Cathepsin C is critical for the release of other cathepsins from lysosomes in leucyl-leucine methyl ester-triggered apoptosis

Lysosomal detergents are lipophilic bases, which remain captured in acidic vesicles and can induce lysosomal membrane permeabilization (LMP). LMP is a point-of-no-return, followed by cell death, usually mediated by cathepsins. L-Leucyl-leucine methyl ester (LLOMe) is synthetical lysosomotropic detergent, which showed extremely good results in preventing the graft-versus-host disease in vivo by successfully eliminating immune cells of the donor. The mechanism of compound action is dependent on the transferase activity of cathepsin C, which leads to the formation of LeuLeu oligomers with membranolytic potential, leading to lysosomal leakage, release of cathepsins into the cytosol and triggering of cell death. However, the exact mechanism of action and interconnection between different cathepsins were never clarified. Using several different cell lines containing different levels of individual cathepsins we have shown that incubation of cells with LLOMe leads to a very early increase in lysosomal pH, indicative of LMP, followed by release of cathepsins into the cytosol and subsequent cell death. Both the increase in lysosomal pH and cell death could be prevented by the cathepsin C selective inhibitor GlyPheDMK. In contrast, the broad spectrum cathepsin inhibitor E64d, efficiently prevented cell death, but only partially lysosomal pH increase. According to the results we can speculate that cathepsin C is required for the exit of lysosomal proteins, including the cathepsins, into the cytosol, where the latter then trigger cell death in the next step. This mechanism was confirmed using primary cells double deficient for cathepsins B and L, and cells deficient for the major cytosolic inhibitor of cathepsins stefin B. The different sensitivity of cells to LLOMe is a combination of the levels of different cathepsins, stefin B, as well as other apoptotic proteins, including Bcl2 homologs.