Дисертації з теми "PIP5K1"

La formation des particules du VIH-1 résulte de l'assemblage des précurseurs Gag sur le feuillet interne de la membrane plasmique (MP) des cellules infectées. Les protéines Gag sont spécifiquement adressées à la MP grâce à des interactions entre le domaine MA et le PI(4,5)P2. Cette étude décrit le rôle des phosphadidylinosito1-4-phosphate 5-kinase de type 1 (PIP5K1alpha, beta et sigma) au cours des étapes tardives du VIH-1 dans le modèle celllulaire HeLa. Nous avons démontré que la PIP5K1alpha est l'enzyme principalement impliquée dans la production du PI(4,5P2. En suivant le trafic de Gag à la MP. Leur inhibition respective induit l'accumulation des précurseurs viraux dans les compartiments intracellulaires distincts et diminue la libération des pseudo-particules Gag dans les deux cas. L'ensemble de nos résultats démontre pour la première fois l'importance de l'activité des PIP5K1alpha et sigma dans l'assemblage et le bourgeonnement du VIH-1 et fournit de nouveaux éléments utiles à la compréhension des étapes tardives du cycle de multiplication viral
The production pocesses of HIV-1 particle of HIV-1 particles results from the assembly of Gag Precursors at the inner leaflet of the plasma membrane (PM) of infected cells. Gag proteins are specifically targeted to PM through interactions between MA domain and PI(4,5)P2. This study describes the role of phosphatidylinositol-4-phosphate 5-kinase type 1 (PIP5K1alpha, beta et sigma) in the late stages of HIV-1 in the context of HeLa cells. We determined that PIP5K1alpha is the principal producer of cellular PI(4,5)P2. By using a confocal microscopy approach, we followed the Gag proteins trafficking and showed that only alpha and y isoforms are required for the correct targeting of Gag to PM. Their respective inhibition leads to the accumulation of viral precursors at distinct intracellular comprtements, and decreases the release of Gag pseudoparticles in both cases. Altogether, our results highlight for the first time the crucial role PIP5K1alpha and sigma in the HIV-1 assembly and budding and provide new insights for a better understanding of the late stages of the virus replication cycle

La conductivité hydraulique racinaire (Lpr) traduit la capacité de transport d’eau de la racine. Lors de son trajet du sol vers le xylème, l’eau diffuse au sein de l’apoplasme ou au travers des cellules (voie de cellule-à-cellule). Au niveau de l’endoderme, la diffusion apoplasmique de l’eau est bloquée par le cadre de Caspari et des lamelles de subérine. La voie de cellule-à-cellule dépend principalement de l’activité des aquaporines régulées en partie par des interactions protéiques. Ce travail caractérise de nouveaux interactants de l’aquaporine racinaire PIP2;1 : le récepteur kinase RKL1 et 4 protéines de fonction inconnue appartenant à la sous-famille 1 des Casparian Strip membrane domain Protein Like (CASPL1) (CASPL1-B1/B2/D1/D2). RKL1 est exprimée dans l’endoderme, est capable d’interagir physiquement avec PIP2;1 et stimule in vitro le transport d’eau par l’aquaporine. Cependant, l’inactivation de RKL1 n’affecte pas la Lpr sans que cela ne puisse être expliqué par une redondance fonctionnelle avec son plus proche homologue, RLK902. Une étude bibliographique suggère que l’interaction entre RKL1 et PIP2;1 interviendrait dans une voie de signalisation en réponse à une attaque pathogène. Concernant les CASPL, D1 est exprimé dans tous les tissus, alors que B1, B2 et D2 semblent uniquement exprimés dans des territoires subérisés. Ce profil suggère une implication de B1, B2 et D2 dans une régulation des aquaporines et de la subérisation. Au niveau moléculaire, D2, malgré son interaction physique avec PIP2;1, ne module pas le transport d’eau par l’aquaporine. En revanche, B1 interagit préférentiellement avec PIP2;1 sous une forme phosphorylée et stimule le transport d’eau par l’aquaporine. Au niveau de la plante entière, l’inactivation d’un ou deux gènes CASPL n’affecte ni la Lpr., ni la subérisation. Par contre, l’inactivation de PIP2;1 et PIP2;2 révèle un effet inhibiteur de ces aquaporines sur la subérisation. Cette étude a permis de décrire de nouveaux mécanismes originaux de régulation des aquaporines. Elle pose également, la question de l’existence d’une relation entre les transports d’eau par la voie apoplasmique et par les aquaporines
The root hydraulic conductivity (Lpr) reflects the water transport capacity of the root. During its transfer from the soil to the xylem, water can diffuse in the apoplasm or through the cells (cell-to-cell pathway). At the endodermis, the apoplastic diffusion of water is blocked by the Casparian Strip and suberin lamellae. The cell-to-cell pathway mainly relies on aquaporin activity which can be regulated by protein interactions. This study aims at characterizing new interactants of the root aquaporin PIP2;1: the receptor kinase RKL1 and 4 proteins of unknown function belonging to the Casparian Strip membrane domain Protein Like 1 sub-family (CASPL1-B1/B2/D1/D2). RKL1 is expressed in the endodermis, can physically interact with PIP2;1 and stimulates its water transport function in vitro. However a loss-of-function of RKL1 does not affect the Lpr., independently of a putative functional redundancy with its closest homolog RLK902. Concerning CASPL, D1 is expressed in every tissue of the root whereas B1, B2 and D2 appear to be specifically expressed in suberized tissues. This suggests a putative role of these isoforms in aquaporin regulation and suberisation. At the molecular level, D2 does not modulate PIP2;1 water transport activity despite a physical interaction between the two partners. By contrast, B1 interacts with PIP2;1 preferentially in its phosphorylated form and enhances the water transport activity of the aquaporin. At the plant level, disrupting one or two CASPL genes neither impact the Lpr nor affect the suberisation. However, the loss of function of both PIP2;1 and PIP2;2 reveals a negative effect of these aquaporins on suberisation. In conclusion, this study, uncovered novel regulation mechanisms of aquaporins. It also raises the question of the existence of a putative relationship between water transport by the apoplastic pathway and by aquaporins

Class I phosphoinositide 3-kinases (PI3Ks) generate the essential lipid second messenger PtdIns(3,4,5)P3 which plays a key role in the regulation of numerous cellular processes including cell growth and survival, gene transcription, cytoskeletal organisation and glucose metabolism through its downstream effectors and in particular the serine/threonine protein kinase Akt. Therefore, the PI3K/Akt signalling plays a critical regulatory role in diverse cellular processes and its dysregulation is implicated in the pathogenesis of many human diseases including cancer and type 2 diabetes. Overexpression of phosphatidylinositol-5-phosphate 4-kinase β (PIP4Kβ), a lipid kinase which phosphorylates the poorly understood phosphoinositide phospholipid PtdIns5P to produce PtdIns(4,5)P2, has been demonstrated to lead to the attenuation of PtdIns(3,4,5)P3 levels and decreased Akt phosphorylation in insulin-stimulated cells. Conversely, mice lacking PIP4Kβ exhibit increased insulin-induced Akt phosphorylation, suggesting a potential role of PIP4Ks in the regulation of PI3K/Akt signalling. The aim of this project was to investigate the potential role of PIP4Kα, the most active isoform among PIP4Ks, in the regulation of PI3K/Akt signalling and whether its substrate, PtdIns5P, is involved in this regulation.While YM201636, an inhibitor of PIKfyve an enzyme believed to be involved in PtdIns5P production, markedly reduced Akt phosphorylation in PDGF-stimulated NIH/3T3 cells, contrary to expectations, overexpression of PIP4Kα in NIH/3T3 and HeLa S3 cells had no effect on PtdIns(3,4,5)P3 levels or Akt phosphorylation upon stimulation with PDGF and IGF-1 respectively. However, PtdIns(3,4,5)P3 levels were significantly decreased in insulin-stimulated L6 myotubes overexpressing PIP4Kα, indicating a possible cell type specific modulation of PI3K signalling by PIP4Kα. Interestingly, despite the decreased PtdIns(3,4,5)P3 levels, insulin-stimulated Akt phosphorylation remained unaffected in PIP4Kα expressing L6 myotubes. PIP4Kα overexpression also resulted in increased levels of PtdIns(3,4)P2. This suggests an increased 5-dephosphorylation of PtdIns(3,4,5)P3 through the action of one or more 5-phosphatases. Although the precise 5-phosphatase(s) are not known, the data indicate that this cannot be SHIP2 which has previously been implicated in the regulation of PtdIns(3,4,5)P3 levels in insulin signalling. Taken together, the data presented in this thesis indicate a role for PIP4Kα in PI3K signalling in a cell type specific manner. This might have important physiological implications.

La polarité cellulaire est un processus fondamental qui contrôle les spécificités fonctionnelle et physiologique de la plupart des cellules eucaryotes. Cette asymétrie intracellulaire repose sur l’existence de compartiments membranaires distincts, à la fois dans leur composition en protéines mais également en phosphatidyl-inositols (PIs). Ainsi, la mise en place et le maintien de la localisation asymétrique de modules multi-protéiques associés notamment aux protéines PAR sont essentiels pour l’élaboration des domaines de polarité cellulaire. Durant ma thèse, j’ai étudié les relations entre les protéines de polarité et les PIs dans le contrôle de la polarité cellulaire. Plus particulièrement, en utilisant la chambre ovarienne de Drosophile, j’ai cherché à caractériser la suite d’évènements qui en amont régule l’activité de la PIP5K, Skittles (SKTL), qui produit le PI(4,5)P2 et à caractériser les mécanismes moléculaires qui lient le PI(4,5)P2, SKTL et les protéines PAR dans le contrôle et le maintien de la polarité cellulaire. J’ai contribué à caractériser l’importance de PI(4,5)P2 majoritairement produit par SKTL, dans le maintien de la polarité apico-basale et lors de la morphogenèse des cellules folliculaires de la chambre ovarienne. Le PI(4,5)P2 assure la localisation apicale de PAR3 et le maintien des jonctions adhérentes, sans affecter la localisation de PAR1. Par une méthode de quantification précise, j’ai ensuite démontré dans l’ovocyte que SKTL et le PI(4,5)P2, probablement grâce au trafic vésiculaire, étaient requis pour à la fois l’accumulation à l’antérieur de PAR3 et son exclusion au postérieur qui se fait à partir du stade 9B. L’accumulation antérieure de PAR3 est également dépendante d’un transport Dynéine dépendant et de la kinase IKKε tandis que son exclusion postérieure dépendant des phosphorylations par PAR1. Enfin, j’ai également étudié les modifications post traductionnelles de SKTL et leur importance dans la polarité cellulaire. J’ai identifié la présence de palmitoylation et de phosphorylations dont certaines impliquent la kinase PAR1 et la phosphatase PP1. Ces phosphorylations pourraient avoir un lien avec le rôle de SKTL dans le trafic vésiculaire. Ces résultats permettent donc d’élucider certains mécanismes cellulaires qui contrôlent la mise en place et le maintien de la polarité des cellules en liant les PIs et les protéines PAR
Cell polarity is a fundamental process that controls cell’s functional and physiological specificities. This process relies on membranous compartments differently composed both on proteins and on phosphatidyl-inositols (PIs). Indeed, through their asymmetric localization, polarity proteins, such as the PAR proteins, are essentials to establish and maintain polarity of the cells. During my PhD, I studied the interplay between the polarity proteins and the PIs. Using the Drosophila egg chamber, as a model, I aimed to characterized the upstream events that regulate the PI(4,5)P2 producing kinase (PIP5K), Skittles (SKTL), activity and localization. I also studied the downstream molecular process that link the PI(4,5)P2, SKTL and the PAR proteins in cell polarity. I contributed to the characterization of the importance of PI(4,5)P2, mainly produced by SKTL in maintaining the apical-basal polarity and during the morphogenesis of the follicle cells. The PI(4,5)P2 is ensuring PAR3 and adherens junctions but not PAR1 proper localizations. Next, through a precise quantification method, I showed that SKTL and the PI(4,5)P2, probably via vesicular traffic, were also ensuring PAR3 proper localizations (anterior accumulation and stage 9B posterior exclusion) in the oocyte. PAR3 accumulation also relies on a Dynein mediated transport and the IKKε kinase while its posterior exclusion relies on PAR1 phosphorylation. Finally, I studied SKTL post translational modifications and their relevance on cell polarity. I identified palmitoylation and phosphorylations that are regulated by the kinase PAR1 and the phosphatase PP1. SKTL phosphorylations seem to be related to its role on the vesicular traffic. Altogether these results clarify some mechanisms involving both PIs and PAR proteins in cell polarity maintaining and establishment

Mycobacterium abscessus (M. abscessus) is a rapidly growing mycobacterium which is able to generate different problems in human infections, such as chronic lung diseases, pulmonary diseases and skin infection. M. abscessus is considered as the first emergent opportunistic pathogen and the number of the infections due to this pathogen increases each year. Since the natural multidrug resistance and the absence of specific treatment to fight it, M. abscessus has become a serious problem of the public health. In this context, an original approach of a phenotypic screen was performed and allowed to identify a new piperidinol-based molecule, PIPD1, which exhibits a potent activity against M. abscessus. The goal of this project was to synthesize and characterize some PIPD1-analogs in order to identify the pharmacophore of PIPD1 and develop a Structure-Activity Relationship (SAR) study.

Type I PIP kinases (phosphatidylinositol 4-phosphate 5-kinases, PIP5Ks) catalyse the majority of cellular synthesis of PI(4,5)P2. To date, three mammalian isoforms (r1, r2, r3) have been found. PIP5KIr is subject to complex C-terminal splice variation, enhancing its transcriptional diversity through evolution and producing at least 5 known spliceoforms in the mammals. This study addresses several important questions. (1) Several remarkable differences have been discovered between the neuronal splice variant PIP5KIr_i3 and its close variant, Ir_i2, whose peptide lacks a 26-AA insert near its C-terminus. This study attempts to map these behavioural differences onto motifs within the peptide insert. Furthermore, a site of point mutation is identified near the activation loop, which amplifies the above differences. (2) This study documents properties of the more recently discovered PIP5KIr_i3, about which relatively little is known, for example, the regulation of its subcellular localisation, kinase activity and post-translational modifications. By site-directed mutagenesis and examining more closely several crucial motifs, insight is gained into the putative relationship between the enzyme’s phosphorylation state, cellular localisation, lipid kinase activity and autophosphorylation. (3) The discovery of a new PIP5KIr splice variant, Ir_v6, is described. First discovered in rodents, PIP5KIr_i6 encompasses the 26-AA insert of Ir_i3, but lacks the common C-terminus of Ir_i2 and Ir_i3 which contains peptide motifs that have several roles in vivo. A polyclonal antibody against the C-terminus of Ir_i6 was also developed. Preliminary characterisation of Ir_i6 demonstrates a similar subcellular localisation, but a wider expression profile than its close relative, Ir_i3, suggesting potentially differential functions across tissues and at various developmental stages. (4) The existence of Ir_v3 and Ir_v6 is also confirmed in humans. In light of recent findings of other novel human spliceoforms, this is shown to be a case of intra-exonic splicing producing “alternative 5’ splice site” exons in the human. Overall, this thesis should help to better understand the regulation and physiological roles of PIP5KIr and, specifically, its different splice variants.

Dissertação de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepreneurship
Plants are exposed to a wide variety of biotic and abiotic stresses that disturb their development and productivity, such as extreme temperatures that can gravely affect the plant morphology, physiology and metabolism. On its turn, plants have developed responses to cope with heat stress, a process termed thermotolerance. Basal thermotolerance refers to the innate capacity of plants to survive when exposed to high temperatures while acquired thermotolerance involves a prior acclimation to a moderate level of heat. Plants’ perception of the heat stress involves multiple pathways and signalling molecules such as the phosphoinositides, which are derived from the structural membrane lipid phosphatidylinositol. Previous work on phospholipid signalling of heat stress have demonstrate a dramatic increase in PtdIns(4,5)P2, as a consequence of an increase in PIP5K activity, and by 32Pphospholipid labelling it was discovered to involve PIP5K7 and PIP5K8 (Mishkind et al., 2009; Munnik lab, unpublished). These kinases are associated to the PtdIns(4,5)P2 generation pathway, throughout the phosphorylation of the head group of another phosphoinositide, PtdInds4P. The principal aim of this work is understanding what are the role of these kinases in heat signalling. For that, T-DNA insertion knock-out mutants (single and double pip5k7 and pip5k8 mutants) have been generated to study the role of these genes in basal- and acquired-heat tolerance and evaluation of the gene expression analysis by Promoter-GUS assays. For evaluating seedlings survival to heat, 3 days-old seedlings were subjected to a heat shock of 44 °C for 36 min, for basal thermotolerance, or 45 °C for 2.5 h with a prior treatment of 37 °C for 1 h, for acquired thermotolerance. Root elongation and GUS-staining analysis were performed with 6 days-old seedlings subjected to 40 °C for 30 min. Seed germination was assessed after seeds being subjected to a heat shock of 50 °C for 2 hours. Phenotypic analysis has showed that pip5k8 loss-offunction seedlings exhibit a better recovery from heat shock, compared to wild-type ones, with greener leaves and a more developed root system. The better performance of the pip5k8 loss-of-function mutants in basal thermotolerance may indicate that it is not the PIP5K8 but the PIP5K7 that is involved in the tolerance to heat stress for basal thermotolerance. This was also corroborated by the histologic GUS-staining analysis of the pip5k7 promoter, being displayed an intensive blue staining in non-vascular cells of the cotyledons, which was not observed for untreated seedlings. For acquired thermotolerance, it was observable a phenotype for the pip5k7 loss-of-function seedlings, that showed higher survival from the heat shock than the wild-type. The higher viability of pip5k7-1 loss-of-function mutant suggest that the PIP5K8 is involved in acquired thermotolerance of young seedlings, promoting an improvement of seedlings survival. Looking at the germination patterns after subjecting the seeds to heat treatments (basal and acquired), the pip5k8 loss-of-function seeds showed a percentage of germination significantly inferior than the wild-type ones. This phenotype may indicate a role for PIP5K8 in heat tolerance during seed germination, either for basal and acquired thermotolerance. The approaches used in this work to investigate the PIP5K7 and PIP5K8 function have disclosed the role of these PIP5K isoforms in heat-stress responses (basal and acquired thermotolerance) during seed germination and early plant development. Further research on the expression of these kinases by Q-PCR will strengthen the current knowledge about their role in heat signalling.
As plantas estão constantemente sujeitas a uma diversidade de stresses que perturbam o seu desenvolvimento e produtividade, como é o caso de temperaturas extremas que podem afetar a morfologia, fisiologia e metabolismo das plantas. Estas, por sua vez, foram desenvolvendo respostas para lidar com o stresse pelo calor excessivo, num processo designado por termotolerância. A termotolerância basal refere-se à capacidade inata das plantas sobreviverem quando expostas a temperaturas elevadas, enquanto a termotolerância adquirida envolve uma aclimação prévia ao calor. A perceção das plantas ao stresse pelo calor envolve múltiplas vias e moléculas sinalizadoras, tais como os fosfoinositídeos que derivam do lípido estrutural de membrana fosfatidilinositol. Trabalhos anteriores na sinalização fosfolipídica do stresse pelo calor demonstraram um aumento substancial de PtdIns(4,5)P2 como consequência de um aumento da atividade da PIP5K e, através de ensaios com fosfolípidos marcados com 32P, foi descoberto que envolve a PIP5K7 e a PIP5K8 (Mishkind et al., 2009; Munnik lab, não publicado). Estas cinases estão associadas à via de formação de PtdIns(4,5)P2. O principal objetivo deste trabalho é perceber o papel destas cinases na sinalização do calor. Para isso foram gerados mutantes (T-DNA insertion knock-outs) mutados no gene pip5k7, no pip5k8 e em ambos, para estudar o seu papel na termotolerância basal e adquirida, bem como avaliar a sua expressão através de um ensaio de promotor-GUS. Para avaliar a sua sobrevivência ao calor, plântulas com 3 dias foram sujeitas a 44 °C por 36 min, para termotolerância basal, ou 45 °C por 2,5 h com tratamento prévio de 37 °C por 1 h, para termotolerância adquirida. A análise do crescimento da raiz e expressão do promotor-GUS foi realizada em plantas com 6 dias sujeitas a 40 °C por 30 min. A germinação das sementes foi avaliada após terem sido sujeitas a 50 °C por 2 h. A análise fenotípica mostrou que as plântulas mutadas no pip5k8 apresentaram uma melhor recuperação do choque térmico, comparativamente às selvagens, apresentando folhas mais clorofilinas e um sistema radicular mais desenvolvido. A melhor performance destas plântulas nos ensaios de termotolerância basal sugere que não é a PIP5K8 mas a PIP5K7 que está envolvida na tolerância basal ao stress pelo calor. Isto é corroborado pela análise histológica (GUSstaining) do promotor de pip5k7, onde foi exibido um intenso sinal nas células não vasculares dos cotilédones que não foi observado em plântulas não tratadas. Nos ensaios de termotolerância adquirida foi observado que as plântulas mutadas no pip5k7 apresentaram uma maior resistência ao choque térmico do que as selvagens. A maior viabilidade destas plântulas sugere que a PIP5K8 está envolvida na termotolerância adquirida, promovendo maior sobrevivência. Relativamente à germinação das sementes após choque térmico (avaliando a termotolerância basal e adquirida), verificou-se que sementes mutadas no pip5k8 apresentaram uma % germinação significativamente inferior à das não mutadas. Este fenótipo sugere um papel para a PIP5K8 na tolerância ao calor durante a germinação, quer para termotolerância basal, quer para adquirida. As estratégias utilizadas neste trabalho permitiram desvendar o papel das isoformas da PIP5K – PIP5K7 e PIP5K8 – nas respostas ao stresse pelo calor (termotolerância basal e adquirida) durante a germinação e desenvolvimento inicial da planta. Investigação adicional na expressão destas isoformas, nomeadamente através de ensaios Q-PCR, servirão para aprofundar o conhecimento atual sobre o seu papel na sinalização do stresse pelo calor.

碩士
亞洲大學
生物科技學系碩士班
98
Enhancer of zeste homolog 2 (EZH2), a polycomb group (PcG) protein, regulates stem cells renewal and differentiation. Phosphatidylinositol-4-phosphate 5-kinase, type I gamma (PIP5K1C) plays an important role in synthesis of phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2]. Phospholipase C (PLC) hydrolyzes PI(4,5)P2 into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), in which IP3 is an intracellular messenger to activate calcium signaling. Change in intracellular calcium concentration is required and critical in neuronal differentiation. However, whether EZH2 modulates intracellular calcium signaling and how it regulates neuronal differentiation of human mesenchymal stem cells (hMSCs) are still unclear. In the present study, we successfully induce hMSCs to differentiate into neuronal lineage by detecting the expression of neuronal markers. Knockdown of EZH2 dramatically increases the level of intracellular calcium by 14-fold, and also evokes the expression of PIP5K1C. EZH2 binds the promoter of PIP5K1C in hMSCs and then dissociates after neuronal differentiation. Knockdown of PIP5K1C significantly reduces intracellular calcium release in EZH2-silenced cells, and disrupts neuronal differentiation of hMSCs, indicating that EZH2 regulates intracellular calcium through PIP5K1C, and thus affects neuronal differentiation of hMSCs. Here, we provide the first evidence to show in proliferating hMSCs, EZH2 negatively regulates intracellular calcium. After induction of neuronal differentiation, EZH2 departs from the promoter of PIP5K1C, activates its expression, and evokes intracellular calcium signaling, leading to differentiate hMSCs into neuronal lineages.