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）：本研究的长期目标是阐明乙醇对中枢神经系统中环磷酸腺苷（cAMP）信号通路影响的分子和细胞机制。腺苷酸环化酶（AC）的活性，产生cAMP的酶，是增强的乙醇在AC异构体特异性的方式与乙醇浓度相关。这种选择性表明，在cAMP生成系统中，AC是乙醇作用的主要靶标。在先前的资助下，我们已经确定了7型AC（AC7）的三个离散区域，这些区域对于乙醇对其活性的影响（乙醇响应结构域）非常重要，以及这些区域内的氨基酸残基可能负责乙醇的增强作用。我们现在建议通过测试乙醇通过直接与AC分子在特异性结合位点相互作用来增强AC活性的假设来继续这个项目。在特定目标1中，在cAMP蓄积试验中使用一系列突变型AC7，我们将鉴定乙醇响应结构域中负责乙醇效应的关键氨基酸残基，并确定每个残基的哪些理化性质是重要的。在具体目标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的催化结构域的三维结构，包括乙醇响应结构域，确定参与与乙醇相互作用的关键氨基酸残基，并使用NMR光谱检查乙醇对AC7构象的影响。这两个特定目标中提出的研究将相互补充，以回答以下问题：1）乙醇响应结构域中的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)与乙醇结合的关键残基是什么，它们的功能贡献是什么？3)乙醇是否改变了乙醇响应域的结构和动力学？如果是，哪些氨基酸残基参与了乙醇诱导的构象变化，这些残基的功能贡献是什么？我们将获得的知识是至关重要的阐明乙醇调节活性的AC的机制。该研究为今后开发以AC分子为靶点的药物提供了理论依据。在这项研究中采用的方法也可以适用于酒精研究领域的其他重要蛋白质的研究。 公共卫生相关性环磷酸腺苷（cAMP）信号转导被认为是人类酒精中毒发展和易感性的一个促成因素。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起作用。这类研究对于合理开发药物干预酒精滥用和酒精中毒非常重要。