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

项目摘要/摘要 泛素系统的蛋白质分解作用在多种生物过程中起着重要作用。 泛素依赖的N-末端规则通路的底物包括具有不稳定N-末端的蛋白质 残留物。这是DK39520赠款的续签申请，目前是其支持的第24个年头。其中两个 在前一支持期间的几个发现是N-端规则路径的新功能 作为传感器，通过不同的机制，检测血红素和一氧化氮。另一项发现是 一类构成N-末端规则的全新氨酰转移酶的鉴定和分析 原核生物中的途径。本更新应用的焦点是生理底物， 泛素连接酶在酵母和哺乳动物N-端规则通路中的机制和功能。因此 从酵母N-末端规则途径的机制和功能看DK39520标题的(过期)变化 泛素连接、N-端规则通路的机制和功能。这个新标题更多 准确描述我们的研究范围(包括模式生物的范围) 39520丹麦克朗赠款。具体目标： 1)酿酒酵母MGT1的研究，这是我们最近发现的一种关键的DNA修复酶 N末端规则的生理底物S Ub连接酶Ubr1。将这些见解扩展到MGMT，a 哺乳动物DNA修复酶(MGT1的对应物)，以及哺乳动物的N端规则？S Ub连接酶。 2)我们在体内发现N-末端规则底物的新方法的系统应用。这 该方法基于改变的遗传密码的使用，这是一种可紫外线激活的CLPs的衍生物(靶向 N-末端规则途径的组成部分)，以及完整细菌或真核细胞中的UV-交联性。 3)酵母和哺乳动物UbR家族Ub连接酶的研究，包括它们的生理功能 底物；UBr1晶体结构的测定；以及对发现的进一步探索 N末端调节通路是血红素和一氧化氮的感受器。 4)对最近发现的精氨基转移酶(R-转移酶)ATE1的进一步调查 特异性地与哺乳动物细胞中的一小部分mRNAs结合。 5)N-端规则通路脱酰胺分支的新成分和新功能：分离， 小鼠谷氨酰胺特异性N-末端酰胺酶NTAQ1的克隆和分子遗传学分析。

Project Summary/Abstract 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 renewal application for the DK39520 grant, currently in its 24th 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. A 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 new title more accurately describes the scope (including the range of model organisms) of our research supported by the DK39520 grant. Specific Aims: 1) Studies of S. cerevisiae MGT1, a key DNA repair enzyme that we recently identified as a physiological substrate of the N-end rule¿s Ub ligase UBR1. Extension of these insights to MGMT, a mammalian DNA repair enzyme (the counterpart of MGT1), and to N-end rule¿s Ub ligases of mammals. 2) Systematic applications of our new in vivo method for discovering N-end rule substrates. This method is based on the use of altered genetic code, a UV-activatable derivative of ClpS (the targeting component of N-end rule pathway), and UV-crosslinking in intact bacterial or eukaryotic cells. 3) Studies of the yeast and mammalian UBR-family Ub ligases, including their physiological substrates; determination of the crystal structure of UBR1; and further exploration of the discoveries that the N-end rule pathway is a sensor of heme and nitric oxide. 4) Further investigations of the recent discovery that the arginyl-transferase (R-transferase) ATE1 specifically binds to a small subset of mRNAs in mammalian cells. 5) New components and functions of the deamidation branch of the N-end rule pathway: isolation, cloning and molecular genetic dissection of mouse NTAQ1, a glutamine-specific N-terminal amidase.