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

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发挥着重要作用。泛素依赖性 N 端规则途径的底物包括具有不稳定 N 端残基的蛋白质。这是 DK39520 补助金续展申请的重新提交，目前已进入第 25 个支持年头。之前支持期间的几个发现中的两个是 N 端规则通路作为血红素和一氧化氮的传感器的新功能，通过不同的机制。另一个发现是鉴定和分析了一类全新的氨酰基转移酶，它是原核生物 N 端规则途径的基础。本次更新申请的重点之一是酵母和哺乳动物 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（逾期）从“酵母 N 端规则途径的力学和功能”更改为“N 端规则途径的泛素连接、机制和功能”。这个标题更准确地描述了我们由 DK39520 资助支持的研究范围（包括模式生物的范围）。更新的具体目标：1) 探索酿酒酵母 MGT1（一种关键的 DNA 修复酶）是两种不同的 Ub 连接酶 UBR1 和 UFD4 的生理底物的发现。将这些见解扩展到 MGMT（酵母 MGT1 的哺乳动物对应物）。 2）开发和应用一种新方法，称为N-降解决定子捕获（NDC），用于发现N-末端规则途径的生理底物。该方法基于改变的遗传密码、可光激活的 ClpS（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 UV 交联。 3）酿酒酵母UBR1和小鼠UBR家族Ub连接酶N端规则途径的研究，包括它们的晶体结构，进一步探索它们最近发现的与血红素的相互作用，以及它们的生理底物。 4）N端规则途径的脱酰胺分支：小鼠NTAQ1（一种谷氨酰胺特异性N端酰胺酶，NtQ-酰胺酶）的分离、克隆和分子遗传学解剖。目前重新提交的现有拨款续展申请包含大多数 Aims 的大量实验性更新（在过去 6-7 个月内产生）。公共健康相关性 本次更新申请中拟议的研究部分基于我们最近的发现，将阐明酵母和哺乳动物（包括人类）中受调节的蛋白质降解的机制和功能。这些系统（包括泛素系统）的先天性或后天性缺陷是许多人类疾病（包括癌症和神经退行性综合症）的主要原因。深入详细地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）提供更好的治疗方法。

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.