Defining the mechanism of protein translocation using a bacterial exotoxin as a model system

使用细菌外毒素作为模型系统定义蛋白质易位机制

• 批准号: 10040017
• 负责人: Michael J. Sheedlo
• 金额: $ 9.39万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10040017
• 关键词: Academic Medical Centers; Address; Algorithm Design; Area; Automobile Driving; Award; Bacterial Toxins; Bacterial exotoxin; Binding; Biochemical; Biological Assay; Biological Models; Cell Line; Cell membrane; Cells; Clostridium difficile; Complex; Confocal Microscopy; Coupled; Cryoelectron Microscopy; Cues; Data; Data Collection; Development; Environment; Family; Flow Cytometry; Foundations; In Vitro; Institution; Intoxication; Knowledge; Label; Life; Ligand Binding; Ligand Binding Domain; Ligands; Maintenance; Mammalian Cell; Mediating; Membrane; Mentors; Modeling; Modernization; Molecular Chaperones; Molecular Conformation; N-terminal; Organism; Pathway interactions; Play; Positioning Attribute; Process; Protein translocation; Proteins; Pyrenes; Regulatory Element; Research; Research Personnel; Resolution; Role; Sampling; Structural Biologist; Structure; System; Techniques; Time; Toxin; Training; Transferase; Work; anthrax toxin; base; career; computerized data processing; experience; experimental study; graduate student; insight; novel; pathogenic bacteria; post-doctoral training; programs; skills; structural biology; structured data; tissue culture; tissue/cell culture; tool

PROJECT SUMMARY The mechanisms underlying the complex process of protein translocation across a plasma membrane have been notoriously difficult to define. Currently, few model systems exist in which this process has been adequately described, with a serious lack of progress being made on this subject in recent years. One of the factors that has led to a lack of development in this area is the absence of structural data which describe key intermediates that occur during protein translocation. This is largely due to complications in obtaining pure, homogenous, samples in these states. With the advent of high-resolution cryo electron microscopy (cryo-EM) and the development of sophisticated data processing algorithms that are designed to tackle these issues, we are now in a position to make significant progress in addressing this question. Under this award, I will describe the fundamental process of protein translocation using a secreted bacterial toxin known as the Clostridioides difficile transferase toxin (CDT) as a model system. The experiments I propose here will build upon my strong foundation of structural and biochemical training as well as the preliminary data that I have collected on this subject, including five distinct structures of CDT that I have already solved by cryo-EM. These structures will be used to guide the development of novel tools and assays to rigorously describe CDT translocation mechanisms. I will address the outstanding questions of how pore formation is regulated and determine what factors drive protein translocation in this model system leading to the elucidation of several structures of key translocation intermediates that exist during this process. These structures will be probed both in vitro and in tissue culture to define protein translocation mechanisms in the context of intoxication. The cell based assays I am proposing will form a crucial component of my one additional year of postdoctoral training under this award as guided by Dr. Borden Lacy during which time I will gain expertise in the maintenance and processing of mammalian tissue culture cell lines for a variety of downstream applications. I will use this base knowledge to build my expertise in confocal microscopy and flow cytometry, taking advantage of the strong research environment at commitment to training at Vanderbilt University Medical Center. The training that I will receive under this award will add a new facet to the independent research program that I intend to develop at a R1 institution. The focus of my lab will be to continue probing the phenomenon of protein translocation mechanisms using bacterial secretion systems as a tool. I believe my extensive structural biology and biochemical background coupled with my extensive experience as a mentor to graduate students make me well suited for such a role.

项目摘要 跨质膜蛋白质易位过程的复杂过程的基础机制 众所周知，很难定义。当前，很少有模型系统存在此过程 充分描述了，近年来对此主题的进展不足。中的一个 导致该领域缺乏发展的因素是缺乏描述关键的结构数据 蛋白质易位期间发生的中间体。这主要是由于获得纯净的并发症 这些状态中的样品均匀。随着高分辨率冷冻电子显微镜（Cryo-EM）的出现 以及旨在解决这些问题的复杂数据处理算法的开发，我们 现在可以在解决这个问题方面取得重大进展。根据这个奖项，我将描述 使用分泌细菌毒素的蛋白质易位的基本过程称为梭状芽胞杆菌 艰难梭菌转移酶毒素（CDT）作为模型系统。我在这里提出的实验将建立在我的强大基础上 我在此收集的结构和生化培训以及初步数据的基础 受试者，包括我已经由Cryo-EM解决的五个不同的CDT结构。这些结构将是 用于指导新工具和测定的开发，以严格描述CDT易位机制。 我将解决如何调节孔形成的杰出问题，并确定哪些因素驱动 该模型系统中的蛋白质易位，导致钥匙易位的几种结构阐明 在此过程中存在的中间体。这些结构将在体外和组织培养中探测 在中毒的背景下定义蛋白质易位机制。我提出的基于细胞的测定 在该奖项下，在我的另外一年的博士后培训中，这是由博士指导的重要组成部分 Borden Lacy在此期间，我将获得哺乳动物组织维护和加工的专业知识 各种下游应用的培养细胞系。我将利用这种基础知识来建立我的专业知识 共聚焦显微镜和流式细胞仪，利用承诺的强大研究环境 在范德比尔特大学医学中心进行培训。我将根据该奖项获得的培训将增加一个新的 我打算在R1机构制定的独立研究计划的方面。我实验室的重点 要继续使用细菌分泌系统探测蛋白质易位机制的现象 作为工具。我相信我广泛的结构生物学和生化背景以及我的广泛 作为研究生的导师经验使我非常适合这样的角色。