Ubiquitin Ligases, Mechanisms and Functions of the N-End Rule Pathway

N 端规则通路的泛素连接、机制和功能

• 批准号: 7777747
• 负责人: ALEXANDER J VARSHAVSKY
• 金额: $ 65.15万
• 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 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777747
• 关键词: Amidohydrolases; Animal Model; Biological Process; Caring; Cells; Cloning; Complex; Crystallization; DNA Repair Enzymes; Defect; Development; Dissection; Enzymes; Escherichia coli; Eukaryota; Family; Figs - dietary; Genetic Code; Glutamine; Grant; Heme; Human; In Vitro; Investigation; Laboratories; Ligase; MGMT gene; Malignant Neoplasms; Mammals; Mechanics; Medical; Methods; Molecular Genetics; Mouse Strains; Mus; N-terminal; Nerve Degeneration; Nitric Oxide; O(6)-Methylguanine-DNA Methyltransferase; Oxidation-Reduction; Pathway interactions; Physiological; Play; Prokaryotic Cells; Proteins; Proteolysis; Research Support; Role; Saccharomyces cerevisiae; Structure; Syndrome; System; Techniques; Transferase; Ubiquitin; Update; Work; Yeasts; amidase; base; crosslink; deamidation; human disease; in vivo; inhibitor/antagonist; insight; protein degradation; public health relevance; sensor; ubiquitin ligase

DESCRIPTION (provided by applicant): Proteolysis by the ubiquitin system plays major roles in a multitude of biological processes. Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. This is the resubmission of renewal application for the DK39520 grant, currently in its 25th year of support. Two of several discoveries during the preceding period of support are the new functions of the N-end rule pathway as a sensor, through different mechanisms, of both heme and nitric oxide. Another discovery is the identification and analysis of an entirely new class of aminoacyl-transferases that underlie the N-end rule pathway in prokaryotes. One focus of the present renewal application is on physiological substrates, mechanisms and functions of ubiquitin ligases in both yeast and mammalian N-end rule pathways. Hence the (overdue) change of DK39520 title, from "Mechanics and Functions of the Yeast N-End Rule Pathway" to "Ubiquitin Ligases, Mechanisms and Functions of the N-End Rule Pathway". This title more accurately describes the scope (including the range of model organisms) of our research supported by the DK39520 grant. Updated Specific Aims: 1) Exploration of the discovery that S. cerevisiae MGT1, a key DNA repair enzyme, is a physiological substrate of two distinct Ub ligases, UBR1 and UFD4. Extension of these insights to MGMT, the mammalian counterpart of yeast MGT1. 2) Development and applications of a new method, termed the N-degron capture (NDC), for discovering physiological substrates of the N-end rule pathway. This method is based on an altered genetic code, a photoactivatable ClpS (the recognition component of the E. coli N-end rule pathway), and target- selective UV-crosslinking in intact cells or in vitro. 3) Studies of S. cerevisiae UBR1 and the mouse UBR-family Ub ligases of the N-end rule pathway, including their crystal structures, further explorations of their recently discovered interactions with heme, as well as their physiological substrates. 4) The deamidation branch of the N-end rule pathway: isolation, cloning and molecular genetic dissection of the mouse NTAQ1, a glutamine-specific N-terminal amidase, NtQ-amidase. The present resubmission application for the renewal of the present grant contains substantial experimental updates (produced during the last 6-7 months) of most Aims. PUBLIC HEALTH RELEVANCE The proposed studies in this renewal application, based in part on our recent discoveries, will illuminate the mechanisms and functions of regulated protein degradation in yeast and mammals, including humans. Either inborn or acquired defects in these systems, which include the ubiquitin system, are a major cause of many human diseases, including cancer and neurodegenerative syndromes. Understanding, in depth and detail, the mechanisms and functions of the ubiquitin system will result in better therapies for these and other medical problems, including currently intractable ones.

描述（由申请人提供）：泛素系统的蛋白水解在多种生物过程中起主要作用。泛素依赖性N端规则途径的底物包括具有不稳定N末端残基的蛋白质。这是DK39520赠款的续签申请，目前已获得25年的支持。在前面的支持期间，几个发现中有两个是通过不同机制，血红素和一氧化氮的不同机制作为传感器的新功能。另一个发现是对原核生物N端规则途径构成的全新氨基酰基转移酶的识别和分析。本更新应用的一个重点是酵母和哺乳动物N端规则途径中泛素连接酶的生理底物，机制和功能。因此，DK39520标题的（逾期）从“酵母N端规则途径的力学和功能”到“ N-End Rule pathway的泛素连接酶，机制和功能”的“机械和功能”。此标题更准确地描述了DK39520赠款支持的我们研究的范围（包括模型生物的范围）。更新的特定目的：1）探索发现的发现，即酿酒酵母MGT1是一种关键的DNA修复酶，是两个不同UB连接酶UBR1和UFD4的生理底物。将这些见解扩展到酵母MGT1的哺乳动物对应物Mgmt。 2）一种新方法的开发和应用，称为N-Degron捕获（NDC），用于发现N端规则途径的生理底物。该方法基于改变的遗传密码，可光活化的CLP（大肠杆菌N端规则途径的识别成分）以及完整细胞或体外的目标 - 选择性紫外线链接。 3）酿酒酵母UBR1和N端规则途径的小鼠UBR-Family UB连接酶，包括它们的晶体结构，进一步探索了它们最近发现的与血红素的相互作用及其生理底物。 4）N端规则途径的脱氨酸分支：小鼠NTAQ1的分离，克隆和分子遗传解剖是一种谷氨酰胺特异性的N末端amidase，NTQ-酰胺酶。本授予赠款的续签申请中，大多数目标中包含大量实验更新（在过去6-7个月中产生）。公共卫生相关性的这一续签应用中提出的研究部分基于我们最近的发现，将阐明酵母和包括人类在内的酵母和哺乳动物中受调节蛋白质降解的机制和功能。这些系统中的先天或获得的缺陷，包括泛素系统，是许多人类疾病的主要原因，包括癌症和神经退行性综合症。在深入和细节上，了解泛素系统的机制和功能将为这些和其他医学问题（包括目前棘手的问题）提供更好的疗法。