RESUMO
Cystic Fibrosis (CF) is present due to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, the most frequent variant being p.phe508del. The CFTR protein is a chloride (Cl-) channel which is defective and almost absent of cell membranes when the p.Phe508del mutation is present. The p.Phe508del-CFTR protein is retained in the endoplasmic reticulum (ER) and together with inflammation and infection triggers the Unfolded Protein Response (UPR). During the UPR, the Activating Transcription Factor 6 (ATF6) is activated with cleavage and then decreases the expression of p.Phe508del-CFTR. We have previously shown that the inhibition of the activation of ATF6 alleviates the p.Phe508del-CFTR defects in cells overexpressing the mutated protein. In the present paper, our aim was to inhibit the cleavage of ATF6, and thus its activation in a human bronchial cell line with endogenous p.Phe508del-CFTR expression and in bronchial cells from patients, to be more relevant to CF. This was achieved by inhibiting the protease MBTP1 which is responsible for the cleavage of ATF6. We show here that this inhibition leads to increased mRNA and p.Phe508del-CFTR expression and, consequently, to increased Cl-efflux. We also explain the mechanisms linked to these increases with the modulation of genes when MBTP1 is inhibited. Indeed, RT-qPCR assays show that genes such as HSPA1B, CEBPB, VIMP, PFND2, MAPK8, XBP1, INSIG1, and CALR are modulated. In conclusion, we show that the inhibition of MBTP1 has a beneficial effect in relevant models to CF and that this is due to the modulation of genes involved in the disease.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Pró-Proteína Convertases , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fatores de Transcrição , Serina EndopeptidasesRESUMO
Disorders of the autosomal dominant polycystic kidney disease (ADPKD) spectrum are characterized by the development of kidney cysts and progressive kidney function decline. PKD1 and PKD2, encoding polycystin (PC)1 and 2, are the two major genes associated with ADPKD; other genes include IFT140, GANAB, DNAJB11, and ALG9. Genetic testing remains inconclusive in â¼7% of the families. We performed whole-exome sequencing in a large multiplex genetically unresolved (GUR) family affected by ADPKD-like symptoms and identified a monoallelic frameshift variant (c.703_704delCA) in ALG5. ALG5 encodes an endoplasmic-reticulum-resident enzyme required for addition of glucose molecules to the assembling N-glycan precursors. To identify additional families, we screened a cohort of 1,213 families with ADPKD-like and/or autosomal-dominant tubulointerstitial kidney diseases (ADTKD), GUR (n = 137) or naive to genetic testing (n = 1,076), by targeted massively parallel sequencing, and we accessed Genomics England 100,000 Genomes Project data. Four additional families with pathogenic variants in ALG5 were identified. Clinical presentation was consistent in the 23 affected members, with non-enlarged cystic kidneys and few or no liver cysts; 8 subjects reached end-stage kidney disease from 62 to 91 years of age. We demonstrate that ALG5 haploinsufficiency is sufficient to alter the synthesis of the N-glycan chain in renal epithelial cells. We also show that ALG5 is required for PC1 maturation and membrane and ciliary localization and that heterozygous loss of ALG5 affects PC1 maturation. Overall, our results indicate that monoallelic variants of ALG5 lead to a disorder of the ADPKD-spectrum characterized by multiple small kidney cysts, progressive interstitial fibrosis, and kidney function decline.
Assuntos
Cistos , Rim Policístico Autossômico Dominante , Cistos/genética , Fibrose , Humanos , Rim/patologia , Mutação/genética , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Sequenciamento do ExomaRESUMO
The aminopyrrolidine amide PF-429242 is a specific inhibitor of the Site-1 Protease which is responsible for the cleavage, and thus the activation of the Activating Transcription Factor6 that down regulates many genes, during the Unfolded Protein Response. We hypothesized that PF-429242 could be used to prevent the ATF6-dependent down regulation of some genes. We chose the CFTR gene encoding the CFTR chloride channel as a model because it is down-regulated by ATF6 in Cystic Fibrosis. We evaluated the action of PF-429242 in human bronchial cells expressing the most frequent mutation of CFTR (p.Phe508del) found in patients. We observed that PF-429242 increases the synthesis of the mRNA and the protein encoded by the CFTR gene harbouring the mutation. We also observed that PF-429242 alleviates the defects of the p.Phe508del-CFTR channel in human Cystic Fibrosis cells. Our results suggest that aminopyrrolidine amide is a potential therapeutic target for Cystic Fibrosis that could also have beneficial effects in other diseases involving CFTR, such as the Chronic Obstructive Pulmonary Disease.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Amidas/farmacologia , Amidas/uso terapêutico , Cloretos/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Transporte de ÍonsRESUMO
In cystic fibrosis (CF), p.Phe508del is the most frequent mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The p.Phe508del-CFTR protein is retained in the ER and rapidly degraded. This retention likely triggers an atypical Unfolded Protein Response (UPR) involving ATF6, which reduces the expression of p.Phe508del-CFTR. There are still some debates on the role of the UPR in CF: could it be triggered by the accumulation of misfolded CFTR proteins in the endoplasmic reticulum as was proposed for the most common CFTR mutation p.Phe508del? Or, is it the consequence of inflammation and infection that occur in the disease? In this review, we summarize recent findings on UPR in CF and show how infection, inflammation and UPR act together in CF. We propose to rethink their respective role in CF and to consider them as a whole.
Assuntos
Fibrose Cística/complicações , Fibrose Cística/microbiologia , Infecções/complicações , Inflamação/complicações , Resposta a Proteínas não Dobradas , Animais , Humanos , Modelos Biológicos , Processamento de Proteína Pós-TraducionalRESUMO
We previously reported a 40-transcripts signature marking the normal mucosa to colorectal adenocarcinoma transition. Eight of these mRNAs also showed splicing alterations, including a specific intron 3 retention in tissue metalloprotease inhibitor I (TIMP1), which decreased during the early steps of colorectal cancer progression. To decipher the mechanism of intron 3 retention/splicing, we first searched for putative RNA binding protein binding sites onto the TIMP1 sequence. We identified potential serine arginine rich splicing factor 1 (SRSF1) and heterogeneous nuclear RiboNucleoProtein A1 (hnRNPA1) binding sites at the end of intron 3 and the beginning of exon 4, respectively. RNA immunoprecipitation showed that hnRNPA1, but not SRSF1 could bind to the corresponding region in TIMP1 pre-mRNA in live cells. Furthermore, using a TIMP1-based ex vivo minigene approach, together with a plasmon resonance in vitro RNA binding assay, we confirmed that hnRNPA1 could indeed bind to wild type TIMP1 exon 4 pre-mRNA and control TMP1 intron 3 splicing, the interaction being abolished in presence of a mutant sequence that disrupted this site. These results indicated that hnRNPA1, upon binding to TIMP1 exon 4, was a positive regulator of intron 3 splicing. We propose that this TIMP1-intron 3 + transcript belongs to the class of nuclear transcripts with "detained" introns, an abundant molecular class, including in cancer.
Assuntos
Neoplasias do Colo/genética , Ribonucleoproteína Nuclear Heterogênea A1/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Processamento Alternativo , Sítios de Ligação/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/metabolismo , Éxons , Células HCT116 , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Íntrons , Ligação Proteica/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride (Cl-) channel regulated by protein kinases, phosphatases, divalent cations and by protein-protein interactions. Among protein-protein interactions, we previously showed that Annexin A5 (AnxA5) binds to CFTR and is involved in the channel localization within membranes and in its Cl- channel function. The deletion of phenylalanine at position 508 (F508del) is the most common mutation in CF which leads to an altered protein (F508del-CFTR) folding with a nascent protein retained within the ER and is quickly degraded. We previously showed that AnxA5 binds to F508del-CFTR and that its increased expression due to a Gonadoliberin (GnRH) augments Cl- efflux in cells expressing F508del-CFTR. The aim of the present work was to use the GnRH analog buserelin which is already used in medicine. Human nasal epithelial cells from controls and CF patients (F508del/F508del) were treated with buserelin and we show here that the treatment alleviates Cl- channel defects in CF cells. Using proteomics we highlighted some proteins explaining this result. Finally, we propose that buserelin is a potential new pharmaceutical compound that can be used in CF and that bronchus can be targeted since we show here that they express GnRH-R.
Assuntos
Busserrelina/farmacologia , Cloretos/metabolismo , Fibrose Cística/metabolismo , Mucosa Nasal/efeitos dos fármacos , Estudos de Casos e Controles , Células Cultivadas , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Transporte de Íons , Mucosa Nasal/metabolismoRESUMO
Cystic Fibrosis is the most common lethal autosomal recessive disorder in the white population, affecting among other organs, the lung, the pancreas and the liver. Whereas Cystic Fibrosis is a monogenic disease, many studies reveal a very complex relationship between genotype and clinical phenotype. Indeed, the broad phenotypic spectrum observed in Cystic Fibrosis is far from being explained by obvious genotype-phenotype correlations and it is admitted that Cystic Fibrosis disease is the result of multiple factors, including effects of the environment as well as modifier genes. Our objective was to highlight new modifier genes with potential implications in the lung, pancreatic and liver outcomes of the disease. For this purpose we performed a system biology approach which combined, database mining, literature mining, gene expression study and network analysis as well as pathway enrichment analysis and protein-protein interactions. We found that IFI16, CCNE2 and IGFBP2 are potential modifiers in the altered lung function in Cystic Fibrosis. We also found that EPHX1, HLA-DQA1, HLA-DQB1, DSP and SLC33A1, GPNMB, NCF2, RASGRP1, LGALS3 and PTPN13, are potential modifiers in pancreas and liver, respectively. Associated pathways indicate that immune system is likely involved and that Ubiquitin C is probably a central node, linking Cystic Fibrosis to liver and pancreatic disease. We highlight here new modifier genes with potential implications in Cystic Fibrosis. Nevertheless, our in silico analysis requires functional analysis to give our results a physiological relevance.
Assuntos
Fibrose Cística/genética , Genes Modificadores , Hepatopatias/genética , Pancreatopatias/genética , Simulação por Computador , Ciclinas/genética , Fibrose Cística/complicações , Bases de Dados Genéticas , Estudos de Associação Genética , Genótipo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Hepatopatias/complicações , Mutação , Proteínas Nucleares/genética , Pancreatopatias/complicações , Fenótipo , Fosfoproteínas/genéticaRESUMO
Cystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca2+) homeostasis. In this study, we discovered that the Ca2+ binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca2+ calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca2+ concentration. In addition, reducing Calumenin expression in CF cells results in a partial restoration of CFTR activity, highlighting a potential function of Calumenin in CFTR maturation. These findings demonstrate a pivotal role for Calumenin in CF cells, providing insights into how modulation of Calumenin expression or activity may be used as a potential therapeutic tool to correct defects in F508del-CFTR.
Assuntos
Brônquios/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Homeostase , Brônquios/patologia , Células Cultivadas , HumanosRESUMO
Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Polaridade Celular , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Citosol/metabolismo , Células Epiteliais/citologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Nucleosídeo NM23 Difosfato Quinases/química , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Sistema Respiratório/citologia , Adulto JovemRESUMO
Cystic Fibrosis is due to mutations in the CFTR gene. The missense mutation G551D (approx. 5% of cases) encodes a CFTR chloride channel with normal cell surface expression but with an altered chloride channel activity, leading to a severe phenotype. Our aim was to identify specific interacting proteins of G551D-CFTR which could explain the channel defect. Wild-type CFTR (Wt-CFTR) was co-immunoprecipitated from stably transfected HeLa cells and resolved by 2D gel electrophoresis. Among the detected spots, one was expressed at a high level. Mass Spectrometry revealed that it corresponded to actin which is known to be involved in the CFTR's channel function. To assess whether actin could be involved in the altered G551D-CFTR function, its basal expression was studied. Because actin expression was the same in wt- and in G551D-CFTR expressing cells, its interaction with both wt- and G551D-CFTR was studied by co-immunoprecipitation, and we found that a higher amount of actin was bound onto G551D-CFTR than onto Wt-CFTR. The role of actin upon wt- and G551D-CFTR function was further studied by patch-clamp experiments after cytochalasin D treatment of the cells. We found a decrease of the very weak currents in G551D-CFTR expressing cells. Because a higher amount of actin is bound onto G551D-CFTR than onto Wt-CFTR, it is likely to be not involved in the mutated CFTR's defect. Nevertheless, because actin is necessary to maintain the very weak global currents observed in G551D-CFTR expressing HeLa cells, we conclude that more actin is necessary to maintain G551D-CFTR in the plasma membrane than for Wt-CFTR.
Assuntos
Actinas/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Eletroforese em Gel Bidimensional , Células HeLa , Humanos , Imunoprecipitação , Espectrometria de Massas , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Polimorfismo de Nucleotídeo Único , Ligação ProteicaRESUMO
The cystic fibrosis transmembrane regulator (CFTR) is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF) patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs). We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with IDPs and is hypothesized to regulate F508del-CFTR folding by electrostatic effects. This work provides useful insights for designing optimized synthetic structural correctors of CFTR mutant proteins in the future.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Dobramento de Proteína , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Mutação , Filogenia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Conformação Proteica , Transporte Proteico , Eletricidade EstáticaRESUMO
Cystic fibrosis (CF), the most common autosomal recessive disease in Caucasians, is due to mutations in the CFTR gene. F508del, the most frequent mutation in patients, impairs CFTR protein folding and biosynthesis. The F508del-CFTR protein is retained in the endoplasmic reticulum (ER) and its traffic to the plasma membrane is altered. Nevertheless, if it reaches the cell surface, it exhibits a Cl(-) channel function despite a short half-life. Pharmacological treatments may target the F508del-CFTR defect directly by binding to the mutant protein or indirectly by altering cellular proteostasis, and promote its plasma membrane targeting and stability. We previously showed that annexine A5 (AnxA5) directly binds to F508del-CFTR and, when overexpressed, promotes its membrane stability, leading to the restoration of some Cl(-) channel function in cells. Because Gonadotropin-Releasing Hormone (GnRH) increases AnxA5 expression in some cells, we tested it in CF cells. We showed that human epithelial cells express GnRH-receptors (GnRH-R) and that GnRH induces an AnxA5 overexpression and an increased Cl(-) channel function in F508del-CFTR cells, due to an increased stability of the protein in the membranes. Beside the numerous physiological implications of the GnRH-R expression in epithelial cells, we propose that a topical use of GnRH is a potential treatment in CF.
Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Anexina A5/genética , Anexina A5/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Células Cultivadas , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/patologia , Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Receptores LHRH/genética , Receptores LHRH/metabolismoRESUMO
Inherited thrombocytopenia is a heterogeneous group of disorders characterized by a reduced number of blood platelets. Despite the identification of nearly 20 causative genes in the past decade, approximately half of all subjects with inherited thrombocytopenia still remain unexplained in terms of the underlying pathogenic mechanisms. Here we report a six-generation French pedigree with an autosomal dominant mode of inheritance and the identification of its genetic basis. Of the 55 subjects available for analysis, 26 were diagnosed with isolated macrothrombocytopenia. Genome-wide linkage analysis mapped a 10.9 Mb locus to chromosome 14 (14q22) with a LOD score of 7.6. Candidate gene analysis complemented by targeted next-generation sequencing identified a missense mutation (c.137GA; p.Arg46Gln) in the alpha-actinin 1 gene (ACTN1) that segregated with macrothrombocytopenia in this large pedigree. The missense mutation occurred within actin-binding domain of alpha-actinin 1, a functionally critical domain that crosslinks actin filaments into bundles. The evaluation of cultured mutation-harboring megakaryocytes by electron microscopy and the immunofluorescence examination of transfected COS-7 cells suggested that the mutation causes disorganization of the cellular cytoplasm. Our study concurred with a recently published whole-exome sequence analysis of six small Japanese families with congenital macrothrombocytopenia, adding ACTN1 to the growing list of thrombocytopenia genes.
Assuntos
Actinina/genética , Plaquetas/patologia , Genes Dominantes , Mutação de Sentido Incorreto , Trombocitopenia/genética , Trombocitopenia/patologia , Actinina/metabolismo , Adolescente , Adulto , Idoso , Animais , Plaquetas/ultraestrutura , Medula Óssea/patologia , Células COS , Chlorocebus aethiops , Família , Feminino , França , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Agregação Plaquetária , Contagem de Plaquetas , Análise de Sequência de DNA , Trombocitopenia/metabolismo , Adulto JovemRESUMO
Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in the Caucasian population. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To date, over 1910 mutations have been identified in the CFTR gene. Among these mutations, the CF-causing missense mutation G551D-CFTR (approx. 5% of cases) encodes for a CFTR chloride channel with normal expression on the cell surface. Nevertheless, it is associated with severe disease due to its altered channel activation. The aim of the present study was to identify specific interacting proteins of G551D-CFTR. Co-immunoprecipitated proteins with G551D-CFTR were resolved by 2D-gel electrophoresis (2-DE). Mass Spectrometry revealed that calumenin was present in the protein complex linked to G551D-CFTR. Despite its basal expression was not modified in G551D-CFTR expressing cells when compared to Wt-CFTR expressing cells, it was more abundant in the G551D-CFTR complex detected by immunoprecipitation. The calumenin-CFTR interaction was also shown by Surface Plasmon Resonance and further confirmed by computational analysis of the predicted calumenin's partners. Because in our cellular model calumenin was found in the endoplasmic reticulum (ER) by immunofluorescence experiments, we suggest that calumenin is likely involved in the mutated CFTR's maturation. In conclusion, we showed for the first time that calumenin binds to CFTR and that it is increased in the G551D-CFTR complex. We suggest that it may be involved in the physiopathology of G551D-CFTR and that G551D-CFTR may follow a specific maturation and trafficking pathway. We also hypothesize that UPR may be triggered independently of the retention of G551D-CFTR in the ER because Grp78/Bip expression is increased in the cells. Finally, we propose here that Calumenin is a new CFTR chaperone.
Assuntos
Substituição de Aminoácidos/genética , Proteínas de Ligação ao Cálcio/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Mutação de Sentido Incorreto/genética , Proteômica/métodos , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Células HeLa , Humanos , Espectrometria de Massas , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Resposta a Proteínas não DobradasRESUMO
The level of circulating anxA5 is correlated to various diseases such as acute myocardial infarction, trauma, thrombosis, inflammation and in some cancers. Our aim was to assess whether a direct approach using surface plasmon resonance (SPR) could be easily used to provide a rapid and cheap alternative to detect anxA5 in blood samples in human. Our results indicate that SPR permits to detect and quantify circulating anxA5 in serum with a minimum time of manipulation. Furthermore, we report here, for the first time, that the level of circulating anxA5 is significantly higher in male than in female (5.43 (± 0.02) and 4.41 (± 0.2)ng/ml, respectively). In conclusion, we found that SPR can be used to rapidly quantify anxA5 in patients and that a gender difference has to be taken into account to explain gender differences in the prevalence of some diseases.
Assuntos
Anexina A5/sangue , Caracteres Sexuais , Ressonância de Plasmônio de Superfície/métodos , Anexina A5/imunologia , Anticorpos Imobilizados/imunologia , Calibragem , Serviços Médicos de Emergência , Feminino , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
In cystic fibrosis (CF), the most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del-CFTR protein, is misfolded and retained in the endoplasmic reticulum (ER). We previously showed that the unfolded protein response (UPR) may be triggered in CF. Since prolonged UPR activation leads to apoptosis via the calcium-calpain-caspase-12-caspase-3 cascade and because apoptosis is altered in CF, our aim was to compare the ER stress-induced apoptosis pathway between wild type (Wt) and F508del-CFTR expressing cells. Here we show that the calcium-calpain-caspase-12-caspase-3 cascade is altered in F508del-CFTR expressing cells. We propose that this alteration is involved in the altered apoptosis triggering observed in CF.
Assuntos
Apoptose , Calpaína/metabolismo , Caspase 12/metabolismo , Caspase 3/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/enzimologia , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Modelos Biológicos , Tapsigargina/farmacologiaRESUMO
Cystic fibrosis (CF) is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In CF, the most common mutant DeltaF508-CFTR is misfolded, is retained in the ER and is rapidly degraded. If conditions could allow DeltaF508-CFTR to reach and to stabilize in the plasma membrane, it could partially correct the CF defect. We have previously shown that annexin V (anxA5) binds to both the normal CFTR and the DeltaF508-CFTR in a Ca(2+)-dependent manner and that it regulates the chloride channel function of Wt-CFTR through its membrane integration. Our aim was to extend this finding to the DeltaF508-CFTR. Because some studies show that thapsigargin (Tg) increases the DeltaF508-CFTR apical expression and induces an increased [Ca(2+)](i) and because anxA5 relocates and binds to the plasma membrane in the presence of Ca(2+), we hypothesized that the Tg effect upon DeltaF508-CFTR function could involve anxA5. Our results show that raised anxA5 expression induces an augmented function of DeltaF508-CFTR due to its increased membrane localization. Furthermore, we show that the Tg effect involves anxA5. Therefore, we suggest that anxA5 is a potential therapeutic target in CF.
Assuntos
Anexina A5/fisiologia , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Mutação , Anexina A5/genética , Anexina A5/metabolismo , Western Blotting , Cálcio/metabolismo , Linhagem Celular Tumoral , AMP Cíclico/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Humanos , Imunoprecipitação , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Tapsigargina/farmacologia , TransfecçãoRESUMO
Cystic fibrosis (CF) is the most common Caucasian autosomal recessive disease. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, which is a chloride (Cl(-)) channel. The most common mutation leads to a missing phenylalanine at position 508 (DeltaF508). The DeltaF508-CFTR protein is misfolded and retained in the endoplasmic reticulum and may trigger the unfolded protein response (UPR). Furthermore, CF is accompanied by inflammation and infection, which are also involved in the UPR. To date, the UPR transducer ATF6 and ER stress sensor Grp78 have been used as UPR markers. Therefore, our aim was to study the activation of ATF6 and Grp78 in transfected human epithelial cells expressing the DeltaF508-CFTR protein, and we showed that they are activated in these cells. We investigated the effect of exogenous UPR inducers thapsigargin (Tg) and tunicamycin (Tu) on Grp78 and ATF6 expression. Whereas the cells reacted to the UPR induction, we show a difference in the electrophoretic pattern of ATF6. The Grp78/ATF6 complex was previously described, but its stability during UPR is controversial. Using co-immunoprecipitation we show that it is stable in DeltaF508-CFTR-expressing cells and is maintained under UPR conditions. Finally, using siRNA, we show that decreased ATF6 expression induces increased cAMP-dependent halide flux through DeltaF508-CFTR due to its increased membrane localization. Therefore, our results suggest that UPR may be triggered in CF and that ATF6 may be a therapeutic target.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Retículo Endoplasmático/patologia , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Vetores Genéticos , Humanos , Ligação Proteica/efeitos dos fármacos , Dobramento de Proteína , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Tapsigargina/farmacologia , Transfecção , Tunicamicina/farmacologiaRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-activated chloride channel, which is regulated by protein-protein interactions. The extent to which CFTR is regulated by these interactions remains unknown. Annexin V is overexpressed in cystic fibrosis (CF), and given the functional properties of annexin V and CFTR we considered whether they are associated and if so whether this has implications for CFTR function. Using co-immunoprecipitation and overlay experiments, we show that annexin V is associated with nucleotide-binding domain 1 (NBD1) of CFTR. Surface plasmon resonance (SPR) indicated different KD values in the absence and presence of both calcium and ATP, suggesting that this interaction is calcium- and ATP-dependent. Using an siRNA approach and overexpression, we showed that CFTR chloride channel function and its localization in the cell membranes were dependent on annexin V expression. We concluded that annexin V is necessary for normal CFTR chloride channel activity. Furthermore, we show that CFTR and annexin V are partially co-distributed in normal epithelial cells in human bronchi. In conclusion, we show for the first time that annexin V is associated with CFTR and is involved in its function.
Assuntos
Anexina A5/biossíntese , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Traqueia/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Anexina A5/química , Anexina A5/genética , Cálcio/química , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/química , Membrana Celular/genética , AMP Cíclico/química , AMP Cíclico/genética , AMP Cíclico/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/química , Regulação da Expressão Gênica/genética , Humanos , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Ressonância de Plasmônio de Superfície , Traqueia/químicaRESUMO
Variations in the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor (PSTI)) are associated with chronic pancreatitis. We have recently determined the functional consequences of three missense mutations that occurred within the signal peptide sequence of PSTI by Western blotting analysis of wild-type and mutant PSTI expressed in Chinese hamster ovary cells. Here, this approach was extended to analyze seven missense mutations (p.N34S, p.G48E, p.D50E, p.Y54H, p.P55S, p.R65Q and p.R67C) occurring within the mature peptide of PSTI. This analysis enabled us to classify these missense mutations into three categories. The first category comprises the p.N34S and p.P55S polymorphisms, both of which occur in evolutionarily non-conserved residues, involve amino-acid substitutions with similar physicochemical properties, and do not cause any significant reduction in terms of PSTI mature peptide expression. The second category contains only the p.R65Q missense mutation, which occurs in a well-conserved residue, involves the substitution of a positively charged amino acid by a non-charged one, and causes a approximately 60% reduction of protein expression. The third category comprises p.G48E, p.D50E, p.Y54H, and p.R67C, all of which occur in strictly conserved residues, involve charged amino acids, and cause complete or nearly complete loss of PSTI expression. Having excluded the possibility that the reduced protein expression may have resulted from reduced transcription or unstable mRNA, we surmise that these missense mutations probably cause intracellular retention of their respective mutant proteins. This is suggestive of a potential unifying pathological mechanism underlying both the signal peptide and mature peptide mutations.