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: 8460772
• 负责人: Masami Yoshimura
• 金额: $ 24.95万
• 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 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8460772
• 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; Health; 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; 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.

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）是一种产生cAMP的酶，其活性被药理学上相关浓度的乙醇以AC异构体特异性的方式增强。这种选择性表明，在cAMP生成系统中，AC是乙醇作用的主要目标。在此之前的资助下，我们已经确定了7型AC （AC7）的三个离散区域（乙醇响应域）对乙醇对其活性的影响很重要，以及这些区域内的氨基酸残基，这些氨基酸残基可能对乙醇的增强作用负责。我们现在建议继续这个项目，通过测试乙醇通过在特定结合位点直接与AC分子相互作用来增强AC活性的假设。在Specific Aim 1中，我们将在cAMP积累试验中使用一系列突变AC7s，确定乙醇反应区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物理化学性质是重要的。在Specific Aim 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，确定与乙醇相互作用的关键氨基酸残基，并利用核磁共振光谱研究乙醇对AC7构象的影响。两个Specific Aims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是重要的，这些残基在蛋白质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反应域的结构和动力学？如果是这样，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？我们将获得的知识对于阐明乙醇调节AC活性的机制至关重要。本研究将为未来针对AC分子的药物开发提供合理的基础。本研究中采用的方法也可以适用于酒精研究领域中其他重要蛋白质的研究。

项目成果

Identification of ethanol responsive domains of adenylyl cyclase.

腺苷酸环化酶的乙醇反应域的鉴定。

• DOI: 10.1111/j.1530-0277.2006.00219.x
• 发表时间: 2006
• 期刊: Alcoholism, clinical and experimental research
• 作者: Yoshimura,Masami;Pearson,Susan;Kadota,Yoichi;Gonzalez,CristinaE
• 通讯作者: Gonzalez,CristinaE

The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase.

酒精对哺乳动物腺苷酸环化酶重组蛋白的影响。

• DOI: 10.1016/j.bbrep.2017.03.011
• 发表时间: 2017
• 期刊: Biochemistry and biophysics reports
• 影响因子: 2.7
• 作者: Qualls-Creekmore,Emily;Gupta,Ratna;Yoshimura,Masami
• 通讯作者: Yoshimura,Masami

Role of an adenylyl cyclase isoform in ethanol's effect on cAMP regulated gene expression in NIH 3T3 cells.

• DOI: 10.1016/j.bbrep.2016.08.025
• 发表时间: 2016-12-01
• 期刊: Biochemistry and biophysics reports
• 影响因子: 2.7
• 作者: Hill, Rebecca A;Xu, Wu;Yoshimura, Masami
• 通讯作者: Yoshimura, Masami

Conversion of red fluorescent protein into a bright blue probe.

• DOI: 10.1016/j.chembiol.2008.08.006
• 发表时间: 2008-10-20
• 期刊: Chemistry & biology
• 作者: Subach OM;Gundorov IS;Yoshimura M;Subach FV;Zhang J;Grüenwald D;Souslova EA;Chudakov DM;Verkhusha VV
• 通讯作者: Verkhusha VV

Real-time monitoring of intracellular cAMP during acute ethanol exposure.

• DOI: 10.1111/acer.12133
• 发表时间: 2013-09
• 期刊: Alcoholism, clinical and experimental research
• 作者: Gupta R;Qualls-Creekmore E;Yoshimura M
• 通讯作者: Yoshimura M