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症）、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。这些保护性蛋白中最重要的是Nrf 2，主要的抗氧化转录因子。氧化应激激活Nrf 2以诱导抗氧化酶和恢复氧化还原稳态的因子的表达。然而，关于Nrf 2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存在悬而未决的问题。本申请的研究解决了这一关键问题，并提供了新的见解UPS如何有助于内源性抗氧化防御系统。这项工作的基础是我们最近发现的E2泛素结合酶，UbcM 2，是一种新的组成部分的内源性氧化应激反应途径。这些发现导致了我们的总体假设，即UbcM 2实际上是一个多功能的E2。它作为一种氧化还原传感器，以增强Nrf 2的细胞保护活性，它在蛋白质降解和E3连接酶调节中起着关键作用。将追求三个具体目标来定义UbcM 2的各种功能。战略目标1：检验UbcM 2是促进Nrf 2的细胞保护活性的氧化还原传感器蛋白的假设; Sp Aim#2：检验UbcM 2调节Keap 1核输入和Nrf 2对其在抗氧化基因启动子中的同源响应元件的亲和力的假设。Keap 1是靶向Nrf 2降解的底物衔接子; Sp Aim#3：检验UbcM 2调节一类称为cullin-RING E3连接酶（CRL）的UPS酶上的底物衔接子交换的假设。此外，确定UbcM 2控制polyUb链合成的分子决定因素。拟议的实验利用一组互补的细胞培养方法（例如，siRNA，显微注射/活细胞视频显微术，转录测定），生物化学和生物物理方法（例如，重组下拉、体外泛素化测定、质谱法、NMR光谱法）和氧化应激的体内小鼠模型。这些研究将共同：（i）定义UbcM 2调节Nrf 2稳定性和激活的分子机制，（ii）探索Ub E2可以作为氧化还原传感器介导应激反应途径的新想法，（iii）弥合我们对CRL如何交换底物接头的理解。这些信息将通过确定E2功能和细胞抗氧化防御之间的新关系来影响UPS和氧化应激领域。 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（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）。我们已经确定了一种称为UbcM 2的酶，它作为氧化还原传感器发挥作用，并提高内源性抗氧化系统的有效性。本申请的目的是阐明UbcM 2在对抗氧化损伤中发挥作用的机制，最终目的是评估这种蛋白质作为治疗佐剂的合法性。

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.