Action of Ethanol on Cyclic AMP Signal Transduction

乙醇对环磷酸腺苷信号转导的作用

• 批准号: 7583410
• 负责人: Masami Yoshimura
• 金额: $ 29.44万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7583410
• 关键词: Acute; Adenylate Cyclase; Affinity; Alcohol abuse; Alcoholism; Alcohols; Amino Acids; Animals; Bacteria; Behavioral; Binding; Binding Sites; Biological Assay; C2 Domain; Catalytic Domain; Chronic; Complement; Cyclic AMP; Development; Enzymes; Ethanol; Funding; Future; Genetic Determinism; Goals; Grant; Human; Individual; Intervention; Knowledge; Location; Molecular; Molecular Conformation; Mutagenesis; Mutate; NMR Spectroscopy; Neuraxis; Physiological; Play; Predisposition; Property; Protein Isoforms; Proteins; Recombinant Proteins; Recombinants; Research; Role; Series; Signal Pathway; Signal Transduction; Solutions; Structure; System; Testing; adenylyl cyclase 7; alcohol effect; alcohol exposure; alcohol research; alcohol response; base; biological systems; design; drug development; mutant; problem drinker; public health relevance; three dimensional structure

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the molecular and cellular mechanisms underlying the effects of ethanol on the cyclic adenosine monophosphate (cAMP) signaling pathway in the central nervous system. The activity of adenylyl cyclase (AC), the enzyme that generates cAMP, is enhanced by pharmacologically relevant concentrations of ethanol in an AC isoform-specific manner. This selectivity indicates that within a cAMP generating system, AC is a primary target of ethanol's action. With previous support from this grant, we have identified three discrete regions of type7 AC (AC7) important for the effect of ethanol on its activity (ethanol responsive domains), as well as the amino acid residues within these regions that are potentially responsible for the enhancing effect of ethanol. We now propose to continue this project by testing the hypothesis that ethanol enhances AC activity by directly interacting with AC molecules at specific binding site(s). In Specific Aim 1, using a series of mutant AC7s in the cAMP accumulation assay, we will identify crucial amino acid residues in the ethanol responsive domains that are responsible for the effect of ethanol and determine which physicochemical properties of each residue are important. In Specific Aim 2, we will design and produce recombinant AC7 proteins using a bacterial expression system. We will determine the three dimensional structure of the catalytic domains of AC7 including the ethanol responsive domains, identify key amino acid residues involved in the interaction with ethanol, and examine the effect of ethanol on the conformation of AC7 using NMR spectroscopy. Studies proposed in the two Specific Aims will complement each other to answer the following questions: 1) Which amino acid residues in the ethanol responsive domains are important for ethanol's effect, and what are the locations of these residues in the three dimensional structure of the protein? 2) What are the key residues involved in binding to ethanol, and what are their functional contributions? 3) Does ethanol change the structure and dynamics of the ethanol responsive domains? If so, which amino acid residues are involved in ethanol-induced conformational change(s), and what is the functional contribution of those residues? The knowledge we will obtain is crucial for elucidating the mechanism by which ethanol modulates the activity of AC. The proposed research will provide a rational basis for future drug development targeting AC molecules. The approach employed in this research can also be adapted to the study of other proteins important in the alcohol research field. PUBLIC HEALTH RELEVANCE Cyclic adenosine monophosphate (cAMP) signal transduction has been postulated to be a contributing factor to the development of and predisposition to alcoholism in humans. The information we will obtain through the proposed research will help us to understand how alcohol acts on biological systems at the molecular level. This type of research is very important for rational drug development for the intervention of alcohol abuse and alcoholism.

描述（由申请人提供）：这项研究的长期目标是阐明乙醇对中枢神经系统中环状腺苷单磷酸（CAMP）信号通路的影响的分子和细胞机制。产生cAMP的酶（AC）的活性通过AC同工型特异性方式的药理相关乙醇浓度增强。这种选择性表明，在营地生成系统中，AC是乙醇作用的主要目标。在该赠款的先前支持下，我们已经确定了三种AC型AC（AC7）的三个离散区域，对于乙醇对其活性的影响（乙醇反应域）以及这些区域内的氨基酸残基有潜在地负责乙醇的增强作用。我们现在建议通过测试乙醇通过在特定结合位点直接与AC分子相互作用的假设来继续该项目。在特定的目标1中，使用CAMP积累测定中的一系列突变AC7，我们将在乙醇反应型域中确定关键的氨基酸残基，这些残基负责乙醇的作用，并确定每个残基的哪些物理化学性质很重要。在特定的目标2中，我们将使用细菌表达系统设计和生产重组AC7蛋白。我们将确定AC7催化结构域的三维结构，包括乙醇反应域，鉴定与乙醇相互作用涉及的关键氨基酸残基，并使用NMR光谱检查检查乙醇对AC7构象的影响。在两个具体目的中提出的研究将相互补充以回答以下问题：1）乙醇反应域中哪些氨基酸残基对乙醇的作用很重要，这些残基在蛋白质的三维结构中的位置是什么？ 2）与乙醇结合涉及哪些关键残基，其功能贡献是什么？ 3）乙醇会改变乙醇反应域的结构和动力学吗？如果是这样，哪些氨基酸残基参与乙醇诱导的构象变化，这些残基有什么功能贡献？我们将获得的知识对于阐明乙醇调节AC活性的机制至关重要。拟议的研究将为未来的针对AC分子的药物开发提供合理的基础。这项研究中采用的方法也可以适用于对酒精研究领域重要的其他蛋白质的研究。 公共卫生相关性循环腺苷单磷酸（CAMP）信号转导已被认为是人类酒精中毒的发展和倾向的一个因素。我们将通过拟议的研究获得的信息将有助于我们了解酒精如何在分子水平上对生物系统作用。这种研究对于酗酒和酒精中毒的理性药物开发非常重要。