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，用于将泛素连接至蛋白质。此外，与泛素不同但相关的多肽，称为泛素样蛋白或Ubls，也可以与蛋白质缀合。与每个Ubl的连接具有独特的机制和功能后果。SUMO（在酵母中由SMT 3编码）是高度分化的Ubl。SUMO连接系统在许多生物体中具有重要作用，包括对人类生物学的重要贡献。SUMO与泛素一样，以前体形式合成，需要对C-末端肽进行酶促加工;此外，泛素和SUMO与蛋白质的连接都是可逆的。专门的蛋白酶负责这些切割反应。在这项资助的支持下，我们的实验室一直在分析两种去泛素化酶（DUBs），最近，一类新的SUMO特异性半胱氨酸蛋白酶，ULP，这是从DUBs的主要序列不同。这项工作的大部分已经进行了酵母 但是我们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水平上了解DUB和ULP所起的生理和机制作用。在此更新申请中，所提出的实验集中在酵母中的SUMO修饰（“sumoylation”），如在上一个循环中所述，以及两种酵母去小泛素化酶Ulp 1和Ulp 2对SUMO系统功能的贡献。ULP的突变对生长和分裂都有很强的影响，而Ulp 1和SUMO本身一样，对细胞周期的进展至关重要。在SUMO系统的元件中，我们在本基金的最后一个周期中发现了SUMO靶向泛素连接酶（STUbLs），它可以介导polySUMO修饰蛋白的降解。在这次更新中，我们将探索这一关键和高度相互关联的新特征。 监管系统，包括ULP和STUBL。我们最近的Ulp 1和Ulp 2研究结果也出乎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，我们提出了以下三个方面的实验：（1）鉴定STUbL Slx 5-Slx 8的体内SUMO依赖性底物，并表征它们在体内和体外的（聚）SUMO化的结果;（2）研究SUMO介导的肌醇生物合成途径调节的机制基础以及与ER未折叠蛋白反应的联系;和（3）确定SUMO蛋白酶Ulp 2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.