Control of redox regulators by the ubiquitin system

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易感性。氧化应激被广泛认为是一系列病理状况的主要因素，包括某些癌症、神经退行性疾病（如帕金森病和阿尔茨海默病）、糖尿病视网膜病变和年龄相关性黄斑变性。泛素（Ub）蛋白水解系统（UPS）通过降解受损蛋白和调节细胞保护蛋白来保护细胞免受氧化应激。在这些保护性蛋白中，最重要的是Nrf2，一种主要的抗氧化转录因子。氧化应激激活Nrf2诱导抗氧化酶和恢复氧化还原稳态的因子的表达。然而，Nrf2被隔离在其靶基因的启动子上直到氧化还原稳态恢复的机制仍然是一个悬而未决的问题。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泛素偶联酶UbcM2是内源性氧化应激反应途径的新组成部分。这些发现导致了我们的首要假设，即UbcM2实际上是一个多功能E2。它作为氧化还原传感器增强Nrf2的细胞保护活性，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。目的1：验证UbcM2是一种促进Nrf2细胞保护活性的氧化还原传感器蛋白的假设；Sp目标#2：验证UbcM2调控Keap1核输入的假设，以及Nrf2对其在抗氧化基因启动子中的同源反应元件的亲和力。Keap1是靶定Nrf2降解的底物适配器；和Sp目标#3：验证UbcM2调节一类被称为cullin-RING E3连接酶（CRLs）的UPS酶的底物适配器交换的假设。此外，还鉴定了控制UbcM2合成polyb链的分子决定因素。拟议的实验利用了一套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，重组拉下，体外泛素化测定，质谱，核磁共振波谱），以及体内小鼠氧化应激模型。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UbcM2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）弥合我们对CRLs交换底物适配器的理解空白。通过确定E2功能与细胞抗氧化防御之间的新关系，这些信息将影响UPS和氧化应力场。

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.