Functions of Ubiquitin-Containing Proteins

描述（由申请人提供）： 泛素（Ub）是一种76个氨基酸残基的蛋白质，存在于细胞中，可以是游离的，也可以与许多其他蛋白质结合。由Ub-蛋白酶体系统调节的细胞内蛋白质降解在众多生物过程中起着核心作用，包括细胞生长和分化、信号转导和对应激的反应。一种普遍存在的Ub/蛋白酶体依赖性蛋白水解系统是N-末端规则途径。N-末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。由该赠款（GM31530）支持的研究，目前在其第22个年度的资助，已经产生了几个重要的见解N端规则途径的机制和功能，如进度报告中所述。在过去的4年里，这笔赠款支持了我们对小鼠N端规则途径的研究。本更新中描述的研究目标是继续这些研究，并进一步推进对哺乳动物Ub系统的N端规则途径和相关方面的理解。具体目标： 1)小鼠品系（和它们衍生的细胞）的构建和功能分析，其中在胚胎发生期间或出生后的特定细胞系中，ATE1编码的Arg-tRNA-蛋白转移酶（R转移酶）的表达被选择性地和有条件地消除（或诱导）。 2)小鼠ATE1（-/-）细胞染色体稳定性分析及凋亡调控 3)小鼠NTAN 1（-/-）、UBRI（-/-）、UBR2（-/-）、UBR3（-/-）和双突变[UBRI（-/-）UBR2（-/-）]细胞中染色体稳定性和细胞凋亡调节的分析。 4)ATEL相关电路的识别（即，涉及N末端腺苷酸化的回路），通过鉴定小鼠基因，所述小鼠基因的表达在ATE1（-/-）胚胎的胚胎发育过程中显著改变，特别是在+/+胚胎中ATE1表达的强烈尖峰（在E8.5天达到峰值）时 5)进一步鉴定哺乳动物N-末端规则途径的生理底物。该目标的项目包括基于标签的共沉淀-质谱方法，以识别小鼠UBR 1，UBR 2和UBR 3 Ub连接酶（E3蛋白）的特异性配体。

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).