Mechanisms of reversible DUB oxidation in genome stability pathways

 描述(申请人提供)：环境因素引起的基因组稳定通路的失调是人类细胞肿瘤发生的主要罪魁祸首；蛋白质泛素化是一种可逆的翻译后信号传递过程，对维持这一过程至关重要。 监管。目标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)。人们推测，细胞中的大多数蛋白质在某种程度上都受到泛素的调控和修饰；然而，由于细胞中存在大量的DUB，许多修饰只是短暂存在的，很难捕获。在人类基因组中编码了近百个DUB，其中许多具有未知的功能(S)和调节。DUBS在维持基因组稳定性方面起着至关重要的作用，它们的异常表达与肿瘤的发生有关。哺乳动物细胞中DUBS的调控以及这如何影响基因组稳定途径仍不清楚。DUBS是半胱氨酸(Cys)蛋白水解酶的一个大家族，但有关DUBS的酶活性如何受环境因素或细胞内信号引起的氧化应激调节的基本信息尚不清楚。我们最近发现，活性氧物种(ROS)的爆发通过催化半胱氨酸残基的氧化可逆地钝化特定的DUB。重要的是，作为Fanconi贫血(FA)基因组稳定途径和增殖细胞核抗原介导的跨损伤合成(TLS)的关键调节因子，Dub USP1在氧化应激下被可逆地失活。我们假设半胱氨酸蛋白酶家族的DUBS在人类细胞中作为ROS传感器，ROS介导的DUB失活是微调健康和疾病中基因组稳定途径的关键机制。在这里，我们建议以USP1为模型系统剖析DUB调控的不同机制，然后将分析扩展到其他基于Cys和金属的DUB。我们将通过一套独特的基于生化和质谱学的分析方法来捕捉和表征可逆氧化形式的DUBS(S)，从而阐明DUB氧化的机制和功能。我们将通过产生保持活性但对可逆氧化具有抵抗力的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。最后，我们将使用一种独特的方法收集 并研究受氧化应激调节的新的泛素化物种。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理，并可能为开发针对DUBS的新型疗法提供指导。

 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.