Mechanistic Investigation of Proteostasis at the Outer Mitochondrial Membrane

线粒体外膜蛋白质稳态的机制研究

• 批准号: 10873385
• 负责人: Matthew Lee Wohlever
• 金额: $ 5.17万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10873385
• 关键词: Mechanistic; Investigation; Proteostasis; Outer; Mitochondrial

Mechanism of Msp1 Mediated Protein Extraction from the Mitochondrial Membrane As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain homeostasis of the proteome (proteostasis). Declines in mitochondrial function and/or proteostasis have been linked to cancer, aging, cardiovascular, and neurodegenerative diseases. A poorly understood aspect of mitochondrial proteostasis is the removal of membrane proteins from the lipid bilayer. This is due to the technical challenges of reconstituting this process in vitro. We have overcome this technical barrier by developing a simple, but powerful reconstituted system with the AAA+ (ATPase Associated with cellular Activities) protein Msp1. Anchored in the outer mitochondrial membrane (OMM), Msp1 maintains mitochondrial proteostasis by removing unwanted proteins from the lipid bilayer. Mutations in Msp1 or the human homologue ATAD1 lead to compromised mitochondrial function, impaired fear conditioning, severe encephalopathy, and early death. Despite its clear physiological importance, there are many important unanswered questions regarding Msp1 activity. How does Msp1 interact with other quality control components to maintain membrane proteostasis? What is the full range of substrates extracted by Msp1/ATAD1? How is this regulated? These are particularly pressing questions given that our collaborator, Jared Rutter (HHMI, University of Utah), has preliminary genetic evidence that ATAD1 may regulate apoptosis by extracting BH3-only proteins from the OMM. We will use an unbiased proteomic approach and our in vitro extraction assay to directly test this paradigm shifting hypothesis and examine the molecular details for how this process is regulated. Because our reconstituted system overcomes key technical barriers that have hampered previous attempts to study the extraction of membrane proteins from a lipid bilayer, we will also utilize our system to draw foundational conclusions about how key factors such as membrane fluidity, substrate structure, and ATP hydrolysis rates affect this essential cellular process. This work will test an exciting new hypothesis for apoptotic regulation, provide a comprehensive picture of Msp1/ATAD1 function in mitochondrial biology, and uncover new insights into the fundamental process of membrane protein extraction.

Msp 1介导的线粒体膜蛋白提取机制 线粒体作为氧化磷酸化和细胞凋亡的调控中心，在人类的免疫系统中起着重要的作用。 健康适当的线粒体功能取决于一个强大的质量控制系统，以维持体内平衡， 蛋白质组（Proteostasis）线粒体功能和/或蛋白质稳态的下降与癌症有关， 衰老、心血管和神经退行性疾病。线粒体的一个知之甚少的方面 蛋白质稳定是从脂质双层中除去膜蛋白。这是由于技术挑战 在体外重建这个过程。我们已经克服了这一技术障碍， 与AAA+（ATP酶与细胞活动相关）蛋白Msp 1的强大重建系统。 Msp 1锚定在线粒体外膜（OMM），通过以下方式维持线粒体蛋白质稳态： 从脂质双层中除去不需要的蛋白质。Msp 1或人类同源物ATAD 1的突变导致 受损的线粒体功能，受损的恐惧条件反射，严重的脑病和早死。 尽管它在生理上的重要性很明显，但关于Msp 1还有许多重要的问题没有回答。 活动Msp 1如何与其他质量控制组分相互作用以维持膜蛋白质稳态？ Msp 1/ATAD 1提取的全部底物是什么？这是如何监管的？这些是特别 鉴于我们的合作者Jared Rutter（HHMI，犹他州大学）已经初步遗传 这表明ATAD 1可能通过从OMM中提取BH 3-only蛋白来调节细胞凋亡。我们将使用 无偏的蛋白质组学方法和我们的体外提取试验直接测试这种范式转变假设 并研究这一过程是如何调控的分子细节。因为我们重建的系统 克服了阻碍以前研究膜提取的关键技术障碍， 蛋白质从脂质双层，我们也将利用我们的系统得出基本结论，如何关键 诸如膜流动性、底物结构和ATP水解速率等因素影响这种基本的细胞内 过程这项工作将测试一个令人兴奋的细胞凋亡调节新假设，提供全面的 Msp 1/ATAD 1在线粒体生物学中的功能，并揭示了对线粒体生物学基础的新见解。 膜蛋白提取过程。