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.

真核细胞被分割成膜隔间，提高了反应效率，并隔离了有毒中间体。这种组织需要细胞器之间的沟通来协调细胞的新陈代谢程序。受调控的营养转运体运输和细胞器接触部位的营养交换维持了跨膜的代谢物平衡。这项研究的重点是一类选择性的蛋白质运输适配器，被称为α-拦截素，以及它们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-拦阻蛋白是蛋白质运输的主要调节者，控制细胞信号反应中膜蛋白的定位。在缺乏α-阻滞剂的情况下，营养转运体被保留在细胞表面，但研究尚未确定α-阻滞剂的丧失如何影响细胞生理。该项目测试了阿尔法抑制素调节营养转运体以维持代谢物平衡和线粒体功能的全球假设。这项研究将改变我们对α-arrestin功能的理解，明确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。将开发一种以课程为基础的本科生研究经验，以阿尔法抑制素为范例教授细胞和分子生物学，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，不同背景的学生将被招募到研究团队中。被称为“皮特工具包”的研究驱动的课程模块将与高中教师合作开发，并在宾夕法尼亚州的公立高中部署。这个项目将促进我们对阿尔法抑制素如何作为维持营养平衡的哨兵的理解，并确定这一过程的中断是如何导致细胞器功能障碍的。初步数据表明，维持细胞中特定氨基酸的平衡需要特定的阿尔法抑制素。这可能是因为每个α-arrestin都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，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，这似乎是导致细胞毒性的原因。令人惊讶的是，来自O‘Donnell实验室的初步研究表明，在阿尔法抑制素功能障碍的细胞中，氨基酸过量会导致线粒体碎裂和线粒体活性降低。线粒体可能是氨基酸水平的风向标，因为氨基酸中间体穿梭于线粒体内外，这可能会使细胞器对氨基酸失衡敏感。在这个项目中，将绘制阿尔法抑制素受损的细胞的代谢变化图，并定义代谢产物平衡改变所需的营养转运体。使用高含量的共聚焦显微镜，我们将确定阿尔法-抑制素如何调控营养转运蛋白的运输，以及这种运输如何与线粒体功能联系起来。机器学习的自动化量化管道将有助于定义转运体本地化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现。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。