Alcohol Binding Site(s) in Protein Kinase C Epsilon

描述(申请人提供)：酗酒和酗酒导致世界范围内的重大健康问题。确定靶点(S)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。这项建议的总体目标是确定信号转导蛋白蛋白激酶C epsilon(PKCE)上的酒精结合部位，并确定这些部位的二级结构。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，但慢性酒精暴露会导致PKC表达和/或功能的升高。相反，PKC亚型表达的改变会影响饮酒和对酒精的行为反应。在活体研究中 与野生型相比，PKC缺失的小鼠显示出更少的酒精消耗，令人鼓舞的体外初步数据表明，乙醇抑制了PKC的活性。需要检验的具体假设是，在PKC的C1域中有一个酒精的位置。这项建议的第一个目的是通过使用荧光PKC激活剂与醇的变构相互作用来识别醇的结合，并通过使用新型光反应醇来精确地确定PKCeC1与醇之间的接触点。当共价结合到结合位点(S)内的氨基酸残基上时，标记的蛋白质将被蛋白水解酶切割，然后用质谱仪对所产生的多肽进行氨基酸测序，以确定蛋白激酶C上的乙醇位点(S)。第二个目的是通过突变分析和高分辨X射线结晶学来表征已确定的结合部位(S)。这将导致产生酒精敏感性改变的PKC突变体的最终目标，这可以在细胞或小鼠的研究中利用，以测试PKC直接参与乙醇的作用。本研究的意义在于建立和鉴定PKC中的酒精结合部位，并培育具有酒精敏感性改变的突变体，这将有助于了解酒精的作用机制。

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.