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 泛素与胞内连接的保守酶系统 蛋白质，这对广泛的生物过程至关重要。 与泛素不同但与泛素相关的多肽-UBLS-也可以 与其他蛋白质连接。UBL具有独特的功能，与泛素不同，UBL 似乎不以蛋白酶体降解的蛋白质为目标。中的缺陷 这些通路与包括癌症在内的各种疾病有关， 免疫系统功能障碍和阿尔茨海默病。泛素和UBL都是 作为前体合成，需要C-末端的酶处理 多肽或氨基酸；此外，泛素和UbL都附着于 蛋白质是可逆的。专门化和高度保守的蛋白酶是 负责这些加工反应。脱泛素酶(DUB) 酵母家族中的酿酒酵母一直是目前研究的焦点 格兰特。在试图确定这一大群人中是否有任何成员 蛋白水解酶可能会处理被称为Smt3或SUMO-1的UbL，这是一类新的 发现了与任何DUB无关的蛋白酶。这些酶具有很高的 保守并在细胞周期进程和细胞生长中起关键作用。这个 该项目的长期目标是从分子水平上了解 Dubs和Smt3-裂解所起的生理和机制作用 酵素。 在本申请中，概述了涉及两个相关领域的实验。 首先，名为Doa4的酵母Dub的工作将继续进行。Doa4是一个中心 泛素系统的酶和doa4突变体在一系列 生理途径。为了理解这些缺陷，有几个机械论 将探讨Doa4行动的各个方面。在应用程序的第二部分中， 有人提议用实验来检验最近发现的酵母。 Smt3特异的蛋白酶，Ulp1和Smt4/Ulp2，使用分子遗传和 生化方法。具体地说，他们建议：1)隔离和 描述在突变中积累到高水平的泛素化物种的特征 DOA4细胞；2)分析DOA4和两者之间的机械联系 蛋白酶体与空泡蛋白分选/内吞途径；3)鉴定 将ulp1和smt4突变体中的Smt3-蛋白偶联，并确定 这些蛋白质Smt3修饰的功能后果；4)表征 Ulp1的生化和分子遗传学调控及其特异性 接近了。

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.