Cathepsin B-like and cell death in the unicellular human pathogen Leishmania

Abstract

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H2O2), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

Functional Analysis of Leishmania Cyclopropane Fatty Acid Synthetase

“…However, from 48 hr onwards, the CFAS null parasites were significantly more tolerant to hydrogen peroxide than the wild type or CFAS complemented parasites (Figure 6D). Interestingly, a similar phenotype has been observed in L. mexicana parasites null for cysteine proteinase C which, when exposed to 1 mM hydrogen peroxide for 60 min (a dose that induces cell death in these cells [28]), show better survival than wild type cells. The explanation of these data is currently unclear.…”

Apoptotic Marker Expression in the Absence of Cell Death in Staurosporine-Treated Leishmania donovani

“…The absence of conserved caspases from the parasite genome might be compensated for by Leishmania metacaspases, as their expression in yeast can functionally replace the yeast metacaspase YCA1 in PCD (35), and their overexpression in Leishmania correlates with enhanced sensitivity to oxidant-induced parasite death (36,37). Furthermore, a recent study linked a cathepsin B-like protease to Leishmania cell death, which can cleave the canonical caspase substrate and was likely monitored in our study (22). An intriguing observation was the absence of cell death despite pleiotropic inhibition of the parasite kinome and ATP-dependent enzymatic activities in staurosporine-treated parasites.…”

“…To better define the mode of action of staurosporine on L. donovani promastigotes, we evaluated the extent of caspase 3/7-like protease activity in the presence of this inhibitor. Despite the absence of conserved caspases in Leishmania, corresponding proteolytic activity has been recently linked to a cathepsin B-like protein involved in parasite cell death (22). Promastigotes from a logarithmic culture (5 ϫ 10 6 cells/ml) were incubated in the presence of 0.261 and 0.428 M staurosporine, 26 and 46 M miltefosine, and 0.111 M amphotericin B at 26°C.…”

Papain-like peptidases: structure, function, and evolution

“…Plant homologs have been shown to be involved in the hypersensitive response, a form of programmed cell death in response to pathogen invasion, basal defense mechanisms, and plant senescence (51,52) . Similarly, the cathepsin B homolog CPC from Leishmania species is involved in programmed cell death (53) . In this unicellular parasite, cathepsin B has also been found to be involved in the parasite stage of its life cycle within macrophages (54) .…”