Functions and Mechanisms of Deubiquitinating Enzymes

描述(申请人提供)：真核细胞有一个高度保守的酶系统，用于连接泛素到蛋白质。此外，不同于泛素但与泛素相关的多肽，称为泛素样蛋白或UBL，也可以与蛋白质结合。对每个UbL的结扎具有独特的机制和功能后果。相扑(在酵母中由SMT3编码)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至关重要的作用，包括对人类生物学的重要贡献。相扑和泛素一样，是以前体形式合成的，需要对C末端的多肽进行酶处理；此外，泛素和相扑与蛋白质的结合都是可逆的。这些裂解反应是由特殊的蛋白酶负责的。在这笔赠款的赞助下，我们的实验室一直在分析去泛素化酶(DUBS)，以及最近一类新的相扑特异性半胱氨酸蛋白酶，ULP，它们在初级序列上与DUBS不同。这项工作的大部分都是用酵母进行的。 但是我们一直在研究的蛋白质从酵母到人类都是保守的。该项目的长期目标是从分子上了解DUBS和ULP所发挥的生理和机制作用。在这次更新应用中，拟议的实验主要集中在酵母中的相扑修饰(“相扑”)，就像上一个周期中的情况一样，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。在这笔赠款的最后一个周期中，我们发现了相扑系统的一些元素，其中包括相扑靶向泛素连接酶(STUbL)，它可以调节PolySUMO修饰的蛋白质的降解。在这次更新中，我们将探索这一关键和高度相互关联的 监管制度，包括ULP和STUbL。我们最近的Ulp1和Ulp2研究结果也出人意料地将我们引向了生物调节的几个特定领域。基于这些新的发现，我们建议在以下三个领域进行实验：(1)在体内鉴定STUbL SLX5-SLX8依赖相扑的底物，并表征它们在体内和体外(多)SUMO化的后果；(2)研究相扑调节肌醇生物合成途径的机制基础以及与内质网未折叠蛋白反应的联系；以及(3)确定相扑蛋白酶Ulp2和PolySUMO链的功能相互作用，包括在基因沉默和转录控制中的潜在作用。

DESCRIPTION (provided by applicant): Eukaryotic cells have a highly conserved enzymatic system for the ligation of ubiquitin to proteins. Moreover, polypeptides distinct from but related o ubiquitin, called ubiquitin-like proteins or Ubls, can also be conjugated to proteins. Ligation to each Ubl has unique mechanistic and functional consequences. SUMO (encoded by SMT3 in yeast) is a highly divergent Ubl. The SUMO ligation system has crucial roles in many organisms, including important contributions to human biology. SUMO, like ubiquitin, is synthesized in precursor form, requiring enzymatic processing of a C-terminal peptide; furthermore, both ubiquitin and SUMO attachment to proteins is reversible. Specialized proteases are responsible for these cleavage reactions. Under the auspices of this grant, our laboratory has been analyzing both deubiquitylating enzymes (DUBs), and more recently, a novel class of SUMO-specific cysteine proteases, the ULPs, which are distinct in primary sequence from the DUBs. Most of this work has been conducted with the yeast Saccharomyces cerevisiae, but the proteins we have been studying are conserved from yeast to humans. The long-term objective of the project is to gain a molecular understanding of the physiological and mechanistic roles played by DUBs and ULPs. In this renewal application, the proposed experiments are concentrated on SUMO modification ("sumoylation") in yeast, as was true in the last cycle, and on the contributions of the two yeast desumoylating enzymes, Ulp1 and Ulp2, to the function of the SUMO system. Mutation of either ULP has strong effects on growth and division, and Ulp1, like SUMO itself, is essential for cell- cycle progression. Among the elements of the SUMO system we uncovered in the last cycle of this grant were the SUMO-targeted ubiquitin ligases (STUbLs), which can mediate the degradation of polySUMO-modified proteins. In this renewal, we will explore novel features of this crucial and highly interconnected regulatory system, including the ULPs and STUbLs. Results from our recent Ulp1 and Ulp2 studies have also unexpectedly directed us toward several specific areas of biological regulation. Based on these new findings, we propose experiments in the following three areas: (1) Identification of in vivo SUMO-dependent substrates for the STUbL Slx5-Slx8 and the characterization of the consequences of their (poly)SUMOylation both in vivo and in vitro; (2) Investigation of the mechanistic basis of SUMO- mediated regulation of the inositol biosynthetic pathway and links to the ER unfolded protein response; and (3) Determination of the functional interplay of the SUMO protease Ulp2 and polySUMO chains, including potential roles in gene silencing and transcriptional control.