A Na, K-ATPase (NKA) é uma proteína de membrana encontrada em organismos eucariotos multicelulares cuja atividade e funções já são amplamente discutidas na literatura. Sua unidade funcional corresponde a um heterodímero formado por duas subunidades , com regiões transmembrana. Espécies multiméricas como dímeros e tetrâmeros dessa enzima também são conhecidos por exercer atividade enzimática. As interações lipídio-proteína são intrínsecas para a NKA, por tal motivo, proteolipossomos constituídos de DPPC e DPPC:DPPE foram preparados por co-solubilização. Como controle, lipossomos de mesma composição foram produzidos por extrusão e/ou sonicação. Para as imagens de AFM, as amostras foram fixadas com glutaraldeído, para proteção mecânica e contra desidratação das vesículas. Para lipossomos de DPPC as imagens topográficas de AFM das vesículas apresentaram formato oval, superfície perfeitamente lisa e diâmetro médio de 151 + 46 nm, enquanto as vesículas de composição DPPC:DPPE, apesar de lisas, tiveram cantos pontiagudos e diâmetro médio de 98 + 28 nm. Imagens de fase de ambas as composições não apresentaram qualquer indicativo de diferenças na composição química, provavelmente devido à natureza de carga neutra dos dois fosfolipídios. As imagens de fase por AFM para os proteolipossomos tanto de DPPC-NKA, quanto DPPC:DPPE-NKA, revelaram resultados inéditos na literatura, onde a inserção da NKA aparece como nítidas regiões transições de fase de composição química distinta quando comparadas com os lipossomos. No entanto, as mudanças de fase são diferentes entre as composições estudadas, aparecendo como manchas escuras circulares para DPPC-NKA e mais visíveis como interstícios brilhantes para composição de DPPC:DPPE-NKA. As vesículas de DPPC-NKA apresentaram diâmetro médio de 390 + 326 nm e, nas imagens de topografia tridimensionais, protusões de 38 a 115 nm correspondentes às regiões de mudanças de fase, que, indicaram o diâmetro dos microdomínios relacionados à proteína. Já nas imagens para DPPC:DPPE-NKA o diâmetro médio dos proteolipossomos foi de 189 + 156 nm, e as protusões apareceram entre os interstícios, variando de 20 a 66 nm. O estudo de DSC dos lipossomos revelou que a concentração de glutaraldeído nas condições das análises de AFM, em torno de 5% (v/v), afetam as características físico-químicas para as composições com DPPE. A AFM foi eficiente para confirmar a reinserção da NKA em proteolipossomos pelas imagens de fase, e, para medir o diâmetro dos microdomínios pelas imagens de topografia. / Na, K-ATPase (NKA) is a membrane protein present in eukaryotic multicellular organisms. Its functions and activity are already widely described in the literature. Its minimal functional structure is a heterodimer of two main subunits , with transmembrane domains. However, dimers and tetramers of the enzyme are also known to have enzymatic activity. Since there are intrinsic lipid-protein interactions, NKA proteoliposomes composed of DPPC and DPPC:DPPE (1:1 molar ratio) were prepared by the co-solubilization method and liposomes of the same compositions were obtained by extrusion and/or sonication to be used as control. The samples to the AFM study were prepared using glutaraldehyde to protect the vesicles from mechanical shocks and dehydration. Liposomes composed of DPPC and DPPC:DPPE (1:1 molar ratio) were prepared by extrusion and sonication, respectively, as control. The topographical images for DPPC liposomes showed vesicles with an oval shape and smoothed surfaces with a mean diameter of 151 + 46 nm. DPPC:DPPE vesicles also presented smoothed surfaces, but with pointed corners and mean diameter of 98 + 28 nm. Phase images for both lipid compositions showed no differences in chemical composition. For DPPC:DPPE samples, this can be explained by the neutral net charge of both lipids. The proteoliposomes observed in the AFM phase images showed darker and large circular spots in the vesicles. These spots represent delays in the phase oscillation of the AFM probe and are associated with different chemical composition. The phase changes showed the reconstitution of the NKA in the proteoliposomes. When compared with topographical images, this spots matched protrusions. The mean diameter of DPPC-NKA proteoliposomes determined by AFM was 390 + 326 nm. In the three-dimensional topographical images of composition, protrusions from 38 to 115 nm near the areas of different phases indicate the diameters of the NKA microdomains. The phase changes for DPPC:DPPE-NKA appeared as bright interstices with the protrusions of the topographical images in between them. The size of these protrusions ranged from 20 to 66 nm and the mean diameter of the proteoliposomes was 189 + 156 nm. The DSC liposomes data showed that the glutaraldehyde concentration used in the AFM analysis affect the physical chemistry properties of the samples with DPPE. AFM proved to be an efficient method to confirm the reconstitution of into proteoliposomes with phase images and to determine the diameter of the protein microdomains with the topographical images.

A novel truncate of Influenza A M2 protein (residues 22-62), incorporated into a uniquely tailored proteoliposome proton uptake assay, demonstrated proton flux more characteristic of an ion transporter than a traditional ion "channel." The liposome paradigm was essential for testing the conductance activity of this M2 truncate at a range of extraphysiological pHs appropriate for channel vs. transport function determination. In addition to transporter-typical proton flux, M2(22-62) showed the key characteristics of functional integrity: selective proton uptake into liposomes and block of uptake by amantadine. Two sets of proteoliposome proton flux assays were carried out, Set 1 at pH values of 6.5, 6.0. 5.5, 5.0, and 4.5; Set 2 at pH values of 6.25, 6.0, 5.75, 5.5, 5.25, 5.0, and 4.75. Observed flux rates followed a proton transport saturation curve similar to that observed in mouse erythroleukemia cells1. Proton transport was maximal at pH 5.5 in Set 1 (139 H+/second/tetramer) and at pH 5.75 in Set 2 (43 H+/second/tetramer). Amantadine block was strongest at pH 5.5 in Set 1 and 6.25 in Set 2, and apparent desensitization of the protein severely reduced proton flux and amantadine sensitivity below pH 5.5 in both sets of experiments. Decreased external pH increased proton uptake with an apparent pKa of 6 (Set 1) or 6.5 (Set 2). These data indicate acid activation of M2(22-62) between pH 5.5-6, optimal amantadine block between pH 5.5-6.25, and a loss of peptide functionality between pH 5.9-4.7.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis, a Gram-negative organism belonging to the order Rickettsiales, is responsible for an emerging infectious disease in humans, the human monocytic ehrlichiosis. E. chaffeensis also infects several other vertebrate hosts including dogs, goats, coyotes and white tailed deers. This organism is transmitted by an infected tick, Amblyomma americanum. The exact pathogenic mechanisms involved for the persistence of the pathogen in vertebrate hosts are still unclear. E. chaffeensis protein expression varies significantly in vertebrate and tick hosts. Differentially expressed proteins include the immunodominant outer membrane proteins encoded by the p28-Omp multigene locus. The p28-Omp 14 is expressed primarily in tick cells and the p28-Omp 19 is the major expressed protein in macrophages both under in vitro and in vivo conditions. The objective of this study is to prepare recombinant proteins and use them to assess the secondary structures and protein functions. The protein sequences were analyzed with the aid of bioinformatics programs to make structural predictions. The analysis suggested the presence of eight β barrel structures for both the p28-Omp proteins. The coding sequence of the p28-Omp genes were cloned and over expressions of proteins in in E. coli was accomplished by using the plasmid expression construct, pET28. The proteins were purified to near homogeneity and used to refold using detergents to mimic native protein structure in the bacterial outer membrane. Refolding of proteins was analyzed by two methods; SDS-PAGE and Circular Dichroism. The Circular dichroism spectroscopy analysis suggested the formation of β-sheet structures of proteins in micelles formed with the detergents. β-sheet structures may have been formed with the hydrophobic domains of the protein imbedded in the micelles. The hydrophilic segments (predicted by bio informatics analysis) may be exposed to the aqueous phase. The recombinant proteins were also iii used to prepare proteoliposomes and tested for the porin activity. The analysis demonstrated the porin activity for both p28-Omp 14 and 19 recombinant proteins by using mono-, di- and tetra- saccharides as well as for amino acid L-glutamine. This study forms the basis for initiating studies to compare the structural difference between the two differentially expressed proteins of E. chaffeensis.

A biomineralização óssea é um processo complexo e multifatorial sendo um grande desafio para a ciência à compreensão dos seus mecanismos regulatórios. Este processo é mediado pela liberação de vesículas da matriz (MVs), as quais surgem das superfícies de osteoblastos e são secretadas no local específico do início da biomineralização. MVs têm a capacidade de acumular altas concentrações de íons Ca2+ e fosfato (Pi), proporcionando um microambiente adequado para a formação inicial e propagação dos cristais de hidroxiapatita. Especial atenção deve ser dada a duas proteínas: Anexina V (AnxA5) e Fosfatase Alcalina (TNAP). As anexinas são as proteínas mais abundantes detectadas nas MVs e responsáveis pela formação de canais de cálcio. TNAP apresenta atividade fosfomonohidrolítica, produzindo Pi a partir, principalmente, de pirofosfato (PPi) e ATP. O enfoque deste projeto foi produzir e caracterizar proteolipossomos com diferentes composições lipídicas de dipalmitoil fosfatidilcolina (DPPC) e dipalmitoil fosfatidilserina (DPPS) contendo TNAP e AnxA5, e manter a funcionalidade das proteínas após incorporação nos sistemas miméticos. Foi possível incorporar AnxA5 em DPPC-proteolipossomos (11,64 µg/mL), mas na presença de DPPS houve um aumento significativo de AnxA5 incorporada (25,79 µg/mL) a DPPC:DPPS 10%-proteolipossomos (razão molar). A presença das proteínas nos proteolipossomos compostos por DPPC e DPPC:DPPS 5, 10 e 15% (razão molar) foi confirmada por SDS-PAGE e Immunoblotting. Melhores rendimentos de incorporação das duas proteínas foram obtidos quando ambas foram incorporadas concomitantemente. DPPC-proteolipossomos e DPPC:DPPS 10%-proteoliposomos revelaram conter 75% de AnxA5 e 25% de TNAP em concentração de proteína. A presença de DPPS não afetou significativamente as porcentagens de proteínas incorporadas. Os parâmetros cinéticos da TNAP na hidrólise de diferentes substratos fisiológicos (ATP, ADP e PPi) foram determinados na presença e ausência de AnxA5, em pH fisiológico, e para os diversos sistemas lipídicos. A melhor eficiência catalítica da enzima foi obtida para sistemas contendo 10% de DPPS (razão molar) (kcat/K0.5= 183,02; 776,06 e 657,08 M-1.s-1, respectivamente). A TNAP apresentou maior especificidade para a hidrólise de PPi quando comparado com ATP e ADP. Estudos utilizando Calorimetria Diferencial de Varredura (DSC) mostraram que o aumento da concentração de DPPS em DPPC-lipossomos proporcionou um progressivo alargamento no pico de transição de fase, diminuição na t1/2 e H. A pré-transição de fase só foi detectada até a concentração de 15% de DPPS em DPPC. Para 20% de DPPS e acima, observou-se uma segregação lateral de fase com a formação de possíveis microdomínios ricos em DPPS. A interação da AnxA5 com DPPC-lipossomos e DPPC:DPPS 10%-lipossomos resultou em uma redução nos valores de H (de 8,73 para 5,68 e 8,43 para 5,37 Kcal.mol-1, respectivamente). Quando a TNAP está presente nos proteolipossomos, este efeito é ainda maior. A AnxA5 incorporada em DPPC-proteolipossomos e DPPC:DPPS 10%-proteolipossomos (razão molar) foi capazes de mediar o influxo de 45Ca2+ para dentro das vesículas (~ 800 nmol Ca2+) quando utilizados faixas de concentração de cálcio em níveis fisiológicos (~2 mM). A presença da TNAP nos proteolipossomos não afetou o influxo de Ca2+ mediado pela AnxA5. Entretanto, a presença da AnxA5 afetou significativamente os parâmetros cinéticos da TNAP para os diferentes substratos. Estudos com vesículas unilamelares gigantes (GUVs) também confirmaram a inserção funcional da AnxA5 em vesículas constituídos de dioleoil fosfatidilcolina (DOPC) e DOPC:DPPS 10% (razão molar). O principal efeito causado pela AnxA5 na morfologia das GUVs foi a perda de contraste óptico devido a formação de poros nas membranas das vesículas. Neste caso, a presença de DPPS não proporcionou mudanças significativas para a incorporação da AnxA5. TNAP quando inserida em GUVs provocou intensa flutuação e excesso de área das vesículas com formação de filamentos. A presença do DPPS provavelmente dificulta a inserção da TNAP à membrana das GUVs. Quando há microdomínios lipídicos heterogêneos na composição de GUVs compostas por DOPC:Colesterol:Esfingomielina (8:1:1) e DOPC:Colesterol:Esfingomielina:Gangliosídeo (7:1:1:1) (razão molar), a inserção da TNAP provocou uma maior segregação lateral de fase evidenciada por imagens com fluorescência. A presença da TNAP e AnxA5 em DPPC:DPPS 10%-proteolipossomos proporcionou mudanças significativas nas propriedades mecânicas visco-elásticas dos proteolipossomos detectadas por imagens de Microscopia de Força Atômica. Assim, no presente trabalho foi possível obter uma inédita metodologia para a formação de proteolipossomos contendo TNAP e AnxA5 concomitantemente, os quais apresentaram uma reconstituição funcional das proteínas, apresentando capacidade de captar Ca2+ para dentro das vesículas e habilidade de hidrolisar fosfosubstratos em sua superfície. / Bone biomineralization is a multifactorial and complex process, being a challenge for the science the understanding of their regulatory mechanisms. This process is mediated by the release of matrix vesicles (MVs), structures which arise by budding from osteoblast and chondroblast surface and are secreted in the specific site where biomineralization begins. MVs have the ability of accumulating high concentrations of Ca2+ and Pi ions, providing an adequate microenvironment for the initial formation and propagation of hydroxyapatite crystals. Two protein families present in MVs merit special attention: Annexins and Phosphatases. The annexins were the most abundant proteins detected in MVs and are responsible for the Ca2+-channels formation (especially AnxA5). Tissue-nonspecific alkaline phosphatase (TNAP) exhibits phosphomonohydrolytic activity, producing Pi mainly from PPi and ATP. Such proteins regulate the formation of calcium phosphate crystals, acting directly in the bone mineralization process. The goal of this project was to produce and characterize proteoliposomes with different lipid compositions of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) harboring TNAP and AnxA5, keeping the functions of both proteins after their incorporation into the mimetic systems. AnxA5 was able to incorporate into DPPC-proteoliposomes (11.64 µg/mL), but the presence of DPPS increased significantly the AnxA5 incorporation (25.79 µg/mL) into DPPC:DPPS 10%-proteoliposomes. The presence of both proteins into DPPC and DPPC:DPPS 5, 10 and 15% (molar ratios) proteoliposomes was confirmed by SDS-PAGE and Immunoblotting analysis. Better yield of TNAP and AnxA5 incorporation was observed when both proteins were reconstituted simultaneously. DPPC-proteoliposomes and DPPC:DPPS 10%-proteoliposomes (molar ratio) incorporated about 75% of AnxA5 and 25% of TNAP (protein concentration). DPPS presence did not affect significantly the yield of incorporation of both proteins. The kinetic parameters for the hydrolysis of different physiological substrates (ATP, ADP and PPi) by TNAP were determined in the presence and absence of AnxA5, at physiological pH, for the different systems. The best catalytic efficiencies were achieved with proteoliposomes containing DPPS 10% (molar ratio) (kcat/K0.5= 183.02; 776.06 and 657.08 M-1.s-1 for ATP, ADP and PPi, respectively), condition that also favored PPi hydrolysis by TNAP when compared to ATP and ADP hydrolysis. Studies by Differential Scanning Calorimetry (DSC) showed that the increasing DPPS concentrations in the DPPC-liposomes resulted in a progressive broadening of the phase transition peaks and decreased t1/2 and H values. The pre-transition was detected only in concentrations up to DPPS 15% in DPPC. Phase lateral segregation can be observed for DPPS 20% and above, suggesting the formation of DPPS-rich microdomains. The interaction of AnxA5 with DPPC and DPPC:DPPS 10%-liposomes resulted in a decrease of H values (from 8.73 to 5.68 and from 8.43 to 5.37 Kcal.mol-1, respectively). When TNAP was present in the proteoliposomes, this effect was even greater. AnxA5 incorporated into DPPC and DPPC:DPPS 10%-proteoliposomes (molar ratio) was able to mediate 45Ca2+-influx (~ 800 nmol Ca2+) into the vesicles at physiological Ca2+-concentrations (~ 2 mM), and this process was not affected by the presence of TNAP in the systems. However, AnxA5 affected significantly the hydrolysis of substrates by TNAP. Studies with Giant Unilamellar Vesicles (GUVs) also confirmed the functional reconstitution of AnxA5 in dioleoylphosphocholine (DOPC) and DOPC:DPPS 10% (molar ratio) vesicles. The main effect caused by AnxA5 in the GUVs morphology was the formation of pores in the vesicles membrane. In this case, DPPS presence did not affect the AnxA5 incorporation. The presence of TNAP in GUVs caused a several fluctuation, indicating that the vesicles acquired an excess of area and undergoes sequential budding transitions. It is suggested that the presence of DPPS makes the TNAP insertion into the GUVs membrane difficult. With the presence of heterogeneous lipid microdomains in GUVs composed of DOPC, Cholesterol (Chol), Sphingomyelin (SM) and Ganglioside (GM1) in the proportions DOPC:Chol:SM 8:1:1 and DOPC:Chol:SM:GM1 7:1:1:1 (molar ratios), the TNAP insertion caused a greater phase lateral segregation, evidenced by fluorescence analysis. Atomic Force Microscopy (AFM) analysis indicated that the presence of both proteins into DPPC:DPPS 10%-proteoliposomes (molar ratio) caused significant changes in the visco-elastic mechanical properties of the vesicles. In conclusion, the present work describes the synthesis of proteoliposomes harboring TNAP and AnxA5 concomitantly, with the functional reconstitution of both proteins, with the ability to transport Ca2+ into the vesicles and hydrolyze phosphosubstrates on their surface.

A biomineralização óssea é um processo complexo e multifatorial sendo um grande desafio para a ciência à compreensão dos seus mecanismos regulatórios. Este processo é mediado pela liberação de vesículas da matriz (MVs), as quais surgem das superfícies de osteoblastos e são secretadas no local específico do início da biomineralização. MVs têm a capacidade de acumular altas concentrações de íons Ca2+ e fosfato (Pi), proporcionando um microambiente adequado para a formação inicial e propagação dos cristais de hidroxiapatita. Especial atenção deve ser dada a duas proteínas: Anexina V (AnxA5) e Fosfatase Alcalina (TNAP). As anexinas são as proteínas mais abundantes detectadas nas MVs e responsáveis pela formação de canais de cálcio. TNAP apresenta atividade fosfomonohidrolítica, produzindo Pi a partir, principalmente, de pirofosfato (PPi) e ATP. O enfoque deste projeto foi produzir e caracterizar proteolipossomos com diferentes composições lipídicas de dipalmitoil fosfatidilcolina (DPPC) e dipalmitoil fosfatidilserina (DPPS) contendo TNAP e AnxA5, e manter a funcionalidade das proteínas após incorporação nos sistemas miméticos. Foi possível incorporar AnxA5 em DPPC-proteolipossomos (11,64 µg/mL), mas na presença de DPPS houve um aumento significativo de AnxA5 incorporada (25,79 µg/mL) a DPPC:DPPS 10%-proteolipossomos (razão molar). A presença das proteínas nos proteolipossomos compostos por DPPC e DPPC:DPPS 5, 10 e 15% (razão molar) foi confirmada por SDS-PAGE e Immunoblotting. Melhores rendimentos de incorporação das duas proteínas foram obtidos quando ambas foram incorporadas concomitantemente. DPPC-proteolipossomos e DPPC:DPPS 10%-proteoliposomos revelaram conter 75% de AnxA5 e 25% de TNAP em concentração de proteína. A presença de DPPS não afetou significativamente as porcentagens de proteínas incorporadas. Os parâmetros cinéticos da TNAP na hidrólise de diferentes substratos fisiológicos (ATP, ADP e PPi) foram determinados na presença e ausência de AnxA5, em pH fisiológico, e para os diversos sistemas lipídicos. A melhor eficiência catalítica da enzima foi obtida para sistemas contendo 10% de DPPS (razão molar) (kcat/K0.5= 183,02; 776,06 e 657,08 M-1.s-1, respectivamente). A TNAP apresentou maior especificidade para a hidrólise de PPi quando comparado com ATP e ADP. Estudos utilizando Calorimetria Diferencial de Varredura (DSC) mostraram que o aumento da concentração de DPPS em DPPC-lipossomos proporcionou um progressivo alargamento no pico de transição de fase, diminuição na t1/2 e H. A pré-transição de fase só foi detectada até a concentração de 15% de DPPS em DPPC. Para 20% de DPPS e acima, observou-se uma segregação lateral de fase com a formação de possíveis microdomínios ricos em DPPS. A interação da AnxA5 com DPPC-lipossomos e DPPC:DPPS 10%-lipossomos resultou em uma redução nos valores de H (de 8,73 para 5,68 e 8,43 para 5,37 Kcal.mol-1, respectivamente). Quando a TNAP está presente nos proteolipossomos, este efeito é ainda maior. A AnxA5 incorporada em DPPC-proteolipossomos e DPPC:DPPS 10%-proteolipossomos (razão molar) foi capazes de mediar o influxo de 45Ca2+ para dentro das vesículas (~ 800 nmol Ca2+) quando utilizados faixas de concentração de cálcio em níveis fisiológicos (~2 mM). A presença da TNAP nos proteolipossomos não afetou o influxo de Ca2+ mediado pela AnxA5. Entretanto, a presença da AnxA5 afetou significativamente os parâmetros cinéticos da TNAP para os diferentes substratos. Estudos com vesículas unilamelares gigantes (GUVs) também confirmaram a inserção funcional da AnxA5 em vesículas constituídos de dioleoil fosfatidilcolina (DOPC) e DOPC:DPPS 10% (razão molar). O principal efeito causado pela AnxA5 na morfologia das GUVs foi a perda de contraste óptico devido a formação de poros nas membranas das vesículas. Neste caso, a presença de DPPS não proporcionou mudanças significativas para a incorporação da AnxA5. TNAP quando inserida em GUVs provocou intensa flutuação e excesso de área das vesículas com formação de filamentos. A presença do DPPS provavelmente dificulta a inserção da TNAP à membrana das GUVs. Quando há microdomínios lipídicos heterogêneos na composição de GUVs compostas por DOPC:Colesterol:Esfingomielina (8:1:1) e DOPC:Colesterol:Esfingomielina:Gangliosídeo (7:1:1:1) (razão molar), a inserção da TNAP provocou uma maior segregação lateral de fase evidenciada por imagens com fluorescência. A presença da TNAP e AnxA5 em DPPC:DPPS 10%-proteolipossomos proporcionou mudanças significativas nas propriedades mecânicas visco-elásticas dos proteolipossomos detectadas por imagens de Microscopia de Força Atômica. Assim, no presente trabalho foi possível obter uma inédita metodologia para a formação de proteolipossomos contendo TNAP e AnxA5 concomitantemente, os quais apresentaram uma reconstituição funcional das proteínas, apresentando capacidade de captar Ca2+ para dentro das vesículas e habilidade de hidrolisar fosfosubstratos em sua superfície. / Bone biomineralization is a multifactorial and complex process, being a challenge for the science the understanding of their regulatory mechanisms. This process is mediated by the release of matrix vesicles (MVs), structures which arise by budding from osteoblast and chondroblast surface and are secreted in the specific site where biomineralization begins. MVs have the ability of accumulating high concentrations of Ca2+ and Pi ions, providing an adequate microenvironment for the initial formation and propagation of hydroxyapatite crystals. Two protein families present in MVs merit special attention: Annexins and Phosphatases. The annexins were the most abundant proteins detected in MVs and are responsible for the Ca2+-channels formation (especially AnxA5). Tissue-nonspecific alkaline phosphatase (TNAP) exhibits phosphomonohydrolytic activity, producing Pi mainly from PPi and ATP. Such proteins regulate the formation of calcium phosphate crystals, acting directly in the bone mineralization process. The goal of this project was to produce and characterize proteoliposomes with different lipid compositions of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) harboring TNAP and AnxA5, keeping the functions of both proteins after their incorporation into the mimetic systems. AnxA5 was able to incorporate into DPPC-proteoliposomes (11.64 µg/mL), but the presence of DPPS increased significantly the AnxA5 incorporation (25.79 µg/mL) into DPPC:DPPS 10%-proteoliposomes. The presence of both proteins into DPPC and DPPC:DPPS 5, 10 and 15% (molar ratios) proteoliposomes was confirmed by SDS-PAGE and Immunoblotting analysis. Better yield of TNAP and AnxA5 incorporation was observed when both proteins were reconstituted simultaneously. DPPC-proteoliposomes and DPPC:DPPS 10%-proteoliposomes (molar ratio) incorporated about 75% of AnxA5 and 25% of TNAP (protein concentration). DPPS presence did not affect significantly the yield of incorporation of both proteins. The kinetic parameters for the hydrolysis of different physiological substrates (ATP, ADP and PPi) by TNAP were determined in the presence and absence of AnxA5, at physiological pH, for the different systems. The best catalytic efficiencies were achieved with proteoliposomes containing DPPS 10% (molar ratio) (kcat/K0.5= 183.02; 776.06 and 657.08 M-1.s-1 for ATP, ADP and PPi, respectively), condition that also favored PPi hydrolysis by TNAP when compared to ATP and ADP hydrolysis. Studies by Differential Scanning Calorimetry (DSC) showed that the increasing DPPS concentrations in the DPPC-liposomes resulted in a progressive broadening of the phase transition peaks and decreased t1/2 and H values. The pre-transition was detected only in concentrations up to DPPS 15% in DPPC. Phase lateral segregation can be observed for DPPS 20% and above, suggesting the formation of DPPS-rich microdomains. The interaction of AnxA5 with DPPC and DPPC:DPPS 10%-liposomes resulted in a decrease of H values (from 8.73 to 5.68 and from 8.43 to 5.37 Kcal.mol-1, respectively). When TNAP was present in the proteoliposomes, this effect was even greater. AnxA5 incorporated into DPPC and DPPC:DPPS 10%-proteoliposomes (molar ratio) was able to mediate 45Ca2+-influx (~ 800 nmol Ca2+) into the vesicles at physiological Ca2+-concentrations (~ 2 mM), and this process was not affected by the presence of TNAP in the systems. However, AnxA5 affected significantly the hydrolysis of substrates by TNAP. Studies with Giant Unilamellar Vesicles (GUVs) also confirmed the functional reconstitution of AnxA5 in dioleoylphosphocholine (DOPC) and DOPC:DPPS 10% (molar ratio) vesicles. The main effect caused by AnxA5 in the GUVs morphology was the formation of pores in the vesicles membrane. In this case, DPPS presence did not affect the AnxA5 incorporation. The presence of TNAP in GUVs caused a several fluctuation, indicating that the vesicles acquired an excess of area and undergoes sequential budding transitions. It is suggested that the presence of DPPS makes the TNAP insertion into the GUVs membrane difficult. With the presence of heterogeneous lipid microdomains in GUVs composed of DOPC, Cholesterol (Chol), Sphingomyelin (SM) and Ganglioside (GM1) in the proportions DOPC:Chol:SM 8:1:1 and DOPC:Chol:SM:GM1 7:1:1:1 (molar ratios), the TNAP insertion caused a greater phase lateral segregation, evidenced by fluorescence analysis. Atomic Force Microscopy (AFM) analysis indicated that the presence of both proteins into DPPC:DPPS 10%-proteoliposomes (molar ratio) caused significant changes in the visco-elastic mechanical properties of the vesicles. In conclusion, the present work describes the synthesis of proteoliposomes harboring TNAP and AnxA5 concomitantly, with the functional reconstitution of both proteins, with the ability to transport Ca2+ into the vesicles and hydrolyze phosphosubstrates on their surface.

L’efflux actif, qui permet aux bactéries d’exporter les antibiotiques vers le milieu extérieur est l’un des mécanismes majeurs de résistance aux antibiotiques. L’une des pompes d’efflux de Pseudomonas aeruginosa, MexA-MexB-OprM, est constituée de trois protéines : i) MexA, une protéine membranaire de fusion qui se trouve dans le périplasme ; ii) MexB qui se trouve dans la membrane interne et qui reconnaît l’antibiotique et initie son transport grâce à la force protomotrice et iii) OprM un canal qui se trouve dans la membrane externe. Durant ma thèse, j’ai mis au point un test fonctionnel pour MexA et MexB. Ce test est basé sur la coreconstitution de ces protéines avec la bactériorhodopsine, une protéine membranaire qui génère un gradient de proton après activation par la lumière. L’activité de MexB est suivie de manière indirecte via la mesure du pH. En mesurant le pH à l’intérieur des liposomes, on peut connaître l’activité de MexB puisque ce dernier utilise la force protomotrice pour transporter ses substrats. Une mesure fiable du pH peut être obtenue grâce à la pyranine dont la fluorescence varie avec le pH. Grâce à ce test, j’ai prouvé que MexB possède une activité basale qui ne dépend pas de la présence de substrat et que l’activité de MexB devient optimale quand cette dernière est reconstituée en présence de MexA. Dans un deuxième temps, j’ai mis au point un test fonctionnel pour la pompe d’efflux entière. Pour cela, je prépare deux types distincts de protéoliposomes. Dans le premier type de liposome, j’encapsule de la pyranine, (pour suivre l’activité de MexB) et un substrat de MexB qui est un agent intercalant de l’ARN. Ce substrat est faiblement fluorescent dans un environnement aqueux et fortement fluorescent lorsqu’il est intercalé dans l'ARN. MexB et MexA sont reconstitués dans ces liposomes. Dans le deuxième type de liposomes, je reconstitue OprM et j’encapsule de l’ARN. Ces deux types de liposomes sont alors mélangés. Lorsque la pompe s’assemble et qu’il y a un transport actif à travers cette dernière, deux phénomènes sont observés: la diminution de la fluorescence de la pyranine (car MexB fait entrer des protons dans le premier type de liposome pour transporter le substrat) et l’augmentation de la fluorescence du substrat car ce dernier s’intercale dans l’ARN se trouvant dans le deuxième type de liposome. En mélangeant les deux types de liposomes, j’obtiens une preuve de la reconstitution in vitro de la pompe entière et j’ai mis en évidence qu’OprM s’ouvre en présence de MexA et MexB et que sa présence augmente l’activité de MexB. / Among the various mechanisms developed by the bacteria to counter to the effect of antibiotics, active efflux is on the front line. In Pseudomonas aeruginosa, a Gram negative bacteria, efflux transporters are organized as multicomponent systems where MexB, the pump located in the inner membrane, works in conjunction with MexA, a periplasmic protein, and OprM, an outer membrane protein. MexB is a proton motive force-dependent pump with broad substrate specificity. During my PhD, I have designed an original activity assay for MexB and MexA. The pump is coreconstituted into proteoliposomes together with bacteriorhodopsin (BR), a light-activated proton pump. In this system, upon illumination with visible light, the photo-induced proton gradient created by the BR is shown to be coupled to the active transport of substrates through the pump. The activity of MexB is monitored indirectly. Since MexB uses the protomotive force to transport antibiotics, one can determine substrate transport though MexB by monitoring the pH inside the liposomes. For that purpose, pyranine, a fluorescent probe whose fluorescence yield increases with increasing pH, is encapsulated inside the liposomes. This test makes the investigation of the pump possible. In the absence of MexA, MexB has a basal activity which is not substrate-dependent. Once MexB is reconstituted together with MexA, its activity is specific and substrate-dependent. Then I worked on the reconstitution of the whole efflux pump. For this, I prepare two different kinds of liposomes: i) Liposomes with reconstituted MexA and MexB in which pyranine and a nucleic acid intercalating agent are encapsulated, ii) Liposomes with reconstituted OprM and encapsulated RNA. The activity of MexB is monitored thanks to the addition of EthB, a substrate of MexB, that is poorly fluorescent in aqueous medium and highly fluorescent when intercalated into RNA. Upon generation of a pH gradient, I observe two concomitant phenomena: the decrease of pyranine fluorescence, as MexB is using protons to transport the substrate, and the increase of the fluorescence of the RNA intercalating agent as a result of its interaction with RNA. I have successfully assembled the efflux pump and monitored transport through it from one liposome to the other. I have demonstrated that OprM needs to interact with MexA and MexB in order to open and that MexB activity is accelerated when the pump is assembled.

No description available.

<p>When drug molecules are passively absorbed through the cell membrane in the small intestine, the first key step is partitioning of the drug into the membrane. Partition data can therefore be used to predict drug absorption. The partitioning of a solute can be analyzed by drug partition chromatography on immobilized model membranes, where the chromatographic retention of the solute reflects the partitioning. The aims of this thesis were to develop the model membranes used in drug partition chromatography and to study the effects of different membrane components and membrane structures on drug partitioning, in order to characterize drug–membrane interactions.</p><p>Electrostatic effects were observed on the partitioning of charged drugs into liposomes containing charged detergent, lipid or phospholipid; bilayer disks; proteoliposomes and porcine intestinal brush border membrane vesicles (BBMVs), and on the retention of an oligonucleotide on positive liposomes. Biological membranes are naturally charged, which will affect drug partitioning in the human body.</p><p>Proteoliposomes containing transmembrane proteins and cholesterol, BBMVs and bilayer disks were used as novel model membranes in drug partition chromatography. Partition data obtained on proteoliposomes and BBMVs demonstrated how cholesterol and transmembrane proteins interact with drug molecules. Such interactions will occur between drugs and natural cell membranes. In the use of immobilized BBMVs for drug partition chromatography, yet unsolved problems with the stability of the membrane were encountered. A comparison of partition data obtained on bilayer disks with data on multi- and unilamellar liposomes indicated that the structure of the membrane affect the partitioning. The most accurate partition values might be obtained on bilayer disks.</p><p>Drug partition data obtained on immobilized model membranes include both hydrophobic and electrostatic interactions. Such partition data should preferably be used when deriving algorithms or computer programs for prediction of drug absorption.</p>

When drug molecules are passively absorbed through the cell membrane in the small intestine, the first key step is partitioning of the drug into the membrane. Partition data can therefore be used to predict drug absorption. The partitioning of a solute can be analyzed by drug partition chromatography on immobilized model membranes, where the chromatographic retention of the solute reflects the partitioning. The aims of this thesis were to develop the model membranes used in drug partition chromatography and to study the effects of different membrane components and membrane structures on drug partitioning, in order to characterize drug–membrane interactions. Electrostatic effects were observed on the partitioning of charged drugs into liposomes containing charged detergent, lipid or phospholipid; bilayer disks; proteoliposomes and porcine intestinal brush border membrane vesicles (BBMVs), and on the retention of an oligonucleotide on positive liposomes. Biological membranes are naturally charged, which will affect drug partitioning in the human body. Proteoliposomes containing transmembrane proteins and cholesterol, BBMVs and bilayer disks were used as novel model membranes in drug partition chromatography. Partition data obtained on proteoliposomes and BBMVs demonstrated how cholesterol and transmembrane proteins interact with drug molecules. Such interactions will occur between drugs and natural cell membranes. In the use of immobilized BBMVs for drug partition chromatography, yet unsolved problems with the stability of the membrane were encountered. A comparison of partition data obtained on bilayer disks with data on multi- and unilamellar liposomes indicated that the structure of the membrane affect the partitioning. The most accurate partition values might be obtained on bilayer disks. Drug partition data obtained on immobilized model membranes include both hydrophobic and electrostatic interactions. Such partition data should preferably be used when deriving algorithms or computer programs for prediction of drug absorption.

Les membranes cellulaires jouent un rôle important dans l’absorption des médicaments et la distribution de ceux-ci dans le corps humain. Elles contiennent différents transporteurs membranaires qui sont responsables des profils pharmacocinétiques, d’innocuité et d’efficacité des xénobiotiques. Lors du développement d’un médicament, il s’avère donc indispensable, de prédire l’interaction des nouveaux composés avec les transporteurs présents dans l’organisme. Le but du projet de recherche est de créer un nouvel outil pour étudier le comportement de la glycoprotéine-P (P-gp), un transporteur membranaire responsable du rejet de nombreux composés, sur différents médicaments. Pour cela, un modèle non cellulaire est développé en utilisant des protéoliposomes : des liposomes dans lesquels des transporteurs sont incorporés. La méthodologie consiste tout d’abord à produire, extraire et purifier la protéine d’intérêt à partir de deux systèmes d’expression : MDCK-MDR1 (cellules de chien transfectées avec le gène humain MDR1) et Pichia pastoris (levures) fin de déterminer les avantages et les limites de ces deux types cellulaires. Différentes méthodes de reconstitution dans des protéoliposomes ont ensuite été testées avec la P-gp obtenue. Puis, l’activité ATPasique de la P-gp reconstituée a été évaluée en présence de différents substrats. Les protocoles de culture cellulaire, d’extraction et de purification des deux systèmes d’expression ont été implémentés avec succès au sein du laboratoire. Les résultats montrent que les rendements obtenus sont supérieurs avec les levures qu’avec les cellules de mammifère. En outre, Pichia pastoris offre les avantages d’être facile et rapide à cultiver et peu couteux. Les premiers résultats d’activité ATPasique obtenus avec la P-gp reconstituée en protéoliposomes étaient prometteurs mais n’ont pas été reproduits en raison de la dégradation de la protéine membranaire. Les prochaines études du projet porteront sur un autre transporteur membranaire de la famille ABC, BCRP, une protéine de plus petite taille qui devrait montrer une plus grande stabilité pour mener à bien les tests. / Cellular membranes play an important role in the absorption and distribution of drugs in the human body. They contain different membrane transporters, which are responsible for the pharmacokinetic properties of drugs, as well as the safety and efficiency of their diffusion. When developing a new drug, it is thus of utmost importance to study the way that it will interact with the transporters present within the body. The aim of this study was to evaluate a new tool for measuring permeability in order to understand the function and mecanisms of P-glycoprotein (P-gp). P-gp is a transporter that is responsible for the rejection of many different compounds found in various drugs. This study thus seeks to use proteoliposomes to develop non-cellular models of membrane permeability including efflux and uptake transporters. This novel model of permeability will be utilized to study the underlying mechanisms of membrane permeability to xenobiotics. The human P-gp was produced, extracted and purified using two different expression systems: MDCK-MDR1 cells (Madin-Darby canine kidney cells transfected with the human MDR1 gene) and Pichia pastoris. Both expression systems were studied in order to compare the strengths and weaknesses of each system. We then tested different methods of reconstituting the P-gp into protéoliposomes. Finally, we measured the level of ATPase activity using different substrates. The protocols of cell culture, extraction and purification of both expression systems were accomplished in a laboratory during this study. These results demonstrated that expressing P-gp using yeast was more effective than that of mammalian cells. Furthermore, working with Pichia pastoris offers multiple advantages: expressing P-gp was easier, faster and cheaper than working with mammalian cells. The first measurements of ATPase activity using reconstituted P-gp proteoliposomes were promising, however they proved difficult to reproduce due to the possible degradation of the membrane protein.Further studies in this project will look to evaluate another ABC membrane transporter, BCRP. This smaller protein should prove to be more stable than P-gp, facilitating experimentation.