3-hydroxy-3-methylglutaryl coenzyme A reductase is sterol-dependently cleaved by cathepsin L-type cysteine protease in the isolated endoplasmic reticulum.
We have recently shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an endoplasmic reticulum (ER) membrane protein, is degraded in ER membranes prepared from sterol pretreated cells and that such degradation is catalyzed by a cysteine protease within the reductase membrane domain. The use of various protease inhibitors suggested that degradation of HMG-CoA reductase in vitro is catalyzed by a cathepsin L-type cysteine protease. Purified ER contains E-64-sensitive cathepsin L activity whose inhibitor sensitivity was well matched to that of HMG-CoA reductase degradation in vitro. CLIK-148 (cathepsin L inhibitor) inhibited degradation of HMG-CoA reductase in vitro. Purified cathepsin L also efficiently cleaved HMG-CoA reductase in isolated ER preparations. To determine whether a cathepsin L-type cysteine protease is involved in sterol-regulated degradation of HMG-CoA reductase in vivo, we examined the effect of E-64d, a membrane-permeable cysteine protease inhibitor, in living cells. While lactacystin, a proteasome-specific inhibitor, inhibited sterol-dependent degradation of HMG-CoA reductase, E-64d failed to do so. In contrast, degradation of HMG-CoA reductase in sonicated cells was inhibited by E-64d, CLIK-148, and leupeptin but not by lactacystin. Our results indicate that HMG-CoA reductase is degraded by the proteasome under normal conditions in living cells and that it is cleaved by cathepsin L leaked from lysosomes during preparation of the ER, thus clarifying the apparently paradoxical in vivo and in vitro results. Cathepsin L-dependent proteolysis was observed to occur preferentially in sterol-pretreated cells, suggesting that sterol treatment results in conformational changes in HMG-CoA reductase that make it more susceptible to such cleavage.
Moriyama
T
T
Research Institute for Food Science, Kyoto University, Gokasho, Japan. moriyama@soya.food.kyoto-u.ac.jp
Wada
M
M
Urade
R
R
Kito
M
M
Katunuma
N
N
Ogawa
T
T
Simoni
R D
RD
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002457:Cell Extracts; D002460:Cell Line; D002463:Cell Membrane Permeability; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D010450:Endopeptidases; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D004852:Epoxy Compounds / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D013010:Sonication; D013261:Sterols / Q000494:pharmacology*
C402823:CLIK 148; D002457:Cell Extracts; D004852:Epoxy Compounds; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D011725:Pyridines; D013261:Sterols; C067713:lactacystin; D006903:Hydroxymethylglutaryl CoA Reductases; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; D046988:Proteasome Endopeptidase Complex; D007930:Leucine; C024974:E 64; D000111:Acetylcysteine