MECHANICS AND FUNCTIONS OF THE N-END RULE PATHWAY

N 端规则路径的机制和功能

• 批准号: 7176060
• 负责人: ALEXANDER J VARSHAVSKY
• 金额: $ 63.17万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-02-11 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7176060
• 关键词: Amidohydrolases; Bacteria; Biochemical Genetics; Biological Process; Cells; Dissection; Genetic; Grant; Half-Life; Head; Homologous Gene; Laboratories; Mammals; Mechanics; Mitochondria; N-terminal; Organism; Pathway interactions; Peptides; Physiological; Play; Progress Reports; Proteins; Proteolysis; Regulation; Research; Role; Saccharomyces cerevisiae; Signal Transduction; Stress; System; Techniques; Ubiquitin; amidase; base; cell growth; in vivo; inhibitor/antagonist; insight; multicatalytic endopeptidase complex; mutant; protein degradation; response

Ubiquitin is a 76-residue protein that exists in cells either free or conjugated to many other proteins. Selective degradation of proteins by the ubiquitin/proteasome-dependent pathways plays a role in a multitude of biological processes, including cell growth and differentiation, signal transduction and responses to stress. One ubiquitin/proteasome-dependent proteolytic system is the N-end rule pathway, identified by the laboratory in 1986. The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is present in all organisms examined, from mammals to bacteria. Studies supported by the present grant ("Mechanics and Functions of the N-end Rule Pathway", DK39520), currently in its 11th year, have yielded significant insights into the N-end rule's functions and mechanisms, as described in the Progress Report. The objective of the research described in this renewal application is to advance the understanding of the N-end rule pathway and related aspects of the ubiquitin system. Specific aims are: 1) Regulation of peptide import by the N-end rule pathway: biochemical and genetic studies; 2) A fusion-based screen for physiological substrates of the N-end rule pathway in S. cerevisiae; 3) Isolation and analysis of regulators of the N-end rule pathway; 4) Screens for mutants whose viability requires the presence of the N-end r; 5) The use of suppression subtractive hybridization to identify new functions of the N-end rule pathway; 6) Analysis of a mitochondrial function of the Ntalp N-terminal amidase in S. cerevisiae; 7) A biochemico-genetic approach to identification of physiological N-end rule substrates; 8) A "double-headed" inhibitor of the N-end rule pathway; 9) The "two-ubiquitin" technique and its application to the problem of cotranslational proteolysis; and 10) Biochemical and genetic dissection of the UBRI-encoded Nrecognin and Ubr2p, a recently identified homolog of Ubrlp in S. cerevisiae.

泛素是存在于细胞中的76个残基的蛋白质，其以游离形式或与许多其他蛋白质缀合。通过泛素/蛋白酶体依赖性途径选择性降解蛋白质在许多生物过程中发挥作用，包括细胞生长和分化、信号转导和对应激的反应。一种泛素/蛋白酶体依赖的蛋白水解系统是N-末端规则途径，由实验室在1986年鉴定。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。N端规则途径存在于从哺乳动物到细菌的所有生物体中。目前资助的研究（“N端规则途径的机制和功能”，DK 39520），目前已进入第11个年头，对N端规则的功能和机制产生了重要的见解，如进度报告所述。本更新申请中描述的研究目的是促进对N端规则途径和遍在蛋白系统相关方面的理解。具体目标是：1）N-末端规则途径对肽输入的调控：生化和遗传学研究; 2）基于融合的S.酿酒酵母; 3）分离和分析N-末端规则途径的调节子; 4）筛选其生存力需要N-末端r存在的突变体; 5）使用抑制消减杂交来鉴定N-末端规则途径的新功能; 6）分析S.酿酒酵母; 7）鉴定生理性N-末端规则底物的生物化学-遗传学方法; 8）N-末端规则途径的“双头”抑制剂; 9）“双泛素”技术及其在共翻译蛋白质水解问题中的应用;和10）UBRI编码的Nrecognin和Ubr 2 p的生物化学和遗传学解剖，Ubr 2 p是最近在S.啤酒。