48 research outputs found

    Modélisation moléculaire de complexes Tubuline-Ligand

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    Les microtubules sont des polymÚres cylindriques de tubuline-ab, membres du cytosquelette eucaryote. Ils possÚdent une dynamique intrinsÚque nécessaire à de nombreuses fonctions cellulaires telle que la mitose. L hydrolyse du nucléotide GTP dans les polymÚres de tubuline-ab ainsi que les interactions entre la tubuline et les protéines partenaires ou les molécules à visées pharmacologiques, jouent un rÎle critique sur la dynamique des microtubules. Durant cette thÚse, des approches de modélisation moléculaire ont été utilisées pour mieux appréhender les interactions tubuline-ligand à l échelle atomique et contribuer au développement de nouvelles molécules actives. Des simulations de dynamiques moléculaires ont été réalisées pour étudier l effet de différents nucléotides dans la tubuline-b sur la structure et la dynamique du protofilament de tubuline. Nous proposons un rÎle du résidu aE254 dans la coordination du magnésium catalytique. Nous observons également des changements conformationnels aux interfaces latérales et un réarrangement de structure aux interfaces longitudinales qui peuvent affecter la stabilisation du microtubule. Des travaux menés au laboratoire ont montré que la colchicine et le carbendazime se fixent dans des poches voisines dans la sous-unité tubuline-b et inhibent la prolifération cellulaire. Nous avons proposé un site de fixation du carbendazime dans les complexes tubuline-colchicine à l aide de l amarrage moléculaire et de simulations de dynamiques moléculaires. Ces expériences ont mené au design de molécules hybrides composées des noyaux colchicine et carbendazime reliés par un linker. Une de ces molécules hybrides a été synthétisée et testée avec succÚs sur des lignées de cellules HeLa. Enfin, nous avons construit des peptides cycliques dérivées d I19L, un peptide anti-microtubule identifié au laboratoire. Des simulations de dynamique moléculaire et des calculs d énergie libre de liaisons ont permis d évaluer ces peptides. Enfin, des mutations ont été proposées afin d optimiser l interaction entre le meilleur peptide et la tubuline.Microtubules are cylindrical polymers of ab-tubulin heterodimers, members of the eukaryotic cytoskeleton. They possess an intrinsic dynamics which is necessary to any cellular functions such as the mitosis. It has long been recognized that GTP hydrolysis in ab-tubulin polymers plays a critical role in this dynamics as well as the interactions between tubulin and the protein partners or the drugs. In this thesis, molecular modeling approaches are applied to three theoretical studies to gain insight at the atomic scale about tubulin-ligand interactions and to contribute to the development of new active compounds. Molecular dynamics simulations were used to study the effect of the different nucleotide states at b-tubulin on the protofilament structure and dynamics. We propose a role for residue aE254 in catalytic magnesium coordination. We also observe conformational changes and structure rearrangement at lateral and longitudinal interfaces that can affect the microtubule stabilization. Previous work carried out in the laboratory showed that colchicine and carbendazime bind neighboring pockets in the b-tubulin subunit and inhibit cell proliferation. We proposed a binding site of carbendazime on the tubulin-colchicine complex, using docking and molecular dynamics simulation, which lead to the design of hybrid molecules composed of both colchicines and carbendazime moieties attached with a linker. One of these hybrid molecules has been synthesized and successfully tested on HeLa cells. Finally, we designed four cyclic peptides based on I19L, an anti-microtubule peptide identified at the laboratory. Molecular dynamic simulations and binding free energy calculations were used to evaluate these peptides. Mutations were then proposed on the best peptide to increase its interactions with tubulin.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

    Molecular organization of the human serotonin transporter at the air/water interface

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    AbstractThe serotonin transporter (SERT) is the target of several important antidepressant and psychostimulant drugs. It has been shown that under defined conditions, the transporter spread at the air/water interface was able to bind its specific ligands. In this paper, the interfacial organization of the protein has been assessed from dynamic surface pressure and ellipsometric measurements. For areas comprising between 10 400 and 7100 Å2/molecule, ellipsometric measurements reveal an important change in the thickness of the SERT film. This change was attributed to the reorientation of the transporter molecules from a horizontal to their natural predictive transmembrane orientation. The thickness of the SERT film at 7100 Å2/molecule was found to be approximately equal to 84 Å and coincided well with the theoretical value estimated from the calculations based on the dimensions of α-helices containing membrane proteins. These data suggest that the three-dimensional arrangement of the SERT may be represented as a box with lengths dz=83–85 Å and dy or dx=41–47 Å

    MOL#8268 1 Title Page. The natural mutation encoding a C-terminus truncated 5-HT 2B receptor is a gain of proliferative functions

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    Number of tables, 1 Number of references, 31 Number of words in the abstract, 230 Number of words in the Introduction, 744 Number of words in the Discussion. 1172 List of non-standard abbreviations receptor was identified in one patient diagnosed with pulmonary hypertension after intake of the anorexigen dexfenfluramine. Although reported to generate a lack of function, this C-terminus truncated 5-HT 2B receptor should somehow affect transduction pathways relevant to pulmonary hypertension. In our study, we investigated putative modifications in transduction of the R393X mutated 5-HT 2B receptor. In stably transfected cells, we confirmed the loss of IP 3 stimulation due to the G αq uncoupling, despite conserved ligand affinity between the normal and mutated receptors. We also observed a partial loss of nitric oxide synthase stimulation. However, the truncated R393X receptor presented (i) a strong gain of efficacy in cell proliferation as assessed by mitogen-activated protein kinase activity and thymidine incorporation, (ii) a preferential coupling to G α13 as shown by blocking antiserum, and (iii) an apparent lack of internalization upon agonist stimulation as observed by confocal microscopy. This work demonstrates that, in the 5-HT 2B receptor, the C-terminus including the palmitoylation and phosphorylation sites is absolutely required for proper transduction and internalization. For the first time, we show that the lack of C-terminus can generate a switch of coupling to G α13 , a reduced NO synthase activation and an increase in cell proliferation. All these modifications are relevant in pathophysiological vasoconstriction. MOL#8268 4 Introduction

    Hippocampal radial glial subtypes and their neurogenic potential in human fetuses and healthy and Alzheimer's disease adults

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    Neuropathological conditions might affect adult granulogenesis in the adult human dentate gyrus. However, radial glial cells (RGCs) have not been well characterized during human development and aging. We have previously described progenitor and neuronal layer establishment in the hippocampal pyramidal layer and dentate gyrus from embryonic life until mid-gestation. Here, we describe RGC subtypes in the hippocampus from 13 gestational weeks (GW) to mid-gestation and characterize their evolution and the dynamics of neurogenesis from mid-gestation to adulthood in normal and Alzheimer's disease (AD) subjects. In the pyramidal ventricular zone (VZ), RGC density declined with neurogenesis from mid-gestation until the perinatal period. In the dentate area, morphologic and antigenic differences among RGCs were observed from early ages of development to adulthood. Density and proliferative capacity of dentate RGCs as well as neurogenesis were strongly reduced during childhood until 5 years, few DCX+ cells are seen in adults. The dentate gyrus of both control and AD individuals showed Nestin+ and/or GFAPÎŽ+ cells displaying different morphologies. In conclusion, pools of morphologically, antigenically, and topographically diverse neural progenitor cells are present in the human hippocampus from early developmental stages until adulthood, including in AD patients, while their neurogenic potential seems negligible in the adult. Key words: adult neurogenesis, hippocampus, human fetal brain, neurogenesis, radial glial cell

    Elucidation of the ATP7B N-Domain Mg2+-ATP Coordination Site and Its Allosteric Regulation

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    The diagnostic of orphan genetic disease is often a puzzling task as less attention is paid to the elucidation of the pathophysiology of these rare disorders at the molecular level. We present here a multidisciplinary approach using molecular modeling tools and surface plasmonic resonance to study the function of the ATP7B protein, which is impaired in the Wilson disease. Experimentally validated in silico models allow the elucidation in the Nucleotide binding domain (N-domain) of the Mg2+-ATP coordination site and answer to the controversial role of the Mg2+ ion in the nucleotide binding process. The analysis of protein motions revealed a substantial effect on a long flexible loop branched to the N-domain protein core. We demonstrated the capacity of the loop to disrupt the interaction between Mg2+-ATP complex and the N-domain and propose a role for this loop in the allosteric regulation of the nucleotide binding process

    Contribution de la modélisation moléculaire à l'étude de pathologies humaines (Application au transporteur ATP7B et au récepteur 5HT2B)

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    Pas de résumé en françaisPas de résumé en anglaisPARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

    Porous yet dense matrices: using ice to shape collagen 3D cell culture systems with increased physiological relevance

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    International audiencePorous yet dense collagen matrices obtained by ice templating allow for long-term 3D cell culture with enhanced physiological relevance

    Characterizing single sinonasal squamous cell carcinoma using dielectrophoresis and electrorotation

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    International audienceWe show the capture and analysis single cells, in particular human sinonasal squamous cell carcinomas (SCC), by the combination of di-electrophoresis (DEP) force and electrorotation, within a microfluidic device. A set of 4 planar polynomial electrodes was employed to perform nDEP trapping of two lines tumor cell with different invasivity, named NC5 and NC7. Once captured at the center of the electrodes set, electrorotation was served to extract the rotational speed's spectra. From the spectra, their electrophysiological properties can be estimated
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