Dimeric Peptide Inhibitor Interacting with Neuronal-SNARE Complex

Abstract

The neuronal SNAREs form a protein complex that mediates Ca2⁺-triggered neurotransmitter release into the synaptic cleft. t-SNAREs (Syntaxin-1 and SNAP-25) and v-SNARE (Synaptobrevin-2) interact with each other to form a SNARE complex. The regulation of neurotransmitter release is essential for neuronal functions. For this reason, the SNARE complex is regarded as an attractive target for neurologic drugs. Previously, many small synthetic peptides that mimic the amino acid sequences of the SNARE motif were developed as inhibitors of neurotransmitter release. However, these studies have not focused on the linker region of SNAREs, although the linker region also plays a critical role in SNARE complex assembly at the plasma membrane. Thus, I attempted to target the amino acid sequences of the synaptobrevin-2 linker region and designed its mimicking peptides, Syb-linker and Di-Syb-linker. Here, we identified the activity of these peptides to inhibit SNARE complex formation and SNARE-mediated neurotransmitter release. To compare the inhibitory effect of two peptides, synthetic peptides were tested by using in vitro reconstitution of SDS-resistant SNARE complex, neuronal SNARE-mediated yeast vacuole fusion test, and the binding affinity of peptides was identified by isothermal titration calorimetry. In all cases, Di-Syb-linker exhibited better inhibitory effect on SNARE complex formation than Syb-linker. The results provide the possibility that the peptides mimic the linker region of SNARE proteins, as well as, the SNARE motif can be used as a drug to control neurotransmitter release. These results also suggest the multimeric form of the SNARE-derived peptide is a more effective inhibitor of SNARE complex formation.