Mapping PP1c and Its Inhibitor 2 Interactomes Reveals Conserved and Specific Networks in Asexual and Sexual Stages of Plasmodium

Abstract

Malaria parasites require multiple phosphorylation and dephosphorylation steps to drive signaling pathways for proper differentiation and transformation. Several protein phosphatases, including protein phosphatase 1 (PP1), one of the main dephosphorylation enzymes, have been shown to be indispensable for the Plasmodium life cycle. The catalytic subunit of PP1 (PP1c) participates in cellular processes via dynamic interactions with a vast number of binding partners that contribute to its diversity of action. In this study, we used Plasmodium berghei transgenic parasite strains stably expressing PP1c or its inhibitor 2 (I2) tagged with mCherry, combined with the mCherry affinity pulldown of proteins from asexual and sexual stages, followed by mass spectrometry analyses. Mapped proteins were used to identify interactomes and to cluster functionally related proteins. Our findings confirm previously known physical interactions of PP1c and reveal enrichment of common biological processes linked to cellular component assembly in both schizonts and gametocytes to biosynthetic processes/translation in schizonts and to protein transport exclusively in gametocytes. Further, our analysis of PP1c and I2 interactomes revealed that nuclear export mediator factor and peptidyl-prolyl cis-trans isomerase, suggested to be essential in P. falciparum, could be potential targets of the complex PP1c/I2 in both asexual and sexual stages. Our study emphasizes the adaptability of Plasmodium PP1 and provides a fundamental study of the protein interaction landscapes involved in a myriad of events in Plasmodium, suggesting why it is crucial to the parasite and a source for alternative therapeutic strategies.

Characterization of GEXP15 as a Potential Regulator of Protein Phosphatase 1 in Plasmodium falciparum

“…Although PfPP1 was not detected in the PfGEXP15 IP/MS, the likelihood of this interaction via the RVxF motif was demonstrated by the use of complementary approaches such as Y2H, GST pull-down, and pull-down experiments (this study), confirming previous findings [ 8 , 9 ]. Supporting this is the fact that in P. berghei parasites, PbGEXP15 was also detected among the top PP1-interacting proteins in both schizont and gametocyte stages [ 9 ]. Further, the reciprocal IP/MS identified PbPP1 after PbGEXP15 immunoprecipitation [ 7 ].…”

“…Mammalian cells have 200 identified regulatory subunits that contribute to the specificity, location, and level of phosphatase activity of PP1c [ 5 , 6 ]. In Pf and in Plasmodium berghei (Pb), PP1c has been shown to have numerous potential regulatory partners, with hundreds of interacting proteins identified through yeast two-hybrid (Y2H) and immunoprecipitation experiments combined with mass spectrometry analysis [ 7 , 8 , 9 ].…”

Characterization of thePlasmodium bergheiregulatory AAA-ATPase subunit Rpt3 as an activator of Protein Phosphatase 1: direct and indirect evidence

“…Regarding the second mo f RKIEF, it is conserved among all Plasmodium species as well as among mammals (Homo sapiens, Mus musculus), yeast (Saccharomyces cerevisiae) and plants (Arabidopsis thaliana -Uniprot Reference A0A178UAH2). The presence of two puta ve RVxF mo fs in PbRpt3 may explain its detec on in PbPP1c interactome [15,16], and likely suggests that it could be a direct interactor of the phosphatase.…”

“…More recently, our analyses based on a proteomic approach conducted in Plasmodium berghei (Pb) have iden fied some of the 19S AAA-ATPase and non-ATPase subunits as poten al interactors of the parasite PP1 cataly c subunit (PP1c) [15,16]. This parasite, causa ve agent of malaria, undergoes rapid growth and cell division in an oxida vely stressed environment.…”

“…A common feature of most of the PP1c regulators is that they contain a RVxF sequence as the primary PP1c-binding mo f [31]. In Plasmodium, numerous poten al regulatory partners have been iden fied in global analyses of PP1c interactome [15,16] . This includes both conserved and specific regulators [15,[32][33][34][35].…”