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Identification of Alcohol Binding Site(s) in Protein Kinase C Epsilon

蛋白激酶 C Epsilon 中醇结合位点的鉴定

基本信息

批准号：
7561403
负责人：
Joydip Das
金额：
$ 20.18万
依托单位：
UNIVERSITY OF HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-06-15 至 2009-05-31
项目状态：
已结题

项目摘要

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）：酗酒和酗酒在全世界范围内造成重大健康问题。确定目标并在分子水平上阐明其作用机制对于制定有效的预防措施是必要的。该提案的总体目标是识别信号转导蛋白、蛋白激酶 C epsilon (PKCe) 上的酒精结合位点，并确定这些位点的二级结构。有证据表明，急性酒精暴露会调节 PKC 活性并改变单个 PKC 同工酶的亚细胞分布，但长期暴露于乙醇会导致 PKC 表达和/或功能升高。相反，PKC 同工型表达的改变会影响饮酒和对酒精的行为反应。在体内研究时与野生型相比，PKC 缺失小鼠的酒精消耗量减少，令人鼓舞的初步体外数据表明乙醇会抑制 PKC 活性。要测试的具体假设是 PKC 的 C1 结构域中有一个醇位点。该提案的第一个目标是通过使用荧光 PKC 激活剂与醇的变构相互作用来识别醇结合，并使用新型光反应醇来精确确定 PKCeC1 和醇之间的接触点。共价连接到结合位点内的氨基酸残基后，标记的蛋白质将被蛋白水解切割，然后通过质谱法对生成的肽进行氨基酸测序，以识别 PKC 上的醇位点。第二个目标是通过突变分析和高分辨率 X 射线晶体学来表征已识别的结合位点。这些将导致产生具有改变的酒精敏感性的 PKC 突变体的最终目标，该突变体可用于细胞或小鼠研究中，以测试 PKC 在乙醇作用中的直接参与。我的研究的意义在于建立和表征 PKC 中的酒精结合位点，并开发具有改变酒精敏感性的突变体，这将有助于理解酒精作用机制。