Novel roles of PDK4 in regulating mitochondrial protein phosphorylation, carbon flux and metabolic resilience

摘要 这个项目旨在了解线粒体碳运输和生物能量学是如何受到 丙酮酸脱氢酶激酶4(PDK4)，一种对营养和能量提示高度反应的蛋白质 作为潜在的治疗靶点而受到广泛关注的一种药物。PDK4是丙酮酸家族的一员 使线粒体丙酮酸磷酸化和失活的脱氢酶激酶(PDK1-4) 脱氢酶复合体(PDC)。通过将丙酮酸转化为乙酰辅酶A，PDC将糖酵解连接到 三羧酸循环(TCAC)，产生ATP合成所需的还原当量。值得注意的是，PDK4 是对急性能量应激反应最强烈的诱导基因/蛋白质之一--例如禁食， 运动和食用高脂肪的食物。急性暴露于脂肪酸也会强烈地诱导PDK4 和/或激活PPAR转录因子家族的其他配体。这一非凡的水平 营养/能量诱导的调节是PDK4独有的(与PDKs1-3相比)，增加了这样一种可能性 PDK4具有独特的代谢功能。这一建议的重大理念创新和中心前提 源于来自我们实验室的新的令人兴奋的证据，即PDK4可以磷酸化和调节蛋白质 在PDC之外。初步研究使用基于质谱学的蛋白质组学来评估磷酸化- 从PDK4基因过表达或去除的小鼠的心脏和/或骨骼肌中提取蛋白质组。 总的来说，这些发现支持一种工作模型，其中PDK4磷酸化并调节 线粒体酶和蛋白质对脂质应激的反应。因此，该项目试图检验假设 PDK4在介导脂质诱导的线粒体蛋白磷酸化过程中起着核心作用 PDC，它反过来以赋予新陈代谢弹性的方式调节碳运输和生物能量学。 为了验证这一假设，我们将结合功能增强和功能丧失的老鼠模型和几种最先进的模型 分子图谱工具(基于质谱学的蛋白质组学、磷酸蛋白质组学、代谢组学和稳定性 同位素代谢通量分析)，一个复杂的线粒体诊断平台，以及全面的 生理评估以描述PDK4相互作用组及其关键的生理功能。

ABSTRACT This project aims to understand of how mitochondrial carbon trafficking and bioenergetics are regulated by pyruvate dehydrogenase kinase 4 (PDK4), a protein that is highly responsive to nutrient and energetic cues and one that has received much attention as a potential therapeutic target. PDK4 is a member of a family of pyruvate dehydrogenase kinase enzymes (PDK1-4) that phosphorylate and inactive the mitochondrial pyruvate dehydrogenase complex (PDC). By converting pyruvate to acetyl-CoA, the PDC connects glycolysis to the tricarboxylic acid cycle (TCAC), which generates reducing equivalents needed for ATP synthesis. Notably, PDK4 is one of the most robustly induced genes/proteins in response to acute energy stresses–such as fasting, exercise and consumption of a high fat meal. PDK4 is also strongly induced by acute exposure to fatty acids and/or other ligands that activate the PPAR family of transcription factors. This remarkable level of nutrient/energy-induced regulation is unique to PDK4 (as compared with PDKs1-3), raising the possibility that PDK4 has distinct metabolic functions. The major conceptual innovation and central premise of this proposal stems from new and exciting evidence from our laboratory that PDK4 phosphorylates and regulates proteins beyond the PDC. Preliminary studies used mass spectrometry-based proteomics to assess the phospho- proteome of hearts and/or skeletal muscles from mice in which the PDK4 gene was overexpressed or ablated. In aggregate, the findings support a working model wherein PDK4 phosphorylates and regulates multiple mitochondrial enzymes and proteins in response to lipid stress. Accordingly, the project seeks to test hypothesis that PDK4 plays a central role in mediating lipid-induced phosphorylation of mitochondrial proteins beyond the PDC, which in turn modulates carbon trafficking and bioenergetics in manner that confers metabolic resilience. To test this hypothesis, we will combine gain- and loss-of-function mouse models with several state-of-the-art molecular profiling tools (mass spectrometry-based proteomics, phospho-proteomics, metabolomics and stable isotope metabolic flux analysis), a sophisticated mitochondrial diagnostics platform, and comprehensive physiological assessments to delineate the PDK4 interactome and its critical physiological functions.