Mitochondrial metabolite compartmentalization in health and disease

健康和疾病中的线粒体代谢物区室化

• 批准号: 10643941
• 负责人: Shingo Kajimura
• 金额: $ 87.5万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10643941
• 关键词: Adipocytes; Biological; Biological Process; Brown Fat; CRISPR interference; CRISPR-mediated transcriptional activation; Carrier Proteins; Cells; Cues; Defect; Diabetes Mellitus; Disease; Enzymes; Escherichia coli; Eukaryotic Cell; Goals; Health; Heart; Human; Inner mitochondrial membrane; Insulin Resistance; Knowledge; Liposomes; Mammals; Maps; Mediating; Metabolic; Metabolic Diseases; Mitochondria; Mitochondrial Matrix; Mitochondrial Membrane Protein; Molecular; Mus; Nature; Obesity; Organelles; Orphan; Pathologic; Physiological; Process; Protein Family; Recombinant Proteins; Regulation; Role; Stress; System; Work; Yeasts; disease phenotype; flexibility; in vivo; metabolomics; reconstitution; stem

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.

项目摘要 真核细胞存储并利用不同细胞器中的代谢物 - 称为亚细胞代谢物 分隔。不同的代谢酶和底物的不同池提供了另一层灵活性 代谢物利用率，从而可以适应各种内在的提示和外部压力。在 转弯，过程中的缺陷与代谢性疾病有关，包括肥胖，胰岛素抵抗和 糖尿病。代谢物分隔的关键调节剂之一是线粒体转运蛋白：一个大的 载体蛋白的数量，其中许多属于SLC25A蛋白家族，介导 在不可渗透的线粒体内膜上的代谢产物，并控制其在 线粒体基质。但是，绝大多数线粒体SLC25A载体蛋白是“孤儿” 转运蛋白，即它们的特定底物和生物学功能仍然未知。 缺乏我们的知识主要是由于许多线粒体膜蛋白不能是 在常规实验系统中正确重构，即使用重组蛋白的脂质体 在大肠杆菌或酵母中。为了避免此问题，我们开发了一个强大的实验平台，以实现系统性 使用棕色脂肪来表征哺乳动物线粒体转运蛋白，这是线粒体最多的脂肪之一 富集的细胞。我们将Crispri和Crispra系统纳入了永生的棕色脂肪细胞中，以至于 我们可以在小鼠和人类中获得基本无限量的“设计师线粒体”。通过使用 新系统，我的实验室最近将SLC25A44确定为哺乳动物中的第一个线粒体BCAA转运蛋白， 该领域的一个谜（Yoneshiro等人自然2019）。 该建议旨在生成线粒体SLC25A代谢物转运蛋白的完整功能图 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师线粒体，并确定孤儿SLC25A载体的特定底物 蛋白质。我们将进一步确定孤儿SLC25A转运蛋白的物理和病理作用 体内，重点是代谢性疾病。该应用程序产生的工作将建立概念 了解线粒体代谢物隔室的分子调节的框架，也是 为逆转疾病表型提供了新的路线图，该表型源于此类过程中的缺陷。