Functions of Ubiquitin-Containing Proteins

描述(由申请人提供)： 泛素(Ub)是一种含76个残基的蛋白质，存在于细胞内，或与许多其他蛋白质结合或以游离形式存在。Ub-蛋白酶体系统调节细胞内蛋白质的降解，在包括细胞生长和分化、信号转导和对应激反应在内的众多生物学过程中发挥着核心作用。一个普遍存在的Ub/蛋白酶体依赖的蛋白分解系统是N-端规则途径。N-端规则将蛋白质在体内的半衰期与其N-端残基的同一性联系起来。由这笔赠款(GM31530)支持的研究，目前处于其第22个供资年，如进度报告中所述，对N末端规则途径的机制和功能产生了几个重要的见解。在过去的4年里，这笔资金支持了我们对小鼠N末端规则通路的研究。本次更新描述的研究目的是继续这些研究，并进一步促进对哺乳动物Ub系统的N-端规则通路和相关方面的理解。具体目标： 1)小鼠品系(及其衍生细胞)的构建和功能分析，在该品系中，在特定的细胞系中，ATE1编码的Arg-tRNA-蛋白转移酶(R转移酶)的表达在胚胎发生期间或出生后选择性地和有条件地被取消(或诱导)。 2)小鼠ATE1(-/-)细胞染色体稳定性分析及细胞凋亡调控。 3)分析小鼠Ntan 1(-/-)、Ubr1(-/-)、Ubr2(-/-)、Ubr3(-/-)和双突变[Ubr1(-/-)Ubr2(-/-)]细胞的染色体稳定性及其对细胞凋亡的调控。 4)通过鉴定在ATE1(-/-)胚胎发育过程中，尤其是在+/+胚胎中ATE1表达在E8.5天达到高峰时，其表达显著改变的小鼠基因，鉴定ATEL依赖的回路(即涉及N末端精氨酸化的回路) 5)进一步鉴定哺乳动物N-末端规则通路的生理底物。这一目标的项目包括一种基于标签的共沉淀-质谱学方法来鉴定小鼠UBR1、UBR2和UBR3Ub连接酶(E3蛋白)的特定配体。

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).