A protein microarray-based analysis of S-nitrosylation.
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.
Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Matthew W
MW
Foster
Michael T
MT
Forrester
Jonathan S
JS
Stamler
D019732:Amide Synthases / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D026403:S-Nitrosothiols / Q000378:metabolism*; D013379:Substrate Specificity; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000378:metabolism; D015003:Yeasts
D011506:Proteins; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D013884:Thiosulfate Sulfurtransferase; D017027:Protein Tyrosine Phosphatases; D043222:Ubiquitin Thiolesterase; D019732:Amide Synthases; C038479:NAD+ synthase; D003545:Cysteine