Collaborative Research: Alpha-arrestins' impact on cellular physiology

合作研究：α-抑制蛋白对细胞生理学的影响

• 批准号: 2321624
• 负责人: Allyson O'Donnell
• 金额: $ 106.88万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2321624&HistoricalAwards=false
• 关键词: Collaborative; Research; Alpha; arrestins; impact

Eukaryotic cells are partitioned into membrane compartments, increasing reaction efficiencies and sequestering toxic intermediates. This organization necessitates communication between organelles to coordinate the cell’s metabolic program. Regulated nutrient transporter trafficking and exchange of nutrients at organelle contact sites maintain metabolite balance across membranes. This research focuses on a class of selective protein trafficking adaptors, known as the alpha-arrestins, and their role in maintaining nutrient balance and cell physiology. Conserved from yeast to man, alpha-arrestins are master regulators of protein trafficking, controlling membrane protein localization in response to cell signaling. In the absence of alpha-arrestins, nutrient transporters are retained at the cell surface, but studies have not yet defined how loss of alpha-arrestins impacts cell physiology. This project tests the global hypothesis that alpha-arrestins regulate nutrient transporters to maintain metabolite balance and mitochondrial function. This research will transform our understanding of alpha-arrestin function, defining their impact on metabolism and mitochondrial function. These research goals are intertwined with teaching and outreach objectives designed to train the next generation of scientists and broaden STEM participation. A course-based undergraduate research experience that uses alpha-arrestins as a paradigm to teach cell and molecular biology will be developed, providing 120 undergraduates with research experience. Graduate, undergraduate, and high school students will be mentored on independent research projects, and students from diverse backgrounds will be recruited to the research team. Research-driven lesson modules called ‘Pitt Kits’ will be developed in collaboration with high school teachers and deployed across Pennsylvanian public high schools. This project will advance our understanding of how alpha-arrestins act as sentinels to maintain nutrient balance and define how disruption of this process leads to organelle dysfunction. Preliminary data demonstrate that specific alpha-arrestins are required to maintain the balance of select amino acids in the cell. This is likely because each alpha-arrestin traffics a unique set of amino acid transporters. Amino acid imbalance is toxic and work from the Hughes lab has begun to define the underlying mechanisms for this toxicity, which had remained elusive for decades. In each case, excess amino acid leads to mitochondrial dysfunction, which appears to drive the cytotoxicity. Strikingly, initial studies from the O’Donnell lab show that in cells with dysfunctional alpha-arrestins, amino acid excess causes mitochondrial fragmentation and reduced mitochondrial activity. Mitochondria may act as bellwethers for amino acid levels as amino acid intermediates shuttle in and out of mitochondria, and this may sensitize this organelle to amino acid imbalance. During this project, the metabolic changes in cells where alpha-arrestins are impaired will be mapped and the nutrient transporters required for alteration in metabolite balance will be defined. Using high-content, confocal microscopy we will determine how alpha-arrestins regulate nutrient transporter trafficking and how this trafficking is linked to mitochondrial function. A machine-learning, automated quantification pipeline will help define transporter localization changes and mitochondrial form and function changes, ensuring that the data generated are rigorous and reproducible.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

真核细胞分为膜室，提高反应效率和隔离有毒中间体。该组织在细胞器之间进行必要的沟通以协调细胞的代谢程序。调节的营养转运蛋白运输和细胞器接触部位的营养交换保持了整个膜的代谢物平衡。这项研究重点是一类选择性蛋白运输适配器，称为α-甲蛋白，及其在维持营养平衡和细胞生理学中的作用。从酵母到人的保守，α-甲蛋白是蛋白质运输的主要调节剂，可以响应细胞信号传导来控制膜蛋白定位。在缺乏α-arr链蛋白的情况下，营养转运蛋白保留在细胞表面，但研究尚未定义α-art骨的丧失如何影响细胞生理。该项目检验了一个全球假设，即α-art骨调节营养转运蛋白以维持代谢物平衡和线粒体功能。这项研究将改变我们对α-arrestin功能的理解，定义它们对代谢和线粒体功能的影响。这些研究目标与旨在训练下一代科学家并扩大STEM参与的教学和外展目标交织在一起。将开发基于课程的本科研究经验，该研究经验将使用alpha-arrestins作为范式来教授细胞和分子生物学，从而提供120名具有研究经验的本科生。将在独立的研究项目上考虑研究生，本科和高中生，并将招募来自潜水员的学生。研究驱动的课程模块称为“ Pitt Kits”，将与高中教师合作开发，并在宾夕法尼亚州公立高中部署。该项目将促进我们对α-arthestins作为哨兵如何保持养分平衡并定义这一过程的破坏导致有机功能障碍的方式的理解。初步数据表明，需要特定的α-arrestin来维持细胞中精选的氨基酸的平衡。这可能是因为每个α-arrpha-arrestin运输都是独特的氨基酸转运蛋白。氨基酸失衡是有毒的，休斯实验室的工作已经开始定义这种毒性的基本机制，数十年来一直难以捉摸。在每种情况下，超过氨基酸会导致线粒体功能障碍，这似乎驱动了细胞毒性。令人惊讶的是，来自O'Donnell实验室的初步研究表明，在功能失调的α-arrpha-arrestin的细胞中，氨基酸过量会导致线粒体碎片和线粒体活性降低。线粒体可以充当氨基酸水平的铃铛，因为氨基酸中间班车进出线粒体，这可能会感觉到这种细胞器对氨基酸不平衡。在此项目中，将映射α-art骨的代谢变化，并将定义代谢物平衡改变所需的养分转运蛋白。使用高含量的共聚焦显微镜，我们将确定α-钟RESTIN如何调节营养转运蛋白运输量以及该运输如何与线粒体功能相关。机器学习的自动定量管道将有助于定义转运蛋白定位变化以及线粒体形式和功能变化，以确保生成的数据是严格且可复制的。该奖项反映了NSF的法定任务，并被认为是通过使用基金会的知识分子和更广泛的影响来审查Criteria来通过评估来获得支持的珍贵的。