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）：这项研究的长期目标是降低脆弱细胞对慢性氧化应激的有害作用的敏感性。氧化应激被广泛接受为一系列病理状况的主要贡献者，包括某些癌症，神经退行性疾病（例如帕金森氏病和阿尔茨海默氏病），糖尿病性视网膜病和与年龄相关的黄斑变性。泛素（UB）蛋白水解系统（UPS）通过降解受损蛋白质并调节细胞保护蛋白来防御细胞免受氧化应激。这些保护蛋白中的最重要的是NRF2，是主要的抗氧化转录因子。氧化应激激活NRF2，以诱导抗氧化酶的表达和恢复氧化还原稳态的因素。然而，关于将NRF2隔离在其靶基因启动子上的机制上，直到恢复了氧化还原稳态。该申请的研究解决了这一关键问题，并提供了有关UPS如何对内源性抗氧化剂防御系统做出贡献的新见解。这项工作的基础是我们最近发现的，E2泛素结合酶UBCM2是内源性氧化应激反应途径的新成分。这些发现导致了我们的总体假设，即UBCM2实际上是多功能的E2。它是增强NRF2的细胞保护活性的氧化还原传感器，并且在蛋白质降解和E3连接酶调节中起关键作用。将追求三个具体目标来定义UBCM2的各种功能。 SP AIM＃1：测试UBCM2是一种氧化还原传感器蛋白的假设，可促进NRF2的细胞保护活性。 SP AIM＃2：测试UBCM2调节KEAP1核进口的假设以及NRF2对抗氧化基因启动子中的相关响应元件的亲和力。 KEAP1是针对NRF2降解的底物适配器；和SP AIM＃3：测试UBCM2在称为Cullin-Ring E3连接酶（CRLS）的UPS酶上调节底物适配器的假设。另外，确定通过UBCM2控制多核链合成的分子决定簇。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/实时细胞视频显微镜，转录测定），生化和生物物理方法（例如重组下拉，，体外尿液，体外尿液中的渗透分析，泛素渗透分析，质谱分析，NMR，NMR光谱）和VIV型。这些研究将共同​​：（i）定义UBCM2调节NRF2稳定性和激活的分子机制，（ii）探讨了一个新颖的想法，即UB E2S可以用作氧化还原传感器来介导应力响应途径，以及（iii）在我们对CRL如何交换CRL交换substrate substrate substrate apapters中的桥梁差距。这些信息将通过确定E2功能与细胞抗氧化剂防御之间的新关系来影响UPS和氧化应激场。 公共卫生相关性：氧化应激被广泛认为是许多疾病的主要病因因素（例如糖尿病性视网膜病，与年龄相关的黄斑变性，癌症，帕金森氏病）。我们已经确定了一种称为UBCM2的酶，该酶充当氧化还原传感器，并提高了内源性抗氧化剂系统的有效性。本应用的目标是阐明UBCM2在对抗氧化损伤中起作用的机制，以评估该蛋白作为治疗辅助的合法性的最终目标。