Human Tryptophanyl-tRNA Synthetase-A Functional Analysis

人色氨酰-tRNA合成酶-A功能分析

• 批准号: 6756981
• 负责人: JERRY D JOHNSON
• 金额: $ 22.13万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6756981
• 关键词: SDS polyacrylamide gel electrophoresis; acylation; aminoacid tRNA ligase; clinical research; electrofocusing; enzyme activity; enzyme substrate; human tissue; immunoprecipitation; microarray technology; peptide library; phosphorylation; protein kinase; protein protein interaction; protein structure function; site directed mutagenesis; tissue /cell culture; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): All organisms possess a family of enzymes known collectively as aminoacyl-tRNA synthetases that covalently attach amino acids to cognate tRNAs capable of inserting them in the correct positions during mRNA-directed protein synthesis. The biochemical properties of theses enzymes are sufficiently different between prokaryotes and eukaryotes that they are attractive targets for antibiotic development. The tryptophan specific enzyme (WRS) from mammals is unique in that it is also an autophosphorylating protein kinase. Further is the only aminoacyl-tRNA synthetase gene sensitive to regulation by gamma-interferon. These properties strongly suggest a regulatory role for this enzyme in addition to it's housekeeping function of aminoacylation. The research to be accomplished is intended to determine the function(s) of the unique protein kinase/autophosphorylation activities of the human WRS. The specific aims are to 1) determine whether phosphorylation affects catalytic properties by assaying the kinetics of aminoacylation and protein phosphation with the phospho and dephospho enzyme as well as mutants with the site of phosphorylation, S467, mutated to either Asp or Ala. 2) Characterize the affects of tRNA on the autophosphorylation and exogenous protein kinase activities of human WRS. 3) Identify protein-protein interactions of HsWRS with potential kinase substrates using the yeast two-hybrid system, co-immunoprecipitation from human cells in culture transfected with normal and mutant forms of WRS, and in vitro phosphorylation of peptide libraries. 4) determine the effect(s) of overexpression of normal HsWRS and mutants with altered kinase and phosphorylation properties on global and gene-specific transcription and translation in transfected mammalian cells 5) identify site-specific mutations in HsWRS that selectively inactive the kinase activity. These mutants will be used to enhance the resolution of experiments done in specific aims 1-4. Results from these experiments will determine how the kinase activities are regulated, what other proteins are modified, and what structural features of the enzyme are responsible for these properties.

描述（由申请人提供）： 所有生物体都拥有一个统称为氨酰基-tRNA 合成酶的酶家族，它们将氨基酸共价连接到同源 tRNA 上，从而能够在 mRNA 指导的蛋白质合成过程中将它们插入到正确的位置。这些酶的生化特性在原核生物和真核生物之间存在很大差异，因此它们是抗生素开发的有吸引力的目标。来自哺乳动物的色氨酸特异性酶 (WRS) 的独特之处在于它也是一种自磷酸化蛋白激酶。 此外，它是唯一对 γ-干扰素调节敏感的氨酰基-tRNA 合成酶基因。这些特性强烈表明该酶除了氨酰化的管家功能之外还具有调节作用。待完成的研究旨在确定人类 WRS 的独特蛋白激酶/自磷酸化活性的功能。具体目标是 1) 通过分析磷酸化和去磷酸化酶以及磷酸化位点 S467 突变为天冬氨酸或丙氨酸的氨酰化和蛋白质磷酸化的动力学，确定磷酸化是否影响催化特性。2) 表征tRNA 对人 WRS 自身磷酸化和外源蛋白激酶活性的影响。 3) 使用酵母双杂交系统、转染正常和突变形式 WRS 的培养物中的人类细胞的免疫共沉淀以及肽文库的体外磷酸化，鉴定 HsWRS 与潜在激酶底物的蛋白质-蛋白质相互作用。 4) 确定正常 HsWRS 和激酶和磷酸化特性改变的突变体的过表达对转染哺乳动物细胞中整体和基因特异性转录和翻译的影响 5) 识别 HsWRS 中选择性失活激酶活性的位点特异性突变。 这些突变体将用于提高特定目标 1-4 中所做实验的分辨率。这些实验的结果将确定如何调节激酶活性、修饰哪些其他蛋白质以及酶的哪些结构特征导致这些特性。