Linking Gene Regulation to Metabolism

将基因调控与新陈代谢联系起来

• 批准号: 8420434
• 负责人: MICHAEL R BRENT
• 金额: $ 32.53万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-06 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8420434
• 关键词: 5' -AMP-activated protein kinase; Aerobic; Affect; Automobiles; Biomass; Carbon; Cell Respiration; Cells; Charge; Communication; Complex; Cyclic AMP-Dependent Protein Kinases; DNA Binding; Diabetes Mellitus; Disease; Energy Metabolism; Enzyme Gene; Enzymes; Equilibrium; Fermentation; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Glucose; Goals; Growth; Health; Individual; Internet; Intervention; Knowledge; Lead; Light; Link; Logic; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Metabolic; Metabolic Pathway; Metabolism; Methods; Modeling; Monitor; Outcome; Output; Pathway interactions; Production; Protein Kinase; Regulation; Research Design; Role; Saccharomyces cerevisiae; Shunt Device; Sirolimus; System; Testing; Therapeutic Intervention; Trees; Yeast Model System; Yeasts; cancer cell; improved; insight; interest; link protein; man; member; mutant; organizational structure; research study; sugar; transcription factor

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.

描述（由申请人提供）：许多健康问题是由中枢代谢失调引起的，或依赖于中枢代谢失调。例如，侵袭性癌细胞将碳从有氧呼吸转向厌氧能量生产和生物质。从酵母到人类，碳命运的三个主要调节因子是保守的：蛋白激酶A（PKA）、AMP活化蛋白激酶（AMPK）和雷帕霉素靶点（TOR）。该项目的长期目标是了解哺乳动物细胞中这三个主调节因子及其下游转录因子（TF）之间如何分配碳命运的责任。在本研究中，我们将研究AMPK和PKA在模式酵母Saccharomycescerevisiae中对碳命运的调控。相当多的DNA结合TF作为末端效应AMPK和PKA是已知的。每个TF在几个途径中调节编码酶的基因，并且每个途径由几个TF调节。在任何给定的途径中，有些基因受单个已知TF的调控，有些受几个TF的调控，有些则不受任何TF的调控。该提案的结果将是理解AMPK和PKA如何通过协调这些末端效应物TF的活动来调节碳命运的重要一步。目的1阐明AMPK和PKA对基因表达和碳命运的影响。为了实现这一目标，我们将用生长限制性葡萄糖供应（其中AMPK是活性的而PKA不是）或过量葡萄糖供应（其中PKA是活性的而AMPK不是）培养酵母，监测碳命运，并进行基因表达谱分析。我们还将用突变株进行这些实验，在这些突变株中，我们可以控制AMPK和PKA的活化水平，而不依赖于葡萄糖的利用率。目的2定量分析TF在AMPK和PKA对基因表达和碳命运影响中的作用。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，其中我们可以独立地控制AMPK和PKA激活，并且12个下游TF之一已经被删除。目的3建立PKA和AMPK通过效应转录因子与代谢产物相关的定量模型。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。我们还将估计代谢通量通量使用通量平衡分析，并构建一个模型的酶基因表达代谢通量，因此碳命运。综合起来，这两个模型将对基因表达和碳如何受到调控系统干预的影响进行定量预测。最后，我们将通过使用已删除单个酶或已删除成对TF的菌株来测试这些预测。