Mechanism of botulinum neurotoxin transport across membranes

项目概要 肉毒杆菌神经毒素 (BoNT) 是人类已知的毒性最强的物质之一，可导致生命 威胁性的神经麻痹性疾病肉毒杆菌中毒。潜在的重大公共卫生影响 故意释放，加上批准的疫苗或疗法的缺乏，导致了分类 BoNT 作为第 1 层、A 类精选药物。矛盾的是，BoNT 的高度特异性作用使它们 针对数量不断增加且异质性的人类疾病提供卓越的治疗方法，这些疾病的特点是 周围神经末梢过度活跃。尽管最近在理解结构方面取得了许多进展- BoNT 的功能关系，神经毒素重链 (HC) 能够通过的分子事件 轻链（LC）穿过内吞囊泡膜的易位仍然不明确。 了解 pH 驱动的神经毒素展开和易位的机制不仅具有内在价值， 还解决了膜蛋白组装和稳定性的一般生物物理问题。网站- 神经毒素的选择性荧光标记与一系列生化、光谱和 将采用分子方法来检验中心假设，即 BoNT 的膜插入 是一个受调节的过程，包含蛋白质易位形成之前的关键中间状态 频道。目标 1 将确定受体结合 (HCR) 结构域在形成 膜插入通道。具体来说，目标 1 将检验 HCR 域作为 环境 pH 值和膜成分传感器，确保通道形成在正确的位置发生 地点和时间。目标 2 将提出这样的假设：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 开发暴露后疗法和改进的药物以治疗神经元 失调。

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.