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

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


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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / metabolism*
  • Brain / ultrastructure
  • Cell Line
  • Cyclic GMP / analogs & derivatives*
  • Cyclic GMP / chemistry*
  • Cyclic GMP / metabolism
  • Cyclic GMP / pharmacology
  • Cysteine / genetics
  • Cysteine / metabolism*
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Mutagenesis, Site-Directed
  • Neuroblastoma / pathology
  • Nitric Oxide Synthase Type I / metabolism
  • Protein Processing, Post-Translational
  • Rats
  • Rats, Wistar
  • Reactive Oxygen Species / metabolism
  • SNARE Proteins / genetics
  • SNARE Proteins / metabolism*
  • Signal Transduction / drug effects
  • Synaptosomal-Associated Protein 25 / genetics
  • Synaptosomal-Associated Protein 25 / metabolism*
  • Synaptosomes / metabolism


  • 8-nitroguanosine 3',5'-cyclic monophosphate
  • Reactive Oxygen Species
  • SNARE Proteins
  • Synaptosomal-Associated Protein 25
  • Nitric Oxide Synthase Type I
  • Cyclic GMP
  • Cysteine