Contribution à l’étude de l’ARN polymérase II de Plasmodium falciparum

L’ARN polymérase II de Plasmodium falciparum, parasite responsable de la forme la plus grave du paludisme, catalyse la transcription des gènes codant les protéines. Nous avons identifié, dans le génome de cet eucaryote apicomplexe, des séquences codant potentiellement les douze sous-unités de l’enzyme parasitaire. Ces séquences ont été clonées par reconstitution génique. Nous avons caractérisé génétiquement l’ARN polymérase II de Plasmodium falciparum en réalisant des tests de complémentation fonctionnelle des sous-unités de ce complexe enzymatique. Nos résultats indiquent que les sous-unités PfRPB4-5-7-9 et 12 codent des protéines capables de remplacer leurs homologues chez la levure Saccharomyces cerevisiae. Ces sous-unités sont bien des orthologues des protéines de levure. De fait, nous avons construit des lignées de levures exprimant de façon stable des protéines de Plasmodium falciparum. Par ailleurs, Les sous-unités PfRPB3-6-8-10 et 11 ne complémentent pas. Nous avons construit des souches viables de levures dont le site de fixation de l’alpha-amanitine sur leur ARN polymérase II est «plasmodifié» ou «humanisé». Un crible de chimiothèque réalisé à l’aide de ces levures ne nous a pas permis d’identifier des molécules capables d’inhiber sélectivement les levures «plasmodifiées». Cependant nous avons mis au point un test cellulaire simple et dont la mise en oeuvre est aisée et permettra l’identification de molécules capables de bloquer l’activité transcriptionnelle de l’enzyme du parasite. De telles drogues constitueront le point de départ vers la mise au point d’une nouvelle classe d’antipaludiques. Enfin, des expériences de double hybride chez la levure ont permis de montrer que la sous-unité hRPB11a de l’ARN polymérase II humaine, contrairement à son homologue de levure, est capable former un homodimère. Ceci est confirmé par des profils de diffraction, à une résolution de 3,4 Å, de cristaux de hRPB11. Ces derniers résultats constituent une contribution importante aux efforts de la communauté scientifique pour l’élucidation de la structure tridimensionnelle de l’ARN polymérase II humaine.The parasite Plasmodium falciparum is the causative agent of the most burdensome form of human malaria. His RNA polymerase II (RNAPII) is responsible for transcription of protein coding genes. The sequencing of the full genome of the parasite enabled us to recover a complete set of genes sequences encoding the putative RNAPII subunits of the parasite. We have cloned those subunits by genetic reconstitution. We investigated the functional conservation of the Plasmodium RNAPII subunits using a genetic test in the yeast Saccharomyces cerevisiae. The subunits PfRPB4-5-7-9 and 12 can complement defective yeast mutants. Those results demonstrate that those subunits are orthologs of yeast conterparts. We establish then some stable yeast strains expressing Plasmodium proteins. No interspecific complementation was observed for the subunits PfRPB3-6-8-10-11. We construct viable yeast strain where the residus implicated in the interaction of the mushrom toxin alpha-amanitin where substituted by their homologs in Plasmodium and human. We screen those strains with a chemical compounds library. We don’t find any drug enable to distinguish the modified strains from wild-type one. But this screen, consisting in a simple cellular test and easy to perform, could permit to identify drugs that inhibit the transcriptional activity of the parasite enzyme. Those chemical compounds will the fist step towards the discovery of a potential new class of antimalarials drugs. In this work, we also study the human RNA polymerase II (RNAPII) hRPB11a subunit. we performed an interaction analysis using the two hybrid-system in yeast. The results confirmed that hRPB11a was indeed capable of interacting with itself. We were able to crystallise the purified hRPB11a subunit. The X-ray diffraction patterns at 3,4 Å resolution allows to infer the structure of an homodimer of the hRPB11a protein

Contribution à l étude de l ARN polymérase II de Plasmodium falciparum

L ARN polymérase II de Plasmodium falciparum, parasite responsable de la forme la plus grave du paludisme, catalyse la transcription des gènes codant les protéines. Nous avons identifié, dans le génome de cet eucaryote apicomplexe, des séquences codant potentiellement les douze sous-unités de l enzyme parasitaire. Ces séquences ont été clonées par reconstitution génique. Nous avons caractérisé génétiquement l ARN polymérase II de Plasmodium falciparum en réalisant des tests de complémentation fonctionnelle des sous-unités de ce complexe enzymatique. Nos résultats indiquent que les sous-unités PfRPB4-5-7-9 et 12 codent des protéines capables de remplacer leurs homologues chez la levure Saccharomyces cerevisiae. Ces sous-unités sont bien des orthologues des protéines de levure. De fait, nous avons construit des lignées de levures exprimant de façon stable des protéines de Plasmodium falciparum. Par ailleurs, Les sous-unités PfRPB3-6-8-10 et 11 ne complémentent pas. Nous avons construit des souches viables de levures dont le site de fixation de l alpha-amanitine sur leur ARN polymérase II est plasmodifié ou humanisé . Un crible de chimiothèque réalisé à l aide de ces levures ne nous a pas permis d identifier des molécules capables d inhiber sélectivement les levures plasmodifiées . Cependant nous avons mis au point un test cellulaire simple et dont la mise en oeuvre est aisée et permettra l identification de molécules capables de bloquer l activité transcriptionnelle de l enzyme du parasite. De telles drogues constitueront le point de départ vers la mise au point d une nouvelle classe d antipaludiques. Enfin, des expériences de double hybride chez la levure ont permis de montrer que la sous-unité hRPB11a de l ARN polymérase II humaine, contrairement à son homologue de levure, est capable former un homodimère. Ceci est confirmé par des profils de diffraction, à une résolution de 3,4 Å, de cristaux de hRPB11. Ces derniers résultats constituent une contribution importante aux efforts de la communauté scientifique pour l élucidation de la structure tridimensionnelle de l ARN polymérase II humaine.The parasite Plasmodium falciparum is the causative agent of the most burdensome form of human malaria. His RNA polymerase II (RNAPII) is responsible for transcription of protein coding genes. The sequencing of the full genome of the parasite enabled us to recover a complete set of genes sequences encoding the putative RNAPII subunits of the parasite. We have cloned those subunits by genetic reconstitution. We investigated the functional conservation of the Plasmodium RNAPII subunits using a genetic test in the yeast Saccharomyces cerevisiae. The subunits PfRPB4-5-7-9 and 12 can complement defective yeast mutants. Those results demonstrate that those subunits are orthologs of yeast conterparts. We establish then some stable yeast strains expressing Plasmodium proteins. No interspecific complementation was observed for the subunits PfRPB3-6-8-10-11. We construct viable yeast strain where the residus implicated in the interaction of the mushrom toxin alpha-amanitin where substituted by their homologs in Plasmodium and human. We screen those strains with a chemical compounds library. We don t find any drug enable to distinguish the modified strains from wild-type one. But this screen, consisting in a simple cellular test and easy to perform, could permit to identify drugs that inhibit the transcriptional activity of the parasite enzyme. Those chemical compounds will the fist step towards the discovery of a potential new class of antimalarials drugs. In this work, we also study the human RNA polymerase II (RNAPII) hRPB11a subunit. we performed an interaction analysis using the two hybrid-system in yeast. The results confirmed that hRPB11a was indeed capable of interacting with itself. We were able to crystallise the purified hRPB11a subunit. The X-ray diffraction patterns at 3,4 Å resolution allows to infer the structure of an homodimer of the hRPB11a protein.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

A genetic analysis of Plasmodium falciparum RNA polymerase II subunits in yeast

RNA polymerase II is an essential nuclear multi subunit enzyme that transcribes nearly the whole genome. Its inhibition by the alpha-amanitin toxin leads to cell death. The enzyme of Plasmodium falciparum remains poorly characterized. Using a complementation assay in yeast as a genetic test, we demonstrate that five Plasmodium putative RNA polymerase subunits are indeed functional in vivo. The active site of this enzyme is built from the two largest subunits. Using site directed mutagenesis we were able to modify the active site of the yeast RNA polymerase II so as to introduce Plasmodium or human structural motifs. The resulting strains allow the screening of chemical libraries for potential specific inhibitors

Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

International audienceThe proapoptotic protein, prostate apoptosis response-4 (Par-4), acts as a tumor suppressor in prostate cancer cells. The serine/threonine kinase casein kinase 2 (CK2) has a well-reported role in prostate cancer resistance to apoptotic agents or anticancer drugs. However, the mechanistic understanding on how CK2 supports survival is far from complete. In this work, we demonstrate both in rat and humans that (i) Par-4 is a new substrate of the survival kinase CK2 and (ii) phosphorylation by CK2 impairs Par-4 proapoptotic functions. We also unravel different levels of CK2-dependent regulation of Par-4 between species. In rats, the phosphorylation by CK2 at the major site, S124, prevents caspase-mediated Par-4 cleavage (D123) and consequently impairs the proapoptotic function of Par-4. In humans, CK2 strongly impairs the apoptotic properties of Par-4, independently of the caspase-mediated cleavage of Par-4 (D131), by triggering the phosphorylation at residue S231. Furthermore, we show that human Par-4 residue S231 is highly phosphorylated in prostate cancer cells as compared with their normal counterparts. Finally, the sensitivity of prostate cancer cells to apoptosis by CK2 knockdown is significantly reversed by parallel knockdown of Par-4. Thus, Par-4 seems a critical target of CK2 that could be exploited for the development of new anticancer drugs

HSP70 sequestration by free α-globin promotes ineffective erythropoiesis in β-thalassaemia

International audienceβ-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-T