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:8133717
- 负责人:
- 金额:$ 35.41万
- 依托单位:
- 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 2012-08-31
- 项目状态:已结题
- 来源:
- 关键词:Active SitesAmino AcidsAntigensArchitectureAutomobile DrivingBacillus anthracisBacteriaBacterial ToxinsBindingBiologicalCaliberCellsChemistryComplexCrystallographyCytosolCytotoxinDependencyElectron MicroscopyElectrophysiology (science)EnzymesGoalsHealthHeterophile AntigensHydrophobic InteractionsHydrophobicityImmuneIn VitroIntegral Membrane ProteinKineticsKnowledgeLipid BilayersMeasuresMembraneMethodsModelingMolecularMolecular ChaperonesMolecular MotorsNormal CellPathogenesisPeptidesPhysiologicalPhysiologyPlayProcessProtein translocationProteinsReactionReagentRecordsResearchRoleSiteSpectrum AnalysisStretchingStructureSurfaceTechnologyTestingThermodynamicsToxinX-Ray Crystallographyanthrax lethal factoranthrax toxinbiophysical chemistrycancer cellchemical kineticscytotoxicdesigndriving forceedema factorflexibilityfunctional groupgraspin vivolight scatteringmicrobialmutantnovelpathogenpolypeptideprotein foldingprotein transportstemthree dimensional structuretooltranslocasevoltage
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 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(申请人提供):要发挥作用,蛋白质必须在细胞中正确定位,特别是在被膜双层内部分隔的细胞中。蛋白质类、膜包埋的转运体,称为转位酶通道,可以通过一种称为跨膜蛋白转位的过程来跨膜转运蛋白质。转位酶通道在微生物发病机制中也扮演着关键的功能角色,因为宿主细胞的脂质双层膜起到了强大的第一道防线的作用,将病原体与其胞浆隔离开来。例如,炭疽芽孢杆菌分泌一种由三种蛋白质组成的毒素,称为炭疽毒素,它由保护性抗原(PA)、致死因子(LF)和水肿因子(EF)组成。PA组装成转位酶通道,形成一条穿过宿主细胞内体膜双层的狭窄通道,但该通道如此狭窄,以至于LF和EF以未折叠的多肽链的形式穿过它。一旦进入靶细胞的胞浆,LF和EF就会重新折叠,然后催化反应,扰乱细胞的正常生理。蛋白质去折叠和跨膜转位的研究激发了生物物理问题,广泛应用于可溶分子马达的研究,这些马达可以解开、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?转位通道中的什么结构特征决定了复杂的能量格局,它引导着一条化学复杂的、未折叠的链穿过狭窄的通道?然而,跨膜蛋白转位的生物物理化学特征一直是具有挑战性的,几乎所有转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,能够自发地插入到脂质双层膜中。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)我们将详细分析描述炭疽毒素转位酶通道如何展开其底物蛋白的热力学和动力学机制,探索伴侣样活性部位表面在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轮易位模型。(3)将利用光谱学、电生理学、电子显微镜和结晶学来研究PA通道的结构和组装。相关性:蛋白质转位机制的知识不仅对于开发中和毒素的新方法具有实际意义,而且对于利用毒素作为将异源抗原和细胞毒素运送到免疫细胞和癌细胞的先进技术也具有重要意义。公共卫生相关性:本应用的范围包括对细胞蛋白质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,一种由炭疽杆菌分泌的三蛋白细菌毒素。我们正在寻求对毒素跨膜转运机制的生物物理了解,该机制允许其细胞毒性货物进入哺乳动物宿主细胞。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(1)
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Bryan Andrew Krantz其他文献
Bryan Andrew Krantz的其他文献
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{{ truncateString('Bryan Andrew Krantz', 18)}}的其他基金
Physical Principles of Bacterial Toxin Translocation across Membranes
细菌毒素跨膜转运的物理原理
- 批准号:
9186499 - 财政年份:2008
- 资助金额:
$ 35.41万 - 项目类别:
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 - 财政年份:2008
- 资助金额:
$ 35.41万 - 项目类别:
Physical Principles of Bacterial Toxin Translocation across Membranes
细菌毒素跨膜转运的物理原理
- 批准号:
8603829 - 财政年份:2008
- 资助金额:
$ 35.41万 - 项目类别:
Physical Principles of Bacterial Toxin Translocation across Membranes
细菌毒素跨膜转运的物理原理
- 批准号:
8993597 - 财政年份:2008
- 资助金额:
$ 35.41万 - 项目类别:
Physical Principles of Bacterial Toxin Translocation across Membranes
细菌毒素跨膜转运的物理原理
- 批准号:
7904038 - 财政年份:2008
- 资助金额:
$ 35.41万 - 项目类别:
Physical Principles of Bacterial Toxin Translocation across Membranes
细菌毒素跨膜转运的物理原理
- 批准号:
8505865 - 财政年份:2008
- 资助金额:
$ 35.41万 - 项目类别:
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 - 财政年份:2008
- 资助金额:
$ 35.41万 - 项目类别:
Physical Principles of Bacterial Toxin Translocation across Membranes
细菌毒素跨膜转运的物理原理
- 批准号:
8784181 - 财政年份:2008
- 资助金额:
$ 35.41万 - 项目类别:
Protein Unfolding During Anthrax Toxin Translocation
炭疽毒素易位过程中蛋白质的折叠
- 批准号:
6835445 - 财政年份:2004
- 资助金额:
$ 35.41万 - 项目类别:
Protein Unfolding During Anthrax Toxin Translocation
炭疽毒素易位过程中蛋白质的折叠
- 批准号:
6909009 - 财政年份:2004
- 资助金额:
$ 35.41万 - 项目类别:
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