基于酿酒酵母细胞构建靶向囊泡相关膜蛋白2的肉毒神经毒素检测系统

Construction of a yeast cell-based assay system for synaptobrevin-2- targeting botulinum neurotoxins

摘要:

肉毒神经毒素(botulinum neurotoxins, BoNTs)是一种神经毒性蛋白质，可水解参与突触囊泡融合的可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体(soluble N-ethylmaleimide factor attachment protein receptor, SNARE)。其中，囊泡相关膜蛋白(synaptobrevin-2, Sb2)是BoNTs主要靶向的SNARE蛋白之一。本研究旨在开发一种基于酵母的BoNTs检测系统，用于分析靶向Sb-2的BoNTs。鉴于SNARE蛋白在真核生物中的高度保守性，Snc1和Snc2是酿酒酵母中Sb-2的同源物，本研究构建了功能性的SNARE嵌合蛋白，其中Snc2中的SNARE结构域的一部分被Sb-2的相应区域所取代，从而Snc2/Sb2-4嵌合蛋白可以被BoNTs所识别。由于Snc1和Snc2是酵母产孢所必需的，因此在携带Snc2/Sb2-4嵌合蛋白的snc1∆snc2∆酵母中，靶向Sb-2蛋白的BoNTs (包括BoNT/B在内)表达后显著降低了孢子的形成效率。然而，在野生型酵母中，BoNTs表达的影响可以忽略不计。产孢效率在本研究中被用作检验BoNTs活性的指标。这种方法的优点在于可以通过荧光比色法来评估酵母的产孢效率，进而反映出BoNTs的活性。最终，本研究基于酵母细胞构建的体系不仅能够简单便捷地分析BoNTs，还能够用于挖掘未鉴定的BoNTs和BoNTs抑制剂。鉴于BoNTs广泛应用于临床和美容领域，该体系有望为发现和优化具有应用价值的BoNTs提供有力支持。

Abstract:

Botulinum neurotoxins (BoNTs) are neurotoxic proteins that can hydrolyze soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) involved in the synaptic vesicle fusion. Synaptobrevin-2 (Sb-2) is a SNARE targeted by BoNTs. This study aims to develop a yeast cell-based assay system for Sb-2-targeting BoNTs. SNAREs are conserved in eukaryotes and Snc1 and Snc2 are homologues of Sb-2 in Saccharomyces cerevisiae. We constructed a functional chimeric SNARE in which a part of Snc2 was replaced with that of Sb-2. The Snc2/Sb2-4 chimera, instead of Snc1 and Snc2, was recognized and cleaved by BoNTs. Since Snc1 and Snc2 are required for sporulation of yeast cells, in snc1∆snc2∆cells harboring the Snc2/Sb2-4 chimera, the sporulation efficiency was significantly decreased by expression of Sb-2-targeting BoNTs including BoNT/B. However, in wild-type yeast cells, the effects of BoNTs expression were negligible. Thus, sporulation efficiency was used as an indicator of the activity of BoNTs in this study. One advantage of this method is that BoNTs activities can be assessed by colorimetric measurement of sporulation efficiency. Our yeast cell-based BoNTs assay provides easy and rapid analysis of BoNTs, which is useful to characterize and engineer the neurotoxins. Furthermore, the assay will be useful for mining of uncharacterized BoNTs and BoNTs inhibitors. BoNTs are widely used for clinical and cosmetic purposes; thus our assay would be useful to find and modify useful BoNTs.