Function of SecA2 in Mycobacterium tuberculosis protein export

描述(申请人提供)：结核分枝杆菌是结核病的病原体。结核病每年导致约200万人死亡，世界上约三分之一的人口感染结核病。结核病每年新感染近1%的世界人口。结核病是艾滋病毒/艾滋病患者死亡的主要原因。虽然结核病通常可以用抗生素治疗，但全球抗击结核病的一个严重问题是出现了耐多药结核病菌株。为了开发新药的合理靶点，必须更好地了解分枝杆菌的生理学。结核分枝杆菌等病原菌分泌蛋白质以逃避宿主防御机制并在宿主细胞中生存，使蛋白质输出成为合理的药物靶点。这项工作的长期目标是表征依赖SEC的结核分枝杆菌蛋白输出。涉及二聚体SecA和SecYEG的SEC依赖的易位途径被用于许多蛋白质的出口。SecA是一种存在于所有细菌中的必需蛋白质，是一种ATPase，通过SecYEG膜转位机制提供蛋白质输出所需的能量。最近，包括结核分枝杆菌在内的几种病原微生物被发现携带两种SecA蛋白，SecA1和SecA2。SecA1对一般蛋白的输出是必需的，而SecA2对某些毒力蛋白的分泌是特异的。然而，为什么这些微生物需要两个SecA蛋白还不清楚。由于SecA蛋白通常是特定微生物所特有的，因此SecA1和SecA2为药物开发提供了良好的靶点。因此，我们建议对这些SECA蛋白在结核分枝杆菌蛋白输出中的功能进行研究。我们将使用生化和生物物理技术，包括ATPase结合和水解分析，分析性超速离心法来监测亚基结合，并建立体外膜结合和转位试验来鉴定SECA蛋白。这笔R03奖金的具体问题是：1)。SecA1和SecA2是否具有相似的结合和水解ATP的能力？2)SecA1和SecA2是否相互作用？SecA1和SecA2如何与膜转运机制相互作用？这些实验旨在对结核分枝杆菌中两种SecA蛋白的功能进行深入的表征。结核分枝杆菌是结核病的病原体。结核病每年导致约200万人死亡，世界上约三分之一的人口感染结核病。结核病每年新感染近1%的世界人口。结核病是艾滋病毒/艾滋病患者死亡的主要原因。虽然结核病通常可以用抗生素治疗，但全球抗击结核病的一个严重问题是出现了耐多药结核病菌株。为了开发新药的合理靶点，必须更好地了解分枝杆菌的生理学。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is the causative agent for the disease tuberculosis (TB). TB kills about 2 million people each year and around a third of the world's population is infected with TB. TB newly infects nearly 1% of the world's population each year. TB is a leading cause of death among people with HIV/AIDS. Though TB can normally be treated with antibiotics, a serious problem in the worldwide fight against TB is the emergence of multi-drug resistant strains of TB. In order to develop logical targets for new drugs, the physiology of Mycobacteria must be better understood. Pathogenic bacteria such as M. tuberculosis secrete proteins in order to evade host defense mechanisms and survive in host cells, making protein export a logical drug target. The long-term goal of this work is to characterize the Sec-dependent protein export of M. tuberculosis. The Sec-dependent translocation pathway that involves dimeric SecA and SecYEG is used for export of many proteins. SecA, an essential protein found in all bacteria, is an ATPase and provides the energy used in the export of proteins through the SecYEG membrane translocation machinery. Recently, several pathogenic microorganisms, including M. tuberculosis, have been discovered to carry two SecA proteins, SecA1 and SecA2. SecA1 is essential for general protein export while SecA2 is specific for secretion of some virulence proteins. However, why these microorganisms require two SecA proteins is not understood. Because SecA proteins are generally specific to a particular microorganism, SecA1 and SecA2 provide good targets for drug development. Therefore, we propose to characterize the function of each of these SecA proteins in protein export from M. tuberculosis. We will characterize the SecA proteins using biochemical and biophysical techniques including ATPase binding and hydrolysis assays, analytical ultracentrifugation to monitor subunit associations, and develop in vitro membrane binding and translocation assays for M. tuberculosis proteins. The specific questions for this R03 grant are: 1). Do SecA1 and SecA2 possess similar abilities to bind and hydrolyze ATP?; 2) Do SecA1 and SecA2 interact with each other?; and 3.) How do SecA1 and SecA2 interact with membrane translocation machinery? These experiments are designed to initiate an in depth characterization of the function of both SecA proteins in M. tuberculosis. Mycobacterium tuberculosis is the causative agent for the disease tuberculosis (TB). TB kills about 2 million people each year and around a third of the world's population is infected with TB. TB newly infects nearly 1% of the world's population each year. TB is a leading cause of death among people with HIV/AIDS. Though TB can normally be treated with antibiotics, a serious problem in the world-wide fight against TB is the emergence of multi-drug resistant strains of TB. In order to develop logical targets for new drugs, the physiology of Mycobacteria must be better understood.