Role of the proteolytic hierarchy between cathepsin L, cathepsin D and caspase-3 in regulation of cellular susceptibility to apoptosis and autophagy.

The present investigation was undertaken to measure the relative abilities of pro-death versus pro-survival proteases in degrading each other and to determine how this might influence cellular susceptibility to death. For this, we first carried out in vitro experiments in which recombinant pro-death proteases (caspase-3 or cathepsin D) were incubated with the pro-survival protease (cathepsin L) in their respective optimal conditions and determined the effects of these reactions on enzyme integrity and activity. The results indicated that cathepsin L was able to degrade cathepsin D, which in turn cleaves caspase-3, however the later enzyme was unable to degrade any of the cathepsins. The consequences of this proteolytic sequence on cellular ability to undergo apoptosis or other types of cell death were studied in cells subjected to treatment with a specific inhibitor of cathepsin L or the corresponding siRNA. Both treatments resulted in suppression of cellular proliferation and the induction of a cell death with no detectable caspase-3 activation or DNA fragmentation, however, it was associated with increased accumulation of cathepsin D, cellular vaculolization, expression of the mannose-6-phosphate receptor, and the autophagy marker LC3-II, all of which are believed to be associated with autophagy. Genetic manipulations leading either to the gain or loss of cathepsin D expression implicated this enzyme as a key player in the switch from apoptosis to autophagy. Overall, these findings suggest that a hierarchy between pro-survival and pro-death proteases may have important consequences on cell fate.