Delineating phosphorylation-mediated regulation of mitochondrial function

描绘磷酸化介导的线粒体功能调节

• 批准号: 10713378
• 负责人: Natalie Niemi
• 金额: $ 38.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713378
• 关键词: Address; Affect; Biochemical; Cells; Citric Acid Cycle; Clinical; Complement; Complex; Cues; Data; Energy Metabolism; Eukaryotic Cell; Event; Functional disorder; Genetic Identity; Homeostasis; Human Pathology; Individual; Ions; Knock-out; Knockout Mice; Knowledge; Link; Maps; Mediating; Mediator; Metabolic; Metabolic dysfunction; Metabolism; Mitochondria; Mitochondrial Proteins; Modification; Mus; Nutrient; Organelles; Perinatal mortality demographics; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Production; Protein Import; Protein phosphatase; Proteins; Regulation; Reproducibility; Role; Testing; Work; clinically relevant; human disease; mitochondrial dysfunction; new therapeutic target; pharmacologic; protein function; therapeutic target; virtual

Abstract Mitochondria are complex organelles found in virtually all eukaryotic cells. These organelles orchestrate diverse functions such as energy expenditure, nutrient selection, and ion homeostasis, and do so through the coordination of over 1,000 mitochondria-resident proteins. Most of these mitochondrial proteins are phosphorylated under select physiological conditions, but surprisingly little has been done to characterize these organellar modifications. Motivated by the observation that mitochondria house numerous protein phosphatases, we predicted that regulated protein phosphorylation may play a larger role in organellar homeostasis than is currently appreciated. Indeed, our studies show that the knockout of one mitochondrial phosphatase, Pptc7, leads to stark metabolic dysfunction culminating in fully penetrant perinatal lethality in mice. This surprisingly severe pathophysiology indicates that proper management of protein phosphorylation is requisite for mitochondrial homeostasis. Despite these data, it remains unclear how mitochondrial proteins become phosphorylated and the extent to which individual phosphorylation events contribute to mitochondrial function. We will begin to address these gaps in knowledge by mapping the full breadth of substrates of matrix-localized kinases and by testing the cellular compartment in which mitochondrial-destined proteins become phosphorylated. These studies will begin to address longstanding questions as to the mechanisms enabling mitochondrial protein phosphorylation as well as and the genetic identities of its regulators. To complement this work, we will utilize mechanistic, hypothesis-driven approaches to test the effects of phosphorylation on two proteins, Timm50 and Idh2. These two proteins are reproducibly hyperphosphorylated in Pptc7 KO conditions, suggesting they drive at least a subset of the stark phenotypes associated with the knockout of this phosphatase. Furthermore, these two proteins play key roles in mitochondrial protein import and TCA cycle-mediated metabolism and their regulation would likely have broad influence on mitochondrial function. We will test the effects of Timm50 and Idh2 phosphorylation at the biochemical level (determining how this modification affects protein functions), at the cellular level (determining how modulation of these phosphorylation events affect organellar processes such as protein import and metabolic flux), and at the organismal level (testing how phosphorylation of these proteins may mediate pathophysiology – particularly of phenotypes manifested in Pptc7 KO mice). Collectively, this work will link kinases to mitochondrial function and will establish a workflow to delineate the functions of individual phosphorylation events from the biochemical to the physiological level. As kinases are druggable and have had positive clinical impact in human disease, these studies may uncover novel therapeutic targets through which we can resolve mitochondrial dysfunction found across human pathologies.

抽象的 线粒体是几乎所有真核细胞中都存在的复杂细胞器。这些细胞器协调不同的 能量消耗、营养选择和离子稳态等功能，并通过 协调 1,000 多种线粒体驻留蛋白。这些线粒体蛋白大部分是 在选定的生理条件下被磷酸化，但令人惊讶的是，很少有人对这些进行表征 细胞器修饰。受到线粒体中含有大量蛋白磷酸酶的观察的启发， 我们预测，受调节的蛋白质磷酸化可能在细胞器稳态中发挥比其更大的作用 目前赞赏。事实上，我们的研究表明，一种线粒体磷酸酶 Pptc7 的敲除， 导致严重的代谢功能障碍，最终导致小鼠完全渗透性围产期死亡。这令人惊讶 严重的病理生理学表明，适当管理蛋白质磷酸化对于 线粒体稳态。尽管有这些数据，但仍不清楚线粒体蛋白如何变成 磷酸化以及单个磷酸化事件对线粒体功能的贡献程度。 我们将通过绘制矩阵局部化基底的全部宽度来开始解决这些知识差距 激酶并通过测试细胞区室，其中线粒体目的地蛋白质变成 磷酸化。这些研究将开始解决有关机制的长期存在的问题 线粒体蛋白磷酸化及其调节因子的遗传特性。为了补充这一点 在工作中，我们将利用机械的、假设驱动的方法来测试磷酸化对两种物质的影响 蛋白质、Timm50 和 Idh2。这两种蛋白在 Pptc7 KO 条件下可重复地过度磷酸化， 这表明它们至少驱动了与这种磷酸酶敲除相关的明显表型的一个子集。 此外，这两种蛋白质在线粒体蛋白质输入和 TCA 循环介导中发挥关键作用 代谢及其调节可能对线粒体功能产生广泛影响。我们将测试 Timm50 和 Idh2 磷酸化在生化水平上的影响（确定这种修饰如何影响 蛋白质功能），在细胞水平（确定这些磷酸化事件的调节如何影响 细胞器过程，例如蛋白质输入和代谢通量），以及在有机体水平（测试如何 这些蛋白质的磷酸化可能介导病理生理学——尤其是 Pptc7 中表现的表型 KO小鼠）。总的来说，这项工作将把激酶与线粒体功能联系起来，并将建立一个工作流程 从生化水平到生理水平描述单个磷酸化事件的功能。作为 激酶是可药物化的，并且对人类疾病产生了积极的临床影响，这些研究可能会发现新的 通过治疗目标，我们可以解决人类病理中发现的线粒体功能障碍。