IDENTIFICATION AND CHARACTERIZATION OF A PLASMODIUM VIVAX INHIBITOR OF CYSTEINE PROTEASES

Abstract

Plasmodial cysteine proteases are important enzymes involved in the hemoglobin degradation pathway that supplies essential amino acids necessary for the growth and development of malaria parasites. In Plasmodium vivax, cysteine proteases from the vivapain subfamily are known to play a pivotal role in this process. However, little is known about their functional regulation.In this study, a proteinaceous inhibitor of cysteine proteases (ICP) was identified from the genome of P. vivax. Sequence analysis of the P. vivax ICP (PvICP) revealed the presence of a cysteine protease inhibitor domain with four predicted haemoglobinase-interaction motifs at its C-terminus (PvICPc), prompting investigation of its influence on the vivapain. Using solubly expressed recombinant proteins, the PvICPc was found to inhibit the proteolytic activity of vivapain 4 against synthetic dipeptide substrates in fluorometric assays. The inhibitory effect of PvICPc was further shown to extend to biological processes, as the native hemoglobinase and auto-cleavage activities of vivapain 4 were abrogated. Interestingly, biocomputational analysis also revealed the presence of a putative signal peptide at the N-terminal of PvICP, suggestive of its potential secretion out of P. vivax into the human host. Presence of PvICPc in HepG2 cells was further found to protect HepG2 cells from tert-butyl hydroperoxide induced cell death in vitro. However, PvICPc appear to mediate this process independent of the pro-apoptotic proteases from the human caspase family as suggested by results from the fluorometric assays. Nonetheless, recombinant PvICPc was found to inhibit the proteolytic activities of human cathepsins. Overall, this study has provided evidence on the presence of a functional cysteine protease inhibitor in P. vivax and offered clues on the potential endogenous and exogenous functions of PvICPc.