Biochemical Characterization of Two Aminopeptidases Involved in Hemoglobin Catabolism in the Food Vacuole of Plasmodium falciparum
Ragheb, Daniel Raafat Tadros
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The parasite Plasmodium falciparum is the causative agent of the most severe form of human malaria. During its intraerythocytic life cycle, P. falciparum transports red blood cell contents to its acidic organelle, known as the food vacuole, where a series of proteases degrade a majority of the host hemoglobin. Two metalloaminopeptidases, PfAPP and PfA-M1, have been previously localized to the food vacuole (in addition to distinct secondary locations for each), implicating them in the final stages of hemoglobin catabolism. Prior genetic work has determined these enzymes are necessary for efficient parasite proliferation, highlighting them as potential anti-malarial drug targets. This study presents the biochemical basis for the catalytic roles of these two enzymes in the hemoglobin degradation pathway. PfAPP, an aminopeptidase P homolog, is specific for hydrolyzing the N-termini of peptides containing penultimate prolines. PfA-M1 is a member of the expansive M1 family of proteases and exhibits a broad specificity towards substrates. The two enzymes are ubiquitous, found in organisms across all kingdoms of life. Their presence in an acidic environment is unique for aminopeptidase P proteins and rare for M1 homologs. Our immunolocalization results have confirmed the dual distribution of these two enzymes in the parasite. Vacuolar targeting was found to be associated with the Plasmodium specific N-terminal extension found in the PfA-M1 sequence by yellow fluorescent protein fusion studies. Kinetic analysis of recombinant forms of PfAPP and PfA-M1 revealed both enzymes are stable and catalytically efficient in the substrate rich, acidic environment of the parasite food vacuole. In addition, mutagenic exploration of the PfA-M1 active site has determined a residue important in dictating substrate specificity among homologs of the same family. These results provide insight into the parasite's functional recruitment of these enzymes to deal with the final stages of hemoglobin catabolism and necessary considerations for inhibitor design.
- Doctoral Dissertations