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.
Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases in vivo, and to detect even the slightest variations of their activities. Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i) fluorophores and (ii) linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties.
Keywords NMR spectroscopy; Plasmodium falciparum; PP1 inhibitor 2; protein phosphatase type 1; protein-protein interaction Correspondence
Protein phosphatase 1 (PP1c) is one of the main phosphatases whose function is shaped by many regulators to confer a specific location and a selective function for this enzyme. Here, we report that eukaryotic initiation factor 2β of Plasmodium falciparum (PfeIF2β) is an interactor of PfPP1c. Sequence analysis of PfeIF2β revealed a deletion of 111 amino acids when compared to its human counterpart and the presence of two potential binding motifs to PfPP1 (29FGEKKK34, 103KVAW106). As expected, we showed that PfeIF2β binds PfeIF2γ and PfeIF5, confirming its canonical interaction with partners of the translation complex. Studies of the PfeIF2β-PfPP1 interaction using wild-type, single and double mutated versions of PfeIF2β revealed that both binding motifs are critical. We next showed that PfeIF2β is able to induce Germinal Vesicle Break Down (GVBD) when expressed in Xenopus oocytes, an indicator of its capacity to regulate PP1. Only combined mutations of both binding motifs abolished the interaction with PP1 and the induction of GVBD. In P. falciparum, although the locus is accessible for genetic manipulation, PfeIF2β seems to play an essential role in intraerythrocytic cycle as no viable knockout parasites were detectable. Interestingly, as for PfPP1, the subcellular fractionation of P. falciparum localized PfeIF2β in cytoplasm and nuclear extracts, suggesting a potential effect on PfPP1 in both compartments and raising the question of a non-canonical function of PfeIf2β in the nucleus. Hence, the role played by PfeIF2β in blood stage parasites could occur at multiple levels involving the binding to proteins of the translational complex and to PfPP1.
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