title: The critical role of nitric oxide signaling, via protein S-guanylation and nitrated cyclic GMP, in the antioxidant adaptive response.
abstract: A nitrated guanine nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (>40 microm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys(434) of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response.
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan.
authors.forename: Shigemoto
authors.initials: S
authors.lastname: Fujii
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Sawa
authors.forename: Hideshi
authors.initials: H
authors.lastname: Ihara
authors.forename: Kit I
authors.initials: KI
authors.lastname: Tong
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Ida
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Okamoto
authors.forename: Ahmed Khandaker
authors.initials: AK
authors.lastname: Ahtesham
authors.forename: Yu
authors.initials: Y
authors.lastname: Ishima
authors.forename: Hozumi
authors.initials: H
authors.lastname: Motohashi
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Takaaki
authors.initials: T
authors.lastname: Akaike
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000737:chemistry*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D005910:Glioma / Q000378:metabolism; D006147:Guanine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D018384:Oxidative Stress; D011499:Protein Processing, Post-Translational; D051381:Rats; D017382:Reactive Oxygen Species; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: C525106:8-nitroguanosine 3',5'-cyclic monophosphate; D000975:Antioxidants; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D006147:Guanine; D006152:Cyclic GMP