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

Abstract

BackgroundIt is clear that the coordinated and reciprocal actions of kinases and phosphatases are fundamental in the regulation of development and growth of the malaria parasite. Protein Phosphatase type 1 is a key enzyme playing diverse and essential roles in cell survival. Its dephosphorylation activity/specificity is governed by the interaction of its catalytic subunit (PP1c) with regulatory proteins. Among these, inhibitor-2 (I2) is one of the most evolutionarily ancient PP1 regulators. In vivo studies in various organisms revealed a defect in chromosome segregation and cell cycle progression when the function of I2 is blocked.ResultsIn this report, we present evidence that Plasmodium falciparum, the causative agent of the most deadly form of malaria, expresses a structural homolog of mammalian I2, named PfI2. Biochemical, in vitro and in vivo studies revealed that PfI2 binds PP1 and inhibits its activity. We further showed that the motifs 12KTISW16 and 102HYNE105 are critical for PfI2 inhibitory activity. Functional studies using the Xenopus oocyte model revealed that PfI2 is able to overcome the G2/M cell cycle checkpoint by inducing germinal vesicle breakdown. Genetic manipulations in P. falciparum suggest an essential role of PfI2 as no viable mutants with a disrupted PfI2 gene were detectable. Additionally, peptides derived from PfI2 and competing with RVxF binding sites in PP1 exhibit anti-plasmodial activity against blood stage parasites in vitro.ConclusionsTaken together, our data suggest that the PfI2 protein could play a role in the regulation of the P. falciparum cell cycle through its PfPP1 phosphatase regulatory activity. Structure-activity studies of this regulator led to the identification of peptides with anti-plasmodial activity against blood stage parasites in vitro suggesting that PP1c-regulator interactions could be a novel means to control malaria.

Identification of a Plasmodium falciparum inhibitor‐2 motif involved in the binding and regulation activity of protein phosphatase type 1

“…In a previous study, we clearly showed the implication of K 12 TISW 16 (‘RVxF’ motif, motif 1) using biochemical and functional approaches , supporting the NMR data presented here. In order to further inspect the contribution of F 95 KEKRK 100 (‘FxxR/KxR/K’ motif, motif 2) present in the region D94–T117 immobilized by PfPP1 interaction, we mapped the interaction site(s) of PfI2 mutated on motif 2 (F95, K98 and K100 were replaced by A) with PfPP1 using NMR spectroscopy under the same conditions as for the wild‐type PfI2 protein.…”

“…). These data showed that motif 2 is also essential for the regulation of PfPP1 activity by PfI2, as demonstrated previously for motif 1 .…”

“…Clarification of HYNE functional role awaits further studies with I2 from other species ( Chlamydomonas reinhardtii , Leishmania major , Trypanosoma brucei ) where HYNE is absent . In P. falciparum , although our mutation studies showed involvement of the HYNE motif in the function of PfI2, the synthetic corresponding peptide did not exhibit any effect on parasite growth, unlike the ‘RVxF’ corresponding peptide . This may be due to the capacity of native PfI2 to displace the HYNE corresponding peptide.…”

“…We observed that the microinjected PfI2 was able to interact with XePP1 and to induce GVBD. When K 12 TISW 16 was deleted or mutated PfI2 was injected, no GVBD was observed, demonstrating the importance of the ‘RVxF’ motif in PfI2 regulatory activity . In this study, we examined the ability of the F 95 KEKRK 100 motif to affect PfI2 function.…”

GardeniajasminoidesEncodes an Inhibitor-2 Protein for Protein Phosphatase Type 1

“…I-2 was first identified as one of two heat-stable proteins from rabbits that can inhibit the activity of PP1 [9]. I-2 like activity has since been found in diverse organisms, from GLC8 in yeast to I-2 in Rabbit, Caenorhabditis elegans, Drosophila, Plasmodium falciparum, and humans [5,13,16]. A plenty of evidence indicated that I-2 function was regulated by phosphorylation.…”