Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1

Abstract

Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization.

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

“…5 A and B). Further sequence analysis of the accumulated Hb-derived oligopeptides revealed that the majority were likely poor substrates for DPAP1, the other essential aminopeptidase with broad substrate specificity found in the DV (47). We thus hypothesized that PfA-M1 was necessary for proteolysis of these oligopeptides.…”

Anti-malaria drug development targeting the M1 alanyl and M17 leucyl aminopeptidases

“…For example, a cytoplasmic aspartyl aminopeptidase (PfM18AAP), that is the only malaria aminopeptidase capable of cleaving aspartic and glutamic acids from the N-termini of peptides, is also present in single copy and is essential for parasite viability. 55 Also in consideration are the post-prolyl aminopeptidase (PfAPP) 32 and dipeptidyl peptidase (cathepsin C) 25,26 that are located in the DV and central to the later stages of hemoglobin degradation.…”

“…13,18,19 However, knockout experiments have shown that the DV plasmepsins and falcipains 2 and 2′ are individually not essential to parasite survival, and because of this redundancy between the function of these enzyme classes it has proved difficult to develop drugs that effectively block the digestive process within the DV. [21][22][23][24] Peptides produced in the DV may be further processed to dipeptides by a dipeptidyl aminopeptidase I, an orthologue of mammalian cathepsin C. 25,26 Small peptides and dipeptides may also be reduced to free aminoacids within the DV and/or transported to the parasite cytosol for processing by aminopeptidases. 20,[27][28][29][30][31][32][33] Aminopeptidases are essential in releasing aminoacids from haemoglobin-derived peptides and are thus central to the growth and development of malaria parasites in the erythrocyte.…”

Development of Plasmodium falciparum Protease Inhibitors in the Past Decade (2002–2012)

“…Once FLN converts short globin polypeptides into oligopeptides consisting of 5-10 amino acids, dipeptidyl amino peptidase 1 (DPAP1), a lysosomal exopeptidase, sequentially cleaves oligopeptide substrates from their N terminus into dipeptides [87,130]. Attempts to disrupt the DPAP1 gene have been unsuccessful, which suggests that the enzyme makes an important contribution to hemoglobin catabolism during the intraerythrocytic cycle [131].…”

“…Attempts to disrupt the DPAP1 gene have been unsuccessful, which suggests that the enzyme makes an important contribution to hemoglobin catabolism during the intraerythrocytic cycle [131]. While DPAP1 is one of the three related DPAP enzymes encoded in the parasite genome, current evidence suggests that only DPAP1 resides in the food vacuole [130]. DPAP1 is also potentially a good drug target because the closest human homologue is cathepsin C, a protease that is not essential in mammals [130,132,133].…”