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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, 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.

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Figure 1
Figure 2: Immunocytochemical analysis of endogenous guanine nitration in mouse macrophages.
Figure 3: Identification of 8-nitro-cGMP and related compounds formed in activated RAW 264.7 cells.
Figure 4: Reactions of 8-nitro-cGMP with sulfhydryls of GSH.
Figure 5: Protein S-guanylation in cells and Keap1 induced by 8-nitro-cGMP.
Figure 6: Modulation of vascular tone by 8-nitro-cGMP.
Figure 7: Generation of 8-nitro-cGMP–stimulated superoxide from P450 reductase and NOS isoforms.

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Acknowledgements

We thank J.B. Gandy for her excellent editing of the manuscript. Thanks are also due to K. Kaneko and Y. Ishima (Kumamoto University), and Y. Unno (University of Shizuoka) for their technical assistance; T. Honda and Z. Liu (Nagoya University) for 2′-O-succinyl-8-nitro-cGMP preparation; H. Taguchi (Tohoku University) for technical assistance with NMR experiments; K. Ichimori (AIST Kansai) and D.J. Stuehr (The Cleveland Clinic) for providing various recombinant NOSs; K.I. Tong (University of Tsukuba) for preparation of recombinant Keap1; and T. Yoshimura, K. Yoshida and A. Molla for reading our paper to evaluate its concepts and interdisciplinary accessibility. This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and from the Ministry of Health, Labor and Welfare of Japan.

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Contributions

T.S. performed chemical and biochemical analyses. M.H.Z., T.O. and T.A. performed immunocytochemistry. Y.T. and S.K.-M. performed organ bath experiments. H.I. performed tandem MS experiments. A.K. and M.Y. produced recombinant Keap1 and its antibody. S.F. performed western blot analyses. H.A. synthesized 8-nitro-cGMP and related compounds. T.A. designed and analyzed experiments and wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Takaaki Akaike.

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Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods (PDF 2200 kb)

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Sawa, T., Zaki, M., Okamoto, T. et al. Protein S-guanylation by the biological signal 8-nitroguanosine 3′,5′-cyclic monophosphate. Nat Chem Biol 3, 727–735 (2007). https://doi.org/10.1038/nchembio.2007.33

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