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

摘要 该项目旨在了解线粒体碳运输和生物能量学是如何被调节的。 丙酮酸脱氢酶激酶4（PDK 4），一种对营养和能量线索高度响应的蛋白质， 作为一个潜在的治疗靶点，它受到了广泛的关注。PDK 4是丙酮酸激酶家族的成员， 磷酸化和失活线粒体丙酮酸的脱氢酶激酶（PDK 1 -4） 脱氢酶复合物（PDC）。通过将丙酮酸转化为乙酰辅酶A，PDC将糖酵解连接到葡萄糖。 三羧酸循环（TCAC），产生ATP合成所需的还原当量。值得注意的是，PDK 4 是响应急性能量应激（例如禁食）的最强烈诱导的基因/蛋白质之一， 运动和高脂肪饮食。PDK 4也被急性暴露于脂肪酸强烈诱导 和/或激活转录因子的PPAR家族的其它配体。这种惊人的 营养/能量诱导的调节是PDK 4所独有的（与PDKs 1 -3相比），提高了 PDK 4具有独特的代谢功能。本提案的主要概念创新和中心前提是 源于我们实验室的新的令人兴奋的证据，即PDK 4磷酸化和调节蛋白质 在PDC之外。初步研究使用基于质谱的蛋白质组学来评估磷酸化， 来自小鼠的心脏和/或骨骼肌的蛋白质组，其中PDK 4基因过表达或消除。 总的来说，这些发现支持了一个工作模型，其中PDK 4磷酸化并调节多个 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 PDK 4在介导脂质诱导的线粒体蛋白磷酸化中发挥着核心作用， PDC，其反过来以赋予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，我们将联合收割机结合功能获得和丧失小鼠模型与几种最先进的 分子分析工具（基于质谱的蛋白质组学、磷酸化蛋白质组学、代谢组学和稳定的 同位素代谢通量分析），一个复杂的线粒体诊断平台， 生理学评估以描绘PDK 4相互作用组及其重要生理功能。

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.