title: Effect and cellular site of action of cysteine protease inhibitors on the cholesterol esterification pathway in macrophages and Chinese hamster ovary cells.
abstract: Stimulation of intracellular cholesterol esterification, which is catalyzed by the enzyme acyl-CoA:cholesterol O-acyltransferase (ACAT), by atherogenic lipoproteins in macrophages is a key step in the development of atheroma foam cells. Since other aspects of intracellular cholesterol metabolism involve proteolytic reactions, we looked for evidence of intracellular proteolysis in the stimulation of the cholesterol esterification pathway. When macrophages and CHO cells were incubated with the cysteine protease inhibitor N-acetylleucylleucylnorleucinal (ALLN), the ability of beta-very-low-density lipoprotein (beta-VLDL) and free cholesterol-rich liposomes to stimulate cholesterol esterification was inhibited by 60-90%. Epoxysuccinylleucylamido-3-methylbutane ethyl ester (EST), a cysteine protease inhibitor structurally different from ALLN, also inhibited beta-VLDL-induced cholesterol esterification in CHO cells. The inhibitory effect of the protease inhibitors could not be explained by decreased net expansion of cellular cholesterol pools, inhibition of lipoprotein cholesteryl ester hydrolysis, or blockage of cholesterol trafficking through the lysosomal pathway. Furthermore, stimulation of cholesterol esterification by 25-hydroxycholesterol and sphingomyelinase was not inhibited by ALLN, indicating that ALLN is not acting as a direct ACAT inhibitor in the cells, and suggesting that the ALLN effect is specific for methods of stimulating cholesterol esterification that expand cellular cholesterol pools. Previous studies have shown that inhibition of protein synthesis (e.g., by cycloheximide) stimulates cholesterol esterification in macrophages and CHO cells, suggesting the presence of a short-lived protein inhibitor of cholesterol esterification. Herein, we show that, when added after cycloheximide, ALLN does not inhibit cycloheximide-induced cholesterol esterification in either cell type. The data in this report are consistent with a novel model in which a proteolytic reaction mediates the stimulation of cholesterol esterification specifically by expanded cellular cholesterol pools. The apparent protease-dependent step is not dependent upon lysosomal trafficking of cholesterol and is proximal to the ACAT enzyme itself; it may function by cleaving an endogenous inhibitor of the interaction of expanded cellular cholesterol pools with ACAT.
authors.affiliation: Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Schissel
authors.forename: N
authors.initials: N
authors.lastname: Beatini
authors.forename: X
authors.initials: X
authors.lastname: Zha
authors.forename: F R
authors.initials: FR
authors.lastname: Maxfield
authors.forename: I
authors.initials: I
authors.lastname: Tabas
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000187:drug effects* / Q000378:metabolism; D002788:Cholesterol Esters / Q000378:metabolism*; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004951:Esterification; D005260:Female; D006888:Hydroxycholesterols / Q000494:pharmacology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008079:Lipoproteins, VLDL / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D008856:Microscopy, Fluorescence; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology
chemical_list: D002788:Cholesterol Esters; D015853:Cysteine Proteinase Inhibitors; D006888:Hydroxycholesterols; D007976:Leupeptins; D008079:Lipoproteins, VLDL; C063261:acetylleucyl-leucyl-norleucinal; C007997:25-hydroxycholesterol; D003513:Cycloheximide; D013108:Sphingomyelin Phosphodiesterase; D007930:Leucine; C108192:aloxistatin