Delineating phosphorylation-mediated regulation of mitochondrial function

摘要 线粒体是一种复杂的细胞器，几乎存在于所有的真核细胞中。这些细胞器协调了 功能，如能量消耗，营养选择和离子稳态，并通过 协调超过1,000种寄生于果蝇的蛋白质。这些线粒体蛋白质中的大多数是 在选择的生理条件下磷酸化，但令人惊讶的是，很少有人做表征这些 细胞器修饰由于观察到线粒体内有许多蛋白磷酸酶， 我们预测，受调节的蛋白质磷酸化在细胞器内环境稳定中可能比在细胞内环境稳定中起更大的作用。 目前赞赏。事实上，我们的研究表明，一种线粒体磷酸酶Pptc 7的敲除， 导致明显的代谢功能障碍，最终导致小鼠的完全渗透性围产期死亡。这个令人惊讶 严重的病理生理学表明，蛋白质磷酸化的适当管理是必要的， 线粒体内稳态尽管有这些数据，仍然不清楚线粒体蛋白质是如何成为 磷酸化的程度以及单个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的基底的全部宽度来解决这些知识差距。 激酶，并通过测试细胞区室，在该细胞区室中， 磷酸化这些研究将开始解决长期存在的问题， 线粒体蛋白磷酸化以及其调节剂的遗传特性。为了配合这一点 工作，我们将利用机械，假设驱动的方法来测试磷酸化对两个 Timm 50和Idh 2蛋白。这两种蛋白质在Pptc 7 KO条件下可重复地过度磷酸化， 这表明它们驱动与该磷酸酶敲除相关的Stark表型的至少一个子集。 此外，这两种蛋白在线粒体蛋白输入和TCA循环介导的细胞凋亡中起关键作用。 代谢及其调节可能对线粒体功能具有广泛的影响。我们将测试 Timm 50和Idh 2磷酸化在生物化学水平上的影响（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，如蛋白质输入和代谢通量），并在生物体水平（测试如何 这些蛋白质的磷酸化可能介导病理生理学-特别是Pptc 7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， 描绘了从生物化学到生理水平的各个磷酸化事件的功能。作为 激酶是可药物化的，并且在人类疾病中具有积极的临床影响，这些研究可能揭示新的 通过这些治疗靶点，我们可以解决人类病理学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.