Alcohol Binding Site(s) in Protein Kinase C Epsilon

蛋白激酶 C Epsilon 中的醇结合位点

• 批准号: 7082723
• 负责人: Joydip Das
• 金额: $ 25.16万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-15 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7082723
• 关键词: X ray crystallography; alcoholism /alcohol abuse; allosteric site; binding sites; biological signal transduction; chemosensitizing agent; diacylglycerols; enzyme activity; ethanol; fluorescent dye /probe; gene expression; gene mutation; intermolecular interaction; mass spectrometry; mutant; phorbols; protein binding; protein kinase C; protein signal sequence; protein structure function; proteolysis

DESCRIPTION (provided by applicant): Alcoholism and alcohol abuse cause major health problems worldwide. Defining the target(s) and elucidating the mechanism of its action at the molecular level is necessary to develop effective prevention. The overall goal of this proposal is to identify alcohol binding sites on a signal transducing protein, protein kinase C epsilon (PKCe), and to determine the secondary structure of these sites. Evidence indicates acute alcohol exposure modulates PKC activity and alters subcellular distribution of individual PKC isoenzymes, but chronic exposure to ethanol leads to an elevation of PKC expression and/or function. Conversely, alteration in the expression of PKC isoforms influences alcohol consumption and behavioral responses to alcohol. While in vivo studies with PKC null mice showed decreased alcohol consumption compared to the wild type, encouraging preliminary in vitro data suggest ethanol inhibits PKC activity. The specific hypothesis to be tested is that there is a site for alcohols in the C1 domain of PKC. The first aim of this proposal is to identify alcohol binding by using fluorescent PKC activators for its allosteric interaction with alcohols and by the use of novel photoreactive alcohols to precisely determine the contact points between the PKCeC1 and alcohols. Upon covalent attachment to the amino acid residues within the binding site(s), the labeled protein will be proteolytically cleaved followed by amino acid sequencing of the generated peptide by mass spectrometry to identify the alcohol site(s) on PKC. The second aim is to characterize the identified binding site(s) by mutational analysis and high resolution X-ray crystallography. These will lead to the final aim of generating PKC mutants with altered alcohol sensitivity which could be exploited in studies in cells or mice to test for the direct involvement of PKC in ethanol's action. The significance of my studies is to establish and characterize alcohol binding site in PKC and to develop mutants with altered alcohol sensitivity which will contribute to understanding the mechanism of alcohol action.

描述（由申请人提供）：酗酒和酒精滥用在世界范围内造成重大健康问题。在分子水平上明确其作用靶点并阐明其作用机制是制定有效预防措施的必要条件。本提案的总体目标是确定信号转导蛋白，蛋白激酶C ε (PKCe)上的醇结合位点，并确定这些位点的二级结构。有证据表明，急性酒精暴露可调节PKC活性并改变单个PKC同工酶的亚细胞分布，但慢性酒精暴露可导致PKC表达和/或功能升高。相反，PKC异构体表达的改变影响酒精消耗和对酒精的行为反应。而体内研究