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: 10026975
• 负责人: Matthew Lee Wohlever
• 金额: $ 37.63万
• 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 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10026975
• 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介导的蛋白质从线粒体膜提取的机理 作为氧化磷酸化和凋亡调节中心，线粒体在人类中起着至关重要的作用 健康。适当的线粒体功能取决于强大的质量控制系统，以维持 蛋白质组（蛋白质结构）。线粒体功能和/或蛋白质症的下降与癌症有关， 衰老，心血管和神经退行性疾病。线粒体的一个知识不足的方面 蛋白质症是从脂质双层中去除膜蛋白。这是由于技术挑战 在体外重新建立了这一过程。我们通过开发一个简单但 使用AAA+（与细胞活性相关的ATPase）蛋白MSP1的强大重构系统。 MSP1锚定在外部线粒体膜（OMM）中，通过 从脂质双层中去除不需要的蛋白质。 MSP1或人类同源物ATAD1中的突变导致 线粒体功能受损，恐惧调节，严重的脑病和早期死亡。 尽管具有明显的生理重要性，但有关MSP1的重要问题有许多重要的问题 活动。 MSP1如何与其他质量控制组件相互作用以维持膜蛋白质量？ MSP1/ATAD1提取的全范围是什么？这是如何受到监管的？这些尤其是 鉴于我们的合作者Jared Rutter（犹他大学HHMI）有初步的遗传 ATAD1可能通过从OMM中提取仅BH3的蛋白来调节凋亡的证据。我们将使用 无偏的蛋白质组学方法和我们的体外提取测定法直接检验此范式转移假设 并检查该过程如何调节的分子细节。因为我们的重组系统 克服了关键的技术障碍，这些障碍阻碍了先前研究膜提取的尝试 来自脂质双层的蛋白质，我们还将利用我们的系统得出有关如何关键的基础结论 膜流动性，底物结构和ATP水解速率等因素会影响这种必需的细胞 过程。这项工作将检验一个令人兴奋的凋亡法规的新假设，提供全面的 线粒体生物学中MSP1/ATAD1功能的图片，并发现了对基本的新见解 膜蛋白提取的过程。