Defining the roles of ubiquitination during the environmental stress response

 描述(申请人提供)：本提案的目标是研究蛋白质泛素化调节细胞对氧化应激的抵抗力的机制。暴露于氧化剂的增加和有毒蛋白质聚集体的积累是几种神经退行性疾病的原因，如帕金森氏症和阿尔茨海默氏症。氧化应激是一种非常突出的应激类型，由暴露于各种环境因素，如电离辐射、热和污染物引起。氧化应激损伤包括脂质、DNA和蛋白质在内的几种生物分子，可能导致细胞死亡。为了防止细胞死亡和疾病进展，细胞使用泛素化作为信号，通过蛋白酶体移除受损的蛋白质，从而防止聚集。然而，泛素化的许多非降解作用已经被描述，当不同的赖氨酸(K)泛素链与靶蛋白结合时，这些作用是通过不同的赖氨酸(K)泛素链来传递信号的。以酿酒酵母为模型，我们发现非常规的K63多泛素链强烈且动态地积累，并影响细胞在氧化应激下的生存能力。利用创新的蛋白质组学方法，我们确定了许多核糖体蛋白作为K63泛素化的靶点。然而，K63泛素化调节细胞活力的机制仍不清楚。因此，在Aim1中，将采用系统范围的方法来研究K63泛素化如何影响蛋白质表达(K99)。在目标2中，将进行相互作用分析，以确定调节K63泛素途径在氧化应激(K99/R00)反应中的特异性的机制。此外，已经证明哺乳动物细胞在氧化应激下积累K63泛素，但这一氧化还原途径的进化保守性完全未知。在目标3中，氧化还原K63泛素系统将被带到哺乳动物细胞中，通过将这一途径映射到神经细胞(R00)的应激反应来研究整体的保守性。该项目的结果将揭开依赖于选择性泛素化提高细胞对压力耐受性的新机制。我的整个职业生涯专注于研究泛素蛋白酶体系统应对压力的不同调控方面。在该奖项的K99指导阶段，我将获得基因组学和计算技术方面的培训，这些培训将使我能够研究由泛素介导的另一层细胞信号。纽约大学为该项目K99阶段的发展提供了智力环境和多学科设施的理想结合。我的长期职业目标是建立我的独立研究小组，将系统范围的方法与分子生物学和遗传学方法相结合，以解码泛素化信号以应对压力。这个奖项为我实现这一目标提供了一个独特的机会，因为它使我的导师和委员会能够为我过渡到独立提供精确的培训和职业指导。了解多种泛素在细胞应激反应中的作用，以及每个不同途径的特异性，将为许多损伤相关疾病的治疗和早期诊断提供开创性的见解。

 DESCRIPTION (provided by applicant): The goal of this proposal is to investigate the mechanisms by which protein ubiquitination regulates cellular resistance to oxidative stress. Increased exposure to oxidants combined to the accumulation of toxic protein aggregates is the cause of several neurodegenerative diseases such as Parkinson's and Alzheimer's. Oxidative stress is a very prominent type of stress induced by exposure to diverse environmental factors such as ionizing radiation, heat, and pollutants. Oxidative stress damages several biomolecules including lipids, DNA and proteins, potentially resulting in cell death. To prevent cell death and disease progression, cells use ubiquitination as the signal for removing damaged proteins through the proteasome and thus preventing aggregation. However, many non-degradative roles for ubiquitination have been described which are signaled by different lysine (K) ubiquitin chains when conjugated to target proteins. Using yeast S. cerevisiae as a model, we found that the unconventional K63 polyubiquitin chain accumulates strongly and dynamically, and impacts cellular viability in response to oxidative stress. Using innovative proteomics approaches, we identified many ribosome proteins as targets of K63 ubiquitination. However, the mechanisms by which K63 ubiquitination regulates cellular viability are still unknown. Therefore, in Aim1 system-wide approaches will be employed to investigate how K63 ubiquitination affects protein expression (K99). In Aim 2, interaction analysis will be performed to define the mechanisms that regulate the specificity of the K63 ubiquitin pathway in response to oxidative stress (K99/R00). Further, it has been demonstrated that mammalian cells accumulate K63 ubiquitin in response to oxidative stress, but the evolutionary conservation of this redox pathway is completely unknown. In Aim 3, the redox K63 ubiquitin system will be taken to mammalian cells to investigate overall conservation by mapping this pathway to the stress response in neuronal cells (R00). The outcome of this project will unravel novel mechanisms to increase cell tolerance to stress depending on selective ubiquitination. My entire career focused on studying different regulatory aspects of the ubiquitin proteasome system in response to stress. During the K99 mentored phase of this award, I will obtain training in genomics and computational techniques that will enable the investigation of one more layer of cellular signaling mediated by ubiquitin. NYU offers an ideal combination of intellectual environment and multidisciplinary facilities for th development of the K99 phase of this project. My long-term career goal is to establish my independent research group that will combine systems-wide methodologies with molecular biology and genetic approaches to decode the ubiquitination signals in response to stress. This award offers a unique opportunity to achieve this goal by enabling precise training and career guidance from my mentors and committee for my transition to independence. Understanding the multiple ubiquitin roles in cellular response to stress and the specificity of each distinct pathwa will provide groundbreaking insights into innovative strategies for treatment and early diagnosis of many damage-related disorders.