Mechanisms of Glucose Signal Transduction in Yeast

酵母中葡萄糖信号转导机制

基本信息

批准号：
8094292
负责人：
Martin C Schmidt
金额：
$ 34.98万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-07-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8094292
关键词：
5&apos -AMP-activated protein kinase Alleles Am 80 American Binding Biochemical Biological Biological Assay Biological Models Cells Complex Diabetes Mellitus Drug usage Epidemic GLC2 protein Genes Glucose Health Human In Vitro Incidence Ligands Malignant Neoplasms Maps Mediating Modeling Molecular Mutagenesis Mutation Non-Insulin-Dependent Diabetes Mellitus Obesity Orthologous Gene Overweight Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Play Property Protein Dephosphorylation Protein Kinase Protein phosphatase Proteins Regulation Role Saccharomyces cerevisiae Signal Pathway Signal Transduction Sirolimus Site Site-Directed Mutagenesis Stress Testing Therapeutic Yeasts gain of function human FRAP1 protein in vivo inhibitor/antagonist inorganic phosphate loss of function mutant new therapeutic target novel protein B research study small molecule

项目摘要

DESCRIPTION (provided by applicant): The incidence of obesity has increased dramatically over the last fifty years. Over half of all Americans are over- weight and almost one quarter are classified as clinically obese. The health ramifications of this epidemic of obesity are seen in the concomitant increase in the incidence of type 2 diabetes. An estimated 20 million Americans have diabetes with 80% being type 2. The most commonly prescribed therapeutic drugs used for type 2 diabetes target the AMP-activated protein kinase (AMPK). Therefore, understanding how AMPK is normally regulated is of great significance to human health. This proposal uses baker's yeast as its model system to study the regulation of the yeast AMPK. In both yeast and human cells, AMPK is activated under conditions of energy stress. Past studies from our lab have shown that the activation of Snf1, the yeast AMPK, involves at least two steps: phosphorylation of the Snf1 activation loop and activation mediated by the gamma subunit of the Snf1 complex. More recently, we have shown that the first step, control of activation loop phosphorylation, is not regulated at the level of phosphate addition. Surprisingly, it is the dephosphorylation step that responds to cellular energy status. Specific aim 1 of this proposal will determine the molecular mechanism by which glucose regulates the dephosphorylation of Snf1. The protein phosphatase which acts on Snf1 will be purified and characterized. We will determine whether small molecule ligands determine the rate of Snf1 dephosphorylation. Specific aim 2 will investigate the role of the gamma subunit plays in activation of Snf1 kinase complex. The mammalian gamma subunit is thought to be the site of AMP binding. Whether the yeast gamma subunit binds AMP or some other ligand is not known. Site directed and random mutagenesis will be used to identify the residues and regions of the gamma subunit that are important for regulation of Snf1. Mutations in the gamma subunit will be characterized genetically and biochemically. Finally, we have uncovered evidence that Snf1 is a potential regulator of the TOR kinase. In humans, mTOR (target of rapamycin) is a conserved protein kinase that is an important new therapeutic target for many cancers. Specific aim 3 will determine the role of the Snf1 in controlling TOR activity. PUBLIC HEALTH RELEVANCE: The incidence of obesity and type 2 diabetes have dramatically increased in the last fifty years. A human protein called AMPK is the site of action for the most widely prescribed drug used to treat type 2 diabetes. This project will elucidate the normal regulation of AMPK in baker's yeast and may reveal additional targets for novel therapeutics.

描述（由申请人提供）：过去五十年来，肥胖症的发病率急剧增加。超过一半的美国人体重超重，近四分之一被归类为临床肥胖。这种肥胖流行病对健康的影响体现在随之而来的 2 型糖尿病发病率的增加。据估计，有 2000 万美国人患有糖尿病，其中 80% 为 2 型糖尿病。治疗 2 型糖尿病最常用的处方治疗药物针对的是 AMP 激活蛋白激酶 (AMPK)。因此，了解AMPK的正常调控方式对人类健康具有重要意义。该提案使用面包酵母作为模型系统来研究酵母 AMPK 的调节。在酵母和人类细胞中，AMPK 在能量应激条件下都会被激活。我们实验室过去的研究表明，Snf1（酵母 AMPK）的激活至少涉及两个步骤：Snf1 激活环的磷酸化和 Snf1 复合物的 γ 亚基介导的激活。最近，我们发现第一步，即激活环磷酸化的控制，不受磷酸盐添加水平的调节。令人惊讶的是，正是去磷酸化步骤对细胞能量状态做出了反应。该提案的具体目标 1 将确定葡萄糖调节 Snf1 去磷酸化的分子机制。作用于 Snf1 的蛋白磷酸酶将被纯化和表征。我们将确定小分子配体是否决定 Snf1 去磷酸化的速率。具体目标 2 将研究 γ 亚基在 Snf1 激酶复合物激活中的作用。哺乳动物的 γ 亚基被认为是 AMP 结合位点。酵母γ亚基是否结合AMP或其他一些配体尚不清楚。定点和随机诱变将用于识别对 Snf1 调节重要的 γ 亚基残基和区域。伽玛亚基的突变将从遗传和生物化学角度进行表征。最后，我们发现了 Snf1 是 TOR 激酶的潜在调节因子的证据。在人类中，mTOR（雷帕霉素靶点）是一种保守的蛋白激酶，是许多癌症的重要新治疗靶点。具体目标 3 将确定 Snf1 在控制 TOR 活性中的作用。公共卫生相关性：过去 50 年来，肥胖和 2 型糖尿病的发病率急剧增加。一种名为 AMPK 的人类蛋白质是用于治疗 2 型糖尿病的最广泛处方药物的作用位点。该项目将阐明面包酵母中 AMPK 的正常调节，并可能揭示新疗法的其他靶点。