Functions of Ubiquitin-Containing Proteins

含泛素蛋白质的功能

• 批准号: 6740182
• 负责人: ALEXANDER J VARSHAVSKY
• 金额: $ 55.81万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6740182
• 关键词: acid aminoacid ligase; active sites; amidases; amination; aminoacid tRNA ligase; aminoacyltransferase; aminopeptidase; apoptosis; arginine; cell differentiation; chemical stability; clinical research; enzyme activity; enzyme mechanism; enzyme substrate; gene targeting; genetically modified animals; laboratory mouse; ligands; posttranslational modifications; proteasome; protein degradation; protein localization; ubiquitin

DESCRIPTION (provided by applicant): Ubiquitin (Ub) is 76-residue protein that exists in cells either free or conjugated to many other proteins. Regulated degradation of intracellular proteins by the Ub-proteasome system plays a central role in an astounding multitude of biological processes, including cell growth and differentiation, signal transduction, and responses to stress. One universally present Ub/proteasome-dependent proteolytic system is the N-end rule pathway. The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Studies supported by this grant (GM31530), currently in its 22rid year of funding, have yielded several important insights into the mechanisms and functions of the N-end rule pathway, as described in the Progress Report. Over the last 4 years, this grant supported our studies of the mouse N-end rule pathway. The objective of research described in the present renewal is to continue these studies, and to further advance the understanding of the N-end rule pathway and related aspects of the mammalian Ub system. SPECIFIC AIMS: 1) Construction and functional analyses of mouse strains (and cells derived from them) in which the expression of the ATE1 encoded Arg-tRNA-protein transferases (R transferases) is selectively and conditionally abolished (or induced) in specific cell lineages during embryogenesis, or postnatally. 2) Analysis of chromosome stability and regulation of apoptosis in mouse ATE1(-/-) cells. 3) Analysis of chromosome stability and regulation of apoptosis in mouse NTAN 1(-/-), UBRI(-/-), UBR2(-/-), UBR3(-/-), and double-mutant [UBRI(-/-)UBR2(-/-)] cells. 4) Identification of ATEl-dependent circuits (i.e., the circuits that involve N terminal arginylation) through the identification of mouse genes whose expression is significantly altered during embryonic development in ATE1(-/-) embryos, specifically at the time of a strong spike of ATE1 expression in +/+ embryos that peaks on day E8.5 5) Further identification of physiological substrates of the mammalian N-end rule pathway. The projects of this Aim include a tag-based coprecipitation-mass spectrometry approach to identifying specific ligands of the mouse UBR1, UBR2 and UBR3 Ub ligases (E3 proteins).

描述（由申请人提供）： 泛素（UB）是76种残基蛋白，存在于自由或与许多其他蛋白质结合的细胞中。 UB-蛋白酶体系统对细胞内蛋白的调节降解在令人震惊的多种生物过程中起着核心作用，包括细胞生长和分化，信号转导以及对压力的反应。一种普遍存在的UB/蛋白酶体依赖性蛋白水解系统是N端规则途径。 N端规则将蛋白质的体内半衰期与其N末端残基的身份联系起来。该赠款（GM31530）目前在其22RID年度中支持的研究已经对N端规则途径的机制和功能产生了一些重要的见解，如进度报告中所述。在过去的四年中，该赠款支持我们对小鼠N端规则途径的研究。当前续签中描述的研究的目的是继续这些研究，并进一步促进对N端规则途径和哺乳动物UB系统相关方面的理解。具体目的： 1）小鼠菌株（以及从中得出的细胞）的构建和功能分析，其中ATE1编码的ARG-tRNA-蛋白质转移酶（R转移酶）在胚胎发生期间有序地废除特定细胞谱系中的（或诱导）。 2）分析小鼠ATE1（ - / - ）细胞中凋亡的染色体稳定性和调节。 3）分析小鼠NTAN 1（ - / - ），UBRI（ - / - ），UBR2（ - / - ），UBR3（ - / - ）和双重突击剂[UBRI（ - - - - - UBR2（ - ）UBR2（ - ）UBR2（ - ）UBR2（ - - ）]细胞中，小鼠NTAN 1（ - / - ），UBRI（ - / - ）中凋亡的调节分析。 4）通过鉴定小鼠基因的鉴定，在ATE1（ - / - ）胚胎中，在Ate1（ - / - ）胚胎中的胚胎发育过程中，特定在ATE1强烈峰值表达在 +/ + embryos强烈的 +/ +/ +/embryos峰时，鉴定出ATE的表达在ATE1（ - / - ）胚胎中的胚胎发育过程中，其表达在 +/ +/ + embryos强烈峰值时，其表达在ATE1（ - / - ）胚胎中发生了重大改变，鉴定了ATEL依赖性电路（即涉及N末端精氨酸的电路）。 5）进一步鉴定哺乳动物N端规则途径的生理底物。此目标的项目包括一种基于标签的共沉淀 - 质谱法，以识别小鼠UBR1，UBR2和UBR3 UB连接酶（E3蛋白）的特定配体。