title: Inhibition of degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo by cysteine protease inhibitors.
abstract: 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulatory enzyme of cholesterol biosynthesis and is located in the endoplasmic reticulum (ER). A fusion protein, HMGal, consisting of the membrane domain of HMG-CoA reductase fused to Escherichia coli beta-galactosidase and expressed in Chinese hamster ovary (CHO) cells from the SV40 promoter, was previously constructed and was found to respond to regulatory signals for degradation in a similar fashion to the intact HMG-CoA reductase. Degradation of both HMG-CoA reductase and HMGal in CHO cells was enhanced by addition of mevalonate or low density lipoprotein (LDL). In this report we show that 2 cysteine protease inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methioninal (ALLM), completely inhibit the mevalonate- or LDL-accelerated degradation of HMG-CoA reductase and HMGal and also block the basal degradation of these enzymes. It has been shown that in vitro these protease inhibitors inhibit the activities of Ca(2+)-dependent neutral proteases as well as lysosomal proteases, including cathepsin L, cathepsin b, and cathepsin D. However, the mevalonate-accelerated degradation of HMG-CoA reductase and HMGal is not affected by lysosomotropic agents, suggesting that the site of action of these inhibitor peptides in preventing the degradation is not the cathepsins. In brefeldin A-treated cells, where protein export from the ER is blocked, ALLN is still effective in inhibiting the degradation of HMG-CoA reductase and HMGal. These results indicate the involvement of non-lysosomal Ca(2+)-dependent proteases in the basal and the accelerated degradation of HMG-CoA reductase and HMGal. Enzymatic assays in vitro and immunoblot analyses have revealed calpain- and calpastatin-like proteins in CHO cells. The activities and the amount of these proteins do not change under conditions of enhanced degradation, indicating that the levels of these proteins are not subject to mevalonate regulation.
authors.affiliation: Department of Biological Sciences, Stanford University, California 94305-5020.
authors.forename: S
authors.initials: S
authors.lastname: Inoue
authors.forename: S
authors.initials: S
authors.lastname: Bar-Nun
authors.forename: J
authors.initials: J
authors.lastname: Roitelman
authors.forename: R D
authors.initials: RD
authors.lastname: Simoni
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C063261:acetylleucyl-leucyl-norleucinal; C069861:calpain inhibitor 2; D004533:Egtazic Acid; D002738:Chloroquine; D006903:Hydroxymethylglutaryl CoA Reductases; D001616:beta-Galactosidase; D002118:Calcium