Clusters of malaria parasites growing inside human red blood cells.

Mike Blackman : Subtilisin-like proteases of the malaria merozoite

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Proteases are known to play important roles during the life cycle of the malaria parasite. We are working on a family of malarial subtilisin-like serine proteases (subtilases) that are involved in host cell invasion and exit.

Images

Molecular structure of the P. falciparum SUB1 catalytic domain

Figure 1: Molecular structure of the P. falciparum SUB1 catalytic domain.

 

A malaria merozoite serine protease

Figure 2: PfSUB1: a malaria merozoite serine protease.

Activity of PfSUB2

Figure 3: Activity of PfSUB2.

On the left is an immunofluorescence image showing the surface location of AMA1 (green) on released P. falciparum merozoites. On the right is a schematic depicting proteolytic shedding of AMA1 and another merozoite surface protein complex called MSP1. The protease PfSUB2 mediates release of both proteins by cleaving them at sites close to the membrane.

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The predicted catalytic serine, histidine and aspartic acid residues (black) are shown in ball-and-stick representation

SUB1 was the first malarial subtilase to be identified (Blackman et al., 1998). It is highly conserved across the genus and plays a critical role in the blood-stage life cycle.

We have shown that SUB1 has a central regulatory role in release of the parasites from the host cell. SUB1 is discharged from merozoite organelles known as exonemes into the parasitophorous vacuole space, where it is responsible for processing a family of papain-like proteins known as SERA, triggering a cascade of proteolytic events which culminate in host cell rupture and parasite egress (Yeoh et al., 2007).

SUB1 is also involved in remodelling of several essential merozoite surface proteins in preparation for invasion of a new cell (Koussis et al., 2009).

Recent work has also shown that SUB1 is essential for release of merozoites from liver stages of the malarial life cycle (Suarez et al., 2013).

Current investigations of SUB1 are focused on dissecting the regulation, timing and significance of these proteolytic events, and identifying inhibitors of SUB1 that have potential for development as drugs.

Surface representation of the P. falciparum SUB1 active site, showing the substrate binding cleft with a bound 10 amino acid peptide substrate derived from the physiological substrate SERA5.

SUB2 (Hackett et al., 1999) is a large membrane-bound subtilase which, like SUB1, is expressed in blood-stage merozoites and is found in all Plasmodium species. SUB2 is the prime candidate for the 'sheddase' which clips important surface proteins such as MSP1 and AMA1 from the surface of the merozoite during erythrocyte invasion (Harris et al., 2005). We are actively pursuing the function of this protease, using biochemical approaches and genetic modification of P. falciparum.