The Impact of Mitochondrial Pyruvate Carriers on Metabolism and Subcellular Dynamics

线粒体丙酮酸载体对代谢和亚细胞动力学的影响

• 批准号: 10455536
• 负责人: Therese Kichuk
• 金额: $ 5.18万
• 依托单位: PRINCETON UNIVERSITY
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10455536
• 关键词: Affect; Animal Model; Autophagocytosis; Biochemical; Biological Assay; Branched-Chain Amino Acids; Cells; Chronic; Clinical; Clinical Data; Clinical Research; Clinical Trials; Communication; Conflict (Psychology); Critical Thinking; Data; Development; Diabetes Mellitus; Diagnosis; Disease; Drug Targeting; Endoplasmic Reticulum; Engineering; Experimental Models; Exposure to; Foundations; Functional disorder; Gas Chromatography; Gene Deletion; Generations; Genetic; Goals; High Pressure Liquid Chromatography; Human; Hyperglycemia; Image; In Vitro; Insulin; Integral Membrane Protein; Knock-out; Knowledge; Link; Mentors; Metabolic dysfunction; Metabolism; Microfluidics; Microscopy; Mitochondria; Molecular; Molecular Target; Monitor; Morphology; Mus; Nerve Degeneration; Non-Insulin-Dependent Diabetes Mellitus; Nutrient; Organelles; Parkinson Disease; Pathologic; Patients; Persons; Physicians; Plant Roots; Population; Production; Proteins; Pyruvate; Recycling; Reporter; Research; Research Project Grants; Respiration; Role; Saccharomyces cerevisiae; Scientist; Site; Solid; Subcellular structure; Techniques; Therapeutic; Training; Treatment Efficacy; Universities; Vacuole; Valine; Western Blotting; Work; Yeasts; base; career; clinically relevant; dimer; experimental study; high risk; improved; in vivo; instrumentation; insulin secretion; insulin sensitizing drugs; monomer; non-diabetic; novel; novel therapeutics; overexpression; pyruvate carrier; uptake

Project Summary Diabetes mellitus affects over 400 million people worldwide. The majority of this affected population is diagnosed with type 2 diabetes (T2DM). Recent clinical studies have demonstrated that patients with T2DM are at higher risk than non-diabetic patients for Parkinson’s disease (PD) and shared subcellular pathologic features indicate that these disorders have common mechanistic underpinnings. Clinical trials have begun to investigate the therapeutic benefit of various T2DM treatments in the context of PD. A new generation of insulin sensitizers engineered to inhibit mitochondrial pyruvate carriers (MPCs) has shown therapeutic promise in experimental models of T2DM and PD. As MPCs are a drug target in the treatment of both disorders, further study of these transmembrane proteins could uncover a mechanistic link between T2DM and PD. MPCs are highly conserved between yeast and humans and therefore this study proposes to take advantage of the simplicity and genetic malleability of the model organism Saccharomyces cerevisiae. This project will provide a deeper understanding of the role of MPCs in regulating cellular metabolism, organelle dynamics, and mitophagy. My first aim will investigate the hypothesis that MPCs are responsible for the transport of branched- chain amino acid (BCAA) metabolites, specifically α-ketoisovalerate (KIV). To achieve this goal I will first engineer yeast strains with altered MPC monomer expression. Isolated mitochondria from these strains will be subjected to biochemical assays and gas chromatography instrumentation will be used to determine the resulting substrate and product concentrations. The second hypothesis investigated by this study is that the lack of functional MPCs will increase mitochondrial tethering to the endoplasmic reticulum and vacuole within yeast. To evaluate intracellular organelle dynamics and morphology I will employ fluorescent reporters and microscopy techniques. The third aim will explore the hypothesis that MPC inhibition decreases mitochondrial recycling and ATP production. Mitochondrial degradation and ATP production will be investigated by employing imaging, immunoblotting, and respiration assays. This project will clarify the downstream effects of MPC inhibition, thereby helping to uncover the molecular basis for the link between T2DM and PD. By providing a better understanding of the impact of MPC inhibition on cellular metabolism, organelle dynamics, and mitochondrial function, this study will inform the development of novel therapeutics for both disorders. The proposed research project will be conducted at Princeton University under the guidance of a superbly suited team of mentors (Sponsor: Dr. José Avalos, Co-sponsor: Dr. Coleen Murphy, Collaborators: Dr. Clifford Brangwynne and Dr. Daniel Cohen). The enclosed proposal contains a training plan to improve knowledge of scientific techniques, enhance critical thinking, and refine communication of scientific material. Additionally, this plan provides opportunities for clinical continuity. Each component of this study was crafted to provide a solid foundation for an independent research career as a physician-scientist.

项目摘要 糖尿病影响全世界超过4亿人。大多数受影响的人口是 被诊断患有2型糖尿病（T2 DM）。最近的临床研究表明，T2 DM患者 与非糖尿病患者相比，帕金森病（PD）的风险更高， 这些特征表明，这些疾病具有共同的机制基础。临床试验已经开始， 研究PD背景下各种T2 DM治疗的治疗获益。新一代 抑制线粒体丙酮酸载体（MPCs）的胰岛素增敏剂已显示出治疗前景 在T2 DM和PD的实验模型中。由于MPC是治疗这两种病症的药物靶标，因此进一步 对这些跨膜蛋白的研究可以揭示T2 DM和PD之间的机制联系。MPC是 在酵母和人类之间高度保守，因此这项研究提出利用 模式生物酿酒酵母的简单性和遗传可塑性。该项目将提供一个 更深入地了解MPC在调节细胞代谢，细胞器动力学和 线粒体自噬我的第一个目标是研究MPC负责运输分支- 链氨基酸（BCAA）代谢产物，特别是α-酮异戊酸（KIV）。为了实现这一目标，我将首先 用改变的MPC单体表达工程化酵母菌株。从这些菌株中分离的线粒体将 进行生化分析和气相色谱仪将用于确定 得到的底物和产物浓度。本研究调查的第二个假设是， 功能性MPC的缺乏将增加线粒体与内质网和内空泡的束缚， 酵母为了评估细胞内细胞器的动力学和形态学，我将使用荧光报告基因， 显微镜技术。第三个目标将探讨MPC抑制降低线粒体 回收和ATP生产。线粒体降解和ATP的产生将通过使用 成像、免疫印迹和呼吸测定。本项目将阐明MPC的下游效应 抑制，从而有助于揭示T2 DM和PD之间联系的分子基础。通过提供 更好地了解MPC抑制对细胞代谢，细胞器动力学的影响， 线粒体功能，这项研究将为这两种疾病的新疗法的发展提供信息。的 拟议中的研究项目将在普林斯顿大学的指导下进行， 导师团队（赞助人：José Avalos博士，共同赞助人：Coleen Murphy博士，合作人：Clifford博士 Brangwynne和丹尼尔科恩博士）。所附建议书包含一项培训计划， 科学技术，提高批判性思维，并完善科学材料的沟通。而且这个 计划为临床连续性提供了机会。这项研究的每个组成部分都是为了提供一个坚实的 作为一个独立的研究事业作为一个物理学家科学家的基础。

项目成果

Using MitER for 3D analysis of mitochondrial morphology and ER contacts.

• DOI: 10.1016/j.crmeth.2023.100692
• 发表时间: 2024-01-22
• 期刊: CELL REPORTS METHODS
• 作者: Kichuk, Therese;Dhamankar, Satyen;Malani, Saurabh;Hofstadter, William A.;Wegner, Scott A.;Cristea, Ileana M.;Avalos, Jose L.
• 通讯作者: Avalos, Jose L.