsuda et al, 2005). Moreover, although mitochondrial release of cytochrome c in mammalian cells initiates formation of the Apaf-1 apoptosome complex and activation of caspases (Cain et al, 2002), there is disagreement over the importance of cytochrome c for promoting cell death in flies (Zimmermann et al, 2002; Arama et al, 2003, 2006; Dorstyn et al, 2004; Mendes et al, 2006). The cytochrome c debate notwithstanding, there are additional mitochondrial proteins in mammals that play a role in promoting apoptosis, including the dual IAP antagonist and serine protease, Omi/HtrA2 (Hegde et al, 2001; Martins et al, 2001; Suzuki et al, 2001; Verhagen et al, 2001). In our studies, we set out to determine if the Drosophila homologue of Omi might likewise participate in cell death. We found that dOmi was highly homologous to hOmi, particularly within the serine protease domain, and that its expression was developmentally regulated. dOmi was imported into fly mitochondria and processed in situ, resulting in the removal of its MTS and exposure of two distinct IBMs. The mature forms of dOmi were then released into the cytoplasm following stress, through both caspase-dependent and -independent processes. However, once in the cytosol, dOmi induced cell death in S2 cells and in the developing fly eye, primarily through proteolytic degradation of DIAP1 and likely other substrates.

Indeed, catalytically inactive 79-dOmi^S266A and 92-dOmi^S266A failed to induce significant apoptosis, which was somewhat surprising, given that both forms of dOmi selectively bound to the BIR2 domain in DIAP1 and displaced the initiator caspase DRONC. In particular, the affinity of 79-dOmi for BIR2 (K_d0.27 M) was lower than that observed for Rpr-IBM (K_d0.036 M), but was slightly higher than that observed for mature Smac with XIAP-BIR3 (K_d0.42 M) (Liu et al, 2000; Wu et al, 2001). So why did dOmi require its proteolytic activity to induce cell death, rather than inducing rapid IBM-dependent apoptosis? Notably, unlike other fly IAP antagonists, which exhibit partial preference for either the BIR1 or BIR2 domains, dOmi completely failed to bind the BIR1 domain in DIAP1 and did not displace the active effector caspase DrICE. Thus, it is possible that the continued inhibition of DrICE by DIAP1 was sufficient to inhibit cell death. There is precedence for such a scenario in mammals, as we have previously shown that XIAP mutants that fail to bind and inhibit caspase-9 can still prevent apoptosis through inhibition of caspase-3 alone (Bratton et al, 2002).

One of the primary differences between fly and mammalian IAP antagonists relates to their abilities to independently induce apoptosis. Indeed, Rpr, Hid, and Grim induce robust cell death in both cultured cells and flies (Kornbluth and White, 2005), whereas overexpression of mature Smac in the cytoplasm of mammalian cells generally fails to induce apoptosis in the absence of an accompanying prodeath stimulus (Du et al, 2000; Creagh et al, 2004). A potential explanation for these results may involve their relative capacities to induce RING-dependent autoubiquitinylation upon binding to IAPs. Indeed, while many IAP antagonists in the fly induce DIAP1 autoubiquitinylation, Smac appears to suppress XIAP autoubiquitinylation (Creagh et al, 2004). In our studies, dOmi failed to induce or suppress DIAP1 autoubiquitinylation upon binding to its BIR2 domain. Thus, in the absence of dOmi's proteolytic activity, DIAP1 may again be free to maintain its inhibition of DrICE via its BIR1 domain. By contrast, given that DIAP1 can protect cells by targeting active DRONC for proteosomal degradation (Wilson et al, 2002), it is also plausible that DIAP1 might regulate cell death, in part by, promoting the turnover of dOmi. Hay and co-workers have previously reported that the DIAP1 binding mutant, DRONC (F118E), induces significantly more cell death than wild-type DRONC, when expressed in the developing fly eye (Chai et al, 2003), and correspondingly, we found that 92-dOmi consistently produced a more severe phenotype than 79-dOmi, in accordance with their relative affinities for DIAP1.

Others have reconciled such differences between the mammalian and fly IAP antagonists by arguing that, in contrast to the Apaf-1caspase-9 apoptosome complex, the DARKDRONC apoptosome complex is constitutively active. Consequently, DIAP1 is required to continuously ubiquitinylate DRONC and mediate its turnover in order to prevent cell death (Muro et al, 2002). In this model, Rpr, Hid, or Grim need only displace this active DRONC, in order to promote the activation of effector caspases and induce apoptosis. However, recent studies suggest that, at least for Rpr and Grim, the C-terminus of these IAP antagonists play important roles in promoting both mitochondrial injury and/or inhibition of protein translation (Claveria et al, 2002; Holley et al, 2002). These alternative functions for Rpr and Grim may be necessary to first initiate caspase activation, after which the IBMs serve to displace these active caspases from DIAP1. Therefore, it could be that binding of dOmi to DIAP1-BIR2 per se does not induce apoptosis, because in the absence of another stimulus, there may be very little active DRONC to displace. In any event, regardless of whether dOmi induces cell killing solely through its proteolytic activity, or functions as a pure IAP antagonist in certain contexts, our studies suggest that mitochondria may play a far more important role in apoptosis in the fly than previously thought.

Bacterial and fly expression constructs

Full-length and truncated dOmi constructs were PCR amplified from an EST (AT14262; BDGP), using Pfu polymerase (Stratagene), and cloned into pRmHa3-myc (EcoRI–BamHI), pUAS (EcoRI–XhoI), or pET21b (NdeI–XhoI, Novagen) vectors for expression in Drosophila S2 cells, flies, and Escherichia coli strain BL21(DE3), respectively. Active-site (S266A), IBM, and cleavage-site mutations were introduced by site-directed mutagenesis (QuikChange^®, Stratagene). Similarly, the fly caspases, DRONC (residues 1–139) and full-length DrICE, were PCR amplified from ESTs (LD28292 and GH24292; BDGP) and cloned into the NdeI–XhoI and NcoI–XhoI sites of pET21b and pET28b, respectively. Full-length DIAP1 was generated by SOE-PCR using an EST (L49440) and a thread construct (kindly provided by Dr Colin S Duckett). DIAP1 and various truncations were then PCR amplified and cloned into pIE1-HA (BamHI–NotI, Novagen) or pGEX-4T-1 (EcoRI–NotI, Pharmacia) vectors for expression in S2 cells and E. coli, respectively.

Cell culture, transfections, and cell death assays

Drosophila S2 cells were routinely cultured at 28°C in HyQ SFX-Insect medium (Hyclone) supplemented with Glutamax (20 mM, Invitrogen). For transfections, 3 10⁶ cells were transfected (Cellfectin, 10 l, Invitrogen) with pRmHa3-myc plasmid DNA (1.5–2.0 g) encoding either wild-type, truncated, or mutant 79-dOmi or 92-dOmi proteins. For cell death experiments, cells were also cotransfected with pPAC-3-GFP (0.5 g) and then split 24 h post-transfection into multiwell-12 plates. Protein expression was then induced with CuSO₄ (0.7 mM), in the presence or absence of Z-VAD-fmk (50 M, Biomol). Cell death was assessed by flow cytometry (Beckman-Coulter FC500; _ex/_em=488/525 nm) at various time points by quantifying the percentage of intact GFP⁺ cells in the induced versus uninduced cell populations (i.e. [1-(GFP⁺_induced/GFP⁺_uninduced)] 100). The expression levels of dOmi and the various mutants were confirmed by Western blotting with a mouse anti-myc antibody (9B11, Cell Signaling).

Drosophila genetics

Transgenic flies were generated by the Transgenic Fly Core Facility of the Cutaneous Biology Research Center at Massachusetts General Hospital. Seven lines for UAS-79-dOmi and nine lines for UAS-92-dOmi were crossed to GMR-gal4 and scored for lethality and eye phenotypes, at 25°C. To score for suppression by p35, flies of the genotype UAS-p35/GMR-gal4; UAS-dOmi/TM6B were compared to SM1/GMR-gal4; UAS-dOmi/TM6B.

We thank Dr Janice Fischer for helpful advice, Dr John Sisson for fly embryos and antibodies to BiP, and Professor Hung-wen Liu and Zhihua Tao for recombinant PARP. This work was supported in part by start-up funds from The University of Texas at Austin, a grant from The American Cancer Society (RSG-05-029-01-CCG), and a Research Starter Grant from the Pharmaceutical Research and Manufacturers of America Foundation (all to SBB); BP was supported by the Massachusetts Biomedical Research Council Tosteson postdoctoral fellowship, and KW was supported in part by NIH grant GM55568.

Arama E, Agapite J, Steller H (2003) Caspase activity and a specific cytochrome c are required for sperm differentiation in Drosophila. Dev Cell 4: 687–697 | Article | PubMed | ISI | ChemPort |

Arama E, Bader M, Srivastava M, Bergmann A, Steller H (2006) The two Drosophila cytochrome c proteins can function in both respiration and caspase activation. EMBO J 25: 232–243 | Article | PubMed | ISI | ChemPort |

Bratton SB, Lewis J, Butterworth M, Duckett CS, Cohen GM (2002) XIAP inhibition of caspase-3 preserves its association with the Apaf-1 apoptosome and prevents CD95- and Bax-induced apoptosis. Cell Death Differ 9: 881–892 | Article | PubMed | ISI | ChemPort |

Cain K, Bratton SB, Cohen GM (2002) The Apaf-1 apoptosome: a large caspase-activating complex. Biochimie 84: 203–214 | Article | PubMed | ISI | ChemPort |

Chai J, Yan N, Huh JR, Wu JW, Li W, Hay BA, Shi Y (2003) Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination. Nat Struct Biol 10: 892–898 | Article | PubMed | ISI | ChemPort |

Claveria C, Caminero E, Martinez AC, Campuzano S, Torres M (2002) GH3, a novel proapoptotic domain in Drosophila Grim, promotes a mitochondrial death pathway. EMBO J 21: 3327–3336 | Article | PubMed | ISI | ChemPort |

Creagh EM, Murphy BM, Duriez PJ, Duckett CS, Martin SJ (2004) Smac/DIABLO antagonizes ubiquitin ligase activity of inhibitor of apoptosis proteins. J Biol Chem 279: 26906–26914 | Article | PubMed | ISI | ChemPort |

Danial NN, Korsmeyer SJ (2004) Cell death: critical control points. Cell 116: 205–219 | Article | PubMed | ISI | ChemPort |

Dorstyn L, Mills K, Lazebnik Y, Kumar S (2004) The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells. J Cell Biol 167: 405–410 | Article | PubMed | ISI | ChemPort |

Du C, Fang M, Li Y, Li L, Wang X (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102: 33–42 | Article | PubMed | ISI | ChemPort |

Fuentes-Prior P, Salvesen GS (2004) The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem J 384: 201–232 | Article | PubMed | ISI | ChemPort |

Goyal L, McCall K, Agapite J, Hartwieg E, Steller H (2000) Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function. EMBO J 19: 589–597 | Article | PubMed | ISI | ChemPort |

Hay BA, Wassarman DA, Rubin GM (1995) Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83: 1253–1262 | Article | PubMed | ISI | ChemPort |

Hays R, Wickline L, Cagan R (2002) Morgue mediates apoptosis in the Drosophila melanogaster retina by promoting degradation of DIAP1. Nat Cell Biol 4: 425–431 | Article | PubMed | ISI | ChemPort |

Hegde R, Srinivasula SM, Zhang Z, Wassell R, Mukattash R, Cilenti L, DuBois G, Lazebnik Y, Zervos AS, Fernandes-Alnemri T, Alnemri ES (2001) Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein–caspase interaction. J Biol Chem 277: 432–438 | Article | PubMed | ISI | ChemPort |

Holley CL, Olson MR, Colon-Ramos DA, Kornbluth S (2002) Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition. Nat Cell Biol 4: 439–444 | Article | PubMed | ISI | ChemPort |

Igaki T, Miura M (2004) Role of Bcl-2 family members in invertebrates. Biochim Biophys Acta 1644: 73–81 | Article | PubMed | ISI | ChemPort |

Jin S, Kalkum M, Overholtzer M, Stoffel A, Chait BT, Levine AJ (2003) CIAP1 and the serine protease HTRA2 are involved in a novel p53-dependent apoptosis pathway in mammals. Genes Dev 17: 359–367 | Article | PubMed | ISI | ChemPort |

Kanuka H, Sawamoto K, Inohara N, Matsuno K, Okano H, Miura M (1999) Control of the cell death pathway by Dapaf-1, a Drosophila Apaf-1/CED-4- related caspase activator. Mol Cell 4: 757–769 | Article | PubMed | ISI | ChemPort |

Kornbluth S, White K (2005) Apoptosis in Drosophila: neither fish nor fowl (nor man, nor worm). J Cell Sci 118: 1779–1787 | Article | PubMed | ISI | ChemPort |

Li W, Srinivasula SM, Chai J, Li P, Wu JW, Zhang Z, Alnemri ES, Shi Y (2002) Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi. Nat Struct Biol 9: 436–441 | Article | PubMed | ISI | ChemPort |

Lisi S, Mazzon I, White K (2000) Diverse domains of THREAD/DIAP1 are required to inhibit apoptosis induced by REAPER and HID in Drosophila. Genetics 154: 669–678 | PubMed | ISI | ChemPort |

Liu Z, Sun C, Olejniczak ET, Meadows RP, Betz SF, Oost T, Herrmann J, Wu JC, Fesik SW (2000) Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature 408: 1004–1008 | Article | PubMed | ISI | ChemPort |

Martins LM, Iaccarino I, Tenev T, Gschmeissner S, Totty NF, Lemoine NR, Savopoulos J, Gray CW, Creasy CL, Dingwall C, Downward J (2001) The serine protease Omi/HtrA2 regulates apoptosis by binding XIAP through a Reaper-like motif. J Biol Chem 277: 439–444 | Article | PubMed | ISI | ChemPort |

Means JC, Muro I, Clem RJ (2006) Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system. Cell Death Differ 13: 1222–1234 | Article | PubMed | ISI | ChemPort |

Meier P, Silke J, Leevers SJ, Evan GI (2000) The Drosophila caspase DRONC is regulated by DIAP1. EMBO J 19: 598–611 | Article | PubMed | ISI | ChemPort |

Mendes CS, Arama E, Brown S, Scherr H, Srivastava M, Bergmann A, Steller H, Mollereau B (2006) Cytochrome c–d regulates developmental apoptosis in the Drosophila retina. EMBO Rep 7: 933–939 | Article | PubMed | ISI | ChemPort |

Muro I, Hay BA, Clem RJ (2002) The Drosophila DIAP1 protein is required to prevent accumulation of a continuously generated, processed form of the apical caspase DRONC. J Biol Chem 277: 49644–49650 | Article | PubMed | ISI | ChemPort |

Olson MR, Holley CL, Gan EC, Colon-Ramos DA, Kaplan B, Kornbluth S (2003) A GH3-like domain in reaper is required for mitochondrial localization and induction of IAP degradation. J Biol Chem 278: 44758–44768 | Article | PubMed | ISI | ChemPort |

Ryoo HD, Bergmann A, Gonen H, Ciechanover A, Steller H (2002) Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nat Cell Biol 4: 432–438 | Article | PubMed | ISI | ChemPort |

Salvesen GS, Duckett CS (2002) IAP proteins: blocking the road to death's door. Nat Rev Mol Cell Biol 3: 401–410 | Article | PubMed | ISI | ChemPort |

Senoo-Matsuda N, Igaki T, Miura M (2005) Bax-like protein Drob-1 protects neurons from expanded polyglutamine-induced toxicity in Drosophila. EMBO J 24: 2700–2713 | Article | PubMed | ISI | ChemPort |

Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, Takahashi R (2001) A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell 8: 613–621 | Article | PubMed | ISI | ChemPort |

Tenev T, Zachariou A, Wilson R, Paul A, Meier P (2002) Jafrac2 is an IAP antagonist that promotes cell death by liberating Dronc from DIAP1. EMBO J 21: 5118–5129 | Article | PubMed | ISI | ChemPort |

Varkey J, Chen P, Jemmerson R, Abrams JM (1999) Altered cytochrome c display precedes apoptotic cell death in Drosophila. J Cell Biol 144: 701–710 | Article | PubMed | ISI | ChemPort |

Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ, Vaux DL (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102: 43–53 | Article | PubMed | ISI | ChemPort |

Verhagen AM, Silke J, Ekert PG, Pakusch M, Kaufmann H, Connolly LM, Day CL, Tikoo A, Burke R, Wrobel C, Moritz RL, Simpson RJ, Vaux DL (2001) HtrA2 promotes cell death through its serine protease activity and its ability to antagonise inhibitor of apoptosis proteins. J Biol Chem 277: 445–454 | Article | PubMed | ISI

Wilson R, Goyal L, Ditzel M, Zachariou A, Baker DA, Agapite J, Steller H, Meier P (2002) The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis. Nat Cell Biol 4: 445–450 | Article | PubMed | ISI | ChemPort |

Wing JP, Schreader BA, Yokokura T, Wang Y, Andrews PS, Huseinovic N, Dong CK, Ogdahl JL, Schwartz LM, White K, Nambu JR (2002) Drosophila Morgue is an F box/ubiquitin conjugase domain protein important for grim-reaper mediated apoptosis. Nat Cell Biol 4: 451–456 | Article | PubMed | ISI | ChemPort |

Wing JP, Schwartz LM, Nambu JR (2001) The RHG motifs of Drosophila Reaper and Grim are important for their distinct cell death-inducing abilities. Mech Dev 102: 193–203 | Article | PubMed | ISI | ChemPort |

Wu JW, Cocina AE, Chai J, Hay BA, Shi Y (2001) Structural analysis of a functional DIAP1 fragment bound to grim and hid peptides. Mol Cell 8: 95–104 | Article | PubMed | ISI | ChemPort |

Yan N, Wu JW, Chai J, Li W, Shi Y (2004) Molecular mechanisms of DrICE inhibition by DIAP1 and removal of inhibition by Reaper, Hid and Grim. Nat Struct Mol Biol 11: 420–428 | Article | PubMed | ISI | ChemPort |

Yang QH, Church-Hajduk R, Ren J, Newton ML, Du C (2003) Omi/HtrA2 catalytic cleavage of inhibitor of apoptosis (IAP) irreversibly inactivates IAPs and facilitates caspase activity in apoptosis. Genes Dev 17: 1487–1496 | Article | PubMed | ISI | ChemPort |

Yoo SJ, Huh JR, Muro I, Yu H, Wang L, Wang SL, Feldman RM, Clem RJ, Muller HA, Hay BA (2002) Hid, Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms. Nat Cell Biol 4: 416–424 | Article | PubMed | ISI | ChemPort |

Zachariou A, Tenev T, Goyal L, Agapite J, Steller H, Meier P (2003) IAP-antagonists exhibit non-redundant modes of action through differential DIAP1 binding. EMBO J 22: 6642–6652 | Article | PubMed | ISI | ChemPort |

Zhou L, Steller H (2003) Distinct pathways mediate UV-induced apoptosis in Drosophila embryos. Dev Cell 4: 599–605 | Article | PubMed | ISI | ChemPort |

Zimmermann KC, Ricci JE, Droin NM, Green DR (2002) The role of ARK in stress-induced apoptosis in Drosophila cells. J Cell Biol 156: 1077–1087 | Article | PubMed | ISI | ChemPort |