The hepatic cytosolic cholesteryl ester hydrolase (hCEH) purified in this laboratory exhibits properties of a regulatory enzyme, providing a potentially important control point for regulation of precursor pools for bile acid synthesis, biliary cholesterol secretion and membrane cholesterol. The long-term objective of this research is elucidation of the role of CEH in regulation of hepatic free cholesterol pools as they relate to cholesterol and bile acid metabolism and cholesterol homeostasis. A lambda gt11 cDNA library from cholestyramine treated rat liver will be screened with anti-hCEH and cDNA or oligonucleotide probes developed in this laboratory. Positive cDNA inserts will be cloned and sequenced to obtain complete mRNA and amino acid sequences to be compared with CEH isoenzymes from other organs. The hCEH will be further characterized with respect to pseudokinetic properties, as a function of substrate properties and various conditions and potential modulators with physiological relevance, such as bile acids, fatty acids, cholesterol, other amphiphiles, triacylglycerol, metal ions and protein constituents of cytosol. Mechanisms for regulation of hCEH will be characterized in rats, cultured hepatocytes and Hep G2 cells by measuring hCEH mass and specific activity and hCEH mRNA levels with Western and Northern blot analyses using anti-hCEH and a nick translated cDNA probe, under various physiological conditions or pharmacological treatments known to alter cholesterol or bile acid metabolism. Levels of protein phosphorylation will be measured where enzyme specific activities are altered and activity will be correlated with phosphorylation of specific sites. Effects of manipulation of hCEH and perturbation of free cholesterol on other key enzymes of cholesterol metabolism (acyl-CoA:cholesterol acyltransferase, HMG-CoA reductase, cholesterol 7alpha-hydroxylase), on bile acid synthesis, biliary cholesterol secretion and hepatic free and esterified cholesterol will be measured with a view to manipulation of free cholesterol pools and measurement of effects on these various activities. CEH inhibitory proteins will be isolated, sequenced and characterized in order to determine their mechanism of action and evaluate their potential physiological role in regulation of CEH. These studies will fill a significant gap in understanding of cholesterol metabolism and homeostasis and may identify a site for intervention in derangements of cholesterol/bile acid metabolism or to alter membrane properties.
