Functions and Mechanisms of Deubiquitinating Enzymes

描述（由申请人提供）：真核生物具有高度保守的泛素（Ub）与蛋白质连接的酶系统。此外，与Ub不同但相关的多肽，称为Ub样蛋白或Ubls，也可以连接到蛋白质上。与每个Ubl的连接具有独特的机制和功能后果。SUMO（small Ub-related modifier）是一种高度分化的Ubl，SUMO连接系统在许多生物体中起着至关重要的作用，包括对人类生物学的重要贡献。Ub和SUMO与蛋白质的结合都可以在体内迅速逆转，并且专门的蛋白酶负责这些切割反应。PI一直在分析去泛素化酶（DUB）家族，主要是在酵母酿酒酵母中，第一个SUMO特异性蛋白酶ULP是在PI的实验室中发现的。ULP类蛋白酶是小鼠胚胎发生所必需的，并且在几种人类癌症中过表达.因此，这些酶已成为药物开发的有吸引力的靶标。 该项目的长期目标是获得DUBs和ULP在体内发挥的生理和机制作用的分子理解。在这个更新的应用程序中，建议的实验集中在酵母中的SUMO修饰和两个酵母desumoylating酶，Ulp1和Ulp2，SUMO系统功能的贡献。ULP的突变对生长和分裂有很强的影响，而Ulp1和SUMO本身一样，对细胞周期的进展至关重要。从广义上讲，目标是双重的：确定Ulp 1和Ulp 2酶的关键调控功能的分子基础，并阐明这些SUMO蛋白酶的分子特征，这些特征是它们在特异性和活性方面的巨大差异的原因。最近关于Ulp1和Ulp2的数据将研究导向了生物调节的几个特定领域。基于这些新发现，我们提出了以下目的：（1）研究Ulp 1在核孔复合体中的功能，特别是在前mRNA核滞留中的作用：（2）确定Ulp 2的新的调控特征，特别是在染色质调控中的作用，并确定Ulp 2非催化结构域在其体内调控中的作用;和（3）研究SUMO与底物的连接如何明显促进它们被异二聚体Hex3-Slx8 Ub连接酶泛素化。 公共卫生相关性：人类细胞的生长和行为，就像几乎所有复杂的生物体一样，是由小的专门蛋白质（称为泛素样蛋白质）与其他蛋白质的快速连接和去除控制的。已知控制这些过程的酶的缺陷会导致人类发育异常、神经退行性疾病和许多不同形式的癌症。该项目旨在加深我们对将某些泛素样蛋白从其伴侣中分离出来的酶的理解，其长期目标是开发治疗癌症和其他疾病患者的疗法。

DESCRIPTION (provided by applicant): Eukaryotes have a highly conserved enzymatic system for the ligation of ubiquitin (Ub) to proteins. Moreover, polypeptides distinct from but related to Ub, called Ub-like proteins or Ubls, can also be attached to proteins. Ligation to each Ubl has unique mechanistic and functional consequences. SUMO (small Ub-related modifier) is a highly divergent Ubl, and the SUMO ligation system has crucial roles in many organisms, including important contributions to human biology. Both Ub and SUMO attachment to proteins can be rapidly reversed in vivo, and specialized proteases are responsible for these cleavage reactions. The PI has been analyzing the deubiquitinating enzyme (DUB) family, primarily in the yeast Saccharomyces cerevisiae, and the first SUMO-specific proteases, the ULPs, were discovered in the PI's laboratory. ULP- class proteases are essential for embryogenesis in the mouse and are overexpressed in several human cancers. Therefore these enzymes have emerged as attractive targets for drug development. The long-range objective of the project is to gain a molecular understanding of the physiological and mechanistic roles played by DUBs and ULPs in vivo. In this renewal application, the proposed experiments are concentrated on SUMO modification in yeast and on the contributions of the two yeast desumoylating enzymes, Ulp1 and Ulp2, to SUMO system function. Mutation of either ULP has strong effects on growth and division, and Ulp1, like SUMO itself, is essential for cell-cycle progression. In broad terms, the goals are two-fold: Determine the molecular basis for key regulatory functions of the Ulp1 and Ulp2 enzymes and elucidate the molecular features of these SUMO proteases that are responsible for their dramatic differences in specificity and activity. Recent data on Ulp1 and Ulp2 have directed the studies into several specific areas of biological regulation. Based on these new findings, the following Aims are proposed: (1) Examine the function of Ulp1 at the nuclear pore complex, particularly its role in pre-mRNA nuclear retention; (2) Determine novel regulatory features of Ulp2, especially its role in chromatin regulation, and determine the contributions of Ulp2 noncatalytic domains to its in vivo regulation; and (3) Examine how SUMO attachment to substrates apparently promotes their ubiquitination by the heterodimeric Hex3-Slx8 Ub ligase. PUBLIC HEALTH RELEVANCE: The growth and behavior of human cells, like those of virtually all complex organisms, is controlled by rapid attachment and removal of small specialized proteins (called ubiquitin-like proteins) to and from other proteins. Defects in the enzymes that control these processes are known to cause human developmental abnormalities, neurodegenerative disorders, and many different forms of cancer. This project aims to deepen our understanding of the enzymes that detach certain ubiquitin-like proteins from their partners, with the long-term goal of developing therapies to treat patients suffering from cancer and other diseases.