The goal of the proposed research project is to understand and to inhibit the function of an unusual, highly specialized digestive organelle which is critical to the growth and maturation of the human malaria parasite, Plasmodium falciparum. This organism causes disease in several hundred million people, death in millions of children each year, and Is of concern to over one million travelers per year in this country alone. The parasite grows by catabolizing host erythrocyte hemoglobin and using the resulting amino acids for protein synthesis. This degradative process takes place in an acidic digestive vacuole which appears to have evolved specifically for proteolysis of hemoglobin and utilization of the amino acid and heme components. A number of events take place within the digestive vacuole, but remain essentially uncharacterized. The first event is the targeting of newly synthesized proteases to the digestive vacuole. How this targeting occurs during vacuole biogenesis Is unclear. The hemoglobin is then Ingested from the erythrocyte cytoplasm and transported to the vacuole in small vesicles. These transport vesicles have to be broken down by an enzyme or lytic agent without harming the digestive vacuole wall. The mechanism of lysis remains undetermined. Next, the hemoglobin thus released is degraded in an ordered fashion by a series of proteases which are just beginning to be characterized. During hemoglobin digestion, the heme ho Is converted from ferrous to ferric, which generates a large load of electrons. The mode of utilization of these electrons is undetermined. The hemes are then linked together in a crystalline lattice, called hemozoin pigment. Its formation and function are unclear. This proposal aims to unravel the metabolic pathways of the digestive vacuole, in order to understand and interfere with the processes of this vacuole which are central to the parasitic function of the malaria organism. In vitro labeling of infected erythrocytes, subcellular fractionation to analyze pure digestive vacuoles, protein purification, immunocytochemistry, and enzyme kinetic analysis are the principal techniques to be used in this endeavor. The proximal goal is to design selective Inhibitors of the digestive vacuole aspartic protease. Preliminary studies have shown that this enzyme is the key protease which initiates malaria hemoglobin catabolism. The enzyme differs in specificity from its mammalian counterparts. The way is now clear to design a new class of antimalarials. The long-term objective is to define unique features of vacuolar metabolism which will serve as targets for development of other chemotherapeutic agents.
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