Glutathione levels and BAX activation during apoptosis due to oxidative stress in cells expressing wild-type and mutant cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is characterized by chronic inflammation and an imbalance in the concentrations of alveolar and lung oxidants and antioxidants, which result in cell damage. Modifications in lung glutathione concentrations are recognized as a salient feature of inflammatory lung diseases such as cystic fibrosis, and glutathione plays a major role in protection against oxidative stress and is important in modulation of apoptosis. The cystic fibrosis transmembrane conductance regulator (CFTR) is permeable to Cl−, larger organic ions, and reduced and oxidized forms of glutathione, and the ΔF508 CFTR mutation found in cystic fibrosis patients has been correlated with impaired glutathione transport in cystic fibrosis airway epithelia. Because intracellular glutathione protects against oxidative stress-induced apoptosis, we studied the susceptibility of epithelial cells (HeLa and IB3–1) expressing normal and mutant CFTR to apoptosis triggered by H2O2. We find that cells with normal CFTR are more sensitive to oxidative stress-induced apoptosis than cells expressing defective CFTR. In addition, sensitivity to apoptosis could be correlated with glutathione levels, because depletion of intracellular glutathione results in higher levels of apoptosis, and glutathione levels decreased faster in cells expressing normal CFTR than in cells with defective CFTR during incubation with H2O2. The pro-apoptotic BCL-2 family member, BAX, is also activated faster in cells expressing normal CFTR than in those with mutant CFTR under these conditions, and artificial glutathione depletion increases the extent of BAX activation. These results suggest that glutathione-dependent BAX activation in cells with normal CFTR represents an early step in oxidative stress-induced apoptosis of these cells

CSN5 binds to misfolded CFTR and promotes its degradation

AbstractCystic fibrosis is mainly caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The aim of this study was to find cellular proteins interacting with CFTR and regulating its processing. We have used a genetic screen in yeast to identify such proteins and identified CSN5 that interacted with the third cytoplasmic loop of CFTR. CSN5 is the 5th component of the COP9 signalosome, a complex of eight subunits that shares significant homologies to the lid subcomplex of the 26S proteasome and controls the stability of many proteins. The present study shows that CSN5 associates with the core-glycosylated form of CFTR and suggests that this association targets misfolded CFTR to the degradative pathway. Identifying CSN5 as a new component of the degradative pathway is an important step towards the goal of unraveling the sorting between misfolded and correctly folded CFTR proteins

COMMD1-Mediated Ubiquitination Regulates CFTR Trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis

Combined Computational-Experimental Analyses of CFTR Exon Strength Uncover Predictability of Exon-Skipping Level.

International audienceWith the increased number of identified nucleotide sequence variations in genes, the current challenge is to classify them as disease causing or neutral. These variants of unknown clinical significance can alter multiple processes, from gene transcription to RNA splicing or protein function. Using an approach combining several in silico tools, we identified some exons presenting weaker splicing motifs than other exons in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. These exons exhibit higher rates of basal skipping than exons harboring no identifiable weak splicing signals using minigene assays. We then screened 19 described mutations in three different exons, and identified exon-skipping substitutions. These substitutions induced higher skipping levels in exons having one or more weak splicing motifs. Indeed, this level remained under 2% for exons with strong splicing motifs and could reach 40% for exons having at least one weak motif. Further analysis revealed a functional exon splicing enhancer within exon 3 that was associated with the SR protein SF2/ASF and whose disruption induced exon skipping. Exon skipping was confirmed in vivo in two nasal epithelial cell brushing samples. Our approach, which point out exons with some splicing signals weaknesses, will help spot splicing mutations of clinical relevance

Deciphering an isolated lung phenotype of NKX2-1 frameshift pathogenic variant

Backgroundto perform a functional analysis of a new NK2 homeobox 1 (NKX2-1) variant (c.85_86del denominated NKX2-1DEL) identified in a family presenting with isolated respiratory disease, in comparison to another frameshift variant (c.254dup denominated NKX2-1DUP) identified in a subject with classical brain-lung-thyroid syndrome.Methodspathogenic variants were introduced into the pcDNA3-1(+)-wt-TTF1 plasmid. The proteins obtained were analyzed by western blot assay. Subcellular localization was assessed by confocal microscopy in A549 and Nthy cells. Transactivation of SFTPA, SFTPB, SFTPC, and ABCA3 promoters was assessed in A549 cells. Thyroglobulin promoter activity was measured with the paired box gene 8 (PAX8) cofactor in Nthy cells.ResultsThe two sequence variants were predicted to produce aberrant proteins identical from the 86th amino acid, with deletion of their functional homeodomain, including the nuclear localization signal. However, 3D conformation prediction of the conformation prediction of the mutant protein assumed the presence of a nuclear localization signal, a bipartite sequence, confirmed by confocal microscopy showing both mutant proteins localized in the nucleus and cytoplasm. Transcriptional activity with SFTPA, SFTPB, SFTPC, ABCA3 and thyroglobulin promoters was significantly decreased with both variants. However, with NKX2-1DEL, thyroglobulin transcriptional activity was maintained with the addition of PAX8.ConclusionThese results provide novel insights into understanding the molecular mechanism of phenotypes associated with NKX2-1 pathogenic variants

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies

COMMD1-Mediated Ubiquitination Regulates CFTR Trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis

Contribution à l'étude du rôle de COMMD1 dans la physiopathologie de la mucoviscidose

La mucoviscidose (CF, Cystic Fibrosis) est la maladie génétique la plus fréquente dans les populations d origine caucasienne. Les malades présentent une symptomatologie variée, dominée par une bronchopneumopathie chronique obstructive due à des sécrétions de mucus abondantes et anormalement épaisses et une réponse inflammatoire chronique excessive. La mucoviscidose résulte de mutations dans le gène codant la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator), dont la plus fréquente est la délétion d une phénylalanine en position 508 (F508del) qui est à l origine d un adressage défectueux et d une fonction altérée de la protéine. Afin d identifier différents partenaires moléculaires de CFTR qui participent à son processus de maturation, à son trafic à la membrane plasmique ou à sa fonction, un criblage double hybride de levure a été effectué en utilisant la troisième boucle intra-cytoplasmique de CFTR (ICL3) comme appât. A l issue de ce criblage, 14 clones indépendants ont pu être identifiés dont la protéine COMMD1 qui a initialement été décrite comme un régulateur de l homéostasie du cuivre, de l absorption sodique et de la voie de signalisation NF-?B. L objectif principal de ce travail a été de déterminer quel pouvait être l impact de la protéine COMMD1 sur la maturation et le trafic intracellulaire de CFTR afin de déterminer le rôle de cette protéine dans la physiopathologie de la mucoviscidose. Nous avons montré que la protéine COMMD1 est un nouveau partenaire cytoplasmique du canal CFTR, qui régule le trafic intracellulaire de ce canal par inhibition de l ubiquitinylation probablement au niveau des endosomes d endocytose et de recyclage. Notre étude permet de proposer une nouvelle voie de trafic de CFTR via un modèle d ubiquitinylation régulé par COMMD1. Au cours de cette étude, nous avons également identifié le récepteur 1 de la transferrine (TFR1) comme un nouveau partenaire de COMMD1 dont le mécanisme de régulation semble similaire à celui proposé pour CFTR. Dans un second temps, nous nous sommes intéressés aux propriétés inhibitrices de COMMD1 dans la réaction inflammatoire. COMMD1 a été décrit comme le prototype d une nouvelle famille de protéines jouant un rôle dans l inhibition de la voie de signalisation NF-kB. Nous avons observé que la distribution subcellulaire de COMMD1 est différente dans les cellules CF et non-CF. Nous avons mis en évidence que la surexpression de COMMD1 dans les cellules épithéliales bronchiques CF, qui présentent une inflammation excessive, pouvait restaurer un niveau d inflammation comparable aux cellules non-CF. COMMD1 est impliquée dans plusieurs processus cellulaires altérés dans la physiopathologie de la mucoviscidose, affectant le trafic du canal CFTR, l absorption de sodium et la réponse inflammatoire. Comprendre comment moduler d une part le processus d absorption/sécrétion des ions par le trafic de canaux ioniques et d autre part, l inflammation des cellules CF par rapport aux non-CF, devrait permettre d identifier de nouvelles pistes thérapeutiques. Ces traitements permettraient à la fois de réduire la réaction inflammatoire exacerbée, sans nuire à l activité essentielle de défense contre les pathogènes, et d améliorer la sécrétion de fluide chez les patients atteints de mucoviscidoseCystic fibrosis is mainly caused by mutations interfering with the biosynthetic folding of the CFTR protein. The aim of this study was to find proteins able to interact with CFTR and modify its processing. We have identified COMMD1 as a new CFTR partner. COMMD1 is a regulator of copper homeostasis and sodium uptake through interaction with ENaC, as well as the prototype of a new protein family that plays a role in inhibiting NF-?B signalling Co-immunoprecipitation experiments showed that COMMD1 associates with endogenous CFTR in HT29 cells and with F508del-CFTR in heterologously expressing epithelial cells. COMMD1 sub-cellular distribution is both nuclear and cytoplasmic, and more precisely in vesicular cytoplasmic compartments, as assessed by immunocytochemical microscopy. Further studies showed COMMD1 partial codistribution with an early endosomal compartments (TfR). COMMD1 is not involved in CFTR processing (C band) but wt-CFTR cell surface expression was halfreduced when COMMD1 expression was silenced. Unlike F508del-CFTR in temperature rescue, COMMD1 over-expression increased 15% wt-CFTR cell surface expression. Assessment of CFTR ubiquitination showed that COMMD1 over-expression strongly decreased CFTR ubiquitination therefore increasing CFTR cell surface expression. Finally, these data indicate that COMMD1 vesicular compartment is involved in CFTR trafficking through inhibition of CFTR ubiquitination. Understanding how COMMD1 modulation modifies transepithelial transport and inflammation in CF versus non CF cells should give new therapeutic clues to reduce exacerbated inflammation and improve fluid secretion in CF patientsPARIS-EST-Université (770839901) / SudocSudocFranceF

Variabilité phénotypique et épissage (combinaison d'analyses in vitro et in silico du gène CFTR)

Depuis plusieurs décennies, l étude des conséquences des mutations pathogéniques a permisnon seulement de définir l origine de nombreuses maladies génétiques humaines, héréditaires ou non,mais également de contribuer à l interprétation de la variabilité phénotypique inter-individus au seind une pathologie donnée. Les mutations induisant une perte de fonction de la protéine synthétisée ouune protéine incomplète ont été et sont encore les plus étudiées. Avec l introduction des technologiesde séquençage à haut-débit, le nombre de variants faux-sens, silencieux ou introniques détectés auniveau des gènes humains augmente de façon continue. Distinguer les variations nucléotidiques quivont modifier significativement un phénotype de celles qui seront neutres est un véritable défi pour larecherche.De nombreuses variations nucléotidiques à signification clinique inconnue ont été identifiéesparmi les près de 2000 mutations décrites au niveau du gène CFTR (Cystic Fibrosis Transmembraneconductance Regulator) responsables de la mucoviscidose. Certaines de ces variations vont avoir unimpact sur les transcrits ARN modifiant leur qualité et leur quantité et par conséquent l expression dugène CFTR. Elles vont notamment affecter l épissage de l ARN pré-messager en altérant des signauxreconnus par la machinerie cellulaire et perturber la fidélité de ce mécanisme. Malgré un recul de plusde 20 ans dans la description de la corrélation génotype-phénotype dans cette maladie, de nombreuxphénotypes inattendus et atypiques sont observés. Ils peuvent être dus à des facteurs autres que desvariations génotypiques, mais sans l accès direct aux transcrits des individus porteurs de tels variants,il est difficile de mesurer les conséquences de ces variations sur la synthèse et la maturation de l ARN.L objectif de ce travail de thèse a été de montrer, par des approches in vitro etbioinformatiques, comment des variations nucléotidiques au sein du gène CFTR peuvent impacter surl épissage de l ARN pré-messager. Ce travail a permis dans un premier temps la découverte demécanismes d épissage inattendus modificateurs de phénotypes sévères attendus pour une mutationnon-sens. En effet, par un épissage alternatif subtil de trois nucléotides au niveau d un site d épissageen tandem, le codon stop se retrouve délété et aboutit à des transcrits fonctionnels expliquant lesphénotypes modérés. Dans un second temps, nous avons montré que plusieurs mutations non-sensportées par le même exon 15 impactaient différemment sur l épissage de l ARN en modifiantsignificativement le taux d inclusion de cet exon. La connaissance préalable des ratios de transcritsincluant ou non l exon peut améliorer l efficacité des traitements correcteurs de codons stopprématurés en les combinant avec des traitements modulateurs de l épissage augmentant le taux detranscrits correctibles. Dans un troisième et dernier temps, une combinaison d analyses in silico et invitro des exons du gène CFTR a permis de détecter des exons porteurs de signaux d épissage plusfaibles les rendant plus sensibles à des mutations exoniques d épissage. Des mutations faux-sens auniveau de l exon 3 ont par exemple été montrées comme favorisant l exclusion de cet exon, diminuantle taux de transcrits fonctionnels.L ensemble de ces travaux a contribué à comprendre les conséquences de mutations au niveaude l épissage du gène CFTR sur les variations du phénotype. La connaissance améliorée des variationspossibles du phénotype rattaché à un génotype donné permettra non seulement de prédire l évolutionde la maladie, mais également d ajuster et de proposer des thérapies personnalisées selon la mutationportée par le patient.Over the past decades, studying the consequences of pathogenic mutations has allowed not only todefine the origin of several genetic diseases, but also to contribute to understand the phenotypicvariability between individuals within a disease. The CFTR gene was extensively analyzed since 1989,but among the over 1,900 mutations identified, the current challenge is to classify them as diseasecausingor neutral. These variants of unknown clinical significance (UVs) can alter multiple processes,from gene transcription to RNA splicing or protein function. The CFTR gene needs to include intactversions of all its 27 exons to be functional, and any mutation affecting its splicing process will reducethe amount of functional full-length transcripts. Previous studies have shown that mutations withinsome CFTR exons increase exon skipping. We hypothesized that a number of UVs occurring in otherCFTR exons could indeed affect splicing. By combining in vitro and bioinformatics approaches, weshowed how nucleotide variations within the CFTR gene could impact on the splicing of its premRNA.This work enabled us to provide the first experimental evidence of a premature terminationcodon removal by alternative splicing at a NAGNAG acceptor splice site. This unexpected phenotypemodifyingmechanism explains the much milder phenotype severity than expected for a nonsensemutation. The correction of premature termination codons (PTCs) by agents that promote readthroughrepresents a promising emerging tool for the treatment of many genetic diseases. Having demonstratedthat nonsense mutation could cause aberrant splicing, we postulated that the efficiency of thereadthrough treatment could be due not only to the stop codon itself but also to the amount ofcorrectible transcripts. We showed that a subset of nonsense mutations within the CFTR exon 15 has adifferent impact on the splicing efficiency by modifying the inclusion rate of this exon anddemonstrated that the total amount of transcripts together with the splicing profile should be assessedto anticipate and improve efficacy of readthrough therapy in CF patients. Finally, in order to anticipatethe occurrence of splicing-causing mutations, we used a combination of in silico and in vitroanalyses of all CFTR exons and pinpointed those harboring weak splicing signals, which render themmore sensitive to exonic splicing mutations.All these studies contribute to expand our knowledge on the phenotypic variability due to alternativesplicing of the CFTR gene. These studies will lead not only to predict the evolution of a disease, butalso to adjust therapy according to the mutation of each patient.PARIS-EST-Université (770839901) / SudocPARIS12-Bib. électronique (940280011) / SudocSudocFranceF

Rôle des interactions moléculaires dans le processus de maturation de la protéine CFTR (implications de CSN5 et de la voie du signalosome)

La mucoviscidose, maladie génétique la plus fréquente dans les populations d origine caucasienne, résulte de mutations dans le gène codant la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator). La plus fréquente des mutations de CFTR est une délétion du codon correspondant à la phénylalanine 508 (F508del) qui est à l origine d un adressage défectueux et d une fonction altérée de la protéine. L objectif de ce travail a été d identifier des protéines interagissant avec CFTR et pouvant jouer un rôle dans son adressage, son recyclage à la membrane ou sa fonction. Pour cela, un crible double hybride de levure a été réalisé avec la troisième boucle cytoplasmique de CFTR comme appât. Ce test a permis d identifier 14 nouveaux partenaires dans la levure. L interaction de CFTR avec quatre d entre eux a ensuite été confirmée dans des modèles cellulaires humains. Le travail a été orienté ensuite vers une étude approfondie du rôle de l un de ces partenaires, CSN5, dans le processus de maturation de CFTR. Nous avons montré que CSN5, cinquième sous-unité du signalosome, interagit avec la forme mal repliée de CFTR et l envoie vers la dégradation via le protéasome. Notre étude permet de proposer un modèle de dégradation de CFTR où le signalosome constitue un nouveau point de contrôle du processus de maturation de CFTR. Par ailleurs, nous avons montré dans deux systèmes cellulaires que CFTR était NEDDylé, cette nouvelle modification post-traductionnelle étant dépendante de CSN5. Dans un dernier temps, nous avons étudié le rôle de CSN5 dans la régulation de gènes dans un contexte CF grâce à une puce ADN. L extinction de CSN5 dans des cellules épithéliales bronchiques IB3-1 (F508del/W1282X), entraîne une dérégulation de près de 400 gènes dont nombre sont impliqués dans les voies de l inflammation et de réponse au stress. Au niveau protéique, CSN5 est également impliqué dans la régulation négative de la production de cytokines pro-inflammatoires IL-8 via une stimulation par le TNFa. CSN5 interagit avec une variété de molécules de signalisation en régulant leur stabilité et peut également diriger les cellules vers une voie de réponse au stress (UPR) ou l apoptose. Identifier CSN5 comme un composant de la voie de dégradation est une étape importante dans la compréhension du processus de maturation de la protéine CFTR, impliquant potentiellement une étape de NEDDylation. De plus, CSN5 pourrait être impliqué dans le mécanisme de réponse inflammatoire exacerbée observé chez les patients CF.Cystic Fibrosis is the most common genetic disease in Caucasians and is caused by mutations in the CFTR gene (Cystic Fibrosis Transmembrane conductance Regulator). The most frequent mutation is the deletion of Phenylalanine 508 (F508del) that produces a protein with processing and functional defects. The aim of our work was to identify new CFTR partners, implicated in its processing, recycling or function. We realized a yeast two hybrid screening with the third cytoplasmic loop of CFTR as a bait. We identified 14 new CFTR partners in yeast with this approach. The interaction between CFTR and 4 of these proteins was confirmed in human cellular models. We then studied the role of CSN5 in CFTR processing. We showed that CSN5, the fifth subunit of signalosome, associates with misfolded CFTR and targets it to the proteasome-dependant degradation pathaway. We propose an additional degradation pathway of CFTR implicating the signalosome as a new processing checkpoint. We also showed in two different cellular models that CFTR is NEDDylated and that this new post-translational modification is CSN5-dependant. In a second study, we investigated the role of CSN5 in gene regulation in CF cells using DNA microarrays. IB3-1 cells (F508del/W1282X) treated with siCSN5 presented a deregulation of more than 400 genes, most of them implicated in inflammatory and stress response pathways. CSN5 was also found to be a negative regulator of pro-inflammatory cytokine IL-8 production in response to TNFa stimulation. CSN5 interacts with a variety of signalling molecules and regulates their stability, and is also a key molecule that directs cells towards apoptosis or stress response (UPR). Identifying CSN5 as a new component of the network of the degradative pathway is an important step towards the goal of unraveling the sorting between misfolded and correctly folded CFTR proteins, possibly implying a NEDDYlation step. Moreover, CSN5 could be considered in the mechanisms of excessive inflammatory response observed in CF patients.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF