title: Identification of S-nitrosylated targets of thioredoxin using a quantitative proteomic approach.
abstract: Reversible protein cysteine nitrosylation (S-nitrosylation) is a common mechanism utilized in signal transduction and other diverse cellular processes. Protein denitrosylation is largely mediated by cysteine denitrosylases, but the functional scope and significance of these enzymes are incompletely defined, in part due to limited information on their cognate substrates. Here, using Jurkat cells, we employed stable isotope labeling by amino acids in cell culture (SILAC), coupled to the biotin switch technique and mass spectrometry, to identify 46 new substrates of one denitrosylase, thioredoxin 1. These substrates are involved in a wide range of cellular functions including cytoskeletal organization, cellular metabolism, signal transduction, and redox homeostasis. We also identified multiple S-nitrosylated proteins that are not substrates of thioredoxin 1. A verification of our principal findings was made in a second cell type (RAW264.7 cells). Our results point to thioredoxin 1 as a major protein denitrosylase in mammalian cells and demonstrate the utility of quantitative proteomics for large-scale identification of denitrosylase substrates.
authors.affiliation: Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Moran
authors.initials: M
authors.lastname: Benhar
authors.forename: J Will
authors.initials: JW
authors.lastname: Thompson
authors.forename: M Arthur
authors.initials: MA
authors.lastname: Moseley
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Stamler
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling; D019169:Jurkat Cells; D051379:Mice; D008962:Models, Theoretical; D040901:Proteomics; D026403:S-Nitrosothiols / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D026403:S-Nitrosothiols; D013879:Thioredoxins