RAS-LIKE GTP-BINDING PROTEINS--REGULATION AND FUNCTION

RAS 样 GTP 结合蛋白——调控和功能

• 批准号: 3200702
• 负责人: IAN G MACARA
• 金额: $ 16.66万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-15 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3200702
• 关键词: Escherichia coli; G protein; affinity chromatography; antibody; enzyme mechanism; gene expression; gene mutation; genetic regulatory element; guanine nucleotide binding protein; guanosine triphosphate; guanosinetriphosphatases; immunoprecipitation; intracellular transport; laboratory rabbit; molecular cloning; oncogenes; posttranslational modifications; protein purification; protein structure function; site directed mutagenesis; transcription factor; transfection; vesicle /vacuole

The goal of this project is to determine the mechanisms that regulate the activities of small, ras-like GTP binding proteins, and to investigate possible functions. A family exists of >20 such proteins, of unknown function, with 20-60% homology to the Ki-ras protein, that probably operate as "molecular switches'. Their medical importance is underlined by the discovery that one member, rap1 (or Krev), suppresses transformation by ras, and another, rho is a target for botulinum toxin. Multiple factors control the switch-states of these proteins, catalyzing GDP/GTP exchange or GTPase activity, or inhibiting GDP release. The ras proteins are also extensively modified, covalently. It is important to understand the functions and control of these GTP-binding proteins. The main focus will be on the rab subclass. The project is divided into 3 main sections: 1. Characterization. Specific antibodies are required against rab peptides and recombinant rab proteins expressed i E. coli. These reagents will be used to determine tissue distribution, subcellular localization and covalent modifications of the rab proteins. The hypothesis will be tested that different C-terminal consensus sequences control modification and localization. 2. Regulation. A ras-specific guanine nucleotide releasing factor has been discovered, that could function as an "on switch". The existence of similar factors specific to the rab proteins will be investigated. Other factors, that control rab GTPase activity (GAPS) will also be identified and purified, to determine control mechanisms. 3. Function. Three approaches will be used. (a) Affinity columns will be prepared from recombinant rab proteins and used to purify factors that bind specifically to the GTP-states of the proteins. Such factors are potential targets (b) The transforming potential, and transformation-suppressing activity of the rab proteins will be measured, to define the specificity of the properties of ras Krev. (c) Finally, the hypothesis will be tested, in collaboration with other laboratories, that the rab proteins normally function to control exocytosis and/or interorganelle transport, using in vitro assays.

这个项目的目标是确定调节 小分子ras样GTP结合蛋白的活性研究 可能的功能。存在一个未知的&gt；20个这样的蛋白质家族 功能，与Ki-ras蛋白有20%-60%的同源性，可能起作用 分子开关。它们在医学上的重要性通过 发现一个成员Rap1(或Krev)通过以下方式抑制变换 RAS和另一种Rho是肉毒杆菌毒素的目标。多重因素 控制这些蛋白质的开关状态，催化GDP/GTP交换或 GTPase活性，或抑制GDP释放。Ras蛋白也是 广泛修饰，共价。重要的是要了解 这些GTP结合蛋白的功能和调控。主要焦点将是 在Rab的子类上。该项目分为3个主要部分： 1.人物塑造。需要针对Rab多肽的特异性抗体 重组Rab蛋白在大肠杆菌中得到表达。这些试剂将是 用于确定组织分布、亚细胞定位和 Rab蛋白的共价修饰。这一假设将得到检验。 不同的C-末端共有序列控制修饰和 本地化。 2.监管。一种ras特异性的鸟嘌呤核苷酸释放因子 一旦被发现，它就可以起到“开关”的作用。的存在 将对Rab蛋白特有的类似因素进行研究。其他 控制Rab GTP酶活性的因素(GAP)也将被确定 并进行纯化，以确定控制机制。 3.功能。将使用三种方法。(A)亲和度栏将为 由重组Rab蛋白制备，用于纯化结合因子 特别是与蛋白质的GTP状态有关。这样的因素是潜在的 目标(B)转化潜力和转化抑制 将测量Rab蛋白的活性，以确定 Ras krev的性质。(C)最后，将在#年检验该假设 与其他实验室合作，Rab蛋白正常 控制胞吐和/或细胞器间转运的功能，使用 体外试验。