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

项目总结/摘要 泛素系统的蛋白水解在许多生物过程中起着重要作用。 泛素依赖性N-末端规则途径的底物包括具有不稳定N-末端的蛋白质。 残基这是DK 39520补助金的续期申请，目前已进入支持的第24个年头。两 在前一个支持阶段的几个发现是N端规则途径的新功能 作为血红素和一氧化氮的传感器，通过不同的机制。另一个发现是 N-末端规则下一类全新的氨酰转移酶的鉴定和分析 原核生物中的途径。本更新申请的焦点在于生理底物， 泛素连接酶在酵母和哺乳动物N-末端规则途径中的机制和功能。因此 DK 39520的名称（过期）变更，从《酵母N端规则途径的机制和功能》 泛素连接酶，N端规则途径的机制和功能。新标题更多 准确描述了我们的研究范围（包括模式生物的范围）， DK 39520拨款。具体目标： 1)研究S。cerevisiae MGT 1是一种关键的DNA修复酶，我们最近发现它是一种 N端规则Ub连接酶UBR 1的生理底物。将这些见解扩展到MGMT， 哺乳动物DNA修复酶（MGT 1的对应物），以及哺乳动物的N-末端规则Ub连接酶。 2)我们的新体内方法用于发现N端规则底物的系统应用。这 该方法基于使用改变的遗传密码，一种ClpS的UV可激活衍生物（靶向 N-末端规则途径的组分）和完整细菌或真核细胞中的UV交联。 3)酵母和哺乳动物UBR家族Ub连接酶的研究，包括它们的生理活性， 基板; UBR 1的晶体结构的测定;和进一步探索的发现， N-末端规则途径是血红素和一氧化氮传感器。 4)对最近发现的乙酰转移酶（R-转移酶）ATE 1的进一步研究 特异性结合哺乳动物细胞中的一小部分mRNA。 5)N端规则途径脱酰胺分支的新组分和功能：分离， 克隆和小鼠NTAQ 1，谷氨酰胺特异性N-末端酰胺酶的分子遗传学解剖。

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.