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 i s 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. ? ? ?
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