Mechanisms of reversible DUB oxidation in genome stability pathways

基因组稳定性途径中可逆 DUB 氧化的机制

• 批准号: 9145183
• 负责人: Tony Tung Huang
• 金额: $ 38.14万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9145183
• 关键词: Active Sites; Affect; Allosteric Regulation; Biochemical; Biological Assay; Biological Models; Cancer Etiology; Caspase; Cell physiology; Cells; Chemicals; Chronic; Cysteine; DNA Repair; Data; Defect; Deubiquitinating Enzyme; Deubiquitination; Development; Disease; Equilibrium; Family; Fanconi' s Anemia; Genome Stability; Genomic Instability; Health; Human; Human Genome; In Vitro; Link; Maintenance; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Mediating; Modification; Monoubiquitination; Normal Cell; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Pharmacotherapy; Play; Proteins; Reactive Oxygen Species; Regulation; Resistance; Role; Signal Transduction; Site; Ubiquitin; Ubiquitination; Work; base; cancer therapy; cell growth; environmental agent; in vivo; mutant; neoplastic cell; novel; oxidation; response; sensor; signal processing; therapeutic target; tumorigenesis; ubiquitin-protein ligase

 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.

 描述（由申请人提供）：环境因素引起的基因组稳定性途径失调是人类细胞肿瘤发生的主要原因；蛋白质泛素化是一种可逆的翻译后信号传导过程，对于维持这种作用至关重要 规定。目标蛋白的泛素化状态是通过两种相反力量之间的微妙平衡来实现的：泛素 E3 连接酶和去泛素化酶 (DUB)。据推测，细胞中的大多数蛋白质在某个时刻都受到泛素的调节和修饰。然而，由于细胞中存在大量 DUB，许多修饰仅短暂存在且难以捕获。人类基因组中编码了近百个 DUB，其中许多具有未表征的功能和调控。 DUB 在维持基因组稳定性中发挥着重要作用，其异常表达与肿瘤发生有关。哺乳动物细胞中 DUB 的调节及其如何影响基因组稳定性途径仍不清楚。 DUB 构成半胱氨酸 (Cys) 蛋白酶大家族，但缺乏关于环境因素或细胞内信号引起的氧化应激如何调节 DUB 酶活性的基本信息。我们最近发现，活性氧 (ROS) 的爆发通过催化 Cys 残基的氧化而可逆地灭活特定的 DUB。重要的是，DUB USP1 是范可尼贫血 (FA) 基因组稳定性通路和 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 USP1 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 Cys 和金属的 DUB。我们将通过使用一套独特的生化和质谱分析来捕获和表征 DUB 的可逆氧化形式，从而阐明 DUB 氧化的机制和功能。我们将通过生成保持活性但抵抗可逆氧化的 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，我们将采用独特的方法来收集 并研究受氧化应激调节的新型泛素化物种。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理，并可能为开发针对 DUB 的新型疗法提供指导。