Lecithin:cholesterol acyltransferase (LCAT) is the major cholesterol esterifying activity of human plasma. It is important in the remodeling of nascent high density lipoproteins (HDL), which are rich in free cholesterol, into mature HDL in which most of the cholesterol is in its esterified form. This process is important in the transport of cholesterol from peripheral tissues to the liver where a portion of the cholesterol if converted to bile salts. This """"""""reverse cholesterol transport"""""""" is an important step in prevention and regression of atherosclerosis. This project will focus upon two important areas; LCAT regulation and structure- function relationships. The first step will be to investigate in rats, isolated rat hepatocytes, and human hepatoma cells the influence that diets which are known to affect plasma LCAT levels have on LCAT mRNA, synthesis, and secretion. The effects of dietary triglycerides containing n-3 fatty acids, which alter plasma LCAT levels in man, on plasma LCAT levels, LCAT secretion, and turnover will be compared with those of control rats given conventional chow or triglycerides with n-9 fatty acids. Cultures of human hepatoma cells will be used to directly compared the effects of n-3 and n-6 fatty acids in albumin complexes and in model reassembled HDL in which the test fatty acids are located at the sn-2 position of phosphatidylcholine. The effects of the free cholesterol and cholesteryl ester contents of the model HDL will also be determined. The second step will be to use site- directed mutagenesis and monoclonal antibodies to unambiguously identify some of the functional regions of LCAT and to determine the molecular basis of its substrate specificity, which is different in different mammalian species. These studies will delineate the mechanism by which LCAT forms cholesteryl esters, better localize the region(s) responsible for binding to lipoprotein surfaces, and reveal differences in the active site region of LCAT from different species that have different substrate specificities.
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