Delineating phosphorylation-mediated regulation of mitochondrial function

摘要 线粒体是一种复杂的细胞器，几乎存在于所有真核细胞中。这些细胞器编排了不同的 功能，如能量消耗、营养选择和离子动态平衡，并通过 1,000多个线粒体驻留蛋白的协调。这些线粒体蛋白中的大多数是 在特定的生理条件下被磷酸化，但令人惊讶的是，几乎没有人对这些进行表征 细胞器的修饰。由于观察到线粒体中含有大量的蛋白磷酸酶， 我们预测，调节蛋白的磷酸化在细胞器内稳态中可能发挥比现在更大的作用。 目前很受欢迎。事实上，我们的研究表明，线粒体磷酸酶Pptc7， 导致严重的代谢功能障碍，最终导致小鼠围产期完全穿透性死亡。令人惊讶的是 严重的病理生理表明，蛋白质磷酸化的适当管理是必要的 线粒体动态平衡。尽管有这些数据，但目前仍不清楚线粒体蛋白是如何变成 磷酸化以及个别磷酸化事件对线粒体功能的贡献程度。 我们将开始通过绘制矩阵本地化衬底的全部宽度来解决这些知识差距 通过测试线粒体目标蛋白所在的细胞室 磷酸化。这些研究将开始解决长期存在的问题，即使 线粒体蛋白磷酸化及其调节者的遗传特性。为了补充这一点 工作中，我们将使用机械的、假设驱动的方法来测试磷酸化对两个 蛋白质、Timm50和IDH2。这两种蛋白质在Pptc7KO条件下可重复过度磷酸化， 这表明它们至少驱动与这种磷酸酶基因敲除相关的STARK表型的一个子集。 此外，这两种蛋白在线粒体蛋白输入和TCA循环介导中起关键作用。 代谢及其调节可能会对线粒体功能产生广泛的影响。我们将测试 TIMM50和IDH2在生化水平上的磷酸化效应(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，如蛋白质输入和代谢流)，以及在生物体水平(测试如何 这些蛋白的磷酸化可能介导病理生理学，特别是Pptc7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，以 从生化水平到生理水平描述各个磷酸化事件的功能。AS 激酶是可以下药的，对人类疾病有积极的临床影响，这些研究可能会发现新的 我们可以通过治疗靶点来解决人类病理中发现的线粒体功能障碍。

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.