The goal of the project is to understand the function of the S14 protein, which bears no similarity to other sequenced proteins and has not been purified. The S14 gene is expressed only in tissues with high rates of fatty acid formation, including liver, fat and mammary gland. Levels of mRNA S14 are rapidly regulated by stimuli which govern the rate of fat synthesis in these tissues, including thyroid hormone, glucagon, glucocorticoids, dietary carbohydrate, and circadian factors. In each case, levels of the mRNA change concordantly with the effects of these stimuli on fat synthesis in vivo. We have extended our knowledge to the level of the protein itself by raising antisera to synthetic peptides corresponding to portions of the predicted S14 protein amino acid sequence. The antisera allowed localization of the protein to cytosol, elucidation of its electrophoretic characteristics, and the observation that its levels are regulated in accordance with those of mRNA S14. We have recently used the antisera to identify the S14 protein in an adipogenic cell line. We now propose to exploit the antisera to probe for the function of this novel protein, which we believe may be of general importance in the regulation of fat metabolism. This will be accomplished by assessing the effects of removal of the protein from in vitro lipogenic systems, and of adding purified S14 protein to such systems. Binding of lipids to purified S14 protein will also be tested. Purification will be by immunoaffinity chromatography. As a complimentary approach, we propose to introduce regulable S14 genes into lipid-synthesizing cultured cells to determine the effects of expression and deletion of the protein on fat metabolism in living cells. Antisera will be used to quantitate the protein in these experiments. These studies should enhance our understanding of this novel protein and provide a means of determining its precise biological function. Such knowledge may ultimately be applicable to the regulation of lipid metabolism in diabetes mellitus and obesity.
