UBIQUITIN-DEPENDENT PROTEOLYSIS IN MAMMALIAN CELLS

选择性蛋白质降解在调节许多 细胞过程，包括正常和异常的细胞周期进程 生长和分化，应激反应，以及消除 受损或异常的蛋白质。 然而，人们对此知之甚少。 蛋白水解途径的底物选择性机制 真核生物 本研究的目的是探讨 哺乳动物细胞的底物选择的调节和机制， N-末端规则途径，一种泛素依赖性蛋白水解途径， 底物选择性由底物的特性决定 蛋白质的氨基末端残基。 具体目标摘要： (1)确定N-末端规则途径在增殖中的选择性 和分化小鼠红白血病细胞，以直接检测 推测（贡达等人，1989年），泛素依赖的选择性 红系细胞中的蛋白质降解是分化特异性的。 (2)为了克隆编码E3 α蛋白的cDNA，其中一种泛素- 在N-末端规则中控制底物选择的蛋白质连接酶 途径; (3)过量表达和纯化E3 α蛋白用于生化分析; (4)为了从功能上剖析E3 α蛋白，使用两种有限的 蛋白水解和突变基因产物的表达; (5)鉴定编码其他E3蛋白的基因， 在泛素系统中的选择，通过与编码 E3 α 拟议的工作将检验关于下列因素的作用的具体假设： 红细胞中泛素依赖的蛋白质水解与N-末端规则 分化，并将启动底物的分子分析 在哺乳动物细胞中通过泛素系统进行选择。 了解 需要调节选择性蛋白水解来理解调节 细胞生理学的许多正常和异常方面，包括 衰老、致癌、生长和分化。

Selective protein degradation plays a direct role in regulating many cellular processes, including cell-cycle progression, normal and aberrant growth and differentiation, the stress response, and the elimination of damaged or otherwise abnormal proteins. However, little is known about the mechanisms governing the substrate selectivity of proteolytic pathways in eucaryotes. The objective of the research proposed here is to explore in mammalian cells the regulation and mechanism of substrate selection by the N-end rule pathway, a ubiquitin-dependent proteolytic pathway whose substrate selectivity is governed by the identity of the substrate protein's amino-terminal residue. Summary of specific aims: (1) To determine the selectivity of the N-end rule pathway in proliferating and differentiating murine erythroleukemia cells, to directly test the conjecture (Gonda et al., 1989) that the selectivity of ubiquitin-dependent protein degradation in erythroid cells is differentiation specific. (2) To clone cDNAs encoding the E3alpha protein, one of the ubiquitin- protein ligases which governs substrate selection in the N-end rule pathway; (3) To overexpress and purify the E3alpha protein for biochemical analyses; (4) To functionally dissect the E3alpha protein, using both limited proteolysis and expression of mutant gene products; (5) To identify genes encoding other E3 proteins that govern substrate selection in the ubiquitin system, via homology with the cDNAs encoding E3alpha The proposed work will test specific hypotheses regarding the role of ubiquitin-dependent proteolysis and the N-end rule in erythroid differentiation, and will initiate the molecular analysis of substrate selection by the ubiquitin system in mammalian cells. Understanding the regulation of selective proteolysis is needed to understand the regulation of many normal and abnormal aspects of cellular physiology, including aging, carcinogenesis, growth and differentiation.