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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

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.

摘要