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.
BACKGROUND
Reversible 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.


RESULTS
We 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.


CONCLUSIONS
The 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.
Kumar
Rajinder
R
Department of Biochemistry and Molecular Biology (MSB 2370), University of South Alabama, College of Medicine, 307 University Blvd, Mobile, AL 36688-0002, USA. rkumar@usamail.usouthal.edu
Adams
Brian
B
Oldenburg
Anja
A
Musiyenko
Alla
A
Barik
Sailen
S
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000187:drug effects / Q000378:metabolism; D005784:Gene Amplification; D020868:Gene Silencing / Q000187:drug effects; D016778:Malaria, Falciparum; D016296:Mutagenesis; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects*; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology*
D018076:DNA, Complementary; D034741:RNA, Small Interfering; D010749:Phosphoprotein Phosphatases