Characterization and localization of Plasmodium falciparum homolog of prokaryotic ClpQ/HslV protease.
The beta subunits (beta1, beta2, and beta5) of 20S proteasome and HslV/ClpQ are ATP-dependent threonine proteases present in eukaryotes and prokaryotes, respectively that control levels of key regulatory proteins in the cell. The orthologue of prokaryotic HslV protease in Plasmodium falciparum (PfHslV) is a novel drug target candidate that has no homolog in the human host. In the present study, the PfHslV was expressed, localized and biochemically characterized. The recombinant PfHslV harbored threonine protease specific activity as well as chymotrypsin like and peptidyl glutamyl peptide hydrolase activities. All the three activities could be inhibited by respective specific inhibitors. The protein was localized in the cytosol of the parasite as a soluble protein by Western immunoblotting of parasite fractions and by immuno-fluorescence microscopy. Activity of the protease in the parasite was ascertained by following the degradation of GFP in a transgenic parasite line expressing fusion protein of GFP and Arc-repressor gene, a known target of HslV protease in the prokaryotes. A model structure of PfHslV was constructed based on the crystal structure of Escherichia coli HslV to assess the structural homology. Availability of the structure model of PfHslV may facilitate identification or designing of novel and specific drugs against PfHslV. The in vitro protease assays with recombinant PfHslV and the transgenic parasite line generated in the present study may be exploited in the screening of novel inhibitors to evaluate their anti-malarial activity.
Ramasamy
Gowthaman
G
Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Gupta
Dinesh
D
Mohmmed
Asif
A
Chauhan
Virander S
VS
D049069:ATP-Dependent Proteases / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D000953:Antigens, Protozoan / Q000276:immunology / Q000378:metabolism; D018360:Crystallography, X-Ray; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000378:metabolism; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D049069:ATP-Dependent Proteases