Physical Principles of Bacterial Toxin Translocation across Membranes

描述（由申请人提供）：为了发挥作用，蛋白质必须正确定位在细胞中，特别是在由膜双层内部分隔的细胞中。蛋白质的膜嵌入转运蛋白（称为转位酶通道）可以通过称为跨膜蛋白易位的过程跨膜运输蛋白质。转位酶通道在微生物发病机制中也发挥着关键的功能作用，因为宿主细胞的脂质双层膜起着强大的第一道防线的作用，将病原体与其细胞质隔离。例如，炭疽杆菌会分泌一种三蛋白毒素，称为炭疽毒素，由保护性抗原（PA）、致死因子（LF）和水肿因子（EF）组成。 PA 组装成易位酶通道，形成穿过宿主细胞内体膜双层的狭窄通道，但该通道非常狭窄，以至于 LF 和 EF 作为未折叠的多肽链穿过它。一旦进入靶细胞的胞质溶胶，LF 和 EF 就会重新折叠，然后催化破坏细胞正常生理的反应。 蛋白质展开和跨膜易位的研究探索了令人兴奋的生物物理问题，这些问题广泛应用于可溶性分子马达的研究，这些分子马达展开、分解和降解蛋白质。稳定的底物蛋白如何在细胞中展开？易位酶通道中的哪些结构特征决定了引导化学复杂的展开链穿过通道狭窄范围的复杂能量景观？然而，跨膜蛋白易位的生物物理化学特征一直难以表征，并且许多易位通道的三维结构尚不清楚。细菌毒素，如炭疽毒素，特别适合这些研究，因为它们携带自己的转位酶通道机制，能够自发插入脂质双层膜。我们将把用于研究蛋白质如何折叠和展开的光谱工具与平面脂质双层电生理学结合起来。我们最终感兴趣的是这些系统如何作为质子梯度驱动的棘轮发挥作用，解折叠酶活性位点或多肽夹如何稳定展开中间体，这些夹位点如何门控和非门控。我们的总体目标是定义力传导和基于棘轮的展开和易位的分子机制。相关性：了解蛋白质易位机制不仅对于开发中和毒素的新方法具有实际意义，而且对于先进技术也具有重要意义，这些技术利用毒素作为异源抗原和细胞毒素进入免疫细胞和癌细胞的递送载体。

DESCRIPTION (provided by applicant): To function, a protein must be correctly localized in the cell, especially in ones that are internally compartmentalized by membrane bilayers. Proteinaceous, membrane-embedded transporters, called translocase channels, can traffic proteins across membranes by a process known as transmembrane protein translocation. Translocase channels also play key functional roles in microbial pathogenesis, because a host cell's lipid bilayer membrane functions as a formidable, first line of defense, isolating the pathogen from its cytosol. The bacterium, Bacillus anthracis, for example, secretes a three-protein toxin, called anthrax toxin, which is composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA assembles into a translocase channel, forming a narrow passageway across the host cell's endosomal membrane bilayer, but the channel is so narrow that LF and EF traverse it as unfolded polypeptide chains. Once inside the target cell's cytosol, LF and EF refold and then catalyze reactions that disrupt the cell's normal physiology. Studies of protein unfolding and transmembrane translocation probe exciting biophysical questions, which apply broadly to the studies of soluble molecular motors, which unfold, disassemble, and degrade proteins. How is a stable substrate protein unfolded in the cell? What structural features in the translocase channel determine the complex energy landscape that guides a chemically- complex, unfolded chain through the narrow confines of the channel? The biophysical chemistry of transmembrane protein translocation, however, has been challenging to characterize, and the three-dimensional structures of many translo- case channels are unknown. Bacterial toxins, like anthrax toxin, are particularly well-suited for these studies, because they carry their own translocase-channel machinery, which is able to spontaneously insert into lipid bilayer membranes. We will couple the spectroscopic tools used to study how proteins fold and unfold with planar lipid bilayer electrophysiology. We are ultimately interested in how these systems function as proton-gradient driven ratchets, how the unfoldase active sites or polypeptide clamps stabilize unfolding intermediates, how these clamp sites gate and ungate. Our overall goal is to define the molecular mechanism of force transduction and ratchet-based unfolding and translocation. Relevance: Knowledge of protein translocation mechanisms are of practical importance not only to developing novel methods to neutralize the toxin but also to advancing technologies, which exploit toxins as delivery vehicles for heterologous antigens and cytotoxins into immune and cancer cells.