SNAREs pp 163-173 | Cite as

SNAP-25 S-Guanylation and SNARE Complex Formation

  • Yusuke Kishimoto
  • Takaaki Akaike
  • Hideshi IharaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)


8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), which is the second messenger in nitric oxide/reactive oxygen species redox signaling, covalently binds to protein thiol groups (called S-guanylation) and exerts various biological functions. Synaptosomal associated protein 25 (SNAP-25), a member of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, plays an important role in the process of membrane fusion. We previously showed that SNAP-25 is S-guanylated at cysteine 90. In addition, we revealed that S-guanylation of SNAP-25 increases SNARE complex formation, but decreases the affinity of SNARE complex for complexin. Since SNAP-25 plays a critical role in regulating exocytosis, it is important to elucidate the physiological or pathophysiological meanings of S-guanylation of this protein. Here we describe a protocol for detecting 8-nitro-cGMP and S-guanylated proteins in cells by immunocytochemistry, and methods to detect SNARE complex in 8-nitro-cGMP-treated cells.

Key words

8-Nitro-cGMP SNAP-25 SNARE complex Nitric oxide Redox signal 


  1. 1.
    Nishida M, Kumagai Y, Ihara H, Fujii S, Motohashi H, Akaike T (2016) Redox signaling regulated by electrophiles and reactive sulfur species. J Clin Biochem Nutr 58(2):91–98. Scholar
  2. 2.
    Sawa T, Ihara H, Ida T, Fujii S, Nishida M, Akaike T (2013) Formation, signaling functions, and metabolisms of nitrated cyclic nucleotide. Nitric Oxide 34:10–18. Scholar
  3. 3.
    Sawa T, Zaki MH, Okamoto T, Akuta T, Tokutomi Y, Kim-Mitsuyama S, Ihara H, Kobayashi A, Yamamoto M, Fujii S, Arimoto H, Akaike T (2007) Protein S-guanylation by the biological signal 8-nitroguanosine 3′,5′-cyclic monophosphate. Nat Chem Biol 3(11):727–735. Scholar
  4. 4.
    Fujii S, Sawa T, Ihara H, Tong KI, Ida T, Okamoto T, Ahtesham AK, Ishima Y, Motohashi H, Yamamoto M, Akaike T (2010) The critical role of nitric oxide signaling, via protein S-guanylation and nitrated cyclic GMP, in the antioxidant adaptive response. J Biol Chem 285(31):23970–23984. Scholar
  5. 5.
    Ihara H, Kasamatsu S, Kitamura A, Nishimura A, Tsutsuki H, Ida T, Ishizaki K, Toyama T, Yoshida E, Abdul Hamid H, Jung M, Matsunaga T, Fujii S, Sawa T, Nishida M, Kumagai Y, Akaike T (2017) Exposure to electrophiles impairs reactive persulfide-dependent redox signaling in neuronal cells. Chem Res Toxicol 30(9):1673–1684. Scholar
  6. 6.
    Kunieda K, Tsutsuki H, Ida T, Kishimoto Y, Kasamatsu S, Sawa T, Goshima N, Itakura M, Takahashi M, Akaike T, Ihara H (2015) 8-nitro-cGMP enhances SNARE complex formation through S-guanylation of Cys90 in SNAP25. ACS Chem Neurosci 6(10):1715–1725. Scholar
  7. 7.
    Brunger AT (2005) Structure and function of SNARE and SNARE-interacting proteins. Q Rev Biophys 38(1):1–47. Scholar
  8. 8.
    Jahn R, Fasshauer D (2012) Molecular machines governing exocytosis of synaptic vesicles. Nature 490(7419):201–207. Scholar
  9. 9.
    Mohrmann R, Dhara M, Bruns D (2015) Complexins: small but capable. Cell Mol Life Sci 72(22):4221–4235. Scholar
  10. 10.
    Kishimoto Y, Kunieda K, Kitamura A, Kakihana Y, Akaike T, Ihara H (2017) 8-nitro-cGMP attenuates the interaction between SNARE complex and complexin through S-guanylation of SNAP-25. ACS Chem Neurosci 9:217. Scholar
  11. 11.
    Di Stasi AM, Mallozzi C, Macchia G, Maura G, Petrucci TC, Minetti M (2002) Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins. J Neurochem 82(2):420–429. Scholar
  12. 12.
    Meffert MK, Calakos NC, Scheller RH, Schulman H (1996) Nitric oxide modulates synaptic vesicle docking fusion reactions. Neuron 16(6):1229–1236. Scholar
  13. 13.
    Ramos-Miguel A, Beasley CL, Dwork AJ, Mann JJ, Rosoklija G, Barr AM, Honer WG (2015) Increased SNARE protein-protein interactions in orbitofrontal and anterior cingulate cortices in schizophrenia. Biol Psychiatry 78(6):361–373. Scholar
  14. 14.
    Wittig I, Braun HP, Schagger H (2006) Blue native PAGE. Nat Protoc 1(1):418–428. Scholar
  15. 15.

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Yusuke Kishimoto
    • 1
  • Takaaki Akaike
    • 2
  • Hideshi Ihara
    • 1
    Email author
  1. 1.Department of Biological Science, Graduate School of ScienceOsaka Prefecture UniversitySakaiJapan
  2. 2.Department of Environmental Medicine and Molecular ToxicologyTohoku University Graduate School of MedicineSendaiJapan

Personalised recommendations