UBIQUITIN-DEPENDENT PROTEOLYSIS IN MAMMALIAN CELLS

选择性蛋白质降解在调节许多 细胞过程，包括细胞周期进程、正常和异常 生长和分化、应激反应和消除 受损的或其他异常的蛋白质。然而，人们对此知之甚少 蛋白质降解途径底物选择性的调控机制 真核生物。这里提出的研究的目的是探索在 哺乳动物细胞对底物选择的调控及其机制 N-末端规则途径，泛素依赖的蛋白分解途径，其 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)测定N-末端规则通路在增殖过程中的选择性 和分化小鼠红白血病细胞，以直接测试 推测(Gonda et al.，1989)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)克隆E3α蛋白的编码基因。 在N-端规则中控制底物选择的蛋白质连接酶 途径； (3)高效表达和纯化E3α蛋白，用于生化分析； (4)对E3α蛋白进行功能分离，利用两者有限的 蛋白降解和突变基因产物的表达； (5)确定编码控制底物的其他E3蛋白的基因 泛素系统中的选择，通过与cDNA编码的同源性 E3Alpha 这项拟议中的工作将检验关于 红系泛素依赖的蛋白分解和N-端规则 分化，将启动底物的分子分析 哺乳动物细胞中泛素系统的选择。了解 需要对选择性蛋白分解的调节才能理解这种调节。 细胞生理的许多正常和不正常的方面，包括 衰老、癌变、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.