Protein Transport by the Bacterial Tat Machinery

细菌 Tat 机器的蛋白质运输

• 批准号: 7736387
• 负责人: SIEGFRIED M MUSSER
• 金额: $ 27.04万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7736387
• 关键词: Address; Animals; Arginine; Bacteria; Biochemical; Biological Assay; Cells; Complex; Cytoplasm; Defect; Development; Drug Delivery Systems; Dyes; Environment; Escherichia coli; Fluorescence; Fluorescence Spectroscopy; Foundations; Future; Grant; Growth; Health; Human; In Vitro; Investigation; Ions; Kinetics; Knowledge; Lead; Lipid Bilayers; Lipids; Membrane; Membrane Lipids; Membrane Proteins; Metabolic; Methods; Modeling; Molecular; Motor; Mycobacterium tuberculosis; Organism; Pathway interactions; Permeability; Physiological; Plants; Process; Property; Protein Precursors; Protein translocation; Proteins; Proteome; Research; Resolution; Role; Shapes; Signal Transduction; Sorting - Cell Movement; Structure; System; Testing; Thylakoids; Time; Tuberculosis; Twin Multiple Birth; Vesicle; Virulence Factors; Work; antimicrobial drug; aqueous; base; cofactor; macromolecule; numb protein; protein complex; protein expression; protein transport; public health relevance; receptor; research study; single molecule; tat Protein; therapeutic protein

DESCRIPTION (provided by applicant): Protein targeting and transport across lipid bilayers is a fundamental energy-requiring process in all organisms. Up to approximately half of the proteins in an organism's proteome are inserted into or transported across membranes by protein translocation systems, or translocons. Many distinct types of translocation systems exist that allow large protein molecules to cross membranes without compromising the membranes' role as a permeability barrier to ions, metabolic intermediates, and other macromolecules. In order to further expand our knowledge of the molecular mechanisms that exist to translocate large molecules across membranes, the proposed research will examine the bacterial twin-arginine translocation (Tat) export system. The Tat system transports fully-folded and assembled proteins. The number of proteins transported by the Tat system is highly species dependent, ranging from none to many (> 100). The absence of a functional Tat system often leads to growth defects in the host bacterium. Further, the Tat machinery is responsible for the export of numerous bacterial virulence factors of human health significance. In a particularly dramatic example, a functional Tat system is required for the growth of Mycobacterium tuberculosis, the causative agent of tuberculosis. Transport by the Tat system minimally requires three proteins, TatA, TatB and TatC. The dominant hypothesis is that a TatBC complex acts as a receptor, which recognizes the presequence of transport substrates, and a TatA oligomer provides a gated pore through which the cargo protein crosses the membrane bilayer. To further understand the basic mechanistic principles governing transport via the Tat system, we will: (1) probe precursor interactions with the lipid and the translocon, by attaching a fluorescence dye to the presequence; (2) construct a kinetic model of transport using a real-time, fluorescence-based transport assay with 1 s time resolution; (3) investigate the role of cargo size and shape on transport rate and transport efficiency; (4) determine the influence of the TatA to TatBC ratio on Tat transport efficiency, transport rate and cargo size restrictions; and (5) develop a single molecule Tat transport assay. These investigations are expected to substantially increase our understanding of how cargos are recognized by the Tat system, and what types of cargos can be translocated. In addition, they will further elucidate the role of the translocon components and the conformational changes required for transport. This characterization of the basic properties of the Tat translocation system will provide an essential foundation for future work, such as the possibility of developing drugs that target the Tat system, or for the utilization of the Tat system in biotechnological applications, such as th expression of protein therapeutics. PUBLIC HEALTH RELEVANCE: The Tat machinery is responsible for the transport of numerous bacterial virulence factors of human health significance, and the absence of a functional Tat system often leads to growth defects in the host bacterium. Consequently, a better understanding of the Tat system is expected to help lead to the development of new antimicrobial drugs. Further, the ability of the Tat system to transport fully-folded protein complexes suggests that it will eventually find utility in the expression of protein therapeutics.

描述（由申请方提供）：蛋白质靶向和跨脂质双层转运是所有生物体中的基本能量需求过程。在生物体的蛋白质组中，多达大约一半的蛋白质通过蛋白质易位系统或translocons插入或转运穿过膜。存在许多不同类型的易位系统，其允许大蛋白质分子穿过膜而不损害膜作为离子、代谢中间体和其它大分子的渗透性屏障的作用。为了进一步扩展我们对跨膜转运大分子的分子机制的认识，拟议的研究将检查细菌双精氨酸转运（达特）输出系统。达特系统转运完全折叠和组装的蛋白质。由达特系统转运的蛋白质数量高度依赖于物种，范围从零到多（> 100）。功能性达特系统的缺乏通常导致宿主细菌的生长缺陷。此外，达特机器负责输出许多对人类健康有重要意义的细菌毒力因子。在一个特别引人注目的例子中，结核病的病原体结核分枝杆菌的生长需要功能性达特系统。通过达特系统的转运最低限度地需要三种蛋白质，TatA、TatB和TatC。主要假设是TatBC复合物充当受体，识别转运底物的前序列，而TatA低聚物提供门控孔，货物蛋白通过该孔穿过膜双层。为了进一步理解控制通过达特系统转运的基本机械原理，我们将：（1）通过将荧光染料连接到前序列上来探测前体与脂质和易位子的相互作用;（2）使用具有1 s时间分辨率的基于荧光的实时转运测定来构建转运的动力学模型;（3）研究货物尺寸和形状对转运速率和转运效率的作用;（4）确定TatA与TatBC的比率对达特转运效率、转运速率和货物尺寸限制的影响;和（5）开发单分子达特转运测定。这些调查预计将大大增加我们对达特系统如何识别货物以及哪些类型的货物可以转运的了解。此外，他们将进一步阐明的作用的translocon组件和运输所需的构象变化。达特易位系统的基本性质的这种表征将为未来的工作提供必要的基础，例如开发靶向达特系统的药物的可能性，或者为达特系统在生物技术应用中的利用，例如蛋白质治疗剂的表达。公共卫生关系：达特机制负责运输许多对人类健康有重要意义的细菌毒力因子，并且功能性达特系统的缺乏通常导致宿主细菌中的生长缺陷。因此，更好地了解达特系统有望有助于开发新的抗菌药物。此外，达特系统转运完全折叠的蛋白质复合物的能力表明，它最终将在蛋白质治疗剂的表达中找到用途。