By buffering cytosolic calcium, mitochondria can shape the magnitude and duration of intracellular calcium transients, which in turn govern key physiological events. Although controlled uptake of calcium into the matrix influences the rate of ATP production, excess calcium within the matrix triggers non-specific permeabilization of the mitochondrial inner membrane, resulting in cell death. Despite its importance in cellular physiology, the molecular identity of the mitochondrial calcium uniporter remained a mystery for nearly five decades. Recently, an approach inspired by comparative genomics was used to identify two proteins required for high-capacity mitochondrial calcium uptake. These include MICU1, an EF-hand protein that may function as a regulatory component by sensing calcium, and MCU, the channel-forming subunit of the uniporter. In this work, I explore two distinct areas within the growing field of molecular mitochondrial calcium biology. First, I discuss the identification of a new protein, MICU1-paralog EFHA1, and present data that implicates it in mitochondrial calcium uptake. Subsequently, I describe efforts to establish an in vitro system to characterize the channel activity of MCU, including my contribution to the development of a liposome-based assay for calcium transport and preliminary work aimed at reconstituting MCU transport activity in proteoliposomes.

La proteína ykbA de "Bacillus subtilis", purificada y reconstituida en proteoliposomas (PLs), presenta actividad de intercambio de L-serina, L-threonina y L-aminoácidos aromáticos. Por ello la renombramos como SteT (Serine/threonine exchanger Transporter). Los estudios cinéticos de la actividad de SteT son compatibles con un mecanismo secuencial de intercambio. SteT se agrupa filogenéticamente dentro de la familia LAT. Por todo ello, SteT es el primer homólogo procariota de la familia LAT que ha sido identificado y caracterizado. Proponemos SteT como modelo procariota para el estudio funcional y estructural de la familia LAT.Los estudios estructurales de SteT mediante electroforesis no desnaturalizante y TEM con tinción negativa y con criofractura en PLs indican que el intercambiador es funcional como monómero. SteT tiene apariencia de donut elíptico con diámetros exteriores de ~6 y ~7 nm. El residuo C291 en el segmento transmembrana (TM) VIII de SteT es la única diana de inactivación por MTSET y de activación por DTT. El sustrato L-serina protege de la inactivación por MTSET. Por tanto, el sustrato bloquea directamente o provoca un cambio conformacional que bloquea la accesibilidad de MTSET al residuo. El segmento TM VIII presenta dos caras funcionalmente diferenciables con periodicidad de hélice α. Una cara está formada por los residuos I285, I288, L292, K295 y F299 que mutados a cisteína muestran inhibición por MTSET y son insensibles a DTT. La otra cara, adyacente a la anterior, está formada por los residuos S287, G290, G294 y S298 que mutados a cisteína muestran activación por DTT y son insensibles a MTSET. Los residuos I284 y C291 poseen características de ambas ya que se inactivan por MTSET y se activan por DTT. DTT activa ~20 veces SteT cysless G294C en PLs, muy probablemente incrementado la Vmáx sin afectar la KM aparente. Esta activación es protegible por el sustrato L-serina con una EC50 similar a su KM aparente. Por tanto, el sustrato bloquea directamente o provoca un cambio conformacional que bloquea la accesibilidad de DTT al residuo. La mutación K295C, tanto en su entorno cysless como wt, genera un transportador ~50 y ~260 veces más rápido que SteT y SteT cysless respectivamente. La mutación K295G provoca un efecto muy inferior y la mutación K295L rinde un transportador inactivo. Estos datos sugieren que no hay una relación directa entre el tamaño de la cadena lateral en la posición 295 y la actividad de SteT, si no que la presencia de cisteína es la causante del aumento de actividad.Los residuos I285 y K295 parecen presentar interacción con la cadena lateral del sustrato ya que las mutaciones I285C y K295C producen un cambio dramático en el patrón de cis-inhibición. En estos mutantes la mayoría de los aminoácidos estudiados inhiben ampliamente el transporte de L-serina, con la excepción de L-prolina, L-hidroxiprolina, L-glutamato y glicina. En este sentido, SteT cysless K295C presenta un claro cambio de especificidad de sustrato ya que, a diferencia de SteT, transporta L-arginina. El hecho de que I285 y K295 disten ~16 Å indicaría la existencia de al menos dos lugares de unión independientes de la cadena lateral del sustrato, contradiciendo el modelo de "Alternating access". / ykbA, a "Bacillus subtilis" protein was purified and reconstituted in proteoliposomes (PLs) showing a L-serine, L-threonine and aromatic L-aminoacids exchange activity. We rename it as SteT (Serine/threonine exchanger Transporter). The kinetic studies of SteT activity are compatible with a sequential mechanism of exchange. SteT belongs philogenetically to LAT family, so SteT is the first prokaryotic member of LAT family that has been identified and characterized. The structural studies of SteT using native electrophoresis, negative staining and freeze fracture TEM in PLs show that it is functional as monomer. SteT looks like an elliptic donut with external diameters of ~6 and ~7 nm. The residue C291, located in the transmembrane segment (TM) VIII of SteT, is the unique target of inactivation by MTSET and of activaction by DTT. L-serine protects the MTSET inactivation. The TM segment VIII has two differentiable faces with α-helix periodicity. One is formed by I285, I288, L292, K295 and F299. These residues mutated to cysteine show MTSET inhibition and DTT insensitivity. The other, adjacent to the first, is formed by S287, G290, G294 and S298. These residues mutated to cysteine show DTT activation and MTSET insensitivity. The residues I284 and C291 have MTSET inhibition and DTT activation. DTT activates ~20 fold SteT cysless G294C in PLs. This activation is protected by L-serine with an EC50 similar to its apparent KM. The mutation K295C, in cysless and wt environments, causes a transporter ~50 and ~260 fold faster than SteT and SteT cysless respectively. The mutation K295G causes a lower effect and the mutation K295L yields and inactive transporter. These data suggest that the presence of cysteine is the cause of increased activity. The residues I285 and K295 seem to have interaction with the substrate's side chain because the I285C and K295C mutations cause a dramatic change in the cis-inhibition pattern. In this sense, SteT cysless K295C shows a clear change of substrate specificity because it transports L-arginine despite SteT. I285 and K295 are ~16 Å away, indicating the existente of at least two independent binding sites for the substrate side chain, contradicting the Alternating access model.

A mineralização do esqueleto começa dentro de vesículas matriz derivadas de células (MVs); então, os minerais se propagam para a matriz de colágeno extracelular. A fosfatase alcalina não específica de tecido (TNAP) degrada o pirofosfato inorgânico (PPi), um potente inibidor da mineralização, quee contribui com Pi (Fosfato) de ATP para iniciar a mineralização. Em comparação com a membrana plasmática, as MVs são ricas em Colesterol (Chol) (32%) e TNAP, mas como o Chol influencia a atividade da TNAP ainda não está claro. Nós reconstituímos TNAP em lipossomos de dipalmitoilfosfatidilcolina (DPPC), dimiristoilfosfocolina (DMPC) dioleoilfosfatidilcolina (DOPC) combinada com Chol ou seus derivados Colestenona (Achol) e Ergosterol (Ergo). Comparamos as propriedades cinéticas: Velocidade máxima de hidrólise (Vmax), constante de afinidade (k0,5), cooperatividade (n) e eficiência catalítica (kcat / k0,5) da TNAP para os substratos fisiológicos ATP e PPi, quando o TNAP é incorporada nestes diferentes microambientes lípidicos. O DPPC mais 36% de esteróis em lipossomos aumentaram a atividade catalítica do TNAP em relação ao ATP. A presença de Chol também aumentou a propagação de minerais em 3,4 vezes. A eficiência catalítica da TNAP em relação ao ATP foi quatro vezes menor nos proteolipossomos de DOPC, em comparação aos proteolipossomos do DPPC. Os proteolipossomos DOPC também aumentaram a biomineralização 2,8 vezes em comparação com os proteolipossomos de DPPC. O TNAP catalisou a hidrólise do ATP mais eficientemente no caso do proteolipossomo consistindo em DOPC com 36% de Chol. O mesmo comportamento surgiu com Achol e Ergo. A organização do lipídio e a estrutura do esterol influenciaram a tensão superficial (), a atividade fosfohidrolítica do TNAP na monocamada e a eficiência catalítica do TNAP nas bicamadas. Membranas na fase L (Achol) proporcionaram melhores parâmetros cinéticos em relação às membranas na fase Lo (Chol e Ergo). a presença de SM ou Chol: SM 90:10 (mol%), proteolipossomos DPPC não alterou os valores de eficiência catalítica, para a hidrólise do ATP. No entanto, estes proteolipossomos aumentaram a propagação mineral em cerca de 4,5 e 8 vezes, respectivamente, em comparação com DPPC puro. O aumento na eficiência catalítica para proteolipossomos contendo DMPC: SM 90:10 e DMPC: Chol: SM 80:10:10 (% molar) foi observado. Em conclusão, as propriedades físicas e a organização lateral de lipídios em proteolipossomas são cruciais para o controle. propagação mineral mediada pela atividade da TNAP durante a mineralização / Mineralization of the skeleton starts within cell-derived matrix vesicles (MVs); then, minerals propagate to the extracellular collagenous matrix. Tissuenonspecific alkaline phosphatase (TNAP) degrades inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and contributes Pi (Phosphate) from ATP to initiate mineralization. Compared to the plasma membrane, MVs are rich in Cholesterol (Chol) (32%) and TNAP, but how Chol influences TNAP activity remains unclear. We have reconstituted TNAP in liposomes of dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphocholine (DMPC) dioleoylphosphatidylcholine (DOPC) combined with Chol or its derivatives Cholestenone (Achol) and Ergosterol (Ergo). We compare the kinetic properties: maximum rate of hydrolysis (Vmax), affinity constant (k0,5), cooperativity (n) and catalytic efficiency (kcat / k0,5) of TNAP for the physiological substrates ATP and PPi, when TNAP is incorporated in these different microenvironments lipids. DPPC plus 36% sterols in liposome increased the catalytic activity of TNAP toward ATP. The presence of Chol also increased the propagation of minerals by 3.4-fold. The catalytic efficiency of TNAP toward ATP was fourfold lower in DOPC proteoliposomes as compared to DPPC proteoliposomes. DOPC proteoliposomes also increased biomineralization by 2.8-fold as compared to DPPC proteoliposomes. TNAP catalyzed the hydrolysis of ATP more efficiently in the case of the proteoliposome consisting of DOPC with 36% Chol. The same behavior emerged with Achol and Ergo. The organization of the lipid and the structure of the sterol influenced the surface tension (), the TNAP phosphohydrolytic activity in the monolayer, and the TNAP catalytic efficiency in the bilayers. Membranes in the L phase (Achol) provided better kinetic parameters as compared to membranes in the Lo phase (Chol and Ergo). The presence of SM or Chol:SM 90:10 (mol%), DPPC-proteoliposomes did not alter the catalytic efficiency values, for the ATP hydrolysis. However, these proteoliposomes increased the mineral propagation by about 4.5 and 8-fold, respectively, compared to neat DPPC. The increase in catalytic efficiency for proteoliposomes containing DMPC:SM 90:10 and DMPC:Chol:SM 80:10:10 (mol%) was observed. In conclusion, the physical properties and the lateral organization of lipids in proteoliposomes are crucial to control mineral propagation mediated by TNAP activity during mineralization

A mineralização do esqueleto começa dentro de vesículas matriz derivadas de células (MVs); então, os minerais se propagam para a matriz de colágeno extracelular. A fosfatase alcalina não específica de tecido (TNAP) degrada o pirofosfato inorgânico (PPi), um potente inibidor da mineralização, quee contribui com Pi (Fosfato) de ATP para iniciar a mineralização. Em comparação com a membrana plasmática, as MVs são ricas em Colesterol (Chol) (32%) e TNAP, mas como o Chol influencia a atividade da TNAP ainda não está claro. Nós reconstituímos TNAP em lipossomos de dipalmitoilfosfatidilcolina (DPPC), dimiristoilfosfocolina (DMPC) dioleoilfosfatidilcolina (DOPC) combinada com Chol ou seus derivados Colestenona (Achol) e Ergosterol (Ergo). Comparamos as propriedades cinéticas: Velocidade máxima de hidrólise (Vmax), constante de afinidade (k0,5), cooperatividade (n) e eficiência catalítica (kcat / k0,5) da TNAP para os substratos fisiológicos ATP e PPi, quando o TNAP é incorporada nestes diferentes microambientes lípidicos. O DPPC mais 36% de esteróis em lipossomos aumentaram a atividade catalítica do TNAP em relação ao ATP. A presença de Chol também aumentou a propagação de minerais em 3,4 vezes. A eficiência catalítica da TNAP em relação ao ATP foi quatro vezes menor nos proteolipossomos de DOPC, em comparação aos proteolipossomos do DPPC. Os proteolipossomos DOPC também aumentaram a biomineralização 2,8 vezes em comparação com os proteolipossomos de DPPC. O TNAP catalisou a hidrólise do ATP mais eficientemente no caso do proteolipossomo consistindo em DOPC com 36% de Chol. O mesmo comportamento surgiu com Achol e Ergo. A organização do lipídio e a estrutura do esterol influenciaram a tensão superficial (), a atividade fosfohidrolítica do TNAP na monocamada e a eficiência catalítica do TNAP nas bicamadas. Membranas na fase L (Achol) proporcionaram melhores parâmetros cinéticos em relação às membranas na fase Lo (Chol e Ergo). a presença de SM ou Chol: SM 90:10 (mol%), proteolipossomos DPPC não alterou os valores de eficiência catalítica, para a hidrólise do ATP. No entanto, estes proteolipossomos aumentaram a propagação mineral em cerca de 4,5 e 8 vezes, respectivamente, em comparação com DPPC puro. O aumento na eficiência catalítica para proteolipossomos contendo DMPC: SM 90:10 e DMPC: Chol: SM 80:10:10 (% molar) foi observado. Em conclusão, as propriedades físicas e a organização lateral de lipídios em proteolipossomas são cruciais para o controle. propagação mineral mediada pela atividade da TNAP durante a mineralização / Mineralization of the skeleton starts within cell-derived matrix vesicles (MVs); then, minerals propagate to the extracellular collagenous matrix. Tissuenonspecific alkaline phosphatase (TNAP) degrades inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and contributes Pi (Phosphate) from ATP to initiate mineralization. Compared to the plasma membrane, MVs are rich in Cholesterol (Chol) (32%) and TNAP, but how Chol influences TNAP activity remains unclear. We have reconstituted TNAP in liposomes of dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphocholine (DMPC) dioleoylphosphatidylcholine (DOPC) combined with Chol or its derivatives Cholestenone (Achol) and Ergosterol (Ergo). We compare the kinetic properties: maximum rate of hydrolysis (Vmax), affinity constant (k0,5), cooperativity (n) and catalytic efficiency (kcat / k0,5) of TNAP for the physiological substrates ATP and PPi, when TNAP is incorporated in these different microenvironments lipids. DPPC plus 36% sterols in liposome increased the catalytic activity of TNAP toward ATP. The presence of Chol also increased the propagation of minerals by 3.4-fold. The catalytic efficiency of TNAP toward ATP was fourfold lower in DOPC proteoliposomes as compared to DPPC proteoliposomes. DOPC proteoliposomes also increased biomineralization by 2.8-fold as compared to DPPC proteoliposomes. TNAP catalyzed the hydrolysis of ATP more efficiently in the case of the proteoliposome consisting of DOPC with 36% Chol. The same behavior emerged with Achol and Ergo. The organization of the lipid and the structure of the sterol influenced the surface tension (), the TNAP phosphohydrolytic activity in the monolayer, and the TNAP catalytic efficiency in the bilayers. Membranes in the L phase (Achol) provided better kinetic parameters as compared to membranes in the Lo phase (Chol and Ergo). The presence of SM or Chol:SM 90:10 (mol%), DPPC-proteoliposomes did not alter the catalytic efficiency values, for the ATP hydrolysis. However, these proteoliposomes increased the mineral propagation by about 4.5 and 8-fold, respectively, compared to neat DPPC. The increase in catalytic efficiency for proteoliposomes containing DMPC:SM 90:10 and DMPC:Chol:SM 80:10:10 (mol%) was observed. In conclusion, the physical properties and the lateral organization of lipids in proteoliposomes are crucial to control mineral propagation mediated by TNAP activity during mineralization

How organelles acquire their unique shapes is a fundamental question of cell biology. The peripheral endoplasmic reticulum (ER) consists of a vast network of membrane sheets and tubules, the formation of which requires homotypic membrane fusion. Previous studies suggest that the dynamin-like GTPase, atlastin (ATL), mediates ER fusion, but the mechanism by which this occurs is unclear. In this study, I investigate 1) the role of dimerization and conformational changes in the N-terminal domain of ATL, 2) how the C-terminal amphipathic helix and the transmembrane domain of ATL cooperate with the N-terminal domain, and 3) the formation of cis and trans ATL dimers in the fusion mechanism. ATL has a cytosolic N-terminal domain, consisting of a GTPase domain and three-helix bundle (3HB), followed by two transmembrane segments (TMs) and a cytosolic C-terminal tail (CT). Crystal structures of ATL and biochemical experiments suggest that nucleotide-dependent dimerization between ATL molecules sitting in different membranes can tether the membranes together. A subsequent conformational change triggered by GTP hydrolysis could pull the membranes toward one another for fusion. This mechanism is supported by in vitro membrane tethering and fusion assays using vesicles containing full-length Drosophila ATL. The CT and TMs of ATL are also required for efficient membrane fusion. A synthetic peptide corresponding to a conserved amphipathic helix in the CT can act in trans to restore the fusion activity of a tailless ATL mutant. We characterize CT mutants to show that the C-terminal helix promotes fusion by perturbing the lipid bilayer. The TMs of ATL also mediate nucleotide-independent oligomerization, which may allow ATL molecules in the same membrane to synchronously undergo the conformational change leading to fusion. Lastly, we show that continuous GTP hydrolysis is required for membrane tethering, occasionally resulting in fusion. The N-terminal cytosolic domain mediates trans dimer formation between ATL molecules on different membranes. GTP binding induces dimerization through the GTPase domains and 3HBs. We propose that GTP hydrolysis and phosphate release are required not just to drive fusion, but also to dissociate cis dimers that form on the same membrane, thus allowing ATL molecules to form trans dimers.

Sterine sind essentielle Komponenten der Zellmembran, deren Konzentration und Lokalisierung genau kontrolliert wird. Die Hefe Saccharomyces cerevisiae ist ein fakultativ anaerober Organismus, der in Abwesenheit von Sauerstoff auxotroph für Sterine wird. Die Proteine Aus1p und Pdr11p gehören zur Familie der ABC Proteine und spielen eine wichtige Rolle in diesem Prozess, da die gleichzeitige Deletion beider Protein die Aufnahme von Sterinen unter anaeroben Wachstumsbedingungen blockiert.In dieser Arbeit wurde das Gen AUS1 in voller Länge kloniert. Methoden für die Extraktion und Reinigung dieses Transporters wurden entwickelt, damit dieser detailliert charakterisiert werden kann. Mit Hilfe von Detergenzien wurde das Protein löslich gemacht und zeigte ATP-Bindung und -Hydrolyse. Die ATP-Hydrolyse konnte durch die Mutation eines konservierten Lysins zu Methionin im Walker A Motif verhindert. Genauso konnte die ATP-Hydrolyse auch durch klassische Inhibitoren von ABC Transportern inhibiert werden. Nach der Rekonstitution von Aus1p in Proteoliposomen wurde die ATPase Aktivität spezifisch durch Phosphatidylserin in einer stereoselektiven Weise stimuliert.Zusätzlich konnte gezeigt werden, dass Änderungen im zellulären PS Spiegel die Aus1p-abhängige Aufnahme von Sterin an die Membran beeinflussen. Diese Ergebnisse schlagen eine für die Aktivität des Transporters wichtige, direkte Interaktion zwischen Aus1p und PS vor.Da es sich bei der Aufnahme von Sterin um einen komplexen Prozess handelt, könnten Komponenten exisitieren, die mit Aus1p interagieren. Der Hefestamm, der die Immunpräzipitation von Aus1p mit seinem Interaktionspartner ermöglicht, wurde erzeugt und der Einfluß von Mannoproteinen auf Sterinaufnahme wurde getestet. Außerdem wurde eine Methode entwickelt, mit der Aus1p in Giant Unilamellar Vesicles rekonstituiert werden kann. Mit diesen Liposomen kann das Verhalten und die Aktivität von Aus1p in Membranen mit einer komplexen Lipidzusammensetzung untersucht werden. / Sterols are essential components of cellular membranes and their concentration and localization are tightly controlled. Saccharomyces cerevisiae is a facultative anaerobic organism which becomes auxotrophic for sterols in the absence of oxygen. However, the precise mechanism of sterol uptake remains to be revealed. Two proteins belonging to ABC protein family, Aus1p and Pdr11p were proposed to play a critical role in this process as simultaneous deletion of both of them blocks sterol uptake under anaerobiosis. In the present work, the full length AUS1 gene was cloned. An extraction and purification procedures were then developed to allow for detailed characterization of the transporter. The detergent solubilized protein was shown to bind and hydrolyse ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1p was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. Furthermore, it was demonstrated that Aus1p-dependent sterol uptake, but not Aus1p expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1p and PS which is critical for the activity of the transporter. Because of the complexity of sterol incorporation process efforts were made to identify additional components of the sterol uptake machinery that interact with Aus1p protein. The yeast strain allowing for immunopercipitation of Aus1p with its interaction partners was generated and previously proposed influence of mannoproteins on the sterol uptake was tested. Additionally, method was developed to reconstitute Aus1p protein into Giant Unilamellar Vesicles. These liposomes can be used further for testing of the behaviour and activity of Aus1p in the membranes with complex lipid composition.

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 protéines / This 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 proteins

No description available.

La Granulomatose Septique Chronique (CGD) est une maladie génétique rare de l’immunodéficience innée affectant les cellules phagocytaires (neutrophiles, macrophages). Elle est causée par des mutations dans les sous-unités du complexe NADPH oxydase formé du cytochrome b558 membranaire (NOX2 associé à p22phox) et de facteurs cytosoliques (p47phox, p67phox et p40phox). La déficience de ce complexe enzymatique va conduire à l’absence de formation de formes réactives de l’oxygène (FRO) microbicides et donc à l’apparition d’infections graves et récurrentes très tôt dans l’enfance. La chimioprophylaxie à vie permet de protéger ces patients mais peut être responsable d’effets indésirables. La seule thérapie curative est la transplantation de moelle osseuse mais tous les patients ne peuvent en bénéficier, et la thérapie génique n’est pas encore envisageable. Il y a donc un manque réel de nouvelles thérapies pour cette maladie. Cependant pour développer de nouveaux traitements, il faut disposer de modèles physiopathologiques pertinents. Or, les modèles existants sont imparfaits ou manquants. Le but de notre travail est donc de produire des modèles cellulaires et animaux de la CGD pour développer dans un second temps, une nouvelle approche thérapeutique basée sur l’utilisation de protéoliposomes.Grâce à leurs propriétés de pluripotence et d’auto-renouvellement à l’infini, les cellules souches pluripotentes induites (iPS) sont un outil puissant pour la modélisation physiopathologique. Ainsi, à partir de fibroblastes de patients atteints de CGD reprogrammés en cellules iPS, nous avons mis au point un protocole efficace de différenciation hématopoïétique in vitro en neutrophiles et macrophages. Nous avons montré que ces cellules phagocytaires sont matures et reproduisent parfaitement le phénotype déficient en FRO des patients CGD. Nous avons donc obtenu des modèles cellulaires pertinent modélisant trois formes génétiques de CGD, la CGD liée à l’X et deux formes autosomiques récessives, CGDAR22 et CGDAR47.Nous avons ensuite réalisé la preuve du concept de l’efficacité de protéoliposomes thérapeutiques sur les macrophages modélisés de la forme CGDX, la forme génétique la plus fréquente (70 % des cas) due à l’absence du cytochrome b558 membranaire (NOX2/p22phox). Grâce à une collaboration avec la start-up Synthelis SAS, des liposomes contenant le cytochrome b558 au niveau de la membrane lipidique ont été produits dans un système d’expression acellulaire basé sur l’utilisation d’extraits d’Escherichia coli. Ces liposomes NOX2/p22phox sont capables de reconstituer une enzyme NADPH oxydase fonctionnelle in vitro et de délivrer le cytochrome b558 à la membrane plasmique des macrophages CGDX qui présentent alors une restauration de l’activité NADPH oxydase avec la production de FRO.Enfin, nous nous sommes proposés de générer des souris dites « humanisées » par transplantation de cellules souches hématopoïétiques CD34+ capables de prise de greffe et de reconstitution hématopoïétique dans des souris immunodéficientes. A partir de cellules iPS saines, nous avons réussi à produire des cellules hématopoïétiques CD34+ possédant un potentiel hématopoïétique in vitro. Cependant, malgré des résultats encourageants, aucune prise de greffe in vivo n’a pu être réellement confirmée à ce jour.Pour conclure, nous avons donc montré au cours de ce projet, la production de modèles cellulaires de trois formes génétiques de CGD à partir de cellules iPS. Puis le modèle de macrophages CGDX nous a permis de faire la preuve de l’efficacité d’une nouvelle thérapie in vitro, une « enzymothérapie substitutive liposomale », qui pourrait à terme, offrir une alternative thérapeutique pour le traitement des infections aigües pulmonaires des patients CGD réfractaires aux traitements antibiotiques et antifongiques conventionnels. / Chronic Granulomatous Disease (CGD) is a rare inherited pathology of the innate immune system that affects the phagocytic cells (neutrophils, macrophages). This disease is caused by mutations in the subunits of the NADPH oxidase complex composed of the membrane cytochrome b558 (NOX2 associated with p22phox) and the cytosolic components (p47phox, p67phox et p40phox). Dysfunction in this enzymatic complex leads to the absence of microbicidal reactive oxygen species (ROS) and therefore to the development of recurrent and life-threatening infections in early childhood. Life-long prophylaxis is used to protect these patients but it may be responsible for side effects. Bone marrow transplantation is the only curative treatment but it can not be proposed to all the patients. In addition, gene therapy is not possible up to now. So there is a real lack of new therapies for this disease. However, to develop new therapeutic approaches, relevant physiopathological models must be available. Actually, existing models are imperfect or missing. Thus, the goal of our work is to produce cellular and animal models of CGD to develop a new proteoliposome-based therapy.Induced pluripotent stem cells (iPSCs) are a powerful tool for physiopathologic modeling due to their pluripotency and self-renewal properties. Using CGD patient-specific iPSCs regrogrammed from fibroblasts, we developped an efficient protocol for in vitro hematopoietic differentiation into neutrophils and macrophages. We showed that the phagocytic cells produced are mature and reproduce the ROS-deficient phenotype found in CGD patients. Thus, we obtained relevant cellular models for three genetic forms of CGD: X-linked CGD and the two autosomal recessive forms AR22CGD and AR47CGD.Then, we demonstrated the proof-of-concept of the efficacy of therapeutic proteoliposomes on X-CGD iPS-derived macrophages. Indeed, X-CGD is the main form of the disease (70% of cases) and is caused by the absence of the membrane cytochrome b558 (NOX2/p22phox). Thanks to a collaboration with the start-up Synthelis SAS, liposomes integrating the cytochrome b558 into lipid bilayers were produced in an E. coli-based cell-free protein expression system. These NOX2/p22phox liposomes were able to reconstitute a functional NADPH oxidase enzyme in vitro and to deliver the cytochrome b558 at the plasma membrane of X-CGD macrophages, leading to restore the NADPH oxidase activity with a ROS production.Finally, we proposed to generate « humanized » mice models with a human immune system after transplantation of CD34+ hematopoietic stem cells able to engraft and reconstitute long-term hematopoiesis in immunodeficient mice. Using healthy iPSCs, we successfuly produced CD34+ hematopoietic cells with in vitro hematopoietic potential. However, no in vivo engraftment was really confirmed yet.In conclusion, during this project, we produced cellular models of three genetic forms of CGD using patient-specific iPSCs. Then, X-CGD macrophages were used to demonstrate in vitro the efficacy of a new therapy. This « liposomal replacement enzymotherapy » could, in the future, represents a curative alternative against life-threatening lung infections refractory to conventional antibiotic and antifungal therapy.

Proteolytic processing of the amyloid precursor protein (APP) may lead to the formation of the Abeta peptide, the major constituent of amyloid plaques in Alzheimer`s disease. The full-length APP is a substrate for at least 2 different (alpha and beta) proteases ("secretases"). The beta-secretase, BACE, cleaves APP in the first step of processing leading to the formation of the neurotoxic Abeta. BACE competes for APP with alpha-secretase, which cleaves APP within its Abeta sequence, thus precluding Abeta formation. It is thus important to understand how is the access of the alpha- and beta-secretase to APP regulated and how are the individual activities of these secretases modulated. Both these regulatory mechanisms, access to substrate and direct activity modulation, can be determined by the lipid composition of the membrane. Integral membrane proteins (like APP and BACE), can be viewed as solutes in a two-dimensional liquid membrane, and as such their state, and biological activity, critically depend on the physico-chemical character (fluidity, curvature, surface charge distribution, lateral domain heterogeneity etc.) of the lipid bilayer. These collective membrane properties will influence the activity of embedded membrane proteins. In addition, activity regulation may involve a direct interaction with a specific lipid (cofactor or co-structure function). Interactions of membrane proteins are furthermore affected by lateral domain organization of the membrane. Previous results had suggested that the regulation of the activity of the alpha- and beta-secretases and of their access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have purified recombinant human full-length APP and BACE to homogeneity, and reconstituted them in large (~100nm, LUVs) and giant (10-150microm, GUVs) unilamellar vesicles. Using a soluble peptide substrate mimicking the beta-cleavage site of APP, we have examined the involvement of individual lipid species in modulating BACE activity in LUVs of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1.cerebrosides, 2.anionic glycerophospholipids, 3. cholesterol. Furthermore, we have co-reconstituted APP and BACE together in LUVs and demonstrated that BACE cleaves APP at the correct site, generating the beta-cleaved ectodomain identical to that from cells. We have developed an assay to quantitatively follow the beta-cleavage in proteoliposomes, and we have shown that the rate of cleavage in total brain lipid proteoliposomes is higher than in phosphatidylcholine vesicles. We have also studied partitioning of APP and BACE in GUVs between liquid ordered (lo) and liquid disordered (ld) phases. In this system, significant part of the BACE pool (about 20%) partitions into the lo phase, and its partitioning into lo phase can be further enhanced by cross-linking of membrane components. Only negligible fraction of APP can be found in the lo phase. We continue to study the behavior of co-reconstituted APP and BACE in GUVs The work presented in this thesis has yielded some interesting results and raised further questions. One of the important assignments of this project will in the next stage be the characterization of the impact of membrane domain organization on the beta-cleavage. Different domain arrangements that can be hypothesized in cell membranes can be modeled by varying the degree of phase fragmentation in proteoliposomes comprising reconstituted APP and BACE.