MOLECULAR MECHANISMS OF SIGNALING IN E COLI CHEMOTAXIS

大肠杆菌趋化性信号传导的分子机制

• 批准号: 6180358
• 负责人: Robert B. Bourret
• 金额: $ 25.85万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180358
• 关键词: Escherichia coli; X ray crystallography; active sites; bacterial genetics; bacterial proteins; biological signal transduction; chemotaxis; gene mutation; genetic regulation; nuclear magnetic resonance spectroscopy; phosphorylation; protein structure function

Cells use signal transduction pathways to convert external stimuli into an internal form that can generate an appropriate response. In both prokaryotic and eukaryotic organisms this vital task is frequently accomplished by a cascade of transient protein phosphorylation and dephosphorylation events. A fundamental understanding of the mechanism of phosphoryl group transfer among proteins, of the regulation of this process, and of the impact phosphorylation has on protein activity is thus of broad interest The long term objective behind this application is to define the molecular mechanisms employed in bacterial signal transduction. The existence in bacteria of a widespread family of "two-component regulatory systems" that utilize an apparently common signal transduction mechanism, together with the superior experimental accessibility offered by bacteria, suggests this objective is feasible. The present application takes advantage of the best understood two- component system, that governing chemotaxis by Escherichia coli. The phosphorylated form of the CheY protein interacts with the flagellar motor to control swimming behavior. CheY obtains phosphoryl groups from either the CheA kinase or small molecules such as acetyl phosphate, and releases phosphoryl groups by either a self-catalyzed route or with the assistance of the CheZ protein. The mechanism of each of these reactions is unknown. The four specific aims of this project are to determine (i) how phosphorylation activates CheY, and the mechanisms of the CheY (ii) phosphorylation, (iii) autodephosphorylation, and (iv) CheZ-mediated dephosphorylation reactions. An integrated genetic, biochemical, and physical approach is proposed. The primary strategy will be to deduce the critical features of CheY signal transduction by isolating and thoroughly characterizing informative mutant CheY proteins. Numerous mutants are already in hand. Schemes are described to construct or identify additional cheY mutations that either test the current model of activation, are analogous to mutations that affect related signal transduction proteins, or alter the ability of CheY to support the various reactions in which it participates. Mutant CheY proteins with interesting in vivo phenotypes will be examined in further detail using appropriate in vitro assays chosen from a large battery of established biochemical and physical tests, up to and including complete structural determination by X-ray crystallography or multidimensional proton NMR. Two aspects of this basic research proposal are directly relevant to health issues. First, regulatory systems highly analogous to chemotaxis but far less well understood control expression of virulence factors by a variety of bacterial pathogens (e.g. Bordetella pertussis, Pseudomonas aeruginosa, Staphylococcus aureus). A detailed understanding of CheY function could facilitate design of therapeutic agents effective against infection by such bacteria. Second, fundamental insights applicable to eukaryotic signal transduction processes are anticipated. There is ample precedent for inappropriate signaling resulting in pathologies such as cancer.

细胞使用信号转导途径将外部刺激转化为 可以产生适当响应的内部形式。在这两个中 原核和真核生物这一至关重要的任务经常 通过一系列瞬态蛋白磷酸化和 去磷酸化事件。对机制的基本理解 蛋白质之间的磷酸群转移，对此调节 过程，以及磷酸化对蛋白活性的影响是 引起广泛关注的该应用程序背后的长期目标是 定义细菌信号转导中使用的分子机制。 广泛家族的细菌中存在“两个组成部分 使用明显常见的信号转导的调节系统” 机构，以及提供的卓越实验可访问性 通过细菌，这表明这个目标是可行的。 本应用程序利用了最佳理解的优势 - 组件系统，通过大肠杆菌控制趋化性。这 Chey蛋白的磷酸化形式与鞭毛电动机相互作用 控制游泳行为。 Chey从任何一个 CHEA激酶或小分子，例如乙酰磷酸盐，并释放 通过自我催化的路线或辅助的磷酸组组 Chez蛋白。这些反应中的每一个的机制尚不清楚。 该项目的四个具体目标是确定（i） 磷酸化激活chey和chey的机制（ii） 磷酸化，（iii）自磷酸化和（iv）Chez介导的 去磷酸化反应。 提出了一种综合的遗传，生化和物理方法。这 主要策略将是推断Chey信号的关键特征 通过隔离和彻底表征信息性突变体的转导 chey蛋白。许多突变体已经在手中。描述了方案 构建或识别其他测试的Chey突变 当前的激活模型类似于影响的突变 相关信号转导蛋白，或改变chey的能力 支持其参与的各种反应。突变的chey 将进一步检查具有有趣体内表型的蛋白质 细节使用适当的体外测定法 已建立的生化和物理测试，直至和包括完整 X射线晶体学或多维的结构测定 质子NMR。 这项基础研究建议的两个方面与 健康问题。首先，调节系统高度类似于趋化性 但是，通过A 各种细菌病原体（例如Bordetella buttussis，pseudomonas 铜绿，金黄色葡萄球菌）。对Chey的详细理解 功能可以促进有效的治疗剂设计 这种细菌感染。第二，适用于 预计真核信号转导过程。有很多 不适当的信号传导的先例，导致病理 癌症。