Physical Principles of Bacterial Toxin Translocation across Membranes

细菌毒素跨膜转运的物理原理

• 批准号: 7684261
• 负责人: Bryan Andrew Krantz
• 金额: $ 36.51万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7684261
• 关键词: Active Sites; Amino Acids; Anthrax disease; Antigens; Architecture; Automobile Driving; Bacillus (bacterium); Bacillus anthracis; Bacteria; Bacterial Toxins; Binding; Biological; Caliber; Cells; Chemistry; Complex; Crystallography; Cytosol; Cytotoxin; Dependency; Electron Microscopy; Electrophysiology (science); Enzymes; Goals; Heterophile Antigens; Hydrophobic Interactions; Hydrophobicity; Immune; In Vitro; Integral Membrane Protein; Kinetics; Knowledge; Lipid Bilayers; Measures; Membrane; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Motors; Normal Cell; Pathogenesis; Peptides; Physiological; Physiology; Play; Process; Protein translocation; Proteins; Reaction; Reagent; Records; Research; Role; Site; Spectrum Analysis; Stretching; Structure; Surface; Technology; Testing; Thermodynamics; Toxin; X-Ray Crystallography; anthrax lethal factor; anthrax toxin; biophysical chemistry; cancer cell; chemical kinetics; cytotoxic; design; driving force; edema factor; flexibility; functional group; grasp; in vivo; light scattering; microbial; mutant; novel; pathogen; polypeptide; protein folding; protein transport; public health relevance; stem; three dimensional structure; tool; translocase; voltage

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.

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 组装成转位酶通道，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合物、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。 (1)我们将详细分析热力学和动力学机制，描述炭疽毒素的易位酶通道如何展开其底物蛋白，探索PA通道中类伴侣活性位点表面的作用。 (2) 我们将通过对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轮易位模型。 (3) 将利用光谱学、电生理学、电子显微镜和晶体学来研究 PA 通道的结构和组装。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。公共健康相关性：本申请的范围涵盖细胞蛋白质解折叠和运输问题的结构/功能研究。我们将重点关注炭疽毒素，这是一种由炭疽杆菌分泌的三蛋白细菌毒素。我们正在寻求对该毒素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。