Characterization of falcilysin, a dual specificity metalloprotease active in Plasmodium falciparum

The apicomplexan parasite Plasmodium falciparum is the causative agent of the deadliest form of human malaria, responsible for over 2 million deaths annually. Afflicted individuals suffer dramatic fevers, chills, organ damage, and even coma. These symptoms are tied to the asexual development of the parasite within erythrocytes. While residing within erythrocytes, parasites degrade hemoglobin to provide the raw material for intraerythrocytic parasite development and growth. Hemoglobin catabolism occurs via a semi-ordered cascade of proteases within the acidic food vacuole of the parasite. Falcilysin (FLN) was first identified as a metalloprotease involved in degrading small globin peptides. The current studies aimed to characterize the enzyme and its role in hemoglobin degradation; but the data strongly suggest that falcilysin is not a typical globinase.; Immunofluorescence and electron microscopy studies confirm the presence of falcilysin within the food vacuole and localize the enzyme to vesicular structures elsewhere within the parasite. FLN associates with membranes in an alkaline-extraction sensitive manner, consistent with the behavior of a peripheral membrane protein. Additionally, several lines of evidence indicate that falcilysin is not proteolytically processed during maturation.; Random peptide libraries used to identify preferred FLN substrate sequences documented different substrate preferences for the acidic and neutral pH conditions that the enzyme likely encounters in its dual localizations. Kinetic analyses of optimized substrates dramatically confirm the pH-dependent substrate selectivity. A model is proposed in which a pH-dependent switch in substrate selection is linked to a change in protonation of the enzyme active site. This is the first time such a pH-dependent dual substrate specificity has been described for a protease.; Studies with substituted benzo-hydroxamic acids reveal that falcilysin can be inhibited in vitro and in vivo. This, in combination with gene disruption experiments that failed to recover fln integrants, argue that falcilysin may be a necessary gene and therefore a good drug target. In toto, this thesis expands and redefines the view of falcilysin. The cell biological and biochemical data argue that this enzyme, while important for hemoglobin degradation, also has a role beyond the food vacuole.