Functions and Mechanisms of Deubiquitinating Enzymes

描述（由申请人提供）：真核细胞具有高度保守的酶系统，用于将泛素（Ub）连接至细胞蛋白。与Ub不同但相关的多肽，称为Ub样蛋白或Ubls，也可以与其他蛋白质连接。与每个Ubl的连接具有独特的机制和功能后果。SUMO（酵母中的Smt 3）是一种高度分化的Ubl，SUMO连接系统具有许多关键功能，包括对人类健康和生物学的重要贡献。Ub和SUMO与蛋白质的结合都可以被专门的蛋白酶迅速逆转。在分析酿酒酵母中的去泛素化酶（Dub）家族的同时，发现了一个新的SUMO特异性半胱氨酸蛋白酶家族，其一级序列与Dub无关。这些Ubl-specific proteins（Ulps）在细胞周期进程、染色体稳定性和生长中具有重要功能。 该项目的长期目标是从分子水平上了解Dubs和Ulps所起的生理和机制作用。在该更新申请中，所提出的实验集中于酵母中的SUMO修饰（“SUMO化”）和两种酵母去小泛素化酶Ulpl和Ulp 2/Smt 4对SUMO系统的功能的贡献。具体目标是： 1.通过一系列生物化学、遗传学和蛋白质组学方法鉴定酵母中的SUMO结合蛋白，尤其是必需蛋白。 2.确定酵母中依赖于SUMO的基本生理过程，并确定脱糖基化酶对这些过程的机械贡献。 3.验证已鉴定底物的SUMO化，定位其SUMO连接位点，并分析特定SUMO蛋白修饰的后果。 4.确定Ulpl和U之间特异性和活性差异的分子基础|β 2去糖基化酶。

DESCRIPTION (provided by applicant): Eukaryotic cells have a highly conserved enzymatic system for the ligation of ubiquitin (Ub) to cellular proteins. Polypeptides distinct from but related to Ub, called Ub-like proteins or Ubls, can also be ligated to other proteins. Ligation to each Ubl has unique mechanistic and functional consequences. SUMO (Smt3 in yeast) is a highly divergent Ubl, and the SUMO ligation system has many crucial functions, including important contributions to human health and biology. Both Ub and SUMO attachment to proteins can be rapidly reversed by specialized proteases. While analyzing the deubiquitinating enzyme (Dub) family in the yeast Saccharomyces cerevisiae, a novel family of SUMO-specific cysteine proteases unrelated in primary sequence to the Dubs was discovered. These Ubl-specific proteases (Ulps) have important functions in cell cycle progression, chromosome stability, and 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 Ulps. In this renewal application, the proposed experiments are concentrated on SUMO modification ("sumoylation") in yeast and on the contributions of the two yeast desumoylating enzymes, Ulpl and Ulp2/Smt4, to the function of the SUMO system. The specific aims are to: 1. Identify SUMO-conjugated proteins in yeast, especially essential ones, by an array of biochemical, genetic, and proteomic approaches. 2. Identify essential physiological processes in yeast that depend on SUMO and determine the mechanistic contributions of the desumoylating enzymes to these processes. 3. Verify the sumoylation of identified substrates, locate their sites of SUMO attachment, and analyze the consequences of specific SUMO-protein modifications. 4. Determine the molecular basis for the differences in specificity and activiity between the Ulpl and U|p2 desumoylating enzymes.