Antioxidant effect of a nitrated cyclic nucleotide functioning as an endogenous electrophile.

We describe here physiological formation of a unique nitrated cyclic nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) and its potent antioxidant activity. Our earlier studies revealed the NO-dependent guanine nitration in several types of cells. In fact, we identified physiological formation and functions of 8-nitro-cGMP, which is the first demonstration of a new second messenger derived from cGMP in mammals since the discovery of cGMP more than 40 years ago. Using immunocytochemical methods, we confirmed 8-nitro-cGMP formation in cultured macrophages, hepatocyte-like cells, adipocytes, and endothelial cells, depending on NO production. We further verified 8-nitro-cGMP formation via HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP as an electrophile reacts efficiently with sulfhydryls of proteins to generate a novel post-translational modification, which we call protein S-guanylation. Particular intracellular proteins can readily undergo S-guanylation by 8-nitro-cGMP. 8-Nitro-cGMP regulates the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. More importantly, we clarified that S-guanylation of Keap1 is involved in potent antioxidant effects mediated by 8-nitro-cGMP, by inducing oxidative stress-response genes such as heme oxygenase-1. Our discovery of 8-nitro-cGMP and its unique antioxidant effects thus shed light on new areas of oxidative stress research. Protein S-guanylation induced by 8-nitro-cGMP thus may have important implications in pharmaceutical chemistry and development of therapeutics for many diseases.