Linking Gene Regulation to Metabolism

描述(申请人提供)：许多健康问题是由中枢新陈代谢失调引起的，或依赖于中枢新陈代谢失调。例如，侵袭性癌细胞将碳从有氧呼吸转移到无氧能量生产和生物量。从酵母菌到人类，碳命运的三个主要调节因子是：蛋白激酶A(PKA)、AMP激活的蛋白激酶(AMPK)和雷帕霉素靶标(TOR)。这个项目的长期目标是了解在哺乳动物细胞中，碳命运的责任是如何在这三个主调控因子及其下游的转录因子(TF)之间分配的。在这个应用中，我们将研究AMPK和PKA在模式酵母酿酒酵母中对碳命运的调节。相当多的DNA结合因子作为末端效应器AMPK和PKA是已知的。每条转铁蛋白在几条途径中调节编码酶的基因，每条途径都受几条转铁蛋白的调节。在任何给定的途径中，有些基因受一个已知的转运蛋白调控，有些受多个转运蛋白调控，有些则不受任何转运蛋白调控。这项提议的结果将是朝着理解AMPK和PKA如何通过协调这些末端效应因子的活动来调控碳命运的重要一步。目的1阐明AMPK和PKA对基因表达和碳归宿的影响。为了实现这一目标，我们将利用限制生长的葡萄糖供应(其中AMPK是活性的，而PKA不是)或过量的葡萄糖供应(其中PKA是活性的，AMPK不是)来培养酵母，监测碳的命运，并进行基因表达谱分析。我们还将对突变菌株进行这些实验，在这些实验中，我们可以控制AMPK和PKA的激活水平，而不依赖于葡萄糖的可获得性。目的2量化各效应因子Tf在介导AMPK和PKA对基因表达和碳命运的影响中的作用。为了实现这一目标，我们将使用突变体进行类似于目标1的实验，在这些突变体中，我们可以独立控制AMPK和PKA的激活，并且12个下游TF中的一个已经被删除。目的3建立通过效应因子将PKA和AMPK与代谢产物联系起来的定量模型。为了实现这一目标，我们将构建AMPK和PKA下游基因调控的定量模型。我们还将使用通量平衡分析来估计代谢通量，并构建代谢通量上的酶基因表达模型，从而构建碳命运模型。综上所述，这两个模型将对基因表达和碳排放将如何受到监管系统干预的影响进行定量预测。最后，我们将使用单个酶被删除或成对的TF被删除的菌株来测试这些预测。

DESCRIPTION (provided by applicant): Many health problems result from, or are dependent on, disregulation of central metabolism. For example, aggressive cancer cells shunt carbon away from aerobic respiration toward anaerobic energy production and biomass. The three master regulators of carbon fate are conserved from yeast to man: Protein Kinase A (PKA), AMP Activated Protein Kinase (AMPK), and Target of Rapamycin (TOR). The long term goal of this project is to understand how the responsibility for carbon fate is divided among these three master regulators and the transcription factors (TFs) downstream of them in mammalian cells. In this application, we will study the regulation of carbon fate by AMPK and PKA in the model yeast Saccharomyces cerevisiae. Quite a few of the DNA-binding TFs that serve as end effectors AMPK and PKA are known. Each TF regulates genes encoding enzymes in several pathways and each pathway is regulated by several TFs. Within any given pathway, some genes are regulated by a single known TF, some by several, and some by none. The outcome of this proposal will be a significant step toward understanding how AMPK and PKA regulate carbon fate by coordinating the activities of these end effector TFs. Aim 1 Elucidate the influence of AMPK and PKA on gene expression and carbon fate. To achieve this aim, we will grow yeast with growth-limiting glucose supplies (in which AMPK is active and PKA is not) or excess glucose supplies (in which PKA is active and AMPK is not), monitor carbon fate, and carry out gene expression profiling. We will also carry out these experiments with mutant strains in which we can control the activation level of the AMPK and PKA independently of glucose availability. Aim 2 Quantify the role of each effector TF in mediating the influence of AMPK and PKA on gene expression and on carbon fate. To achieve this aim, we will carry out experiments like those of Aim 1 using mutants in which we can control AMPK and PKA activation independently and one of 12 downstream TFs has been deleted. Aim 3 Build a quantitative model linking PKA and AMPK to metabolic outcomes via effector TFs. To achieve this aim, we will construct a quantitative model of gene regulation downstream of AMPK and PKA. We will also estimate metabolic fluxes using flux balance analysis and construct a model of enzyme gene expression on metabolic fluxes and hence carbon fate. Taken together, these two models will make quantitative predictions about how both gene expression and carbon would be affected by interventions in the regulatory system. Finally, we will test these predictions by using strains in which individual enzymes have been deleted or pairs of TFs have been deleted.