FUNCTIONS AND MECHANISMS OF DEUBIQUITINATING ENZYMES

描述：（改编自申请人的摘要）真核细胞具有高度的 用于将泛素连接到细胞内的保守酶系统 蛋白质，这对广泛的生物过程至关重要。 与泛素不同但相关的多肽- Ubls -也可以是 与其他蛋白质连接。Ubls具有独特的功能，与泛素不同， 似乎不靶向蛋白质以被蛋白酶体降解。缺陷 这些途径与包括癌症在内的多种疾病有关， 免疫系统功能障碍和阿尔茨海默病泛素和Ubls都是 作为前体合成，需要酶促处理C-末端 肽或氨基酸;此外，泛素和Ubl两者都附着于 蛋白质是可逆的。专门的和高度保守的蛋白酶是 负责这些加工反应。去泛素化酶（Dub） 酿酒酵母（Saccharomycescerevisiae）中的家族一直是目前研究的重点 格兰特.在试图确定这一大群人中是否有人 蛋白酶可能处理称为Smt 3或SUMO-1的Ubl，这是一类新的蛋白酶， 发现了与任何Dub无关的蛋白酶。这些酶是高度 在细胞周期进程和细胞生长中起关键作用。的 该项目的长期目标是获得对分子的了解 Dubs和Smt 3-裂解所起的生理和机械作用 内切酶 在本申请中，概述了解决两个相关领域的实验。 首先，称为Doa 4的酵母Dub的工作将继续进行。Doa 4是一个中心 泛素系统的酶，和doa 4突变体在一系列的缺陷， 生理途径。为了理解这些缺陷，一些机械的 将探讨Doa 4行动的各个方面。在申请的第二部分中， 实验提出，检查最近发现的酵母 Smt 3特异性蛋白酶，Ulp 1和Smt 4/Ulp 2，使用分子遗传学和 生物化学方法。具体而言，他们建议：1）隔离和 表征在突变体中积累到高水平的泛素化物质 doa 4细胞; 2）分析Doa 4与两种细胞之间的机制联系， 蛋白酶体和空泡蛋白分选/内吞途径; 3）鉴定 来自ulp 1和smt 4突变体的Smt 3-蛋白缀合物，并确定 这些蛋白质的Smt 3修饰的功能后果; 4）表征 Ulp 1基因表达调控及其特异性研究 接近。

DESCRIPTION: (adapted from applicant's abstract) Eukaryotic cells have a highly conserved enzymatic system for the ligation of ubiquitin to intracellular proteins, which is crucial for a wide range of biological processes. Polypeptides distinct from but related to ubiquitin - Ubls - can also be ligated to other proteins. Ubls have unique functions and, unlike ubiquitin, do not appear to target proteins for degradation by the proteasome. Defects in these pathways are being linked to a variety of disorders including cancer, immune system dysfunction, and Alzheimer's disease. Ubiquitin and Ubls are all synthesized as precursors, requiring enzymatic processing of C-terminal peptides or amino acids; furthermore, both ubiquitin and Ubl attachment to proteins is reversible. Specialized and highly conserved proteases are responsible for these processing reactions. The deubiquitinating enzyme (Dub) family in the yeast Saccharomyces cerevisiae has been the focus of the present grant. While trying to determine whether any members of this large group of proteases might process the Ubl called Smt3 or SUMO-1, a novel class of proteases unrelated to any Dub was discovered. These enzymes are highly conserved and play key roles in cell cycle progression and cell growth. The long-term objective of the project is to gain a molecular understanding of the physiological and mechanistic roles played by Dubs and by the Smt3-cleaving enzymes. In this application, experiments that address two related areas are outlined. First, work on the yeast Dub called Doa4 will continue. Doa4 is a central enzyme of the ubiquitin system, and doa4 mutants are defective in an array of physiological pathways. To understand these defects, several mechanistic aspects of Doa4 action will be explored. In the second part of the application, experiments are proposed that examine the recently discovered yeast Smt3-specific proteases, Ulp1 and Smt4/Ulp2, using both molecular genetic and biochemical approaches. Specifically, they propose to: 1) isolate and characterize the ubiquitinated species that accumulate to high levels in mutant doa4 cells; 2) analyze the mechanistic connections between Doa4 and both the proteasome and the vacuolar-protein-sorting/endocytosis pathway; 3) identify the Smt3-protein conjugates from ulp1 and smt4 mutants and determine the functional consequences of Smt3 modification of these proteins; 4) characterize the regulation and specificity of Ulp1 by biochemical and molecular genetic approaches.