Biochemical characterization of unusual cysteine protease of P. falciparum, metacaspase-2 (MCA-2)

[Display omitted] •PfMCA-2 has arginine/lysine substrate specificity.•Effector caspase inhibitor, Z-FA-FMK found to be a potent inhibitor of PfMCA-2 affecting PfMCA-2 expression and parasite growth.•PfMCA-2 cleaves its natural substrate, TSN and Z-FA-FMK inhibits PfMCA-2 mediated TSN fragmentation.•In presence of Z-FA-FMK, a significant population of parasite displayed signs of apoptotic cell death. Earlier studies on Plasmodium apoptosis revealed the presence of proteases with caspases like- activity, which are known as “metacaspases”. Although this family of cysteine proteases is structurally similar to caspases with Cys-His dyad but their evolutionary significance and functional relevance remains largely unknown. These proteases are considered to be an important target against malaria due to their absence in humans. In this report, we have biochemically characterized metacaspase-2 (PfMCA-2) of P.falciparum. Enzymatic assay showed that PfMCA-2 efficiently cleaved arginine/lysine specific peptide, but not caspase-specific substrate. Consistently, PfMCA-2 activity was sensitive to effector caspases inhibitor, Z-FA-FMK, and mildly inhibited by aprotinin and E-64. However, general caspase inhibitors such as Z-VAD-FMK and Z-DEVD-FMK had no effect on PfMCA-2 activity. Z-FA-FMK inhibits parasite growth with an IC50 value of 2.7 μM along with the notable morphological changes. PfMCA-2 specifically expressed in schizonts and gametocyte stages and there was a notable depletion of PfMCA-2 expression in Z-FA-FMK treated schizonts and gametocytes stages of parasite. Notably, PfMCA-2 cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease) and the proteolysis of PfTSN did not occur after treatment with the Z-FA-FMK. The production of large amount of reactive oxygen species in presence of Z-FA-FMK caused oxidative stress which in turn leads to loss of cell viability. The oxidative stress further generates positive feedback for the occurrence of cell death in term of phosphatidylserine externalization and DNA fragmentation in vitro.