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

• 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
• 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

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.

摘要 单粒子cryoEM结构测定现在是一种广泛使用的方法，其揭示了详细的 广泛的生物系统的潜在机制。高分辨率单粒子cryoEM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生物功能。 这些因素会导致疾病。通过对细胞机器的结构研究获得的见解大大提高了 受益于药物发现工作，并扩大了我们对耐药性和治疗方法的了解 复发然而，成功的单粒子cryoEM结构确定仍然依赖于 用于成像的高度均匀、生化稳定的样品的生产和纯化。在这里，我们计划 利用单粒子cryoEM技术的独特优势-最少的样品要求和 对高度异构数据进行结构表征的卓越能力-超越传统的 approach.这种研究的优先权已经由先前的高分辨率cryoEM结构设定， 从单细胞中提取的可溶性或膜相关蛋白质的异质混合物中测定 裂解物。我们计划扩展这些方法来阐明内源性哺乳动物线粒体的结构， 配合物特别是，这项工作开发的方法将建立一个途径， 研究来自线粒体肌病患者的线粒体复合物。线粒体 骨骼肌细胞的功能障碍可能具有严重的病理结果，并且与多种 肌肉萎缩性疾病和许多神经肌肉疾病。美国每5000人中就有一人 由于基因突变引起的线粒体肌病，虽然已经在 了解这些疾病的遗传学，我们缺乏一个潜在的分子描述的具体 造成病理学的扰动。直接观察携带线粒体复合物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 为了能够详细评估参与人线粒体中的内源性复合物， 蛋白质稳定和线粒体OXPHOS系统，而不需要广泛的蛋白质纯化。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 丰富的复合物结构。我们将进一步开发这一管道，以生产高分辨率的结构， 线粒体复合物和相互作用伙伴从不同的线粒体亚室，提供 线粒体肌病相关突变如何干扰蛋白质的重要分子见解 结构和功能。这些结果将通过直接的方法促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，揭示了以前从未见过的线粒体 组装，为疾病状态提供分子解释，并为未来的治疗奠定基础。