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Aims: 8-nitroguanosine 3′,5′-cyclic monophosphate (8-Nitro-cGMP) is a nitrated derivative of cGMP that is formed via cross-talk of reactive oxygen species formed by NADPH oxidase 2 and mitochondria. This nitrated nucleotide can function as a unique electrophilic second messenger in regulation of redox signaling by inducing a post-translational modification of protein thiols via cGMP adduction (protein S-guanylation). With S-guanylation proteomics, we investigated endogenous mitochondrial protein S-guanylation. Results: We developed a new mass spectrometry (MS)-based proteomic method—S-guanylation proteomics—which comprised two approaches: (i) direct protein digestion followed by immunoaffinity capture of S-guanylated peptides that were subjected to liquid chromatography–tandem MS (LC-MS/MS); and (ii) two-dimensional (2D)-gel electrophoretic separation of S-guanylated proteins that were subjected to in-gel digestion, followed by LC-MS/MS. We thereby identified certain mitochondrial proteins that are S-guanylated endogenously during immunological stimulation, including mortalin and 60-kDa heat-shock protein (HSP60). Mortalin and HSP60 were recently reported to regulate mitochondrial permeability-transition pore (mPTP) opening, at least partly, by interacting with cyclophilin D, an mPTP component. Our data revealed that immunological stimulation and 8-nitro-cGMP treatment induced mPTP opening in a cyclophilin D-dependent manner. Innovation and Conclusion: Our S-guanylation proteomic method determined that mitochondrial HSPs may be novel targets for redox modification via protein S-guanylation that participates in mPTP regulation and mitochondrial redox signaling. Antioxid. Redox Signal. 20, 295–307.

The biological significance of nitrated guanine derivatives, especially 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), has become evident. Therefore it is important to determine the presence and relative abundance of 8-nitro-cGMP formed in cells and tissues. In the present study, we performed immunocytochemistry with monoclonal antibodies specific for 8-nitroguanine (clone NO2-52) and 8-nitro-cGMP (clone 1G6) in rat C6 glioma cells and rat primary cultured astrocytes. Immunocytochemistry utilizing the anti-8-nitro-cGMP monoclonal antibody (1G6) indicated that immunostaining increased markedly in C6 cells expressing increased amounts of inducible nitric oxide synthase (iNOS) after treatment with lipopolysaccharide (LPS) plus cytokines. Treatment of C6 cells with inhibitors for NOS and soluble guanylate cyclase (sGC) completely nullified the elevated 1G6 immunoreactivity. These results were consistent with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Immunocytochemistry performed using NO2-52 also showed that treatment of cells with inhibitors for NOS and sGC completely nullified the elevated immunoreactivity; this indicated that 8-nitro-cGMP is a major component of 8-nitroguanine derivatives produced in cells. Similar results were obtained in the primary astrocytes stimulated with LPS plus cytokines. Because immunocytochemistry is a conventional, powerful, and fairly straightforward method for determining the presence, localization, and relative abundance of an antigen of interest in cultured cells, anti-8-nitroguanine (NO2-52) and anti-8-nitro-cGMP (1G6) antibodies could be useful tools for analyzing nitrated guanine nucleotides, especially 8-nitro-cGMP, by means of immunocytochemistry.

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.