...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate
【24h】

A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate

机译：酵母检测探针探查B型肉毒神经毒素与其SNARE底物之间的相互作用

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The seven functionally distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are dichains consisting of light chain (LC) with zinc-dependent endoprotease activity connected by one disulfide bond to heavy chain with neuronal-cell translocation and receptor-binding domains. LC-mediated proteolysis of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and consequent inhibition of synaptic vesicle fusion to the presynaptic membrane of human motor neurons are responsible for flaccid paralysis associated with botulism. LC endoproteolysis is complex, requiring highly extended SNARE sequences at the surface of intracellular membranes and prompting our development of a genetically amenable assay to monitor the interaction between BoNT/LC and its SNARE substrate. Using BoNT serotype B as a model, the assay employs a chimeric SNARE protein where a portion of neuronal synaptobrevin (Sb) is fused to Snc2p, a Sb ortholog required for protein secretion from yeast cells. Regulated expression of serotype B-LC in yeast leads to cleavage of the chimera and a conditional growth defect. To assess utility of this assay for monitoring SNARE protein cleavage, we growth-selected chimeric SNARE mutations that inhibited proteolysis. When these mutations were introduced into Sb and examined for cleavage, substrate residues located near and distal to the cleavage site were important, including residues positioned near the Sb transmembrane domain, an unexplored aspect of BoNT cell intoxication. Additional mutations were positioned in a nine-residue SNARE motif, supporting a previously assigned role for this motif in LC recognition and providing proof of principle for the application of yeast-based technology to study intracellular BoNT/LC endoproteases.
机译：肉毒杆菌神经毒素（BoNT）的七个功能独特的血清型（A-G）是由具有锌依赖性内切蛋白酶活性的轻链（LC）通过一个二硫键连接到具有神经元细胞易位和受体结合结构域的重链的双链组成。 LC介导的可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白的蛋白水解以及对人运动神经元突触前膜的突触小泡融合的抑制作用是与肉毒中毒相关的松弛性麻痹的原因。 LC内蛋白水解是复杂的，需要在细胞内膜表面高度扩展的SNARE序列，并促使我们开发一种遗传学上适用的检测方法，以监测BoNT / LC及其SNARE底物之间的相互作用。使用BoNT血清型B作为模型，该测定法使用嵌合的SNARE蛋白，其中一部分神经元突触素（Sb）与Snc2p融合，Snc2p是酵母细胞分泌蛋白所需的Sb直向同源物。酵母中血清型B-LC的表达调控导致嵌合体裂解和条件性生长缺陷。为了评估该方法用于监测SNARE蛋白裂解的效用，我们选择了生长抑制蛋白水解的嵌合SNARE突变。当将这些突变引入Sb并检查其裂解情况时，位于裂解位点附近和远端的底物残基很重要，包括位于Sb跨膜结构域附近的残基，这是BoNT细胞中毒的一个未探索的方面。其他突变位于9个残基的SNARE基序中，以支持该基序先前在LC识别中的作用，并为应用基于酵母的技术研究细胞内BoNT / LC内蛋白酶提供了原理证明。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号