Effects of cysteine protease inhibitors on rabbit cathepsin D maturation.
To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed (24 h) to media containing leupeptin (0-10 mM), E 64 (0-10 mM), or chloroquine (0-50 microM). Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D (Mr 53,000) within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D (Mr 48,000). Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate (not detectable in control fibroblasts). Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation.
Samarel
A M
AM
Department of Medicine, Loyola University, Stritch School of Medicine, Maywood 60153.
Ferguson
A G
AG
Decker
R S
RS
Lesch
M
M
D000818:Animals; D002402:Cathepsin D / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011817:Rabbits; D013462:Sulfur Radioisotopes
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D013462:Sulfur Radioisotopes; D002738:Chloroquine; D008715:Methionine; D002402:Cathepsin D; D007930:Leucine; C024974:E 64