Physical Principles of Bacterial Toxin Translocation across Membranes

描述（由申请人提供）：为了发挥作用，蛋白质必须在细胞中正确定位，特别是在那些被膜双层内部分隔的细胞中。蛋白质，膜嵌入的转运体，称为转运酶通道，可以通过一个被称为跨膜蛋白质易位的过程将蛋白质运送到膜上。跨位点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是一道强大的第一道防线，将病原体与其细胞质分离开来。例如，炭疽芽孢杆菌分泌一种由保护性抗原（PA）、致死因子（LF）和水肿因子（EF）组成的三蛋白毒素，称为炭疽毒素。PA组装成一个转位酶通道，在宿主细胞的内体膜双分子层上形成一个狭窄的通道，但通道是如此狭窄，以至于LF和EF作为未展开的多肽链穿过它。一旦进入目标细胞的细胞质，LF和EF就会重新折叠，然后催化破坏细胞正常生理机能的反应。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，这些马达可以展开、分解和降解蛋白质。一个稳定的底物蛋白是如何在细胞中展开的？跨位通道的哪些结构特征决定了复杂的能量格局，从而引导化学上复杂的、未展开的链通过狭窄的通道？然而，跨膜蛋白易位的生物物理化学特征一直具有挑战性，并且几乎所有易位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转运酶通道机制，能够自发地插入脂质双分子层膜。我们将把用于研究蛋白质折叠和展开的光谱工具与平面脂质双分子层电生理学结合起来。(1)我们将详细分析炭疽毒素转位酶通道展开底物蛋白的热力学和动力学机制，探索类伴侣活性位点表面在PA通道中的作用。(2)我们将通过人工设计多肽底物的整体和单通道电生理学研究来剖析布朗棘轮易位模型。(3) PA通道的结构和组装将使用光谱学、电生理学、电子显微镜和晶体学进行研究。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具有实际意义，而且对开发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也具有重要意义。公共卫生相关性：本应用的范围涵盖细胞蛋白展开和运输问题的结构/功能研究。我们将重点介绍炭疽毒素，一种由炭疽芽孢杆菌分泌的三蛋白细菌毒素。我们正在寻求对毒素的跨膜易位机制的生物物理学理解，该机制允许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.