Functions and Mechanisms of Deubiquitinating Enzymes

描述(申请人提供)：真核生物有一个高度保守的酶系统，用于连接泛素(Ub)到蛋白质。此外，与Ub不同但与Ub相关的多肽，称为Ub样蛋白或UBL，也可以附着在蛋白质上。对每个UbL的结扎具有独特的机制和功能后果。相扑(Small Ub-Related Modify)是一种高度分化的UbL，相扑连接系统在许多生物体中发挥着重要作用，包括对人类生物学的重要贡献。在体内，Ub和相扑与蛋白质的结合都可以迅速逆转，而特殊的蛋白酶负责这些切割反应。国际酿酒协会一直在分析去泛素酶(DUB)家族，主要是在酵母酿酒酵母中，第一个相扑特异的蛋白酶，ULPS，是在国际酿酒协会的实验室中发现的。ULP类蛋白是小鼠胚胎发育所必需的，在几种人类癌症中过度表达。因此，这些酶已成为药物开发的有吸引力的靶点。 该项目的长期目标是从分子上了解DUBS和ULPs在体内发挥的生理和机制作用。在这一更新应用中，拟议的实验主要集中在酵母中的相扑修饰以及两种酵母脱苏糖化酶Ulp1和Ulp2对相扑系统功能的贡献。任何一种ULP的突变都会对生长和分裂产生强烈的影响，而Ulp1就像相扑本身一样，对细胞周期的进展是必不可少的。从广义上讲，目标有两个：确定Ulp1和Ulp2酶关键调控功能的分子基础，并阐明导致它们特异性和活性显著差异的这些相扑蛋白酶的分子特征。关于Ulp1和Ulp2的最新数据将研究引向了生物调控的几个特定领域。基于这些新的发现，提出了以下目标：(1)研究Ulp1在核孔复合体中的功能，特别是它在前mRNA核保持中的作用；(2)确定Ulp2的新调节功能，特别是它在染色质调节中的作用，并确定Ulp2非催化结构域在体内对其调节的贡献；(3)研究SUMO与底物的结合如何通过异二聚体Hex3-SLx8 Ub连接酶明显促进它们的泛素化。 与公共健康相关：与几乎所有复杂生物体一样，人类细胞的生长和行为是由小的专门蛋白质(称为泛素样蛋白)快速附着和从其他蛋白质上或从其他蛋白质上移除所控制的。控制这些过程的酶的缺陷已知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伴侣中分离出来的酶的理解，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.