title: A substrate trapping approach identifies proteins regulated by reversible S-nitrosylation.
abstract: Protein S-nitrosylation, the nitric oxide-mediated posttranslational modification of cysteine residues, has emerged as an important regulatory mechanism in diverse cellular processes. Yet, knowledge about the S-nitrosoproteome in different cell types and cellular contexts is still limited and many questions remain regarding the precise roles of protein S-nitrosylation and denitrosylation. Here we present a novel strategy to identify reversibly nitrosylated proteins. Our approach is based on nitrosothiol capture and enrichment using a thioredoxin trap mutant, followed by protein identification by mass spectrometry. Employing this approach, we identified more than 400 putative nitroso-proteins in S-nitrosocysteine-treated human monocytes and about 200 nitrosylation substrates in endotoxin and cytokine-stimulated mouse macrophages. The large majority of these represent novel nitrosylation targets and they include many proteins with key functions in cellular homeostasis and signaling. Biochemical and functional experiments in vitro and in cells validated the proteomic results and further suggested a role for thioredoxin in the denitrosylation and activation of inducible nitric oxide synthase and the protein kinase MEK1. Our findings contribute to a better understanding of the macrophage S-nitrosoproteome and the role of thioredoxin-mediated denitrosylation in nitric oxide signaling. The approach described here may prove generally useful for the identification and exploration of nitroso-proteomes under various physiological and pathophysiological conditions.
authors: From the ‡From the Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel;
authors: Shani
authors: S
authors: Ben-Lulu
authors: §Smoler Proteomics Center and Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors: Tamar
authors: T
authors: Ziv
authors: §Smoler Proteomics Center and Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors: Arie
authors: A
authors: Admon
authors: From the ‡From the Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel;
authors: Pnina
authors: P
authors: Weisman-Shomer
authors: From the ‡From the Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel; benhar@tx.technion.ac.il.
authors: Moran
authors: M
authors: Benhar
mesh_terms: D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D009569:Nitric Oxide / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D026403:S-Nitrosothiols / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
chemical_list: D011506:Proteins; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D013879:Thioredoxins; D007371:Interferon-gamma; C021315:S-nitrosocysteine; D003545:Cysteine