Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.

Abstract

Clostridial neurotoxins inhibit neurotransmitter release by selective and specific intracellular proteolysis of synaptobrevin/VAMP, synaptosomal‐associated protein of 25 kDa (SNAP‐25) or syntaxin. Here we show that in binary reactions synaptobrevin binds weakly to both SNAP‐25 and syntaxin, and SNAP‐25 binds to syntaxin. In the presence of all three components, a dramatic increase in the interaction strengths occurs and a stable sodium dodecyl sulfate‐resistant complex forms. Mapping of the interacting sequences reveals that complex formation correlates with the presence of predicted alpha‐helical structures, suggesting that membrane fusion involves intermolecular interactions via coiled‐coil structures. Most toxins only attack the free, and not the complexed, proteins, and proteolysis of the proteins by different clostridial neurotoxins has distinct inhibitory effects on the formation of synaptobrevin‐syntaxin‐SNAP‐25 complexes. Our data suggest that synaptobrevin, syntaxin and SNAP‐25 associate into a unique stable complex that functions in synaptic vesicle exocytosis.

Adrenal chromaffin cells contain functionally different SNAP‐25 monomers and SNAP‐25/syntaxin heterodimers

“…In previous investigations resistance to SDS as a signature of high stability has only been observed in complexes of higher order, that is in SNAP-25/syntaxin/synaptobrevin heterotrimers [9,40]. Heterotrimeric complexes have been found in solubilized adrenal chromaffin and pheochromocytoma cells [13,36,41], in detergent extracts of brain and upon incubation of the recombinant proteins in media containing nonionic detergents [3,5,9,39,40].…”

“…Heterotrimeric complexes have been found in solubilized adrenal chromaffin and pheochromocytoma cells [13,36,41], in detergent extracts of brain and upon incubation of the recombinant proteins in media containing nonionic detergents [3,5,9,39,40]. These complexes, formed in the presence of detergents in the aforementioned studies, are assumed to represent intermediates generated during docking of synaptic vesicles and chromaffin vesicles to the plasmalemmal target membrane, a process predicted by the SNARE hypothesis [2,3,22].…”

Botulinum neurotoxin light chains inhibit both Ca2+‐induced and GTP analogue‐induced catecholamine release from permeabilised adrenal chromaffin cells

“…Recombinant botulinum neurotoxins were expressed as His6-tagged fusion proteins [38] from plasmids which were a generous gift from Dr Heiner Neimann (Tubingen, Germany). Proteins were purified from Escherichia coli M15 [pREP4] (Qiagen) after induction of expression with 0.5 mM isopropyl-D-thiogalactopyranoside for 2.5-5 h, cell lysis by ultrasonication and affinity chromatography on Ni-NTA-agarose.…”

Innocuous full-length botulinum neurotoxin targets and promotes the expression of lentiviral vectors in central and autonomic neurons

“…42 Interestingly, a relatively higher frequency of spontaneous synaptic currents in MTR-S25-expressing neurons exposed to BoNT/E-treated as compared with BoNT/A-treated neurons suggests that MTR-S25 counteracts against BoNT/E inhibition more effectively. The latter, perhaps, reflects the fact that BoNT/Etruncated S25 fails to assemble with synaptobrevin/VAMP and syntaxin into stable SDS-resistant complexes [43][44][45] unlike BoNT/A-cleaved S25, which retains limited functionality [45][46][47] impeding the replacement of truncated S25 by its toxin-resistant recombinant variant. The cleavage of endogenous SNAREs by perduring active proteases could explain the partial protection of synaptic functions by MTR-S25 from BoNT/A and BoNT/E, whereas the lack of arresting protection of synaptic activity observed with naive and MTR-S25-expressing neuronal cultures exposed to BoNT/C1 accords with its cleavage of additional SNARE protein syntaxins.…”