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: Shigemoto Fujii, Tomohiro Sawa, Hideshi Ihara, Kit I Tong, Tomoaki Ida, Tatsuya Okamoto, Ahmed Khandaker Ahtesham, Yu Ishima, Hozumi Motohashi, Masayuki Yamamoto, Takaaki Akaike
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 285 Issue 31 Pg. 23970-84 (Jul 30 2010) ISSN: 1083-351X [Electronic] United States
• PMID: 20498371 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• 8-nitroguanosine 3',5'-cyclic monophosphate
• Antioxidants
• Reactive Oxygen Species
• Nitric Oxide
• Guanine
• Cyclic GMP

Topics

• Animals
• Antioxidants (chemistry)
• Cell Line, Tumor
• Chromatography, Liquid (methods)
• Cyclic GMP (analogs & derivatives, chemistry, metabolism)
• Glioma (metabolism)
• Guanine (chemistry)
• Mass Spectrometry (methods)
• Nitric Oxide (chemistry, metabolism)
• Oxidative Stress
• Protein Processing, Post-Translational
• Rats
• Reactive Oxygen Species
• Tandem Mass Spectrometry (methods)