Plasmodium falciparum metacaspase-2 capture its natural substrate in a non-canonical way

Programmed cell death (PCD) is a multi-step process initiated by a set of proteases, which interacts and cleaves diverse proteins, thus modulating their biochemical and cellular functions. In metazoans, PCD is mediated by proteolytic enzymes called caspases, which triggered cell death by proteolysis of human Tudor staphylococcus nuclease (TSN). Non-metazoans lack a close homologue of caspases but possess an ancestral family of cysteine proteases termed 'metacaspases'. Studies supported that metacaspases are involved in PCD, but their natural substrates remain unknown. In this study, we performed the Plasmodium falciparum TSN (PfTSN) cleavage assay using wild and selected mutants of P. falciparum metacaspases-2 (PfMCA-2) in vitro and in vivo. Interestingly, PfMCA-2, cleaved a phylogenetically conserved protein, PfTSN at multiple sites. Deletion or substitution mutation in key interacting residues at the active site, Cys157 and His205 of PfMCA-2, impaired its enzymatic activity with the artificial substrate, z-GRR-AMC. However, the mutant Tyr224A did not affect the activity with z-GRR-AMC but abolished the cleavage of PfTSN. These results indicated that the catalytic dyad, Cys157 and His205 of PfMCA-2 was essential for its enzymatic activity with an artificial substrate, whereas Tyr224 and Cys157 residues were responsible for its interaction with the natural substrate and subsequent degradation of PfTSN. Our results suggested that MCA-2 interacts with TSN substrate in a non-canonical way using non-conserved or conformationally available residues for its binding and cleavage. In future, it would be interesting to explore how this interaction leads to the execution of PCD in the Plasmodium.