Human obesity and hyperlipidemia are major health factors affecting the body's susceptibility to a variety of diseases such as heart attack, stroke, and diabetes. Long-chain fatty acids are the main constituents of fats and lipids. Humans, like other animals, derive their fatty acids from dietary sources and by de novo synthesis from acetyl-CoA, malonyl-CoA, and NADPH, a reaction catalyzed by the multifunctional enzyme, fatty acid synthase (FAS). In humans, the liver is the main site for fatty acid synthesis, a process affected by many dietary, hormonal, and genetic factors. Because our knowledge of FAS is limited to studies of the enzyme in animal tissues, bacteria, and yeast, little is known about the human enzyme. Currently, we are studying the structure-function of chicken liver FAS, and we have cloned and sequenced the cDNA coding for the subunit protein. Segments of the cDNA coding for partial catalytic activities will be cloned and expressed in heterologous systems like Escherichia coli or yeast. The recombinant protein will be isolated and characterized. Experiments involving site-directed mutagenesis of specific amino acid residues at the putative catalytic sites will bc carried out to gain insight into the structure-function of the partial activities. Studies of the structure of the recombinant thioesterases and their mutant derivatives are planned using various physical techniques. We plan to study human FAS and its regulation in Hep G2 cells, a well-differentiated human hepatoblastoma cell line. We have prepared FAS from Hep G2 cells and plan to study its properties, kinetics, and partial reactions. A cDNA fragment that encodes the NH2-terminal end of FAS has been isolated and would be used to isolate the 5'untranslated region of the FAS mRNA. Genomic clones containing the regulatory regions of the human FAS gene will be isolated, sequenced, and mapped for the transcription initiation site. The cis-active elements involved in the regulation of the FAS gene will be identified using luciferase or CAT as reporter genes. The roles of hormones such as insulin, triiodothyronine, glucagon, and cAMP in the expression of the FAS gene and in the regulation of FAS mRNA transcription, stability, and translation will be investigated. These studies ultimately would lead to the isolation and characterization of the trans-acting factors involved in the regulation of FAS gene expression. In the proposed studies, various biochemical, cloning, molecular biological, and tissue culturing techniques will be employed.
