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ACS Chem Neurosci. 2015 Oct 21;6(10):1715-25. doi: 10.1021/acschemneuro.5b00196. Epub 2015 Aug 11.

8-Nitro-cGMP Enhances SNARE Complex Formation through S-Guanylation of Cys90 in SNAP25.

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Department of Biological Science, Graduate School of Science, Osaka Prefecture University , Osaka 599-8531, Japan.
Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University , Kumamoto 860-8556, Japan.
Department of Environmental Health Sciences and Molecular Toxicology, Graduate School of Medicine, Tohoku University , Miyagi 980-8575, Japan.
Quantitative Proteomics Team, Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology , Tokyo 135-0064, Japan.
Department of Biochemistry, Kitasato University School of Medicine , Kanagawa 252-0374, Japan.


Nitrated guanine nucleotide 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) generated by reactive oxygen/nitrogen species causes protein S-guanylation. However, the mechanism of 8-nitro-cGMP formation and its protein targets in the normal brain have not been identified. Here, we investigated 8-nitro-cGMP generation and protein S-guanylation in the rodent brain. Immunohistochemistry indicated that 8-nitro-cGMP was produced by neurons, such as pyramidal cells and interneurons. Using liquid chromatography-tandem mass spectrometry, we determined endogenous 8-nitro-cGMP levels in the brain as 2.92 ± 0.10 pmol/mg protein. Based on S-guanylation proteomics, we identified several S-guanylated neuronal proteins, including SNAP25 which is a core member of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex. SNAP25 post-translational modification including palmitoylation, phosphorylation, and oxidation, are known to regulate neurotransmission. Our results demonstrate that S-guanylation of SNAP25 enhanced the stability of the SNARE complex, which was further promoted by Ca(2+)-dependent activation of neuronal nitric oxide synthase. Using site-directed mutagenesis, we identified SNAP25 cysteine 90 as the main target of S-guanylation which enhanced the stability of the SNARE complex. The present study revealed a novel target of redox signaling via protein S-guanylation in the nervous system and provided the first substantial evidence of 8-nitro-cGMP function in the nervous system.


8-Nitroguanosine 3′,5′-cyclic monophosphate; SNAP25; SNARE complex; neuronal nitric oxide synthase; protein S-guanylation; redox signaling

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