STUDIES ON SELECTIVE PROTEIN TURNOVER

摘要 使用一种新的方法，最近发现， 识别细胞内的氨基末端残基 蛋白质介导蛋白质的代谢稳定性， 显然也有可能调节其稳定性（1）。 本提案所述研究的目的是 探索这一见解的含义（“N端规则”途径 ① ①）。 具体目标概述： 1)用除以下之外的测试蛋白质构建泛素融合体 β-半乳糖苷酶（β gal），特别是与小鼠的融合物 二氢叶酸还原酶（DHFR）。 这些泛蛋白融合体用于 通过N-末端蛋白水解规则解决靶向机制 通路 2)确定是否使用beta定义的N端规则 当用不相关的测试蛋白质定义时，gal是相同的。 3)确定是否使用了 酵母酿酒cerevisiae与E.蛙大肠杆菌 卵母细胞和培养的哺乳动物细胞。 4)在靶向的， 多重泛素化蛋白。 5)分离纯化及分子遗传学分析 来自酵母的泛素特异性加工。 该蛋白酶用于 原核生物中N-末端规则的直接测试（E. coli）。 6)利用一种新的筛选方法分离酵母突变体， 选择性蛋白质周转 7)体外选择性蛋白质周转机制的研究 泛素-β gal融合体（1）和类似的定义明确的 印刷受体. 8)基因调控的生化和分子遗传学分析 和氨基酸的翻译后缀合的功能， 蛋白质的氨基末端。 对选择性蛋白质周转的详细机制理解， 仍然是一个遥远的目标，预计将证明是有益的，如果不是必要的 为了解决许多目前尚未解决的生物学问题， 从调节细胞周期进程到分化、应激 反应、致癌作用和衰老。

Abstract Using a new approach, it has been recently found that the recognition of an amino-terminal residue in an intracellular protein mediates the metabolic stability of the protein and apparently also the potential for regulation of its stability (1). The objective of the research described in this proposal is to explore the implications of this insight (the "N-end rule" pathway (1)). Summary of Specific Aims: 1) Construction of ubiquitin fusions with test proteins other than beta-galactosidase (beta gal), in particular fusions with the mouse dihydrofolate reductase (DHFR). Use of these ubiquitin fusions to address the mechanism of targeting by the N-end rule proteolytic pathway. 2) Determination of whether the N-end rule as defined with beta gal is the same when defined with unrelated test proteins. 3) Determination of whether the N-end rule as defined in the yeast S. cerevisiae is the same when defined in E. coli, frog oocytes and cultured mammalian cells. 4) Mapping positions of ubiquitin moieties within a targeted, mulitply ubiquitinated protein. 5) Isolation, purification and molecular genetic analysis of the ubiquitin-specific processing from yeast. Use of this protease for direct testing of the N-end rule in prokaryotes (E. coli). 6) Use of a novel screening approach to isolate yeast mutants in selective protein turnover. 7) Mechanistic studies on selective protein turnover in vitro using both ubiquitin-beta gal fusions (1) and analogous well-defined substrates. 8) Biochemical and molecular genetic analyses of the regulation and function of posttranslational conjugation of amino acids to amino-termini of proteins. Detailed mechanistic understanding of selective protein turnover, still a distant goal, is expected to prove helpful if not essential for solving many of the currently outstanding biological problems, from regulation of cell cycle progression to differentiation, stress responses, carcinogenesis and aging.