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• W2024674223 abstract "X-linked inhibitor of apoptosis (XIAP) is a potent antagonist of caspase apoptotic activity. XIAP also functions as an E3 ubiquitin ligase, targeting caspases for degradation. However, molecular pathways controlling XIAP activities remain unclear. Here, we report that nitric oxide (NO) reacts with XIAP by S-nitrosylating its RING domain (forming SNO-XIAP), thereby inhibiting E3 ligase and antiapoptotic activity. NO-mediated neurotoxicity and caspase activation have been linked to several neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. We find significant SNO-XIAP formation in brains of patients with these diseases, implicating this reaction in the etiology of neuronal damage. Conversely, S-nitrosylation of caspases is known to inhibit apoptotic activity. Unexpectedly, we find that SNO-caspase transnitrosylates (transfers its NO group) to XIAP, forming SNO-XIAP, and thus promotes cell injury and death. These findings provide insights into the regulation of caspase activation in neurodegenerative disorders mediated, at least in part, by nitrosative stress. X-linked inhibitor of apoptosis (XIAP) is a potent antagonist of caspase apoptotic activity. XIAP also functions as an E3 ubiquitin ligase, targeting caspases for degradation. However, molecular pathways controlling XIAP activities remain unclear. Here, we report that nitric oxide (NO) reacts with XIAP by S-nitrosylating its RING domain (forming SNO-XIAP), thereby inhibiting E3 ligase and antiapoptotic activity. NO-mediated neurotoxicity and caspase activation have been linked to several neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. We find significant SNO-XIAP formation in brains of patients with these diseases, implicating this reaction in the etiology of neuronal damage. Conversely, S-nitrosylation of caspases is known to inhibit apoptotic activity. Unexpectedly, we find that SNO-caspase transnitrosylates (transfers its NO group) to XIAP, forming SNO-XIAP, and thus promotes cell injury and death. These findings provide insights into the regulation of caspase activation in neurodegenerative disorders mediated, at least in part, by nitrosative stress. XIAP is S-nitrosylated in neurodegenerative conditions S-nitrosylation of XIAP downregulates its ubiquitin E3 ligase activity S-nitrosylation of XIAP attenuates its antiapoptotic activity S-nitrosylated caspase-3 transnitrosylates (transfers its NO group to) XIAP Neuronal cell injury and death are prominent features of neurodegenerative disorders such as Alzheimer's, Huntington's, and Parkinson's diseases (Mattson, 2000Mattson M.P. Apoptosis in neurodegenerative disorders.Nat. Rev. Mol. Cell Biol. 2000; 1: 120-129Crossref PubMed Scopus (1189) Google Scholar, Friedlander, 2003Friedlander R.M. Apoptosis and caspases in neurodegenerative diseases.N. Engl. J. Med. 2003; 348: 1365-1375Crossref PubMed Scopus (726) Google Scholar). While acute fulminant insults result in osmotic swelling and necrosis, chronic degenerative disorders can produce apoptotic cell death (Ankarcrona et al., 1995Ankarcrona M. Dypbukt J.M. Bonfoco E. Zhivotovsky B. Orrenius S. Lipton S.A. Nicotera P. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function.Neuron. 1995; 15: 961-973Abstract Full Text PDF PubMed Scopus (1639) Google Scholar, Bonfoco et al., 1995Bonfoco E. Krainc D. Ankarcrona M. Nicotera P. Lipton S.A. Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures.Proc. Natl. Acad. Sci. USA. 1995; 92: 7162-7166Crossref PubMed Scopus (1837) Google Scholar), often mediated by the caspase family of cysteine proteases (Chan and Mattson, 1999Chan S.L. Mattson M.P. Caspase and calpain substrates: roles in synaptic plasticity and cell death.J. Neurosci. Res. 1999; 58: 167-190Crossref PubMed Scopus (365) Google Scholar, Lu et al., 2000Lu D.C. Rabizadeh S. Chandra S. Shayya R.F. Ellerby L.M. Ye X. Salvesen G.S. Koo E.H. Bredesen D.E. A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor.Nat. Med. 2000; 6: 397-404Crossref PubMed Scopus (334) Google Scholar). During degenerative processes, for example, in Alzheimer's disease, activated caspases may induce proteolysis of β-amyloid precursor protein (APP) or synaptic proteins, which may contribute to synaptic dysfunction and neuronal cell death. Inhibitors of apoptosis proteins (IAPs) represent important regulators of apoptosis through their ability to associate with active caspases and repress their catalytic activity (Eckelman et al., 2006Eckelman B.P. Salvesen G.S. Scott F.L. Human inhibitor of apoptosis proteins: why XIAP is the black sheep of the family.EMBO Rep. 2006; 7: 988-994Crossref PubMed Scopus (629) Google Scholar, Salvesen and Duckett, 2002Salvesen G.S. Duckett C.S. IAP proteins: blocking the road to death's door.Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1531) Google Scholar). In particular, XIAP interacts with active caspases-3/-7/-9 in the cytosol and is thought to be the most potent endogenous caspase inhibitor among the IAPs. XIAP harbors three copies of the baculovirus IAP repeat (BIR) domain and one RING domain. Characteristic BIR and RING folds contain zinc ions coordinated by histidine and cysteine residues. Biochemical and structural analyses indicate that BIR domains and their flanking sequences bind and inhibit the catalytic activity of apoptotic caspases (Fuentes-Prior and Salvesen, 2004Fuentes-Prior P. Salvesen G.S. The protein structures that shape caspase activity, specificity, activation and inhibition.Biochem. J. 2004; 384: 201-232Crossref PubMed Scopus (643) Google Scholar). Additionally, the RING domain of XIAP can act as an E3 ligase, functioning in ubiquitination and subsequent degradation of heterologous substrates in vivo (caspases and other IAP proteins) as well as XIAP itself (MacFarlane et al., 2002MacFarlane M. Merrison W. Bratton S.B. Cohen G.M. Proteasome-mediated degradation of Smac during apoptosis: XIAP promotes Smac ubiquitination in vitro.J. Biol. Chem. 2002; 277: 36611-36616Crossref PubMed Scopus (245) Google Scholar, Schile et al., 2008Schile A.J. García-Fernández M. Steller H. Regulation of apoptosis by XIAP ubiquitin-ligase activity.Genes Dev. 2008; 22: 2256-2266Crossref PubMed Scopus (155) Google Scholar, Suzuki et al., 2001Suzuki Y. Nakabayashi Y. Takahashi R. Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death.Proc. Natl. Acad. Sci. USA. 2001; 98: 8662-8667Crossref PubMed Scopus (526) Google Scholar, Vaux and Silke, 2005Vaux D.L. Silke J. IAPs, RINGs and ubiquitylation.Nat. Rev. Mol. Cell Biol. 2005; 6: 287-297Crossref PubMed Scopus (507) Google Scholar, Yang et al., 2000Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli.Science. 2000; 288: 874-877Crossref PubMed Scopus (836) Google Scholar). Nitric oxide (NO) is also known to contribute to neuronal cell damage and death when present at excessive levels, but can promote neuronal survival under physiological conditions (Beckman, 1990Beckman J.S. Ischaemic injury mediator.Nature. 1990; 345: 27-28Crossref PubMed Scopus (136) Google Scholar, Dawson et al., 1991Dawson V.L. Dawson T.M. London E.D. Bredt D.S. Snyder S.H. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures.Proc. Natl. Acad. Sci. USA. 1991; 88: 6368-6371Crossref PubMed Scopus (2061) Google Scholar, Lipton et al., 1993Lipton S.A. Choi Y.B. Pan Z.H. Lei S.Z. Chen H.S. Sucher N.J. Loscalzo J. Singel D.J. Stamler J.S. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds.Nature. 1993; 364: 626-632Crossref PubMed Scopus (2240) Google Scholar). NO exerts its effects in large part through stimulation of guanylate cyclase or via protein S-nitrosylation, representing the covalent attachment of NO to cysteine thiol or, more properly, thiolate anion (Hess et al., 2005Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Protein S-nitrosylation: purview and parameters.Nat. Rev. Mol. Cell Biol. 2005; 6: 150-166Crossref PubMed Scopus (1604) Google Scholar, Stamler et al., 1997Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. (S)NO signals: translocation, regulation, and a consensus motif.Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar). S-nitrosylation has recently emerged as an important regulator of redox signaling, comparable in controlling protein function to other posttranslational modifications such as phosphorylation or acetylation. Physiological levels of NO can be neuroprotective, in part, via S-nitrosylation-mediated inhibition of N-methyl-d-aspartate (NMDA)-type glutamate receptor activity and caspase activity (Choi et al., 2000Choi Y.B. Tenneti L. Le D.A. Ortiz J. Bai G. Chen H.S. Lipton S.A. Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation.Nat. Neurosci. 2000; 3: 15-21Crossref PubMed Scopus (361) Google Scholar, Dimmeler et al., 1997Dimmeler S. Haendeler J. Nehls M. Zeiher A.M. Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.J. Exp. Med. 1997; 185: 601-607Crossref PubMed Scopus (764) Google Scholar, Lipton et al., 1993Lipton S.A. Choi Y.B. Pan Z.H. Lei S.Z. Chen H.S. Sucher N.J. Loscalzo J. Singel D.J. Stamler J.S. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds.Nature. 1993; 364: 626-632Crossref PubMed Scopus (2240) Google Scholar, Mannick et al., 1999Mannick J.B. Hausladen A. Liu L. Hess D.T. Zeng M. Miao Q.X. Kane L.S. Gow A.J. Stamler J.S. Fas-induced caspase denitrosylation.Science. 1999; 284: 651-654Crossref PubMed Scopus (691) Google Scholar, Melino et al., 1997Melino G. Bernassola F. Knight R.A. Corasaniti M.T. Nistico G. Finazzi-Agro A. S-nitrosylation regulates apoptosis.Nature. 1997; 388: 432-433Crossref PubMed Scopus (365) Google Scholar, Tenneti et al., 1997Tenneti L. D'Emilia D.M. Lipton S.A. Suppression of neuronal apoptosis by S-nitrosylation of caspases.Neurosci. Lett. 1997; 236: 139-142Crossref PubMed Scopus (144) Google Scholar). However, excess NO production in neurons results in activation of cell death signaling cascades that underlie many neurodegenerative disorders (Dawson et al., 1991Dawson V.L. Dawson T.M. London E.D. Bredt D.S. Snyder S.H. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures.Proc. Natl. Acad. Sci. USA. 1991; 88: 6368-6371Crossref PubMed Scopus (2061) Google Scholar, Lipton et al., 1993Lipton S.A. Choi Y.B. Pan Z.H. Lei S.Z. Chen H.S. Sucher N.J. Loscalzo J. Singel D.J. Stamler J.S. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds.Nature. 1993; 364: 626-632Crossref PubMed Scopus (2240) Google Scholar, Huang et al., 1994Huang Z. Huang P.L. Panahian N. Dalkara T. Fishman M.C. Moskowitz M.A. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase.Science. 1994; 265: 1883-1885Crossref PubMed Scopus (1426) Google Scholar, Leist et al., 1997Leist M. Volbracht C. Kühnle S. Fava E. Ferrando-May E. Nicotera P. Caspase-mediated apoptosis in neuronal excitotoxicity triggered by nitric oxide.Mol. Med. 1997; 3: 750-764Crossref PubMed Google Scholar, Gu et al., 2002Gu Z. Kaul M. Yan B. Kridel S.J. Cui J. Strongin A. Smith J.W. Liddington R.C. Lipton S.A. S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death.Science. 2002; 297: 1186-1190Crossref PubMed Scopus (811) Google Scholar, Hara et al., 2005Hara M.R. Agrawal N. Kim S.F. Cascio M.B. Fujimuro M. Ozeki Y. Takahashi M. Cheah J.H. Tankou S.K. Hester L.D. et al.S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding.Nat. Cell Biol. 2005; 7: 665-674Crossref PubMed Scopus (824) Google Scholar). Recent evidence directly links S-nitrosylation to protein misfolding and neuronal cell death (Chung et al., 2004Chung K.K. Thomas B. Li X. Pletnikova O. Troncoso J.C. Marsh L. Dawson V.L. Dawson T.M. S-nitrosylation of parkin regulates ubiquitination and compromises parkin's protective function.Science. 2004; 304: 1328-1331Crossref PubMed Scopus (623) Google Scholar, Yao et al., 2004Yao D. Gu Z. Nakamura T. Shi Z.Q. Ma Y. Gaston B. Palmer L.A. Rockenstein E.M. Zhang Z. Masliah E. et al.Nitrosative stress linked to sporadic Parkinson's disease: S-nitrosylation of parkin regulates its E3 ubiquitin ligase activity.Proc. Natl. Acad. Sci. USA. 2004; 101: 10810-10814Crossref PubMed Scopus (436) Google Scholar, Uehara et al., 2006Uehara T. Nakamura T. Yao D. Shi Z.Q. Gu Z. Ma Y. Masliah E. Nomura Y. Lipton S.A. S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration.Nature. 2006; 441: 513-517Crossref PubMed Scopus (718) Google Scholar). Here, we report that S-nitrosylation of the RING domain of XIAP decreases its E3 ubiquitin ligase activity both in vitro and in intact cells, thereby blocking its ability to inhibit apoptosis by degrading caspases. We found that S-nitrosylated XIAP (SNO-XIAP) accumulates in neurons stimulated with pathophysiologically relevant levels of NMDA and in the brains of patients exhibiting neurodegeneration. Moreover, transnitrosylation of XIAP by SNO-caspase provides an additional mechanism for proapoptotic signaling. These results indicate that SNO-XIAP regulates caspase activity and contributes to neuronal injury or death in a number of neurodegenerative diseases. Since we and others have found that the E3 ubiquitin ligase, parkin, is S-nitrosylated via cysteine thiol in its RING domain (Chung et al., 2004Chung K.K. Thomas B. Li X. Pletnikova O. Troncoso J.C. Marsh L. Dawson V.L. Dawson T.M. S-nitrosylation of parkin regulates ubiquitination and compromises parkin's protective function.Science. 2004; 304: 1328-1331Crossref PubMed Scopus (623) Google Scholar, Yao et al., 2004Yao D. Gu Z. Nakamura T. Shi Z.Q. Ma Y. Gaston B. Palmer L.A. Rockenstein E.M. Zhang Z. Masliah E. et al.Nitrosative stress linked to sporadic Parkinson's disease: S-nitrosylation of parkin regulates its E3 ubiquitin ligase activity.Proc. Natl. Acad. Sci. USA. 2004; 101: 10810-10814Crossref PubMed Scopus (436) Google Scholar), we asked whether another RING domain-containing E3 ligase, XIAP, was also a target of S-nitrosylation. To answer this question, we employed a specific fluorescence assay for S-nitrosothiols (Gu et al., 2002Gu Z. Kaul M. Yan B. Kridel S.J. Cui J. Strongin A. Smith J.W. Liddington R.C. Lipton S.A. S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death.Science. 2002; 297: 1186-1190Crossref PubMed Scopus (811) Google Scholar, Wink et al., 1999Wink D.A. Kim S. Coffin D. Cook J.C. Vodovotz Y. Chistodoulou D. Jourd'heuil D. Grisham M.B. Detection of S-nitrosothiols by fluorometric and colorimetric methods.Methods Enzymol. 1999; 301: 201-211Crossref PubMed Scopus (55) Google Scholar) to detect SNO-XIAP in vitro. Incubation of a GST-XIAP fusion protein with a physiological NO donor, S-nitrosocysteine (SNOC), induced S-nitrosothiol formation, indicating the presence of SNO-XIAP (Figure 1A ). To ascertain whether the RING domain of XIAP was S-nitrosylated, we performed mapping studies using truncated forms of XIAP. The SNO-fluorescence assay indicated that XIAP fragments containing BIR2 or BIR2/3 domains were not S-nitrosylated, whereas a fragment expressing the BIR2/3 and RING domains (BIR2-3-RING) was S-nitrosylated, suggesting that the ubiquitin-associated (UBA) or RING domain of XIAP contains the principal S-nitrosylated residue(s) (Figure 1B). We then asked whether XIAP is S-nitrosylated in intact cells using the NO-biotin switch method, a modified immunoblot to detect S-nitrosothiols (Jaffrey et al., 2001Jaffrey S.R. Erdjument-Bromage H. Ferris C.D. Tempst P. Snyder S.H. Protein S-nitrosylation: a physiological signal for neuronal nitric oxide.Nat. Cell Biol. 2001; 3: 193-197Crossref PubMed Scopus (1184) Google Scholar). After exposing neuroblastoma SH-SY5Y cells to SNOC, we detected S-nitrosylation of endogenous XIAP using a specific anti-XIAP antibody (Figures 1C and S1). Next, using the NO-biotin switch assay after expressing XIAP fragments in intact cells, we found that the RING domain of XIAP is the predominant location of S-nitrosylated residues (Figures 1D and S1B), confirming our earlier finding on recombinant preparations in vitro. In order to identify the principal site of S-nitrosylation in the RING domain of XIAP, we employed three complementary approaches, using nuclear magnetic resonance (NMR) techniques to direct additional NO-biotin switch assays and mass spectrometry analyses. We labeled recombinant RING domain (residues 432–497 of XIAP) with 15N and then acquired 2D 1H-15N transverse relaxation-optimized spectroscopy (TROSY)-NMR spectra to reveal the target cysteine of S-nitrosylation by chemical shift mapping (Pervushin et al., 1997Pervushin K. Riek R. Wider G. Wüthrich K. Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution.Proc. Natl. Acad. Sci. USA. 1997; 94: 12366-12371Crossref PubMed Scopus (1973) Google Scholar). Comparison of the NMR spectra between native and SNOC-treated RING domains revealed small but reproducible chemical shifts of several peaks corresponding to the backbone 1H-15N groups of amino acid residues that were located in close proximity to Cys450 and Cys471 (Figures 2, S2A, and S2B). These TROSY-NMR experiments were consistent with the notion that among the seven cysteines in the RING domain, either Cys450 or Cys471 (or possibly both) were the target cysteine residues for S-nitrosylation. Moreover, these experiments suggested that formation of S-nitrosothiol on the RING domain induced minor conformational perturbations to proximate amino acid residues (e.g., Lys448, Leu449, Ile458, and Leu468) and that nitrosylation did not result in substantial unfolding of the RING (Figures 2D and S2B). Since we also found that alanine substitution of two of these residues (Lys448 or Leu449) resulted in a decrease in E3 ligase activity (Figure S2C), it is tempting to speculate that S-nitrosylation-induced conformational changes can affect the function of the RING domain. Consistent with this notion, we found that exposure to SNOC could effect release of zinc from the RING domain (Figure S2D). To further investigate the specific site of S-nitrosylation on XIAP under physiological conditions, we next expressed WT-XIAP, XIAP(C450H), or XIAP(C471H) in HEK293 cells stably expressing neuronal NO synthase (HEK-nNOS) (Bredt et al., 1991Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase.Nature. 1991; 351: 714-718Crossref PubMed Scopus (2147) Google Scholar). This approach allowed us to detect S-nitrosylation of XIAP engendered by endogenous NO. We chose histidine to replace cysteine in these constructs because similar to cysteine, histidine can coordinate the zinc ion in the RING structure. The NO-biotin switch assay revealed that the C450H mutation significantly diminished endogenous NO-mediated S-nitrosylation, pointing to Cys450 in the RING domain as the primary site of S-nitrosylation of XIAP (Figure 3A ). Interestingly, a characteristic acid-base motif for S-nitrosylation (Asp455, Lys448, and Lys451) surrounds the Cys450 residue (Stamler et al., 1997Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. (S)NO signals: translocation, regulation, and a consensus motif.Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar, Hess et al., 2005Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Protein S-nitrosylation: purview and parameters.Nat. Rev. Mol. Cell Biol. 2005; 6: 150-166Crossref PubMed Scopus (1604) Google Scholar). Next, we performed a high-resolution Orbitrap mass spectrometry analysis of the WT XIAP-RING domain after exposure to SNOC in order to determine if S-nitrosylation of XIAP occurs at Cys450. To keep this posttranslational modification intact during the acquisition of MS/MS spectra, we employed an innovative top-down approach together with electron transfer dissociation (ETD) technology, which, compared to conventional collision-induced dissociation methods, allows us to preserve labile modifications on larger peptides (Han et al., 2008Han X. Aslanian A. Yates 3rd, J.R. Mass spectrometry for proteomics.Curr. Opin. Chem. Biol. 2008; 12: 483-490Crossref PubMed Scopus (452) Google Scholar, Mikesh et al., 2006Mikesh L.M. Ueberheide B. Chi A. Coon J.J. Syka J.E. Shabanowitz J. Hunt D.F. The utility of ETD mass spectrometry in proteomic analysis.Biochim. Biophys. Acta. 2006; 1764: 1811-1822Crossref PubMed Scopus (438) Google Scholar). Consistent with the results of our NO-biotin switch assay, the LTQ Orbitrap XL-ETD MS/MS data detected an NO adduct predominantly at Cys450 of the XIAP-RING domain (Figures 3B and S3). Taken together, these results strongly suggest that S-nitrosylation occurs on the RING domain of XIAP, specifically at Cys450, and that formation of SNO-XIAP at this site modulates the surrounding local conformation or chemical environment. Caspase activation and excessive NO generation have been associated with several human neurodegenerative conditions, including Parkinson's with diffuse Lewy body disease (DLBD), Alzheimer's disease (AD), and Huntington's disease (HD). Our findings from in vitro and cell-based experiments raised the possibility that XIAP activity could be affected by S-nitrosylation in these disorders. Therefore, to determine whether XIAP is S-nitrosylated in humans in vivo, we performed an NO-biotin switch assay with brain extracts prepared from postmortem brains of patients with sporadic DLBD, AD, HD, and control brains (patients who died of non-CNS disorders) (Uehara et al., 2006Uehara T. Nakamura T. Yao D. Shi Z.Q. Gu Z. Ma Y. Masliah E. Nomura Y. Lipton S.A. S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration.Nature. 2006; 441: 513-517Crossref PubMed Scopus (718) Google Scholar). We observed that these brains contained significantly higher levels of SNO-XIAP than controls (Figures 3C and S3C and Table S1), supporting our hypothesis that SNO-XIAP levels are positively correlated with disease pathogenesis. Interestingly, we did not find elevated levels of S-nitrosylated caspase-3 in these diseased brains (Figure S3D and Table S1). In addition to XIAP, other inhibitors of apoptosis include cIAP1 and cIAP2. However, SNO-cIAP1 and SNO-cIAP2 were undetectable under our conditions (Figures S3E and S3F and Table S1). Taken together, these results are in accord with previous findings that downregulation of XIAP but not cIAP1 or cIAP2 is essential in regulating neuronal apoptosis (Potts et al., 2003Potts P.R. Singh S. Knezek M. Thompson C.B. Deshmukh M. Critical function of endogenous XIAP in regulating caspase activation during sympathetic neuronal apoptosis.J. Cell Biol. 2003; 163: 789-799Crossref PubMed Scopus (117) Google Scholar). Recent studies have shown that XIAP and caspases are substrates for XIAP-mediated ubiquitination in vitro and in intact cells (Morizane et al., 2005Morizane Y. Honda R. Fukami K. Yasuda H. X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO.J. Biochem. 2005; 137: 125-132Crossref PubMed Scopus (116) Google Scholar, Schile et al., 2008Schile A.J. García-Fernández M. Steller H. Regulation of apoptosis by XIAP ubiquitin-ligase activity.Genes Dev. 2008; 22: 2256-2266Crossref PubMed Scopus (155) Google Scholar, Shin et al., 2003Shin H. Okada K. Wilkinson J.C. Solomon K.M. Duckett C.S. Reed J.C. Salvesen G.S. Identification of ubiquitination sites on the X-linked inhibitor of apoptosis protein.Biochem. J. 2003; 373: 965-971Crossref PubMed Scopus (43) Google Scholar, Suzuki et al., 2001Suzuki Y. Nakabayashi Y. Takahashi R. Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death.Proc. Natl. Acad. Sci. USA. 2001; 98: 8662-8667Crossref PubMed Scopus (526) Google Scholar, Yang et al., 2000Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli.Science. 2000; 288: 874-877Crossref PubMed Scopus (836) Google Scholar). Our NMR data suggested that NO could perturb the structural or chemical environment of the RING domain in a region that would be expected to affect ubiquitin enzymatic activity. Thus, we asked whether S-nitrosylation of the XIAP-RING domain affects its E3 ligase activity. Initially, we examined autoubiquitination of XIAP in vitro to confirm a direct effect of NO on XIAP E3 ubiquitin ligase activity. When recombinant GST-XIAP was incubated in vitro with ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2 (UbcH5b), and ubiquitin (Ub), a smear of proteins representing polyubiquitinated XIAP on immunoblots was identified by reaction with anti-ubiquitin antibody (Figures 4A and S4A). Pretreatment of recombinant XIAP with SNOC starting 30 min prior to addition of E1, E2, and Ub proteins markedly reduced XIAP autoubiquitination, suggesting that NO directly interferes with XIAP E3 ligase activity. Although NO dissipates from SNOC quickly (within ∼5 min under our experimental conditions), we found that S-nitrosylation of cysteine thiol on XIAP and certain other proteins lasts for more than 30 min (Gu et al., 2002Gu Z. Kaul M. Yan B. Kridel S.J. Cui J. Strongin A. Smith J.W. Liddington R.C. Lipton S.A. S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death.Science. 2002; 297: 1186-1190Crossref PubMed Scopus (811) Google Scholar, Lei et al., 1992Lei S.Z. Pan Z.H. Aggarwal S.K. Chen H.S. Hartman J. Sucher N.J. Lipton S.A. Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex.Neuron. 1992; 8: 1087-1099Abstract Full Text PDF PubMed Scopus (671) Google Scholar). This time frame allowed us to exclude a direct effect of NO on E1, E2, or ubiquitin, since all the NO from SNOC was dissipated by the time these proteins were added to the reaction milieu, supporting the notion that NO directly inactivated the E3 ligase activity of XIAP (Figures 4A and S4A). Additionally, control treatment with inactive SNOC (old SNOC from which NO had been dissipated) had no effect on autoubiquitination (Figures 4A and S4A). To further verify the effect of NO on XIAP, we compared the E3 ligase activity of WT-XIAP before and after nitrosylation in HEK293T cells. HEK293T cells transfected with myc-tagged XIAP and hemagglutinin (HA)-tagged ubiquitin exhibited a significant, time-dependent decrease in XIAP autoubiquitination after exposure to SNOC (Figures 4B, S4B, and S4C). Lysates of cells transfected with XIAPΔRING did not exhibit autoubiquitination. Next, we asked if S-nitrosylation of XIAP affected its function in a pathophysiologically relevant manner. Since a contribution of XIAP autoubiquitination to antiapoptotic activity has not been clearly demonstrated (Shin et al., 2003Shin H. Okada K. Wilkinson J.C. Solomon K.M. Duckett C.S. Reed J.C. Salvesen G.S. Identification of ubiquitination sites on the X-linked inhibitor of apoptosis protein.Biochem. J. 2003; 373: 965-971Crossref PubMed Scopus (43) Google Scholar, Yang et al., 2000Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli.Science. 2000; 288: 874-877Crossref PubMed Scopus (836) Google Scholar), we studied whether XIAP-mediated ubiquitination of caspases-3 and -9, which has recently been reported to be important in modulating apoptosis in vivo (Schile et al., 2008Schile A.J. García-Fernández M. Steller H. Regulation of apoptosis by XIAP ubiquitin-ligase activity.Genes Dev. 2008; 22: 2256-2266Crossref PubMed Scopus (155) Google Scholar), was affected by NO. We found that pretreatment of XIAP with SNOC resulted in a decrease in polyubiquitination of caspases-3 and -9 in vitro (Figures 4C and 4D). Next, to verify that SNO-XIAP manifests decreased E3 ligase activity in intact cells, we cotransfected HEK293T cells with various combinations of XIAP, ubiquitin, and catalytic mutants of caspases-3 or -9. We and others had previously shown that NO can inhibit activity of caspases via S-nitrosylation of the catalytic cysteine (Choi et al., 2000Choi Y.B. Tenneti L. Le D.A. Ortiz J. Bai G. Chen H.S. Lipton S.A. Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation.Nat. Neurosci. 2000; 3: 15-21Crossref PubMed Scopus (361) Google Scholar, Dimmeler et al., 1997Dimmeler S. Haendeler J. Nehls M. Zeiher A.M. Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.J. Exp. Med. 1997; 185: 601-607Crossref PubMed Scopus (764) Google Scholar, Hoffmann et al., 2001Hoffmann J. Haendeler J. Zeiher A.M. Dimmeler S. TNFalpha and oxLDL reduce protein S-n" @default.
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• W2024674223 date "2010-07-01" @default.
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• W2024674223 title "Transnitrosylation of XIAP Regulates Caspase-Dependent Neuronal Cell Death" @default.
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• W2024674223 doi "https://doi.org/10.1016/j.molcel.2010.07.002" @default.
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