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Inhibitor of Apoptosis Proteins Are Substrates for the Mitochondrial Serine Protease Omi/HtrA2

2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês

10.1074/jbc.c300240200

1083-351X

Srinivasa M. Srinivasula, Sanjeev Gupta, Pinaki Datta, Zhijia Zhang, R.P. Hegde, Naeun Cheong, Teresa Fernandes‐Alnemri, Emad S. Alnemri,

Cassava research and cyanide

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 demonstrated that Omi/HtrA2 could degrade 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, our 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. 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 demonstrated that Omi/HtrA2 could degrade 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, our 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. The genes for inhibitor of apoptosis proteins (IAPs) 1The abbreviations used are: IAP, inhibitor of apoptosis protein; BIR, baculovirus IAP repeat; IBM, IAP-binding motif; DMEM, Dulbecco's modified Eagle's medium; siRNA, small interfering RNA; DEVD, Asp-Glu-Val-Asp; AFC, amino-4-trifluoromethylcoumarin; AMC, amino-4-methylcoumarin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. were originally identified in the genome of baculoviruses based on the ability of their gene products to protect infected host cells from virus-induced apoptosis (1Crook N.E. Clem R.J. Miller L.K. J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar). Cellular homologues of the viral IAPs have been identified in insects, nematodes, yeast, and mammals (2Miller L.K. Trends Cell Biol. 1999; 9: 323-328Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 3Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2303) Google Scholar). All IAPs contain one or more conserved domains, referred to as baculovirus IAP repeats (BIRs), that are essential for inhibition of apoptosis (reviewed in Refs. 4Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1504) Google Scholar and 5Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1590) Google Scholar). The BIR domains and the linker regions between them bind directly to caspases and inhibit their activity. Some IAPs, such as human XIAP, c-IAP1, and c-IAP2, and Drosophila DIAP1 and DIAP2, also contain C-terminal RING domains. The RING domain is important for ubiquitination and proteosome degradation of IAPs and IAP-associated proteins (reviewed in Refs. 5Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1590) Google Scholar and 6Martin S.J. Cell. 2002; 109: 793-796Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The antiapoptotic activity of IAPs is regulated by a group of proteins that bind to the BIR domains of IAPs via a N-terminal conserved 4-residue IAP-binding motif (IBM) (reviewed in Refs. 5Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1590) Google Scholar and 7Shi Y. Cell Death Differ. 2002; 9: 93-95Crossref PubMed Scopus (75) Google Scholar). In Drosophila melanogaster, five IBM-containing proteins known as Reaper, Hid, Grim, Sickle, and Jafrac2 have been identified as direct IAP-binding proteins (5Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1590) Google Scholar, 8Tenev T. Zachariou A. Wilson R. Paul A. Meier P. EMBO J. 2002; 21: 5118-5129Crossref PubMed Scopus (78) Google Scholar). These proteins promote caspase activation by disrupting caspase-IAP complexes and/or inducing autoubiquitination and degradation of IAPs, thus preventing IAPs from inhibiting caspases (4Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1504) Google Scholar, 5Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1590) Google Scholar, 6Martin S.J. Cell. 2002; 109: 793-796Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7Shi Y. Cell Death Differ. 2002; 9: 93-95Crossref PubMed Scopus (75) Google Scholar). In mammals two functional homologues of the Drosophila proteins, known as Smac/Diablo (9Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2974) Google Scholar, 10Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1998) Google Scholar) and Omi/HtrA2 (11Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar, 12Verhagen A.M. Silke J. Ekert P.G. Pakusch M. Kaufmann H. Connolly L.M. Day C.L. Tikoo A. Burke R. Wrobel C. Moritz R.L. Simpson R.J. Vaux D.L. J. Biol. Chem. 2002; 277: 445-454Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 13Martins L.M. Iaccarino I. Tenev T. Gschmeissner S. Totty N.F. Lemoine N.R. Savopoulos J. Gray C.W. Creasy C.L. Dingwall C. Downward J. J. Biol. Chem. 2002; 277: 439-444Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 14Suzuki Y. Imai Y. Nakayama H. Takahashi K. Takio K. Takahashi R. Mol. Cell. 2001; 8: 613-621Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 15van Loo G. van Gurp M. Depuydt B. Srinivasula S.M. Rodriguez I. Alnemri E.S. Gevaert K. Vandekerckhove J. Declercq W. Vandenabeele P. Cell Death Differ. 2002; 9: 20-26Crossref PubMed Scopus (284) Google Scholar) have been identified. Both Smac/Diablo and Omi/HtrA2 are synthesized as precursor proteins with N-terminal mitochondrial localization signal peptides that are removed during maturation in the mitochondria to expose their N-terminal IBM. During apoptosis both proteins are released from the intermembrane space of the mitochondria into the cytoplasm and promote caspase activation and apoptosis by binding to the BIR3 domain of XIAP (11Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar, 12Verhagen A.M. Silke J. Ekert P.G. Pakusch M. Kaufmann H. Connolly L.M. Day C.L. Tikoo A. Burke R. Wrobel C. Moritz R.L. Simpson R.J. Vaux D.L. J. Biol. Chem. 2002; 277: 445-454Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 13Martins L.M. Iaccarino I. Tenev T. Gschmeissner S. Totty N.F. Lemoine N.R. Savopoulos J. Gray C.W. Creasy C.L. Dingwall C. Downward J. J. Biol. Chem. 2002; 277: 439-444Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 14Suzuki Y. Imai Y. Nakayama H. Takahashi K. Takio K. Takahashi R. Mol. Cell. 2001; 8: 613-621Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 15van Loo G. van Gurp M. Depuydt B. Srinivasula S.M. Rodriguez I. Alnemri E.S. Gevaert K. Vandekerckhove J. Declercq W. Vandenabeele P. Cell Death Differ. 2002; 9: 20-26Crossref PubMed Scopus (284) Google Scholar). Unlike Smac/Diablo, Omi/HtrA2 is a serine protease with a high degree of homology to the bacterial heat inducible serine protease HtrA/DegP (16Clausen T. Southan C. Ehrmann M. Mol. Cell. 2002; 10: 443-455Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, 17Maurizi M.R. Nat. Struct. Biol. 2002; 9: 410-412Crossref PubMed Scopus (16) Google Scholar). In bacteria DegP aids in the degradation or refolding of misfolded or unfolded proteins in the periplasmic space. The high degree of similarity between bacterial HtrA/DegP and mammalian Omi/HtrA2 suggests that the later might also have a similar function in the mitochondria. However, very little is known about the protease activity or substrates of Omi/HtrA2 after its release from the mitochondria during cell death. In this report we provide evidence demonstrating for the first time that IAPs are substrates for Omi/HtrA2 and their degradation could be a mechanism by which Omi/HtrA2 activates caspases and induces cell death. Cell Culture—Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) (HeLa cells) or DMEM/F-12 (293T cells) or RPMI 1640 (MCF-7 cells) (Invitrogen), supplemented with 10% fetal bovine serum, 200 μg·ml–1 penicillin and 100 μg·ml–1 streptomycin sulfate. Expression Constructs—Expression constructs for Omi/HtrA2 variants and IAPs were described before (11Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). RNA Interference—Cells were seeded in 6-well or 12-well plates and then transfected with nonspecific small interfering RNA (siRNA) duplex (UGU UGU UUG AGG GGA ACG G TT) or Omi-specific siRNA duplex (GGC AAG GGG AGU UUG UUG UTT) using Oligofectamine (Invitrogen) as per manufacturer's recommendations. 48 h after the transfection cells were either treated with TRAIL (100 ng/ml) for 6 h or etoposide (100 μm) for 24 h. Cells were collected for immunoblot analysis. A duplicate set of cells was stained with annexin V and propidium iodide as per the manufacturer's recommendations (Clontech) to assay for cell death. Dead cells in each field (annexin V and/or propidium iodide-positive) were counted using fluorescence microscopy. The total number of cells (live plus dead) in each field was counted by light microscopy. The percentage of cell death in each experiment was calculated from the values of dead cells divided on total cells. Data represent mean values ± S.D. of three independent experiments. In Vitro Protease Assay—The protease activity of Omi was assayed with in vitro translated 35S-labeled XIAP, c-IAP1, or c-IAP2 as substrates. Bacterially expressed Omi/HtrA2 was purified on Talon affinity resins and then incubated with 35S-labeled proteins in buffer A (20 mm HEPES, pH 7.4, 10 mm KCl, 1.5 mm MgCl2,1mm EDTA, and 1 mm EGTA with protease inhibitors in a total 20-μl reaction. The cleavage products were analyzed by SDS-PAGE and visualized by autoradiography. Endogenous Omi/HtrA2 was isolated by immunoprecpitation from 293T S100 extracts. The S100 extracts were incubated with Omi polyclonal antibody (made in the laboratory) for 1 h. After incubation, the Omi-antibody complexes were bound to protein G-Sepharose and washed several times. The protein G-Sepharose-bound complexes were incubated with 35S-labeled XIAP in buffer A for 1 h at 37 °C in a total 20-μl reaction. The cleavage products were analyzed as outlined above. Omi/HtrA2-induced Cell Death Assay—The ability of Omi/HtrA2 variants to induce cell death in transfected cells was assayed as described previously (11Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). 293T cells were seeded in 12-well plates at a density of 1 × 106 cells/well and then transfected with 0.4 μg of pEG-FPN1 reporter plasmid (Clontech) and 1.0 μg of empty vector or constructs encoding Omi/HtrA2 variants using the LipofectAMINE™ method. After 36 h of transfection normal and apoptotic GFP-expressing cells were counted using fluorescence microscopy. The percentage of cell death in each experiment was calculated from the values of dead cells divided on total cells. Data represent mean values ± S.D. of three independent experiments. DEVD Cleavage Assay—To assay caspase activity in 293T S-100 extracts (50 mg), an equal amount of the extracts were incubated with 100 μm DEVD-AFC fluorogenic substrate for 60 min at 37 °C. The amount of cleaved AFC was measured using 400 and 505 nm as the excitation and emission wavelengths, respectively. Results were expressed as DEVD cleavage activity in relative fluorescence units. The data represent the average of three independent experiments. Suppression of Omi/HtrA2 by RNA Interference Suppresses Cell Death—Treatment of cancer cell lines with death stimuli causes the release of Omi/HtrA2 from the mitochondria into the cytoplasm (11Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar, 12Verhagen A.M. Silke J. Ekert P.G. Pakusch M. Kaufmann H. Connolly L.M. Day C.L. Tikoo A. Burke R. Wrobel C. Moritz R.L. Simpson R.J. Vaux D.L. J. Biol. Chem. 2002; 277: 445-454Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 13Martins L.M. Iaccarino I. Tenev T. Gschmeissner S. Totty N.F. Lemoine N.R. Savopoulos J. Gray C.W. Creasy C.L. Dingwall C. Downward J. J. Biol. Chem. 2002; 277: 439-444Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 14Suzuki Y. Imai Y. Nakayama H. Takahashi K. Takio K. Takahashi R. Mol. Cell. 2001; 8: 613-621Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 15van Loo G. van Gurp M. Depuydt B. Srinivasula S.M. Rodriguez I. Alnemri E.S. Gevaert K. Vandekerckhove J. Declercq W. Vandenabeele P. Cell Death Differ. 2002; 9: 20-26Crossref PubMed Scopus (284) Google Scholar). To evaluate the role of Omi/HtrA2 in the extrinsic (TRAIL) or intrinsic (etoposide) cell death pathways we used Omi/HtrA2-specific siRNA oligonucleotides to reduce the protein level of Omi/HtrA2 in HeLa and MCF-7 cancer cells (Fig. 1A). Transfection of these cells with Omi/HtrA2-specific siRNA-resulted in almost 40–50% reduction in the extent of TRAIL- or etoposide-induced cell death compared with untransfected or control siRNA-transfected cells (Fig. 1, B and C). These results indicate that Omi/HtrA2 plays an important proapoptotic role in the extrinsic and intrinsic cell death pathways in cancer cell lines. Mature Omi/HtrA2 Can Promote Caspase Activation and Cell Death—Recent results suggest that the serine protease activity of Omi/HtrA2 could be responsible for its potent killing activity (18Li W. Srinivasula S.M. Chai J. Li P. Wu J.W. Zhang Z. Alnemri E.S. Shi Y. Nat. Struct. Biol. 2002; 9: 436-441Crossref PubMed Scopus (251) Google Scholar). To determine whether Omi/HtrA2 protease activity can activate the caspase pathway, we measured caspase activity in 293T cells transfected with constructs encoding mature wildtype or active site mutant Omi/HtrA2 proteins. Expression of mature wild type, but not the active site mutant, Omi/HtrA2 resulted in significant caspase activation (Fig. 2A). This enhanced caspase activation was associated with increased cell death (Fig. 2B). Similar results were observed in MCF-7 and HeLa cell lines (Ref. 11Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar and data not shown). These results suggest that the ability of Omi/HtrA2 to promote caspase activation and cell death in transfected cells is largely dependent on its serine protease activity. Omi/HtrA2 Can Degrade IAPs in Vitro—Western blot analysis revealed that expression of mature wild type Omi/HtrA2, but not an active site mutant protein, results in significant reduction in the amount of XIAP in the transfected cells (Fig. 2C). These results suggest that degradation of XIAP by Omi/HtrA2 could be responsible for this effect. To test this hypothesis we first determined the physiological concentration of Omi/HtrA2 in 293T and HeLa cells by immunoblot analysis with Omi/HtrA2 antibodies. The concentration of Omi/HtrA2 in 293 was found to be ∼150 nm and in HeLa cells ∼60 nm. Next we assayed the activity of physiological amounts of recombinant Omi/HtrA2 with 35S-labeled XIAP, c-IAP1, or c-IAP2 as substrates. As shown in Fig. 3A, physiological amounts of Omi/HtrA2 were able to degrade these IAPs in a dose-dependent manner. Consistent with these results, physiological amounts of endogenous Omi/HtrA2 protein isolated by immunoprecipitation from 293T cells were also able to degrade XIAP in a dose-dependent manner (Fig. 3B). These results indicate that IAPs are physiological substrates for Omi/HtrA2 protein. Next we tested the effect of Omi/HtrA2 on the ability of IAPs to inhibit caspase activation in S100 extracts stimulated with cytochrome c and dATP (Fig. 3C). Incubation of XIAP with mature wildtype Omi resulted in a large dose-dependent reduction in the inhibitory activity of XIAP. Similar results were obtained with the active site mutant Omi/HtrA2-S306A. In contrast, incubation of XIAP with protease active Omi/HtrA2 without its IAP binding motif (ΔAVPS) did not cause significant reduction in the inhibitory activity of XIAP. Western blot analysis showed that XIAP is degraded in the reaction mixtures containing wildtype Omi/HtrA2 and Omi/HtrA2 ΔAVPS, but not Omi/HtrA2-S306A. The IAP binding motif of Omi/HtrA2 seems to enhance its ability to cleave XIAP, because the Omi/HtrA2 ΔAVPS was less efficient in cleaving XIAP. In addition, a distinct pattern of cleavage was observed with the wildtype Omi/HtrA2 compared with Omi/HtrA2 ΔAVPS. Nevertheless, the cleavage products generated by Omi/HtrA2 ΔAVPS were able to inhibit caspases, indicating that cleavage alone might not be sufficient for inactivation of XIAP. These interesting results suggest that the presence of the IAP binding motif enhances the ability of Omi/HtrA2 to cleave and disrupt the association of the cleavage products of XIAP with caspases. In contrast, in the absence of the IAP binding motif, Omi/HtrA2 cleaves XIAP less efficiently at multiple sites and is not able to disrupt the association of the cleavage products with caspases. IAP Degradation during Apoptosis Is Dependent on Omi/HtrA2 Expression Levels—To provide additional support for the role of Omi/HtrA2 in IAP-degradation we decreased the expression levels of Omi/HtrA2 in the mitochondria by transfection of MCF-7 cells with Omi/HtrA2-specific or nonspecific siRNAs and then treated the cells with etoposide for different periods of time. Less IAP degradation was observed in cells transfected with the Omi-specific siRNA compared with the cells transfected with the nonspecific siRNA (Fig. 4). Taken together, these results indicate that Omi/HtrA2 plays a direct role in the degradation of IAPs during cell death. IAP degradation is an important mechanism for caspase activation during developmental and perhaps p53-induded cell death in Drosophila (5Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1590) Google Scholar, 6Martin S.J. Cell. 2002; 109: 793-796Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 19Steller H. Nat. Cell Biol. 2000; 2: E100-E102Crossref PubMed Scopus (18) Google Scholar). Drosophila DIAP1 destruction is stimulated by binding of IAP-binding proteins such as Reaper, which is induced during development or DNA damage, to the BIR2 domain of DIAP1. This binding induces autoubiquitination of DIAP1, which targets it for degradation by the proteosomal pathways. So far, a similar mechanism of IAP destruction has not been identified in mammalian cells during cell death. However, recent results demonstrated that c-IAP1 is cleaved in human HeLa cells during p53-dependent apoptosis by a serine protease (20Jin S. Kalkum M. Overholtzer M. Stoffel A. Chait B.T. Levine A.J. Genes Dev. 2003; 17: 359-367Crossref PubMed Scopus (80) Google Scholar). Based on results showing up-regulation of Omi/HtrA2 mRNA by p53, it was suggested that Omi/HtrA2 could be responsible for the observed degradation of c-IAP1 during p53-dependent apoptosis (20Jin S. Kalkum M. Overholtzer M. Stoffel A. Chait B.T. Levine A.J. Genes Dev. 2003; 17: 359-367Crossref PubMed Scopus (80) Google Scholar). Our biochemical and cellular data indicate that XIAP is also degraded in human cancer cell lines during cell death. Omi/HtrA2 appears to be directly responsible for degradation of XIAP, since increased level of Omi/HtrA2 induces more degradation (Fig. 2C), while decreased level has an opposite effect (Fig. 4). In addition, purified Omi/HtrA2 can directly degrade XIAP and other human IAPs in vitro (Fig. 3). The new results also indicate that the IAP binding motif of Omi/HtrA2 is important for efficient cleavage of IAPs at distinct sites and for disruption of the association between caspases and the IAP fragments. In the absence of the IAP-binding motif, Omi/HtrA2 can still cleave IAPs with less efficiency but is unable to disrupt the association between caspases and the IAP fragments. Thus, the presence of the IAP-binding motif enhances the ability of Omi/HtrA2 to find and destroy IAPs after its release from the mitochondria into the cytosol during mammalian cell death. Although the level of XIAP was reduced by the transfected wild type Omi/HtrA2, there were no XIAP fragments detectable in the cellular extracts of the transfected cells (Fig. 2B). In contrast multiple fragments were detectable in vitro after incubation of Omi/HtrA2 with IAPs (Fig. 3). These interesting results suggest that in cells, the Omi/HtrA2-generated IAP fragments could be targeted for further degradation by the N-end rule degradation pathway. This is consistent with recent results, which demonstrated that caspase-cleavage of the Drosophila DIAP1 makes it unstable and targets it for degradation by the N-end rule degradation pathway (21Ditzel M. Wilson R. Tenev T. Zachariou A. Paul A. Deas E. Meier P. Nat. Cell Biol. 2003; 5: 467-473Crossref PubMed Scopus (206) Google Scholar). Thus Omi/HtrA2 could promote apoptosis in mammalian cells by two mechanisms. One mechanism relies on its IAP-binding motif to bind to IAPs and disrupts association of active caspases with IAPs, and the other mechanism relies on its protease activity to cleave bound IAPs and targets them for further degradation by the proteosomal pathways. However, both mechanisms might be necessary for efficient killing by Omi/HtrA2. This could explain why the active site mutant Omi/HtrA2-S306A can disrupts IAP-caspase association and promote caspase activation in vitro (Fig. 3C), while it is not able to induce efficient cell death or caspase activation in transfected cells (Fig. 2). Ironically, the similarity between Omi/HtrA2 and the bacterial survival chaperone-protease DegP/HtrA suggests that Omi/HtrA2 could have an important survival function in the mitochondria (16Clausen T. Southan C. Ehrmann M. Mol. Cell. 2002; 10: 443-455Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, 17Maurizi M.R. Nat. Struct. Biol. 2002; 9: 410-412Crossref PubMed Scopus (16) Google Scholar). Omi/HtrA2 may protect the mitochondria against cellular stresses by recognizing unfolded or misfolded proteins in the intermembrane space and helps refold or degrade them. If this is the case, then loss of this protein could lead to accumulation of damaged proteins in the intermembrane space of the mitochondria over a long period of time. This is likely to be deleterious to mitochondrial function and increase sensitivity of cells to stress-induced cell death. These possibilities are currently under investigation. Addendum—While this paper was under review, Yang et al. (22Yang Q.H. Church-Hajduk R. Ren J. Newton M.L. Du C. Genes Dev. 2003; 17: 1487-1496Crossref PubMed Scopus (277) Google Scholar) reported that IAPs are substrates for Omi/HtrA2. Their paper presents essentially the same results as ours.