Identification of a non-exported Plasmepsin V substrate that functions in the parasitophorous vacuole of malaria parasites.

In the manuscript, the authors investigate the role of the protease Plasmepsin V in the parasite-host interaction. Whereas processing by Plasmepsin V was previously thought to target a protein for export into the host cell, the authors now show that there are proteins cleaved by this protease that are not exported but instead function at the host-parasite interface. This changes the view of this protease, which turns out to have a much broader role than anticipated. The result shows that the protease may have a function much more similar to that of related organisms. The authors also investigate the requirements for protein export by analyzing exported and non-exported proteins and find commonalities between the proteins of each set that further our understanding of the requirements for protein export

Deciphering the Role of Protein Phosphatases in Apicomplexa: The Future of Innovative Therapeutics?

Parasites belonging to the Apicomplexa phylum still represent a major public health and world-wide socioeconomic burden that is greatly amplified by the spread of resistances against known therapeutic drugs. Therefore, it is essential to provide the scientific and medical communities with innovative strategies specifically targeting these organisms. In this review, we present an overview of the diversity of the phosphatome as well as the variety of functions that phosphatases display throughout the Apicomplexan parasites' life cycles. We also discuss how this diversity could be used for the design of innovative and specific new drugs/therapeutic strategies

Peptides derived from Plasmodium falciparum leucine-rich repeat 1 bind to serine/threonine phosphatase type 1 and inhibit parasite growth in vitro.

International audienceThe biogenesis of protein phosphatase 1 (PP1) holoenzyme in eukaryotes requires diverse regulatory subunit proteins (RSPs) that bind to the highly conserved PP1 catalytic subunit (PP1c) and direct its spatiotemporal activity as well as its specificity. Several studies demonstrated that most RSPs share a canonical common binding motif, the RVXF motif, which is present in ~85% of RSPs and is considered as the main contributor for the interaction to PP1c.1 In Plasmodium falciparum (Pf), our earlier studies revealed that leucine-rich repeat 1 (LRR1), one of the major RSPs of PfPP1 and an ortholog of human and yeast Sds22, lacks the RVXF motif. The amino acids sequence of PfLRR1 exhibits nine leucine-rich repeats (LRRs) and a hydrophobic region at the C-terminal end, known as the LRR cap motif.2 In this work, we identified the PP1-binding peptides of PfLRR1 and examined their capacity to affect Pf growth

Plasmodium falciparum protein phosphatase PP7 is required for early ring-stage development.

We previously reported that the Plasmodium falciparum putative serine/threonine protein phosphatase 7 (PP7) is a high-confidence substrate of the cAMP-dependent protein kinase (PKA). Here we explore the function of PP7 in asexual P. falciparum blood stage parasites. We show that conditional disruption of PP7 leads to a severe growth arrest. We show that PP7 is a calcium-dependent phosphatase that interacts with calmodulin and calcium-dependent protein kinase 1 (CDPK1), consistent with a role in calcium signaling. Notably, PP7 was found to be dispensable for erythrocyte invasion, but was crucial for ring-stage development, with PP7-null parasites arresting shortly following invasion and showing no transition to ameboid forms. Phosphoproteomic analysis revealed that PP7 may regulate certain PKAc substrates. Its interaction with calmodulin and CDPK1 further emphasizes a role in calcium signaling, while its impact on early ring development and PKAc substrate phosphorylation underscores its importance in parasite development. IMPORTANCE: Plasmodium falciparum causes malaria and is responsible for more than 600,000 deaths each year. Although effective drugs are available to treat disease, the spread of drug-resistant parasites endangers their future efficacy. It is hoped that a better understanding of the biology of malaria parasites will help us to discover new drugs to tackle the resistance problem. Our work is focused on the cell signaling mechanisms that control the development of the parasite throughout its complex life cycle. All signal transduction pathways are ultimately regulated by reversible protein phosphorylation by protein kinase and protein phosphatase enzymes. In this study, we investigate the function of calcium-dependent protein phosphatase PP7 and show that it is essential for the development of ring-stage parasites following the invasion of human erythrocytes. Our results contribute to the understanding of the erythrocytic stages of the parasite life cycle that cause malaria pathology

Essential role of GEXP15, a specific Protein Phosphatase type 1 partner, in Plasmodium berghei in asexual erythrocytic proliferation and transmission.

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle

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

BACKGROUND: It 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. RESULTS: In 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 (12)KTISW(16) and (102)HYNE(105) 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. CONCLUSIONS: Taken 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

Plasmodium pseudo-Tyrosine Kinase-like binds PP1 and SERA5 and is exported to host erythrocytes.

Pseudokinases play key roles in many biological processes but they are poorly understood compared to active kinases. Eight putative pseudokinases have been predicted in Plasmodium species. We selected the unique pseudokinase belonging to tyrosine kinase like (TKL) family for detailed structural and functional analysis in P. falciparum and P. berghei. The primary structure of PfpTKL lacks residues critical for kinase activity, supporting its annotation as a pseudokinase. The recombinant pTKL pseudokinase domain was able to bind ATP, but lacked catalytic activity as predicted. The sterile alpha motif (SAM) and RVxF motifs of PfpTKL were found to interact with the P. falciparum proteins serine repeat antigen 5 (SERA5) and protein phosphatase type 1 (PP1) respectively, suggesting that pTKL has a scaffolding role. Furthermore, we found that PP1c activity in a heterologous model was modulated in an RVxF-dependent manner. During the trophozoite stages, PbpTKL was exported to infected erythrocytes where it formed complexes with proteins involved in cytoskeletal organization or host cell maturation and homeostasis. Finally, genetic analysis demonstrated that viable strains obtained by genomic deletion or knocking down PbpTKL did not affect the course of parasite intra-erythrocytic development or gametocyte emergence, indicating functional redundancy during these parasite stages

Deciphering the Role of Protein Phosphatases in Apicomplexa: The Future of Innovative Therapeutics?

Parasites belonging to the Apicomplexa phylum still represent a major public health and world-wide socioeconomic burden that is greatly amplified by the spread of resistances against known therapeutic drugs. Therefore, it is essential to provide the scientific and medical communities with innovative strategies specifically targeting these organisms. In this review, we present an overview of the diversity of the phosphatome as well as the variety of functions that phosphatases display throughout the Apicomplexan parasites’ life cycles. We also discuss how this diversity could be used for the design of innovative and specific new drugs/therapeutic strategies

Identification et caractérisation des inhibiteurs de type 2 et 3 de la phosphatase de type 1 chez Plasmodium falciparum

XLe paludisme (ou malaria) est la parasitose la plus répandue à travers le monde (WHO, 2011).La moitié de la population mondiale est exposée à cette maladie causée par le protozoairePlasmodium. La résistance aux traitements qui se développe chez le parasite représente un véritableobstacle à la mise en place de programmes de lutte globale. Le développement de nouveauxprotocoles thérapeutiques plus efficaces ciblant les apicomplexes Plasmodium passe par uneamélioration de nos connaissances concernant la biologie fondamentale des parasites. Par ce biais,nous pourrons identifier de nouvelles cibles originales visant des mécanismes essentiels etspécifiques au développement du pathogène. Chez Plasmodium falciparum (l’espèce responsable dela forme la plus mortelle de malaria), la phase érythrocytaire qui se déroule chez son hôte humain estrelativement courte (48 heures). Le parasite y subit un grand nombre de changementsmorphologiques nécessitant une différentiation précise, spécifique et régulée au cours du temps.Parmi les éléments pouvant contrôler ces mécanismes, les phénomènes de phosphorylationréversibles semblent être des candidats de choix.Nous avons, dans un premier temps, entrepris la caractérisation de PfI2. Ce travail a permisd’identifier cette protéine comme étant un régulateur négatif de PfPP1 localisé au niveaunucléocytoplasmique et indispensable au développement érythrocytaire du parasite. Des étudesd’interaction in vitro et in vivo ont permis d’identifier un certain nombre de résidus impliqués dans lafixation et la fonction de PfI2. En nous basant sur ces résultats, nous avons entrepris une explorationplus précise des relations structure/fonction du complexe PfI2/PfPP1.Concernant l’identification de PfI3, nous avons récemment publiée dans le Journal ofBiological Chemistry (Frèville A. 2012) un travail montrant que chez le parasite, l’inhibiteur 3 estlocalisé au niveau nucléaire et est essentiel au développement asexuel du pathogène. Desexpériences d’interaction in vitro ont permis de montrer que PfI3 est capable de se fixer in vitro àPfPP1 et ce principalement via le motif primaire RVxF. Chez des levures déplétées de leur inhibiteur3, l’expression épisomique de PfI3 n’a pas permis de restaurer la croissance des cellules. Desexpériences in vitro d’activités phosphatase révèlent une action positive de PfI3 sur PfPP1. Cerésultat, inverse de celui que l’on observe chez ses homologues chez la levure ou l’humain met enévidence une fonction différente et spécifique de PfI3 et font de cette protéine un régulateurnucléaire potentiel de PfPP1 chez Plasmodium falciparum.L’ensemble de ce travail de thèse à permis d’identifier et de caractériser chez Plasmodiumfalciparum deux régulateurs potentiels de la phosphatase de type 1 mais également de mettre enévidence un certain nombre d’éléments spécifiques au fonctionnement et au développement duparasite faisant de ces protéines des cibles thérapeutiques intéressantes

Characterisation and functional studies of Inhibitors 2 and 3 of protein phosphatase type 1 in Plasmodium falciparum

Le paludisme (ou malaria) est la parasitose la plus répandue à travers le monde (WHO, 2011).La moitié de la population mondiale est exposée à cette maladie causée par le protozoairePlasmodium. La résistance aux traitements qui se développe chez le parasite représente un véritableobstacle à la mise en place de programmes de lutte globale. Le développement de nouveauxprotocoles thérapeutiques plus efficaces ciblant les apicomplexes Plasmodium passe par uneamélioration de nos connaissances concernant la biologie fondamentale des parasites. Par ce biais,nous pourrons identifier de nouvelles cibles originales visant des mécanismes essentiels etspécifiques au développement du pathogène. Chez Plasmodium falciparum (l’espèce responsable dela forme la plus mortelle de malaria), la phase érythrocytaire qui se déroule chez son hôte humain estrelativement courte (48 heures). Le parasite y subit un grand nombre de changementsmorphologiques nécessitant une différentiation précise, spécifique et régulée au cours du temps.Parmi les éléments pouvant contrôler ces mécanismes, les phénomènes de phosphorylationréversibles semblent être des candidats de choix.Nous avons, dans un premier temps, entrepris la caractérisation de PfI2. Ce travail a permisd’identifier cette protéine comme étant un régulateur négatif de PfPP1 localisé au niveaunucléocytoplasmique et indispensable au développement érythrocytaire du parasite. Des étudesd’interaction in vitro et in vivo ont permis d’identifier un certain nombre de résidus impliqués dans lafixation et la fonction de PfI2. En nous basant sur ces résultats, nous avons entrepris une explorationplus précise des relations structure/fonction du complexe PfI2/PfPP1.Concernant l’identification de PfI3, nous avons récemment publiée dans le Journal ofBiological Chemistry (Frèville A. 2012) un travail montrant que chez le parasite, l’inhibiteur 3 estlocalisé au niveau nucléaire et est essentiel au développement asexuel du pathogène. Desexpériences d’interaction in vitro ont permis de montrer que PfI3 est capable de se fixer in vitro àPfPP1 et ce principalement via le motif primaire RVxF. Chez des levures déplétées de leur inhibiteur3, l’expression épisomique de PfI3 n’a pas permis de restaurer la croissance des cellules. Desexpériences in vitro d’activités phosphatase révèlent une action positive de PfI3 sur PfPP1. Cerésultat, inverse de celui que l’on observe chez ses homologues chez la levure ou l’humain met enévidence une fonction différente et spécifique de PfI3 et font de cette protéine un régulateurnucléaire potentiel de PfPP1 chez Plasmodium falciparum.L’ensemble de ce travail de thèse à permis d’identifier et de caractériser chez Plasmodiumfalciparum deux régulateurs potentiels de la phosphatase de type 1 mais également de mettre enévidence un certain nombre d’éléments spécifiques au fonctionnement et au développement duparasite faisant de ces protéines des cibles thérapeutiques intéressantes.