Human Tryptophanyl-tRNA Synthetase-A Functional Analysis

人色氨酰-tRNA合成酶-A功能分析

• 批准号: 6877096
• 负责人: JERRY D JOHNSON
• 金额: $ 23.18万
• 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/6877096
• 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）： 所有生物体均具有统称为氨基酰基-TRNA合成酶的酶家族，该酶共价附着在mRNA定向蛋白质的刺激过程中，将氨基酸连接到同志TRNA中，能够将它们插入正确的位置。这些酶的生化特性在原核生物和真核生物之间足够不同，它们是抗生素发育的有吸引力的靶标。哺乳动物的色氨酸特异性酶（WRS）是独一无二的，因为它也是一种自磷酸化蛋白激酶。 进一步是唯一对γ-互化对调节敏感的氨基酰基-TRNA合成酶基因。这些特性强烈暗示了该酶的调节作用，除了其氨基酰化的管家功能。要完成的研究旨在确定人类WR的独特蛋白激酶/自磷酸化活性的功能。具体目的是1）确定磷酸化是否通过测定磷酸和去磷酶的氨基化和蛋白质磷酸化的动力学来影响催化特性，以及具有磷酸化位点的突变体S467，S467，S467，对ASP或ALA的影响。 WRS。 3）使用酵母两杂交系统，通过正常和突变的WRS转染的培养物中的人类细胞的共免疫沉淀与潜在的激酶底物进行蛋白质 - 蛋白质相互作用，以及肽库的体外磷酸化。 4）确定具有改变激酶和磷酸化特性的正常HSWR和突变体对全球和基因特异性转录以及转染的哺乳动物细胞中翻译的过表达的影响5）鉴定在HSWR中选择性不活跃的HSWR中的位点特异性突变。 这些突变体将用于增强在特定目标1-4中进行的实验的分辨率。这些实验的结果将决定如何调节激酶活性，修饰其他蛋白质以及酶的哪些结构特征对这些特性负责。