线粒体中蛋白质原位核磁共振研究方法的建立

Mitochondria are important organelles in eukaryotes. Mitochondrial dysfunction will lead to large numbers of diseases, such as premature senility syndrome, neurodegenerative diseases including Parkinson’s disease and Alzheimer's disease. An important pathway to understand the pathogenesis of these diseases is to carry out in situ studies of proteins in mitochondria, in which nuclear magnetic resonance (NMR) technology has its own unique advantage. At present, in situ NMR studies of proteins in mitochondria still remain a primary stage and systematic investigations are lacking. Therefore, it is urgent to develop the method of protein NMR in mitochondria. In this project, we use α-synuclein, which is tightly related to Parkinson's disease and impairs mitochondrial function, as an object of study, and try using three different methods to import α-synuclein and its mutants with various isotopic labeling such as selective 15N-Leu enrichment, 13C-methyl enrichment of Ile, Leu, Val and Ala, and 19F labeling, into mitochondria. The efficiency of protein imported into mitochondria of these three methods and the advantages and disadvantages of different isotopic labeling are evaluated by acquired NMR data, and then the method of protein NMR in mitochondria will be developed. At the same time, the possible molecular mechanism of mitochondrial dysfunction caused by α-synuclein will be investigated. The project will provide an effective way to observe protein structure, interaction and modification in atom level for in situ studies of proteins in mitochondria, help greatly guide investigators in selecting the high efficient method of proteins imported into mitochondria and the appropriate enrichment or labeling method for protein NMR studies in mitochondria, and be of great significance to protein science and the study of mitochondrial diseases.

线粒体是真核生物细胞中重要的细胞器，线粒体功能障碍将引起诸多疾病，如早衰、神经退行性疾病—帕金森病等。在线粒体中开展蛋白质原位研究是理解这些疾病发生机制的一个重要途径。能提供蛋白质原子水平信息的核磁共振波谱技术应用于线粒体蛋白质原位研究具有独特优势，但目前该领域的发展尚处刚刚起步阶段，非常缺乏系统的研究方法。本项目以引起线粒体功能障碍、与帕金森病紧密相关的α-突触核蛋白为研究对象，采用多种不同方法将该蛋白及其突变体引入到线粒体中，通过研究同位素标记蛋白质进入线粒体的高效实验方法，以及不同标记方法的优缺点，建立起线粒体中蛋白质原位核磁共振研究方法，同时探究α-突触核蛋白引起线粒体功能障碍的可能分子机制。该研究方法的建立能为线粒体中蛋白质的原位研究提供一种原子水平观察蛋白质结构、相互作用、修饰等重要信息的有效途径，从而为线粒体疾病发病机制的研究提供新的研究方法突破。

线粒体是真核生物细胞中重要的细胞器，线粒体功能障碍将引起诸多疾病，如早衰、神经退行性疾病—帕金森病等。在线粒体中开展蛋白质原位研究是理解这些疾病发生机制的一个重要途径。能提供蛋白质原子水平信息的核磁共振波谱技术应用于线粒体蛋白质原位研究具有独特优势，但目前该领域的发展尚处刚刚起步阶段，非常缺乏系统的研究方法。本项目以引起线粒体功能障碍、与帕金森病紧密相关的α-突触核蛋白为研究对象，首先在原核表达系统中实现蛋白质同位素标记，然后采用不同方法将标记蛋白引入线粒体中，并采集核磁共振数据。研究发现在尝试的诸多方法中，电转导方法是最为有效的转入目标蛋白到线粒体内的方法，我们基于该方法将目标蛋白转入大鼠组织（肝或脑）线粒体中，收集一系列核磁共振谱图，建立了线粒体中蛋白质原位核磁共振研究方法。该研究方法的建立为线粒体中蛋白质的原位研究提供一种原子水平观察蛋白质结构、相互作用、修饰等重要信息的有效途径。