Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization

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Development of the TransMembraChip technology membrane biochips for reinsertion and screening of membrane protein agonists antagonists

Ces travaux de thèse concernent le développement d'une biopuce à membranes permettant de réincorporer de manière fonctionnelle une protéine transmembranaire de la famille des récepteurs couplés aux protéines G (RCPG), CXCR4, dans une bicouche lipidique attachée et espacée sur un substrat d'or par pilotis peptidiques (pep-tBLM), sous un format miniaturisé et parallélisé. Le peptide pilotis utilisé, P19-4H, possède une cystéine en position N-terminale pour son greffage covalent sur la surface d'or et quatre résidus Histidine en position C-terminale pour l'attachement par chélation, en présence de Nickel, de protéoliposomes réintégrant CXCR4. La synthèse de cette protéine s'effectue par expression acellulaire sous forme de protéoliposomes, dans une composition lipidique adaptée et en présence d'un lipide chélatant, le DOGS-NTA, à 2% de la quantité molaire totale des lipides. Le peptide AH, un peptide fusogène, est utilisé dans une dernière étape pour fusionner les protéoliposomes attachés. La caractérisation approfondie des protéoliposomes et l'optimisation des conditions expérimentales ont permis d'aboutir à l'attachement robuste des protéoliposomes avec une densité lipidique suffisante pour leur fusion par le peptide AH et la formation d'une pep-tBLM réintégrant CXCR4. Des études de recouvrement de fluorescence après photoblanchiment (FRAP) ont montré que la pep-tBLM réinsérant CXCR4 était fluide, homogène et continue, avec un coefficient de diffusion de 2.10-7 cm2/s. Des études d'interaction entre CXCR4 et un ligand antagoniste, le T22, ont révélé que la protéine s'insère dans la pep-tBLM de manière fonctionnelle et orientée. Le processus de formation de la pep-tBLM a été miniaturisé par microstructuration du support consistant à recouvrir la surface d'or de polystyrène puis à former des micropuits exposant la surface d'or en leur fond. Le peptide P19-4H a été déposé de manière contrôlée dans les micropuits à l'aide d'un robot de dépôt pour former des plots de pep-tBLM intégrant CXCR4. La fonctionnalité de CXCR4 réinsérée dans ces plots de membranes a été attestée par des études d'interaction avec son ligand T22. L'ensemble des étapes de formation, d'optimisation et de miniaturisation des pep-tBLM a été suivi, visualisé et caractérisé en temps réel et sans marquage par la technique d'imagerie par résonance plasmonique de surface (SPRi). La technologie « TransMembraChip » développée au cours de cette thèse représente une méthode de choix pour la réincorporation et l'étude fonctionnelle de protéines transmembranaires dans une composition lipidique adaptée. Les protéines transmembranaires, en particulier les RCPG, représentent des cibles thérapeutiques intéressantes. Ainsi, dans le cadre de la recherche de candidats médicaments pour le traitement de pathologies impliquant des protéines transmembranaires, cette nouvelle génération de biopuce à membranes constitue un outil prometteur adapté au criblage de ligands agonistes ou antagonistes de ces protéinesThis thesis presents the development of a membrane biochip allowing to functionally reincorporate a transmembrane protein of the G-protein coupled receptor (GPCR) family, CXCR4, in a peptide-tethered bilayer lipid membrane (pep-tBLM), in a miniaturized and parallelized format. The peptide tether used, P19-4H, possesses a cysteine in its N-terminal extremity for covalent grafting onto the gold surface and four Histidine residues in its C-terminal extremity for attachment of proteoliposomes integrating CXCR4 by metal-chelate interaction in the presence of nickel. The synthesis of CXCR4 was carried out by cell-free expression in the form of proteoliposomes, in a suitable lipid composition and in the presence of a chelating lipid, DOGS-NTA, at 2% molar ratio. The AH peptide, a fusogenic peptide, was employed in a last step to fuse the attached proteoliposomes. The thorough characterization of proteoliposomes and the optimization of experimental conditions led to the robust attachment of proteoliposomes with sufficient lipid density to perform their fusion by the AH peptide and the formation of a pep-tBLM integrating CXCR4. Fluorescence recovery after photobleaching (FRAP) studies have shown that the pep-tBLM reinserting CXCR4 was fluid, homogeneous and continuous, with a diffusion coefficient of 2 x 10-7 cm2/s. Ligand binding studies between CXCR4 and T22, an antagonist, revealed that the protein was functional and well-oriented in the peptBLM. The formation process of the pep-tBLM was miniaturized by support microstructuration, consisting in covering the gold surface with polystyrene and then, forming microwells exposing the gold surface at their bottom. The P19-4H peptide was spotted in a controlled manner into the microwells to form microspots of pep-tBLM incorporating CXCR4. The functionality of CXCR4 reinserted into these membrane microspots was confirmed by T22 ligand binding studies. All the steps of formation, optimization and miniaturization of the pep-tBLM were monitored, visualized and characterized by surface plasmon resonance imaging (SPRi), a real time and label-free technique for the detection of interactions. The "TransMembraChip" technology developed in this work represents a method of choice for the reincorporation and functional study of transmembrane proteins in a suitable lipid composition. Transmembrane proteins, particularly GPCRs, form interesting therapeutic targets. Thus, in the context of pharmaceutical research of drug candidates for the treatment of pathologies involving transmembrane proteins, this new generation of membrane biochip is a promising tool for screening agonist or antagonist ligands of these protein

A new functional membrane protein microarray based on tethered phospholipid bilayers

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New Tethered Phospholipid Bilayers Integrating Functional G‑Protein-Coupled Receptor Membrane Proteins

Membrane proteins exhibiting extra- and intracellular domains require an adequate near-native lipid platform for their functional reconstitution. With this aim, we developed a new technology enabling the formation of a peptide-tethered bilayer lipid membrane (pep-tBLM), a lipid bilayer grafted onto peptide spacers, by way of a metal–chelate interaction. To this end, we designed an original peptide spacer derived from the natural α-laminin thiopeptide (P19) possessing a cysteine residue in the N-terminal extremity for grafting onto gold and a C-terminal extremity modified by four histidine residues (P19-4H). In the presence of nickel, the use of this anchor allowed us to bind liposomes of variable compositions containing a 2% molar ratio of a chelating lipid, 1,2-dioleoyl-<i>sn</i>-glycero-3-[(<i>N</i>-(5-amino-1-carboxypentyl)­iminodiacetic acid)­succinyl] so-called DOGS-NTA, and to form the planar bilayer by triggering liposome fusion by an α-helical (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A protein. The formation of pep-tBLMs was characterized by surface plasmon resonance imaging (SPRi), and their continuity, fluidity, and homogeneity were demonstrated by fluorescence recovery after photobleaching (FRAP), with a diffusion coefficient of 2.5 × 10^–7 cm²/s, and atomic force microscopy (AFM). By using variable lipid compositions including phos­pha­tid­yl­choline (PC), phos­pha­tid­yl­serine (PS), phos­pha­tid­yl­ethanol­amine (PE), phos­pha­tid­yl­inositol 4,5-bisphosphate (PIP₂), sphin­go­myelin (SM), phos­pha­tidic acid (PA), and cholesterol (Chol) in various ratios, we show that the membrane can be formed independently from the lipid composition. We made the most of this advantage to reincorporate a transmembrane protein in an adapted complex lipid composition to ensure its functional reinsertion. For this purpose, a cell-free expression system was used to produce proteo­lipo­somes expressing the functional C-X-C motif chemokine receptor 4 (CXCR4), a seven-transmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs). We succeeded in reinserting CXCR4 in pep-tBLMs formed on P19-4H by the fusion of tethered proteo­lipo­somes. AFM and FRAP characterization allowed us to show that pep-tBLMs inserting CXCR4 remained fluid, homogeneous, and continuous. The value of the diffusion coefficient determined in the presence of reinserted CXCR4 was 2 × 10^–7 cm²/s. Ligand binding assays using a synthetic CXCR4 antagonist, T22 ([Tyr5,12, Lys7]-poly­phe­musin II), revealed that CXCR4 can be reinserted in pep-tBLMs with functional folding and orientation. This new approach represents a method of choice for investigating membrane protein reincorporation and a promising way of creating a new generation of membrane biochips adapted for screening agonists or antagonists of transmembrane proteins

Further Evidence of Mutational Heterogeneity of the XPC Gene in Tunisian Families: A Spectrum of Private and Ethnic Specific Mutations.

International audienceXeroderma Pigmentosum (XP) is a rare recessive autosomal cancer prone disease, characterized by UV hypersensitivity and early appearance of cutaneous and ocular malignancies. We investigated four unrelated patients suspected to be XP-C. To confirm linkage to XPC gene, genotyping and direct sequencing of XPC gene were performed. Pathogenic effect of novel mutations was confirmed by reverse Transciptase PCR. Mutation screening revealed the presence of two novel mutations g.18246G>A and g.18810G>T in the XPC gene (NG_011763.1). The first is present in one patient XP50NEF, but the second is present in three unrelated patients (XP16KEB, XP28SFA, and XP45GB). These 3 patients are from three different cities of Southern Tunisia and bear the same haplotype, suggesting a founder effect. Reverse Transciptase PCR revealed the absence of the XPC mRNA. In Tunisia, as observed in an other severe genodermatosis, the mutational spectrum of XP-C group seems to be homogeneous with some clusters of heterogeneity that should be taken into account to improve molecular diagnosis of this disease