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: 10465089
• 负责人: Matthew Lee Wohlever
• 金额: $ 32.75万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10465089
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Aging; Apoptosis; Apoptotic; Biological Assay; Biology; Cardiovascular Diseases; Cell Death; Cell physiology; Cessation of life; Encephalopathies; Excision; Foundations; Genetic; Health; Homeostasis; Homologous Gene; Human; Impairment; In Vitro; Investigation; Lead; Link; Lipid Bilayers; Malignant Neoplasms; Mediating; Membrane; Membrane Fluidity; Membrane Proteins; Metabolism; Mitochondria; Mitochondrial Proteins; Molecular; Mutation; Neurodegenerative Disorders; Outer Mitochondrial Membrane; Oxidative Phosphorylation; Physiological; Play; Process; Proteins; Proteome; Proteomics; Quality Control; Regulation; Role; Site; Structure; System; Testing; Universities; Utah; Work; conditioned fear; experimental study; insight; mitochondrial membrane; novel therapeutic intervention; proteostasis; reconstitution

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.

Msp1 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调节的中心，在人类生命活动中发挥着至关重要的作用。 健康。适当的线粒体功能取决于强大的质量控制系统来维持线粒体的稳态 蛋白质组（蛋白质稳态）。线粒体功能和/或蛋白质稳态的下降与癌症有关， 衰老、心血管疾病和神经退行性疾病。线粒体的一个鲜为人知的方面 蛋白质稳态是指从脂质双层中去除膜蛋白。这是由于技术挑战 在体外重建这个过程。我们克服了这一技术障碍，开发了一种简单但 具有 AAA+（与细胞活动相关的 ATP 酶）蛋白 Msp1 的强大重组系统。 Msp1 锚定于线粒体外膜 (OMM)，通过以下方式维持线粒体蛋白质稳态： 从脂质双层中去除不需要的蛋白质。 Msp1 或人类同源物 ATAD1 的突变导致 线粒体功能受损、恐惧调节受损、严重脑病和过早死亡。 尽管 Msp1 具有明显的生理重要性，但仍有许多重要的未解答问题 活动。 Msp1 如何与其他质量控制成分相互作用以维持膜蛋白质稳态？ Msp1/ATAD1 提取的底物全系列是什么？这是如何监管的？这些特别是 鉴于我们的合作者 Jared Rutter（HHMI，犹他大学）已初步掌握了遗传 ATAD1 可能通过从 OMM 中提取 BH3-only 蛋白来调节细胞凋亡。我们将使用一个 无偏见的蛋白质组学方法和我们的体外提取测定直接测试这种范式转变假设 并检查分子细节以了解该过程是如何调节的。因为我们重构的系统 克服了之前阻碍膜提取研究尝试的关键技术障碍 来自脂质双层的蛋白质，我们还将利用我们的系统得出关于如何关键的基本结论 膜流动性、底物结构和 ATP 水解速率等因素影响这一重要的细胞 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 Msp1/ATAD1 在线粒体生物学中的功能图片，并揭示对该基本原理的新见解 膜蛋白提取过程。