title: 8-Nitro-cGMP Enhances SNARE Complex Formation through S-Guanylation of Cys90 in SNAP25.
abstract: 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.
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University , Osaka 599-8531, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Kunieda
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University , Kumamoto 860-8556, Japan.
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Tsutsuki
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Graduate School of Medicine, Tohoku University , Miyagi 980-8575, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Ida
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University , Osaka 599-8531, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kishimoto
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Graduate School of Medicine, Tohoku University , Miyagi 980-8575, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Kasamatsu
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University , Kumamoto 860-8556, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Sawa
authors.affiliation: Quantitative Proteomics Team, Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology , Tokyo 135-0064, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Goshima
authors.affiliation: Department of Biochemistry, Kitasato University School of Medicine , Kanagawa 252-0374, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Itakura
authors.affiliation: Department of Biochemistry, Kitasato University School of Medicine , Kanagawa 252-0374, Japan.
authors.forename: Masami
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Graduate School of Medicine, Tohoku University , Miyagi 980-8575, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Akaike
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University , Osaka 599-8531, Japan.
authors.forename: Hideshi
authors.initials: H
authors.lastname: Ihara
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D002460:Cell Line; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D009447:Neuroblastoma / Q000473:pathology; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism
chemical_list: C525106:8-nitroguanosine 3',5'-cyclic monophosphate; D017382:Reactive Oxygen Species; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D052248:Nitric Oxide Synthase Type I; D006152:Cyclic GMP; D003545:Cysteine
keywords: 8-Nitroguanosine 3′,5′-cyclic monophosphate; SNAP25; SNARE complex; neuronal nitric oxide synthase; protein S-guanylation; redox signaling