Interactive Fly, Drosophila

reaper


EVOLUTIONARY HOMOLOGS

X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP. XIAP associates with the active caspase-9-Apaf-1 (see Drosophila Apaf-1-related-killer) holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp315. Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevents caspase-9 association with XIAP and its concomitant inhibition. The N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs. Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. These studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis (Srinivasula, 2001).

Recent reports suggest that a cross-talk exists between apoptosis pathways mediated by mitochondria and cell death receptors. Mitochondrial events are required for apoptosis induced by the cell death ligand TRAIL (TNF-related apoptosis-inducing ligand) in human cancer cells. The Bax null cancer cells are resistant to TRAIL-induced apoptosis. Bax deficiency has no effect on TRAIL-induced caspase-8 activation and subsequent cleavage of Bid; however, it results in an incomplete caspase-3 processing because of inhibition by XIAP. Release of Smac/DIABLO from mitochondria through the TRAIL-caspase-8-tBid-Bax cascade is required to remove the inhibitory effect of XIAP and allow apoptosis to proceed. Inhibition of caspase-9 activity has no effect on TRAIL-induced caspase-3 activation and cell death, whereas expression of the active form of Smac/DIABLO in the cytosol is sufficient to reconstitute TRAIL sensitivity in Bax-deficient cells. These results show for the first time that Bax-dependent release of Smac/DIABLO, not cytochrome c, from mitochondria mediates the contribution of the mitochondrial pathway to death receptor-mediated apoptosis (Deng, 2002).

The signaling events leading to apoptosis can be divided into two distinct pathways, involving either mitochondria or death receptors. In the mitochondria pathway, death signals lead to changes in mitochondrial membrane permeability and the subsequent release of pro-apoptotic factors involved in various aspects of apoptosis. The released factors include cytochrome c (cyto c), apoptosis inducing factor (AIF), second mitochondria-derived activator of caspase (Smac/DIABLO), and endonuclease G. Cytosolic cyto c forms an essential part of the apoptosis complex 'apoptosome,' which is composed of cyto c, Apaf-1, and procaspase-9. Formation of the apoptosome leads to the activation of caspase-9, which then processes and activates other caspases to orchestrate the biochemical execution of cells. Smac/DIABLO is also released from the mitochondria along with cyto c during apoptosis, and it functions to promote caspase activation by inhibiting IAP (inhibitor of apoptosis) family proteins (Deng, 2002).

The IAP family proteins negatively regulate apoptosis by inhibiting caspase activity directly. Six human IAPs have been discovered. They regulate apoptosis by preventing the action of the central execution phase of apoptosis through direct inhibition of the effector caspase-3 and/or caspase-7. In addition, they prevent initiation of the intrinsic caspase activation cascade by directly inhibiting the apical caspase-9. Structural and biochemical dissection of XIAP, a widely expressed IAP member, reveals that the conserved BIR domains of XIAP mediate both its inhibitory activity on caspases and the protein-protein interaction with Smac/DIABLO. Binding of Smac/DIABLO to XIAP antagonizes caspase-XIAP interaction, thereby promoting apoptosis. Recent studies have shown that XIAP is highly expressed in most human cancer cells and that high levels of XIAP confer tumor resistance to chemotherapy or irradiation (Deng, 2002).

The key regulatory proteins of mitochondria-mediated apoptotosis are the Bcl-2 family of proteins, which can either promote cell survival, as do Bcl-2 and Bcl-xl, or induce cell death, as do Bax and Bak. Bcl-2 and Bcl-xl appear to directly or indirectly preserve the integrity of the outer mitochondrial membrane, thus preventing cyto c release and mitochondria-mediated cell death initiation, whereas the pro-apoptotic proteins Bax and Bak promote cyto c release from mitochondria. Bax has been implicated in apoptosis in many cell types under various conditions. More recently, studies using Bax-deficient human colon cancer cells have provided direct evidence that Bax plays a key role in mediating apoptosis induced by certain anti-cancer agents. The Bax protein exerts at least part of its activity by triggering cyto c release from mitochondria. Bax is in a predominantly cytosolic latent form in healthy cells and translocates to mitochondria after death signal stimulation. Accumulating evidence suggests that Bax translocation is required for its pro-apoptotic function and that regulation of Bax's association with the mitochondrial membrane represents a critical step in the transduction of apoptotic signals (Deng, 2002).

In the death receptor pathway, the apoptotic events are initiated by engaging the tumor necrosis factor (TNF)-family receptors, including TNFR1, Fas, DR-3, DR-4, and DR-5. Upon ligand binding or when overexpressed in cells, TNF receptor family members aggregate, resulting in the recruitment of an adapter protein called FADD. The receptor-FADD complex then recruits procaspase-8. This allows proteolytic processing and activation of the receptor-associated procaspase-8, thereby initiating the subsequent cascade of additional processing and activation of downstream effector caspases (Deng, 2002).

TRAIL/Apo2L (TNF-related apoptosis-inducing ligand TRAIL or Apo2 ligand) is an apoptosis-inducing member of the TNF gene superfamily. Unlike TNF-alpha and FasL, TRAIL appears to specifically kill transformed and cancer cells while leaving normal cells intact. Preclinical experiments in mice and nonhuman primates have shown that administration of TRAIL suppresses tumor growth without apparent systematic cytotoxicity. Therefore, TRAIL represents a promising anti-cancer agent. TRAIL interacts with four cellular receptors that form a distinct subgroup within the TNFR superfamily. Most recent experiments have shown that FADD and procaspase-8 associate with the endogenous TRAIL receptors DR4 and DR5. FADD and caspase-8 are required for TRAIL-induced apoptosis. Thus, TRAIL/Apo2L and FasL appear to engage similar pathways to apoptosis (Deng, 2002).

Although the extrinsic pathway (through the death receptors) and the intrinsic pathway (through the mitochondria) for apoptosis are capable of operating independently, accumulating evidence suggests that a cross-talk between the two pathways exists in cells. The link between death receptor signaling and the mitochondrial pathway comes from the finding that a BH3-domain-only subfamily protein, Bid, is cleaved by active caspase-8. The truncated Bid (tBid) translocates to mitochondria and triggers cyto c release. It has been proposed that tBid regulates cyto c release by inducing the homo-oligomerization of pro-apoptotic family members Bak or Bax. Cells lacking both Bax and Bak, but not cells lacking just one of these components, are completely resistant to tBid-induced cyto c release and apoptosis (Deng, 2002).

Bid appears to link the intrinsic pathway to the cell death receptor-mediated apoptosis. However, the precise mitochondrial events required for this cross-talk remain unclear. The mechanisms of TRAIL-induced apoptosis and the role of mitochondria in the cell death receptor pathway also need further investigation. Using human colon cancer cells defective in Bax function, it has been shown that mitochondrial events are required for TRAIL-induced apoptosis. The reason for this requirement is the presence of negative regulation of caspase cascade by XIAP. Activation of the mitochondrial pathway leads to the release of Smac/DIABLO, which removes XIAP blockage of caspase activation. These results further show that release of Smac/DIABLO, not cyto c, is the key event mediating the contribution of the mitochondrial pathway to the death receptor-mediated apoptosis (Deng, 2002).

Functions of Inhibitors of apoptosis proteins and their interaction the Reaper type proteins

The baculovirus protein p35 inhibits programmed cell death in such diverse animals as insects, nematodes and mammals. p35 protein is a substrate for and inhibitor of the C. elegans cell-death protease CED-3 and a substrate for four CED-3-like vertebrate cysteine protease activities implicated in apoptosis in mammals. A p35 mutation, that greatly reduces p35 activity in vitro as a CED-3 substrate and inhibitor, abolishes p35 activity in vivo in protecting against cell death in C. elegans. Introduction of the CED-3 cleavage site in p35 into the cowpox virus protein crmA, (which inhibits mammalian apoptosis but not programmed cell death in C. elegans), causes crmA to block CED-3-mediated cell death. These observations suggest that p35 may prevent programmed cell death in C. elegans and other species by acting as a competitive inhibitor of cysteine proteases (Xue, 1995).

The baculovirus antiapoptotic protein p35 inhibits the proteolytic activity of human interleukin-1 beta converting enzyme (ICE) and three of its homologs in enzymatic assays. Coexpression of p35 prevents the autoproteolytic activation of ICE from its precursor form and blocks ICE-induced apoptosis. Inhibition of enzymatic activity correlates with the cleavage of p35 and the formation of a stable ICE-p35 complex. The ability of p35 to block apoptosis in different pathways and in distantly related organisms suggests a central and conserved role for ICE-like proteases in the induction of apoptosis (Bump, 1995).

Baculovirus p35 prevents programmed cell death in diverse organisms and encodes a protein inhibitor (P35) of the CED-3/interleukin-1 beta-converting enzyme (ICE)-related proteases. P35 domains have been identified that are necessary for suppression of virus-induced apoptosis in insect cells, the context in which P35 evolved. During infection, P35 is cleaved within an essential domain at or near the site DQMD-87G required for cleavage by CED-3/ICE family proteases. Cleavage site substitution of alanine for aspartic acid at position 87 (D87A) of the P1 residue abolishes P35 cleavage and antiapoptotic activity. Although the P4 residue substitution D84A also causes loss of apoptotic suppression, it does not eliminate cleavage and suggests that P35 cleavage is not sufficient for antiapoptotic activity. Apoptotic insect cells contain a CED-3/ICE-like activity that cleaves in vitro-translated P35 and is inhibited by recombinant wild-type P35 but not P1- or P4-mutated P35. Thus, baculovirus infection directly or indirectly activates a novel CED-3/ICE-like protease inhibited by P35, thereby preventing virus-induced apoptosis. These findings confirm the inhibitory activity of P35 towards the CED-3/ICE protease, including recombinant mammalian enzymes, and are consistent with a mechanism involving P35 stoichiometric interaction and cleavage. P35's inhibition of phylogenetically diverse proteases accounts for its general effectiveness as an apoptotic suppressor (Bertin, 1996).

The 75 kDa tumor necrosis factor receptor (TNFR2) transduces extracellular signals via receptor-associated cytoplasmic proteins. Two of these signal transducers are TRAF1 and TRAF2. Two novel TNFR2-associated proteins, designated c-IAP1 and c-IAP2, are closely related mammalian members of the inhibitor of apoptosis protein (IAP) family originally identified in baculoviruses. The viral and cellular IAPs contain N-terminal baculovirus IAP repeat (BIR) motifs and a C-terminal RING finger. The c-IAPs do not directly contact TNFR2, but rather associate with TRAF1 and TRAF2 through their N-terminal BIR motif-comprising domain. The recruitment of c-IAP1 or c-IAP2 to the TNFR2 signaling complex requires a TRAF2-TRAF1 heterocomplex (Rothe, 1995).

Drosophila activators of apoptosis mapping to the Reaper region function, in part, by antagonizing IAP proteins through a shared RHG motif. Reaper isolated from the Blowfly L. cuprina, triggers 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 binds to DIAP1 but fails 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 (Chen, 2004).

Mammalian IAPs and their interaction proteins with Reaper-type motifs

The inhibitor-of-apoptosis proteins (IAPs) play a critical role in the regulation of apoptosis by binding and inhibiting caspases. Reaper family proteins and Smac/DIABLO use a conserved amino-terminal sequence to bind to IAPs in flies and mammals, respectively, blocking their ability to inhibit caspases and thus promoting apoptosis. The serine protease Omi/HtrA2 has been identified as a second mammalian XIAP-binding protein with a Reaper-like motif. This protease autoprocesses to form a protein with amino-terminal homology to Smac/DIABLO and Reaper family proteins. Full-length Omi/HtrA2 is localized to mitochondria but fails to interact with XIAP. Mitochondria also contain processed Omi/HtrA2, which, following apoptotic insult, translocates to the cytosol, where it interacts with XIAP. Overexpression of Omi/HtrA2 sensitizes cells to apoptosis, and its removal by RNA interference reduces cell death. Omi/HtrA2 thus extends the set of mammalian proteins with Reaper-like function that are released from the mitochondria during apoptosis (Martins, 2002).

Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac. 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 (Verhagen, 2002).

Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis to antagonize inhibitors of apoptosis (IAPs) and contribute to caspase-independent cell death. Omi/HtrA2 directly cleaves various IAPs in vitro, and the cleavage efficiency is determined by its IAP-binding motif, AVPS. Cleavage of IAPs such as c-IAP1 substantially reduces its ability to inhibit and ubiquitylate caspases. In contrast to the stoichiometric anti-IAP activity by Smac/DIABLO, Omi/HtrA2 cleavage of c-IAP1 is catalytic and irreversible, thereby more efficiently inactivating IAPs and promoting caspase activity. Elimination of endogenous Omi by RNA interference abolishes c-IAP1 cleavage and desensitizes cells to apoptosis induced by TRAIL. In addition, overexpression of cleavage-site mutant c-IAP1 makes cells more resistant to TRAIL-induced caspase activation. This IAP cleavage by Omi is independent of caspase. Taken together, these results indicate that unlike Smac/DIABLO, Omi/HtrA2's catalytic cleavage of IAPs is a key mechanism for it to irreversibly inactivate IAPs and promote apoptosis (Yang, 2003).

Inhibitor of apoptosis proteins (IAPs) prevent apoptosis through direct inhibition of caspases. The serine protease HtrA2/Omi has an amino-terminal IAP interaction motif like that found in Reaper, which displaces IAPs from caspases, leading to enhanced caspase activity. The cell death-promoting properties of HtrA2/Omi are not only exerted through its capacity to oppose IAP inhibition of caspases but also through its integral serine protease activity. Peptide libraries were used to determine the optimal substrate sequence for cleavage by HtrA2 and also the preferred binding sequence for its PDZ domain. Using these peptides, it has been show that the PDZ domain of HtrA2/Omi suppresses the proteolytic activity unless it is engaged by a binding partner. Subjecting HtrA2/Omi to heat shock treatment also increases its protease activity. Unexpectedly, binding of X-linked inhibitor of apoptosis protein (XIAP) to the Reaper motif of HtrA2/Omi results in a marked increase in proteolytic activity, suggesting a new role for IAPs. When HtrA2/Omi is released from mitochondria following an apoptotic stimulus, binding to IAPs may switch their function from caspase inhibition to serine protease activation. Thus although IAP overexpression can suppress caspase activation, it could have the opposite effect on HtrA2/Omi-dependent cell death. This, together with the ability of HtrA2/Omi to degrade IAPs, may limit the overall cellular protection that can be provided by these proteins (Martins, 2003).

The mature serine protease Omi/HtrA2 is released from the mitochondria into the cytosol during apoptosis. Suppression of Omi/HtrA2 by RNA interference in human cell lines reduces cell death in response to TRAIL and etoposide. In contrast, ectopic expression of mature wildtype Omi/HtrA2, but not an active site mutant, induces potent caspase activation and apoptosis. In vitro assays have demonstrated that Omi/HtrA2 degrades inhibitor of apoptosis proteins (IAPs). Consistent with this observation, increased expression of Omi/HtrA2 in cells increases degradation of XIAP, while suppression of Omi/HtrA2 by RNA interference has an opposite effect. Combined, these data demonstrate that IAPs are substrates for Omi/HtrA2, and their degradation could be a mechanism by which the mitochondrially released Omi/HtrA2 activates caspases during apoptosis (Srinivasula, 2003).


reaper : Biological Overview | Regulation | Protein Interactions and parallel pathways | Developmental Biology | Effects of Mutation | References

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