title: Proteomic characterization of the cellular response to nitrosative stress mediated by s-nitrosoglutathione reductase inhibition.
abstract: The S-nitrosoglutathione-metabolizing enzyme, GSNO reductase (GSNOR), has emerged as an important regulator of protein S-nitrosylation. GSNOR ablation is protective in models of asthma and heart failure, raising the idea that GSNOR inhibitors might hold therapeutic value. Here, we investigated the effects of a small molecule inhibitor of GSNOR (GSNORi) in mouse RAW 264.7 macrophages. We found that GSNORi increased protein S-nitrosylation in cytokine-stimulated cells, and we utilized stable isotope labeling of amino acids in cell culture (SILAC) to quantify the cellular response to this "nitrosative stress". The expression of several cytokine-inducible immunomodulators, including osteopontin, cyclooxygenase-2, and nitric oxide synthase isoform 2 (NOS2), were decreased by GSNORi. In addition, selective targets of the redox-regulated transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-including heme oxygenase 1 (HO-1) and glutamate cysteine ligase modulatory subunit-were induced by GSNORi in a NOS2- and Nrf2-dependent manner. In cytokine-stimulated cells, Nrf2 protected from GSNORi-induced glutathione depletion and cytotoxicity and HO-1 activity was required for down-regulation of NOS2. Interestingly, GSNORi also affected a marked increase in NOS2 protein stability. Collectively, these data provide the most complete description of the global effects of GSNOR inhibition and demonstrate several important mechanisms for inducible response to GSNORi-mediated nitrosative stress.
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Small Molecule Synthesis Facility and Institute for Genome Sciences and Policy, Duke University Medical Center, Durham, North Carolina 27710, United States.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Foster
authors.forename: Zhonghui
authors.initials: Z
authors.lastname: Yang
authors.forename: David M
authors.initials: DM
authors.lastname: Gooden
authors.forename: J Will
authors.initials: JW
authors.lastname: Thompson
authors.forename: Carol H
authors.initials: CH
authors.lastname: Ball
authors.forename: Meredith E
authors.initials: ME
authors.lastname: Turner
authors.forename: Yongyong
authors.initials: Y
authors.lastname: Hou
authors.forename: Jingbo
authors.initials: J
authors.lastname: Pi
authors.forename: M Arthur
authors.initials: MA
authors.lastname: Moseley
authors.forename: Loretta G
authors.initials: LG
authors.lastname: Que
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D005786:Gene Expression Regulation; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism; D007553:Isotope Labeling; D008264:Macrophages / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D015538:Nitrosation; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D026422:S-Nitrosoglutathione / Q000378:metabolism; D013312:Stress, Physiological / Q000502:physiology*
chemical_list: D016207:Cytokines; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D020543:Proteome; D026422:S-Nitrosoglutathione; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D006419:Heme Oxygenase (Decyclizing); D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent