The most abundant mRNA sequences which accumulate in the small intestinal lining cells (enterocytes) of rat and man encode proteins that are involved in lipid metabolism. These mRNAs specify several apolipoproteins which are components of plasma high density lipoproteins as well as two small cytosolic fatty acid binding proteins (FABP). This proposal is designed to address four questions concerning intestinal lipid metabolism: (1) What are the functions of the FABPs and can they serve as a useful model for analyzing the molecular details of fatty acyl-protein interactions? (2) How is the processing of the primary translation products of the apolipoprotein mRNAs related to the assembly of high density lipoproteins? (3) How are the FABP and apoprotein genes organized and can this information be used to understand their evolution and to define structurally and functionally important peptide domains? (4) Are these genes coordinately expressed and what factors influence their expression? We will perform site specific mutagenesis of cloned cDNAs introduced into (i) prokaryotic expression vectors and (ii) coupled in vitro transcription-translation-translocation systems in order to study structure-function relationships. To facilitate the design and interpretation of the FABP in vitro mutagenesis studies, we will purify wild type mammalian protein from bacterial lysates for high resolution x-ray crystallographic studies. This will generate information about their three-dimensional structure. Gene structure and linkage will be determined by Southern blotting of cellular and cloned genomic DNA as well as by nucleotide sequencing. Cloned cDNAs will be used for hybridization analyses (liquid, Northern and in situ) to quantitate specific mRNA accumulation in a variety of tissues and after metabolic perturbations. These studies should provide insights into the role of the gut epithelium in maintaining lipid homeostasis. They should also provide information about the contribution of this organ to the pathogenesis of atherosclerosis.
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