Developing minimal purification cryo-EM to understand mitochondrial myopathies

开发最小纯化冷冻电镜来了解线粒体肌病

基本信息

批准号：
10732697
负责人：
Gabriel C Lander
金额：
$ 44.41万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10732697
关键词：
Affect Biochemical Biological Process Cell physiology Cells Complex Cryoelectron Microscopy DNA Sequence Alteration Data Collection Defect Development Disease Disease Progression Drug Targeting Drug resistance Exercise Functional disorder Funding Future Genetic Genetic Diseases Human Image Individual Investigation Link Macromolecular Complexes Membrane Proteins Metabolic Methodology Methods Missense Mutation Mitochondria Mitochondrial Myopathies Mitochondrial Proteins Molecular Mus Muscle Muscle Cells Muscle Fibers Muscle Mitochondria Muscle function Muscular Atrophy Mutation Myopathy Neuromuscular Diseases Organelles Outcome Oxidative Phosphorylation Pathologic Pathology Patients Production Prokaryotic Cells Protocols documentation Relapse Resolution Sampling Skeletal Muscle Sorting Structure System Technology Therapeutic Tissues Visualization Wasting Syndrome Work base biological systems cell type data analysis pipeline diagnostic tool drug discovery expectation heterogenous data insight macromolecular assembly mitochondrial dysfunction particle personalized medicine protein function protein purification protein structure proteostasis structural biology

项目摘要

ABSTRACT Single particle cryoEM structure determination is now a widely used methodology that has revealed the detailed mechanisms underlying a wide range of biological systems. High-resolution single particle cryoEM studies have helped us understand how environmental or genetic factors perturb normal biological function, and how these factors can give rise to disease. Insights gained through such structural studies of cellular machinery have greatly benefited drug discovery efforts, as well as expanded our understanding of drug resistance and therapeutic relapse. However, successful single particle cryoEM structure determination continues to be dependent on the production and purification of highly homogeneous, biochemically stable samples for imaging. Here, we plan to harness the unique strengths of single particle cryoEM technologies - minimal sample requirements and an exceptional capacity for structural characterization of highly heterogeneous data - to move beyond this traditional approach. Precedence for such studies have been set by previous high-resolution cryoEM structures that were determined from heterogeneous mixtures of soluble or membrane-associated proteins extracted from single-cell lysates. We plan to extend these approaches to elucidate structures of endogenous mammalian mitochondrial complexes. In particular, the methodologies developed by this work will establish an avenue to perform structural investigation of mitochondrial complexes derived from mitochondrial myopathy patients. Mitochondrial dysfunction in skeletal muscle cells can have severe pathological outcomes, and is associated with a variety of muscle-wasting diseases and numerous neuromuscular disorders. One in 5000 individuals in the U.S. suffers from mitochondrial myopathies due to genetic mutation, and while substantial effort has been placed on understanding the genetics of these diseases, we lack an underlying molecular description of the specific perturbations responsible for pathology. Directly visualizing the endogenous mitochondrial complexes that carry mutations implicated in disease states enables us to inspect how missense mutations impact macromolecular assembly and interactions. We will develop mitochondrial isolation and structure determination methodologies to enable detailed structural assessment of the endogenous complexes involved in human mitochondrial proteostasis and the mitochondrial OXPHOS system, without the need for extensive protein purification. We have shown that mitochondrial lysates can be directly applied to EM grids and imaged to yield high-resolution structures of abundant complexes. We will further develop this pipeline to produce high-resolution structures of mitochondrial complexes and interaction partners from the distinct mitochondrial subcompartments, providing important molecular insights into how mutations associated with mitochondrial myopathies perturb protein structure and function. The results will advance our understanding of skeletal muscle myopathies through direct visualization of the machines involved in disease progression, unveiling never-before-seen mitochondrial assemblies, providing a molecular explanation for disease states, and laying the groundwork for future therapies.

抽象的单颗粒冷冻电镜结构测定现在是一种广泛使用的方法，它揭示了详细的广泛生物系统的潜在机制。高分辨率单粒子冷冻电镜研究帮助我们了解环境或遗传因素如何扰乱正常的生物功能，以及这些因素如何影响正常的生物功能。因素可导致疾病。通过细胞机械的这种结构研究获得的见解极大地促进了有益于药物发现工作，并扩大了我们对耐药性和治疗的理解复发。然而，成功的单粒子冷冻电镜结构测定仍然取决于生产和纯化高度均质、生化稳定的成像样品。在这里，我们计划利用单颗粒冷冻电镜技术的独特优势 - 最少的样品要求和高度异构数据的结构表征的卓越能力 - 超越了这种传统方法。此类研究的优先顺序是由以前的高分辨率冷冻电镜结构确定的由从单细胞中提取的可溶性或膜相关蛋白的异质混合物确定裂解物。我们计划扩展这些方法来阐明内源哺乳动物线粒体的结构复合物。特别是，这项工作开发的方法将为执行结构性研究建立一条途径。对源自线粒体肌病患者的线粒体复合物的研究。线粒体骨骼肌细胞功能障碍可能会产生严重的病理结果，并且与多种疾病相关肌肉萎缩疾病和许多神经肌肉疾病。美国每 5000 人中就有 1 人患有此病由于基因突变引起的线粒体肌病，虽然已经付出了大量努力了解这些疾病的遗传学后，我们缺乏对特定疾病的潜在分子描述造成病理学的扰动。直接可视化携带的内源性线粒体复合物与疾病状态有关的突变使我们能够检查错义突变如何影响大分子组装和相互作用。我们将开发线粒体分离和结构测定方法能够对人类线粒体中涉及的内源复合物进行详细的结构评估蛋白质稳态和线粒体 OXPHOS 系统，无需大量的蛋白质纯化。我们已经表明线粒体裂解物可以直接应用于 EM 网格并成像以产生高分辨率丰富的配合物结构。我们将进一步开发该管道以生产高分辨率结构线粒体复合物和来自不同线粒体亚区室的相互作用伙伴，提供关于与线粒体肌病相关的突变如何扰乱蛋白质的重要分子见解结构和功能。研究结果将通过直接研究增进我们对骨骼肌肌病的理解参与疾病进展的机器的可视化，揭示了前所未见的线粒体组装，为疾病状态提供分子解释，并为未来的治疗奠定基础。