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

描述(由申请人提供)：泛素系统的蛋白质分解作用在许多生物过程中起主要作用。泛素依赖的N-末端规则通路的底物包括具有不稳定N-末端残基的蛋白质。这是重新提交的DK39520赠款的续签申请，目前是其支持的第25个年头。在前一支持时期的几项发现中，有两项是N-末端规则通路的新功能，通过不同的机制，作为血红素和一氧化氮的传感器。另一项发现是鉴定和分析了一类全新的氨基酰转移酶，它是原核生物N端规则通路的基础。目前更新应用的一个焦点是酵母和哺乳动物N-端规则通路中泛素连接酶的生理底物、机制和功能。因此，DK39520的标题从“酵母N-端规则途径的机制和功能”更改为“泛素连接、N-端规则途径的机制和功能”。这个标题更准确地描述了我们由DK39520资助的研究的范围(包括模式生物的范围)。更新的具体目标：1)探索发现酿酒酵母MGT1是一种关键的DNA修复酶，是两种不同的Ub连接酶UBR1和UFD4的生理底物。将这些洞察力扩展到MGMT，即酵母MGT1的哺乳动物对应物。2)一种新的发现N-末端规则通路的生理底物的方法--N-退化捕获(NDC)的发展和应用。这种方法基于改变的遗传密码、可光激活的CLPs(大肠杆菌N-末端规则通路的识别成分)和在完整细胞或体外的靶向选择性紫外光交联。3)酿酒酵母UBR1和小鼠UBR家族N端规则连接酶的研究，包括它们的晶体结构，以及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-末端规则通路的脱酰胺分支：小鼠NTAQ1-谷氨酰胺专一性N-末端酰胺酶NTQ-酰胺酶的分离、克隆和分子遗传学分析。目前重新提交的续期申请包含了MOST AIMS的大量实验更新(在过去6-7个月中产生)。公共卫生相关性部分基于我们最近的发现，在这一更新应用中拟议的研究将阐明酵母和哺乳动物(包括人类)中受调控的蛋白质降解的机制和功能。这些系统中包括泛素系统的先天或后天缺陷，是许多人类疾病的主要原因，包括癌症和神经退行性综合征。深入和详细地了解泛素系统的机制和功能将导致对这些和其他医学问题的更好治疗，包括目前难以解决的问题。

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.