Protein-protein interactions in the erythrocytic stage of Plasmodium falciparum

Smit, Brigit Karen
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Increasing levels of morbidity and mortality due to malaria are being reported, fuelled by the development of drug-resistant strains of Plasmodium falciparum. This indicates that new compounds and drug targets are needed to strengthen the drug arsenal. Concentrating on the discovery of novel drug targets, two P. falciparum genes were selected for study, due to their putative importance in major protein-protein interaction pathways facilitating growth and survival within the erythrocytic stage of the parasite. PFB0150c codes for a putative protein kinase (PK). Biopanning a phage display library had previously shown that the PK interacted with protein 4.1 and spectrin of the host erythrocyte membrane. The catalytic domain of PFB0150c was subcloned with a GST-tag and the recombinant protein was expressed in E. coli. A kinase assay using [γ-32P] ATP showed that the recombinant PK phosphorylated exogenous casein. This enzyme is hypothesised to phosphorylate host RBC membrane proteins and facilitate RBC invasion by merozoites. It may also play a role in intraerythrocytic growth and exit of the parasite. The PK may be multifunctional since the catalytic kinase domain makes up only a small portion of the protein. The additional domains could be good drug targets since they have very low sequence homology to human proteins. PFE1400c codes for a putative adaptor protein complex-1 β1 subunit presumed to be involved in protein trafficking, which is essential to the survival of malaria parasites. The N-terminal adaptin and C-terminal clathrin adaptor appendage domains were subcloned with a histidine- and a GST-tag, respectively, and were expressed in E. coli. The N-terminal adaptin domain was highly conserved, but the smaller C-terminal domain had low homology with the orthologous human protein and has potential as a drug target. Targeting P. falciparum invasion proteins is difficult because they are highly redundant. By inhibiting the protein trafficking complex, transport of invasion proteins to the micronemes and rhoptries would be blocked and they would be unable to reach their final destination and carry out their function. Thus invasion of host RBC by these defective merozoites would be prevented. The current study forms the foundation for future work in characterising the structure and function of these P. falciparum proteins.