The Impact of Mitochondrial Pyruvate Carriers on Metabolism and Subcellular Dynamics

项目摘要 糖尿病影响着全世界超过4亿人。大多数受影响的人口是 被诊断为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，患帕金森氏病(PD)的风险更高，并具有共同的亚细胞病理 这些特征表明，这些疾病有共同的机制基础。临床试验已经开始 在帕金森病的背景下，研究各种T2 DM治疗方法的疗效。新一代的 设计用来抑制线粒体丙酮酸载体(MPC)的胰岛素增敏剂已经显示出治疗的前景 在2型糖尿病和帕金森病的实验模型上。由于MPC是治疗这两种疾病的药物靶点，进一步 对这些跨膜蛋白的研究可能揭示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，因此这项研究建议利用 模式生物酿酒酵母的简单性和遗传延展性。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 有丝分裂。我的第一个目标将调查假设，MPC负责运输分支- 链氨基酸(BCAA)代谢物，特别是α-酮基异戊酸酯(KIV)。为了实现这一目标，我将首先 改造MPC单体表达改变的酵母菌株。从这些菌株中分离出的线粒体将被 通过生化分析和气相色谱仪器来确定 由此产生的底物和产物浓度。这项研究调查的第二个假设是， 缺乏功能的MPC将增加线粒体与内质网和空泡的连接 酵母。为了评估细胞内细胞器的动力学和形态，我将使用荧光记者和 显微镜技术。第三个目标将探索MPC抑制减少线粒体的假设 回收利用和三磷酸腺苷生产。线粒体的降解和ATP的产生将通过使用 成像、免疫印迹和呼吸测试。该项目将阐明MPC的下游影响 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，这项研究将为开发治疗这两种疾病的新疗法提供信息。这个 拟议的研究项目将在普林斯顿大学的一个超级合适的指导下进行 导师团队(赞助商：何塞·阿瓦洛斯博士，联合赞助商：科林·墨菲博士，合作者：克利福德博士 Brangwynne和Daniel Cohen博士)。随函附上的建议书包含一项培训计划，以提高对 科学技术，加强批判性思维，改进科学材料的交流。此外，这一点 计划为临床连续性提供了机会。本研究的每个组成部分都经过精心设计，以提供坚实的 作为一名内科科学家的独立研究生涯的基金会。

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.