protein microarray-based analysis of S-nitrosylation

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 45; pp. 18948 - 18953
• Main Authors: Foster, Matthew W, Forrester, Michael T, Stamler, Jonathan S
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 10-11-2009; National Acad Sciences
• Subjects: Active sites; Amide Synthases - metabolism; Biochemistry; Biological Sciences; Catalysis; Chemical reactions; Cysteine - metabolism; Enzymes; Humans; Nitric oxide; Nitric Oxide - metabolism; Open reading frames; Phosphatases; Protein Array Analysis - methods; Protein Tyrosine Phosphatases - metabolism; Proteins; Proteins - metabolism; Proteomes; Proteomics; Proteomics - methods; Reactivity; S-Nitrosothiols - metabolism; Substrate Specificity; Thiols; Thiosulfate Sulfurtransferase - metabolism; Ubiquitin Thiolesterase - metabolism; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0900729106

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 α-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.