GH3, a novel proapoptotic domain in Drosophila Grim,promotes a mitochondrial death pathway

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. We describe 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 induce programmed cell death efficiently.     

A GH3-like domain in reaper is required for mitochondrial localization and induction of IAP degradation

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. We report here that this domain, although required for IAP destabilization, is not sufficient. Moreover, we have identified a small region of Reaper, similar to the GH3 domain of Grim, 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 was deficient in these properties, these defects could 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.     

HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins

Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac. We now demonstrate that HtrA2, a mammalian homologue of the Escherichia coli heat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin. Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP. HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs. HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3 in vitro and is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death. The proapoptotic activity of HtrA2 in vivo involves both IAP binding and serine protease activity. Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated.     

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.     

NRADD,a novel membrane protein with a death domain involved in mediating apoptosis in response to ER stress

NRADD (neurotrophin receptor alike death domain protein) is a novel protein with transmembrane and cytoplasmic regions highly homologous to death receptors, particularly p75(NTR). However, the short N-terminal domain is unique. Expression of NRADD induced apoptosis in a number of cell lines. The apoptotic mechanism involved the activation of caspase-8 and execution of apoptosis without requiring mitochondrial components. The activation of this death receptor-like mechanism required the N-terminal domain, which is N-glycosylated and needed for subcellular targeting. Deletion of the N-terminal domain produced a dominant-negative form of NRADD that protected neurons and Schwann cells from a variety of endoplasmic reticulum (ER) stressors. NRADD may therefore be a necessary component for generating an ER-induced proapoptotic signal.     

grim promotes programmed cell death of Drosophila microchaete glial cells

The Inhibitor of apoptosis (IAP) antagonists Reaper (Rpr), Grim and Hid are central regulators of developmental apoptosis in Drosophila. Ectopic expression of each is sufficient to trigger apoptosis, and hid and rpr have been shown to be important for programmed cell death (PCD). To investigate the role for grim in PCD, a grim null mutant was generated. grim was not a key proapoptotic gene for embryonic PCD, confirming that grim cooperates with rpr and hid in embryogenesis. In contrast, PCD of glial cells in the microchaete lineage required grim, identifying a death process dependent upon endogenous grim. Grim associates with mitochondria and has been shown to activate a mitochondrial death pathway distinct from IAP antagonization; therefore, the Drosophila bcl-2 genes buffy and debcl were investigated for genetic interaction with grim. Loss of buffy led to microchaete glial cell survival and suppressed death in the eye induced by ectopic Grim. This is the first example of a developmental PCD process influenced by buffy, and places buffy in a proapoptotic role. PCD of microchaete glial cells represents an exceptional opportunity to study the mitochondrial proapoptotic process induced by Grim.     

Drosophila grim induces apoptosis in mammalian cells.

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. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Grim-induced death is antagonized by bcl-2 in a dose-dependent manner, and neither Fas signalling nor p53 are required for grim pro-apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway.     

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.     

Siva-1 and an alternative splice form lacking the death domain, Siva-2, similarly induce apoptosis in T lymphocytes via a caspase-dependent mitochondrial pathway

Siva-1 is a death domain-containing proapoptotic protein identified as an intracellular ligand of CD27 and of the glucocorticoid-induced TNFR family-related gene, which are two members of the TNFR family expressed on lymphoid cells. Although Siva-1 expression is up-regulated in multiple pathological processes, little is known about the signaling pathway underlying the Siva-induced apoptosis. In this study, we investigated the mechanism of the proapoptotic activity of Siva-1 and an alternative splice form lacking the death domain of Siva-1, Siva-2, in T lymphocytes in which Siva proteins, CD27, and glucocorticoid-induced TNFR family-related gene are primarily expressed. Overexpression of Siva proteins triggers a typical apoptotic process manifested by cell shrinkage and surface exposure of phosphatidylserine, and confirmed by ultrastructural features. Siva-induced apoptosis is related to the CD27-mediated apoptotic pathway and results in activation of both initiator and effector caspases. This pathway involves a mitochondrial step evidenced by activation of Bid and cytochrome c release, and is modulated by overexpression of Bcl-2 or Bcl-x(L). The determinants for Siva-induced apoptosis are not contained within the death domain found in the central part of Siva-1, but rather in both the N-terminal and C-terminal regions shared by both Siva proteins. The N-terminal region also participates in the translocation of both Siva proteins into the nuclear compartment. These results indicate that Siva-1 and Siva-2 mediate apoptosis in T lymphocytes via a caspase-dependent mitochondrial pathway that likely involves both cytoplasmic and nuclear events.     

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.     

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.     

Unlike Diablo/smac, Grim promotes global ubiquitination and specific degradation of X chromosome-linked inhibitor of apoptosis (XIAP) and neither cause apoptosis

Grim is a Drosophila inhibitor of apoptosis (IAP) antagonist that directly interferes with inhibition of caspases by IAPs. Expression of Grim, or removal of DIAP1, is sufficient to activate apoptosis in fly cells. Transient expression of Grim in mammalian cells induces apoptosis, arguing for the conservation of apoptotic pathways, but cytoplasmic expression of the mammalian IAP antagonist Diablo/smac does not. To understand why, we compared Grim and Diablo. Although they have the same IAP binding specificity, only Grim promoted XIAP ubiquitination and degradation. Grim also synergized with XIAP to promote an increase in total cellular ubiquitination, whereas Diablo antagonized this activity. Surprisingly, Grim-induced ubiquitination of XIAP did not require the IAP RING finger. Analysis of a Grim mutant that promoted XIAP degradation, but was not cytotoxic, suggests that Grim killing in transient assays is due to a combination of IAP depletion, blocking of IAP-mediated caspase inhibition, and at least one other unidentified function. Unlike transiently transfected cells, inducible mammalian cell lines can sustain continuous expression of Grim and selective degradation of XIAP without undergoing apoptosis, demonstrating that down-regulation and antagonism of IAPs is not sufficient to cause apoptosis of mammalian cells.     

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.     

mE10, a novel caspase recruitment domain-containing proapoptotic molecule

Apoptotic signaling is mediated by homophilic interactions between conserved domains present in components of the death pathway. The death domain, death effector domain, and caspase recruitment domain (CARD) are examples of such interaction motifs. We have identified a novel mammalian CARD-containing adaptor molecule termed mE10 (mammalian E10). The N-terminal CARD of mE10 exhibits significant homology (47% identity and 64% similarity) to the CARD of a gene from Equine Herpesvirus type 2. The C-terminal region is unique. Overexpression of mE10 in MCF-7 human breast carcinoma cells induces apoptosis. Mutational analysis indicates that CARD-mediated mE10 oligomerization is essential for killing activity. The C terminus of mE10 bound to the zymogen form of caspase-9 and promoted its processing to the active dimeric species. Taken together, these data suggest a model where autoproteolytic activation of pro-caspase-9 is mediated by mE10-induced oligomerization.     

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.     

c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.     

A new face on apoptosis: death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins

Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography-tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.     

Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells.

Expression of the proapoptotic protein Bax under the control of a GAL10 promoter in Saccharomyces cerevisiae resulted in galactose-inducible cell death. Immunofluorescence studies suggested that Bax is principally associated with mitochondria in yeast cells. Removal of the carboxyl-terminal transmembrane (TM) domain from Bax [creating Bax (deltaTM)] prevented targeting to mitochondrial and completely abolished cytotoxic function in yeast cells, suggesting that membrane targeting is crucial for Bax-mediated lethality. Fusing a TM domain from Mas70p, a yeast mitochondrial outer membrane protein, to Bax (deltaTM) restored targeting to mitochondria and cytotoxic function in yeast cells. Deletion of four well-conserved amino acids (IGDE) from the BH3 domain of Bax ablated its ability to homodimerize and completely abrogated lethality in yeast cells. In contrast, several Bax mutants which retained ability to homodimerize (deltaBH1, deltaBH2, and delta1-58) also retained at least partial lethal function in yeast cells. In coimmunoprecipitation experiments, expression of the wild-type Bax protein in Rat-1 fibroblasts and 293 epithelial cells induced apoptosis, whereas the Bax (deltaIGDE) mutant failed to induce apoptosis and did not associate with endogenous wild-type Bax protein. In contrast to yeast cells, Bax (deltaTM) protein retained cytotoxic function in Rat-1 and 293 cells, was targeted largely to mitochondria, and dimerized with endogenous Bax in mammalian cells. Thus, the dimerization-mediating BH3 domain and targeting to mitochondrial membranes appear to be essential for the cytotoxic function of Bax in both yeast and mammalian cells.     

Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway

Here we identify a new gene, dark, which encodes a Drosophila homologue of mammalian Apaf-1 and Caenorhabditis elegans CED-4, cell-death proteins. Like Apaf-1, but in contrast to CED-4, Dark contains a carboxy-terminal WD-repeat domain necessary for interactions with the mitochondrial protein cytochrome c. Dark selectively associates with another protein involved in apoptosis, the fly apical caspase, Dredd. Dark-induced cell killing is suppressed by caspase-inhibitory peptides and by a dominant-negative mutant Dredd protein, and enhanced by removal of the WD domain. Loss-of-function mutations in dark attenuate programmed cell deaths during development, causing hyperplasia of the central nervous system, and other abnormalities including ectopic melanotic tumours and defective wings. Moreover, ectopic cell killing by the Drosophila cell-death activators, Reaper, Grim and Hid, is substantially suppressed in dark mutants. These findings establish dark as an important apoptosis effector in Drosophila and raise profound evolutionary considerations concerning the relationship between mitochondrial components and the apoptosis-promoting machinery.