During its replication cycle in red blood cells, the human malaria parasite Plasmodium falciparum ingests and catabolizes up to 75% of the host cell's hemoglobin in an acidic degradative organelle called the food vacuole. Hemoglobin catabolism is required for normal parasite development, and peptidases that participate in this process are highly promising targets for anti-malarial drug development. While the initial steps in the degradation of hemoglobin to oligopeptides have been well studied, the processes by which amino acids are generated have remained ill-defined. We have identified two vacuolar aminopeptidases, P. falciparum aminopeptidase N (PfA-M1) and aminopeptidase P (PfAPP), and propose that hemoglobin is extensively degraded to amino acids in the food vacuole lumen. The goals of this project are to understand, in biochemical and cell biological terms, how the recruitment of PfA-M1 and PfAPP to the parasite's food vacuole has enhanced its ability to degrade hemoglobin.
In Aim 1, we examine whether these two aminopeptidases, which in other eukaryotes are much better catalysts at neutral or basic pH values than at acidic pH, have adapted to function efficiently in an acidic environment. The substrate specificity of PfA-M1, a potential key player in the generation of amino acids from globin peptides, will be profiled at acidic pH in order to define the extent of its contribution to vacuolar peptide catabolism. Characterization of the atomic structure of PfA-M1 in Aim 2 will provide a molecular basis for interpreting the substrate specificities observed in Aim 1. In addition, mutagenic analysis of a key substrate binding site will shed light on the molecular basis of specificity in this enzyme and aid in the design of potent, specific inhibitors.
In Aim 3, the mechanism underpinning the dual targeting of PfAPP to the food vacuole and cytosol will be explored, with a focus on alternate transcription or translation initiation as the most likely candidates. The Plasmodium-specific PfAPP N-terminal extension, which contains the sequence elements specifying dual targeting, will be selectively mutagenized to reveal the presence of targeting and sorting information. The insight gained from these studies will be used to further dissect the vacuolar and cytosolic roles of PfAPP.
Malaria is responsible for the death of 1-2 million people annually. This project examines the roles of two enzymes called aminopeptidases that help the parasite to digest the hemoglobin of its host red blood cell. By understanding how these enzymes work, we hope to discover chinks in the parasite's armor that could be exploited for the development of new anti-malarial drugs.
|Dalal, Seema; Ragheb, Daniel R T; Schubot, Florian D et al. (2013) A naturally variable residue in the S1 subsite of M1 family aminopeptidases modulates catalytic properties and promotes functional specialization. J Biol Chem 288:26004-12|
|Dalal, Seema; Ragheb, Daniel R T; Klemba, Michael (2012) Engagement of the S1, S1' and S2' subsites drives efficient catalysis of peptide bond hydrolysis by the M1-family aminopeptidase from Plasmodium falciparum. Mol Biochem Parasitol 183:70-7|
|Ragheb, Daniel; Dalal, Seema; Bompiani, Kristin M et al. (2011) Distribution and biochemical properties of an M1-family aminopeptidase in Plasmodium falciparum indicate a role in vacuolar hemoglobin catabolism. J Biol Chem 286:27255-65|
|Velmourougane, Geetha; Harbut, Michael B; Dalal, Seema et al. (2011) Synthesis of new (-)-bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase. J Med Chem 54:1655-66|
|Harbut, Michael B; Velmourougane, Geetha; Dalal, Seema et al. (2011) Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases. Proc Natl Acad Sci U S A 108:E526-34|
|Ragheb, Daniel; Bompiani, Kristin; Dalal, Seema et al. (2009) Evidence for catalytic roles for Plasmodium falciparum aminopeptidase P in the food vacuole and cytosol. J Biol Chem 284:24806-15|