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

Kohei Kunieda, Hiroyasu Tsutsuki, Tomoaki Ida, Yusuke Kishimoto, Shingo Kasamatsu, Tomohiro Sawa, Naoki Goshima, Makoto Itakura, Masami Takahashi, Takaaki Akaike, Hideshi Ihara

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19 Citations (Scopus)


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 Ca2+-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.

Original languageEnglish
Pages (from-to)1715-1725
Number of pages11
JournalACS Chemical Neuroscience
Issue number10
Publication statusPublished - 2015 Oct 21


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


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