Defining Novel Roles of Ubiquitin Accumulation During the Mammalian Oxidative Stress Response

定义哺乳动物氧化应激反应中泛素积累的新作用

基本信息

批准号：
10629240
负责人：
Austin O. Maduka
金额：
$ 4.01万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10629240
关键词：
Acute Address Air Pollutants Antibodies Antioxidants Apoptosis Arsenicals Atherosclerosis Autophagocytosis Binding Biochemical CRISPR/Cas technology Cell Death Cell Survival Cell physiology Cells Chronic Kidney Failure Clinical Clustered Regularly Interspaced Short Palindromic Repeats Data Development Disease Disease Progression Endoplasmic Reticulum Environmental Exposure Environmental Risk Factor Enzymes Event Exposure to Foundations Genes Genetic Genetic Transcription Genomics Human Immunofluorescence Immunologic In Vitro Knowledge Laboratories Lead Libraries Malignant Neoplasms Mammalian Cell Mediating Mentors Methods Modification Molecular Mutation Necrosis Nerve Degeneration Outcome Oxidation-Reduction Oxidative Stress Pathway interactions Physicians Population Post-Translational Protein Processing Postdoctoral Fellow Production Proteins Proteomics Reactive Oxygen Species Recovery Regulation Residencies Residual state Resistance Ribosomal Proteins Ribosomes Role Scientist Signal Transduction Small Interfering RNA Stress Structure System Technology Testing Training Transcriptional Regulation Translations Ubiquitin Ubiquitination Ultraviolet Rays Western Blotting Work Yeasts biological adaptation to stress candidate identification experience exposed human population human subject improved in vivo loss of function multicatalytic endopeptidase complex novel oxidation prevent protein degradation response skills small molecule inhibitor symposium tool ubiquitin isopeptidase

Acute Address Air Pollutants Antibodies Antioxidants Apoptosis Arsenicals Atherosclerosis Autophagocytosis Binding Biochemical CRISPR/Cas technology Cell Death Cell Survival Cell physiology Cells Chronic Kidney Failure Clinical Clustered Regularly Interspaced Short Palindromic Repeats Data Development Disease Disease Progression Endoplasmic Reticulum Environmental Exposure Environmental Risk Factor Enzymes Event Exposure to Foundations Genes Genetic Genetic Transcription Genomics Human Immunofluorescence Immunologic In Vitro Knowledge Laboratories Lead Libraries Malignant Neoplasms Mammalian Cell Mediating Mentors Methods Modification Molecular Mutation Necrosis Nerve Degeneration Outcome Oxidation-Reduction Oxidative Stress Pathway interactions Physicians Population Post-Translational Protein Processing Postdoctoral Fellow Production Proteins Proteomics Reactive Oxygen Species Recovery Regulation Residencies Residual state Resistance Ribosomal Proteins Ribosomes Role Scientist Signal Transduction Small Interfering RNA Stress Structure System Technology Testing Training Transcriptional Regulation Translations Ubiquitin Ubiquitination Ultraviolet Rays Western Blotting Work Yeasts biological adaptation to stress candidate identification experience exposed human population human subject improved in vivo loss of function multicatalytic endopeptidase complex novel oxidation prevent protein degradation response skills small molecule inhibitor symposium tool ubiquitin isopeptidase

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项目摘要

ABSTRACT Many harmful environmental exposures humans experience, such as UV radiation, air pollutants, and exposure to arsenic compounds, lead to oxidative stress from the accumulation of reactive oxygen species (ROS) in the cell. A vast array of disease progression results from oxidative stress, such as cancer, neurodegeneration, and atherosclerosis, and chronic kidney disease. In response to oxidative stress, humans rewire cellular processes at the transcriptional, translational, and post-translational level. A central aspect of this response includes the ubiquitin modification, as its global accumulation during oxidative stress is essential for cell survival. These roles that promote cellular defense include proteasome degradation, autophagy, translational control, and antioxidant protein production. However, many roles of the ubiquitination in the context of oxidative stress remain unexplored. My sponsor’s laboratory has begun to uncover new roles for ubiquitination under oxidative stress, such as how mutations preventing specific K63 ubiquitin linkages increase sensitivity to oxidative stress in yeast. Additionally, recent unpublished findings highlight an accumulation of K63 ubiquitin at the endoplasmic reticulum (ER) under oxidative stress in mammalian cells, which is likely to modify ribosomes. This proposal aims to characterize vital roles of the ubiquitin modification within the oxidative stress response in humans, in my first aim by defining the outcome of this localized K63 accumulation, and in my second aim by defining vital roles for ubiquitin enzymes in cell viability through use of a pooled CRISPR loss-of-function screen. For aim one, I will first test the impact of DUB activity, autophagy and proteasome degradation on removing ER K63 ubiquitin during stress recovery by western blot and immunofluorescence. If related to DUB activity, which is my leading hypothesis, I will then use siRNAs to identify a specific ER-resident DUB from a candidate list whose depletion prevents recovery, then determine how redox levels regulates the DUBs through in vitro and in vivo studies. For aim two, I will use sgRNA libraries to target 40/40 E2s, 512/~600 E3s, and 90/102 DUBs to identify candidate enzymes whose sgRNAs become underrepresented under higher ROS levels. Once identified, I will then define the roles of the most underrepresented enzyme by identifying its stress-associated targets of ubiquitination, as well as how oxidation regulates its function. Deeper understanding of ubiquitin’s role in the basic mechanisms of oxidative stress response pathways will inform clinical strategies to mitigate disease progression under ROS accumulation. This proposal will ultimately support my training to become an independent physician-scientist. My training plan includes presenting my findings of this work at multiple conferences, and structured interactions with my mentoring team. With the support of this F30, I will develop the required skill set to successfully transition to my post-doctoral and residency training.

抽象的许多有害的环境暴露于人类的经历，例如紫外线辐射，空气污染物和暴露到砷化合物，导致活性氧（ROS）积累的氧化应激细胞。大量疾病进展来自氧化物胁迫，例如癌症，神经退行性变化和动脉粥样硬化和慢性肾脏疾病。为了响应氧化应激，人类重新布置细胞过程在转录，翻译和翻译后级别。此回应的一个核心方面包括泛素修饰，因为其在氧化应激过程中的全球积累对于细胞存活至关重要。这些角色促进细胞防御包括蛋白酶体降解，自噬，翻译控制和抗氧化剂蛋白质产生。但是，在氧化应激的背景下，泛素化的许多作用仍然存在未探索。我的赞助商的实验室已经开始揭示在氧化应激下泛素化的新作用，例如，防止特定K63泛素连接的突变如何增加对酵母中氧化应激的敏感性。此外，最近未发表的发现突出了内质网处K63泛素的积累（ER）在哺乳动物细胞中的氧化应激下，可能会改变核糖体。该建议旨在表征了人类氧化应激反应中泛素修饰的重要作用通过定义本地化K63积累的结果，并在我的第二个目标中定义至关重要的角色通过使用合并的CRISPR功能丧失屏幕，细胞活力中的泛素酶。对于目标，我会首先测试配音活动，自噬和蛋白酶体降解对去除ER K63泛素的影响通过蛋白质印迹和免疫荧光恢复应力。如果与配音活动有关，这是我的领导假设，然后我将使用siRNA从其部署防止恢复，然后确定氧化还原水平如何通过体外和体内研究来调节DUB。为了目标两个，我将使用SGRNA库来定位40/40 E2，512/〜600 E3和90/102 DUB来识别候选人 SGRNA的酶在较高的ROS水平下的代表性不足。一旦确定，我将定义通过确定其与压力相关的泛素化靶标的最大代表性酶的作用，以及氧化如何调节其功能。对泛素在基本机制中的作用的更深入了解氧化应激反应途径将为临床策略提供信息，以减轻ROS下的疾病进展积累。该建议最终将支持我的培训，成为独立的身体科学家。我的培训计划包括在多个会议上介绍我对这项工作的发现以及结构化的互动与我的心理团队在一起。在此F30的支持下，我将开发所需的技能设置以成功过渡进行我的博士后和居住培训。