The over-all objective of this project is to understand the regulation of the enzyme choline acetyltransferase through both transcriptional and post translational mechanisms. The project involves 5 specific aims. These include the elucidation of the three dimensional structure of the enzyme by x-ray defraction analysis. Crystals of the rat enzyme which defract to 4.5 angstroms have been obtained and will be used for initial structural determination. Crystalization conditions will be optimized to obtain crystals which defract to a higher resolution. Studies on the regulation of the enzyme by post-translational modification will involve an analysis of the use of alternative translational start sites to produce two molecular forms of human ChAT and the potential for one of these enzyme forms to serve as a precursor for the membrane bound form of the enzyme. Regulation of the enzyme by phosphorylation is implicated by the finding that phosphorylated recombinant rat ChAT derived from expression in plant cells exhibits 1/20 the activity of non-phosphorylated recombinant rat ChAT expressed in E. coli. Studies will be conducted on the in vitro phosphorylation of the enzyme and the effect of phosphorylation on activity. The phosphorylation state of the enzyme derived from rat brain will be assessed. Site directed mutagenesis will be used to assess the role of specific arginine, histidine, and cysteine residues in catalysis. The regulation of the enzyme at the transcriptional level will be investigated by studying those elements in the human gene which define cholinergic specific expression. Putative regulatory regions of the gene will be studied by a combination of cell transfection studies and the analysis of transgenic mice. Lastly the transcription factors which bind to the regulatory sequences in the gene will be characterized by gel shift and footprint analysis. The former technique used to isolate and characterize any unique transcription factors whose function will be studied.
