Mitochondrial metabolite compartmentalization in health and disease

项目摘要 真核细胞在不同的细胞器中储存和利用代谢物--称为亚细胞代谢物 分隔化。不同的代谢酶和底物池提供了另一层灵活性 代谢产物的利用，从而允许强大的适应各种内在线索和外部压力。在……里面 反过来，这些过程中的缺陷与代谢紊乱有关，包括肥胖、胰岛素抵抗和 糖尿病。代谢产物区域化的关键调节因素之一是线粒体转运蛋白：一种大型 许多载体蛋白，其中许多属于SLC25A蛋白家族，介导着 代谢产物穿过不透性的线粒体内膜，并控制它们在体内的利用率 线粒体基质。然而，绝大多数线粒体SLC25A载体蛋白都是“孤儿” 转运蛋白，即其特定底物和生物功能仍不清楚。 我们知识的缺乏主要是因为许多线粒体膜蛋白不能 在常规实验系统中正确重组，即使用重组蛋白制备的脂质体 在大肠杆菌或酵母菌中。为了绕过这个问题，我们开发了一个强大的实验平台，使系统 利用棕色脂肪表征哺乳动物线粒体转运体，棕色脂肪是最具代表性的线粒体之一。 浓缩的细胞。我们将CRISPRi和CRISPRA系统整合到永生化的棕色脂肪细胞中，这样 我们基本上可以在老鼠和人类身上获得无限量的“设计线粒体”。通过使用 新系统，我的实验室最近发现SLC25A44是哺乳动物中第一个线粒体支链氨基酸转运蛋白， 该领域长期存在的谜团(Yonehiro等人)。自然2019年)。 该建议旨在生成线粒体SLC25A代谢物转运体的完整功能图谱 哺乳动物。为了实现这一目标，我们计划将最先进的代谢组学和线粒体脂质体应用于 棕色脂肪衍生的设计线粒体，并确定孤儿SLC25A携带者的特定底物 蛋白质。我们将进一步确定孤儿SLC25A转运蛋白在脑内的生理和病理作用 活体，重点是代谢紊乱。这个应用程序所产生的工作将建立一个概念性的 理解线粒体代谢产物区域化的分子调控的框架，以及 为逆转由这些过程中的缺陷引起的疾病表型提供了新的路线图。

Project Summary Eukaryotic cells store and utilize metabolites in different organelles – referred to as subcellular metabolite compartmentalization. Distinct pools of metabolic enzymes and substrates provide another layer of flexibility in metabolite utilization, thereby allowing for robust adaptation to a variety of intrinsic cues and external stress. In turn, defects in the processes are associated with metabolic disorders, including obesity, insulin resistance, and diabetes. One of the critical regulators of metabolite compartmentalization is mitochondrial transporters: a large number of carrier proteins, many of which belong to the SLC25A protein family, mediate the translocation of metabolites across the impermeable mitochondrial inner-membrane and control their availability in the mitochondrial matrix. However, a vast majority of the mitochondrial SLC25A carrier proteins are “orphan” transporters, i.e., their specific substrates and biological functions remain unknown. The lack of our knowledge is primarily due to the fact that many mitochondrial membrane proteins cannot be reconstituted correctly in the conventional experimental system, i.e., liposomes using recombinant proteins made in E. Coli or yeast. To circumvent this issue, we developed a robust experimental platform that enables systemic characterization of mammalian mitochondrial transporters using brown fat, one of the most mitochondria- enriched cells. We incorporated the CRISPRi and CRISPRa system in immortalized brown adipocytes, such that we can obtain essentially unlimited amounts of “designer mitochondria” in mice and humans. By employing the new system, my lab has recently identified SLC25A44 as the first mitochondrial BCAA transporter in mammals, a long-standing mystery in the field (Yoneshiro et al. Nature 2019). This proposal aims to generate a complete functional map of mitochondrial SLC25A metabolite transporters in mammals. To achieve this goal, we plan to apply the state-of-art metabolomics and mitochondrial-liposomes to the brown fat-derived designer mitochondria, and to determine the specific substrates for orphan SLC25A carrier proteins. We will further determine the physiological and pathological roles of orphan SLC25A transporters in vivo, with an emphasis on metabolic disorders. The work resulting from this application will establish a conceptual framework to understand the molecular regulation of mitochondrial metabolite compartmentalization, and also provide a new roadmap for reversing disease phenotypes that stem from defects in such processes.