Grim stimulates Diap1 poly-ubiquitination by binding to UbcD1

Diap1 is an essential Drosophila cell death regulator that binds to caspases and inhibits their activity. Reaper, Grim and Hid each antagonize Diap1 by binding to its BIR domain, activating the caspases and eventually causing cell death. Reaper and Hid induce cell death in a Ring-dependent manner by stimulating Diap1 auto-ubiquitination and degradation. It was not clear that how Grim causes the ubiquitination and degradation of Diap1 in Grim-dependent cell death. We found that Grim stimulates poly-ubiquitination of Diap1 in the presence of UbcD1 and that it binds to UbcD1 in a GST pull-down assay, so presumably promoting Diap1 degradation. The possibility that dBruce is another E2 interacting with Diap1 was examined. The UBC domain of dBruce slightly stimulated poly-ubiquitination of Diap1 in Drosophila extracts but not in the reconstitution assay. However Grim did not stimulate Diap1 poly-ubiquitination in the presence of the UBC domain of dBruce. Taken together, these results suggest that Grim stimulates the poly-ubiquitination and presumably degradation of Diap1 in a novel way by binding to UbcD1 but not to the UBC domain of dBruce as an E2.     

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. Here we report 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.     

sickle, a novel Drosophila death gene in the reaper/hid/grim region, encodes an IAP-inhibitory protein

Inhibitors of apoptosis proteins (IAPs) interact with caspases and inhibit their protease activity, whereas the IAP-inhibitory proteins Smac/DIABLO in mammals and Reaper, Hid, and Grim in flies relieve IAP-mediated inhibition to induce cell death. Here we describe the functional characterization of the novel Drosophila cell death protein Sickle (Skl), which binds to IAPs and neutralizes their apoptotic inhibitory activity. Skl exhibits no sequence homology to Reaper, Hid, Grim, or Smac/DIABLO, except within the 4 residue N-terminal IAP binding motif. Skl interacts with Drosophila and mammalian IAPs and can promote caspase activation in the presence of IAPs. Consistent with these findings, expression of Skl in Drosophila and mammalian cell lines or in Drosophila embryos induces apoptosis. Skl can also synergize with Grim to induce cell death in the Drosophila eye imaginal disc. Based on biochemical and structural data, the N terminus of Skl, like that of the mammalian Smac/DIABLO, is absolutely required for its apoptotic and caspase-promoting activities and its ability to interact with IAPs. These findings point to conservation in the structure and function of the IAP-inhibitory proteins across species and suggest the existence of other family members.     

Molecular mechanisms of DrICE inhibition by DIAP1 and removal of inhibition by Reaper, Hid and Grim

The Drosophila melanogaster inhibitor of apoptosis protein DIAP1 suppresses apoptosis in part through inhibition of the effector caspase DrICE. The pro-death proteins Reaper, Hid and Grim (RHG) induce apoptosis by antagonizing DIAP1 function. However, the underlying molecular mechanisms remain unknown. Here we demonstrate that DIAP1 directly inhibits the catalytic activity of DrICE through its BIR1 domain and this inhibition is countered effectively by the RHG proteins. Inhibition of DrICE by DIAP1 occurs only after the cleavage of its N-terminal 20 amino acids and involves a conserved surface groove on BIR1. Crystal structures of BIR1 bound to the RHG peptides show that the RHG proteins use their N-terminal IAP-binding motifs to bind to the same surface groove, hence relieving DIAP1-mediated inhibition of DrICE. These studies define novel molecular mechanisms for the inhibition and activation of a representative D. melanogaster effector caspase.     

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. We report here that Drosophila Reaper can induce mitochondrial fragmentation by binding to and inhibiting the pro-fusion protein MFN2 and its Drosophila counterpart dMFN/Marf. Our in vitro and in vivo analyses reveal that dMFN overexpression can inhibit cell death induced by Reaper or γ-irradiation. In addition, knockdown of dMFN causes a striking loss of adult wing tissue and significant apoptosis in the developing wing discs. Our 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.     

Reaper-induced dissociation of a Scythe-sequestered cytochrome c-releasing activity.

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. We recently reported that 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. We now show that 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 which is detectably homologous to Grim and Hid is dispensable for Scythe binding.     

Amsacta moorei Entomopoxvirus inhibitor of apoptosis suppresses cell death by binding Grim and Hid

Inhibitor of apoptosis (iap) genes have been identified in the genomes of two independent families of insect viruses, the Baculoviridae and the Entomopoxvirinae. In this report, we examined the functional attributes of the Amsacta moorei entomopoxvirus-encoded IAP protein (AMV-IAP). The binding specificity of the individual baculoviral IAP repeat (BIR) domains of AMV-IAP was investigated by using a random-peptide, phage display library, and sequences similar to the amino termini of proapoptotic Drosophila proteins in the Reaper/Hid/Grim family were identified. Furthermore, the BIR domains of AMV-IAP protein were demonstrated to bind the mammalian IAP inhibitor Smac through the AVPI tetrapeptide sequence, suggesting that the peptide binding pocket and groove found in the insect and mammalian IAPs is conserved in this viral protein. Interaction analysis implicated BIR1 as the high-affinity site for Grim, while BIR2 interacted more strongly with Hid. Both Grim and Hid were demonstrated to interact with AMV-IAP in vivo, and Grim- or Hid-induced cell death was suppressed when AMV-IAP was coexpressed.     

Reaper is regulated by IAP-mediated ubiquitination

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. In this report, we show that 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, we also show that regulation of Reaper by ubiquitination is a significant factor in determining its biological activity. These data demonstrate a novel function for IAPs and suggest that IAPs and Reaper-like proteins mutually control each other's abundance.     

The mitochondrial ARTS protein promotes apoptosis through targeting XIAP

ARTS is an unusual septin-like mitochondrial protein that was originally shown to mediate TGF-beta-induced apoptosis. Recently, we found that ARTS is also important for cell killing by other pro-apoptotic factors, such as arabinoside, etoposide, staurosporine and Fas. In Drosophila, the IAP antagonists Reaper, Hid and Grim are essential for the induction of virtually all apoptotic cell death. We found that mutations in peanut, which encodes a Drosophila homologue of ARTS, can dominantly suppress cell killing by Reaper, Hid and Grim, indicating that peanut acts downstream or in parallel to these. In mammalian cells, ARTS is released from mitochondria upon pro-apoptotic stimuli and then binds to XIAP. Binding of ARTS to XIAP is direct, as recombinant ARTS and XIAP proteins can bind to each other in vitro. ARTS binding to XIAP is specific and related to its pro-apoptotic function, as mutant forms of ARTS (or related septins) that fail to bind XIAP failed to induce apoptosis. ARTS leads to decreased XIAP protein levels and caspase activation. Our data suggest that ARTS induces apoptosis by antagonizing IAPs.     

Structural analysis of a functional DIAP1 fragment bound to grim and hid peptides

The inhibitor of apoptosis protein DIAP1 suppresses apoptosis in Drosophila, with the second BIR domain (BIR2) playing an important role. Three proteins, Hid, Grim, and Reaper, promote apoptosis, in part by binding to DIAP1 through their conserved N-terminal sequences. The crystal structures of DIAP1-BIR2 by itself and in complex with the N-terminal peptides from Hid and Grim reveal that these peptides bind a surface groove on DIAP1, with the first four amino acids mimicking the binding of the Smac tetrapeptide to XIAP. The next 3 residues also contribute to binding through hydrophobic interactions. Interestingly, peptide binding induces the formation of an additional alpha helix in DIAP1. Our study reveals the structural conservation and diversity necessary for the binding of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins.     

Mitochondrial localization of Reaper to promote inhibitors of apoptosis protein degradation conferred by GH3 domain-lipid interactions

Morphological hallmarks of apoptosis result from activation of the caspase family of cysteine proteases, which are opposed by a pro-survival family of inhibitors of apoptosis proteins (IAPs). In Drosophila, disruption of IAP function by Reaper, HID, and Grim (RHG) proteins is sufficient to induce cell death. RHG proteins have been reported to localize to mitochondria, which, in the case of both Reaper and Grim proteins, is mediated by an amphipathic helical domain known as the GH3. Through direct binding, Reaper can bring the Drosophila IAP (DIAP1) to mitochondria, concomitantly promoting IAP auto-ubiquitination and destruction. Whether this localization is sufficient to induce DIAP1 auto-ubiquitination has not been reported. In this study we characterize the interaction between Reaper and the mitochondria using both Xenopus and Drosophila systems. We find that Reaper concentrates on the outer surface of mitochondria in a nonperipheral manner largely mediated by GH3-lipid interactions. Importantly, we show that mitochondrial targeting of DIAP1 alone is not sufficient for degradation and requires Reaper binding. Conversely, Reaper able to bind IAPs, but lacking a mitochondrial targeting GH3 domain (DeltaGH3 Reaper), can induce DIAP1 turnover only if DIAP1 is otherwise targeted to membranes. Surprisingly, targeting DIAP1 to the endoplasmic reticulum instead of mitochondria is partially effective in allowing DeltaGH3 Reaper to promote DIAP1 degradation, suggesting that co-localization of DIAP and Reaper at a membrane surface is critical for the induction of DIAP degradation. Collectively, these data provide a specific function for the GH3 domain in conferring protein-lipid interactions, demonstrate that both Reaper binding and mitochondrial localization are required for accelerated IAP degradation, and suggest that membrane localization per se contributes to DIAP1 auto-ubiquitination and degradation.     

Bifunctional killing activity encoded by conserved reaper proteins

Drosophila activators of apoptosis mapping to the Reaper region function, in part, by antagonizing IAP proteins through a shared RHG motif. We isolated Reaper from the Blowfly L. cuprina, which triggered extensive apoptosis in Drosophila cells. Conserved regions of Reaper were tested in the context of GFP fusions and a second killing activity, distinct from the RHG, was identified. A 20 amino-acid peptide, designated R3, conferred targeting to a focal compartment and promoted membrane blebbing. Killing by the R3 fragment did not correlate with translational suppression or with reduced DIAP1 levels. Likewise, R3-induced cell deaths were only modestly suppressed by silencing of Dronc and involved no detectable association with DIAP1. Instead, a second IAP-binding domain, distinct from the R3, was identified at the C terminus of Reaper that bound to DIAP1 but failed to trigger apoptosis. Collectively, these findings are inconsistent with single effector models for cell killing by Reaper and suggest, instead, that Reaper encodes conserved bifunctional death activities that propagate through distinct effector pathways.     

Hid,Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Here we show that Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis.     

Smac/DIABLO selectively reduces the levels of c-IAP1 and c-IAP2 but not that of XIAP and livin in HeLa cells

The inhibitor of apoptosis (IAP) proteins bind and inhibit caspases via their baculovirus IAP repeat domains. Some of these IAPs are capable of ubiquitinating themselves and their interacting proteins through the ubiquitin-protein isopeptide ligase activity of their RING domain. The Drosophila IAP antagonists Reaper, Hid, and Grim can accelerate the degradation of Drosophila IAP1 and some mammalian IAPs by promoting their ubiquitin-protein isopeptide ligase activity. Here we show that Smac/DIABLO, a mammalian functional homolog of Reaper/Hid/Grim, selectively causes the rapid degradation of c-IAP1 and c-IAP2 but not XIAP and Livin in HeLa cells, although it efficiently promotes the auto-ubiquitination of them all. Smac binding to c-IAP via its N-terminal IAP-binding motif is the prerequisite for this effect, which is further supported by the findings that Smac N-terminal peptide is sufficient to enhance c-IAP1 ubiquitination, and Smac no longer promotes the ubiquitination of mutant c-IAP1 lacking all three baculovirus IAP repeat domains. In addition, different IAPs require the same ubiquitin-conjugating enzymes UbcH5a and UbcH6 for their ubiquitination. Taken together, Smac may serve as a key molecule in vivo to selectively reduce the protein level of c-IAPs through the ubiquitin/proteasome pathway.     

The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis

Members of the Inhibitor of Apoptosis Protein (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 (head involution defective) all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. Here, we show that in vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, we show that 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. Our data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination.     

果蝇蜕皮激素诱导程序性细胞死亡的遗传调控因子

近年来关于果蝇程序性细胞死亡（programmed cell death, PCD）的研究结果表明,在果蝇的变态发育过程中,蜕皮激素与受体结合后诱导转录因子的表达。这些转录因子作为程序性细胞死亡调控网络中的初、次级应答信号,激活凋亡诱导因子Reaper、Hid和Grim的表达。Reaper、Hid和Grim进而阻止凋亡蛋白抑制因子的活性,从而启动半胱氨酸蛋白酶caspase途径,引起细胞凋亡（apoptosis）。该文综述了蜕皮激素诱导的果蝇程序性细胞死亡中各遗传调控因子之间的关系。     

Drosophila BRUCE inhibits apoptosis through non-lysine ubiquitination of the IAP-antagonist REAPER

Active caspases execute apoptosis to eliminate superfluous or harmful cells in animals. In Drosophila, living cells prevent uncontrolled caspase activation through an inhibitor of apoptosis protein (IAP) family member, dIAP1, and apoptosis is preceded by the expression of IAP-antagonists, such as Reaper, Hid and Grim. Strong genetic modifiers of this pathway include another IAP family gene encoding an E2 ubiquitin conjugating enzyme domain, dBruce. Although the genetic effects of dBruce mutants are well documented, molecular targets of its encoded protein have remained elusive. Here, we report that dBruce targets Reaper for ubiquitination through an unconventional mechanism. Specifically, we show that dBruce physically interacts with Reaper, dependent upon Reaper's IAP-binding (IBM) and GH3 motifs. Consistently, Reaper levels were elevated in a dBruce -/- background. Unexpectedly, we found that dBruce also affects the levels of a mutant form of Reaper without any internal lysine residues, which normally serve as conventional ubiquitin acceptor sites. Furthermore, we were able to biochemically detect ubiquitin conjugation on lysine-deficient Reaper proteins, and knockdown of dBruce significantly reduced the extent of this ubiquitination. Our results indicate that dBruce inhibits apoptosis by promoting IAP-antagonist ubiquitination on unconventional acceptor sites.     

Drosophila Bruce can potently suppress Rpr- and Grim-dependent but not Hid-dependent cell death

Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain (E2). 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. Here, we demonstrate, using gain-of-function and deletion alleles, that Drosophila Bruce (dBruce) can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The dBruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. dBruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1 BIR2, be intact. dBruce 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 dBruce can regulate cell death at a novel point.     

An emerging blueprint for apoptosis in Drosophila.

Apoptosis research demonstrates that, even though the multitude of regulatory circuits controlling programmed cell death might diverge, core elements of the 'apoptotic engine' are widely conserved. Therefore, studies in less complex model systems, such as the nematode and the fly, should continue to have a profound impact on our understanding of the process. This review explores genes and molecules that control apoptosis in Drosophila. The death inducers Reaper, Grim and Hid relay signals, possibly through IAPs (inhibitor of apoptosis proteins) and Dark (an Apaf-1/Ced-4 homologue), to trigger caspase function. This animal model promises continued insights into the determinants of cell death in 'naturally occurring' and pathological contexts.