Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

La malaria est la 1ère parasitose mondiale du fait de son taux de morbidité et de mortalité. Elle est responsable de 198 millions de cas dont 584 000 décès en 2013 (OMS). La forme la plus sévère est due à l’apicomplexe Plasmodium falciparum. Etant donné l’absence d’un vaccin efficace et l’augmentation des résistances aux traitements, il est crucial d’approfondir nos connaissances sur la biologie de P. falciparum afin de trouver de nouvelles cibles thérapeutiques. Le cycle de vie complexe avec deux hôtes nécessite une régulation précise et dynamique de l’expression des gènes et des modifications post-traductionnelles. Dans ce contexte, il a été montré que les kinases et les phosphatases, impliquées dans les processus de phosphorylation et de déphosphorylation respectivement, jouent un rôle crucial pour la survie du parasite. Chez les eucaryotes, les phosphatases sont impliquées dans la croissance cellulaire, la différentiation et la division. Parmi elles, PP1, une des principales sérine/thréonine phosphatases, est composée d’une sous-unité catalytique (PP1c) et d’une sous-unité régulatrice. Ces régulateurs sont essentiels et confère à PP1 une localisation, une spécificité et une régulation de son activité. La majorité des régulateurs interagissent avec PP1c via différents motifs tel que le motif RVxF. Chez P. falciparum, PP1 (PfPP1c) est exprimée et semble être essentielle au niveau du stade érythrocytaire, en particulier dans la libération des mérozoïtes infectieux. Pour mieux comprendre la fonction de PfPP1c, nous étudions les régulateurs de PP1 chez le parasite. Nos études précédentes nous ont permis de caractériser 3 régulateurs au niveau moléculaire et fonctionnel. Dans ce contexte, nous avons montré que PfLRR1 et PfI2 inhibent l’activité de PP1 alors que PfI3 l’active. Des études de génétique inverse suggèrent que ces régulateurs sont aussi essentiels que la PP1c elle-même. Récemment, nous avons identifié dans le génome de P. falciparum le facteur d’initiation de la traduction de type 2 sous-unité ß (eIF2ß) qui pourrait être un partenaire/régulateur potentiel de PfPP1. Dans la 1ère partie de cette étude, l’objectif principal a été d’étudier la présence de motifs additionnels de fixation à PfPP1c dans PfI2 et leur impact sur sa fonction. En utilisant la RMN, un troisième motif d’interaction FxxR/KxR/K a été identifié. Ce motif a été montré comme agissant de concert avec le motif canonique RVxF. En effet, la mutation des deux motifs abolie complètement l’interaction avec PfPP1. De plus, en utilisant le modèle d’ovocytes de Xénope, nous avons montré que ces motifs sont nécessaires à PfI2 pour réguler l’activité de PP1. Finalement, l’utilisation d’un peptide dérivé du motif d’interaction FxxR/KxR/K de PfI2 a montré une accumulation dans les érythrocytes infectés et un effet anti-plasmodial a été observé. Dans la 2ème partie de cette étude, nous avons étudié eIF2β, un autre régulateur potentiel de PfPP1. Par des expériences de GST pull-down, nous avons montré l’interaction entre PfeIF2β/PfPP1 et deux motifs d’interaction ont été identifiés : RVxF et FxxR/kxR/K. De plus, en utilisant le modèle d’ovocytes de Xénope, nous avons démontré que PfeIF2ß est impliqué dans la transition G2/M, suggérant un rôle inhibiteur sur l’activité de PP1. La mutation d’un des deux motifs n’empêche pas la formation du complexe alors que la mutation des deux abolie l’interaction avec PP1. Afin de déterminer la fonction de PfeIF2ß in vivo chez Plasmodium, des expériences de génétique inverse ont été réalisées. Nous avons montré l’accessibilité au locus du PfeIF2ß par Knock-in et des expériences d’interruption du gène elf2ß chez Plasmodium falciparum et berghei (espèce spécifique aux rongeurs) sont actuellement en cours afin de déterminer l’essentialité de cette protéine dans le développement du parasite. / Malaria is still the most severe infectious disease in the world because of its high rate of morbidity and mortality. Malaria is responsible for 198 million cases among which 584 000 deaths in 2013 (WHO). The most deadly parasite is the Apicomplexa Plasmodium falciparum. Given the lack of efficient vaccine with long-lasting protection and the increase of resistance against current treatments it is crucial to further deepen our understanding the biology of Plasmodium falciparum to find new means of control. The complex life cycle within two hosts necessitates a highly accurate and dynamic regulation of gene expression and of post translational modifications. In this context, it has been shown that kinases and phosphatases, involved in phosphorylation/dephosphorylation processes respectively, play a key role in parasite survival. In eukaryotes, phosphatases have been shown to be involved in cell growth, differentiation and division. Among them, Protein phosphatase type 1 (PP1) has been reported as one of the major serine/threonine phosphatase proteins involved in diverse cellular functions. PP1 is composed of a single catalytic subunit (PP1c) with a capacity to interact with a high number of regulatory subunits. These regulators are essential as they are key players in different roles of PP1c, including its trafficking, activity and specificity. Most of regulators interact with PP1c via several binding motifs including the RVXF motif. In Plasmodium falciparum, PP1c (PfPP1c) is expressed and seems to be essential for blood stage parasite, in particular merozoïte liberation. To better understand the function of PfPP1c, we investigated the regulators of protein phosphatase type I in this parasite. Our earlier studies have characterized three regulators at the molecular and functional levels. In this context, we have shown that PfLRR1 and PfI2 inhibit PP1 activity while PfI3 activates it. Reverse genetic studies suggested that these regulators are as essential as the PP1c itself. Recently, we found in P. falciparum genome the eukaryotic translation initiation factor 2 subunit ß (eIF2ß) which could be a potential partner/regulator of PfPP1. In the first part of this study, the main objective was to further explore in PfI2 the presence of additional motifs of binding to PfPP1c and their impacts on its function. Using NMR spectroscopy, a third motif was identified: FxxR/KxR/K. This motif has been found to act together with the canonical motif RVxF. Indeed, mutations in both motifs abolished completely the interaction with PfPP1. In addition, using Xenopus oocytes model, we showed that both motifs were necessary for PfI2 to regulate the activity of PP1. Finally the use of a peptide spanning the FxxR/KxR/K motif of PfI2 regulator showed an accumulation in infected erythrocytes and an antiplasmodial effect was observed.In the second part, we investigated eIF2ß as a potential regulator of PfPP1. By GST pull-down assays, we have shown the interaction between PfeIF2ß/PfPP1 and two binding motifs were identified : RVxF and FxxR/KxR/K motifs. Moreover, using Xenopus oocytes model, we demonstrated that PfeIF2ß is involved in G2/M transition, suggesting an inhibitor function of PP1 activity. Mutation of one of two motifs did not prevent the interaction while mutation of both abolished this binding. To gain more insights on the function of PfeIF2ß in Plasmodium, reverse genetic experiments were carried out. We have shown the accessibility of PfeIF2ß locus by Knock-in and we are performing Knock-out experiments on Plasmodium falciparum and berghei (specific species of the rodents) to determine the essentiality of this protein for parasite development.

A proteína serina/treonina fosfatase do tipo 1 (PP1) é a principal serina/treonina fosfatase envolvida na regulação de diversos processos tais como metabolismo, crescimento e divisão celular, síntese protéica e processamento de RNA. A holoenzima PP1 é constituída de uma subunidade catalítica conservada (PP1c) e subunidades reguladoras variáveis. Em mamíferos já foram identificados dezenas de polipeptídeos que associam-se direta ou indiretamente a PP1c, gerando holoenzimas com localizações celulares e especificidades distintas. Entre as proteínas que se associam a PP1c, muitas têm função inibitória como o inibidor-1 (I-1) e o inibidor-2 (I-2). A partir de extratos de micélios de Neurospora crassa foi purificada uma proteína, denominada INc-1, que atua in vitro como inibidor da atividade de fosforilase fosfatase de PP1c e constitui-se no primeiro exemplo de subunidade reguladora da PP1 descrito em fungos filamentosos. INc-1 apresenta diversas características bioquímicas comuns ao I-2 de mamíferos. Seqüências parciais de aminoácidos de três fragmentos proteolíticos obtidos de INc-1 permitiram a identificação de uma ORF (fase aberta de leitura) no genoma de N. crassa que provavelmente codifica INc-1. A análise dessa ORF mostrou que a sequência de aminoácidos do INc-1 é similar a do I-2, especialmente em regiões supostamente envolvidas em sua interação com a PP1c. Neste trabalho descrevemos a clonagem e a expressão em bactérias da sequência codificadora de INc-1. A atividade inibidora de PP1c de duas isoformas recombinantes purificadas, INc-1L e INc-1, foram avaliadas e comparadas. A forma denominada INc-1L apresenta em sua região aminoterminal um segmento de 38 aminoácidos derivado da retenção de um íntron, sem alterar a fase de leitura. Ambas proteínas recombinantes exibiram efeito inibidor sobre a atividade de fosforilase fosfatase de PP1c recombinante, sendo que a IC50 determinada para INc-1L foi de ~50nM e para INc-1 foi de ~11nM, sugerindo que a retenção do segmento de aminoácidos codificado pelo íntron na isoforma INc-1L diminui seu potencial inibitório. Verificamos também que o mRNA de INc-1 é expresso durante o crescimento vegetativo de N.crassa, apresentando níveis máximos na fase exponencial. / Type 1 protein serine/threonine phosphatases (PP1) play important roles in the regulation of many cellular functions including metabolism, cell growth and division, protein synthesis and pre-mRNA splicing. PP1 holoenzyme consists of one highly conserved catalytic subunit (PP1c) and variable regulatory subunits. A number of proteins that interact with PP1c have been described in mammals and the respective holoenzymes present distinct substrate specificity and/or different subcelular localization. Among the proteins that interact with PP1c, there are many with inhibitory effect such as inhibitor-1 (I-1) and inhibitor-2 (1-2). It has been demonstrated that a protein denominated INc-1, purified from Neurospora crassa extracts, specifically inhibits PP1c and has biochemical properties that resemble those of mammalian I-2. INc-1 is the first example of a PP1c regulatory subunit in filamentous fungi. Partial amino acid sequences of INc-1 led to the identification of an ORF (open reading frame) in Neurospora crassa genome which appears to encode INc-1. This ORF shows similarity with mammalian I-2 mainly in regions mapped as sites for interaction with PP1c. In this work we report the cloning and bacterial expression of the coding sequence for INc-1. The PP1c inhibitory activities of two recombinant isoforms, named INc-1L and INc-1, were compared. INc-1L aminoacid sequence presents an in frame segment of 38 residues encoded by an non-processed intron. 80th recombinant proteins showed inhibitory effect against phosphorylase phosphatase activity of recombinant PP1c, with IC50 of ~50nM for INc-1L and ~11nM for INc-1, suggesting that retention of the 38 residue segment decrease the inhibitory potential of INc-1L. We have also verified that INc-1 mRNA is expressed during N.crassa vegetative growth with maximum level at the exponential phase.

A proteína serina/treonina fosfatase do tipo 1 (PP1) é a principal serina/treonina fosfatase envolvida na regulação de diversos processos tais como metabolismo, crescimento e divisão celular, síntese protéica e processamento de RNA. A holoenzima PP1 é constituída de uma subunidade catalítica conservada (PP1c) e subunidades reguladoras variáveis. Em mamíferos já foram identificados dezenas de polipeptídeos que associam-se direta ou indiretamente a PP1c, gerando holoenzimas com localizações celulares e especificidades distintas. Entre as proteínas que se associam a PP1c, muitas têm função inibitória como o inibidor-1 (I-1) e o inibidor-2 (I-2). A partir de extratos de micélios de Neurospora crassa foi purificada uma proteína, denominada INc-1, que atua in vitro como inibidor da atividade de fosforilase fosfatase de PP1c e constitui-se no primeiro exemplo de subunidade reguladora da PP1 descrito em fungos filamentosos. INc-1 apresenta diversas características bioquímicas comuns ao I-2 de mamíferos. Seqüências parciais de aminoácidos de três fragmentos proteolíticos obtidos de INc-1 permitiram a identificação de uma ORF (fase aberta de leitura) no genoma de N. crassa que provavelmente codifica INc-1. A análise dessa ORF mostrou que a sequência de aminoácidos do INc-1 é similar a do I-2, especialmente em regiões supostamente envolvidas em sua interação com a PP1c. Neste trabalho descrevemos a clonagem e a expressão em bactérias da sequência codificadora de INc-1. A atividade inibidora de PP1c de duas isoformas recombinantes purificadas, INc-1L e INc-1, foram avaliadas e comparadas. A forma denominada INc-1L apresenta em sua região aminoterminal um segmento de 38 aminoácidos derivado da retenção de um íntron, sem alterar a fase de leitura. Ambas proteínas recombinantes exibiram efeito inibidor sobre a atividade de fosforilase fosfatase de PP1c recombinante, sendo que a IC50 determinada para INc-1L foi de ~50nM e para INc-1 foi de ~11nM, sugerindo que a retenção do segmento de aminoácidos codificado pelo íntron na isoforma INc-1L diminui seu potencial inibitório. Verificamos também que o mRNA de INc-1 é expresso durante o crescimento vegetativo de N.crassa, apresentando níveis máximos na fase exponencial. / Type 1 protein serine/threonine phosphatases (PP1) play important roles in the regulation of many cellular functions including metabolism, cell growth and division, protein synthesis and pre-mRNA splicing. PP1 holoenzyme consists of one highly conserved catalytic subunit (PP1c) and variable regulatory subunits. A number of proteins that interact with PP1c have been described in mammals and the respective holoenzymes present distinct substrate specificity and/or different subcelular localization. Among the proteins that interact with PP1c, there are many with inhibitory effect such as inhibitor-1 (I-1) and inhibitor-2 (1-2). It has been demonstrated that a protein denominated INc-1, purified from Neurospora crassa extracts, specifically inhibits PP1c and has biochemical properties that resemble those of mammalian I-2. INc-1 is the first example of a PP1c regulatory subunit in filamentous fungi. Partial amino acid sequences of INc-1 led to the identification of an ORF (open reading frame) in Neurospora crassa genome which appears to encode INc-1. This ORF shows similarity with mammalian I-2 mainly in regions mapped as sites for interaction with PP1c. In this work we report the cloning and bacterial expression of the coding sequence for INc-1. The PP1c inhibitory activities of two recombinant isoforms, named INc-1L and INc-1, were compared. INc-1L aminoacid sequence presents an in frame segment of 38 residues encoded by an non-processed intron. 80th recombinant proteins showed inhibitory effect against phosphorylase phosphatase activity of recombinant PP1c, with IC50 of ~50nM for INc-1L and ~11nM for INc-1, suggesting that retention of the 38 residue segment decrease the inhibitory potential of INc-1L. We have also verified that INc-1 mRNA is expressed during N.crassa vegetative growth with maximum level at the exponential phase.