Direct inhibition of caspase 3 is dispensable for the anti-apoptotic activity of XIAP
2001; Springer Nature; Volume: 20; Issue: 12 Linguagem: Inglês
10.1093/emboj/20.12.3114
ISSN1460-2075
AutoresJohn Silke, Paul G. Ekert, Catherine L. Day, Christine J. Hawkins, Manuel Baca, Joanne Chew, Miha Pakusch, Anne M. Verhagen, David L. Vaux,
Tópico(s)ATP Synthase and ATPases Research
ResumoArticle15 June 2001free access Direct inhibition of caspase 3 is dispensable for the anti-apoptotic activity of XIAP John Silke Corresponding Author John Silke The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Paul G. Ekert Paul G. Ekert The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Catherine L. Day Catherine L. Day Institute of Molecular BioSciences, Massey University, Private Bag, 11222 Palmerston North, New Zealand Search for more papers by this author Christine J. Hawkins Christine J. Hawkins Department of Haematology and Oncology, Royal Children's Hospital, Flemington Road, Parkville, 3052 Australia Search for more papers by this author Manuel Baca Manuel Baca The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Joanne Chew Joanne Chew The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Miha Pakusch Miha Pakusch The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Anne M. Verhagen Anne M. Verhagen The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author David L. Vaux David L. Vaux The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author John Silke Corresponding Author John Silke The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Paul G. Ekert Paul G. Ekert The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Catherine L. Day Catherine L. Day Institute of Molecular BioSciences, Massey University, Private Bag, 11222 Palmerston North, New Zealand Search for more papers by this author Christine J. Hawkins Christine J. Hawkins Department of Haematology and Oncology, Royal Children's Hospital, Flemington Road, Parkville, 3052 Australia Search for more papers by this author Manuel Baca Manuel Baca The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Joanne Chew Joanne Chew The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Miha Pakusch Miha Pakusch The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Anne M. Verhagen Anne M. Verhagen The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author David L. Vaux David L. Vaux The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia Search for more papers by this author Author Information John Silke 1, Paul G. Ekert1, Catherine L. Day2, Christine J. Hawkins3, Manuel Baca1, Joanne Chew1, Miha Pakusch1, Anne M. Verhagen1 and David L. Vaux1 1The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, VIC, 3050 Australia 2Institute of Molecular BioSciences, Massey University, Private Bag, 11222 Palmerston North, New Zealand 3Department of Haematology and Oncology, Royal Children's Hospital, Flemington Road, Parkville, 3052 Australia *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:3114-3123https://doi.org/10.1093/emboj/20.12.3114 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info XIAP is a mammalian inhibitor of apoptosis protein (IAP). To determine residues within the second baculoviral IAP repeat (BIR2) required for inhibition of caspase 3, we screened a library of BIR2 mutants for loss of the ability to inhibit caspase 3 toxicity in the yeast Schizosaccharomyces pombe. Four of the mutations, not predicted to affect the structure of the BIR fold, clustered together on the N-terminal region that flanks BIR2, suggesting that this is a site of interaction with caspase 3. Introduction of these mutations into full-length XIAP reduced caspase 3 inhibitory activity up to 500-fold, but did not affect its ability to inhibit caspase 9 or interact with the IAP antagonist DIABLO. Furthermore, these mutants retained full ability to inhibit apoptosis in transfected cells, demonstrating that although XIAP is able to inhibit caspase 3, this activity is dispensable for inhibition of apoptosis by XIAP in vivo. Introduction Apoptosis is a physiological process of cell death common to metazoans (Vaux and Korsmeyer, 1999). Caspases, the key effector proteases of apoptosis, exist in healthy cells as inactive precursor molecules and their activation is, in large part, regulated by proteolytic processing between the p20 and p10 subunits. Autoprocessing of 'upstream' or 'initiator' caspases is facilitated by adaptor molecules such as FADD (Boldin et al., 1996; Muzio et al., 1996) and Apaf-1 (Li et al., 1997; Rodriguez and Lazebnik, 1999). 'Downstream' or 'effector' caspases can be activated following proteolytic processing by initiator caspases (Nicholson and Thornberry, 1997). Nevertheless, in some cases, proteolytic processing might not be required for proteolytic activity (Stennicke et al., 1999), and effector caspases such as caspase 3 can feed back to process upstream caspases such as caspase 8 and caspase 9 (Woo et al., 1999). Inhibitor of apoptosis (IAP) proteins can inhibit apoptosis in both insect and mammalian organisms (Crook et al., 1993; Clem and Miller, 1994; Duckett et al., 1996; Hawkins et al., 1996; Liston et al., 1996). In particular, mutations in the Drosophila IAP locus, thread, result in increased programmed cell death and lethality early in development, demonstrating a fundamental role for DIAP-1 in regulating developmental apoptosis in the fly (Hay et al., 1995; Wang et al., 1999). All IAPs bear baculoviral inhibitory repeats (BIRs), zinc-binding folds of ∼70 amino acids. XIAP/hILP/MIHA, c-iap1/MIHB and c-iap2/MIHC each bear three BIRs followed by a C-terminal RING finger. Certain IAPs interact with either death (Rothe et al., 1995) or BMP receptors (Yamaguchi et al., 1999) in a BIR-dependent fashion, but it is their interaction with caspases that has generated the most interest. XIAP can inhibit caspase 3 in vitro with a Ki of 0.7 nM (Deveraux et al., 1997), and does so predominantly via its second BIR domain (BIR2) (Takahashi et al., 1998). In contrast, c-iap1 and c-iap2 have inhibitory activity for caspases 3 and 7 that is 100- to 1000-fold lower, indicating that they are unlikely to target these caspases in vivo (Roy et al., 1997). The C-terminal fragment of XIAP, containing the BIR3 and RING finger, has also been reported to bind to the initiator caspase 9, although one report suggested an exclusive interaction with procaspase 9 (Deveraux et al., 1999), whereas another described inhibition of active caspase 9 with an IC50 of 10 nM (Sun et al., 2000). Analysis of caspases in mammalian cells is complicated by the presence of many different caspases and caspase regulatory molecules. In contrast, the yeasts Schizo saccharomyces pombe and Saccharomyces cerevisiae do not encode any caspases or caspase inhibitors and thus provide a naïve system in which to test heterologous proteins. Furthermore, because expression of mammalian caspases in yeast is often lethal, they provide a convenient system for analysing caspases and their regulators, allowing libraries to be screened for novel inhibitors, inhibitor mutants or caspase mutants (Ekert et al., 1999; Hawkins et al., 1999; Ryser et al., 1999; Wang et al., 1999; Wright et al., 1999). We performed a functional screen in the yeast S.pombe to identify mutations in the BIR2 of XIAP that prevented inhibition of caspase 3. Full-length XIAP, or a fragment encoding the BIR2 and flanking regions (Takahashi et al., 1998), was able to inhibit caspase 3-mediated death of S.pombe and S.cerevisiae. In a library of random BIR2 domain mutants we identified several mutants that no longer protected, either on their own or in the context of full-length XIAP. However, these XIAP mutants retained the ability to inhibit caspase 9 and to inhibit mammalian cell death induced by UV irradiation. Importantly, the mutants also retained the ability to bind to the mammalian IAP antagonist DIABLO/smac, thus excluding the possibility that the mutant XIAPs inhibited cell death because they had lost the ability to be antagonized by DIABLO/smac (Du et al., 2000; Verhagen et al., 2000). Our results suggest that the primary point of action of XIAP is probably upstream of effector caspases, because abolition of the ability of XIAP to inhibit caspase 3 did not prevent it from protecting cells as efficiently as wild-type protein. Results Autoactivating, but not wild-type caspase 3 is toxic when expressed in S.pombe In order to express active caspase 3 in S.pombe, we inserted the cDNA into the non-integrating plasmid pNeu, a pREP derivative that controls expression of the inserted gene by the full-strength nmt promoter (Maundrell, 1993). This allows caspase 3 expression to be induced by removal of thiamine from the media. While wild-type human caspase 3 does not kill S.pombe significantly because it fails to become processed, caspase 3 variants engineered to autoactivate are lethal (Ekert et al., 1999). Wild-type caspase 3 was not toxic when its expression was induced in S.pombe (Figure 1A, compare C3 with C3mut). However, a caspase 3–β-Gal fusion protein auto activated to a greater extent, probably due to multimer formation mediated by the β-galactosidase moiety, and was toxic to the yeast (Figure 1A and B). Toxicity required the catalytic activity of the caspase because the catalytic site mutant (QAGRG) caspase 3–β-Gal fusion protein was not toxic, and did not autoactivate (Figure 1A and B). This autoactivating caspase displays the same pH dependence as the unmodified enzyme in DEVD-AMC cleavage assays (data not shown), and in other respects behaves similarly to the unmodified enzyme, e.g. it can be inhibited by XIAP (see below). Figure 1.Autoactivating caspase 3 kills S.pombe. (A) Yeast expressing a caspase 3–β-Gal fusion (C3 βGal), a caspase 3 catalytic mutant–β-Gal fusion (C3mut βGal), caspase 3 fused at the N-terminus with the CARD of caspase 2 (CARD C3) (Colussi et al., 1998), caspase 3 (C3) and caspase 3 catalytic mutant (C3mut) under the inducible nmt promoter were plated in serial 10-fold dilutions on solid inducing media. (B) Caspase 3 processing requires the catalytic cysteine and occurs in both caspase 3- and caspase 3–β-Gal fusion-expressing yeast. Yeast were induced in minimal media without thiamine, and proteins were harvested and run on an SDS–polyacrylamide gel, transferred and blotted with anti-caspase 3. Download figure Download PowerPoint Full-length MIHA, or BIR2 plus flanking regions, can inhibit caspase 3-mediated death of S.pombe To test which IAPs were able to inhibit caspase 3-mediated killing of S.pombe, we inserted cDNAs for XIAP, its murine homologue MIHA, c-iap1/MIHB, c-iap2/MIHC and survivin/MIHD into the non-integrating yeast vector pURAS K, which drives expression using the constitutive ADH promoter (Losson and Lacroute, 1983), and pREP, which drives expression from the inducible nmt promoter. Expression of XIAP and MIHA from both the pURAS and pREP vectors was able to suppress caspase 3 toxicity (Figure 2A and data not shown), but neither a construct expressing XIAP BIR1+3, nor any of the other IAPs, were able to do so. Expression of c-iap1, c-iap2, XIAP, XIAP BIR1+3 and XIAP BIR2 was confirmed by western blotting (Figure 2B). Figure 2.The BIR2 and full-length XIAP inhibit caspase 3 toxicity in yeast. (A) Yeast expressing either a caspase 3–β-Gal fusion (C3 βGal), a caspase 3 catalytic mutant–β-Gal fusion (C3mut βGal) or CARD caspase (CARD C3) under the inducible nmt promoter and co-expressing the IAP indicated on a constitutive promoter were plated in serial 10-fold dilutions on solid inducing media. (B) Expression of the IAPs. Yeast were grown in minimal media, and the proteins were extracted, run on SDS–polyacrylamide gels and transferred to nitrocellulose. pURAS vector (lane 1), XIAP (lanes 2 and 3) and XIAP BIR1+3 (lanes 4 and 5) were probed with anti-XIAP, and pURAS vector (lanes 6 and 7) and XIAP BIR2 (lanes 8 and 9) were probed with anti-tetraHis. Likewise, c-iap1 and c-iap2 were probed with anti-c-iap1 and c-iap2, respectively. Download figure Download PowerPoint To confirm further that protection by XIAP was not due to inhibition of caspase activation by the β-galactosidase moiety, we also tested the ability of XIAP to inhibit another construct that uses the caspase recruitment domain (CARD) of caspase 2 to autoactivate caspase 3 (Colussi et al., 1998). XIAP and MIHA were both able to inhibit death mediated by this CARD–caspase 3 construct (Figure 2A). The portion of XIAP including the BIR2 and flanking regions (Takahashi et al., 1998) was also able to inhibit caspase 3-mediated killing of yeast, but a construct without BIR2, BIR1+3, was not able to do so (Figure 2A), confirming that the region of XIAP containing BIR2 and its flanking regions is both necessary and sufficient for inhibition of caspase 3. XIAP BIR2s with mutations to conserved BIR residues, or the N-terminal flanking region, fail to inhibit caspase 3 The BIR2 fragment of XIAP has a Ki against caspase 3 similar to that of the full-length protein (Deveraux et al., 1997; Takahashi et al., 1998), and can inhibit caspase 3-mediated death of yeast (Figure 2A). We therefore generated mutations in this region to identify residues necessary for caspase inhibition. Error-prone PCR with limiting nucleotides was used to generate a library of BIR2 genes with random point mutations. Sequence analysis of individual clones from the library revealed that ∼50% contained a single mutation and 5–10% contained double point mutations. Yeast expressing an inducible caspase 3−β-Gal fusion were transformed with the library and grown on non-inducing selective media. A total of 2200 colonies were picked and replica plated onto solid media with (non-inducing) or without (inducing) thiamine. Colonies that only grew on the non-inducing plates, indicating loss of the ability to counter caspase 3 toxicity, were isolated and the plasmids recovered. Fifteen of the plasmids contained a single point mutation in the BIR2, four had two point mutations, and nine had a single nucleotide deletion (Figure 3A). All single point mutants in non-structural residues were expressed to approximately the same levels as the wild-type protein in yeast, demonstrating that loss of caspase 3 inhibition was not due to lower expression levels (data not shown). Figure 3.Mutations in the BIR2 domain that attenuate its inhibitory function against caspase 3. (A) Sequence alignment of the wild-type BIR domain and those of the mutants mapped onto a linear sequence; the numbering indicates the amino acid position according to full-length XIAP. The BIR2 domain mutants reported in Sun et al. (1999) are shown. Residues that when mutated attenuated BIR2's inhibitory activity against caspase 3 by >200-fold, i.e. L141 and D148, are highlighted in red, and mutants V147, I149 and D151, whose activity was attenuated 11.5-, 10- and 4.3-fold, respectively, are highlighted in green. The single nucleotide deletions that resulted in truncation of the BIR2 domain in yeast and loss of caspase 3 inhibitory potential are indicated with a red asterisk. Point mutations, the point mutants in the BIR2 domain that resulted in loss of caspase 3 inhibition in yeast; Consensus, residues conserved in all BIRs identified to date are in upper case and highlighted in blue, residues conserved in most BIRs are in lower case. (B) Residues that chemically shift when the BIR2 domain is incubated with caspase 3 (Sun et al., 1999). D148A, V147, I149 and D151 are highlighted in orange, mutations identified in this screen are shown in red and indicated with text, and other residues that interact with caspase 3 are shown in pink. (C) The opposing face of BIR2 with the same colour scheme as in (B). Prepared using data given to us by Stephen Fesik with the program RasMol (Sayle and Milner-White, 1995). Download figure Download PowerPoint All deletion mutants had lost the highly conserved BIR2 structure but retained some of the N-terminal amino acids. One of the mutants, F228L, had a frameshift immediately after the last Zn co-ordinating cysteine (Figure 3A), indicating that the whole BIR2 is required for caspase 3 inhibition. Three of the single point mutations (C200R, H220Y and C203R) were in three of the four amino acids responsible for co-ordinating the Zn ion, emphasizing the requirement for a correctly folded BIR domain for caspase 3 inhibition. One of the mutations, R166G, was to the arginine residue conserved in all BIRs. Three of the mutations (T143A, M160T and C203R) were to residues that chemically shift when incubated with active caspase 3 and which are presumed to interact directly with caspase 3 (Figure 3B and C; and Sun et al., 1999). A major group of four single point mutations occurred in the linker region between BIR1 and BIR2 of XIAP, a region that has been shown to be important for inhibition of caspase 3 by the BIR2 domain (Sun et al., 1999). Full-length mutant XIAPs are no longer able to inhibit caspase 3 We then tested whether mutations to BIR2 affected inhibition of caspase 3 by the full-length XIAP protein in S.cerevisiae. In addition to our mutants, we also analysed an XIAP mutant, D148A, described by Sun et al. (1999). The mutants were tested against the caspase 3−β-Gal fusion protein expressed in S.cerevisiae under a glucose-suppressable promoter. Wild-type MIHA, XIAP and the baculoviral p35 all inhibited yeast death caused by caspase 3, and, consistent with our previous result, all the BIR2 mutants had reduced caspase 3 inhibitory activity, even in the context of the full-length protein (Figure 4A). While mutants L140P and V146A retained a small amount of activity in this assay, C200R (a Zn co-ordinating mutant) and the D148A mutant displayed no detectable activity. Figure 4.Full-length XIAPs with mutations in the BIR1–BIR2 linker are attenuated in their ability to inhibit caspase 3. (A) Yeast expressing a caspase 3–β-Gal fusion from the pGALL-inducible vector were co-transformed with full-length XIAP mutants, the baculoviral p35 (p35), wild-type MIHA or XIAP and plated in serial 10-fold dilutions on solid inducing (galactose) and non-inducing (glucose) media. (B) Kis for full-length XIAP, XIAP mutants and the tetrapeptide aldehyde DEVD-CHO against caspase 3, and IC50s for full-length XIAP against caspase 9. (C) Purified XIAP and XIAP mutants separated on a 12% SDS–polyacrylamide gel and stained with Coomassie Blue. (D) XIAP D148A interacts with DIABLO. Purified XIAP and XIAP D148A were used to co-immunoprecipitate bacterially produced DIABLO, which were separated on a 12% SDS–polyacrylamide gel and stained with Coomassie Blue. (E) XIAP mutants are impaired in their ability to interact with caspase 3 in vivo. 293T cells were transiently transfected with plasmids expressing Flag-tagged XIAP, XIAP mutants or TAB1 and caspase 3–β-Gal (C3 βGal). Cell lysates were immunoprecipitated with anti-Flag beads and immunoblotted with anti-caspase 3. Download figure Download PowerPoint To quantitate the changes to the inhibitory constant (Ki) caused by the mutations, we expressed full-length wild-type XIAP and the mutants in Escherichia coli, and partially purified the recombinant proteins (Figure 4C). Analysis of the proteins by size exclusion chromatography indicated that they all existed as high molecular weight complexes (data not shown). These proteins were then characterized for their ability to inhibit caspase 3 in an in vitro DEVD-AMC cleavage assay. In accordance with previously published results (Deveraux et al., 1997), we determined the Ki for wild-type XIAP against caspase 3 to be 0.6 ± 0.1 nM (Figure 4B). Consistent with the results obtained in yeast, XIAP mutants L140P, T143A and V146A had a 10- to 20-fold reduction in their activity, and the D148A mutant had a >500-fold reduced ability to inhibit caspase 3 (Figure 4B). It was not possible to produce recombinant mutants C200R, R166G, M160T or F170S. Wild-type XIAP is itself difficult to produce in vitro and we suspect that mutations that even slightly affect the structure of XIAP affect its stability in vitro. Processed caspase 3 interacts with XIAP (Sun et al., 1999) and to evaluate the effect of the mutants we immunoprecipitated the transiently transfected mutants with the autoactivating caspase 3–β-Gal from mammalian cells. As expected, all mutations interfered with the ability of XIAP to bind caspase 3. Consistent with the in vitro inhibition data, mutants L140P and V146A retained a small amount of caspase 3 binding activity, whereas D148A, M160T, F170S C200R and R166G had significantly lost the ability to bind caspase 3 in this assay. Mutant T143A retained some caspase 3 binding, indicating that the lack of inhibition of caspase 3 is not due to its inability to bind. Full-length mutant XIAPs retain the ability to inhibit caspase 9 and to bind to caspase 9 and DIABLO The ability of the full-length XIAP mutants to inhibit caspase 9 was tested in the S.cerevisiae system. Apaf-1 lacking its WD40 repeats and wild-type procaspases 3 and 9 were all co-expressed together with full-length wild-type or mutant XIAP. In this system, caspase 3 does not autoactivate significantly, but requires processing by Apaf-1-activated caspase 9 for activation and death of the yeast (Hawkins et al., 2001). Death of the yeast in this system is dependent on both caspase 9 and caspase 3, but inhibition of caspase 9 is sufficient to prevent cell death because a BIR3-only construct was able to protect the yeast fully (Figure 5A). Mutants L140P, V146A and T143A protected the yeast cells as well as wild-type XIAP, and the D148A mutant retained significant activity (Figure 5A), whereas C200R, R166G, F170S and M160T were not able to block this caspase 9-mediated death. Figure 5.Full-length XIAPs with mutations in the BIR1–BIR2 linker retain their ability to inhibit caspase 9 and interact with DIABLO. (A) Yeast expressing Apaf-1 −WD40, caspase 9 and caspase 3 were co-transformed with full-length XIAP mutants or a BIR3–eGFP fusion construct, and plated in serial 10-fold dilutions on solid inducing (galactose) and non-inducing (glucose) media. Western blots of the mutants were performed with anti-XIAP. (B) Co-immunoprecipitation of XIAP mutants and caspase 9 from cell lysates. 293T cells were transiently transfected with plasmids expressing Flag-tagged XIAP, XIAP mutants or TAB1 and caspase 9. Cell lysates were immuno precipitated with anti-Flag beads and immunoblotted with anti-caspase 9 and anti-Flag antibodies. p. caspase 9 = processed caspase 9; caspase 9 = full-length caspase 9. (C) Co-immunoprecipitation of XIAP mutants and DIABLO from cell lysates. 293T cells were transiently transfected with plasmids expressing Flag-tagged XIAP, XIAP mutants, CrmA or TAB1 and DIABLO-HA tag. Cell lysates were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA and anti-Flag. unp. DIABLO = unprocessed DIABLO; DIABLO = full-length processed DIABLO (Verhagen et al., 2000). Download figure Download PowerPoint To confirm that mutations in the linker region did not affect interaction with caspase 9, we immunoprecipitated the XIAP mutants from 293T cells co-transfected with full-length caspase 9. In this system, caspase 9 autoactivates, and both processed and unprocessed forms of caspase 9 are present in the lysates (Figure 5B). All XIAP mutants in the BIR2 linker were able to immunoprecipitate processed caspase 9. Mutant D148A showed a slight reduction in its ability to bind caspase 9 (Figure 5B), but the reduction was not as dramatic as for mutant C200R. Mutants M160T, F170S, R166G and C200R all showed reduced binding to processed caspase 9. However, the amount of processed caspase 9 present in the lysates was less, and this was most probably due to increased cell death among cells expressing the mutants (or the negative control TAB1), so that less caspase 9 accumulated. This finding corroborates our observation in yeast that these mutants are less effective at blocking caspase 9. We determined the IC50 of XIAP against caspase 9 in an in vitro LEHD-AMC cleavage assay (Figure 4B). Surprisingly, the IC50s for the full-length protein were higher than for the BIR3 domain alone (Sun et al., 2000). To exclude the possibility that only the BIR2 domain was folded correctly in the bacterially produced XIAP, we performed in vitro precipitations with bacterially produced DIABLO (Figure 4D). Because DIABLO binding is determined to a large extent by the BIR3 of XIAP (Chai et al., 2000), and the bacterially produced XIAP and XIAP D148A bound DIABLO to the same extent, it seems likely that the bacterially produced XIAPs have a correctly folded BIR3. We recently have identified a novel mammalian antagonist of IAPs called DIABLO/smac (Du et al., 2000; Verhagen et al., 2000). DIABLO was identified originally due to its ability to bind to XIAP. The XIAP mutants were therefore tested for their ability to bind to DIABLO in vivo, and, consistent with data from the bacterially produced proteins, all linker mutants behaved indistinguishably from wild-type XIAP in an immunoprecipitation assay (Figure 5C). Both the caspase 9 and DIABLO binding assays demonstrate that the linker mutations generated have not greatly interfered with the structure or other capabilities of XIAP. XIAP mutants that do not inhibit caspase 3, but still inhibit caspase 9 and interact with DIABLO, block UV-induced apoptosis as well as wild-type XIAP To test whether loss of caspase 3 inhibitory activity affected the ability of XIAP to inhibit apoptosis of mammalian cells, we expressed the mutants in NT2 teratocarcinoma cells and exposed them to UV radiation (Figure 6A). Some of the mutants were able to protect against UV-induced apoptosis as efficently as wild-type XIAP, while others were unable to protect, even though all except for mutant C200R were expressed equivalently (Figure 6B). Mutants R166G, F170S and C200R, which contain mutations that probably affect the BIR2 fold, could not inhibit UV-induced death. However, mutants L140P, V146A, T143A and D148A, which no longer inhibit caspase 3, still retained full activity against UV-induced apoptosis. These four independent mutants demonstrate that caspase 3 inhibitory activity is independent of caspase 9 inhibitory activity, and is not required for XIAP to inhibit UV-induced cell death. Figure 6.Full-length XIAPs that retain caspase 9 inhibitory potential inhibit UV-induced cell death. (A) The XIAP mutants (M160T, etc), empty vector (V) and wild-type XIAP (XIAP) were cloned into a pEF vector, and transiently transfected into NT2 cells with a GFP marker plasmid. Cells subsequently were induced to undergo cell death with UV irradiation, and stained with annexin V. The fractions of cells that were positive for GFP and annexin V over GFP-positive cells were expressed as the percentage of annexin V-positive cells. Error bars are two standard errors of the mean for three independent experiments. (B) Extracts of transiently transfected NT2 cells were made, separated on SDS–polyacrylamide gels, transferred to nitrocellulose and probed with anti-Flag antibody. Download figure Download PowerPoint Discussion If caspases are the major effectors of the apoptotic programme, and IAPs function to block caspases, IAPs are in a pivotal position to determine whether a cell undergoes apoptosis or not. Consistent with a key role in the decision process, mutants of the diap-1 locus, thread, in Drosophila, do not develop due to massive ectopic cell death. However, a similar drastic phenotype for mammalian IAP knock-outs has yet to be reported. XIAP can inhibit caspase 3 with a Ki of 0.7 nM (Deveraux et al., 1997). The BIR2 of XIAP plus flanking regions of 60 amino acids has been shown to account for nearly all this caspase 3 inhibitory activity (Takahashi et al., 1998). Further studies have shown that the small region upstream of the conserved BIR2 is required for caspase 3 inhibition (Sun et al., 1999) and, although our studies involved a non-directional approach, they are in accord with this data. First, a structurally intact BIR is required for caspase 3 inhibition by the BIR2 fragment because mutants that had lost any part of the BIR2 were no longer able to inhibit caspase 3. Secondly, the N-terminal linker region is required for caspase 3 inhibition because single point mutants in this region were unable to inhibit caspase 3. Strikingly two of these mutants, V146A and T143A, represented very subtle changes to the primary sequence, yet th
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