Mechanism of botulinum neurotoxin transport across membranes

项目摘要 肉毒神经毒素（BoNT）是人类已知的毒性最强的物质之一， 威胁性神经麻痹性疾病肉毒杆菌中毒可能对公共卫生造成重大影响， 故意释放，加上缺乏批准的疫苗或疗法，导致了分类， BONT作为第1层A类精选代理。巧合的是，BONTs的高度特异性作用使它们 用于日益增长的和异质性的人类疾病的极好的治疗剂，所述疾病的特征在于 周围神经末梢活动过度。尽管最近在了解结构方面取得了许多进展- BoNTs的功能关系，神经毒素重链（HC）能够通过其 转运轻链（LC）穿过内吞囊泡的膜仍然不清楚。 理解pH驱动的神经毒素解折叠和移位的机制不仅具有内在价值， 而且还解决了膜蛋白组装和稳定性所涉及的一般生物物理问题。研究中心- 神经毒素的选择性荧光标记结合一系列生物化学、光谱和 将采用分子方法来检验中心假设，即BoNT的膜插入 是一个受调控的过程，包含在蛋白质易位形成之前的关键中间状态 频道目的1将确定受体结合（HCR）结构域在形成 膜插入通道。具体地说，目的1将检验HCR结构域作为一个功能域发挥作用的假设。 传感器的环境pH值和膜组成，确保通道形成发生在正确的 地点和时间。目的2将阐述BoNT/A通道的形成是通过一系列 界面中间态。完成拟议的研究将为以下方面提供机会： 用于治疗神经元损伤的暴露后治疗剂和改进的药理学试剂的开发 紊乱

PROJECT SUMMARY Botulinum neurotoxins (BoNTs) are among the most toxic agents known to humans and cause the life threatening, neuroparalytic disorder botulism. The potential for major public health impact resulting for an intentional release, combined with the paucity of approved vaccines or therapies has led to the classification of BoNTs as Tier 1, Category A Select Agents. Paradoxically, the highly specific action of BoNTs make them excellent therapeutics for a growing and heterogeneous number of human diseases that are characterized by a hyperactivity of peripheral nerve terminals. Despite many recent advances in understanding the structure- function relationship of BoNTs, the molecular events by which the neurotoxin heavy chain (HC) is able to translocate the light chain (LC) across the membrane of endocytic vesicles remains poorly defined. Understanding the mechanism of pH-driven neurotoxin unfolding and translocation is not only of intrinsic value, but also addresses general biophysical questions underlying membrane protein assembly and stability. Site- selective fluorescence labeling of neurotoxins in conjunction with an array of biochemical, spectroscopic and molecular approaches will be employed to test the central hypothesis stating that membrane insertion of BoNT is a regulated process containing key intermediate states which precede formation of the protein translocating channel. Aim 1 will determine the contribution of the receptor binding (HCR) domain in formation of the membrane inserted channel. Specifically, aim 1 will test the hypothesis that the HCR domain functions as a sensor of environmental pH and membrane composition which ensures channel formation occurs at the correct site and time. Aim 2 will address the hypothesis that formation of the BoNT/A channel occurs through a series of interfacial intermediate states. Completion of the proposed studies will provide opportunities for the development of post-exposure therapeutics and improved pharmacologic agents for the treatment of neuronal disorders.