Lysosomal acid lipase/cholesteryl ester hydrolase is crucial for the intracellular hydrolysis of cholesteryl esters and triglycerides that have been taken up via receptor mediated endocytosis of lipoprotein particles. The process is central to the supply of cholesterol to cells for growth and membrane function and in the regulation of processes that are mediated by cellular cholesterol flux. This project will characterize, at the molecular genetic level, the defects that cause a deficiency of this acid lipase activity in two human diseases, Wolman disease (WD) and cholesteryl ester storage disease (CESD), both inherited as Mendelian autosomal recessive traits, with the long term objective of understanding the biochemical and physiological activities of human lysosomal acid lipase (HLAL) in relationship to normal and aberrant states of cholesteryl ester processing and storage. The deficiencies of acid lipase activity in WD and CESD, with their dramatically different biochemical and clinical phenotypes, provide an """"""""experiment of nature"""""""" for the testing of hypotheses concerning the control of cholesterol metabolism. The observation that CESD patients exhibit premature atherosclerosis suggests the applicability of this """"""""experiment"""""""" to cardiovascular risk in the general population. Despite the recognized importance of HLAL in the regulation of cholesterol metabolism, difficulties with experimental manipulation of the enzyme have limited progress in understanding its control and physiological role. This project will circumvent earlier problems by exploiting the recently isolated HLAL cDNA and genomic DNA clones to test hypotheses concerning the mutations causing WD and CESD: l) Defects in expression of the structural gene for acid lipase are responsible for both WD and CESD throughout the spectrum of phenotypes. This will be done by transducing the cDNA coding for functional HLAL into mutant fibroblasts using retroviral vectors to demonstrate that this structural information is sufficient to correct the deficiency. 2) The different phenotypes arise from mutations at the HLAL structural gene locus that differentially inactivate its triglyceride hydrolase and cholesteryl esterase functions. This will involve identifying the mutations that cause WD and CESD at the nucleotide sequence level by a) characterizing the functional status of mutant HLAL genes; b) determining the structure of the coding and control regions of the normal genomic locus for comparison to the structures of the loci from the mutant cells; and c) recreating the mutated sites in vitro and expressing the constructions.
| Wang, J; Gebre, A K; Anderson, R A et al. (1997) Cloning and in vitro expression of rat lecithin:cholesterol acyltransferase. Biochim Biophys Acta 1346:207-11 |
| Wang, J; Gebre, A K; Anderson, R A et al. (1997) Amino acid residue 149 of lecithin:cholesterol acyltransferase determines phospholipase A2 and transacylase fatty acyl specificity. J Biol Chem 272:280-6 |
