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

Abstract

The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3',5'-cyclic monophosphate (cGMP). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), 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.

Cytoprotective Function of Heme Oxygenase 1 Induced by a Nitrated Cyclic Nucleotide Formed during Murine Salmonellosis

“…8-Nitroguanosine has a redox active property (18,19), which suggests that nucleotide nitration may have a biological function rather than simply being a biological marker. In this context, we recently discovered a nitrated cyclic nucleotide, 8-nitroguanosine 3Ј,5Ј-cyclic monophosphate , which is formed in physiological systems and may be involved in redox activity and signal transduction (20). The signaling function of 8-nitro-cGMP appears to be mediated by its unique reaction with the sulfhydryl group of cysteines, thus forming a protein S-cGMP adduct, which is an important mechanism of posttranslational modification of proteins that we have named protein S-guanylation (20).…”

“…Liver tissues and cultured cells were fixed and cryostat tissue sections were prepared as described earlier (20,45). Immunostaining was achieved by using biotin-conjugated monoclonal mouse Abs for 8-nitro-cGMP (clones 1G6 and 1H7) and by using rabbit polyclonal anti-HO-1 IgG (Stressgen Biotechnologies), as reported in a recent publication (17,20).…”

Nitric Oxide Activates Signaling by c-Raf, MEK, p-JNK, p38 MAPK and p53 in Human Mesenchymal Stromal Cells inhibits their Osteogenic Differentiation by Blocking Expression of Runx2

“…Here NO not only raises the cytoplasmic cGMP concentrations, but in addition generates a novel signalling molecule, the 8-NO 2 -cGMP. A direct, chemical non-enzymatical nitrosylation of cGMP was suggested [66]. This pathway is important especially at higher concentrations of NO, thus explaining in part the bi-phasic effects observed with NO on MSC.…”

“…Recently a novel NO-dependent intracellular signalling pathway was described [66]. Here NO not only raises the cytoplasmic cGMP concentrations, but in addition generates a novel signalling molecule, the 8-NO 2 -cGMP.…”

“…This pathway is important especially at higher concentrations of NO, thus explaining in part the bi-phasic effects observed with NO on MSC. The 8-NO 2 -cGMP then generates cGMPmodified proteins by S-guanylation [66]. This post-translational modification was described with the redox sensor protein, Keap1.…”