Analysis of the interactome of the Ser/Thr Protein Phosphatase type 1 in Plasmodium falciparum

Abstract

BackgroundProtein Phosphatase 1 (PP1) is an enzyme essential to cell viability in the malaria parasite Plasmodium falciparum (Pf). The activity of PP1 is regulated by the binding of regulatory subunits, of which there are up to 200 in humans, but only 3 have been so far reported for the parasite. To better understand the P. falciparum PP1 (PfPP1) regulatory network, we here report the use of three strategies to characterize the PfPP1 interactome: co-affinity purified proteins identified by mass spectrometry, yeast two-hybrid (Y2H) screening and in silico analysis of the P. falciparum predicted proteome.ResultsCo-affinity purification followed by MS analysis identified 6 PfPP1 interacting proteins (Pips) of which 3 contained the RVxF consensus binding, 2 with a Fxx[RK]x[RK] motif, also shown to be a PP1 binding motif and one with both binding motifs. The Y2H screens identified 134 proteins of which 30 present the RVxF binding motif and 20 have the Fxx[RK]x[RK] binding motif. The in silico screen of the Pf predicted proteome using a consensus RVxF motif as template revealed the presence of 55 potential Pips. As further demonstration, 35 candidate proteins were validated as PfPP1 interacting proteins in an ELISA-based assay.ConclusionsTo the best of our knowledge, this is the first study on PfPP1 interactome. The data reports several conserved PP1 interacting proteins as well as a high number of specific interactors to PfPP1. Their analysis indicates a high diversity of biological functions for PP1 in Plasmodium. Based on the present data and on an earlier study of the Pf interactome, a potential implication of Pips in protein folding/proteolysis, transcription and pathogenicity networks is proposed. The present work provides a starting point for further studies on the structural basis of these interactions and their functions in P. falciparum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2571-z) contains supplementary material, which is available to authorized users.

The serine/threonine phosphatases of apicomplexan parasites

“…Surprisingly, by contrast to other eukaryotes, PfI3 is a PP1 activator in P. falciparum and it has an essential role in the growth and survival of blood stage parasites (Freville et al, 2012). A more recent study combined co-affinity purification followed by mass spectrometry (MS), yeast two hybrid (Y2H) screening and in silico analysis for P. falciparum proteins with RVxF motif, which is present in most PP1 regulators, to define the PfPP1 interactome (Hollin et al, 2016). This effort identified 186 potential PP1 interactors in P. falciparum, of which 35 have been validated as PfPP1 interactors in an ELISA based assay (Hollin et al, 2016).…”

“…A more recent study combined co-affinity purification followed by mass spectrometry (MS), yeast two hybrid (Y2H) screening and in silico analysis for P. falciparum proteins with RVxF motif, which is present in most PP1 regulators, to define the PfPP1 interactome (Hollin et al, 2016). This effort identified 186 potential PP1 interactors in P. falciparum, of which 35 have been validated as PfPP1 interactors in an ELISA based assay (Hollin et al, 2016). However, further experimentation is needed to confirm their roles as PfPP1 regulators.…”

The Toxoplasma gondii inhibitor-2 regulates protein phosphatase 1 activity through multiple motifs

“…Functional and structural studies revealed that interactions between PP1 and its regulators are mediated by specific binding sites that are conserved throughout evolution, including the so called RVxF and SILK motifs (Wakula et al, 2003) (Huang et al, 1999). So far, about 200 regulators have been described in humans (Hendrickx et al, 2009) while 100 proteins potentially interact with PP1 in Plasmodium (Hollin et al, 2016).…”

Regulating the regulator: Insights into the cardiac protein phosphatase 1 interactome

“…One recent study implemented all three methods in an effort to map the PP1 interactome in the malaria parasite Plasmodium falciparum . [53] Although this study used all three methods, the overlap between the hits from the different methods was quite poor, mainly due to the fact that their AP-MS protocol yielded too few putative interactors (only 6). Ideally, a work-flow will begin with a large-scale AP/IP-MS screen for endogenous PP1 complexes followed by targeted Y2H experiments to map the binary interactions among the proteins, and finally analyzed using bioinformatics both retrospectively (e.g.…”