Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

Abstract

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.

Distinct cathepsins control necrotic cell death mediated by pyroptosis inducers and lysosome-destabilizing agents

“…14 Cathepsin C is activated early in the endocytic pathway by other lysosomal enzymes, such as cathepsin D. 14,16,47 We have shown that the cathepsin D inhibitor, pepstatin b, specifically blocks LLOMe-induced cell death, but not pyroptotic cell death (Fig. 1B).…”

Multiple functions of pro‐parts of aspartic proteinase zymogens

“…Lysosomal and vacuolar proteinases are representatives of the second type. Human procathepsin D is capable of only partial self processing and activation [30,31] requiring lysosomal cysteine proteinase(s) for the final processing of its pro-part [32]. Similarly, serine vacuolar proteinase B is responsible for the final proteolytic maturation of the zymogen of proteinase A of yeast [33].…”

Cathepsin D—Many functions of one aspartic protease

“…Further proteolytic cleavage that does not result in dissociation of CD globular structure yields the mature active lysosomal protease which is composed of heavy (34 kDa) and light (14 kDa) chains linked by non-covalent interactions [11][12][13]. Cysteine proteases and autocatalytic activity of CD is most-likely involved in pCD/CD processing [14][15][16][17]. Several factors were found to affect CD activation including a lipid second mesenger ceramide and prosaposin [10,18].…”