MOLECULAR MECHANISMS OF SIGNAL TRANDUCTION BY CHEY

细胞利用信号转导途径将外部刺激转化为信号转导途径。 可以生成适当响应的内部表单。无论是 原核生物和真核生物的这一重要任务往往是 通过瞬时蛋白质磷酸化级联反应完成， 去磷酸化事件。基本了解的机制 蛋白质之间的磷酰基转移，这种调节 这一过程，以及磷酸化对蛋白质活性的影响， 这项应用背后的长期目标是 定义了细菌信号转导的分子机制。 在细菌中存在广泛的“双组分”家族， 调节系统”，利用一个明显共同的信号转导 机制，以及提供的上级实验可及性 这表明这个目标是可行的。 本申请利用了最好理解的两个- 组成系统，由大肠杆菌的趋化性。的 磷酸化形式的CheY蛋白与鞭毛马达相互作用 来控制游泳行为。CheY从以下两种物质中获得磷酰基： CheA激酶或小分子如乙酰磷酸， 磷酰基通过自催化途径或在辅助下 CheZ蛋白质。这些反应的机制都是未知的。 该项目的四个具体目标是确定（i）如何 磷酸化激活CheY，并且CheY的机制（ii） 磷酸化，（iii）自身去磷酸化，和（iv）CheZ介导的 去磷酸化反应 一个综合的遗传，生物化学和物理的方法提出。的 主要的策略是推导出CheY信号的关键特征 通过分离和彻底表征信息突变体的转导 CheY蛋白质。很多变种人已经在控制之中。方案描述 构建或鉴定额外的cheY突变， 目前的激活模型，类似于突变， 相关的信号转导蛋白，或改变CheY的能力， 支持它所参与的各种反应。变异CheY 具有感兴趣的体内表型的蛋白质将在进一步的研究中进行检查。 使用从大电池中选择的适当体外试验详细说明 已建立的生化和物理测试，包括完整的 通过X射线晶体学或多维 质子核磁共振 这一基础研究提案的两个方面直接关系到 健康问题。第一，调控系统与趋化性高度相似 但对毒力因子表达的控制还不太了解， 各种细菌病原体（例如百日咳杆菌、假单胞菌 铜绿假单胞菌、金黄色葡萄球菌）。详细了解CheY 功能可以帮助设计有效的治疗药物， 感染这种细菌。第二，适用于以下方面的基本见解 真核生物的信号转导过程是预期的。有充分 不适当信号传导导致病理的先例， 癌

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.