La mucoviscidose est causée par des mutations du gène CFTR (p.Phe508del étant la plus fréquente). Celui-ci code pour la protéine CFTR qui constitue un canal chlorure exprimé à la face apicale des cellules épithéliales. Au niveau du reticulum endoplasmique (RE), le contrôle de qualité conformationnelle oriente la majorité du CFTR en cours de repliement vers une voie de dégradation. Une fraction limitée du WT-CFTR parvient cependant à se replier correctement et peut ensuite progresservers la surface cellulaire, contrairement au Phe508del-CFTR (qui est néanmoins fonctionnel). Lorsque des formes mutées sont exportées à partir du RE, grâce à des traitements correcteurs, elles sont alors instables à la membrane plasmique. Par ailleurs, il a été montré que l'organisation des microfilaments d'actine participe à l'ancrage du canal au cytosquelette et à sa stabilité. Or, la petite GTPase Cdc42 influence la dynamique de nucléation de l'actine fibrillaire. Au cours de nos travaux, nous avons testé l'implication de Cdc42 et de certains de ses effecteurs dans la régulation de WT-CFTR dans des cellules épithéliales bronchiques. Dans ce cadre, la fonction de la voie Cdc42 a été perturbée par des traitements pharmacologiques et par ARN interférence. Les résultats obtenus, principalement par biotinylation de surface, ont permis de proposer que (1) la protéine Cdc42 participe à ladégradation de formes mal repliées de CFTR dans les étapes précoces et tardives de la voie de sécrétion et (2) la voie Cdc42, par son implication dans l'organisation de l'actine F corticale, affecte l’ancrage du canal chlorure au cytosquelette et régule ainsi son recrutement dans des vésicules d'internalisation. / Cystic Fibrosis is caused by CFTR gene mutations (p.Phe508del being the most frequently encountered). The CFTR protein functions as a chloride channel expressed at the plasma membrane of epithelial cells. Its productive folding in the endoplasmicreticulum (ER) is poorly efficient and unfolded proteins are therefore targeted to degradation. Nevertheless, a limited fraction of WT-CFTR acquires a native conformation and then progesses into the secretory pathway. In the case of Phe508del-CFTR, virtually all channels are degraded at this step except through corrector treatments. Under these conditions the mutant remains unstable at the plasma membrane (although it is functionnaly competent). Furthermore, it has been shown that fibrillar actin organization is involved in CFTR tethering to the cytoskeleton and channel stability. Moreover, the small GTPase Cdc42 promotes F actin nucleation. In the present study, we aimed at testing the involvement of Cdc42, and of some of its effectors, in WT-CFTR regulation in epithelial airway cells. In this context, Cdc42 pathway function was altered through pharmacological treatments or siRNAmediated depletions. Our results, mainly obtained via cell surface biotinylation assays, led us to propose that (1) Cdc42 is involved in misfolded CFTR degradation at early and late steps of the secretory pathway, and (2) Cdc42 pathway, through its F actin organization function, affects CFTR anchoring to the cytoskeleton and thus regulates its endocytosis.

La mucoviscidose est la plus fréquente des maladies rares chez la population caucasienne. Cette maladie héréditaire récessive est causée par des mutations du gène Cystic Fibrosis transmembrane conductance regulator (CFTR) qui code pour une protéine localisée au niveau de la membrane apicale des cellules épithéliales. La sévérité du phénotype est déterminée par les classes des mutations et leurs combinaisons en trans, ainsi que par la présence d'allèles complexes. La détermination des effets d'une mutation est essentielle pour avoir une corrélation génotype/phénotype correcte, donner un diagnostic prénatal adapté et permettre aux cliniciens de prescrire le traitement approprié à chaque mutation quand celui-ci sera disponible. Pour cela, nous avons étudié aux niveaux cellulaire et moléculaire les effets de plusieurs mutations qui intéressent le laboratoire : c.1392G>T (p.Lys464Asn), c.3909C>G (p.Asn1303Lys) et c.965T>C (p.Val322Ala). L'effet de ces mutations sur la protéine a été évalué. De plus, l'impact sur l'épissage aberrant des deux premières mutations, seules et dans le cadre de leurs allèles complexes, a été déterminé. Nous avons montré que : 1) la mutation c.1392G>T est de classe V et II et son allèle complexe aggrave l'épissage aberrant, 2) la mutation c.3909C>G appartient à la classe II et l'effet sur l'épissage résulte de son allèle complexe et 3) la mutation familiale c.965T>C est un simple polymorphisme. Ces travaux montrent l'importance de l'étude d'une mutation à différents niveaux cellulaires par l'intermédiaire des analyses in silico, in cellulo et in vivo et soulignent l'effet des allèles complexes qui peuvent moduler l'impact de la mutation seule. / Cystic Fibrosis is the most frequent rare disease in the Caucasian population. This hereditary recessive disease is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that encodes for a protein expressed on the apical membrane of epithelial cells. The mutations classes, their associations in trans and the presence of complex alleles define the phenotype severity. The determination of mutations effects is essential to have a correct genotype/phenotype correlation to give an adapted prenatal diagnosis and to help the clinicians in providing an appropriate treatment when available. In this respect, we have studied on the cellular and molecular levels the effects of several mutations of interest for the laboratory: c.1392G>T (p.Lys464Asn), c.3909C>G (p.Asn1303Lys) et c.965T>C (p.Val322Ala). The effects of these mutations were evaluated on the protein level. Moreover, the impact on aberrant splicing of these first two mutations solely and in the context of their complex alleles was determined. We have demonstrated that: 1) the c.1392G>T mutations belongs to class V and II and its complex allele aggravates the aberrant splicing, 2) the c.3909C>G is a class II mutation and the effect on splicing is due to its complex allele, and 3) the familial c.965T>C mutation is a simple polymorphism. This work highlights the importance to study the CFTR mutation at different cellular levels using in silico, in cellulo and in vivo analyses and emphasizes on the effect of complex allele in modulating the basal impact of a single mutation.

La mucoviscidose est la maladie héréditaire autosomique récessive, rare, létale, la plus fréquente dans la population caucasienne. Cette maladie est causée par des mutations du gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) codant la protéine CFTR. Cette protéine est principalement un canal chlorure (Cl-) AMPc-dépendant localisé dans la membrane apicale des cellules épithéliales. La mutation F508del entraîne un défaut de maturation de la protéine qui est retenue dans le réticulum endoplasmique avant d’être dégradée. Cependant, une faible quantité de protéines malformées échappe à ce système de contrôle et parvient à la membrane plasmique.Des travaux de notre équipe ont montré une augmentation des efflux ioniques dépendants du CFTR dans des lignées cellulaires épithéliales bronchiques (CFBE41o-), exprimant le CFTR sauvage ou le CFTR muté F508del, après un traitement par une hormone: la gonadolibérine (GnRH, Gonadotropin releasing hormone, 1h, 10-9M). Cette augmentation est vraisemblablement due à un nombre plus important de canaux CFTR à la membrane plasmique.L’objectif de cette thèse a été de tester un analogue de la GnRH comme modulateur de l’exportation membranaire et/ou de l’activité canal du CFTR muté, sur des cultures primaires de cellules épithéliales nasales humaines homozygotes pour la mutation F508del. Dans un premier temps, nous avons vérifié la présence du récepteur à la GnRH (R-GnRH) dans notre modèle cellulaire. Puis, nous avons étudié l’effet de l’analogue sur la fonction du CFTR par des techniques d’électrophysiologie. Nous avons observé une augmentation des efflux d’ions Cl- médiés par le canal CFTR après un traitement à l’analogue (2h, 10-12M). Enfin, une étude protéomique nous a permis d’identifier des protéines différentiellement exprimées après traitement. Certaines protéines mises en évidence pourraient appartenir à des voies de signalisation intracellulaires ayant un rôle dans la régulation de la protéine CFTR et être des cibles thérapeutiques.Par ailleurs, le canal CFTR est régulé par le Mg2+ intracellulaire ([Mg2+]i). Le canal TRPM7 est le principal régulateur du [Mg2+]i. La [Mg2+]i a été mesurée et l’expression de TRPM7 vérifiée dans des cellules Hela transfectées avec le CFTR sauvage (wt) ou mutés (G551D et F508del). Nous avons étudié la localisation, la fonction et la régulation de TRPM7 dans nos modèles cellulaires avant de rechercher un possible lien fonctionnel entre le CFTR et TRPM7. Dans les modèles CF, l’expression, la fonction et la localisation du canal TRPM7 sont altérées. Il existerait un lien fonctionnel entre TRPM7 et le CFTR par l’intermédiaire de la diminution du [Mg2+]i impliquant TRPM7 dans la physiopathologie de la mucoviscidose. / Cystic fibrosis is the most common lethal autosomal recessive disease in the Caucasian population. This disease is caused by mutations in the gene encoding the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) protein. This protein is a cAMP-regulated chloride channel expressed at the apical membrane of epithelial cells. The F508del mutation causes a defect in CFTR protein folding preventing its maturation. Some misfolded proteins escape the control system and reach the plasma membrane.We previously showed a rise of CFTR-dependent ion efflux in wt- and F508del-CFTR human bronchial epithelial expressing cells (CFBE41o-) after incubation with GnRH (Gonadotropin releasing hormone; 1h, 10-9M). This increase was probably due to an increased cell surface expression of CFTR.The aim of the present study was to test a GnRH analog as a modulator of CFTR delivery to the plasma membrane and/or activity of CFTR on primary culture of human nasal epithelial cells (F508del/F508del).We checked the GnRH receptor expression in our model. Then, we studied the GnRH effect on CFTR’s function by electrophysiology. We found a significant increase of CFTR dependent chloride efflux in cells pretreated with analogue (2h, 10-12M). Proteomic study enabled us to identify differentially expressed proteins after treatment. Some highlighted proteins could be part of signalling pathways regulating CFTR and could be therapeutic targets.Moreover, CFTR is regulated by intracellular Mg2+ ([Mg2+]i). TRPM7 (Transient Receptor Potential Melastatin 7) is the main channel regulating [Mg2+]i. [Mg2+]i and TRPM7 expression were measured in Hela cells stably expressing wildtype and two CFTR mutants (F508del-CFTR and G551D-CFTR). We studied TRPM7 expression, function and regulation in our cell models before examining a functional link between TRPM7 and CFTR. In CF models, TRPM7 expression, localization and function are altered. There should be a functional link between TRPM7 and CFTR through the decreased [Mg2+]i involving TRPM7 in cystic fibrosis physiopathology.

La mucoviscidose (CF) est la maladie génétique récessive létale la plus fréquente dans la population caucasienne. Elle est caractérisée par une obstruction et des infections des voies respiratoires et une inflammation chronique. La morbidité et la mortalité sont principalement dues à l'atteinte pulmonaire, qui est variable chez les patients, même lorsqu’ils sont porteurs du même génotype. Les facteurs responsables sont multiples : les mutations dans CFTR (le gène responsable de la maladie), les gènes modificateurs, mais aussi les facteurs environnementaux et les modifications épigénétiques. L'objectif principal de ce projet était de déterminer s'il y avait une corrélation entre la méthylation de l'ADN et la sévérité de l'atteinte pulmonaire chez les patients CF. Nous avons obtenu la cohorte METHYLCF (49 patients CF p.Phe508del homozygotes et 24 témoins sains) ainsi qu’une biobanque d'ADN à partir de sang total et de cellules épithéliales nasales (NEC). Les patients CF ont été stratifiés en fonction de leur VEMS, ajusté à l’âge. D’une part, nous avons analysé la méthylation de l'ADN dans CFTR plus 13 gènes modificateurs en utilisant la méthode de conversion au bisulfite et séquençage de nouvelle génération (plateforme 454 Roche). D’autre part, nous avons réalisé une analyse pan-génomique de la méthylation de l'ADN avec la plateforme 450k BeadChip (Illumina). Les sites différentiellement méthylés (DMS) sélectionnés ont été validés par pyroséquençage (PyroMark Q24, Qiagen). Deux gènes modificateurs ont été identifiés comme différentiellement méthylés chez les patients CF par rapport aux témoins: EDNRA dans le sang et HMOX1 dans le sang et dans les NEC. De façon intéressante, dans les NEC, la méthylation de EDNRA, HMOX1 et GSTM3 a été corrélée avec la sévérité de l’atteinte pulmonaire. De plus, de faibles niveaux de méthylation d'ADN dans GSTM3 ont été associés à la présence de l'allèle GSTM3*B, un polymorphisme de séquence qui a un effet protecteur chez les patients CF. Grâce à l'analyse tout-génome, nous avons identifié 1267 DMS, associés à 638 gènes, chez les patients CF par rapport aux témoins, et 187 DMS, associés à 116 gènes, chez les patients CF sévères par rapport aux modérés. Parmi ces gènes, il y a de nombreux gènes importants pour l’adhésion cellulaire et les réponses immunitaire et inflammatoire. Les DMS identifiés sont enrichis dans des régions prédites comme enhancers, pouvant représenter des séquences régulatrices, mais également en régions intergéniques. De façon intéressante, 80 gènes différentiellement méthylés sur 638 étaient différentiellement exprimés (méta-analyse de données transcriptomiques disponibles). Six sur neuf DMS sélectionnés ont été validés et cinq DMS sur six ont été répliqués dans une population indépendante. De plus, 23 DMS, dont 10 intergéniques, étaient corrélés avec le VEMS. Notre étude a montré que la méthylation de l'ADN est profondément modifiée dans le sang et dans les NEC des patients CF. Des faibles changements de méthylation de l'ADN ont été observés dans des gènes modificateurs connus ; des changements de méthylation plus importants ont été observés dans d'autres gènes qui pourraient représenter de nouveaux modificateurs de la fonction pulmonaire. Ensemble, ces gènes pourraient moduler la sévérité de l’atteinte pulmonaire chez les patients CF. / Cystic fibrosis (CF) is the most common life-threatening recessive genetic disease in the Caucasian population. It is characterized by airway obstruction, respiratory infection and inflammation. Morbidity and mortality are mainly due to lung disease, which is variable among CF patients, even for those having the same genotype. Contributing factors are mutations in CFTR (the disease-causing gene), modifier genes, but also environmental factors and epigenetics. The main goal of this project was to determine whether there was a correlation between DNA methylation and the severity of CF lung disease. We built the METHYLCF cohort (49 p.Phe508del homozygous CF patients and 24 healthy controls) and a DNA biobank from whole blood and nasal epithelial cells (NEC). CF patients were stratified accordion to their FEV1% predicted, adjusted to age. We profiled DNA methylation at 14 modifier genes using bisulfite conversion and next-generation sequencing (454 Roche). Genome-wide DNA methylation was analyzed with the 450K Beadchip (Illumina). Selected differentially methylated sites (DMS) were validated by pyrosequencing. Using the candidate modifier gene approach, we showed that two CF modifier genes were differentially methylated in CF patients compared to controls: EDNRA in blood and HMOX1 in blood and NEC. Methylation of EDNRA, HMOX1 and GSTM3 was associated with lung disease severity in NEC. Interestingly, low DNA methylation levels at GSTM3 were associated with the GSTM3*B allele, a polymorphic 3-bp deletion that has a protective effect on CF patients. In addition, through the genome-wide analysis, we identified 1267 DMS, associated with 638 genes, between CF patients and controls and 187 DMS, associated with 116 genes, between severe CF and mild CF patients. DMS were enriched at predicted enhancers, which may represent regulatory sequences, and also at intergenic regions. Gene ontology analyses highlighted cellular processes relevant to CF, i.e. cell adhesion and inflammatory and immune response. Interestingly, 80 out of 638 differentially methylated genes were differentially expressed in publicly available NEC transcriptomic data. Six out of 9 selected DMS were validated and five out of six DMS were replicated in an independent set of patients. Additionally, 23 DMS, 10 of which were intergenic, correlated with FEV1% predicted. Our study has shown that DNA methylation is altered in blood and NEC of CF patients. Small DNA methylation changes were observed at known CF modifier genes; more dramatic DNA methylation changes were found at other genes that may impact lung function. Collectively, these epigenomic variations may lead to different degrees of lung disease severity in CF patients.

70% des mutations identifiées sur le gène responsable de la mucoviscidose correspondent à la délétion de la phénylalanine en position 508 (F508del) de la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Cette mutation est responsable, à 37°C, d'un mauvais repliement, du blocage et de la dégradation rapide de CFTR au niveau du réticulum endoplasmique (RE), et par conséquent de l'absence de sécrétion des ions Cl- au niveau de la membrane apicale des cellules épithéliales. Deux conséquences principales de cette mutation sont un épaississement important du mucus bronchique et une diminution de l'intégrité de la barrière luminale de l'épithélium bronchique. Ces deux phénomènes participent à l'invasion et à l'infection du tissu pulmonaire par des bactéries pathogènes comme Pseudomonas aeruginosa, exacerbant l'inflammation et la destruction tissulaire au niveau des poumons. L'objectif de cette étude a été de déterminer le rôle de deux protéines appartenant à la famille des protéines G hétérotrimériques, G12 et G13, dans la dégradation de la protéine CFTR-F508del ainsi que dans le contrôle des complexes jonctionnels au niveau de l'épithélium bronchique sain et mucoviscidosique. Nos travaux démontrent pour la première fois que dans la mucoviscidose, l'expression des protéines G12 et G13 est faible. Nous avons aussi montré que G12, et non G13, est impliquée dans le contrôle de la dégradation de la protéine CFTR-F508del via les protéines chaperonnes Calnexine et HSP90, et dans la formation et le maintien des jonctions cellulaires bronchiques via E-cadhérine et ZO-1 de manière inverse par rapport à l'épithélium rénale. Ces travaux placent donc G12 comme un acteur non négligeable de la maladie de la mucoviscidose. / F508del, the most frequent mutation found in cystic fibrosis (CF) population, impacts CFTR (Cystic Fibrosis Transmembrane conductance Regulator) trafficking and causes its rapid degradation at the endoplasmic compartment, resulting in a significant decrease in Cl- secretion at the apical membrane of epithelial cells. F508del has two main features, significant thickening of the bronchial mucus and a reduction in the integrity of the luminal barrier of the bronchial epithelium. These two phenomena are involved in the invasion and infection of lung tissue by pathogenic bacteria such as Pseudomonas aeruginosa, exacerbating the inflammation and lung destruction. The objective of this study was to determine the role of two proteins member of the heterotrimeric G proteins family, G12 and G13, in the degradation of the F508del CFTR, and in the control of junctional complexes in the normal and CF bronchial epithelium. Our results show for the first time that G12 and G13 are down expressed in CF. G12, but not G13, is involved in the control of F508del-CFTR degradation through its interaction with Calnexin and HSP90 chaperones. Unlike kidney epithelia cells, G12 promotes the formation and maintenance of cell junctions in the bronchial epithelium by affecting E-cadherine and ZO-1 stability. Altogether, our results set therefore G12 as a significant actor of the CF disease.

Cystic fibrosis (CF) is genetically conditioned, autosomal recessive disease that occurs in the European population with a prevalence of about 1:2500 - 1:1800. In this disease we observe a mutation of the CTFR gene with subsequent fault in chloride channels. Such afflicted individuals usually suffer from chronic respiratory problems, pancreatic insufficiency, high concentration of chloride ions in sweat and obstructive azoospermia. Genetic testing of CFTR gene is indicated in individuals who meet the CF clinical picture and a positive sweat test (increased concentration of chlorides in the sweat). Genetic testing of the CFTR gene is usually done by using commercial kits detecting the most common mutations of the CFTR gene in the Czech Republic. If the testing results are negative, it is further performed an MLPA method that captures the larger deletions and duplications of gene, eventually a sequencing of all exons is. Despite the well-established algorithm of the testing, some patients suffering from symptoms of CF are left without genetic findings. Thanks to development of next generation sequencing, it is possible to make the diagnosis of CF more effective and uncover the variants that were not captured by previous methods.

[pt] A fibrose cística é uma doença genética causada por mutações no gene CFTR, que implica na alteração do transporte de íons pela membrana das células de diversos órgãos, aumentando a quantidade e viscosidade do muco, suor e secreções pancreáticas. A principal consequência são os danos pulmonares, caracterizados por frequentes infecções e insuficiência respiratória. Com isso, vários estudos vêm sendo desenvolvidos ao longo dos anos, com o intuito de descobrir fármacos que possam agir como moduladores do CFTR, melhorando a função pulmonar desses pacientes. O presente trabalho teve o objetivo de sintetizar derivados de 3-fenilindeno[1,2-d][1,2,3]triazol-8(3H)-ona, 3-fenil-3,4- di-hidro-9H-[1,2,3]triazolo[4,5-b]quinolin-9-ona e 3-(1-fenil-1H-1,2,3-triazole-4- carbonil)quinolin-4(1H)-ona caracterizar a relação estrutura atividade desses compostos como potenciadores do CFTR. Para a síntese de 3-fenilindeno[1,2-d][1,2,3]triazol-8(3H)-ona inicialmente utilizou-se a 2 bromoacetofenona para, através do reagente de Gold produzido a partir do cloreto de cianurila, produzir a enaminona. A etapa chave de obtenção dos triazóis foi realizada através de uma reação de cicloadição entre as (E)-3-(dimetilamino)-1-(2-nitrofenil)prop-2-en-1-ona e aril azidas azido-benzeno, 1-azido-4-bromo-benzeno, 1-azido-4- nitrobenzeno e ácido 4-azido-benzóico. Foram obtidos quatro compostos com rendimentos entre 20 por cento e 50 por cento. Foram realizadas tentativas de ciclização intramolecular catalisada por paládio na presença de fosfinas, porém o produto esperado não foi obtido. Para a obtenção da 3-fenil-3,4-di-hidro-9H-[1,2,3]triazolo[4,5-b]quinolin-9-ona obteve-se o intermediário (E)-3-(dimetilamino)-1-(2-nitrofenil)prop-2-en-1-ona, que através de uma reação de cicloadição com a azido-benzeno obteve-se o triazol com rendimento de 30 por cento. A proposta seria a redução do grupo nitro para amina, e posterior substituição da amina por azida, para fazer a arilação intramolecular do triazol via catálise fotoredox. Para a obtenção de 3-(1-fenil-1H-1,2,3-triazole-4-carbonil)quinolin4(1H)-ona foram utilizados anilina e dietil etoximetilenomalonato como precursores para sintetizar 4-oxo-1,4-di-hidroquinolino-3-carboxilato de etilo, que foi obtida com rendimento de 50 por cento. Realizou-se uma reação de adição de alcino seguida da eliminação de etanol, para obter a 3-(1-fenil-1H-1,2,3-triazole-4-carbonil)quinolin-4(1H)-ona com etiniltrimetilsilano e então realizar uma reação click com azidas. Todos os compostos obtidos foram caracterizados por RMN1H e RMN13C. / [en] Cystic fibrosis is a genetic disease caused by mutations in the CFTR gene, which involves changing the transport of ions across the membrane of various organ cells, increasing the amount and viscosity of the mucus, sweat and pancreatic secretions. The main consequence is lung damage, characterized by frequent infections and respiratory failure. Several studies have been developed over the years with the intention of discovering drugs that can act as modulators of CFTR, improving the lung function of these patients. The present work aimed to synthesize derivatives of 3-phenylindeno[1,2-d [1,2,3]triazol-8(3H)-one, 3-phenyl-3,4-dihydro-9H-[1,2,3]triazolo[4,5-b]quinolin-9-one and 3-(1-phenyl-1H1,2,3-triazole-4-carbonyl)quinolin-4(1H)-one to characterize the relationship structure of these compounds as enhancers of CFRT. For the synthesis of 3-phenylindeno[1,2-d][1,2,3]triazol-8(3H)-one, the 2-bromoacetophenone was initially used to produce the (E)-3-(dimethylamino) -1- (2-nitrophenyl) prop-2-en1-one through the Gold reagent obtained from the cyanuric chloride. The key step of obtaining the triazoles was accomplished by a cycloaddition reaction between the (E)-3-(dimethylamino)-1-(2-nitrophenyl) prop-2-en-1-one and the aryl azides azido-benzene, 1-azido-4-bromo-benzene, 1-azido-4-nitrobenzene and 4-azido benzoic acid. Four compounds in yields between 20 percent and 50 percent were obtained. Attempts were made for intramolecular cyclization catalyzed by palladium in the presence of phosphines, but the expected product was not obtained. To obtain 3- phenyl-3,4-dihydro-9H-[1,2,3]triazolo[4,5-b]quinolin-9-one, first It was obtained the intermediate (E)-3- (dimethylamino)-1-(2-nitrophenyl) prop-2-en-1-one through cycloaddition reaction with the azido-benzene gave the triazole in 30 percent yield. The proposal would be the reduction of the nitro group to amine, and subsequent replacement of the amine with azide, to make the intramolecular arylation of triazole via photoredox catalysis. To obtain 3-(1-phenyl-1H-1,2,3-triazole-4-carbonyl)quinolin-4(1H)-one, aniline and diethyl ethoxymethylene malonate were used as precursors to synthesize ethyl 4-oxo-1,4-dihydroquinoline3-carboxylate, which was obtained in 50 percent yield. An addition reaction of alkyne followed by the elimination of ethanol was performed to obtain the 3-(1-phenyl1H-1,2,3-triazole-4-carbonyl)quinolin-4(1H)-one with ethynyltrimethylsilane and then carried out a click reaction with azides. All compounds obtained were characterized by 1H-NMR and 13C-NMR.

Membrane proteins are of undeniable importance for cell physiology across all domains of life and a loss of their function, e.g., due to mutations in their coding sequence, is almost always linked to disease. In humans, mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-gated anion channel in epithelia, give rise to cystic fibrosis (CF). Over 2100 mutations of the CFTR gene are known, however, their disease liability remains mostly undetermined. Causal therapies, i.e., small-molecule drugs that target CFTR itself, have improved the lives of people with the most common mutations (e.g. ΔF508, G551D) over the last decade. In contrast, many rare CF-phenotypic mutations are not eligible for these novel treatments and would benefit from in vitro evaluation of their molecular consequences. In vitro studies of membrane proteins are often complicated by the intrinsic hydrophobicity and aggregation susceptibility of this protein group. However, this can be avoided by using short membrane protein fragments corresponding to the smallest in vivo folding unit of the respective protein at the ER membrane. These model proteins can be easily genetically modified, expressed and purified, making them a suitable tool to pinpoint the effects of mutations. This thesis demonstrates the utility of such a reductionist model system: TM3/4, the second helical hairpin of CFTR’s transmembrane domain 1, was used to study protein folding with a focus on disease-causing missense mutations of CFTR, which may cause CFTR misfolding in vivo. TM3/4 purification was first optimized by using a thioredoxin tag, which allowed heat purification of the fusion protein even after initial purification steps. Optimal heat treatment for maximal protein purity and recovery were determined for TM3/4 and another helical hairpin, ATP synthase subunit c. Moreover, tertiary folding of a CF-phenotypic loop mutation, E217G, introducing a non-native GXXXG interaction motif was analyzed by single-molecule Förster resonance energy transfer (smFRET) in different lipid bilayer conditions, showing unusually increased stability in comparison to wild type (WT) TM3/4. Furthermore, smFRET was used in tandem with circular dichroism and fluorescence spectroscopy to assess the effect of a specific membrane lipid, cholesterol, on TM3/4 variants showing significant changes on secondary but not tertiary structure. Lastly, a mutant library of 13 TM3/4 mutants was established to perform drug screenings with CFTR correctors – a class of small molecules rescuing or preventing misfolding of CFTR. This screening study demonstrated that (i) not all CF-phenotypic missense mutations are locally misfolded at a lipid bilayer comparable to the ER membrane; and (ii) in vitro restoration of a native WT-like conformation of locally misfolded TM3/4 mutants is not only possible but different drug-mutant pairings can be identified related to folding rescue efficiency of a given corrector on a respective mutant. The latter identified drug-mutant pairings may lead to drug repurposing if the effect can be confirmed in cell culture experiments. In conclusion, the TM3/4 minimal model of CFTR and biophysical methods, such as smFRET, proved as versatile tools not only for investigation of mutation and lipid effects on membrane protein folding but also for drug screenings in a disease context.:1 INTRODUCTION 2 THEORETICAL BACKGROUND 2.1 MEMBRANE PROTEINS AND THEIR NATIVE ENVIRONMENTS 2.1.1 Membrane protein families and their role in human health 2.1.2 Fundamental folding models of α-helical membrane proteins 2.1.3 Co-translational folding at the ER supported by the translocon 2.1.4 Folding-relevant interactions within membrane proteins 2.1.5 Biological membranes and lipid classes 2.1.6 Physical properties of lipid bilayers impacting membrane proteins 2.1.7 Membrane models for in vitro studies 2.2 CYSTIC FIBROSIS AND CFTR 2.2.1 Pathology of cystic fibrosis 2.2.2 Structure and function of the CFTR channel 2.2.3 A minimal model of CFTR to study rare CF mutations 2.2.4 Missense mutations within the CFTR segmental model TM3/4 2.2.5 Novel modulator therapies for the treatment of cystic fibrosis 2.3 IN VITRO ASSESSMENT OF MEMBRANE PROTEIN FOLDING 2.3.1 Expression and purification of membrane proteins 2.3.2 Single-molecule FRET in single- and multi-well mode for protein folding 3 HEAT PURIFICATION OF TRX MEMBRANE PROTEIN FUSIONS 3.1 PREAMBLE AND SUMMARY 3.2 RESULTS AND DISCUSSION 4 IMPACT OF A CFTR LOOP MUTATION WITH ATYPICAL STABILITY 4.1 PREAMBLE AND SUMMARY 4.2 RESULTS AND DISCUSSION 5 EFFECTS OF CHOLESTEROL ON LOCAL CFTR FOLDING 5.1 PREAMBLE AND SUMMARY 5.2 RESULTS 5.2.1 Folding of TM3/4 hairpins in the presence of cholesterol 5.2.2 Folding of TM3/4 hairpins in the presence of Lumacaftor 5.2.3 Impact of Lumacaftor on membrane fluidity 5.3 DISCUSSION 6 CFTR CORRECTOR SCREENINGS WITH SINGLE-MOLECULE FRET 6.1 PRESCREENING TO IDENTIFY MISFOLDED TM3/4 VARIANTS 6.2 SCREENING OF MISFOLDED TM3/4 VARIANTS WITH CFTR CORRECTORS 7 CONCLUSIONS 8 OUTLOOK 9 MATERIALS AND METHODS 9.1 CONSTRUCT DESIGN OF HELICAL TRANSMEMBRANE HAIRPINS 9.2 PROTEIN EXPRESSION AND PURIFICATION 9.3 HEAT TREATMENT OF HELICAL TRANSMEMBRANE CONSTRUCTS 9.4 SINGLE-MOLECULE FRET EXPERIMENTS 9.4.1 Labeling of TM3/4 constructs 9.4.2 Liposome preparation and reconstitution of labeled protein constructs 9.4.3 Single-molecule FRET measurements in manual mode 9.4.4 Single-molecule FRET measurements in multi-well screening mode 9.5 CIRCULAR DICHROISM SPECTROSCOPY 9.5.1 Circular dichroism to determine protein heat stability 9.5.2 Circular dichroism to study protein structure in different lipid bilayers 9.6 FLUORESCENCE SPECTROSCOPY 9.6.1 Vesicle leakage assay to test lipid bilayer stability 9.6.2 Examining lipid bilayer fluidity with fluorescent probes 10 APPENDIX 10.1 GENERATION OF A TM3/4 MUTANT LIBRARY 10.2 TM3/4 SCREENINGS WITH CFTR CORRECTORS 10.2.1 SmFRET control screenings and supporting data 10.2.2 Extracted closed state fractions from smFRET screenings 10.2.3 DLS to measure vesicle integrity after corrector addition 11 REFERENCES 12 ACKNOWLEDGEMENTS 13 ERKLÄRUNG GEMÄß §5 ABS. 1 S. 3 DER PROMOTIONSORDNUNG / Membranproteine sind für die Zellphysiologie aller biologischen Domänen von unbestreitbarer Bedeutung und ein Verlust ihrer Funktion, z.B. durch Mutationen in ihrer kodierenden Sequenz, ist fast immer Auslöser von Krankheiten. Beim Menschen führen Mutationen im Gen für den Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), einen ATP-abhängigen Anionenkanal in Epithelien, zu Mukoviszidose (CF). Über 2100 Mutationen des CFTR-Gens sind bekannt – ob jedoch alle Mutationen tatsächlich CF auslösen, ist weitgehend ungeklärt. Kausale Therapien, d.h. niedermolekulare Medikamente, die auf CFTR selbst abzielen, haben in den letzten zehn Jahren die Lebensqualität von Menschen mit den häufigsten Mutationen (z.B. ΔF508, G551D) verbessert. Demgegenüber stehen jedoch viele seltene CF-phänotypische Mutationen, für welche diese neuartigen Behandlungen nicht zugelassen sind, wodurch diese Mutationen von einer In-vitro-Analyse ihrer molekularen Konsequenzen profitieren würden. In-vitro-Untersuchungen von Membranproteinen werden oft durch die intrinsische Hydrophobizität und Aggregationsanfälligkeit dieser Proteine erschwert. Dies kann jedoch vermieden werden, indem kurze Membranproteinfragmente verwendet werden, die der kleinsten in vivo Faltungseinheit des jeweiligen Proteins an der ER-Membran entsprechen. Diese Modellproteine können routiniert genetisch verändert, exprimiert und aufgereinigt werden, was sie zu einem geeigneten Werkzeug macht, um die Auswirkungen von Mutationen zu genau festzustellen. Diese Dissertation demonstriert die Nützlichkeit eines solchen reduktionistischen Modellsystems: TM3/4, das zweite helikale Haarnadel-Motiv der Transmembrandomäne 1 von CFTR, wurde verwendet, um Proteinfaltung mit Schwerpunkt auf krankheitsverursachende Missense-Mutationen von CFTR zu untersuchen, welche eine CFTR-Fehlfaltung in vivo verursachen können. Die TM3/4-Aufreinigung wurde zunächst durch die Verwendung eines Thioredoxin-Tags optimiert, der eine Hitzeaufreinigung des Fusionsproteins auch nach anfänglichen Reinigungsschritten ermöglichte. Die optimale Hitzebehandlung für maximale Proteinreinheit und -ausbeute wurde für TM3/4 und ein weiteres helikales Haarnadelprotein, die ATP-Synthase-Untereinheit c, bestimmt. Weiterhin wurde die tertiäre Faltung einer CF-phänotypischen Mutation, E217G, die ein nicht-natives GXXXG-Interaktionsmotiv einführt, mittels einzelmolekularem Förster-Resonanzenergietransfer (smFRET) in verschiedenen Lipiddoppelschichten analysiert, welche eine ungewöhnlich erhöhte Stabilität im Vergleich zum TM3/4-Wildtyp (WT) zeigte. Darüber hinaus wurde smFRET in Verbindung mit Circulardichroismus und Fluoreszenzspektroskopie verwendet, um die Wirkung eines spezifischen Membranlipids, Cholesterin, auf TM3/4-Varianten zu untersuchen, welches signifikante Auswirkungen auf die sekundäre, aber nicht auf die tertiäre Proteinstruktur hatte. Schließlich wurde eine Mutantenbibliothek von 13 TM3/4-Mutanten eingerichtet, um Wirkstoffscreenings mit CFTR-Korrektoren durchzuführen – einer Klasse kleiner Moleküle, die die Fehlfaltung von CFTR verhindern können. Diese Screening-Studie zeigte, dass (i) nicht alle CF-phänotypischen Missense-Mutationen lokal an einer Lipiddoppelschicht fehlgefaltet sind, die mit der ER-Membran vergleichbar ist; und (ii) die In-vitro-Wiederherstellung einer nativen WT-ähnlichen Konformation von lokal fehlgefalteten TM3/4-Mutanten ist nicht nur möglich, sondern es können auch verschiedene Wirkstoff-Mutanten-Paare identifiziert werden, die mit der Faltungsrettungseffizienz eines Korrektors auf eine bestimmte Mutante zusammenhängen. Die letztgenannten Wirkstoff-Mutanten-Paare können zu Drug-Repurposings führen, wenn die Wirkung in Zellkulturexperimenten bestätigt werden kann. Im Allgemeinen, haben sich das TM3/4-Minimalfaltungsmodell von CFTR sowie biophysikalische Methoden, wie z.B. smFRET, als vielseitige Werkzeuge nicht nur für die Untersuchung von Mutations- und Lipideffekten auf die Membranproteinfaltung, sondern auch für das Screening von Medikamenten im Krankheitskontext erwiesen.:1 INTRODUCTION 2 THEORETICAL BACKGROUND 2.1 MEMBRANE PROTEINS AND THEIR NATIVE ENVIRONMENTS 2.1.1 Membrane protein families and their role in human health 2.1.2 Fundamental folding models of α-helical membrane proteins 2.1.3 Co-translational folding at the ER supported by the translocon 2.1.4 Folding-relevant interactions within membrane proteins 2.1.5 Biological membranes and lipid classes 2.1.6 Physical properties of lipid bilayers impacting membrane proteins 2.1.7 Membrane models for in vitro studies 2.2 CYSTIC FIBROSIS AND CFTR 2.2.1 Pathology of cystic fibrosis 2.2.2 Structure and function of the CFTR channel 2.2.3 A minimal model of CFTR to study rare CF mutations 2.2.4 Missense mutations within the CFTR segmental model TM3/4 2.2.5 Novel modulator therapies for the treatment of cystic fibrosis 2.3 IN VITRO ASSESSMENT OF MEMBRANE PROTEIN FOLDING 2.3.1 Expression and purification of membrane proteins 2.3.2 Single-molecule FRET in single- and multi-well mode for protein folding 3 HEAT PURIFICATION OF TRX MEMBRANE PROTEIN FUSIONS 3.1 PREAMBLE AND SUMMARY 3.2 RESULTS AND DISCUSSION 4 IMPACT OF A CFTR LOOP MUTATION WITH ATYPICAL STABILITY 4.1 PREAMBLE AND SUMMARY 4.2 RESULTS AND DISCUSSION 5 EFFECTS OF CHOLESTEROL ON LOCAL CFTR FOLDING 5.1 PREAMBLE AND SUMMARY 5.2 RESULTS 5.2.1 Folding of TM3/4 hairpins in the presence of cholesterol 5.2.2 Folding of TM3/4 hairpins in the presence of Lumacaftor 5.2.3 Impact of Lumacaftor on membrane fluidity 5.3 DISCUSSION 6 CFTR CORRECTOR SCREENINGS WITH SINGLE-MOLECULE FRET 6.1 PRESCREENING TO IDENTIFY MISFOLDED TM3/4 VARIANTS 6.2 SCREENING OF MISFOLDED TM3/4 VARIANTS WITH CFTR CORRECTORS 7 CONCLUSIONS 8 OUTLOOK 9 MATERIALS AND METHODS 9.1 CONSTRUCT DESIGN OF HELICAL TRANSMEMBRANE HAIRPINS 9.2 PROTEIN EXPRESSION AND PURIFICATION 9.3 HEAT TREATMENT OF HELICAL TRANSMEMBRANE CONSTRUCTS 9.4 SINGLE-MOLECULE FRET EXPERIMENTS 9.4.1 Labeling of TM3/4 constructs 9.4.2 Liposome preparation and reconstitution of labeled protein constructs 9.4.3 Single-molecule FRET measurements in manual mode 9.4.4 Single-molecule FRET measurements in multi-well screening mode 9.5 CIRCULAR DICHROISM SPECTROSCOPY 9.5.1 Circular dichroism to determine protein heat stability 9.5.2 Circular dichroism to study protein structure in different lipid bilayers 9.6 FLUORESCENCE SPECTROSCOPY 9.6.1 Vesicle leakage assay to test lipid bilayer stability 9.6.2 Examining lipid bilayer fluidity with fluorescent probes 10 APPENDIX 10.1 GENERATION OF A TM3/4 MUTANT LIBRARY 10.2 TM3/4 SCREENINGS WITH CFTR CORRECTORS 10.2.1 SmFRET control screenings and supporting data 10.2.2 Extracted closed state fractions from smFRET screenings 10.2.3 DLS to measure vesicle integrity after corrector addition 11 REFERENCES 12 ACKNOWLEDGEMENTS 13 ERKLÄRUNG GEMÄß §5 ABS. 1 S. 3 DER PROMOTIONSORDNUNG

<p>The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease.</p><p>Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells.</p><p>S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. </p><p>Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity.</p><p>Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.</p>

Cystic Fibrosis (CF) is an autosomal recessive disease that arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The deletion of phenylalanine-508 (ΔF508-CFTR) is the most prevalent CF mutation and results in a misfolded protein that fails to exit the endoplasmic reticulum (ER). Previous studies demonstrated that mutation of a di-arginine based ER retention motif (R553AR555) in the first nucleotide binding domain (NBD1) rescues the trafficking defect of ΔF508-CFTR. We hypothesized that if the R553AR555 motif mediates retention of the ΔF508-CFTR protein, peptides that mimic this motif should antagonize mistrafficking mediated by aberrant exposure of the endogenous R553AR555 motif. We generated a peptide bearing the R553AR555 motif (CF-RXR) and conjugated it to the cell penetrating peptide Tat (CPP-CF-RXR) to facilitate intracellular delivery and investigated its efficacy in rescuing the mistrafficking and function of ΔF508-CFTR. Using a variety of biochemical and functional assays we demonstrate that the CPP-CF-RXR peptide is effective at increasing surface expression of ΔF508-CFTR in baby hamster kidney (BHK) and human embryonic kidney (HEK) cell lines. Furthermore, the increased surface expression is accompanied by an increase in its functional expression as a chloride channel. Using Ussing chamber assays, we demonstrate that the CPP-CF-RXR peptide improved ΔF508-CFTR channel function in respiratory epithelial tissues obtained from CF patients. Additionally, we investigated the effects of small molecules on mediating biosynthetic rescue of a ΔF508-CFTR construct bearing the additional mutations R553K and R555K (ΔFRK-CFTR) to inactivate the R553AR555 motif. Interestingly, mutation of the R553AR555 motif exerts an additive effect with correctors VRT-325 and Corrector 4a. Taken together, our data suggests that abnormal accessibility of the RXR motif present in NBD1 is a key determinant of the mistrafficking of the major CF causing mutant.