Physical Principles of Bacterial Toxin Translocation across Membranes

细菌毒素跨膜转运的物理原理

• 批准号: 7533723
• 负责人: Bryan Andrew Krantz
• 金额: $ 36.59万
• 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/7533723
• 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; Localized; Measures; Membrane; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Motors; Normal Cell; Pathogenesis; Peptides; Personal Satisfaction; Physiological; Physiology; Play; Process; Protein translocation; Proteins; Public Health; Rate; 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; functional group; grasp; in vivo; intracellular protein transport; light scattering; microbial; mutant; novel; pathogen; polypeptide; protein folding; protein transport; size; 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）：为了功能，必须正确地将蛋白质定位在细胞中，尤其是在膜双层内部隔离的蛋白质中。蛋白质，膜包裹的转运蛋白，称为转运酶通道，可以通过称为跨膜蛋白易位的过程在膜上交通蛋白质。易位酶通道还在微生物发病机理中起关键功能作用，因为宿主细胞的脂质双层膜充当强大的第一道防线，从其细胞质中分离出来。例如，细菌，炭疽芽孢杆菌分泌一种三蛋白毒素，称为炭疽毒素，该毒素由保护性抗原（PA），致死因子（LF）和水肿因子（EF）组成。 PA聚集到易位酶通道中，在宿主细胞的内体膜双层中形成一条狭窄的通道，但是该通道是如此狭窄，以至于LF和EF在展开的多肽链中横穿它。一旦进入靶细胞的细胞溶胶，LF和EF重新叠加，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探针令人兴奋的生物物理问题的研究，这些问题广泛适用于可溶性分子电机的研究，这些分子电动机的研究，蛋白质展开，拆卸和降解蛋白质。细胞中如何展开稳定的底物蛋白？易位酶通道中的哪些结构特征决定了通过通道的狭窄范围指导化学复杂的，展开的链的复杂能量景观？然而，跨膜蛋白易位的生物物理化学对于表征的表征很具有挑战性，几乎所有易位酶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，因为它们携带了自己的易位酶通道机械，能够自发地将其插入脂质双层膜中。我们将融合用于研究蛋白质如何与平面脂质双层电生理学折叠和展开的光谱工具。 （1）我们将详细分析热力学和动力学机制，这些机制描述了炭疽毒素的易位酶通道如何展开其底物蛋白，从而探索了PA通道中类似伴侣的活性位点表面的作用。 （2）我们将通过人工设计的多肽底物的集合和单渠道电生理研究来剖析布朗棘轮易位模型。 （3）将使用光谱，电生理学，电子显微镜和晶体学来追求PA通道的结构和组装。相关性：蛋白质易位机制的知识不仅对于开发新方法来中和毒素，而且对推进技术的发展，这些方法将毒素作为异源抗原和细胞毒素的递送工具剥夺到免疫细胞和癌细胞。公共卫生相关性：该应用程序的范围涵盖了细胞蛋白展开和运输问题的结构/功能研究。我们将专注于炭疽毒素，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跨膜易位机制的生物物理理解，这使其细胞毒性货物进入哺乳动物宿主细胞。