Molecular Screens for Deubiquitinase Function

描述（由申请人提供）：细胞调节的一个关键方面是维持蛋白质稳态，它为药物干预提供了许多潜在的机会。蛋白质在细胞中不断地合成和降解，它们的细胞水平必须受到严格的调节。泛素-蛋白酶体途径是真核细胞中主要的非溶酶体蛋白水解系统，由多酶级联组成，可修饰和降解多种蛋白质，包括许多关键的调节分子。泛素（Ub）和泛素样蛋白（UBL）对细胞蛋白的修饰在许多生物过程中起着至关重要的作用，包括细胞周期控制、转录激活、信号转导、抗原呈递、囊泡运输和细胞内蛋白水解。泛素化是一个动态的可逆过程，泛素连接酶（E3s）和去泛素酶（DUBs）对这一途径的广泛影响及其选择性负责。DUB酶负责维持细胞内足够的游离Ub池，并调节细胞蛋白质的泛素化状态。这些酶的缺陷似乎在几种疾病的发病机制中发挥作用，包括帕金森病、安格尔曼综合征、宫颈癌和冯希佩尔林道综合征。本研究计划的重点是dub和开发一种新的高通量测定方法，以确定调节这些关键调节酶活性的药物。所提出的分析是基于这样的观察，即某些蛋白质需要一个确定的和游离的氨基末端残基才能发挥功能。Ub/UBL和这些蛋白之间的融合将作为底物，在DUB切割时进行激活。该反应将被优化并适应为高通量格式，最终用于筛选临床相关dub的抑制剂/激活剂。

DESCRIPTION (provided by applicant): A key aspect of cellular regulation that affords a number of potential opportunities for pharmacological intervention is the maintenance of protein homeostasis. Proteins are continuously being synthesized and degraded in cells, and their cellular levels must be strictly regulated. The ubiquitin-proteasome pathway is the major non-lysosomal proteolytic system in eucaryotic cells, consisting of a multi-enzyme cascade that modifies and degrades a diverse array of proteins, including many key regulatory molecules. The modification of cellular proteins by ubiquitin (Ub) and ubiquitin-like proteins (UBL) plays an essential role in a number of biological processes, including cell cycle control, transcriptional activation, signal transduction, antigen presentation, vesicular trafficking, and intracellular proteolysis. Ubiquitination is a dynamic reversible process, with a multitude of ubiquitin ligases (E3s) and deubiquitinases (DUBs) responsible for both the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes are responsible for maintaining adequate pools of free Ub within the cell and for regulating the ubiquitination status of cellular proteins. Defects in these enzymes appear to play a role in the pathogenesis of several diseases, including Parkinson's disease, Angelman's syndrome, cervical cancer, and von Hippel Lindau syndrome. This research proposal focuses on the DUBs and on the development of a novel high-throughput assay to identify agents that modulate the activity of these key regulatory enzymes. The assay being proposed is based on the observation that certain proteins require a defined and free amino-terminal residue in order to be functional. Fusions between Ub/UBL and these proteins will serve as substrates that undergo activation upon DUB cleavage. This reaction will be optimized and adapted to a high-throughput format for eventual use in screens for inhibitors/activators of clinically relevant DUBs.