In the present study, we demonstrate that endogenous levels of NO are capable of S-nitrosylating the RING domain of XIAP, thus suppressing its E3 ligase activity in neuronal cells. Point mutation of the cysteine residue at position 450 (C450H) in the RING domain virtually eliminated S-nitrosylation of full-length XIAP. Since caspase-3 is normally a substrate for XIAP E3 ligase, less caspase-3 is degraded when XIAP is S-nitrosylated and thus caspase-dependent cell death pathways are further activated and contribute to neuronal cell death (). Our finding that SNO-XIAP accumulates in the brains of human patients with neurodegenerative diseases at pathophysiologically relevant levels supports a role for SNO-XIAP in neurodegeneration.
We initially found in SH-SY5Y cells that S-nitrosylation of XIAP not only decreased its E3 ligase activity but also rendered cells susceptible to cell death triggered by Bax. We also demonstrated in primary cerebrocortical neurons that direct reduction of XIAP levels by RNAi led to increased caspase-3 activity and subsequent cell death elicited by NMDA. These findings are consistent with the notion that decreased XIAP activity via nitrosylation leads to SNO-XIAP-mediated caspase activation, which may at least in part underlie the neuronal loss seen in several neurodegenerative diseases. Additionally, we observed evidence for transnitrosylation in this system, resulting in the transfer of NO from SNO-caspase-3 to XIAP, providing another mechanism for SNO-XIAP formation to increase neuronal vulnerability. Mechanistic details of this reaction and its effect on XIAP activity are discussed further in the Supplemental Data
While this work was under review, another group found that XIAP is S-nitrosylated in animal models of PD and human PD brains (Tsang et al., 2009
). These authors reported that very high (500 µM) concentrations of exogenous NO produced S-nitrosylation of cysteine residues in the BIR domains of XIAP to influence caspase binding and activity. We feel that the discrepancy with our observation that XIAP is S-nitrosylated in the RING domain rather than the BIR domains can be explained by our use of lower (physiological) levels of endogenous
NO to characterize the critical protein thiol undergoing reaction; very high (non-physiological) amounts of exogenous NO can react indiscriminately with other protein thiols to produce nitrosylation of virtually any cysteine residue, and therefore may affect BIR domain cysteines in this manner (Tsang et al., 2009
In conclusion, in this study, we demonstrate that NO negatively regulates the antiapoptotic function of XIAP by decreasing E3 ligase activity. We find evidence for specific S-nitrosylation of XIAP (to form SNO-XIAP) and posit that this reaction may be relevant to a number of pathophysiological conditions mediated at least in part by nitrosative or oxidative stress. The elucidation of this pathway to S-nitrosylation and inhibition of XIAP activity also provides a potential target for therapies directed at neurodegenerative disorders and possibly other diseases associated with nitrosative stress.