Protein S-guanylation by the biological signal 8-nitroguanosine 3′,5′-cyclic monophosphate

The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3′,5′-cyclic monophosphate (cGMP, 1). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP, 2), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO.