Physical Principles of Bacterial Toxin Translocation across Membranes

描述（由申请人提供）：蛋白质必须正确定位于细胞中，特别是在由膜双层内部分隔的细胞中，才能发挥功能。蛋白质，膜包埋转运，称为转位酶通道，可以运输蛋白质跨膜的过程称为跨膜蛋白转位。易位酶通道在微生物发病机制中也起着关键的功能作用，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，将病原体与其胞质溶胶隔离开来。例如，炭疽杆菌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组装成移位酶通道，形成穿过宿主细胞的内体膜双层的狭窄通道，但通道如此狭窄，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重新折叠，然后催化破坏细胞正常生理的反应。蛋白质解折叠和跨膜转运的研究是一个激动人心的生物物理学问题，它广泛应用于研究蛋白质解折叠、分解和降解的可溶性分子马达。一个稳定的底物蛋白在细胞中是如何展开的？移位酶通道中的什么结构特征决定了复杂的能量景观，引导化学复杂的未折叠链通过通道的狭窄范围？然而，跨膜蛋白易位的生物物理化学的表征一直具有挑战性，并且几乎所有易位酶通道的三维结构都是未知的。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易位酶通道机制，能够自发插入脂质双层膜。我们将耦合用于研究蛋白质如何折叠和展开与平面脂质双层电生理学的光谱工具。(1)我们将详细分析的热力学和动力学机制，描述了炭疽毒素的转位酶通道如何展开其底物蛋白，探索分子伴侣样，活性位点表面的PA通道中的作用。(2)我们将通过人工设计的多肽底物的整体和单通道电生理学研究来剖析布朗棘齿易位模型。(3)PA通道的结构和组装将采用光谱学，电生理学，电子显微镜和晶体学。相关性：蛋白质易位机制的知识不仅对开发中和毒素的新方法而且对推进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的技术具有实际重要性。公共卫生相关性：本申请的范围涵盖细胞蛋白质解折叠和转运问题的结构/功能研究。我们将重点介绍炭疽毒素，这是一种由炭疽杆菌分泌的三蛋白细菌毒素。我们正在寻求获得一个生物物理的理解毒素的跨膜易位机制，这使得其细胞毒性货物进入哺乳动物宿主细胞。

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 nearly all translocase 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. (1) We will analyze in detail the thermodynamic and kinetic mechanisms, which describe how the translocase channel of anthrax toxin unfolds its substrate proteins, exploring the role of chaperone-like, active-site surfaces in the PA channel. (2) We will dissect Brownian-ratchet translocation models through ensemble and single-channel electrophysiology studies of artificial, designed polypeptide substrates. (3) The structure and assembly of the PA channel will be pursued using spectroscopy, electrophysiology, electron microscopy, and crystallography. 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. PUBLIC HEALTH RELEVANCE: The scope of this application covers a structure/function study of the problem of cellular protein unfolding and transport. We will focus on anthrax toxin, a three-protein, bacterial toxin secreted by Bacillus anthracis. We are seeking to obtain a biophysical understanding of the toxin's transmembrane translocation mechanism, which allows its cytotoxic cargo to enter into mammalian host cells.