Physical Principles of Bacterial Toxin Translocation across Membranes

细菌毒素跨膜转运的物理原理

基本信息

批准号：
8784181
负责人：
Bryan Andrew Krantz
金额：
$ 38.38万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-15 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8784181
关键词：
Active Sites Affinity Anthrax disease Antigens Bacillus anthracis Bacteria Bacterial Toxins Binding Binding Sites Biological Carrier Proteins Cell physiology Cells Complex Cytosol Cytotoxin Diffusion Electrophysiology (science)Elements Epitopes Exhibits Goals Health Heterophile Antigens Immune In Vitro Integral Membrane Protein Kinetics Knowledge Learning Lipid Bilayers Measures Mechanics Membrane Methods Molecular Molecular Conformation Molecular Motors Motion Normal Cell Pathogenesis Peptides Physiology Play Polymers Process Protein translocation Proteins Protons Reaction Role Shapes Site Specific qualifier value Structure Substrate Specificity Surface System Technology Testing Thermodynamics Toxin Virulence Factors Work anthrax lethal factor anthrax toxin base biophysical analysis biophysical chemistry cancer cell cytotoxic design edema factor flexibility grasp in vivo interest microbial novel pathogen polypeptide protein folding protein transport retinal rods three dimensional structure tool translocase unfoldase

项目摘要

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.

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，许多转移病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。最终，我们对这些系统如何充当质子梯度驱动的棘轮，如何稳定展开的中间体，这些夹具位点栅极和Ungate如何稳定展开的中间体。我们的总体目标是定义力转导和基于棘轮的展开和易位的分子机制。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。