MOLECULAR MECHANISMS OF SIGNAL TRANDUCTION BY CHEY

细胞使用信号转导途径将外部刺激转化为 可以生成适当响应的内部形式。在这两个地方 原核生物和真核生物这一重要任务经常 通过一连串的瞬时蛋白磷酸化和 去磷酸化事件。对其作用机制有一个基本的认识 蛋白质间的磷酸基转移，对此的调节 过程，以及磷酸化对蛋白质活性的影响如下 这一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。 细菌中广泛存在的“双组分”家族 利用一种明显常见的信号转导的调控系统 机制，以及提供的卓越的实验可访问性 细菌，表明这一目标是可行的。 本申请利用了最好理解的两个- 控制大肠埃希菌趋化性的成分系统。这个 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从以下任一基团获得磷酰基 Chea激酶或小分子如乙酰磷酸，并释放 通过自催化路线或在辅助下的磷酰基 切兹蛋白的含量。这些反应中的每一个反应的机理都是未知的。 该项目的四个具体目标是确定(I)如何 磷酸化激活Chey，以及Chey的机制(II) 磷酸化，(Iii)自身去磷酸化，和(Iv)CHEZ介导 去磷酸化反应。 提出了一种综合遗传、生化和物理方法。这个 主要策略将是推断Chey信号的关键特征 通过分离和彻底鉴定信息突变体进行转导 乳酪蛋白。许多变种人已经掌握在手中。对方案进行了描述 为了构建或识别其他Chey突变，这些突变要么测试 目前的激活模式类似于影响 相关信号转导蛋白，或改变Chey的能力 支持它参与的各种反应。突变型Chey 具有有趣的体内表型的蛋白质将在下一步进行研究 使用从大电池中选择的适当的体外分析来详细说明 已确定的生化和物理测试，直至并包括完成 用X射线结晶学或多维方法确定结构 质子核磁共振。 这项基础研究提案的两个方面直接与 健康问题。第一，高度类似于趋化作用的监管体系 但远不为人所知的是，毒力因子通过一种 多种细菌病原体(如百日咳波氏杆菌、假单胞菌 铜绿假单胞菌、金黄色葡萄球菌)。对Chey的详细了解 功能可促进设计有效的治疗药物 被这种细菌感染。第二，适用于以下方面的基本见解 真核细胞的信号转导过程是可以预见的。有足够的 不适当的信号导致病理的先例，如 癌症。

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.