Abstract
The Plasmodium falciparum kinome includes a family of four protein kinases (Pfnek-1 to -4) related to the NIMA (never-in-mitosis) family, members of which play important roles in mitosis and meiosis in eukaryotic cells. Only one of these, Pfnek-1, which we previously characterized at the biochemical level, is expressed in asexual parasites. The other three (Pfnek-2, -3 and -4) are expressed predominantly in gametocytes, and a role for nek-2 and nek-4 in meiosis has been documented. Here we show by reverse genetics that Pfnek-1 is required for completion of the asexual cycle in red blood cells and that its expression in gametocytes in detectable by immunofluorescence in male (but not in female) gametocytes, in contrast with Pfnek-2 and Pfnek-4. This indicates that the function of Pfnek-1 is non-redundant with those of the other members of the Pfnek family and identifies Pfnek-1 as a potential target for antimalarial chemotherapy. A medium-throughput screen of a small-molecule library provides proof of concept that recombinant Pfnek-1 can be used as a target in drug discovery.
Highlights
Proteins participating in cell signalling, notably the large families of G-protein-coupled receptors and protein kinases (PKs), constitute a vast reservoir of potential molecular targets for chemotherapy in a variety of diseases
Functions in the life cycle have first been established for a few PKs using a gene knockout approach in the Plasmodium berghei system (Rangarajan et al, 2005, 2006; Reininger et al, 2005, 2009; Tewari et al, 2005), which is more amenable than P. falciparum to genetic manipulation (Carvalho & Menard, 2005)
The only NIMA family member whose mRNA is detectable by microarray throughout the erythrocytic asexual cycle is Pfnek-1, whose enzymatic properties we described previously using a recombinant enzyme (Dorin et al, 2001)
Summary
Proteins participating in cell signalling, notably the large families of G-protein-coupled receptors and protein kinases (PKs), constitute a vast reservoir of potential molecular targets for chemotherapy in a variety of diseases. Functions in the life cycle have first been established for a few PKs using a gene knockout approach in the Plasmodium berghei system (Rangarajan et al, 2005, 2006; Reininger et al, 2005, 2009; Tewari et al, 2005), which is more amenable than P. falciparum to genetic manipulation (Carvalho & Menard, 2005). This culminated in a kinome-wide systematic study identifying all P. berghei PKs with a role in transmission (Tewari et al, 2010). We show that a functional pfnek-1 gene is required for parasite survival and a potential target for chemotherapy, and provide proof of concept that the recombinant enzyme can be used in medium-throughput screening campaigns to identify inhibitors as the first step in the drug discovery process
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