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• W2022135873 abstract "Article1 October 1999free access Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases Quinn L. Deveraux Quinn L. Deveraux The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Eugen Leo Eugen Leo The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Henning R. Stennicke Henning R. Stennicke The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Kate Welsh Kate Welsh The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Guy S. Salvesen Guy S. Salvesen The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author John C. Reed Corresponding Author John C. Reed The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Quinn L. Deveraux Quinn L. Deveraux The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Eugen Leo Eugen Leo The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Henning R. Stennicke Henning R. Stennicke The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Kate Welsh Kate Welsh The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Guy S. Salvesen Guy S. Salvesen The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author John C. Reed Corresponding Author John C. Reed The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Author Information Quinn L. Deveraux1, Eugen Leo1, Henning R. Stennicke1, Kate Welsh1, Guy S. Salvesen1 and John C. Reed 1 1The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:5242-5251https://doi.org/10.1093/emboj/18.19.5242 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Several human inhibitor of apoptosis (IAP) family proteins function by directly inhibiting specific caspases in a mechanism that does not require IAP cleavage. In this study, however, we demonstrate that endogenous XIAP is cleaved into two fragments during apoptosis induced by the tumor necrosis factor family member Fas (CD95). The two fragments produced comprise the baculoviral inhibitory repeat (BIR) 1 and 2 domains (BIR1-2) and the BIR3 and RING (BIR3-Ring) domains of XIAP. Overexpression of the BIR1-2 fragment inhibits Fas-induced apoptosis, albeit at significantly reduced efficiency compared with full-length XIAP. In contrast, overexpression of the BIR3-Ring fragment results in a slight enhancement of Fas-directed apoptosis. Thus, cleavage of XIAP may be one mechanism by which cell death programs circumvent the anti-apoptotic barrier posed by XIAP. Interestingly, ectopic expression of the BIR3-Ring fragment resulted in nearly complete protection from Bax-induced apoptosis. Use of purified recombinant proteins revealed that BIR3-Ring is a specific inhibitor of caspase-9 whereas BIR1-2 is specific for caspases 3 and 7. Therefore XIAP possesses two different caspase inhibitory activities which can be attributed to distinct domains within XIAP. These data may provide an explanation for why IAPs have evolved with multiple BIR domains. Introduction First identified in insect viruses, inhibitor of apoptosis (IAP) family proteins appear to be evolutionarily conserved with apparent homologs identified thus far in mammals, flies, worms and yeast. IAP family members are characterized by a highly conserved ∼70 amino acid domain termed the baculoviral inhibitory repeat (BIR) that can be present as many as three times in some IAPs (reviewed in Clem and Duckett, 1998; LaCasse et al., 1998; Deveraux and Reed, 1999). In addition to BIR domains, some IAPs contain a C-terminal zinc-binding RING motif (Saurin et al., 1996; Borden, 1998). Structure–function studies of IAP family proteins performed to date have uniformly demonstrated a requirement for at least one BIR domain for suppression of apoptosis (reviewed in Clem and Duckett, 1998; LaCasse et al., 1998; Deveraux and Reed, 1999). Other reports have indicated that at least under some conditions baculoviral IAPs require both N-terminal BIR domains and a C-terminal RING domain for their anti-apoptotic function in insect cells (Clem and Miller, 1994; Harvey et al., 1997). The relevance of the RING domain for IAP-mediated suppression of apoptosis, however, appears to depend upon cellular context. The BIR2 domain alone of human XIAP was found to be sufficient for inhibition of apoptosis induced by Fas (CD95) (Takahashi et al., 1998). Likewise, the BIR2 domain from baculovirus Op-IAP or Drosophila D-IAP1 was reported to be sufficient for inhibition of apoptosis induced by the fly apoptosis protein HID in insect cells (Vucic et al., 1998). In a separate study, cell death-suppressing activity mapped to the N-terminal BIR domains in the Drosophila IAPs, D-IAP1 or D-IAP2, and removal of the C-terminal RING domain actually enhanced their ability to suppress developmental programmed cell death and cell death induced by ectopic expression of the fly apoptosis gene reaper in the developing fly eye (Hay et al., 1995). Although the structural requirements for IAP function may vary, numerous reports suggest strongly that at least some IAP family members play a highly conserved role in the regulation of cell death (reviewed in Clem and Duckett, 1998; Deveraux and Reed, 1999). Consistent with this idea, we reported previously that the human IAP family members XIAP, cIAP1 and cIAP2 can directly inhibit specific caspases (Deveraux et al., 1997, 1998; Roy et al., 1997), an evolutionarily conserved family of cysteine proteases that play critical roles in the execution of apoptosis (Salvesen and Dixit, 1997; Thornberry and Lazebnik, 1998). XIAP, cIAP1 and cIAP2 each contain three tandem BIR domains and a single C-terminal RING motif. The BIR domains of XIAP, cIAP1 and cIAP2 are sufficient for both suppression of apoptosis and inhibition of caspase-3 and caspase-7 (Deveraux et al., 1997; Roy et al., 1997). Furthermore, the ability of XIAP to inhibit caspases 3 and 7 was localized to the BIR2 domain alone—supporting the concept that a single BIR domain can be sufficient for suppression of caspases and blockade of apoptosis (Takahashi et al., 1998). Two prototypical pathways for induction of apoptosis in mammalian cells are induced by Fas and Bax. Fas (CD95) is a member of the tumor necrosis factor (TNF) family of apoptosis-inducing receptors that activate pro-caspase-8 and possibly other initiator caspases (Boldin et al., 1996; Muzio et al., 1996; Wallach et al., 1997). caspase-8 then cleaves and activates caspase-3 and other downstream caspases that function as the ultimate effectors of apoptosis. Bax is a pro-apoptotic member of the Bcl-2 family that associates with mitochondria and induces release of cytochrome c (reviewed in Green and Reed, 1998). Once activated by cytochrome c, together with cofactor nucleotide triphosphates (dATP or ATP), apoptosis promoting factor (Apaf-1) then binds and activates pro-caspase-9, which in turn cleaves and activates caspase-3 and other downstream caspases (Li et al., 1997; Reed, 1997; Zou et al., 1997). IAPs block cell death at distinct steps in apoptotic pathways induced by Fas or Bax. XIAP, cIAP1 and cIAP2 interfere with the cytochrome c-mediated activation of caspase-3, which is induced by Bax, by directly inhibiting caspase-9 (Deveraux et al., 1998). Thus, caspase-9 was identified as a new target for the IAPs upstream of caspase-3. In the Fas pathway, which can activate caspase-3 without necessarily involving mitochondria and caspase-9 (Peter and Krammer, 1998; Scaffidi et al., 1998), XIAP and other IAPs suppress the caspase cascade through direct inhibition of active caspase-3 (Deveraux et al., 1998). In this study we observed that during apoptosis induced by the TNF family member Fas, XIAP is cleaved, separating the BIR1-2 domains from the BIR3-Ring domain. Although cleavage of XIAP yields a BIR1-2 fragment that is capable of inhibiting active caspases 3 and 7, as well as apoptosis induced by Fas, ectopically expressed BIR1-2 has significantly reduced potency compared with full-length XIAP. Moreover, the BIR1-2 fragment appears to be susceptible to further degradation by caspases. Thus, cleavage of XIAP may be one mechanism for lowering the threshold of caspase activity necessary for inducing apoptosis. In contrast to BIR1-2, ectopic expression of the BIR3-Ring fragment slightly enhances Fas-induced apoptosis. Surprisingly, however, expression of BIR3-Ring potently inhibited cell death due to Bax. Here we elucidate the mechanism by which BIR3-Ring blocks Bax-induced cell death and compare it with the mode of BIR1-2-mediated inhibition of both the Fas and Bax apoptotic pathways. These results demonstrate unique functions of different BIR domains within XIAP and thus provide a possible explanation for the presence of multiple BIR domains in the IAP family of anti-apoptotic proteins. Results XIAP is cleaved during Fas-induced apoptosis Treatment of Jurkat T cells with antibodies specific for Fas (anti-Fas) results in apoptosis which is preceded by proteolytic processing and activation of pro-caspase-3 (Enari et al., 1996; Cohen, 1997; Games et al., 1998). In a time-dependent manner, we observed that endogenous XIAP is cleaved into at least two fragments during treatment of Jurkat T cells with anti-Fas antibody. Anti-Fas treatment results in depletion of much of the full-length 53 kDa protein and concomitant generation of a 30 kDa fragment that reacts with an anti-XIAP antibody specific for an epitope found within the BIR3-Ring region (Figure 1). Fas-induced proteolysis of XIAP occurred with approximately the same kinetics as processing of pro-caspase-3. Addition of 50 μM zVAD-FMK, a broad spectrum caspase inhibitor, completely prevented anti-Fas-induced cleavage of XIAP, indicating that this is a caspase-dependent event. Figure 1.Endogenous XIAP is cleaved during Fas-mediated apoptosis. CH11 anti-Fas antibody (300 ng/ml) was added to Jurkat T cells in culture at 37°C. Cells were removed at the indicated times, normalized for protein content and analyzed by Western blot analysis. Nitro- cellulose filters were incubated with antibodies specific for the region between the BIR3-Ring domain of XIAP (left panel) or with caspase-3 antisera (right panel). Arrows denote endogenous XIAP and the BIR3-Ring cleavage product (left panel) or the pro- and processed forms of caspase-3 (right panel). The apparently non-specific anti-XIAP immune-reactive molecule migrating slightly larger than XIAP serves as a fortuitous loading control (*). Download figure Download PowerPoint Mapping of XIAP cleavage site Since XIAP cleavage correlates with caspase activation and can be inhibited with caspase inhibitors, 35S-labeled XIAP was translated in vitro and incubated with purified active caspases in an effort to reproduce the cleavage of XIAP observed in intact cells. Incubation of XIAP with purified recombinant caspases 3, 6, 7 or 8 produced at least two ∼30 kDa cleavage fragments (Figure 2A). No cleavage of the in vitro-translated XIAP was detected if caspases were omitted from the reaction. caspase-3 and caspase-7 were the most efficient enzymes with respect to XIAP cleavage, although all caspases tested produced similar XIAP fragments. However, in the caspase-3-treated [35S]XIAP reaction, the slower migrating fragment (later determined to be the BIR1-2 region) was not visible—possibly due to further cleavage. Figure 2.XIAP is cleaved in vitro by purified recombinant caspases. (A) cDNAs encoding full-length XIAP or XIAP mutants XIAPD242E or XIAPD214E were translated in vitro in reticulocyte lysate containing [35S]methionine. The 35S-radiolabeled XIAP, XIAPD242E or XIAPD214E proteins were incubated with 100 nM of the indicated caspases for 1 h at 37°C. Reactions were then resolved on SDS gels and analyzed by fluorography. (B) 293T cells were transfected with myc control, wild-type myc–XIAP (WT) or mutant myc–XIAP(D242E) with or without co-transfection of Fas. Cell samples were analyzed on Western blots using antibodies specific for the BIR3-Ring region of XIAP. Arrows denote the endogenous XIAP, Myc–XIAP transfection products and the XIAP cleavage fragment. Expression levels of MYC–XIAP proteins were similar in cells not expressing Fas (not shown). (C) The diagram at the bottom shows a schematic of the full-length XIAP, mapped cleavage site, BIR1-2 and BIR3-Ring fragments. In experiments similar to those presented in (A), we observed no cleavage of XIAP by recombinant active caspase-9; however, recombinant caspase-9 is ∼10 000-fold less active than the other recombinant caspases tested in these assays. Download figure Download PowerPoint To confirm that caspases cleave XIAP directly and to map the cleavage site, purified recombinant GST–XIAP was incubated with purified active caspase-3 or -7. A 20-fold molar excess of caspase relative to XIAP protein was added in these assays, since at equimolar concentration XIAP completely inhibits caspase-3 or -7 activity and therefore XIAP cleavage. The resulting material was separated on an SDS gel and transferred to PVDF membrane and the faster migrating C-terminal fragment was excised and subjected to N-terminal sequence analysis. Both caspase-3 and -7 treatment of XIAP resulted in identical C-terminal fragments beginning with alanine-243 of XIAP. Based upon these results, we prepared constructs in which aspartic acid-242 in XIAP was replaced by a glutamic acid (XIAPD242E). As a control, in vitro-translated XIAPD214E was prepared and incubated with purified caspase-3. The XIAPD214E protein underwent cleavage akin to the wild-type protein, whereas XIAPD242E was not cleaved when incubated with active caspase-3 under the same conditions (Figure 2A). To further address the idea that XIAP aspartic acid-242 is the relevant caspase cleavage site in vivo, wild-type XIAP and XIAPD242E constructs were expressed by transient transfection in 293T cells. When co-transfected with Fas, the expressed wild-type XIAP protein was cleaved, generating a 30 kDa product that reacts with antibodies specific for the BIR3-Ring region (Figure 2B). In contrast, the expressed XIAPD242E mutant protein was intact and present at comparable levels in both control and Fas-transfected 293T cells. Differential effects of BIR1-2 and BIR3-Ring fragments on apoptotic pathways Since caspase-mediated cleavage can regulate molecules either by disrupting their previous function or by generating a fragment(s) with new activities (reviewed in Salvesen and Dixit, 1997), we wished to examine the effects of each XIAP fragment on apoptotic events induced by ectopic expression of Fas receptor construct (Fas) or the pro-apoptotic protein Bax. To study the potential effects of expression of the BIR1-2 and BIR3-Ring constructs on cell death we chose 293 and 293T cells as a model system, due to their higher transfection efficiency and their apoptotic sensitivity to expression of both Fas and the Bax protein. As expected, co-expression of full-length XIAP or BIR1-2 suppressed Fas-induced cell death, although BIR1-2 was significantly less efficient (∼2- to 3-fold) than full-length XIAP. In contrast, cells expressing the BIR3-Ring fragment showed a slight enhancement (∼10%) of cell death induced by Fas (Figure 3), indicating that the BIR3-Ring fragment lacks anti-apoptotic activity against Fas in these cells. Expression of the non-cleavable mutant XIAPD242E was slightly more protective against Fas-mediated apoptosis than wild-type XIAP. Figure 3.Differential effects of BIR3-Ring and BIR1-2 on Fas and Bax-induced apoptosis in 293 cells. (A) Fas or Bax expression plasmids were co-transfected into 293 cells with the indicated XIAP expression plasmids. Apoptosis (dark bars) or caspase activity (light bars) was measured as described in Materials and methods and the data presented here as a percentage relative to cells transfected with Fas or Bax alone (mean ± SD; n = 2). (B) Western blot analysis of transfected cells using antisera specific for BIR2 (left) or BIR3-Ring (right) of XIAP. The upper arrowhead denotes the transfected full-length XIAP or XIAPD242E expression products. The lower arrow denotes the Fas-induced cleavage products and the expressed BIR1-2 and BIR3-Ring fragments. An asterisk is pictured next to the endogenous full-length XIAP to the right of each panel. All the expression products contain an N-terminal myc sequence. Similar results were obtained in 293T cells. Note that XIAP is not cleaved in cells co-expressing Bax whereas it is readily cleaved in cells co-expressing Fas. Download figure Download PowerPoint Expression of full-length XIAP or BIR1-2 resulted in suppression of Bax-mediated cell death. These results were not surprising since we had previously localized the caspase-3 and -7 inhibitory activity of XIAP to the BIR2 domain which behaves similarly to the BIR1-2 construct with respect to anti-apoptotic activity (Takahashi et al., 1998). Surprisingly, however, co-expression of Bax and the BIR3-Ring construct resulted in nearly complete inhibition of cell death caused by Bax (Figure 3). Thus, in contrast to Fas, apoptosis induced by Bax was potently inhibited by the BIR3-Ring fragment. Independent transfection of constructs expressing the BIR1, BIR3 or Ring domains alone showed no significant inhibition of apoptosis in these assays, although co-expression of Ring with Fas resulted in a slight enhancement of cell death (not shown). Western blot analysis of lysates prepared from the transiently transfected 293 cells confirmed production of the expected proteins and revealed that the failure of the BIR3-Ring fragment to suppress Fas-induced cell death was not due to lower expression levels. Note that during Fas-induced apoptosis the expressed wild-type XIAP, but not XIAPD242E, is cleaved. Under these conditions, antibodies specific for the BIR3-Ring region of XIAP readily detect the endogenous Fas-induced BIR3-Ring cleavage fragment, whereas incubation of the same blot with antisera specific for the BIR1-2 domain reveals only small amounts of the BIR1-2 fragment, suggesting that it may be unstable. Interestingly, XIAP is not cleaved during apoptosis induced by Bax. Recombinant BIR3-Ring suppresses caspase activation induced in vitro by cytochrome c To address the biochemical step at which BIR3-Ring might block the Bax apoptotic pathway, we explored whether recombinant BIR3-Ring protein could inhibit caspase activation induced by cytochrome c and dATP (Cyto-c/dATP) in lysates from cultured cells. Addition of recombinant BIR3-Ring concurrently with or before addition of Cyto-c/dATP to cell lysates suppressed the generation of caspase-3-like protease activity as measured by the hydrolysis of DEVD–7-amino-4-trifluoromethyl coumarin (DEVD–AFC) (Figure 4A). Addition of either BIR3 alone or Ring alone was ineffective at blocking Cyto-c/dATP induction of caspase activity, implying that the combination of these domains is required. While BIR3-Ring suppressed caspase activity when added at the time of, or before the introduction of, Cyto-c/dATP, the addition of BIR3-Ring protein 10 min after the addition of Cyto-c/dATP failed to inhibit caspase activity that had already been generated (Figure 4B). In contrast, addition of full-length XIAP, BIR1-2 or BIR2 recombinant protein still suppressed caspase activity regardless of the timing of Cyto-c/dATP addition. Recombinant BIR1, BIR3 or Ring domain proteins did not affect Cyto-c/dATP-induced caspase activity in these assays, demonstrating the specificity of these results. Consistent with our observation that expression of the BIR3-Ring fragment does not block Fas-induced cell death in intact cells, addition of recombinant BIR3-Ring had no effect upon caspase-8-induced DEVD–AFC cleaving activity in cell lysates, even when added before the addition of active caspase-8 (Figure 4C). In contrast, full-length XIAP, BIR1-2 or BIR2 alone did suppress accumulation of caspase-3-like activity in the caspase-8-treated lysates. Recombinant XIAPD242E or XIAPD242A behaved similarly to recombinant wild-type XIAP in these assays (not shown), further confirming that cleavage of XIAP is not a necessary component of its caspase inhibitory mechanism. Figure 4.Effect of recombinant XIAP and XIAP fragments on Cyto-c/dATP or caspase-8 induction of caspase activity in lysates from 293 cells. Recombinant XIAP or XIAP fragment proteins (2 μM) were added to lysates from 293 cells concurrently with (A) or 10 min after (B) the addition of 10 μM cytochrome c and 1 mM dATP. (C) Recombinant XIAP-derived proteins were added to cell lysates followed by the addition of active caspase-8 (100 nM). Lysates were incubated at 30°C and aliquots containing equivalent amounts of total protein were withdrawn after 10 min and assayed for caspase activity by continuously measuring the release of fluorogenic 7-amino-4-trifluoromethyl coumarin (AFC). Release of AFC from DEVD–AFC (y-axis) was measured from the onset of substrate addition (denoted on the x-axis in seconds). Control lysate samples were incubated with (CNTL) or without (Bkg) Cyto-c/dATP alone (A and B). In (C), caspase-8 alone was designated as control (CNTL). Experiments were repeated three times with similar results. Download figure Download PowerPoint BIR1-2 and BIR3-Ring differentially inhibit caspases Based upon dose–response experiments, BIR3-Ring inhibits Cyto-c/dATP-induced activation of caspase-3 ∼3-fold more efficiently than BIR1-2 or BIR2 alone (Figure 5A and B, and data not shown). However, addition of recombinant BIR1-2 or BIR3-Ring had very different effects on pro-caspase-3 processing induced by Cyto-c/dATP, as revealed by Western blot analysis of these lysates (Figure 5C). In control lysates to which XIAP protein fragments were not added, Cyto-c/dATP induced proteolytic cleavage of the ∼36 kDa pro-caspase-3 protein, producing 17 (p17) and 20 (p20) kDa fragments which react with an anti-caspase-3 antiserum specific for epitopes in the large subunit of caspase-3 (the small p10 subunit is not detected). The p20 and p17 caspase-3 products are alternatively cleaved forms of the large subunit of active caspase-3, which have previously been attributed to autocatalytic processing events that sequentially remove the N-terminal pro-domain from the ∼24 kDa form (large subunit + the pro-domain) (Martin et al., 1996; Deveraux et al., 1997). BIR1-2 did not substantially suppress the Cyto-c/dATP-induced proteolysis of ∼36 kDa pro-caspase-3 whereas BIR3-Ring did preserve the bulk of caspase-3 in its pro-form. The BIR1-2 recombinant protein, however, did arrest the Cyto-c/dATP-induced processing of caspase-3 at an intermediate step, resulting in an accumulation of an ∼24 kDa fragment which we previously determined to be the large subunit with the pro-domain still attached (Deveraux et al., 1997, 1998). The differences in processing patterns seen with BIR1-2 versus the BIR3-Ring fragment were not entirely attributable to potency since even when added at low concentrations (0.2 μM), BIR3-Ring did not cause the accumulation of the p24 caspase-3 processing intermediate, suggesting qualitative differences in the caspase inhibitory properties of BIR1-2 and BIR3-Ring. Figure 5.Comparison of the effect of BIR1-2 and BIR3-Ring on Cyto-c/dATP-induced caspase-3 activation. Various concentrations of recombinant BIR1-2 (A) or BIR3-Ring (B) were added to 293 cell lysates concurrently with 10 μM cytochrome c and 1 mM dATP. Lysates normalized for total protein content were incubated at 30°C and aliquots were removed after 30 min and assayed for the ability to hydrolyze DEVD–AFC using continuous measurements over the time indicated (A and B) or for Western blot analysis using an antiserum specific for caspase-3 (C). (D) Schematic of Cyto-c/dATP-induced processing of caspase-3. Experiments were repeated twice with similar results. Download figure Download PowerPoint As depicted in Figure 5D, the first cleavage of pro-caspase-3 to the p24 subunit in the Cyto-c/dATP pathway is likely made by caspase-9, with subsequent processing of p24 to p20 and p17 dependent upon caspase-3 autocatalytic activity (Martin et al., 1996; Li et al., 1997). Thus, in these assays, BIR1-2 appears to inhibit caspase-3 activity, thereby preventing its maturation beyond the p24 stage of processing, whereas BIR3-Ring functions upstream of pro-caspase-3 by possibly inhibiting caspase-9 or its activation. To further address the mechanism by which BIR3-Ring suppresses Cyto-c/dATP-induced activation of caspase-3, we recovered the recombinant GST fusion proteins following incubation in cell lysates containing Cyto-c/dATP, and analyzed the bound proteins by Western blotting using antisera specific for caspase-3 or caspase-9. BIR1-2 did, indeed, bind to the p24 form of the large subunit of caspase-3, whereas full-length XIAP or BIR3-Ring bound only pro-caspase-9 or active caspase-9 (Figure 6). The inability to detect the pro-form or p24 partially processed form of caspase-3 in GST pull-downs involving full-length XIAP reflects the ability of this protein to suppress caspase-9, thus blocking the effects of Cyto-c/dATP upstream of pro-caspase-3 (Deveraux et al., 1998). Figure 6.Association of recombinant GST–XIAP, GST–BIR1-2 and GST–BIR3-Ring with endogenous caspases 3 and 9 in cell lysates containing Cyto-c/dATP. Cytochrome c was added to cell lysates with or without the addition of the indicated GST fusion proteins. Following incubation at 30°C for 30 min, samples were removed and analyzed by Western blotting using antisera specific for caspase-3 (left panel) or caspase-9 (right panel). In lanes 1–5, lysates were analyzed directly. In lanes 6–8, GST fusion proteins were recovered on glutathione–Sepharose beads and bound caspase-3 or caspase-9 molecules were determined by Western blot analysis. Arrowheads denote the pro- and processed forms of caspase-3 and caspase-9. Note that neither the caspase-3 nor the caspase-9 antiserum reacts with the small subunit of their respective immunogens. Download figure Download PowerPoint BIR3-Ring directly inhibits caspase-9 To provide direct evidence for the biochemical function of BIR3-Ring, we tested its ability to inhibit purified recombinant active caspases. Recombinant BIR1-2, like BIR2 alone, inhibited caspase-3 and caspase-7 but not caspase-9 or other caspases tested (Figure 7 and data not shown). BIR3-Ring, however, displayed no ability to inhibit caspases 3 and 7 or other caspases tested but did inhibit active recombinant caspase-9. Recombinant BIR1, BIR3 or Ring proteins had no significant effect in these assays, demonstrating the specificity of these results. Thus BIR3-Ring is a specific inhibitor of caspase-9, whereas the BIR1-2 fragment (like BIR2 alone) is specific for caspases 3 and 7. Figure 7.Comparison of the inhibitory effects of BIR1-2 and BIR3-Ring on inhibition of caspases. Recombinant active caspases 3 (A), 7 (B) and 9 (C) were incubated with DEVD–AFC (A and B) or LEHD–AFC (C) in the presence or absence of the indicated recombinant XIAP-derived fusion proteins. Reactions were incubated at 37°C for the indicated times with continuous monitoring of AFC release. caspase-3 (A) and caspase-7 (B) were employed at 100 and 300 pM, respectively, using a 20-fold molar excess of XIAP fragments. Recombinant BIR3-Ring did not inhibit caspase-3 or caspase-7 even when present at 100-fold molar excess (A and B). Recombinant ΔCARD-caspase-9 (C) was added at 200 nM due to its lower specific activity, and XIAP-derived proteins were added at 800 nM (4-fold molar excess). However, recombinant BIR1, BIR2, BIR1-2, BIR3 and Ring showed no significant inhibition even at 20-fold molar excess in these assays (C). None of the XIAP-derived fusion proteins inhibited caspase-8 in similar assays (not shown), further demonstrating the specificity of these results. Active caspases were incubated alone as controls (CNTL). Recombinant ΔCARD-caspase-9 was used in these assays due to its greater activity and stability relative to recombinant caspase-9 containing the CARD domain; however, similar results were obtained using the recombinant caspase-9 containing the CARD domain in these assays. Experiments were repeated a minimum of three times with similar results. Download figure Download PowerPoint Discussion When ectopically overexpressed, members of the IAP family have been shown to effectively inhibit a variety of cell death programs (reviewed in Clem and Duckett, 1998; Deveraux and Reed, 1999). For at least some IAPs, their anti-apoptotic potential can be explained by their potent inhibition of caspases 3, 7 and 9, which are often essential for the execution of a" @default.
• W2022135873 created "2016-06-24" @default.
• W2022135873 creator A5030174900 @default.
• W2022135873 date "1999-10-01" @default.
• W2022135873 modified "2023-10-15" @default.
• W2022135873 title "Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases" @default.
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