Functional dissection of a malarial serine protease.
Doctoral thesis, UCL (University College London).
Malaria is a major threat to human health. It is caused by Plasmodium s-pp., a protozoan parasite that belongs to the phylum Apicomplexa. Malaria parasites have a complicated life cycle and clinical symptoms occur during replication of the parasite in erythrocytes. Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) belongs to the subtilisin-like family of serine proteases (subtilases). It is encoded by a single copy gene and is translated as a protein with a molecular mass of about 78 kDa. Attempts to disrupt the gene have not been successful, indicating an essential role in the intraerythrocytic life cycle of the parasite. Recent studies have shown that PfSUB1 is discharged into the parasitophorous vacuole (PV) in the final stages of schizont maturation to mediate proteolytic maturation of an abundant PV protein called SERA5. In this project, an examination of the SERA5 processing sites along with the use of a small library of internally quenched fluorogenic peptides, revealed a consensus PfSUB1 substrate recognition motif that showed a striking resemblance to all known primary processing sites in merozoite surface proteins. The role of PfSUB1 in these processing events was therefore examined. The results showed that PfSUB1 is responsible for the proteolytic processing of merozoite surface proteins 1, 3, 6 and 7 that takes place in the final stages of schizont maturation. Different strategies to obtain a knock-down of the pfsub1 gene by means of reverse genetics were also attempted. Mutagenesis studies were performed to study the structural characteristics of PfSUB1 that are responsible for its substrate specificity, and in an attempt to optimise the expression of soluble recombinant PfSUB1 for future crystallographic studies. The results obtained identify PfSUB1 as a multifunctional processing protease of the malarial PV that plays a key role in both egress and proteolytic remodelling of the developing merozoite in preparation for its release from the confines of the infected cell.
|Title:||Functional dissection of a malarial serine protease|
|Additional information:||Pending digitisation|
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