Mechanism of botulinum neurotoxin transport across membranes

项目总结 肉毒杆菌神经毒素(BoNTs)是人类已知的毒性最大的毒物之一，它会导致 威胁性神经麻痹障碍肉毒杆菌中毒。对公众健康造成重大影响的可能性 故意释放，加上缺乏获得批准的疫苗或治疗方法，导致了对 BoNTs作为第1级，A类精选代理。矛盾的是，BoNTs的高度特异性使它们 对越来越多的、不同种类的人类疾病的极佳治疗方法，这些疾病的特点是 周围神经末梢多动。尽管最近在理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)能够通过的分子事件 轻链(LC)在内吞泡膜上的移位仍然不明确。 了解pH驱动的神经毒素解离和移位的机制不仅具有内在的价值， 而且还解决了膜蛋白组装和稳定性背后的一般生物物理问题。站点- 神经毒素的选择性荧光标记与一系列生化、光谱和 分子方法将被用来检验中心假说，即BONT的膜插入 是一个受调控的过程，其中包含蛋白质转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。具体地说，Aim 1将测试HCR结构域作为一个 环境pH和膜成分传感器，确保在正确的时间形成通道 地点和时间。目标2将解决以下假设，即BONT/A通道的形成是通过一系列 界面中间态。建议的研究完成后，将为 治疗神经元损伤的暴露后治疗药物和改良药理药物的研究进展 精神错乱。

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.