Mechanisms of reversible DUB oxidation in genome stability pathways

 描述（由申请人提供）：由环境因子引起的基因组稳定性途径的失调是人类细胞中肿瘤发生的主要罪魁祸首;蛋白质泛素化，一种可逆的翻译后信号传导过程，对于维持这种稳定性至关重要。 调控靶蛋白的泛素化状态是通过两种相反的力量之间的微妙平衡实现的：泛素E3连接酶和去泛素化酶（DUBs）。据推测，细胞中的大多数蛋白质在某个时候被泛素调节和修饰;然而，由于细胞中存在大量DUB，许多修饰仅短暂存在并且难以捕获。在人类基因组中编码有近100个DUB，其中许多具有未表征的功能和调节。DUB在维持基因组稳定性中起重要作用，并且其异常表达与肿瘤发生有关。DUB在哺乳动物细胞中的调节以及这如何影响基因组稳定性途径仍不清楚。 DUBs构成了一个大家族的半胱氨酸（Cys）蛋白酶，但基本的信息DUBs的酶活性是如何调节由环境因素或细胞内信号引起的氧化应激缺乏。我们最近发现，活性氧簇（ROS）的爆发可逆地通过氧化的催化Cys残基的特异性DUBs。重要的是，DUB USP 1是范可尼贫血（FA）基因组稳定性途径和PCNA介导的跨损伤合成（TLS）的关键调节因子，在氧化应激时可逆失活。我们假设半胱氨酸蛋白酶家族的DUB在人类细胞中充当ROS传感器，并且ROS介导的DUB失活是微调健康和疾病中基因组稳定性途径的关键机制。在此，我们建议使用USP 1作为模型系统来剖析DUB调节的不同机制，然后将分析扩展到其他基于半胱氨酸和金属的DUB。我们将阐明DUB氧化的机制和功能，通过使用一套独特的生物化学和质谱为基础的测定，以捕获和表征可逆氧化形式的DUB。我们将通过产生DUB突变体来研究DUB氧化在DNA修复和细胞生长途径中的作用，这些突变体保持活性，但对可逆氧化具有抗性。最后，我们将采用独特的方法收集 并研究受氧化应激调控的新型泛素化物种。总之，我们的研究将揭示去泛素化和基因组稳定性调节的核心原则，并可能为开发靶向DUB的新型治疗药物提供指导。

 DESCRIPTION (provided by applicant): The deregulation of genome stability pathways caused by environmental agents is a major culprit of tumorigenesis in human cells; protein ubiquitination, a reversible posttranslational signaling process, is essential for maintaining this regulation. The ubiquitination status of a target protein is achieved via a delicate balance between two opposing forces: ubiquitin E3 ligases and deubiquitinating enzymes (DUBs). It has been postulated that the majority of proteins in a cell are regulated and modified by ubiquitin at some point; however, due to the multitude of DUBs present in cells, many of the modifications only exist transiently and are difficult to capture. There are nearly one hundred DUBs encoded in the human genome, many of which have uncharacterized function(s) and regulation. DUBs play an essential role in the maintenance of genomic stability, and their aberrant expression has been linked to tumorigenesis. The regulation of DUBs in mammalian cells and how this affects genome stability pathways remain unclear. DUBs constitute a large family of cysteine (Cys) proteases yet basic information on how the enzymatic activity of DUBs is modulated by oxidative stress caused by either environmental agents or intracellular signals is lacking. We recently discovered that bursts of reactive oxygen species (ROS) reversibly inactivate specific DUBs through the oxidation of the catalytic Cys residue. Importantly, the DUB USP1, a key regulator of the Fanconi Anemia (FA) genomic stability pathway and PCNA-mediated translesion synthesis (TLS), was reversibly inactivated upon oxidative stress. We hypothesize that DUBs of the cysteine protease family act as ROS sensors in human cells and that ROS-mediated DUB inactivation is a critical mechanism for fine-tuning genome stability pathways in both health and disease. Herein, we propose to dissect the different mechanisms of DUB regulation using USP1 as a model system, and then extend the analysis to other Cys- and metallo-based DUBs. We will elucidate the mechanisms and functions of DUB oxidation by using a set of unique biochemical and mass spectrometry-based assays to capture and characterize the reversibly oxidized form(s) of DUBs. We will investigate the role of DUB oxidation in DNA repair and cell growth pathways by generating DUB mutants that remain active, yet resistant to reversible oxidation. Finally, we will employ a unique approach to collect and study novel ubiquitinated species that are regulated by oxidative stress. Together, our studies will reveal core principles of deubiquitination and genome stability regulation, and will likely provide guidance for the development of novel classes of therapeutics that target DUBs.