In vitro and in-cell investigation of the acid-stress chaperone HdeA

酸应激伴侣 HdeA 的体外和细胞内研究

基本信息

批准号：
9249639
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 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9249639
关键词：
Acidity Acids Affect Bacteria Bacterial Proteins Binding Biological Models Biomedical Engineering Buffers Cells Cessation of life Chemicals Crowding Data Dimerization Disease Dissociation Dysentery Environment Future Goals Grant Health Hydrogen Hydrophobicity In Vitro Infection Intestines Investigation Methods Molecular Molecular Chaperones Molecular Conformation Monitor Motion NMR Spectroscopy Nuclear Magnetic Resonance Occupations Organism Pathogenicity Periplasmic Proteins Phase Physiological Play Positioning Attribute Property Protein Conformation Proteins Publications Relaxation Research Research Personnel Role Sampling Side Stomach Structure Techniques Therapeutic Titrations Travel Vaccines Vertebral column Work acid stress analytical tool biological systems biophysical analysis biophysical properties combat design dimer experience experimental study follow-up improved insight interest killings mindfulness monomer pathogenic bacteria periplasm prevent protein folding protein function public health relevance vaccine development

项目摘要

DESCRIPTION (provided by applicant): 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, the ATP- independent chaperone HdeA plays a major role in aiding bacterial survival at low pH. HdeA's mechanism of action is rather unique, in that it is an unfolded monomeric protein in its activated state. Its 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. Once the bacteria enter the intestinal tract, HdeA releases these proteins and refolds into its inactive dimer conformation. Biophysical studies have provided clues that HdeA unfolds below pH 3.0 and interacts with its binding partners using hydrophobic residues found at the dimer interface of the folded protein. However, there is a dearth of data that monitors, in detail, the mechanism of monomerization, unfolding and activation at multiple pH values below 3.0. In addition, the properties of instrinsically disordered proteins are generally not well-understood. Specific aims. We propose to pursue a thorough, atomic-level investigation of the mechanism of activation of HdeA at low pH, using Nuclear Magnetic Resonance (NMR) spectroscopy as our primary analytical tool. Since it is likely that cellular crowding is an important contributor to or understanding of HdeA activity (especially in its unfolded state) we propose to study the mechanism of HdeA activation both in vitro and in-cell. HdeA will also be an excellent model system to improve our understanding of functionality in an intrinsically disordered protein. Our specific aims are to 1) determine the specific structural and dynamic changes that trigger activation of chaperone activities in HdeA in vitro between pH 3.0 and 2.0 and 2) investigate the differences in structural and dynamic changes that occur in HdeA in-cell or in lysate between pH 6.0 and 2.0 compared to HdeA in vitro. NMR experiments will include titrations to monitor chemical shift changes as a function of pH, hydrogen exchange and 3D experiments to structurally characterize HdeA, and spin relaxation experiments to analyze backbone and side chain protein motions in HdeA at multiple timescales and multiple pH values. Health-related significance. Dysentery, caused by intestinal infection by pathogenic bacteria, kills over one million people per year worldwide. If we can understand how HdeA senses and is triggered by pH changes, we can better understand how this type of acid-stress chaperone helps bacteria survive under extreme conditions. Armed with this understanding we will be able to improve targeting for vaccines or other therapeutics that can disable the activities of HdeA and thereby weaken the infectivity of these pathogenic bacteria.

描述（由申请人提供）：致病菌必须穿过胃的高酸性环境才能到达并感染肠道，因此胃是一个重要的屏障，有助于在某些细菌引起疾病之前杀死它们。然而，作为传染性最强的细菌，不依赖 ATP 的伴侣蛋白 HdeA 在帮助细菌在低 pH 条件下生存方面发挥着重要作用。 HdeA 的作用机制相当独特，因为它是一种未折叠的单体蛋白。它的作用是在细胞通过胃的恶劣环境并进入肠道的中性环境时保护其他蛋白质免于错误折叠和聚集。一旦细菌进入肠道，HdeA 就会释放这些蛋白质并重新折叠。生物物理学研究提供了 HdeA 在 pH 3.0 以下展开并利用折叠蛋白二聚体界面上的疏水残基与其结合伙伴相互作用的线索。缺乏详细监测 3.0 以下多个 pH 值下的单体化、解折叠和激活机制的数据。此外，本质上无序的蛋白质的特性通常还不太清楚。具体目标是使用核磁共振 (NMR) 光谱作为我们的主要分析工具，对低 pH 下 HdeA 的激活机制进行彻底的原子水平研究，因为细胞拥挤可能是一个重要的因素。为了或理解 HdeA 活性（特别是在其未折叠状态），我们建议研究体外和细胞内 HdeA 激活的机制，HdeA 也将是一个很好的模型系统，以提高我们对内在功能的理解。我们的具体目标是 1) 确定在 pH 3.0 和 2.0 之间触发 HdeA 体外伴侣活性激活的特定结构和动态变化，以及 2) 研究细胞内 HdeA 中发生的结构和动态变化的差异。或在 pH 6.0 和 2.0 之间的裂解液中，与 HdeA 相比，体外 NMR 实验将包括滴定以监测作为 pH 函数的化学位移变化、氢交换和 3D 实验。对 HdeA 进行结构表征，并通过自旋弛豫实验分析 HdeA 在多个时间尺度和多个 pH 值下的主链和侧链蛋白质运动。与健康相关的意义。由肠道致病菌感染引起的痢疾每年导致全球超过一百万人死亡。如果我们能够了解 HdeA 如何感知并由 pH 值变化引发，我们就能更好地了解这种酸应激伴侣的机制。帮助细菌在极端条件下生存。有了这种认识，我们将能够改进疫苗或其他疗法的靶向性，从而禁用 HdeA 的活性，从而削弱这些致病菌的传染性。