Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

• Published in: The American journal of physiology Vol. 257; no. 6 Pt 1; p. C1069
• Main Authors: Samarel, A M, Ferguson, A G, Decker, R S, Lesch, M
• Format: Journal Article
• Language: English
• Published: United States 01.12.1989
• Subjects: Animals; Cathepsin D - biosynthesis; Cathepsin D - genetics; Cells, Cultured; Chloroquine - pharmacology; Cysteine Proteinase Inhibitors - pharmacology; Fibroblasts - drug effects; Fibroblasts - enzymology; Kinetics; Leucine - analogs & derivatives; Leucine - pharmacology; Leupeptins - pharmacology; Methionine - metabolism; Myocardium - enzymology; Protein Processing, Post-Translational - drug effects; Rabbits; Sulfur Radioisotopes
• ISSN: 0002-9513
• DOI: 10.1152/ajpcell.1989.257.6.c1069

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.