Glucosamine (GlcN) is a naturally occurring derivative of glucose and an over-the-counter food additive. However, the mechanism underlying GlcN action on cells is unknown. In this study, we investigated the effect of GlcN on natural killer (NK) cells. We demonstrate that GlcN affects NK-92 cell cytotoxicity by altering the distribution of cathepsin C, a cysteine protease required for granzyme processing in cytotoxic granules. The relocation of cathepsin C due to GlcN was shown to be accompanied by a decrease in the intracellular enzyme activity and its extracellular secretion. Similarly, the relocation of endosomal aspartic cathepsin E was observed. Furthermore, we elucidated that repositioning of cathepsin C is a consequence of altered signaling pathways of cytotoxic granule movement. The inhibition of phosphorylation upstream and downstream of ERK by GlcN disturbed the polarized release of cytotoxic vesicles. Considerable changes in the ERK phosphorylation dynamics, but not in those of p38 kinase or JNK, were observed in the IL2-activated NK-92 cells. We found decreased phosphorylation of the transcription factor FOXO1 and simultaneous prolonged phosphorylation of ERK as well as its nuclear translocation. Additionally, a protein downstream of the ERK phosphorylation cascade, paxillin, was less phosphorylated, resulting in a diffuse distribution of cytotoxic granules. Taken together, our results suggest that dietary GlcN affects signaling pathway activation of NK-92 immune cells.

Lysosomal cathepsins were previously found to be involved in tumor necrosis factor-α (TNFα)-induced apoptosis. However, there are opposing views regarding their role as either initiators or amplifiers of the signaling cascade as well as the order of molecular events during this process. In this study, we investigated the role of cathepsin D (catD) in TNFα/cycloheximide-induced apoptosis in U937 human monocytic cells. TNFα-induced apoptosis proceeds through caspase-8 activation, processing of the pro-apoptotic molecule Bid, mitochondrial membrane permeabilization, and caspase-3 activation. The translocation of lysosomal catD into the cytosol was a late event, suggesting that lysosomal membrane permeabilization and the release of cathepsins are not required for the induction of apoptosis, but rather amplifies the process through the generation of reactive oxygen species. For the first time, we show that apoptosis is accompanied by degradation of the cysteine cathepsin inhibitor stefin B (StfB). CatD did not exhibit a crucial role in this step. However, this degradation was partially prevented through pre-incubation with the antioxidant N-acetyl cysteine, although it did not prevent apoptosis and its progression. These results suggest that the degradation of StfB, as a response to TNFα, could induce a cell death amplification effect as a result of progressive damage to lysosomes during TNFα treatment. J. Cell. Biochem. 118: 4813-4820, 2017. © 2017 Wiley Periodicals, Inc.

Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin.These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation. K E Y W O R D S apoptosis, Bid, caspases, cathepsin D, cysteine cathepsins, menadione, stefin B

Granzymes A and B are activated by proteolytic removal of their N-terminal dipeptides by cathepsin C (dipeptidyl-peptidase I). However, the possible physiological role of the cleaved dipeptides Glu-Lys and Gly-Glu is not yet understood. In this study, adding either of the two dipeptides to NK-92 cells, resulted in enhanced cytotoxicity toward the targeted K562 cells and increased death rate of the target cells. Cathepsin C is known to generate cytotoxic polymers from various dipeptides, however, in the case of the dipeptides Glu-Lys and Gly-Glu, cathepsin C was unable to polymerize them. Unexpectedly the dipeptides were found to be inhibitors of the transferase activity of cathepsin C (IC50 < 20 mM), and weak competitive inhibitors of the peptidase activity with K i values in the millimolar range. This suggests that the dipeptides can play role in a feedback loop that controls transferase and proteolytic activities of cathepsin C in various biological processes.