Protein Interactions and parallel pathways

Bruce can suppress Reaper dependent cell death

Mammalian Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain. Bruce upregulation occurs in some cancers and contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. However, it is still unknown whether Bruce inhibits apoptosis directly or instead plays some other more indirect role in mediating chemoresistance, perhaps by promoting drug export, decreasing the efficacy of DNA damage-dependent cell death signaling, or by promoting DNA repair. Using gain-of-function and deletion alleles, it has been demonstrated that Drosophila Bruce can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The Bruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. Drosophila Bruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1/Thread BIR2, be intact. Bruce does not block the activity of the apical cell death caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok. Together, these results argue that Bruce can regulate cell death at a novel point (Vernooy, 2002).

In Drosophila, the products of the reaper (rpr), head involution defective (hid), and grim genes are essential activators of caspase-dependent cell death. A genetic screen was carried out for suppressors of Rpr-, Hid-, and Grim-dependent cell death to identify regulators of their activity. Approximately 7000 new insertion lines of the GMREP P element transposon were generated. GMREP contains an engineered eye-specific enhancer sequence (GMR). This sequence is sufficient to drive the expression of linked genes in and posterior to the morphogenetic furrow during eye development. Thus, insertion of GMREP within a region can lead to the eye-specific expression of nearby genes. Each insertion line was crossed to flies that had small eyes due to the eye-specific expression of Rpr (GMR-Rpr flies), Hid (GMR-Hid flies), or Grim (GMR-Grim flies), and the progeny were scored for enhancement or suppression. A number of suppressors were identified. Five lines (GMREP-86A-1–5) mapped to the 86A region, and each strongly suppressed cell death induced by eye-specific expression of Rpr or Grim but not Hid. These lines mapped within a 6-kb interval. A number of other lines were obtained with P-element insertions located in the nearby region. Four of these, EP(3)0359, EP(3)0739, l(3)j8B6, and l(3)06142, mapped within six base pairs of the GMREP-86A-3–5 insertion sites. None of these, nor a fifth nearby line, l(3)06439, acted as a suppressor of GMR-Rpr-, GMR-Grim-, or GMR-Hid-dependent cell death. These results argue that the cell death suppression seen with the GMREP-86A lines was not due to a transposon-induced loss of function, but rather to the GMREP-dependent expression of a nearby gene. All of the GMREP-86A insertions were located 5' to a gene encoding the Drosophila homolog, Bruce, of murine Bruce (also known as Apollon in humans, suggesting this as an obvious candidate. The results of tissue in situ hybridizations with a Drosophila Bruce probe and immunocytochemistry with a Bruce-specific antibody support this possibility. Bruce transcript and protein are expressed at uniform low levels in wild-type eye discs. However, in the GMREP86A lines, they are expressed at high levels in and posterior to the morphogenetic furrow of the eye disc, which is where the GMR element drives expression (Vernooy, 2002).

To demonstrate that Bruce is responsible for the GMREP-86A-dependent suppression of Rpr- and Grim-dependent cell death, levels of the Bruce transcript were specifically downregulated in the eyes of flies carrying a GMR-Rpr transgene as well as a GMREP-86A element. Analysis focussed on one line, GMREP-86A-1, since all five lines behaved similarly with respect to cell death suppression and Bruce overexpression. Flies were generated that carried a Bruce RNA interference (RNAi) construct driven under GMR control (GMR-Bruce-RNAi flies). The eyes of GMR-Bruce-RNAi flies were normal. These animals were crossed to flies in which GMR-Rpr-dependent cell death was suppressed by the presence of the GMREP-86A-1 transposon and progeny from this cross were identified that carried all three transgenes, GMR-Bruce-RNAi, GMR-Rpr, and GMREP-86A-1. It was reasoned that if ectopic expression of Bruce in the eye, driven by the GMREP-86A-1 insertion, was responsible for the suppression of Rpr-dependent cell death, then expression of Bruce-RNAi should downregulate levels of the Bruce sense transcript. This should lead to an attenuation of the GMR-EP-86A-1-dependent suppression of Rpr-dependent cell death, causing a decrease in eye size. Such an attenuation was in fact observed. These observations, in conjunction with those obtained from studies with Bruce deletion mutants, argue that Bruce can suppress Rpr- and Grim-dependent cell death (Vernooy, 2002).

cDNAs encompasing the Bruce coding region were sequenced. This allowed an accurate map to be assembled of the Bruce exon-intron structure, which differs in some respects from that of the BDGP predicted gene. Overall, Bruce is 30% identical to murine Bruce. However, the Bruce N-terminal BIR domain and the C-terminal E2 domain show much higher degrees of homology, 83% and 86% identity, respectively. C. elegans homologs of Bruce were not apparent. Mutations in the Bruce gene were generated by carrying out imprecise excision of a P element, EP3731, located 3' to the Bruce transcript. Two deletions were generated that extended only in one direction, into the 3' end of the Bruce coding region. E12 deleted a relatively small region of the C terminus that includes the E2 domain, while E16 deleted approximately the C-terminal half of the Bruce coding region. Both lines were homozygous viable but male sterile. The possibility that E12 and E16 represent neomorphic mutations in Bruce cannot be excluded. However, the hypothesis that they represent hypomorphs or null mutations is favored, since they had the opposite phenotype of the GMREP-86A Bruce expression lines when in combination with GMR-Rpr, acting as enhancers rather than suppressors of Rpr-dependent cell death in the eye. E12 and E16 also enhanced GMR-Grim, but this effect is much more modest. E12 and E16 have no clear effect on cell death due to expression of Hid (Vernooy, 2002).

These results argue that endogenous Bruce levels, at least in the eye, are sufficient to act as a brake on Rpr-, and to some extent, Grim-dependent cell death. How does Bruce suppress apoptosis? A number of observations argue that Rpr- and Grim-dependent killing proceeds through distinct mechanisms and/or is regulated differently from those activities that is due to Hid. These differences are manifest at multiple points. At the level of DIAP1, point mutations of DIAP1 have effects on Rpr- and Grim-dependent cell death that are the opposite of those due to Hid. In addition, in a Drosophila extract, Hid, but not Rpr and Grim, promotes DIAP1 polyubiquitination. In contrast, in a different set of assays, Rpr and Grim, but not Hid, act as general inhibitors of protein translation. Finally, Rpr and Grim, but not Hid, show strong synergism with the effector caspase DCP-1 in terms of their ability to induce cell death in the eye. Each of these points defines a possible target for Bruce antiapoptotic action (Vernooy, 2002 and references therein).

Because Bruce strongly suppresses cell death induced by Rpr and Grim but not by Hid, one obvious possibility was that Bruce promotes Rpr and Grim ubiquitination and degradation. This hypothesis was tested by generating mutant versions of Grim and Rpr that lacked all lysines, the amino acid to which ubiquitin is added. These genes were introduced into flies under GMR control. GMR-Rpr-lys- and GMR-Grim-lys- flies have small eyes, indicating that these mutant proteins are effective cell death inducers. GMREP-86A-1-dependent Bruce expression suppresses this death very effectively, indicating that Bruce cannot be promoting ubiquitin-dependent degradation of Rpr or Grim. Interestingly, however, Bruce expression does not suppress cell death induced by expression of versions of Rpr (GMR-RprC) or Grim (GMR-GrimC) lacking their N termini, which are required for their IAP-caspase-disrupting interactions with the DIAP1 BIR2. This result is important because it argues that Bruce does not act to regulate this relatively uncharacterized death pathway (Vernooy, 2002).

The N-terminal Bruce BIR lacks a number of residues thought to be important for binding of Rpr, Hid, and Grim to DIAP1 BIR2. Thus, it seems unlikely that GMR-driven expression of Bruce inhibits cell death by simply titrating Rpr and Grim away from interactions with DIAP1 BIR2 as a result of similar interactions with the Bruce BIR. Nonetheless, the high degree of conservation between Bruce and mammalian Bruce in the BIR suggests that it is functionally important. To explore this role further, a fragment of Bruce that contained residues 1–531, including the BIR domain, was expressed under GMR control. Flies carrying this construct, GMR-Bruce-BIR flies, had normal appearing eyes, and in crosses to flies expressing GMR-Rpr, -Hid, or -Grim, GMR-Bruce-BIR did not enhance or suppress these eye phenotypes. These results do not rule out a role for the Bruce BIR in suppressing Rpr- and Grim-dependent cell death. However, they do suggest that the BIR alone is unlikely to mediate this inhibition (Vernooy, 2002).

Bruce overexpression in the eye also does not suppress cell death resulting from GMR-driven expression of the caspase Dronc, which is required for many apoptotic cell deaths in the fly, including those induced by expression of Rpr, Grim, and Hid. Dronc most resembles mammalian caspase-9, and its activation is likely to involve interactions with the Drosophila Apaf-1 homolog Ark. Thus, this result strongly suggests that Bruce does not block Ark-dependent Dronc activation or Dronc activity. This result is also suggested by the observation that decreasing Ark or Dronc in the eye strongly suppressed Hid-dependent cell death, which Bruce does not. A similar lack of cell death suppression is seen in the progeny of crosses between GMR-Bruce flies and flies expressing a second long prodomain caspase, Strica, whose mechanism of activation and normal functions are unknown. Finally, GMREP-86A-1 also fails to suppress the cell death due to GMR-dependent expression of the Drosophila proapoptotic Bcl-2 family member known variously as Debcl, Drob-1, dBorg-1, or Dbok (Vernooy, 2002).

Thus, the Bruce gene is found in mammals and flies, but not in the worm C. elegans. In humans, it is upregulated in some cell lines derived from gliomas and an ovarian carcinoma, and the results of antisense inhibition of Bruce suggest that it contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. The Drosophila homolog of Bruce, can potently inhibit cell death induced by Rpr and Grim but not Hid. In addition, flies with C-terminal deletions that removed the Bruce ubiquitin conjugation domain, or much larger regions of the coding region, acted as dominant enhancers of Rpr- and Grim-dependent, but not Hid-dependent, cell death. Together, these observations clearly demonstrate that Bruce can function as a cell death suppressor. The results with the deletion mutants suggest, but do not prove, that Bruce's death-inhibiting activity requires its function as a ubiquitin-conjugating enzyme. Based on the general conservation of cell death regulatory mechanisms, these results argue that mammalian Bruce is likely to facilitate oncogenesis by directly promoting cell survival in the face of specific death signals. One mechanism by which Rpr, Grim, and Hid promote apoptosis is by binding to DIAP1, thereby blocking its ability to inhibit caspase activity. It will be interesting to determine if mammalian Bruce also inhibits cell death induced by the expression of specific IAP binding proteins (Vernooy, 2002).

How does Bruce inhibit cell death? It does not promote the ubiquitination and degradation of Rpr and Grim directly. However, the possibility cannot be ruled out that Bruce somehow sequesters these proteins from their proapoptotic targets. The fact that it does not inhibit cell death due to Hid or Dronc expression argues that it is unlikely to be acting on core apoptotic regulators such as Ark, Dronc, or DIAP1, which are important for Hid-, Rpr-, and Grim-dependent cell death. An attractive hypothesis is that Bruce, perhaps in conjunction with apoptosis-inhibiting ubiquitin-protein ligases such as DIAP1 or DIAP2, promotes the ubiquitination and degradation of a component specific to Rpr- and Grim-dependent death signaling pathways. What might such a target be? Little is known about how Rpr- and Grim-dependent death signals differ from those due to Hid. However, one possibility is suggested by the recent observation that Rpr and Grim, but not Hid, can inhibit global protein translation. This creates an imbalance between levels of short-lived IAPs and the caspases they inhibit, thereby sensitizing cells to other death signals. Perhaps Bruce targets a protein(s) required for this activity (Vernooy, 2002).

Finally, Bruce is a very large protein, and thus its coding region might be expected to be subject to a relatively high frequency of mutation. Truncation of Bruce through the introduction of a stop codon or a frame shift is thus likely to be a relatively common form of Bruce mutation. The results of the deletion analysis show that C-terminal Bruce truncations act to enhance cell death in response to several different signals. Given this, it will be interesting to determine if human Bruce mutations are associated with a predisposition to pathologies that involve an inappropriate increase in cell death (Vernooy, 2002).


A new activator of apoptosis, grim, maps between two previously identified cell death genes in this region reaper (rpr) and head involution defective (hid). Expression of Grim RNA coincides with the onset of programmed cell death at all stages of embryonic development, whereas ectopic induction of grim triggers extensive apoptosis in both transgenic animals and in cell culture. Cell killing by Grim was blocked by coexpression of p35, a viral product that inactivates ICE-like proteases, and does not require the functions of rpr or hid. The predicted Grim protein shares an amino-terminal motif in common with RPR. However, Grim is sufficient to elicit apoptosis in at least one context, where RPR was not. The grim gene product might thus function in a parallel circuit of cell death signaling that ultimately activates a common set of downstream apoptotic effectors (Chen, 1996).

Reaper (Rpr), Hid, and Grim activate apoptosis in cells programmed to die during Drosophila development. Transient overexpression of Rpr in the lepidopteran SF-21 cell line induces apoptosis. Members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit Rpr-induced apoptosis and physically interact with Rpr through IAP family members' BIR (baculovirus IAP repeat) motifs. Transient overexpression of HID and GRIM also induces apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs block HID- and GRIM-induced apoptosis and also physically interact with them through the BIR motifs of the IAPs. The region of sequence similarity shared by Rpr, Hid, and Grim (the N-terminal 14 amino acids of each protein) is required for the induction of apoptosis by Hid and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of Hid and Grim are sufficient to induce apoptosis and confer IAP binding activity. However, Grim is more complex than HID since the C-terminal 124 amino acids of Grim retain apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within Grim. Coexpression of IAPs with Hid stabilizes Hid levels and results in the accumulation of Hid in punctate perinuclear locations that coincide with IAP localization. The physical interaction of IAPs with Rpr, Hid, and Grim provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins (Vucic, 1998).

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. grim expression is shown to induce apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. The conserved N-terminal domain is not required for either the initiation or the execution of apoptosis by Grim. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. Grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Three clustered aspartate residues near the grim C-terminus (positions 126, 128 and 129) could then be used by a caspase as a target sequence. The downshift predicted by digestion at these sites is between 1 and 1.3 kDa; however, the observed size for the short form is ~4 kDa less than that observed for the intact protein. Nevertheless, C-terminal deletions of the protein provoke downshifts greater than expected, which are in the range of the observed downshift for the proteolysed form of the protein. These results show that besides activating caspases, Grim itself can be processed as a consequence of caspase proteolytic activity. Even though inhibitors of apoptosis OpIAP and DEVD.cho do not block Grim-induced apoptosis, they are both functionally active in repressing endogenous caspases in either treated or transfected cells, since they prevented Grim cleavage. Therefore, it is likely that caspases insensitive to OpIAP and/or DEVD.cho are sufficient to achieve Grim-induced apoptosis (Clavería, 1998).

Grim-induced death is antagonized by bcl-2 in a dose-dependent manner; neither Fas signaling nor p53 are required for Grim pro-apoptotic activity. The sensitivity of the grim-induced death to bcl-2 levels suggests that Grim acts by activating a mitochondrial pathway. To determine the site of Grim action, its subcellular localization was explored in transfected cells prior to the time they show morphological symptoms of apoptosis. Pre-apoptotic fibroblasts simultaneously show Grim in two different cytoplasmic localizations: diffuse cytosolic and a punctate pattern. However, while Grim-transfected cells show an almost exclusive cytosolic localization, cells treated with cell-permeable broad specificity caspase inhibitor zVAD.fmk show predominantly the punctate pattern. The same pattern is observed in Grim-expressing p35-rescued cells. Co-localization of Grim protein with a mitochondrial-specific antibody shows that the punctate pattern corresponds to mitochondria. It is concluded that Grim localizes initially in the cytoplasm but accumulates progressively in the mitochondria, correlating with apoptosis progression. It is possible that Grim translocation to mitochondria is the event that triggers the apoptotic pathway. In that case, the increased mitochondrial localization of grim in zVAD-treated cells would be the result of the apoptosis blockade by zVAD at a point downstream of Grim incorporation in the mitochondria. These results show that Drosophila Grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases (Clavería, 1998).

It is possible that mitochondrial components of the mammalian cellular apoptotic machinery recognize the Drosophila Grim protein and respond by activating the apoptotic programme, as they would after the proper endogenous stimulus. In good agreement with this view, Grim action is counteracted by the overexpression of the anti-apoptotic factor bcl-2, which generally inhibits all mitochondrial death pathways. bcl-2 inhibits Grim-induced death in a dose-dependent manner, suggesting that they mutually antagonize to either promote or inhibit caspase activation. A bcl-2-like molecule has not yet been isolated in Drosophila, but mammalian and nematode members of this family have been shown to rescue ced-3- and Reaper-induced apoptosis in cultured fly cells. One possibility is that Grim promotes cytochrome c release from the mitochondria, as has been shown for Reaper in an in vitro amphibian system. However, Reaper does not induce apoptosis in mouse fibroblasts, arguing for a mechanism of action or regulation different from that of Grim. The activity of caspases insensitive to OpIAP, DEVD.cho and YVAD.cho is sufficient to execute the apoptosis induced by Grim. Caspase 9, which has been shown to transduce apoptotic mitochondrial signals, may be one of those activated by mitochondrial Grim. Nevertheless, group II caspases are activated as well, since Grim cleavage is inhibited by caspase inhibitors specific for this group and it cannot be excluded that activation of group II caspases may also serve to execute Grim-induced apoptosis. Inhibitor of apoptosis proteins (IAPs), the natural inhibitors of group II caspases, have been shown to inhibit apoptosis by directly binding to Grim, Reaper and Hid in a lepidopteran cell line. In contrast, neither OpIAP nor DEVD block Grim-induced death. These results, nevertheless, show that group II caspases are activated, suggesting that some of the Grim-induced caspase activation cascades are similar between mouse fibroblasts and cultured lepidopteran cells, whereas others may differ. It is possible that the particular caspase activation cascade triggered by grim depends more on the cell type in which it is expressed rather than on any intrinsic specificity of its action (Claveria, 1998).

The Drosophila reaper, head involution defective (hid), and grim genes play key roles in regulating the activation of programmed cell death. Two useful systems for studying the functions of these genes are the embryonic CNS midline and adult eye. In this study the Gal4/UAS targeted gene expression system was used to demonstrate that unlike reaper or hid, expression of grim alone is sufficient to induce ectopic CNS midline cell death. In both the midline and eye, grim-induced cell death is not blocked by the Drosophila anti-apoptosis protein Diap2, which does block both reaper- and hid-induced cell death. grim can also function synergistically with either reaper or hid to induce higher levels of midline cell death than those observed for any of the genes individually. Analysis was made of the function of a truncated Reaper-C protein, which lacks the NH2-terminal 14 amino acids that are conserved between Reaper, Hid, and Grim. Ectopic expression of Reaper-C reveals cell killing activities distinct from full length Reaper, and indicates that the conserved NH2-terminal domain acts in part to modulate Reaper activity (Wing, 1998).

The prototype baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) expresses p35, a potent anti cell-death gene that promotes the propagation of the virus by blocking host cell apoptosis. Infection of insect Sf-21 cells with AcMNPV lacking p35 induces apoptosis. This pro-apoptotic property of the p35 null virus was used to screen for genes encoding inhibitors of apoptosis that rescue cells infected with the p35 defective virus. Tn-IAP1, a novel member of the IAP family of cell death inhibitors, is described. Tn-IAP1 blocks cell death induced by p35 null AcMNPV, actinomycin D, and Drosophila cell-death inducers HID and GRIM. Given the conserved nature of the cell death pathway, this genetic screen can be used for rapid identification of novel inhibitors of apoptosis from diverse sources (Seshagiri, 1999).

Grim encodes a protein required for programmed cell death in Drosophila. The Grim N-terminus induces apoptosis by disrupting IAP blockage of caspases; however, N-terminally-deleted Grim retains pro apoptotic activity. This study describes GH3, a 15 amino acid internal Grim domain absolutely required for its proapoptotic activity and sufficient to induce cell death when fused to heterologous carrier proteins. A GH3 homology region is present in the Drosophila proapoptotic proteins Reaper and Sickle. The GH3 domain and the homologous regions in Reaper and Sickle are predicted to be structured as amphipathic alpha-helixes. During apoptosis induction, Grim colocalizes with mitochondria and cytochrome c in a GH3-dependent but N-terminal- and caspase activity-independent manner. When Grim is overexpressed in vivo, both the N-terminal and the GH3 domains are equally necessary, and cooperate for apoptosis induction. The N-terminal and GH3 Grim domains thus activate independent apoptotic pathways that synergize to efficiently induce programmed cell death (Clavería, 2002).

Secondary structure prediction of the Grim protein has identified three regions with a very high probability of conforming to an a-helical structure. These regions have been termed GH1, GH2 and GH3, for Grim Helix 1, 2 and 3. The GH3 domain shows similarity to a region in Reaper and Sickle, both also predicted to conform as an a-helix. The homology region spans the 15 amino acids predicted to conform as an a-helix in Grim, with two 5 amino acid regions of high similarity flanking a 5 amino acid central region with lesser homology. Representation in a helical wheel projection of GH3 residues and of those in the Reaper and Sickle homology regions reveals the amphipathic nature of the predicted a-helices (Clavería, 2002).

Tests were performed to see whether GH3 is important for Grim proapoptotic function by assaying the cell killing ability of several Grim mutants altered in the GH3 domain in Drosophila SL2 cells. Wild-type (WT) Grim induces cell death when overexpressed in this assay. In contrast, a Grim mutant form with a 13 amino acid deletion that removes the GH3 residues most reliably predicted to form an a-helix only marginally induces apoptosis. A 5'-shifted 11 amino acid deletion, such that the 3' part of GH3 was respected, was less effective in eliminating the proapoptotic activity than the complete GH3 deletion. An internal deletion removing four amino acids commonly deleted in the two larger deletions (Delta89-92) also resulted in strong impairment of Grim killing ability, but to a lesser extent than the complete GH3 deletion. The relevance was tested of L89, a conserved residue within positions 89-92, whose hydrophobicity could be relevant for the amphipathic nature of the GH3 domain. A non-conservative replacement of L89 by glutamic acid (L89E) impaired GH3 killing ability nearly to the same level as the Delta89-92 mutant. Semi-conservative replacement of L89 by alanine (L89A) had a mild effect on Grim proapoptotic function. These results show that the GH3 domain is required for Grim proapoptotic function and that L89 is a functionally relevant residue in the domain (Clavería, 2002).

Since N-terminally deleted Grim can still bind IAPs, GH3 domain function could be related to Grim's ability to bind IAPs and inhibit their protective function. Deletion of the Grim N-terminal domain was found to lead to a slight reduction in its ability to bind DIAP2, however, deletion of the GH3 domain, either alone or in combination with the N-terminal deletion, does not impair Grim's ability to bind DIAP2. These results suggest that GH3 activity is unrelated to the IAP inhibitory Grim activity (Clavería, 2002).

To determine whether the GH3 domain could function as a proapoptotic motif itself, a Grim fragment containing the GH3 domain was fused to green fluorescent protein (GFP) as a carrier protein (GH3-GFP) and the ability of this fusion protein to induce apoptosis in Drosophila SL2 cells was tested. GH3-GFP induces cell death with the same efficiency as does the complete Grim-GFP fusion protein. The cell death observed is specific to GH3 domain activity, since it is largely abolished by an L-to-E mutation in the residue equivalent to Grim L89. In all cases, cell death was rescued by coexpression with the baculoviral caspase inhibitor p35. The GH3 domain is therefore sufficient to trigger a specific proapoptotic route in SL2 cells (Clavería, 2002).

Immunocytochemistry was used to identify Grim subcellular localization in Drosophila SL2 cells. In phases previous to any obvious apoptotic phenotype, Grim generally shows a diffuse distribution in the cytoplasm, but also displays rings of stronger Grim staining. Grim rings are mitochondria-associated, as indicated by the presence of a mitochondrial matrix marker, mainly inside the rings, but also in colocalization with strong Grim staining. Grim colocalization with cytochrome c is similar to that observed with the mitochondrial matrix marker but, in addition, Grim and cytochrome c show extensive colocalization in larger dots. These larger cytochrome c dots are not observed in untransfected cells: they increase in size and abundance as apoptosis progresses, and do not colocalize with a mitochondrial marker. No substantial cytochrome c release to cytosol was found. Instead, Grim promotes redistribution of cytochrome c signal in large dots in colocalization with Grim itself (Clavería, 2002).

Grim subcellular localization is dependent on the presence of an intact GH3 domain. GH3-deficient Grim shows no obvious organization in rings associated with mitochondria and does not colocalize with either the mitochondrial marker or cytochrome c. In contrast, deletion of Grim 2-14 amino acids does not alter subcellular distribution of the Grim protein, nor the changes induced in cytochrome c display (Clavería, 2002).

The relevance of the GH3 and N-terminal Grim domains was examined by overexpressing the Grim mutants in transgenic flies using the Gal4-UAS system. To drive Grim expression, the GMR-Gal4 line, which targets expression to the eye disc, and the MS1096-Gal4 line, which directs expression to the wing imaginal disc, were used. Overexpression of WT Grim with either driver causes total or partial lethality in all transgenic lines. These results suggested that leaky expression from both promoters in vital tissues produces sufficient cell death to block development. Surviving adult flies overexpressing WT Grim display a considerable reduction in eye size with the GMR driver, and wing agenesis plus notum reduction and elimination of macro- and microchaetae with the MS1096 driver (Clavería, 2002).

In contrast to these results, overexpression of a GH3-deleted Grim (Delta86-98) results in very low lethality, as well as rescue of the eye, notum and wing phenotypes. Elimination of amino acids 89-92 results in a lesser impairment of Grim killing ability, showing that the 3' part of the GH3 domain is important for proapoptotic function. In correlation with these results, the Delta89-92 mutant rescues the eye phenotype induced by WT Grim, but only partially rescues the more sensitive wing and notum phenotypes. Elimination of amino acids 83-93, which extends the deletion N-terminal to the putative helical domain, does not increase the rescue observed with the 89-92 deletion, suggesting that residues 5' of leucine 89 might be less important for proapoptotic function. The relevance of leucine 89 was again shown by the significant rescue of viability and of eye, notum and wing phenotypes in flies overexpressing the non-conservative L89E substitution. In contrast, the semi-conservative substitution L89A results in mild, but significant, impairment of Grim death induction and targeted tissue deletion (Clavería, 2002).

In accordance with the results observed in cultured cells, mutations of the GH3 domain impairs Grim proapoptotic activity in transgenic flies. Deletion of the N-terminal domain, in contrast to the results observed in cultured cells, results in highly significant elimination of Grim proapoptotic function in vivo. Both viability and appearance of the tissues targeted by the GMR and MS1096 drivers were rescued by the 2-14 deletion to a level similar to that observed for the GH3 deletion. Simultaneous deletion of the N-terminus and amino acids 89-92 of the GH3 domain results in even lower lethality and fewer alterations in the targeted tissues than those induced by each deletion in isolation (Clavería, 2002).

To determine whether the N-terminal and GH3 Grim domains can function independently of each other, tests were performed to see whether the simultaneous expression of independent 2-14- and GH3-deleted Grim proteins could induce apoptosis. Flies were generated carrying independent transgenes for 2-14 and 86-98 Grim deletion mutants driven by MS1096 expression. Whereas males carrying either protein alone showed little or no lethality and no alterations in wing development, double transgenic males simultaneously expressing Delta2-14 and Delta86-98 Grim mutants display severe lethality and reduced wings. Females did not display lethality in any situation, but frequently showed reduced wings in the double transgenics, although not in single transgenics. Functions of both the N-terminal and the GH3 domains are therefore essential for Grim activity in vivo and they independently activate specific death mechanisms that synergize to trigger apoptosis (Clavería, 2002).

Several lines of evidence point to the mitochondrial-cytochrome c pathway as the target of GH3 action. Grim associates with mitochondria in colocalization with cytochrome c and this activity resides in the GH3 domain. In vertebrate cells, Grim targets the mitochondria and induces cytochrome c release in a GH3-dependent and N-terminal-, caspase- and IAP-independent manner, suggesting functional conservation of the pathway. Interestingly, during apoptosis induction by Grim and Reaper in flies, changes in cytochrome c display are observed, rather than its free release to the cytosol as in vertebrates. Grim-expressing cells specifically show large cytoplasmic deposits of cytochrome c at sites where Grim itself is present, but other mitochondrial markers are not. The changes observed in the distribution of cytochrome c may result from its relocation from mitochondria to hypothetical specialized cytoplasmic structures involved in apoptosis induction. Alternatively, the apoptosome might be formed in the vicinity of the mitochondria, and cytochrome c deposits may constitute the remnants of damaged mitochondria, which have lost some of their constitutive components, but retain cytochrome c and Grim. The relevance of the cytochrome c proapoptotic pathway in Drosophila PCD is supported as well by the observation that elimination of Dark, a Drosophila homolog of Apaf-1 that mediates cytochrome c-primed apoptosis, impairs Reaper, Hid and Grim killing in flies. Even though no cytochrome c free release appears to take place in Drosophila cells, it is possible that a mechanism homologous to that of vertebrate cells is activated, but from different subcellular compartments (Clavería, 2002).

The involvement of the GH3 domain in a mitochondrial pathway and its predicted structure, an amphipathic a-helix, resemble the characteristics of the widespread proapoptotic BH3 domain. These similarities could be interpreted as functional homology between the two pathways; however, no association has been detected between Grim and either mammalian (Bcl-2 and Bcl-xL) or insect (Debcl) Bcl-2-family members, as would be expected for a BH3-containing protein. Rather than representing homologous proapoptotic pathways, BH3 and GH3 domains may have converged during evolution to a similar proapoptotic mechanism. Since BH3-containing proteins coexist in Drosophila with GH3-containing proteins, the two pathways may operate in alternative routes, or even cooperate in apoptosis induction, not only in Drosophila, but perhaps also in other species (Clavería, 2002).

Two independent pathways may thus be triggered by Grim; an IAP inhibitory pathway activated by the N-terminal domain, and a mitochondrial-cytochrome c route activated by the GH3 domain. Either pathway could be alternatively or simultaneously promoted by Grim, and the relevance of each may depend on cellular context. The presence of a GH3 homology region in Reaper and Sickle suggests functional conservation of this domain in at least these other two Drosophila proapoptotic proteins. In this context, it is important to consider that Reaper promotes cytochrome c release in a cell-free Xenopus egg extract and does not require the N-terminal domain for this function (Clavería, 2002).

Although Reaper, Hid, Sickle and Grim induce specific apoptotic pathways in vertebrate cells, and in the fly participate in highly conserved routes, such as the p53 and Ras-MAPK pathways, no homolog for these proteins has been yet identified in any other organism. The vertebrate Smac/Diablo protein may, however, represent a functional homolog of the IAP inhibitory pathway. Smac/Diablo can bind to and block the protective effect of IAPs. However, it is unlikely that Smac/Diablo represent homologs of the mitochondrial-cytochrome c pathway. Database searches have failed to identify any protein with sequence similarity to the GH3 domain but, given the restricted sequence conservation among, for example, BH3 family members, this does not exclude conservation of this pathway. Whether vertebrate proapoptotic proteins exist that represent direct or functional homologs of the GH3 proapoptotic activity thus remains to be determined (Clavería, 2002).

Thread and IAPs

Induction of apoptosis in Drosophila requires the activity of three closely linked genes: reaper, hid and grim. The proteins encoded by reaper, hid and grim activate cell death by inhibiting the anti-apoptotic activity of the Drosophila IAP1 (Diap1, also known as Thread) protein. In a genetic modifier screen, both loss-of-function and gain-of-function alleles in the endogenous diap1 gene were obtained, and the mutant proteins were functionally and biochemically characterized. Gain-of-function mutations in diap1 strongly suppress reaper-, hid- and grim-induced apoptosis. Sequence analysis of these diap1 alleles reveals that they are caused by single amino acid changes in the baculovirus IAP repeat domains of Diap1, a domain implicated in binding Reaper, Hid and Grim. Significantly, the corresponding mutant Diap1 proteins display greatly reduced binding of Reaper, Hid and Grim, indicating that Reaper, Hid and Grim kill by forming a complex with Diap1. Collectively, these data provide strong support for the idea that Reaper, Hid and Grim kill by inhibiting DIAP1's ability to antagonize caspase function (Goyal, 2000).

It is thought that the previously proposed function of IAPs upstream of reaper, hid and grim is simply an artifact of unphysiologically high levels of protein expression in heterologous systems. When IAP expression constructs are introduced into cultured cells under the control of strong promoters and at high copy numbers, the levels of proteins expressed far exceed those of the endogenous cellular IAP proteins. Under these unphysiological conditions, cellular IAPs can display properties that do not reflect their normal mechanism of action. In particular, the current results demonstrate that mutant proteins that completely lack anti-apoptotic activity in vivo can still inhibit cell death in vitro as long as they can bind to Reaper, Hid and Grim. Conversely, gain-of-function diap1 alleles that display reduced binding to Reaper, Hid and Grim have strongly increased anti-apoptotic function in vivo, but show reduced protection in heterologous cell transfection assays. These results clearly reveal the limitations of overexpression studies in cultured cells for determining the normal mechanism of action of these proteins in the cell death pathway (Goyal, 2000).

Dronc (Nedd2-like caspase) was isolated through its interaction with the effector caspase drICE (Ice). Ectopic expression of Dronc induces cell death in Schizosaccharomyces pombe, mammalian fibroblasts and the developing Drosophila eye. The caspase inhibitor p35 fails to rescue Dronc-induced cell death in vivo and is not cleaved by Dronc in vitro, making Dronc the first identified p35-resistant caspase. The Dronc pro-domain interacts with Drosophila inhibitor of apoptosis protein 1 (Diap1: known as Thread), and co-expression of DIAP1 in the developing Drosophila eye completely reverts the eye ablation phenotype induced by pro-Dronc expression. In contrast, Diap1 fails to rescue eye ablation induced by Dronc lacking the pro-domain, indicating that interaction of Diap1 with the pro-domain of Dronc is required for suppression of Dronc-mediated cell death. Heterozygosity at the Diap1 locus enhances the pro-Dronc eye phenotype, consistent with a role for endogenous Diap1 in suppression of Dronc activation. Both heterozygosity at the Dronc locus and expression of dominant-negative Dronc mutants suppress the eye phenotype caused by Reaper (Rpr) and Head involution defective (Hid), consistent with the idea that Dronc functions in the Rpr and Hid pathway (Meier, 2000).

The finding that Diap1 directly binds to and inhibits cell death caused by ectopic expression of Dronc, as well as by Rpr, Grim and Hid, underscores the key role played by Diap1 in the regulation of apoptosis in D. melanogaster and raises the possibility that Rpr, Hid or Grim may exert some, or all, of their pro-apoptotic action through displacement of Diap1 from the pro-domain of Dronc, thereby allowing activation of the caspase and consequent cell death. This idea is strongly supported by the successful isolation of Diap1 mutants that display greatly reduced binding for Rpr, Hid and Grim and significantly suppress Rpr, Hid and Grim cell killing. According to this model, IAPs function as 'guardians' of the apoptotic machinery: they act to suppress the chance of spontaneous activation of the intrinsic cell death machinery by neutralizing pro-apoptotic caspases, thereby establishing a buffered threshold that must be either exceeded or neutralized in order to initiate the destruction of a cell (Meier, 2000).

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 drICE and DCP-1 and 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 drICE. 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).

It is interesting to note that th7, which acts as a very strong LOF mutation and seems to show some dominant-negative properties, has only the BIR1 attached to the spacer and ring domains. Thus, despite the extensive homologies between the two BIR domains of the protein, a single BIR is not sufficient for Th/Diap1 function, at least in the presence of an attached ring domain. BIR2 of Th/Diap1 and Op-IAP, as well as the single BIR of survivin, are able to inhibit apoptosis (Lisi, 2000).

Again, on the basis of the model above, the GOF mutations identified would be predicted to bind to caspases, but not to the inducers. The thSL mutation maps to a weakly conserved residue in BIR1 and does not result in increased th protein levels. This suggests that BIR1 is important for Rpr and Grim binding, but not for Hid binding, as Hid activity is unaffected in this mutation. Even in the context of overexpression in the eyes of transgenic flies, this mutant IAP retains some specificity for Rpr and Grim killing. This implies that the simple model of BIR2 binding to the conserved NH2-terminal sequences of Rpr, Grim, and Hid is not accurate, and that other residues in the protein are differentially important for Rpr and Grim, as opposed to Hid binding (Lisi, 2000).

The importance of regions outside of BIR2 for Diap1 activity is supported by the analysis of the GOF1 class of mutations, th6B and th81.03. Both of these mutations suppress Hid killing and would be predicted to inhibit Hid binding. These mutations change conserved cysteines in the ring domain to tyrosines. This suggests that the ring is important for Hid/Diap1 interaction. However, the region of Hid binding to Diap1 and Op-IAP has been mapped to BIR2, while the ring does not show any ability to bind to Hid. In addition, mutations in the ring, including those in conserved cysteines, have little effect on the ability of Op-IAP to protect against Hid killing. These data, together with the finding that both GOF1 mutations are cysteine-to-tyrosine changes, suggest that these mutations might have a novel ability to interfere with binding of Hid to BIR2. In addition, the observation that the GOF1 mutations slightly enhance Rpr and Grim killing suggests that these mutants are less potent inhibitors of caspases. This might result from weaker binding to caspases or from proteins that are slightly less stable. This second attribute would be predicted to enhance killing by any inducer that binds the IAP, but not to have an effect on Hid, which is unable to bind (Lisi, 2000 and references therein).

In conclusion, the data support a model where Rpr, Grim, and Hid interact with Th/Diap1 to induce apoptosis. Mutations that affect killing by Rpr and Grim or by Hid can be isolated, indicating that these inducers interact with Th/Diap1 in different ways. The GOF mutations that have been identified also provide useful tools to examine the roles of IAPs, rpr, grim, and hid during Drosophila development. The other Drosophila IAP homolog, DIAP2, has been shown to selectively inhibit Rpr- and Hid-induced but not Grim-induced death (Lisi, 2000).

In LOF th alleles, a developmental arrest occurs at the blastoderm stage and, subsequently, a synchronous apoptosis of all the nuclei. Earlier reports that homozygous th embryos show no ectopic apoptosis probably reflects the very early stage at which this apoptosis occurs. At this time, a direct requirement for th to block apoptosis cannot be distinguised from a requirement for th in another developmental process. This developmental defect could then result in secondary apoptosis. The latter possibility is reasonable, as many failures in development result in ectopic apoptosis. A BIR containing protein from Caenorhabditis elegans is required for cytokinesis in embryos. However, it is also possible that developmental arrest occurs as a result of the initiation of apoptosis, which is manifest only as DNA damage several hours later (Lisi, 2000).

Does this early requirement for th reflect a need to inhibit apoptosis induced by rpr, grim, and hid? Double mutants of th and Df(3L)H99, the deletion that removes rpr, grim, and hid, show a phenotype similar to th alone. This indicates that Th/Diap1 is not required to suppress zygotic Rpr, Grim, and Hid activity. However, hid and rpr mRNA can be seen in a subset of cells in the blastoderm embryo, as judged by in situ analysis. This may indicate that these gene products are supplied maternally. Th/Diap1 may be required to suppress maternally supplied Rpr, Grim, or Hid. Allelic differences in the stage at which apoptosis begins in the th mutants parallel the general ability of the alleles to inhibit apoptosis induced by Rpr, Hid, and Grim. The strong LOF alleles arrest at the blastoderm stage; the GOF1 alleles arrest much later, and the GOF2 allele is completely viable (Lisi, 2000).

Expression of the cell death regulatory protein Reaper (RPR) in the developing Drosophila eye results in a smaller than normal eye owing to excess cell death. Mutations in thread (th) are dominant enhancers of RPR-induced cell death. thread encodes a protein homologous to baculovirus inhibitors of apoptosis (IAPs), called Drosophila IAP1 (DIAP1). Overexpression of DIAP1 or a related protein, DIAP2, in the eye suppresses normally occurring cell death as well as death due to overexpression of rpr or head involution defective. IAP death-preventing activity localizes to the N-terminal baculovirus IAP repeats, a motif found in both viral and cellular proteins associated with death prevention (Hay, 1995).

The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. iap can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this hypothesis, Drosophila and human genes that encode IAP-like proteins (dILP and hILP) have been isolated. Like IAP, both dILP and hILP contain amino-terminal baculovirus IAP repeats (BIRs) and carboxy-terminal RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several human genes related to iap. Together these data suggest that iap and related cellular genes play an evolutionarily conserved role in the regulation of apoptosis (Ducket, 1996).

IAPs comprise a family of inhibitors of apoptosis found in viruses and animals. In vivo binding studies demonstrate that both baculovirus and Drosophila IAPs physically interact with an apoptosis-inducing protein of Drosophila [Reaper (RPR)] through their baculovirus IAP repeat (BIR) region. Expression of IAPs block RPR-induced apoptosis and results in the accumulation of RPR in punctate perinuclear locations, which coincide with IAP localization. When expressed alone, RPR rapidly disappears from the cells undergoing RPR-induced apoptosis. Expression of P35, a caspase inhibitor, also blocks RPR-induced apoptosis and delays RPR decline, but RPR remains cytoplasmic in its location. Mutational analysis of RPR demonstrates that caspases are not directly responsible for RPR disappearance. The physical interaction of IAPs with RPR provides a molecular mechanism for IAP inhibition of RPR's apoptotic activity (Vucic, 1997a).

Members of the transforming growth factor-beta superfamily bind to two different types of serine/threonine kinase receptors, termed type I and type II. Type I receptors act as downstream components of type II receptors in the receptor complexes. Therefore, intracellular proteins that interact with the type I receptors are likely to play important roles in signaling. Drosophila inhibitor of apoptosis (Diap) 1 has been identified as an interacting protein of Thick veins (Tkv), a Dpp type I receptor. Diap1 associates with Tkv in vivo. The binding region in Diap1 is mapped to its C-terminal RING finger region. Diap2, another Drosophila member of the inhibitor of apoptosis protein family, also interacts with Tkv in vivo. These data suggest that Diap1 and Diap2 may be involved, possibly as negative regulators, in the Dpp signaling pathway, which leads to cell apoptosis. Tkv may induce apoptosis by suppressing the DIAP1 function (Oeda, 1998).

Ectopic expression of Reaper or Grim induces substantial apoptosis in mammalian cells. Reaper- or Grim-induced apoptosis is inhibited by a broad range of caspase inhibitors and by human inhibitor of apoptosis proteins cIAP1 and cIAP2. Additionally, in vivo binding studies demonstrate that both Reaper and Grim physically interacte with human IAPs through a homologous 15-amino acid N-terminal segment. Deletion of this segment from either Reaper or Grim abolishes binding to cIAPs. In vitro binding experiments indicate that Reaper and Grim bind specifically to the BIR domain-containing region of cIAPs, since deletion of this region results in loss of binding. The physical interaction has been further confirmed by immunolocalization. When co-expressed, Reaper or Grim co-localize with cIAP1. However, deletion of the N-terminal 15 amino acids of Reaper or Grim abolishes co-localization with cIAP1, suggesting that this homologous region can serve as a protein-protein interacting domain in regulating cell death. Moreover, by virtue of this interaction, it has been demonstrated that cIAPs can regulate Reaper and Grim by abrogating their ability to activate caspases and thereby inhibit apoptosis. This is the first function attributed to this 15-amino acid N-terminal domain, which is the only region having significant homology between these two Drosophila death inducers (McCarthy, 1998).

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

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

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

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

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

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

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

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

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

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

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

Although Jun amino-terminal kinase (JNK) is known to mediate a physiological stress signal that leads to cell death, the exact role of the JNK pathway in the mechanisms underlying intrinsic cell death is largely unknown. Through a genetic screen, it has been shown that a mutant of Drosophila Tumor-necrosis factor receptor-associated factor 1 (DTRAF1) is a dominant suppressor of Reaper-induced cell death. Reaper modulates the JNK pathway through Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), which negatively regulates DTRAF1 by proteasome-mediated degradation. Reduction of JNK signals rescues the Reaper-induced small eye phenotype, and overexpression of DTRAF1 activates the Drosophila ASK1 (apoptosis signal-regulating kinase 1; a mitogen-activated protein kinase kinase kinase) and JNK pathway, thereby inducing cell death. Overexpresson of DIAP1 facilitates degradation of DTRAF1 in a ubiquitin-dependent manner and simultaneously inhibits activation of JNK. Expression of Reaper leads to a loss of DIAP1 inhibition of DTRAF1-mediated JNK activation in Drosophila cells. Taken together, these results indicate that DIAP1 may modulate cell death by regulating JNK activation through a ubiquitin-proteasome pathway (Kuranaga, 2002).

Cell death in higher organisms is negatively regulated by Inhibitor of Apoptosis Proteins (IAPs), which contain a ubiquitin ligase motif, but how ubiquitin-mediated protein degradation is regulated during apoptosis is poorly understood. Drosophila IAP1 (DIAP1) auto-ubiquitination and degradation is actively regulated by Reaper (Rpr) and UBCD1. Rpr, but not Hid (Head involution defective), promotes significant DIAP1 degradation. Rpr-mediated DIAP1 degradation requires an intact DIAP1 RING domain. Among the mutations affecting ubiquitination, ubcD1 was found to suppresses rpr-induced apoptosis. UBCD1 and Rpr specifically bind to DIAP1 and stimulate DIAP1 auto-ubiquitination in vitro. These results identify a novel function of Rpr in stimulating DIAP1 auto-ubiquitination through UBCD1, thereby promoting its degradation (Ryoo, 2002).

In most cases, apoptotic cell death culminates in the activation of the caspase family of cysteine proteases, leading to the orderly dismantling and elimination of the cell. The IAPs (inhibitors of apoptosis) comprise a family of proteins that oppose caspases and thus act to raise the apoptotic threshold. Disruption of IAP-mediated caspase inhibition has been shown to be an important activity for pro-apoptotic proteins in Drosophila (Reaper, HID, and Grim) and in mammalian cells (Smac/DIABLO and Omi/HtrA2). In addition, in the case of the fly, these proteins are able to stimulate the ubiquitination and degradation of IAPs by a mechanism involving the ubiquitin ligase activity of the IAP itself. The Drosophila RHG proteins (Reaper, HID, and Grim) are themselves substrates for IAP-mediated ubiquitination. This ubiquitination of Reaper requires IAP ubiquitin-ligase activity and a stable interaction between Reaper and the IAP. Additionally, degradation of Reaper can be blocked by mutating its potential ubiquitination sites. Most importantly, regulation of Reaper by ubiquitination has been shown to be a significant factor in determining Reaper biological activity. These data demonstrate a novel function for IAPs and suggest that IAPs and Reaper-like proteins mutually control each other's abundance (Olson, 2003a).

Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction. Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release. Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function. This domain, although required for IAP destabilization, is not sufficient. Moreover, a small region of Reaper, similar to the GH3 domain of Grim, has been identified that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing. Although a mutant Reaper protein lacking the GH3 domain is deficient in these properties, these defects can be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-xL or a homologous region from the pro-apoptotic protein HID. Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells (Olson, 2003b).

Death executioner Bcl-2 homolog

The Bcl-2 family of proteins consists of antiapoptotic and proapoptotic members, both of which control the cell-death decision by regulating such processes as mitochondrial cytochrome c release and caspase activation through adapter protein Apaf-1 (Drosophila homolog: Apaf-1-related-killer), and/or by neutralizing the effects of opposing Bcl-2 family members. The first Drosophila Bcl-2 protein to be described has been termed it Debcl (pronounced debacle) for Death executioner Bcl-2 homolog. After screening a number of UAS-debcl lines, two lines (UAS-debcl#26 on chromosome III and UAS-debcl#18 on chromosome II) were found that, when crossed to GMR-GAL4, give rise to adults with severely ablated eyes. To use this phenotype to examine genetic interactions, a stock was generated containing GMR-GAL4 (2nd chromosome) and UAS-debcl#26. To examine whether the rough eye phenotype is due to the activity of caspases, GMR-p35 was crossed to these flies and the eye phenotype of the progeny was examined. GMR-p35 significantly improves the severe rough eye phenotype of GMR-GAL4; UAS-debcl#26 eyes. These results confirm that Debcl functions in a caspase-dependent fashion upstream of caspase activation (Colussi, 2000).

To determine the involvement of rpr, hid, or grim in the GMR-GAL4; UAS-debcl#26 eye phenotype, these flies were crossed to a deficiency that removes all three genes (Df(3L)H99). If rpr, hid, or grim are rate limiting for Debcl function, then suppression of the GMR-GAL4; UAS-debcl#26 eye phenotype would be expected. However, no significant suppression of this phenotype was observed, suggesting that the GMR-GAL4; UAS-debcl#26 eye phenotype is not dependent on the gene dosage of rpr, hid, or grim (Colussi, 2000).

Next, a test was performed to see whether the inhibitor of apoptosis (IAP) homolog, diap1, genetically interacts with debcl, by examining the GMR-GAL4; UAS-debcl#26 eye phenotype when the dosage of diap1 is halved. Halving the dosage of diap1, using two different deficiencies, results in a strong enhancement of the GMR-GAL4; UAS-debcl#26 eye phenotype. Furthermore, there is a significant reduction in the number of flies expected to contain either of the diap1 deficiencies and GMR-GAL4; UAS-debcl#26. This is possibly due to leaky expression of the GMR-GAL4; UAS-debcl#26 construct in other tissues during development and the enhancement of this effect by reducing the dose of diap1. Thus, diap1 genetically interacts with debcl. No genetic interaction between debcl and diap2 was observed when a diap2 deficiency was crossed with GMR-GAL4; UAS-debcl#26 (Colussi, 2000).

Shaker K+ channel

Drosophila genes reaper, grim, and head-involution-defective (hid) induce apoptosis in several cellular contexts. Voltage-dependent Shaker (Sh) K+ channels open in response to depolarization and subsequently undergo N-type inactivation by a "ball and chain" mechanism. The 20 N-terminal residues of the ShB channel form the inactivation "ball," which is tethered to membrane-spanning channel domains by the following ~200-residue "chain." Inactivation occurs when the N-terminal inactivation ball physically occludes the inner pore of the channel from the cytoplasmic side. Stability of the inactivated state is enhanced by the hydrophobicity of approximately the first 10 residues of the inactivation ball, whereas positively charged amino acids within the following 10 residues promote entry into the inactivated state via electrostatic interactions. Deletions in the distal N terminus of the channel disrupt inactivation, which can be reversed by application of a 20-residue synthetic peptide corresponding to the initial N-terminal sequence of the channel. Ancillary beta subunits in some K+ channel complexes serve to produce N-type inactivation by a similar mechanism. The conserved N-terminal sequences of Reaper, Grim, and Hid resemble those N-terminal Sh K+ channel domains that are involved in inactivation. This sequence similarity led to the hypothesis that Reaper, Grim, and Hid facilitate initiation of apoptosis by inducing inactivation of K+ channels. Sustained inactivation of K+ channels will result in chronic membrane depolarization that may lead to the initiation of the caspase-dependent apoptotic program, perhaps by increasing the level of cytosolic free Ca2+. Synthetic Reaper and Grim N terminus peptides are shown to induce fast inactivation of Shaker-type K+ channels when applied to the cytoplasmic side of the channel that is qualitatively similar to the inactivation produced by other K+ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper also reduced the synthetic peptide's ability to induce channel inactivation, indicating that K+ channel inactivation correlates with apoptotic activity. Coexpression of Reaper RNA or direct injection of full length Reaper protein causes near irreversible block of the K+ channels. These results suggest that Reaper and Grim may participate in initiating apoptosis by stably blocking K+ channels (Avdonin, 1998).

Head involution defective

Deletion of chromosomal region 75C1,2 blocks virtually all programmed cell death (PCD) in the Drosophila embryo. A second gene in this region, head involution defective (hid) plays a similar role in PCD. hid mutant embryos have decreased levels of cell death and contain extra cells in the head. The hid gene expression is sufficient to induce PCD in cell death defective mutants. The hid gene encodes a novel 410-amino-acid protein, and its mRNA is expressed in regions of the embryo where cell death occurs. Ectopic expression of hid in the Drosophila retina results in eye ablation. This phenotype can be suppressed completely by expression of the anti-apoptotic p35 protein from baculovirus, indicating that p35 may act genetically downstream from hid (Grether, 1995).

Interleukin-1 beta converting enzyme (ICE)-like proteases

Genetic studies indicated that the Drosophila protein Reaper controls apoptosis during embryo development. Induction of RPR expression in Drosophila Schneider cells rapidly stimulates apoptosis. RPR-mediated apoptosis is blocked by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), which suggests that an interleukin-1 beta converting enzyme (ICE)-like protease is required for RPR function. RPR-induced apoptosis is associated with increased ceramide production that is also blocked by Z-VAD-fmk, suggesting that ceramide generation requires an ICE-like protease as well. Thus, the intracellular RPR protein uses cell death signaling pathways similar to those used by the vertebrate transmembrane receptors Fas (CD95) and tumor necrosis factor receptor type 1 (Pronk, 1996).

While Caenorhabditis elegans has only a single identified caspase, CED-3, whose activity is absolutely required for all developmental programmed cell deaths, most mammalian cell types express multiple caspases with varying specificities. The fruit fly possesses two known caspases: DCP-1 and drICE. The role of drICE was examined in in vitro apoptosis of the D. melanogaster cell line S2. Cytoplasmic lysates made from S2 cells undergoing apoptosis induced by either reaper expression or cycloheximide treatment contain a caspase activity with DEVD specificity that can cleave p35, lamin DmO, drICE and DCP-1 in vitro, one that can trigger chromatin condensation in isolated nuclei. Immunodepletion of drICE from lysates is sufficient to remove most measurable in vitro apoptotic activity; re-addition of exogenous drICE to such immunodepleted lysates restores apoptotic activity. It is concluded that, at least in S2 cells, drICE can be the sole caspase effector of apoptosis (Fraser 1997).

ced-9, a member of the bcl-2 gene family in Caenorhabditis elegans plays a central role in preventing cell death in worms. Overexpression of human bcl-2 can partially prevent cell death in C. elegans. However, it remains to be elucidated whether ced-9 can regulate cell death when expressed in other organisms. The CED-9 protein is co-localized with BCL-2 in COS cells and Drosophila Schneider's L2 (SL2) cells, suggesting that the site of CED-9 action is located to specific cytoplasmic compartments. Overexpression of ced-9 only poorly protects cells from the death induced by ced-3 in HeLa cells, but ced-9 significantly reduces the cell death induced by ced-3 in Drosophila SL2 cells. Apoptosis of SL2 cells induced by reaper, a Drosophila cell-death gene, is partially prevented by ced-9, bcl-2 and bcl-xL. These results suggest that the signaling pathway that is required for the anti-apoptotic function of bcl-2 family members, including ced-9, is conserved in Drosophila cells. In addition, SL2 cells provide a unique systems for dissecting the main machinery of cell death (Hisahara, 1998).

The cytoplasmic region of Fas, a mammalian death factor receptor, shares a limited homology with Reaper, an apoptosis-inducing protein in Drosophila. Expression in Drosophila cells of either the Fas cytoplasmic region (FasC) or reaper causes cell death. The death process induced by FasC or reaper is inhibited by crmA or p35, suggesting that in both cases the death process is mediated by caspase-like proteases. Both Ac-YVAD aldehyde and Ac-DEVD aldehyde, specific inhibitors of caspase 1- and caspase 3-like proteases, respectively, inhibited the FasC-induced death of Drosophila cells. However, the cell death induced by Reaper is inhibited by Ac-DEVD aldehyde, but not by Ac-YVAD aldehyde. A caspase 1-like protease activity that preferentially recognizes the YVAD sequence gradually increases in the cytosolic fraction of the FasC-activated cells, whereas the caspase 3-like protease activity recognizing the DEVD sequence is observed in the Reaper-activated cells. Partial purification and biochemical characterization of the proteases indicates that there are at least three distinct caspase-like proteases in Drosophila cells that are differentially activated by FasC and Reaper. The conservation of the Fas-death signaling pathway in Drosophila cells, which is distinct from that for Reaper, may indicate that cell death in Drosophila is controlled not only by the Reaper suicide gene, but also by a Fas-like killer gene (Kondo, 1997).

Scythe: a Xenopus Reaper-binding protein

Reaper is a central regulator of apoptosis in D. melanogaster. With no obvious catalytic activity or homology to other known apoptotic regulators, Reaper's mechanism of action has remained obscure. Recombinant Drosophila Reaper protein induces rapid mitochondrial cytochrome c release, caspase activation and apoptotic nuclear fragmentation in extracts of Xenopus eggs. This paper reports a 150 kDa Reaper-interacting protein from Xenopus egg extracts, named Scythe. Scythe is highly conserved among vertebrates and contains a ubiquitin-like domain near its N-terminus. Other than this conserved domain, Scythe shows no other resemblence to proteins in the database. Immunodepletion of Scythe from extracts completely prevents Reaper-induced apoptosis without affecting apoptosis triggered by activated caspases. Moreover, a truncated variant of Scythe lacking the N-terminal domain induces apoptosis even in the absence of Reaper. Since Reaper requires cooperating cytosolic factors to trigger mitochondrial cytochrome c release, it was hypothesized that Scythe might be a cytochrome c-releasing factor. Indeed, addition of truncated Scythe to crude egg extracts accelerated release of cytochrome c from the mitochondria, relative to controls. Truncated Scythe is also able to trigger cytochrome c release when added to a mixture of isolated cytosol and mitochondria. Unlike truncated Scythe, full-length Scythe does not induce mitochondrial cytochrome c release in either crude extract or isolated cytosol; in several experiments, some suppression of cytochrome c release by the full-length Scythe protein has been observed. In contrast to the results obtained in the presence of cytosol, truncated Scythe does not promote cytochrome c release from isolated mitochondria in buffer (in the absence of other cytosolic proteins), even in the presence of recombinant Reaper. These data suggest that other accessory cytosolic factors are required to promote cytochrome c release. Efforts by many researchers have failed to identify a vertebrate reaper homolog using standard molecular cloning techniques. Given the findings reported here, it is hypothesized that Drosophila Reaper triggers apoptosis in Xenopus egg extracts by mimicking an endogenous vertebrate Scythe-activating factor. By analogy to Reaper, such a Scythe-activating factor might be transcriptionally induced in response to external stimuli or in response to developmental cues. Therefore, using Scythe as a bait to search for Reaper-like factors in extracts from appropriately staged or irradiated embryos may provide a means to isolate Reaper-like factors that may not be well conserved at the primary sequence level. It will be equally interesting to determine whether there are Scythe-related proteins acting downstream of Reaper in Drosophila. It is theoretically possible that Reaper accesses an apoptotic pathway in Xenopus egg extracts that is distinct from the one used in flies. It is concluded that Scythe is a novel apoptotic regulator that is an essential component in the pathway of Reaper-induced apoptosis (Thress, 1998).

Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper that is detectably homologous to Grim and Hid is dispensable for Scythe binding (Thress, 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 Apaf-1-related-killer (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. drICE and Dredd appear to be activated downstream of Rpr in Drosophila S2 cells, and drICE is an essential caspase in rpr-induced cell death, consistent with observations that Dapaf-1L and Dapaf-1S activate drICE 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).

In mammals and Drosophila, apoptotic caspases are under positive control via the CED-4/Apaf-1/Dark adaptors and negative control via IAPs (inhibitor of apoptosis proteins). However, the in vivo genetic relationship between these opposing regulators is not known. In this study, it has been demonstrated that a dark mutation reverses catastrophic defects seen in Diap1 mutants and rescues cells specified for Diap1-regulated cell death in development and in response to genotoxic stress. dark function is required for hyperactivation of caspases that occurs in the absence of Diap1. Since the action of dark is epistatic to that of Diap1, these findings demonstrate that caspase-dependent cell death requires concurrent positive input through Apaf-1-like proteins together with disruption of IAP-caspase complexes (Rodriguez, 2002).

Mutations in dark cause a variety of developmental defects, including an enlarged larval CNS. In the absence of dark function, cells specified to die actually survive and also differentiate. The embryonic CNS midline glia is a well-studied cell lineage in the fly embryo and, though only this particular lineage was studied, it is likely that supernumerary cell types in other lineages also persist in dark mutants. Several important conclusions derive from these observations. (1) The persistence of extra cells excludes trivial explanations for reduced TUNEL labeling in dark mutants (e.g. cell death in the absence of TUNEL labeling or redirected cell fates that prevent the specification of PCD) and reinforces models that favor a fundamental role for dark in embryonic PCD. (2) As shown in C.elegans and in studies of Reaper interval Drosophila mutants, rescue from PCD can uncover a cryptic differentiation program. (3) Most important, the same midline glial cells that fail to die in dark mutants also require the induced action of reaper, grim and hid. There is a requirement for dark function in normal PCD when reaper, grim and/or hid are expressed at physiological levels. It is noteworthy that Diap1 and at least two of the cell death initiators (i.e. reaper and hid) are expressed in pools of progenitor cells, which give rise to the embryonic midline glia and also play an important role in specifying the death of these cells. Thus, although midline glia presumably receive death signals from reaper and hid leading to the disruption of Diap1-caspase interactions, these cells still fail to die and are even capable of differentiating normally in the absence of dark function. This inference supports conclusions from epistasis studies indicating that a disruption of caspase-Diap1 interactions alone is insufficient for apoptosis, and suggests that dark functions as a co-effector of cell death signaling along with reaper, grim and/or hid. It is worth noting that the mammalian ortholog of dark, Apaf-1, functions downstream from the point at which mitochondrial factors including cytochrome c are released from mitochondria. If the same holds true for the fly protein, it follows that embryonic midline glia (and perhaps other cell types) can survive and differentiate beyond this mitochondrial 'point of no return' (Rodriguez, 2002).

Additional evidence that PCD requires coordinated dark-dependent caspase activation in conjuction with the release of IAP-mediated caspase inhibition comes from tests of dark mutants under conditions of stress that elicit apoptotic responses by the embryonic cell death initiators. Like the Reaper interval mutants, dark mutants also exhibit profound failures in cell death in response to ionizing radiation. Interestingly, the dark mutation itself does not interfere with the radiation induction of reaper mRNA. Therefore the possibility that resistance to damage-induced apoptosis evident in irradiated dark mutants is caused by a disruption of upstream elements in the signaling pathway can be excluded. Instead, the results raise the possibility that dark, like reaper, may function as an important effector of Drosophila p53-mediated apoptosis. In addition, these findings reinforce an apoptotic requirement for dark (even when reaper is transcriptionally induced) and provide evidence for models where cell death signals do not converge on Diap1 alone. In future studies, it will be interesting to determine the range of damage signals that can engage dark activity, since this locus might also function in a broader range of damage signals beyond those provoked by radiation (Rodriguez, 2002).

In flies, Diap1 is thought to function as a rate-limiting brake on apoptotic cell death. If, however, Diap1 were the most proximal rate-limiting regulator of apoptosis, then the presence or absence of dark function should have no influence on Diap1-dependent effects. If, however, dark and Diap1 exert interdependent functions, then the opposite outcome is predicted. The results from these studies provide consistent and compelling evidence favoring the latter scenario since, wherever tested, the influence of Diap1 upon apoptotic signaling is heavily dependent upon intact dark function. In the darkCD4 mutant, for instance, the Diap15 mutation fails to enhance grim- and hid-induced cell killing in the eye. These experiments examined Diap1 in the heterozygous condition, but the same outcome is also true when Diap1 is tested in the homozygous state. Early in embryonic development, Diap1 homozygotes exhibit catastrophic phenotypes including morphological arrest soon after gastrulation, extensive TUNEL-positive nuclei and hyperactivation of caspases. Each of these defects is profoundly dependent upon dark function since each is dramatically reversed by the dark mutation. In darkCD4; Diap15 double mutant embryos, no evidence is found for widespread apoptosis and, in fact, the large majority of these double mutants proceed through stages of embryogenesis well beyond the point at which single Diap1 mutants arrest. Thus, loss of dark not only suppresses apoptotic deaths that would otherwise occur, but definitively reverses a profound morphogenetic arrest that ensues in the absence of Diap1. Consequently, homozygosity at dark not only prevents the onset of apoptotic markers (i.e. DNA fragmentation and/or caspase activation), but actually preserves cells that are otherwise fated to die when normal checks upon caspases are removed. Rescue from inappropriate apoptosis in this instance is consistent with what was observed in the midline glia but, rather than preserving cells fated for programmed death, rescue occurs even when signaling from IAP antagonists in the Reaper region is bypassed. A similar result, with similar implications, was obtained in the ovary, where ~90% of heteroallelic Diap16/8 mutants show abnormal degeneration of early staged egg chambers and associated sterility. Again, loss of dark function not only suppresses the pathological effect, but actually reverses these degenerative defects to the extent that half of the darkCD4; Diap16/8 double mutant females produce normal egg chambers and are fertile. Thus, in both the ovary and the embryo, loss of dark rescues functional cells from apoptosis caused by misregulated Diap1 (Rodriguez, 2002).

Reversal of Diap1-dependent defects by dark is also evident when caspase activity is directly assayed in early embryos. In contrast to the 'hyperactivated' caspase levels detected in Diap1 single mutant lysates, Diap15; darkCD4 lysates show levels of caspase activity that are suppressed >90% relative to Diap1 mutants. The unusually high caspase activity detected in Diap1 mutant embryos is thought to reflect the action of these proteolytic enzymes unimpeded by native inhibitors. However, studies here demonstrate that removing a negative regulator alone is not sufficient to achieve caspase hyperactivation, since dark is clearly required for this unrestrained activity. It is worth mentioning that the basal DEVDase activity in double mutants is still somewhat elevated compared with control embryos. This might indicate dark-independent caspase activity due to caspase auto-activation in the absence of Diap1 or, since it is possible that the darkCD4 allele used here is not a complete null, it could reflect hypomorphic dark function. Alternatively, since a role for Diap1 in cytokinesis has not been ruled out, it is possible that this residual caspase activity ensues because of secondary developmental defects leading to cell death. Nevertheless, these enzymatic data extend the analyses to the biochemical level in a manner fully consistent with phenotypic studies (Rodriguez, 2002).

Interestingly, since Apaf-1/Dark adaptor proteins act upon procaspases while IAPs preferentially inhibit processed caspases, these results further suggest that pre-existing levels of processed caspases in most cells are probably not high enough to achieve an apoptotic threshold. Instead, a positive cell death stimulus from dark is required for the unusually high levels of caspase activation seen in Diap1-/- embryos. It is also evident that Dark-dependent 'basal' levels of DEVDase are detected in these assays several hours before the onset of embryonic PCD, and therefore authentic effector caspase (e.g. DrICE, Dcp-1) activity occurs even in the absence of overt apoptotic signals. These data indicate that constitutive levels of active effector caspases, not derived from autoproteolysis but instead promoted by Apaf-1-like adaptor proteins, may exist in many and perhaps all viable cells (Rodriguez, 2002).

Taken together, a strict Diap-1-caspase 'liberation' model does not explain sufficiently the evidence described. The action of dark is epistatic to that of Diap1, demonstrating an order of gene action whereby Dark functions either downstream or parallel to Diap1. Therefore, simple derepression of caspases via an IAP inhibitory bridge does not account adequately for epistasis between Diap1 and dark. Put another way, these results support the notion that apoptotic cell death in vivo results from the simultaneous activation of caspases by dark and the derepression of caspases by reaper, grim and/or hid. Accordingly, the findings are inconsistent with models that presume that Diap1 is the sole effector of reaper, grim and hid and that cells are 'pre-loaded' with sufficient levels of IAP-inhibited processed caspases to achieve cell killing. Instead, a 'gas and brake' model is favored whereby positive input from Apaf-1/Dark adaptors, together with removal of IAP inhibition, drives caspase activation to levels that exceed a threshold necessary for apoptosis (Rodriguez, 2002).

RHG motif of Reaper confers distinct cell death-inducing abilities

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

Analyses of R/Grim and G/Reaper chimeras have indicated that the closely related RHG motifs of Reaper and Grim are not functionally interchangeable. Instead, the four amino acid substitutions between their RHG motifs help determine the unique cell killing abilities of Reaper and Grim. For example, unlike Grim, R/Grim resembles Reaper and is unable to induce cell death in the CNS midline. In contrast, one P[UAS-g/reaper] strain induces significant midline cell death, implying that the presence of the Grim RHG motif can confer Grim-like cell killing abilities on Reaper. It is important to note, however, that the identity of the RHG motif does not completely transform the cell killing properties of the chimeras, indicating that other regions of Reaper and Grim proteins are also crucial for their distinct actions. In this regard, like Grim, both R/Grim and G/Reaper are able to act synergistically with Reaper to induce CNS midline cell death (Wing, 2001b).

While the Grim-Reaper proteins do not contain defined structural domains, they each share sequence similarity in the 14 amino acids at their NH2-termini. This RHG (Reaper, Hid, Grim) motif is most similar between Reaper and Grim (71.4% identity), and least similar between Hid and Grim (21.4% identity). The RHG motif plays a key role in interactions between Grim-Reaper proteins and members of the Inhibitor-of-Apoptosis-Protein (IAP) family, including Drosophila Diap1 and Diap2. Like other IAPs, Diap1 and Diap2 both contain related baculovirus IAP repeat (BIR) motifs, as well as a Really Interesting New Gene (RING) finger. Diap1 is an essential cell death regulator and diap1 mutants exhibit early embryonic lethality due to massive ectopic cell death. The functions of Diap2 in regulating cell death are less clear; however, it does share a number of functional properties with Diap1. Diap1 can directly bind caspases and repress their proteolytic activities. Significantly, caspase inhibition by Diap1 is antagonized by Hid, suggesting a double-repression model where the Grim-Reaper proteins promote cell death by binding to Diaps, suppressing their ability to inhibit caspases. Recent studies have indicated that the vertebrate Diablo/SMAC protein also promotes cell death activation by binding to IAPs and suppressing their death inhibitory activities. Thus, IAP suppression may be an evolutionarily conserved cell death regulatory mechanism. In this regard, while Grim-Reaper orthologs have not been identified, the expression of each protein can induce vertebrate cells to die, implying that they may suppress vertebrate IAPs (Wing, 2001b).

As with Reaper or Grim, P[GMR-gal4]-targeted expression of R/Grim or G/Reaper is very effective at inducing cell death. However, the actions of the chimeras are distinct from those of Reaper or Grim. In particular, the cell death phenotypes resulting from R/Grim or G/Reaper expression are completely blocked by Diap1 and partially blocked by Diap2. In contrast, both Diap1 and Diap2 completely block the effects of Reaper expression, but do not affect cell death induced by Grim. Thus, as a result of the presence of the Reaper RHG motif, R/Grim exhibits an increased sensitivity to repression by the Diaps compared with Grim. Similarly, the presence of the Grim RHG motif in G/Reaper results in decreased sensitivity to repression by Diaps compared with Reaper. These results indicated that the sequence differences in the RHG motifs of Reaper and Grim may strongly influence functional interactions with the Diaps (Wing, 2001b).

Diap1, like Diap2, exhibits distinct abilities to repress cell death induced by Reaper, Hid, or Grim. In the CNS midline, Diap1 more effectively blocks Grim-induced cell death than cell death induced by Reaper and Hid. In contrast, when examined in the adult eye, Diap1 is most effective at blocking Reaper-induced cell death, moderately effective against Hid, and ineffective against Grim. Similar results were obtained using the thsl gain-of-function diap1 mutant allele, which represses Reaper-induced eye cell death more effectively than death induced by Hid or Grim. Importantly, these data indicate that Diap1 has distinct, tissue-specific effects on cell death induced by Grim-Reaper proteins, and that these effects differ from those of Diap2. The basis for these functional distinctions are not yet clear. One possibility is that the associations between each Diap and Grim-Reaper protein may differ in strength, or be influenced by specific ancillary factors. Differences have been noted between Diap1 and Diap2 in their ability to bind and repress the actions of certain caspases, and Reaper, Hid, and Grim can act through different downstream caspases. Taken together, these findings suggest potentially complex functional interactions between Grim-Reaper proteins, Diaps, and caspases. It is likely that distinct activities of individual Grim-Reaper and Diap proteins provide enhanced capabilities for regulating cell death processes in different developmental and physiological contexts (Wing, 2001b).

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

A second region of sequence similarity, the 30 amino acid Trp-block, has been identified that is present once in Reaper and Grim, and four times in Hid. The Trp-blocks may be important for the cell death activation capabilities of GrimC and ReaperC, as well as for potentially shared RHG motif-independent activities of native Grim-Reaper proteins. This additional sequence similarity also suggests a modular organization of the Grim-Reaper proteins, where distinct functions may be afforded by the RHG motif and Trp-block. Taken together, the sequence similarities of the Grim-Reaper proteins, as well as the organization and chromosomal location of the corresponding genes, imply that the grim-reaper genes arose from duplication of a common ancestor and have diverged to assume overlapping yet distinct cell death activation functions. It will be of interest to determine the representation of grim-reaper orthologs in other species, information that could provide important insights into the evolution of cell death control mechanisms. This is of particular relevance given that inhibition of IAP activity is likely to constitute a conserved mechanism to regulate cell death activation (Wing, 2001b).

Ceramide and Slipper acts upstream of Reaper

Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity have not been identified. This study identifies ceramide as the activator of Drosophila MLK (Slipper) and ceramide and TNF-alpha are identified as agonists of mammalian MLK3. Slipper and MLK3 are activated by a ceramide analog and bacterial sphingomyelinase in vivo, whereas a low nanomolar concentration of natural ceramide activates them in vitro. Specific inhibition of Slipper and MLK3 significantly attenuates activation of JNK by ceramide in vivo without affecting ceramide-induced p38 or ERK activation. In addition, TNF-alpha also activates MLK3 and evidently leads to JNK activation in vivo. Thus, the ceramide serves as a common agonist of Slipper and MLK3, and MLK3 contributes to JNK activation induced by TNF-alpha (Sathyanarayana, 2002).

There are very few known kinases that are direct targets of ceramide. PKC-zeta, Raf-1, and CAPK (ceramide activated protein kinase) have been shown to be activated by ceramide. These results show that Slipper and MLK3 are targeted by ceramide. Reaper, a Drosophila protein known to cause ceramide generation, induces apoptosis during normal Drosophila development. In addition, overexpression of reaper induces apoptosis in S2 cells. It is therefore speculated that reaper may lead to apoptosis, at least in part, via ceramide-mediated activation of Slipper (Sathyanarayana, 2002).

In conclusion, ceramide is a potent agonist of Drosophila MLK and mammalian MLK3. The specificity of Slipper and MLK3 in mediating only ceramide-induced JNK activation without affecting ceramide-induced activation of ERK and p38 suggests an intriguing mechanism by which a specific MAPKKK can regulate different MAPK pathways in response to various physiological and pathological stimuli. These results also suggest that MLK3 plays a role in TNF-alpha-induced JNK activation. Studies showing that overexpression of MLK3 causes apoptosis and that neuronal cell death can be prevented by inhibition of the MLK family of kinases suggest a role for MLKs in apoptosis of neuronal cells. Since both ceramide and TNF are important triggers of cell death, these studies also indirectly suggest a role for MLK3 in modulating apoptosis. It is speculated that further elucidation of the role of MLKs, and specifically of MLK3, in apoptosis may ultimately facilitate the development of a targeted pharmacological intervention in neurodegenerative disorders such as idiopathic Parkinson's disease and Alzheimer's disease, both of which are associated with dysregulation of apoptosis (Sathyanarayana, 2002).

Reaper protein induces ceramide

Rpr expression induces generation of the lipid second messenger ceramide, and through use of the peptide caspase inhibitor N-benzyloxycarbonyl-VAD-fluoromethylketone(zVAD.fmk ) ceramide generation has been ordered downstream of caspases in SL2 cells. This study evaluates these events in SL2 cells transfected with cDNA for Rpr, with or without the baculovirus caspase inhibitor p35, under the control of the metallothionein promoter. Following copper addition, Rpr protein is detected at 1.5 h and is maximal at 2.5 h. Ceramide generation and caspase activation occurs nearly simultaneously; each is detectable at 2-2.5 h and is maximal at 6 h. Ceramide levels increase from a base line of 5 pmol/nmol lipid phosphorus to a maximum of 10 pmol/nmol lipid phosphorus. Apoptosis, first detected at 4 h, is maximal at 16 h. Co-expression of p35 did not affect Rpr-induced ceramide generation, whereas caspase activation and apoptosis are abolished. In contrast, zVAD.fmk inhibits ceramide generation and apoptosis. These data show that Rpr-induced ceramide generation is upstream or independent of p35-inhibitable caspases and demonstrate differences in the actions of peptide and p35 caspase inhibitors (Bose, 1998).

Loss of mir-14 enhances Reaper-dependent cell death

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).

Reaper 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).

Drosophila IAP antagonists form multimeric complexes to promote cell death

Apoptosis is a specific form of cell death that is important for normal development and tissue homeostasis. Caspases are critical executioners of apoptosis, and living cells prevent their inappropriate activation through inhibitor of apoptosis proteins (IAPs). In Drosophila, caspase activation depends on the IAP antagonists, Reaper (Rpr), Head involution defective (Hid), and Grim. These proteins share a common motif to bind Drosophila IAP1 (DIAP1) and have partially redundant functions. This study shows that IAP antagonists physically interact with each other. Rpr is able to self-associate and also binds to Hid and Grim. The domain involved in self-association has been defined and it was demonstrated to be critical for cell-killing activity in vivo. In addition, Rpr requires Hid for recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo. Both targeting of Rpr to mitochondria and forced dimerization strongly promotes apoptosis. These results reveal the functional importance of a previously unrecognized multimeric IAP antagonist complex for the induction of apoptosis (Sandu, 2010).

This study shows that IAP antagonists undergo self-association and hetero-association that is essential for their full killing activity. Specifically, the physical association between Rpr, Hid, and Grim involves the central helical domain of Rpr. Disrupting this protein-protein interface leads to a significant loss of Rpr’s ability to induce cell death in vivo. The importance of Rpr self-association was revealed by generating enforced Rpr dimers in which the central helical domain of this protein is replaced by defined dimerization motifs. These experiments revealed that enforced parallel, but not anti-parallel dimerization of Rpr (RprLZ) can induce cell death very efficiently in transgenic Drosophila. The resulting cell death occurred by apoptosis and was rescued by the overexpression of the caspase inhibitor p35, or through Rpr-insensitive diap1 alleles. Furthermore, mutants that inhibit the self-association of Rpr have reduced pro-apoptotic activity, providing independent support for the importance of Rpr multimerization. Because an anti-parallel Rpr dimer (RprProP) was not efficiently inducing cell death in transgenic animals, it appears that the IBM motifs of multimeric Rpr have to be in a specific conformation, or at least in close proximity for efficient DIAP1 inactivation. This may occur, for example, by engaging both BIR domains of one DIAP1 molecule in a similar fashion to how SMAC can engage XIAP (Sandu, 2010).

The association of Rpr with the other IAP antagonists Grim and Hid is also reported. Hid is the only IAP antagonist that has a defined mitochondrial targeting sequence at its C terminus and is targeted to the mitochondria by itself; therefore, focus was placed particularly on the interaction between Rpr and Hid. Consistent with previous reports, it was found that Hid consistently localizes to the mitochondria in both human and Drosophila cells. Although it has been previously reported that Rpr localizes to the mitochondria through the GH3-lipid interaction, the current results support an alternative view that Rpr’s ability to translocate to the mitochondria is an indirect consequence of associating with Hid. Specifically, in support of this model, it was shown that Rpr is uniformly distributed in cells when transfected alone in heterologous cells, translocating to the mitochondria only when cotransfected with Hid. It was further shown that the GH3 mutant F34AL35A, unlike wild-type Rpr, does not coimmunoprecipitate with Hid. This is in agreement with previous observations that a GH3 mutant failed to localize to the mitochondria in Drosophila S2 cells (Sandu, 2010).

Rpr induces ubiquitination of DIAP1 in vitro and in HEK293 cells. Unlike Rpr, Hid is not able to perform this function. Thus, the significance of Rpr-Hid interaction might be to bring Rpr at the mitochondrial surface to degrade DIAP1. Although both Rpr and Hid belong to the IAP antagonists family, share a conserved IBM motif, bind DIAP1, and induce cell death, their role in induction of cell death seems to be distinct. In many paradigms Hid appears to be a more potent inducer of cell death than Rpr. It is possible that the primary role of Hid is to assemble a complex at the mitochondrial membrane that recruits Rpr as one the players. The role of Rpr in this complex is to induce DIAP1 ubiquitination. Inability of Hid itself to induce DIAP1 degradation might be related to its larger size (410 amino acids) as compared with Rpr (64 amino acids) or even Grim (138 amino acids). Potentially, the bulkier Hid might interfere with conformational changes in DIAP1 or with the ubiquitin-related transfer process (Sandu, 2010).

In addition, evidence is provided that Rpr is more potent at inducing apoptosis when present at the mitochondrial membrane. When Rpr was fused to the mitochondrial targeting sequence from Hid and expressed in Drosophila eyes, strong cell killing and pupal lethality were observed. Flies dissected from the pupal cases show severely ablated eyes that are reduced to black spots. Even the inactive GH3 mutant F34AL35A, when artificially targeted to the mitochondria using the Hid MTS, induces significant eye ablation. Therefore, Rpr is more potent when present at the mitochondrial membrane. Two possible explanations are considered for this enhanced pro-apoptotic activity: First, Rpr may be more active at the mitochondrial surface because of increased protein stability. Consistent with this idea, cytoplasmic Rpr is not very stable and it was found that Rpr accumulates to higher protein levels when the presence of Hid permits mitochondrial localization. The resulting high local concentration of Rpr may be critical for DIAP1 ubiquitination. As predicted by this model, it was found that Rpr-induced cell death is less efficient when Hid is depleted by RNA knockdown. The model is also in agreement with several previous observations. For example, it has been reported that Rpr and Hid localize to mitochondria and can induce changes of the mitochondrial ultrastructure. This study also showed that inhibition of Rpr localization to mitochondria significantly inhibits cell killing, and that Rpr and Hid act in concert with caspases to promote mitochondrial disruption and Cyt C release. In addition, overexpression of both rpr and hid is required to induce cell death in midline cells of the nervous system, and neither of them kills well individually. This is consistent with the observation that more than one IAP antagonist is expressed and they act synergistically in the dying midline glia cells. Finally, Drosophila salivary gland cell death is preceded by the expression of both rpr and hid, and RNAi knockdown of hid alone is sufficient to block the death of these cells. The second, and not mutually exclusive explanation is that Rpr may be more active at the mitochondria because of local concentration of apoptosis regulators that operate at this surface. It has been previously shown that Dronc and active Drice are present at the mitochondrial membrane, and more recently that mammalian XIAP can translocate to the mitochondrial surface in response to apoptotic stimuli. In addition, mitochondrial proteins involved in energy metabolism have been recently described to modulate caspase activity and cell death in Drosophila cells. Recently, it was shown by coimmunoprecipitation experiments in fly cell culture that Grim interacts with the Bcl-2 family proteins Debcl and Buffy. Thus, Rpr may be part of a higher-order complex at the mitochondria to locally regulate IAP turnover and caspase activity (Sandu, 2010).

Taken together, this study uncovered the role of the Rpr helical domain in self-association and interaction with Hid and Grim. The mechanism of Rpr recruitment to the mitochondria by interaction with Hid was revealed. Most importantly, this study has provided a new concept with respect to IAP antagonist activity in fly, which acts cooperatively by physical interaction rather than by additive cell death output (Sandu, 2010).

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

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

Mitochondrial fusion is regulated by Reaper to modulate Drosophila programmed cell death

In most multicellular organisms, the decision to undergo programmed cell death in response to cellular damage or developmental cues is typically transmitted through mitochondria. It has been suggested that an exception is the apoptotic pathway of Drosophila melanogaster, in which the role of mitochondria remains unclear. Although IAP antagonists in Drosophila such as Reaper, Hid and Grim may induce cell death without mitochondrial membrane permeabilization, it is surprising that all three localize to mitochondria. Moreover, induction of Reaper and Hid appears to result in mitochondrial fragmentation during Drosophila cell death. Most importantly, disruption of mitochondrial fission can inhibit Reaper and Hid-induced cell death, suggesting that alterations in mitochondrial dynamics can modulate cell death in fly cells. This study reports that Drosophila Reaper can induce mitochondrial fragmentation by binding to and inhibiting the pro-fusion protein MFN2 and its Drosophila counterpart dMFN/Marf. These in vitro and in vivo analyses reveal that dMFN overexpression can inhibit cell death induced by Reaper or gamma-irradiation. In addition, knockdown of dMFN causes a striking loss of adult wing tissue and significant apoptosis in the developing wing discs. These findings are consistent with a growing body of work describing a role for mitochondrial fission and fusion machinery in the decision of cells to die (Thomenius, 2011).

Tango7 regulates cortical activity of caspases during reaper-triggered changes in tissue elasticity

Caspases perform critical functions in both living and dying cells; however, how caspases perform physiological functions without killing the cell remains unclear. This study identified a novel physiological function of caspases at the cortex of Drosophila salivary glands. In living glands, activation of the initiator caspase Dronc triggers cortical F-actin dismantling, enabling the glands to stretch as they accumulate secreted products in the lumen. Tango7 (Eukaryotic translation initiation factor 3 subunit m), not the canonical Apaf-1-adaptor Dark, regulates Dronc activity at the cortex; in contrast, dark is required for cytoplasmic activity of dronc during salivary gland death. Therefore, tango7 and dark define distinct subcellular domains of caspase activity. Furthermore, Tango7-dependent cortical Dronc activity is initiated by a sublethal pulse of the inhibitor of apoptosis protein (IAP) antagonist Reaper. The results support a model in which biological outcomes of caspase activation are regulated by differential amplification of IAP antagonists, unique caspase adaptor proteins, and mutually exclusive subcellular domains of caspase activity. Caspases are known for their role in cell death, but they can also participate in other physiological functions without killing the cells. In this study the authors show that unique caspase adaptor proteins can regulate caspase activity within mutually-exclusive and independently regulated subcellular domains (Kang, 2017).

Principles that govern the activation and function of caspases have fallen short in providing an understanding for how these enzymes can be activated to perform both delicate intracellular remodeling in living cells and total destruction in dying cells. This paper provides new insights into the mechanisms that regulate caspase activation by comparing two completely different biological outcomes in the same tissue that both require caspase function. The Drosophila homolog of caspase-9, dronc, is required for dismantling of the cortical F-actin cytoskeleton during salivary gland development -- a role that is distinct from its known function in the salivary gland death response during metamorphosis. By systematically dissecting the regulation of dronc function at the cortex, this study showed that cortical functions of dronc are regulated independently from its cytoplasmic functions. The cytoplasmic functions of activated dronc require the canonical adaptor protein Dark, while the cortical roles of dronc require tango7. In this manner, tango7 and Dark restrict the function of dronc to distinct subcellular domains. Moreover, this study also showed that these two functions can be initiated independently through differential amplification of IAP antagonist expression, providing a model for how lethal and vital roles of caspases can be differentially activated in the same cell. Finally, a new non-apoptotic function was identified for caspases in the control of tissue elasticity to accommodate buildup of secreted products in the lumen of secretory tissues, facilitating their timely release (Kang, 2017).

The results demonstrate that caspases can be activated in distinct, mutually exclusive subcellular domains within a single cell, and that these subcellular domains are generated by use of unique caspase adaptor proteins. Local activation of caspases, as detected by staining with antibodies to activated caspases, has been reported before; however, this study demonstrates that local activation is achieved by targeting caspases to subcellular domains, and this targeting is necessary for subcellular functions of these caspases. Importantly, this study shows that caspases can be activated specifically in one domain without being activated in another, providing a mechanism that allows control of caspase activity with a previously unknown level of subcellular precision. However, the mechanisms that restrict caspase cascades to distinct subcellular compartments remain unclear. It is possible that caspase expression levels are intentionally kept low during non-lethal responses, and localized enrichment mediates subcellular domain-specific activation. For example, if most of the Dronc protein present in the cell localizes to the cortex, then this specific localization may restrict caspase functions to the cortical compartment. This model fits with the results at the end of larval development; however, in dying glands, caspases are independently activated in cortical and cytoplasmic compartments, suggesting that additional mechanisms are in play to restrict caspase activity to the appropriate subcellular compartment. For example, it is possible that caspase cascades occur within a physical complex consisting of initiator caspases, their adaptor proteins, effector caspases, and their substrates. In this model, only one of these proteins, likely the initiator caspase, would need to be subcellularly localized in order to generate a compartment-specific caspase cascade. However, resolution of this possible mechanism will require further studies. This subcellular domain-specific model for caspase activation contrasts with the commonly held belief that activated caspase cascades passively perpetuate themselves and spread throughout the cell, and also opens the possibility that caspases, through specific subcellular localization mediated by adaptor proteins, may play a role in many yet-to-be-identified biological processes (Kang, 2017).

This study demonstrates that differential amplification of IAP antagonists at specific developmental stages determines lethal vs. non-lethal outcomes of caspase activation. In the system used in this study, differential amplification is accomplished through the use of transcription factors that function downstream of a steroid hormone signal. However, caspases must have an ability to 'sense' the magnitude of the IAP antagonist pulse, ensuring that they initiate the appropriate lethal or non-lethal responses. One possible 'sensing' mechanism may involve the aforementioned selectivity of initiator caspase adaptor proteins, like was observed with tango7 and dark. In this model, some adaptor protein complexes would require a lower IAP antagonist threshold for initiator caspase activation than others. However, elucidation of the detailed molecular mechanisms mediating 'sensing' of IAP antagonist expression levels will require further study. Finally, the results indicate that small pulses of IAP antagonist expression are tissue specific, raising the possibility that many more of these pulses are generated in other tissues and developmental stages that have not yet been detected or characterized. The data suggests that non-lethal, physiological functions of caspases may be more widespread than previously thought (Kang, 2017).

These results show that caspases play a novel role during the secretion of glue proteins. Glue proteins are essential to allow a newly formed prepupa to adhere to a solid surface; however, when cortical F-actin dismantling fails, glue precociously 'leaks' onto the surface of the animal. Although precocious expulsion of glue does not appear to have a deleterious effect in the lab, in the wild, it may adversely affect fitness by inhibiting larval movement or reducing the ability of the animal to stick securely to a surface during metamorphosis. Additionally, the results raise the question of whether other exocrine tissues in different species, such as the mammary gland, may utilize caspases in a similar manner to accommodate large amounts of secreted luminal products prior to their release (Kang, 2017).

In conclusion, systematic analysis of vital and lethal responses to caspase activation in the same cells has revealed mechanisms that allow caspases to be activated without killing the cell. The results demonstrate that caspases can be activated in mutually exclusive subcellular domains, where activation of caspases in one domain does not trigger activation of caspases in another domain. These subcellular domains were shown to. e generated by different caspase adaptor proteins. It is likely that yet-to-be-identified adaptor proteins define other subcellular domains and, in so doing, help regulate the many physiological functions of caspases. Moreover, the results demonstrate that some of these subcellular domains have lower thresholds for activation of caspases, thereby allowing sublethal pulses of IAP antagonists to selectively initiate physiological functions of caspases. Together, these results outline a simple conceptual framework for controlling caspase activation during normal development and physiology (Kang, 2017).

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

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