The immediate goal of this project is to identify and fully characterize the structure and function of aspartic proteinases found in the human malaria parasites, Plasmodium falciparum and Plasmodium vivax. Potential antimalarial drugs exist among inhibitors of these enzymes, and the long range goal is to develop a novel class of antimalarial drugs that function by inhibiting the aspartic proteinases of the parasite. These goals will be approached in this proposal by addressing the following specific aims: 1. Clone the genes encoding the aspartic proteinases of P. falciparum and P. vivax, utilizing the complete gene sequences available for plasmepsins I and II of P. falciparum and a recently cloned plasmepsin gene for P. vivax. 2. Characterize the in vivo expression and determine the size and subcellular location of the native aspartic proteinases of both parasite species. Oligonucleotide primers and probes specific for each gene will be used to estimate the steady-state level of mRNA accumulation in each erythrocytic stage. Polyclonal and monoclonal polypeptide-specific antibodies will be used to determine the size of native enzymes on western blots, and used to follow-intracellular synthesis, processing and translocation of the native enzymes in different stages of erythrocytic development via immunoelectron microscopy. Life cycle stages found in the mosquito vector will also be examined by immunoelectron microscopy for the presence of plasmepsins. Monoclonal antibodies will be used to purify the native forms of plasmepsins I and II from P. falciparum for comparative purposes. 3. Characterize the specificity of the malarial aspartic proteinases. Recombinant plasmepsins of P. falciparum and P. vivax will be refolded as necessary, and purified to homogeneity. The activity of each enzyme will be studied and compared to native enzymes from P. falciparum using a panel of oligopeptide substrates and inhibitors constructed for this purpose. These data will provide information on subsite interactions and will be interpreted by reference to a computer built molecular model of the structure of each enzyme, based on homology to structurally-defined proteinases. Site-directed mutagenesis will be used to test hypotheses concerning the critical residues within the active site in comparison to other aspartic proteinases. Protein will be provided to collaborating crystallographers for determination of the structure. Novel peptidomimetics will be constructed to provide possible ligands for crystallography as well as to test for in vivo function. Potential inhibitors will be tested in in vitro culture for antimalarial activity.
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