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: 10393361
• 负责人: Matthew Lee Wohlever
• 金额: $ 0.54万
• 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/10393361
• 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控的中心，在人体内起着至关重要的作用 健康。线粒体的正常功能有赖于强大的质量控制系统来维持体内的动态平衡 蛋白质组(蛋白质平衡)。线粒体功能和/或蛋白稳定性的下降与癌症有关， 衰老、心血管和神经退行性疾病。线粒体的一个鲜为人知的方面 蛋白平衡是将膜蛋白从脂双层中移除。这是由于技术上的挑战。 在体外重建这一过程。我们已经通过开发一种简单的、但 强大的重组系统与AAA+(与细胞活动相关的ATPase)蛋白Msp1。 Msp1锚定在线粒体外膜(OMM)，通过以下方式维持线粒体蛋白平衡 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，恐惧条件反射受损，严重脑病，以及过早死亡。 尽管Msp1具有明显的生理重要性，但仍有许多重要的问题悬而未决。 活动。MSP1如何与其他质量控制成分相互作用以维持膜蛋白平衡？ Msp1/ATAD1提取的所有底物是什么？这是如何监管的？这些是特别的 迫在眉睫的问题是，我们的合作者Jared Rutter(HHMI，犹他大学)具有初步的基因 有证据表明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 无偏蛋白质组学方法和我们的体外提取实验直接验证这一范式转换假说 并检查分子细节，了解这一过程是如何调控的。因为我们重组后的系统 克服了阻碍以前研究膜提取的关键技术障碍 来自脂质双层的蛋白质，我们还将利用我们的系统来得出基本的结论 膜流动性、底物结构和三磷酸腺苷水解率等因素影响这一基本细胞。 进程。这项工作将检验一个令人兴奋的新的细胞凋亡调控假说，提供一个全面的 线粒体生物学中Msp1/ATAD1功能的图景，并揭示基本的 膜蛋白提取工艺。