A protein microarray-based analysis of S-nitrosylation

作者：Foster Matthew W; Forrester Michael T; Stamler Jonathan S^*

来源：Proceedings of the National Academy of Sciences, 2009, 106(45): 18948-18953.

DOI：10.1073/pnas.0900729106

• cysteine
• nitric oxide
• S-nitrosothiol
• thiol

The ubiquitous cellular influence of nitric oxide ( NO) is exerted substantially through protein S-nitrosylation. Whereas NO is highly promiscuous, physiological S-nitrosylation is typically restricted to one or very few Cys residue(s) in target proteins. The molecular basis for this specificity may derive from properties of the target protein, the S-nitrosylating species, or both. Here, we describe a protein microarray-based approach to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). We identify large sets of yeast and human target proteins, among which those with active-site Cys thiols residing at N termini of alpha-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins ( e. g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation ( which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols.

• 出版日期2009-11-10