Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

Abstract

BackgroundReversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level.ResultsWe have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA) led to inhibition of parasite DNA synthesis.ConclusionsThe high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes.

A Novel Class of Dual-family Immunophilins

“…All of the pET15b clones were introduced into Escherichia coli BL21(DE3) pLysS and induced by isopropyl 1-thio-␤-D-galactopyranoside. The soluble N-terminally His-tagged recombinant proteins were purified by Ni 2ϩ -affinity chromatography using the HisBind TM kit (Novagen) (15). The CyP and FKBP domains of TgFCBP57 were similarly expressed and purified.…”

“…RNAi-like effect in the Apicomplexa as well, especially in T. gondii (15,20,29,30). We adopted a similar approach and targeted the TgFCBP57 mRNA by transfecting a dsRNA corresponding to an internal 500-bp region of the coding sequence, which led to successful silencing of TgFCBP57 (Fig.…”

Plasmodium falciparumencodes a conserved active inhibitor-2 for Protein Phosphatase type 1: perspectives for novel anti-plasmodial therapy

“…The use of potent inhibitors of phosphatases showed that P. falciparum predominantly expressed PP1-like activity which appears to control parasite growth and seems to be involved in the release of infectious merozoites [19,20]. In the past decade, a vast body of research has provided converging evidence that the key mechanism of the mode of action of the PP1c subunit resides in the presence of interacting regulators that direct the proper functions of this phosphatase (i.e.…”

Plasmodium falciparum DNA helicase 60

“…In previous studies, dsRNA has been reported to affect the growth of P. falciparum in culture [28]. Some studies have suggested that the inhibition of growth is due to classic RNA interference pathway [29][30][31][32], whereas others have reported that this is due to an antisense effect [32][33][34]. It is noteworthy that on closer examination of the genome of Plasmodium, it was not possible to identify the homologues of the genes of the components of the classic RNA interference pathway [32].…”