Control of redox regulators by the ubiquitin system

泛素系统对氧化还原调节剂的控制

• 批准号: 8106682
• 负责人: Scott M Plafker
• 金额: $ 31.32万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8106682
• 关键词: Address; Adjuvant; Affinity; Age related macular degeneration; Alzheimer' s Disease; Antioxidants; Binding; Biochemical; Biological Assay; CUL3 gene; Cell Culture Techniques; Cell Nucleus; Cell physiology; Cells; Cellular Stress; Chronic; Complex; Cullin Proteins; Custom; Cysteine; DNA Binding Domain; Diabetes Mellitus; Diabetic Retinopathy; Disease; Dominant-Negative Mutation; Effectiveness; Elements; Enzymes; Equilibrium; Failure; Foundations; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Goals; Homeostasis; Human; In Vitro; In Vivo NMR Spectroscopy; Inflammatory; Knockout Mice; Legitimacy; Life; Link; Maintenance; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Methods; Microinjections; Modeling; Molecular; N-terminal; Neurodegenerative Disorders; Nuclear Import; Outcome; Oxidants; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Pathway interactions; Play; Positioning Attribute; Predisposition; Proteins; Publishing; Reagent; Recombinants; Recruitment Activity; Regulation; Reporting; Research; Response Elements; Retina; Retinal Degeneration; Role; Small Interfering RNA; Solutions; System; Testing; Therapeutic; Ubiquitin; Ubiquitin-Conjugating Enzymes; Video Microscopy; Work; base; biological adaptation to stress; design; in vivo; insight; mouse model; mutant; novel; oxidative damage; promoter; protein degradation; research study; response; restoration; sensor; stem; superoxide dismutase 1; transcription factor; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The long-term goal of this research is to reduce the susceptibility of vulnerable cells to the deleterious effects of chronic oxidative stress. Oxidative stress is widely accepted as a primary contributor to a range of pathological conditions including certain cancers, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's), diabetic retinopathy, and age-related macular degeneration. The ubiquitin (Ub) proteolytic system (UPS) defends cells against oxidative stress by degrading damaged proteins and by regulating cytoprotective proteins. Paramount among these protective proteins is Nrf2, the master anti-oxidant transcription factor. Oxidative stress activates Nrf2 to induce the expression of anti-oxidant enzymes and factors that restore redox homeostasis. Yet, open questions remain as to the mechanism(s) by which Nrf2 is sequestered on the promoters of its target genes until redox homeostasis is restored. The studies of this application address this critical issue and offer fresh insights into how the UPS contributes to the endogenous antioxidant defense system. The foundation for this work is our recent discovery that the E2 ubiquitin conjugating enzyme, UbcM2, is a novel component of the endogenous oxidative stress response pathway. These findings have led to our overarching hypothesis that UbcM2 is in fact a multi-functional E2. It functions as a redox sensor to enhance the cytoprotective activity of Nrf2 and it plays critical roles in protein degradation and E3 ligase regulation. Three specific aims will be pursued to define the diverse functions of UbcM2. Sp Aim#1: Test the hypothesis that UbcM2 is a redox sensor protein that promotes the cytoprotective activity of Nrf2; Sp Aim#2: Test the hypotheses that UbcM2 regulates Keap1 nuclear import and the affinity of Nrf2 for its cognate response elements in the promoters of anti-oxidant genes. Keap1 is the substrate adaptor that targets Nrf2 for degradation; and Sp Aim#3: Test the hypothesis that UbcM2 regulates substrate adaptor exchange on a class of UPS enzymes called cullin-RING E3 ligases (CRLs). In addition, identify the molecular determinants governing polyUb chain synthesis by UbcM2. The proposed experiments utilize a complementary set of cell culture approaches (e.g., siRNA, microinjection/live cell video microscopy, transcription assays), biochemical and biophysical methods (e.g., recombinant pulldowns, in vitro ubiquitylation assays, mass spectrometry, NMR spectroscopy), and in vivo mouse models of oxidative stress. Together, these studies will: (i) define the molecular mechanisms by which UbcM2 modulates Nrf2 stability and activation, (ii) explore the novel idea that Ub E2s can function as redox sensors to mediate stress response pathways, and (iii) bridge gaps in our understanding of how CRLs exchange substrate adaptors. This information will impact both the UPS and oxidative stress fields by identifying new relationships between E2 function and cellular anti-oxidant defenses. PUBLIC HEALTH RELEVANCE: Oxidative stress is widely held to be a primary etiological factor for many diseases (e.g., diabetic retinopathy, age-related macular degeneration, cancer, Parkinson's disease). We have identified an enzyme, called UbcM2, which functions as a redox-sensor and boosts the effectiveness of the endogenous anti-oxidant system. The goals of this application are to elucidate the mechanisms by which UbcM2 functions in countering oxidative damage with the ultimate goal of evaluating the legitimacy of this protein as a therapeutic adjuvant.

描述（由申请人提供）：本研究的长期目标是降低脆弱细胞对慢性氧化应激有害影响的敏感性。氧化应激被广泛认为是导致一系列病理状况的主要原因，包括某些癌症、神经退行性疾病（例如帕金森病和阿尔茨海默病）、糖尿病性视网膜病和年龄相关性黄斑变性。泛素 (Ub) 蛋白水解系统 (UPS) 通过降解受损蛋白质和调节细胞保护蛋白来保护细胞免受氧化应激。这些保护性蛋白中最重要的是 Nrf2，它是主要的抗氧化转录因子。氧化应激激活 Nrf2，诱导抗氧化酶和因子的表达，恢复氧化还原稳态。然而，关于 Nrf2 被隔离在其靶基因启动子上直到氧化还原稳态恢复的机制仍然存在悬而未决的问题。该应用的研究解决了这一关键问题，并为 UPS 如何促进内源性抗氧化防御系统提供了新的见解。这项工作的基础是我们最近发现 E2 泛素结合酶 UbcM2 是内源性氧化应激反应途径的一个新成分。这些发现得出了我们的总体假设：UbcM2 实际上是一种多功能 E2。它作为氧化还原传感器增强 Nrf2 的细胞保护活性，并在蛋白质降解和 E3 连接酶调节中发挥关键作用。将追求三个具体目标来定义 UbcM2 的不同功能。 Sp Aim#1：检验 UbcM2 是一种氧化还原传感器蛋白，促进 Nrf2 细胞保护活性的假设； Sp 目标#2：测试 UbcM2 调节 Keap1 核输入的假设以及 Nrf2 与其在抗氧化基因启动子中的同源反应元件的亲和力。 Keap1 是针对 Nrf2 进行降解的底物适配器； Sp 目标#3：测试 UbcM2 调节一类称为 cullin-RING E3 连接酶 (CRL) 的 UPS 酶上的底物接头交换的假设。此外，还确定了控制 UbcM2 多聚泛素链合成的分子决定因素。所提出的实验利用了一组互补的细胞培养方法（例如，siRNA、显微注射/活细胞视频显微镜、转录测定）、生化和生物物理方法（例如，重组下拉、体外泛素化测定、质谱、核磁共振波谱），以及体内氧化应激小鼠模型。总之，这些研究将：（i）定义 UbcM2 调节 Nrf2 稳定性和激活的分子机制，（ii）探索 Ub E2s 可以作为氧化还原传感器介导应激反应途径的新想法，以及（iii）弥补我们对 CRL 如何交换底物适配器的理解。该信息将通过识别 E2 功能和细胞抗氧化防御之间的新关系来影响 UPS 和氧化应激场。 公众健康相关性：氧化应激被广泛认为是许多疾病（例如糖尿病视网膜病变、年龄相关性黄斑变性、癌症、帕金森病）的主要病因。我们发现了一种名为 UbcM2 的酶，它具有氧化还原传感器的作用，可提高内源性抗氧化系统的有效性。本申请的目的是阐明 UbcM2 对抗氧化损伤的机制，最终目标是评估该蛋白质作为治疗佐剂的合法性。