Overview
This year between one and two million people will die from the malaria parasite Plasmodium falciparum and hundreds of millions more will suffer acute infection. With a malaria vaccine many years away, there is an urgent need for novel anti-malarial agents with which to combat emerging drug resistant parasites.

The pathology of severe malaria is a consequence of the multiplication of P. falciparum within human erythrocytes. One remarkable feature of this phase of the life cycle is the parasite's catabolism of large amounts of host hemoglobin within an acidic degradative organelle called the food vacuole. Our goal is to explore the process of hemoglobin degradation from biochemical and cell biological perspectives. In doing so, we aim to better understand the particular adaptations that allow the parasite to thrive in its host, and to characterize essential processes that can be targeted in the search for new anti-malarial drugs.

Peptide catabolism in the food vacuole
One of our main interests is the role of peptidases in the later stages of hemoglobin degradation. We are characterizing the substrate and inhibitor specificities of a recently identified P. falciparum dipeptidyl aminopeptidase (Klemba et al (2004), J. Biol. Chem. 279 43000). This enzyme functions near the end of the hemoglobin catabolic pathway by generating dipeptides from larger peptides and shows promise as a new drug target, as its activity appears to be essential to the parasite.

To understand how dipeptides are hydrolyzed to amino acids, we are currently localizing all putative aminopeptidases that have been identified in a homology-based search of the P. falciparum genome sequence. This is being accomplished by independently tagging each aminopeptidase with green fluorescent protein (GFP). Those aminopeptidases that are implicated in hemoglobin catabolism will be purified or expressed recombinantly for detailed biochemical analysis.

Protein trafficking to the food vacuole
Another major interest is the trafficking of endogenous food vacuole peptidases. The trafficking route of the food vacuole aspartic protease plasmepsin II encompasses both the classical secretory pathway as well as a parasite-specific endocytic pathway (Klemba et al (2004) J. Cell Biol. 164 47). By fusing fragments of plasmepsin II to GFP, we aim to identify specific sequences that direct plasmepsin II to the food vacuole. We are also developing a high-throughput screen for inhibitors of plasmepsin II trafficking.

Wang, F, Krai, P, Deu, E, Bibb, B, Lauritzen, C, Pedersen J, Bogyo M & Klemba, M (2011) Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase I. Molecular and Biochemical Parasitology 175:10-20

Velmourougane, G, Harbut, M, Dalal, S, McGowan, S, Oellig, CA, Whisstock, JC, Klemba, M & Greenbaum, DC (2011) Synthesis of new (-)-Bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase. Journal of Medicinal Chemistry 54:1655-66.

Ragheb, D, Dalal, S, Bompiani, KM, Ray, WK & Klemba, M (2011) Distribution and biochemical properties of an M1-family aminopeptidase in Plasmodium falciparum indicate a role in vacuolar hemoglobin catabolism. Journal of Biological Chemistry 286:27255-65.

Harbut, MB, Velmourougane, G, Dalal, S, Reiss, G, Whisstock, JC, Onder, O, Brisson, D, McGowan, S, Klemba, M, Greenbaum, DC (2011) Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases. Proceedings of the National Academy of Sciences USA 108:E526-34.

Deu, E, Yang, Z, Wang, F, Klemba, M & Bogyo M (2010) Use of activity-based probes to develop high-throughput screening assays that can be performed in complex cell extracts. PLoS One 5:e11985

Ragheb, D, Bompiani, K, Dalal, S & Klemba, M (2009) Evidence for catalytic roles for Plasmodium falciparum aminopeptidase P in the food vacuole and cytosol. Journal of Biological Chemistry 284:24806-15.

Dalal, S & Klemba, M (2007) Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum. Journal of Biological Chemistry 282:35978-87.