Mechanisms of Glucose Signal Transduction in Yeast

酵母中葡萄糖信号转导机制

• 批准号: 7877843
• 负责人: Martin C Schmidt
• 金额: $ 35.35万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7877843
• 关键词: 5' -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; public health relevance; research study; small molecule

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）：在过去的五十年中，肥胖的发生率急剧增加。超过一半的美国人体重超重，将近四分之一被归类为临床上的肥胖。肥胖流行的健康后果在2型糖尿病的发生率的伴随增加中。估计有2000万美国人患有糖尿病，其中80％为2型。用于2型糖尿病的最常见的治疗药物针对AMP激活的蛋白激酶（AMPK）。因此，了解AMPK通常如何受到监管对人类健康具有重要意义。该提案使用贝克的酵母作为其模型系统来研究酵母AMPK的调节。在酵母和人类细胞中，在能量应力条件下，AMPK被激活。我们实验室的过去研究表明，SNF1（酵母AMPK）的激活涉及至少两个步骤：SNF1激活环的磷酸化和由SNF1复合物的伽马亚基介导的激活。最近，我们表明，在磷酸盐添加水平上，对激活环磷酸化的控制不受调节。令人惊讶的是，响应细胞能量状态的是去磷酸化步骤。该提案的具体目的1将决定葡萄糖调节SNF1去磷酸化的分子机制。作用于SNF1的蛋白质磷酸酶将被纯化和表征。我们将确定小分子配体是否确定SNF1去磷酸化的速率。具体目标2将研究伽马亚基在SNF1激酶复合物激活中的作用。哺乳动物伽马亚基被认为是AMP结合的位置。酵母菌亚基是否结合AMP或其他一些配体尚不清楚。定向和随机诱变将用于识别伽马亚基的残基和区域，这些残基和区域对于调节SNF1很重要。伽马亚基中的突变将以遗传和生化为特征。最后，我们发现了SNF1是TOR激酶的潜在调节剂的证据。在人类中，MTOR（雷帕霉素的靶标）是一种保守的蛋白激酶，是许多癌症的重要新治疗靶标。特定的目标3将确定SNF1在控制TOR活动中的作用。公共卫生相关性：在过去的五十年中，肥胖和2型糖尿病的发病率显着增加。一种称为AMPK的人蛋白是用于治疗2型糖尿病的最广泛规定药物的作用部位。该项目将阐明贝克酵母中AMPK的正常调节，并可能揭示新的治疗剂的其他靶标。