Mechanisms of Glucose Signal Transduction in Yeast

描述（由申请人提供）：在过去的50年里，肥胖的发病率急剧增加。超过一半的美国人体重超标，近四分之一的人被归类为临床肥胖。肥胖的流行对健康的影响体现在伴随的2型糖尿病发病率的增加上。据估计，有2000万美国人患有糖尿病，其中80%是2型糖尿病。治疗2型糖尿病最常用的药物是靶向amp活化蛋白激酶（AMPK）。因此，了解AMPK在正常情况下是如何调控的，对人类健康具有重要意义。本研究以面包酵母为模型系统，研究酵母AMPK的调控。在酵母和人类细胞中，AMPK在能量应激条件下被激活。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γ亚基介导的激活。最近，我们已经表明，第一步，控制活化环磷酸化，不受磷酸盐添加水平的调节。令人惊讶的是，正是去磷酸化的步骤对细胞能量状态做出了反应。本提案的具体目标1将确定葡萄糖调节Snf1去磷酸化的分子机制。将对作用于Snf1的蛋白磷酸酶进行纯化和鉴定。我们将确定小分子配体是否决定Snf1去磷酸化的速率。特异性目的2将研究γ亚基在Snf1激酶复合物激活中的作用。哺乳动物γ亚基被认为是AMP结合的位点。酵母γ亚基是否与AMP或其他配体结合尚不清楚。位点定向和随机诱变将用于鉴定对Snf1调控重要的γ亚基的残基和区域。γ亚基的突变将被描述为遗传和生物化学特征。最后，我们发现了Snf1是TOR激酶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。特异性目标3将确定Snf1在控制TOR活性中的作用。公共卫生相关性：在过去的50年里，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的作用部位。该项目将阐明面包酵母中AMPK的正常调控，并可能揭示新的治疗靶点。

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.