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: 10237172
• 负责人: Therese Kichuk
• 金额: $ 5.1万
• 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/10237172
• 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; Immunoblotting; 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; 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; 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; Immunoblotting; 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; 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 糖尿病在全球影响超过4亿人。这些受影响人口的大多数是 被诊断为2型糖尿病（T2DM）。最近的临床研究表明T2DM患者 帕金森氏病（PD）的风险高于非糖尿病患者，并具有共同的细胞内病理 特征表明这些疾病具有常见的机械基础。临床试验已经开始 在PD的背景下，研究各种T2DM治疗的治疗益处。新一代 设计用于抑制线粒体丙酮酸载体（MPC）的胰岛素传感器已显示出治疗前景 在T2DM和PD的实验模型中。由于MPC是两种疾病治疗的药物靶标 对这些跨膜蛋白的研究可以发现T2DM和PD之间的机械联系。 MPC是 酵母与人之间的高度保守，因此这项研究提出了利用 模型生物体苏氏酿酒酵母模型的简单性和遗传性生长。该项目将提供一个 更深入地了解MPC在控制细胞代谢，细胞器动力学和 线粒体。我的第一个目标将调查MPC负责分支运输的假设。 链氨基酸（BCAA）代谢产物，特别是α-酮异估（KIV）。为了实现这个目标，我将首先 MPC单体表达改变的工程酵母菌菌株。这些菌株的孤立线粒体将是 经过生化测定和气相色谱仪的约束将用于确定 产生的底物和产品浓度。这项研究调查的第二个假设是 缺乏功能性MPC会增加线粒体的束缚在内质网和真空中 酵母。为了评估细胞内细胞器动力学和形态，我将使用荧光记者和 显微镜技术。第三个目标将探讨MPC抑制下降线粒体的假设 回收和ATP生产。线粒体降解和ATP生产将通过使用 成像，免疫印迹和呼吸测定。该项目将阐明MPC的下游效应 抑制作用，从而有助于发现T2DM和PD之间联系的分子基础。通过提供一个 更好地了解MPC抑制对细胞代谢，细胞器动力学和 线粒体功能，这项研究将为两种疾病的新疗法开发。 拟议的研究项目将在普林斯顿大学在一个非常适合的指导下在普林斯顿大学进行 导师团队（赞助商：JoséAvalos博士，共同发起人：Coleen Murphy博士，合作者：Clifford博士 Brangwynne和Daniel Cohen博士）。封闭的提案包含一个培训计划，以提高对 科学技术，增强批判性思维并完善科学材料的交流。另外，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坚实的 作为身体科学家独立研究职业的基础。