A Yeast Genetics Approach to Identify and Characterize Mitochondrial Transporters in Coenzyme Q Biosynthesis

项目总结/摘要 线粒体在调节代谢和维持细胞内稳态方面发挥着公认的作用。 线粒体功能障碍已在衰老细胞和生物体中被描述，这表明它可能有助于 与年龄相关的生理完整性下降。然而，我们对线粒体在衰老中的作用的理解是， 由于我们对线粒体过程和蛋白质的不完整描述， 有限的线粒体干预，以改善人类健康。因此，新方法的开发 依赖于对线粒体内发生的生化过程的完整理解。但不少 线粒体过程和蛋白质仍然是未知的或很少被表征。因此，阐明这些 线粒体生物化学中的未知数将使我们更好地了解线粒体功能及其 在衰老过程中的作用。 辅酶Q（CoQ）是一种线粒体脂质，对许多过程至关重要，包括氧化 磷酸化尽管其对代谢和线粒体功能的重要性， CoQ仍然没有完全定义。该途径的缺陷会影响辅酶Q的丰度， 新陈代谢.在动物模型中，辅酶Q丰度的变化已被证明会影响寿命和健康寿命， 尽管具体的影响还不清楚。辅酶Q促进衰老的机制仍然存在 这在很大程度上是模糊的，部分原因是我们对辅酶Q生物合成的知识有限。因此，所提出的研究的目标 就是更好地理解这种生物合成途径。 烯丙基焦磷酸盐（APP）是一类带电的代谢产物，是辅酶Q的前体 疏水尾。考虑到它们的电荷，假设存在APP的转运蛋白。然而，在这方面， 迄今尚未查明这种转运人。因此，拟议的研究将利用酵母遗传学和 APP摄取测定以鉴定和表征该转运蛋白。具体目标是：（1）确定候选人 APP转运蛋白使用辅酶Q依赖表型，和（2）定义线粒体APP的动力学 体外摄取。成功完成拟议目标将有助于深入了解APP的机制 运输，辅酶Q生物合成的一个重要步骤。此外，它还可以进一步研究APP转运蛋白的 在辅酶Q缺乏、代谢功能障碍和衰老中的潜在作用。 拟议的研究将作为与华盛顿大学的Pagliarini博士制定的培训计划的一部分进行， 麦迪逊。它的特点是在生物化学科学的坚实基础，书面和口头的机会， 沟通，强大的导师，大学实验室文化，并在威斯康星大学麦迪逊分校的支持性培训环境。 重要的是，这个计划是通过纵向临床经验与医学院的整合。总之， 该培训计划和研究提案将确保严格的医学和研究教育， 有抱负的物理学家兼科学家

PROJECT SUMMARY/ABSTRACT Mitochondria play a well-established role in regulating metabolism and maintaining cellular homeostasis. Mitochondrial dysfunction has been described in aged cells and organisms, suggesting that it may contribute to the age-related decline in physiologic integrity. However, our understanding of mitochondria’s role in aging is limited by our incomplete characterization of mitochondrial processes and proteins – a fact highlighted by the limited mitochondrial interventions to improve human healthspan. Thus, the development of new approaches relies on a complete understanding of the biochemical processes that occur within mitochondria. However, many mitochondrial processes and proteins remain unknown or poorly characterized. Thus, elucidating these unknowns in mitochondrial biochemistry will allow for a better understanding of mitochondrial function and its role in the aging process. Coenzyme Q (CoQ) is a mitochondrial lipid that is critical for many processes, including oxidative phosphorylation. Despite its importance to metabolism and mitochondrial function, the biosynthetic pathway for CoQ remains incompletely defined. Defects in this pathway affect CoQ abundance and can disrupt cellular metabolism. Changes in CoQ abundance have been shown to affect longevity and healthspan in animal models, though the specific effects are poorly understood. The mechanisms by which CoQ contribute to aging remain largely obscure, in part due to our limited knowledge of CoQ biosynthesis. Thus, a goal of the proposed research is gain a better understanding of this biosynthetic pathway. Allylic pyrophosphates (APPs) are a class of charged metabolites that are precursors for CoQ’s hydrophobic tail. Given their charge, it is hypothesized that there is a transporter protein for APPs. However, such a transporter has not been identified to date. Thus, the proposed research will utilize yeast genetics and an APP uptake assay to identify and characterize this transporter. The specific aims are to (1) identify candidate APP transporters using CoQ-depdent phenotypes, and (2) define the kinetics of mitochondrial APP uptake in vitro. Successful completion of the proposed aims will provide insight into the mechanisms of APP transport, a vital step in CoQ biosynthesis. Additionally, it enables further investigation into the APP transporter’s potential role in CoQ deficiency, metabolic dysfunction, and aging. The proposed research will be carried out as part of a training plan developed with Dr. Pagliarini at UW- Madison. It is characterized by a robust foundation in the biochemical sciences, opportunities for written and oral communication, strong mentorship, a collegial lab culture, and a supportive training environment at UW-Madison. Important to this plan is the integration with medical school through longitudinal clinical experiences. Altogether, this training plan and research proposal will ensure a rigorous medical and research education ideal for an aspiring physician-scientist.