Role of Protein Kinase C regulatory domains in modulating alcohol actions

蛋白激酶 C 调节域在调节酒精作用中的作用

基本信息

批准号：
7940763
负责人：
Joydip Das
金额：
$ 45万
依托单位：
UNIVERSITY OF HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-15 至 2013-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7940763
关键词：
Alcohol abuse Alcohol consumption Alcohol dependence Alcoholism Alcohols Behavioral Binding Binding Sites Brain Butyric Acids C2 Domain Cause of Death Cells Cessation of life Chimera organism Chimeric Proteins Complement component C1s DAG/PE-Binding Domain Data Diazomethane Disease Economic Burden Ethanol Family Gated Ion Channel Hand Knowledge Label Length Ligand Binding Ligands Mass Spectrum Analysis Measures Molecular Mouse Protein Pharmaceutical Preparations Phosphorylation Phosphotransferases Prevention Property Protein Kinase Protein Kinase C Proteins Role Signal Transduction Signal Transduction Pathway Societies Testing Wild Type Mouse alcohol effect alcohol response alcohol-related death analog circumsporozoite protein cost design effective intervention novel novel therapeutics public health relevance receptor response

项目摘要

DESCRIPTION (provided by applicant): Alcoholism and alcohol abuse is a major cause of death (annually 75,000-100,000 deaths in the US) with an economic burden of 184 billion dollars/year in the US. Defining the target(s) and elucidating the molecular mechanism of its action is needed for effective intervention. The objective of this proposal is to define the molecular mechanisms by which alcohols exert their action on intracellular signal transduction pathways in brain. Alcohols are known to alter the expression and activity of Protein Kinase Cs (PKC), a family of kinases mainly expressed in the brain. While PKC epsilon knock-outs showed significant decrease in alcohol consumption and increase in tolerance to ethanol compared to wild type mice, the PKC gamma knock-outs showed significant increase in alcohol consumption and decrease in tolerance. PKC¿ and PKC3 knock-outs also showed opposite properties in regulating responses to GABAA (3-amino butyric acid) receptors. GABAA is a ligand-gated ion channel in brain and believed to be an important target of alcohol. Structurally, PKC3 has its regulatory C1 domain (combination of C1A and C1B) at the N terminus followed by the regulatory C2 domain. On the other hand, PKC¿ has its C2 domain at the N terminus followed by the C1 domain. The C1A and C1B subdomains of epsilon and gamma differ significantly in terms of their ligand binding properties. The central hypothesis to be tested is that the structural and ligand binding differences in the regulatory domains of PKC¿ and PKC3 are responsible for their differential behavioral response to alcohol. The first aim of this proposal is to determine how alcohols regulate PKC¿ and PKC3 activity and if there is an alcohol binding site in their regulatory domains. In order to identify alcohol binding site we will use novel photoactive diazirine analog of alcohols to photolabel the PKC domain/subdomains followed by identification of the labeled residues by mass spectrometry. The second aim is to generate PKC chimeras by swapping the regulatory domains/subdomains between PKC¿ and PKC3 and characterize their alcohol binding properties. This will be achieved by measuring the effect of alcohols on the activities of the chimeric proteins. The third aim is to investigate the effect of alcohols on GABAA-PKC interactions by measuring GABAA current electrophysiologically. The proposal will enhance our current understanding on the molecular mechanism of alcohol action and will provide strategy for designing new therapeutics against alcohol addiction. PUBLIC HEALTH RELEVANCE: Despite huge number of alcohol related deaths (annually 75000-100,000 in the US) and cost to society ($184 billion/yr in the US) very few medications are available for treating alcohol related diseases. To develop new medications and effective prevention it is necessary to define the target and mechanism of its action at the molecular level. The significance of the present study is to establish the role of a signal transducing protein in regulating alcohol actions. This study will enhance the knowledge in developing medication for alcohol addiction.

描述（由适用提供）：在美国，酒精中毒和酗酒是死亡的主要原因（每年75,000-100,000人死亡），在美国，经济燃烧为1840亿美元。为了有效干预，需要定义目标并阐明其作用的分子机制。该提议的目的是定义醇对大脑中细胞内信号转导途径的作用的分子机制。众所周知，醇会改变蛋白激酶CS（PKC）的表达和活性，这是一个主要在大脑中表达的激酶家族。与野生型小鼠相比，PKC Epsilon敲除饮酒量显着降低和对乙醇的耐受性的增加，但PKCγ敲除量表显示出饮酒量显着增加和耐受性降低。 PKCC和PKC3敲除在控制对GABAA（3-氨基丁酸）受体的反应方面也显示出相反的特性。 GABAA是大脑中的配体门控离子通道，被认为是酒精的重要靶标。从结构上讲，PKC3的调节性C1结构域（C1A和C1B的组合）在N末端，然后是调节性C2结构域。另一方面，pKc¿在N末端具有其C2域，然后是C1域。 Epsilon和Gamma的C1a和C1b子域在其配体结合特性方面有显着的不同。要测试的中心假设是，PKC¿和PKC3的调节结构域中的结构和配体结合差异负责其对酒精的行为反应的不同。该提案的第一个目的是确定酒精如何调节PKCC和PKC3活性，以及其调节域中是否存在酒精结合位点。为了鉴定酒精结合位点，我们将使用醇的新型光活性重氮嘧啶类似物来光标记PKC结构域/子域，然后通过质谱鉴定标记的残留物。第二个目的是通过将调节域/子域交换PKCC与PKC3来产生PKC嵌合体，并表征其酒精结合特性。这将通过测量醇对嵌合蛋白活性的影响来实现。第三个目的是通过通过电生理学测量GABAA电流来研究醇对GABAA-PKC相互作用的影响。该提案将增强我们目前对酒精作用分子机制的理解，并将为设计针对酒精成瘾的新疗法提供策略。公共卫生相关性：尽管与酒精相关的死亡人数大量（在美国每年75000-100,000）和社会成本（在美国为1840亿美元/年），很少有药物可用于治疗与酒精有关的疾病。为了开发新的药物和有效的预防，有必要在分子水平上定义其作用的目标和机制。本研究的重要性是确定信号转导蛋白在调节酒精作用中的作用。这项研究将增强开发酒精成瘾药物的知识。