Synergy between acid stress chaperones HdeA and HdeB with clients and their key sites of activity

酸应激伴侣 HdeA 和 HdeB 与客户及其关键活动位点之间的协同作用

基本信息

批准号：
10681291
负责人：
KARIN A CROWHURST
金额：
$ 10.88万
依托单位：
CALIFORNIA STATE UNIVERSITY NORTHRIDGE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10681291
关键词：
Acidity Acids Affect Award Bacteria Bacterial Proteins Binding Binding Sites Biological Models Brucella abortus Cells Cessation of life Circular Dichroism Client Data Disease Dissociation Dysentery Environment Escherichia coli Exposure to Funding Goals Health Human In Vitro Individual Infection Intestines Investigation Isotope Labeling Link Measures Modeling Molecular Chaperones Molecular Conformation Molecular Mechanisms of Action Monitor Mutation NMR Spectroscopy Occupations Periplasmic Proteins Persons Physiological Play Positioning Attribute Process Property Protein Engineering Proteins Publishing Recovery Research Personnel Resolution Role Series Shigella flexneri Site Site-Directed Mutagenesis Stomach Structure Techniques Testing Therapeutic Time Titrations Travel Tryptophan Vaccine Design Visualization Work acid stress biological systems biophysical techniques combat diarrheal disease dimer disulfide bond enteric infection experimental study flexibility improved innovation insight interest molecular dynamics monomer mutant pathogenic bacteria periplasm protein aggregation protein folding protein structure stem synergism targeted treatment vaccine development

项目摘要

PROJECT SUMMARY / ABSTRACT Background. Pathogenic bacteria must travel through the highly acidic environment of the stomach before they can reach and infect the intestines. The stomach is therefore an important barricade which helps to kill many bacteria before they can cause illness. In some of the most infectious bacteria, however, ATP- independent chaperones HdeA and HdeB play major roles in aiding bacterial survival at low pH. Their job is to protect other proteins from misfolding and aggregating as the cell transitions through the harsh environment of the stomach and into the neutral environment of the intestines. HdeB is active at intermediate pH values, while HdeA functions at the low pH typical of stomach acid. Although there are various models available to explain the interplay between the two chaperones, it is still unclear which, if any, is correct. Specific aims. The goal of the proposed work is to use NMR spectroscopy and other biophysical techniques to pursue an in-depth investigation of the apparently synergistic mechanism by which the two chaperone proteins operate and to probe the roles of specific residues that trigger or modify the activation of HdeA or HdeB. Aim #1 is to examine the roles and interactions of HdeA and HdeB with chaperone clients as a function of pH. Isotopic labeling and the unique properties of NMR spectroscopy will be employed to monitor each protein individually within a mixture of HdeA, HdeB and a client protein, thereby providing different vantage points to obtain unprecedented detail. Aim #2 is to probe sites of chaperone activation and stability in HdeA and HdeB using targeted mutations. Here, a variety of techniques will be used to closely assess segments of each protein that have been linked to essential roles in function and/or activation, including a key tryptophan in the dimer interface of HdeB and the disulfide bond in HdeA, which helps to maintain the semi-folded structure believed to be important to its chaperone function. Health-related significance. Dysentery, caused by intestinal infection by pathogenic bacteria, kills at least 350,000 people per year worldwide. If we can elucidate the individual and collective roles of HdeA and HdeB in the presence of client proteins, as well as the mechanistic importance of specific residues or regions, we can better understand how these acid-stress chaperones help bacteria survive under extreme conditions. Improved understanding can inform researchers designing vaccines or other therapeutics that can disable the activities of HdeA and HdeB and thereby weaken the infectivity of these pathogenic bacteria.

项目概要/摘要背景。致病菌必须经过胃部的高酸性环境才能传播。它们可以到达并感染肠道。因此，胃是一个重要的屏障，有助于杀死许多细菌在引起疾病之前。然而，在一些最具传染性的细菌中，ATP- 独立伴侣 HdeA 和 HdeB 在帮助细菌在低 pH 条件下生存方面发挥着重要作用。他们的工作是当细胞在恶劣的环境中过渡时，保护其他蛋白质免于错误折叠和聚集胃并进入肠道的中性环境。 HdeB 在中等 pH 值下具有活性，而 HdeA 在胃酸典型的低 pH 值下发挥作用。尽管有多种模型可以解释两个伴侣之间的相互作用，目前还不清楚哪一个（如果有的话）是正确的。具体目标。拟议工作的目标是使用核磁共振波谱和其他生物物理技术深入研究两个伴侣的明显协同机制蛋白质运作并探测触发或修饰 HdeA 或 HdeA 激活的特定残基的作用哈德B。目标 #1 是检查 HdeA 和 HdeB 与伴侣客户端的角色和交互作为一个函数 pH 值。同位素标记和核磁共振波谱的独特性质将用于监测每个蛋白质在 HdeA、HdeB 和客户蛋白质的混合物中单独存在，从而提供不同的优势点以获得前所未有的细节。目标 #2 是探测 HdeA 中伴侣蛋白激活和稳定性的位点和 HdeB 使用靶向突变。在这里，将使用各种技术来仔细评估与功能和/或激活中的重要作用相关的每种蛋白质，包括关键的色氨酸 HdeB的二聚体界面和HdeA中的二硫键，有助于维持半折叠结构据信对其伴侣功能很重要。与健康相关的意义。由肠道病原菌感染引起的痢疾至少可导致死亡全球每年有 350,000 人。如果我们能够阐明 HdeA 和 HdeB 在客户蛋白的存在，以及特定残基或区域的机械重要性，我们可以更好地了解这些酸胁迫伴侣如何帮助细菌在极端条件下生存。改进了解可以帮助研究人员设计疫苗或其他可以禁用这些活动的疗法 HdeA和HdeB的结合，从而削弱这些病原菌的感染性。