Protein Transport by the Bacterial Tat Machinery

描述(由申请人提供)：蛋白质靶向和跨脂双层运输是所有生物体中一个基本的需要能量的过程。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转运系统或转运子插入或跨膜运输。存在许多不同类型的易位系统，它们允许大的蛋白质分子跨膜，而不影响膜作为离子、代谢中间体和其他大分子的渗透性屏障的作用。为了进一步扩大我们对大分子跨膜转运分子机制的了解，拟议的研究将检查细菌双精氨酸易位(TAT)输出系统。TAT系统运输完全折叠和组装的蛋白质。TAT系统运输的蛋白质数量高度依赖于物种，从无到多不等(&gt；100)。缺乏功能齐全的TAT系统往往会导致宿主细菌的生长缺陷。此外，TAT机制负责出口大量对人类健康具有重要意义的细菌毒力因子。在一个特别戏剧性的例子中，结核病的病原体--结核分枝杆菌的生长需要一个有效的TAT系统。TAT系统的运输最低限度需要三种蛋白质：TATA、TatB和TatC。主要的假设是，TatBC复合体作为受体，识别运输底物的预序列，而TATA寡聚体提供一个门控孔，货物蛋白通过这个孔穿过膜双层。为了进一步了解支配TAT系统运输的基本机制原理，我们将：(1)通过在前序列上附着荧光染料来探测前体与脂质和转位基因的相互作用；(2)使用基于荧光的实时、1 S时间分辨率的运输分析来构建运输动力学模型；(3)研究货物尺寸和形状对运输速率和运输效率的影响；(4)确定TATA和TATBC的比例对TAT运输效率、运输速率和货物尺寸限制的影响；以及(5)建立单分子TAT运输试验。这些调查预计将大大增加我们对TAT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，他们还将进一步阐明转运子成分的作用和运输所需的构象变化。这种对TAT转位系统基本性质的表征将为未来的工作提供必要的基础，例如开发靶向TAT系统的药物的可能性，或为TAT系统在生物技术应用中的利用，如蛋白质疗法的表达。公共卫生相关性：TAT机制负责运输许多对人类健康具有重要意义的细菌毒力因子，而缺乏功能齐全的TAT系统往往会导致宿主细菌的生长缺陷。因此，对TAT系统的更好理解有望有助于开发新的抗微生物药物。此外，TAT系统运输完全折叠的蛋白质复合体的能力表明，它最终将在蛋白质疗法的表达中找到用途。

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.