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.

真核细胞被分成膜区室，提高反应效率并隔离有毒中间体。该组织需要细胞器之间的通信来协调细胞的代谢程序。调节营养转运蛋白的运输和细胞器接触位点的营养物质交换维持跨膜代谢平衡。这项研究的重点是一类选择性蛋白质运输接头，称为α-抑制蛋白，及其在维持营养平衡和细胞生理学中的作用。从酵母到人类，α-抑制蛋白都是保守的，是蛋白质运输的主要调节因子，可响应细胞信号传导控制膜蛋白的定位。在缺乏α-抑制蛋白的情况下，营养转运蛋白保留在细胞表面，但研究尚未确定α-抑制蛋白的损失如何影响细胞生理学。该项目测试了α-抑制蛋白调节营养转运蛋白以维持代谢平衡和线粒体功能的全球假设。这项研究将改变我们对α-抑制蛋白功能的理解，确定它们对新陈代谢和线粒体功能的影响。这些研究目标与旨在培训下一代科学家和扩大 STEM 参与的教学和推广目标交织在一起。将开发基于课程的本科生研究经验，使用α-抑制蛋白作为细胞和分子生物学教学范式，为 120 名本科生提供研究经验。研究生、本科生和高中生将接受独立研究项目的指导，并且来自不同背景的学生将被招募到研究团队中。将与高中教师合作开发名为“皮特套件”的研究驱动型课程模块，并在宾夕法尼亚州公立高中部署。该项目将加深我们对α-抑制蛋白如何充当维持营养平衡的哨兵的理解，并确定这一过程的破坏如何导致细胞器功能障碍。初步数据表明，需要特定的α-抑制蛋白来维持细胞中选定氨基酸的平衡。这可能是因为每个α-抑制蛋白运输一组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确定这种毒性的潜在机制，而这种毒性几十年来一直难以捉摸。在每种情况下，过量的氨基酸都会导致线粒体功能障碍，这似乎会导致细胞毒性。引人注目的是，奥唐纳实验室的初步研究表明，在 α-抑制蛋白功能失调的细胞中，氨基酸过量会导致线粒体断裂并降低线粒体活性。当氨基酸中间体进出线粒体时，线粒体可能充当氨基酸水平的风向标，这可能会使该细胞器对氨基酸不平衡敏感。在该项目期间，将绘制α-抑制蛋白受损的细胞中的代谢变化，并定义改变代谢平衡所需的营养转运蛋白。使用高内涵共焦显微镜，我们将确定α-抑制蛋白如何调节营养转运蛋白的运输以及这种运输如何与线粒体功能相关。机器学习、自动量化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，确保生成的数据严格且可重复。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。