SNAP-25 Contains Non-Acylated Thiol Pairs that can Form Intrachain Disulfide Bonds: Possible Sites for Redox Modulation of Neurotransmission

Abstract

Intrachain disulfide bond formation among the cysteine thiols of SNAP-25, a component of the SNARE protein complex required for neurotransmitter release, has been hypothesized to link oxidative stress and inhibition of synaptic transmission. However, neither the availability in vivo of SNAP-25 thiols, which are known targets of S-palmitoylation, nor the tendency of these thiols to form intrachain disulfide bonds is known. We have examined, in rat brain extracts, both the availability of closely spaced, or vicinal, thiol pairs in SNAP-25 and the propensity of these dithiols toward disulfide bond formation using a method improved by us recently that exploits the high chemoselectivity of phenylarsine oxide (PAO) for vicinal thiols. The results show for the first time that a substantial fraction of soluble and, to a lesser extent, particulate SNAP-25 contain non-acylated PAO-binding thiol pairs and that these thiols in soluble SNAP-25 in particular have a high propensity toward disulfide bond formation. Indeed, disulfide bonds were detected in a small fraction of soluble SNAP-25 even under conditions designed to prevent or greatly limit protein thiol oxidation during experimental procedures. These results provide direct experimental support for the availability, in a subpopulation of SNAP-25, of vicinal thiols that may confer on one or more isoforms of this family of proteins a sensitivity to oxidative stress.

Intraviral interactome of Chikungunya virus reveals the homo-oligomerization and palmitoylation of structural protein TF

“…However, the function of oligomeric state of CHIKV TF remains to be studied. Interestingly, palmitoylation of CHIKV TF reduced the homo-oligomerization of TF, which is consistent with previous reports that less palmitoylation of SNAP-25 and sortilin increased disulfide bonds formation [23,24]. CHIKV TF formed homo-oligomer with an intermolecular disulfide bond at the cysteine 43, which could be palmitoylated.…”

Arsenic Binding to Proteins

“…66,134,196,278,279 They can further be used to ascertain the critical thiols for binding in proteins after site-directed mutagenesis. 134,161,280 …”

Assembly and Comparison of Plasma Membrane SNARE Acceptor Complexes

“…Alternatively, we expressed wild-type SNAP-25 and used alkylation with dodecyl chains to the four native cysteines as described in the Supporting Material. This form of SNAP-25 (d-SNAP-25) closely resembles palmitoylated SNAP-25, which is the predominant form in neurons or SNAP-25-expressing insect cells (16,17). SNARE acceptor complexes were formed from these products by assembly in DPC or in the plane of supported bilayers, respectively, and their activities were compared with SNARE acceptor complexes prepared, with or without the syb49-96 peptide, in more traditional fashion.…”