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Complementary 2D-PAGE and ‘shotgun’ LC–MS/MS approaches were combined to identify medium and low-abundant proteins in sera of Cystic Fibrosis (CF) patients (mild or severe pulmonary disease) in comparison with healthy CF-carrier and non-CF carrier individuals aiming to gain deeper insights into the pathogenesis of this multifactorial genetic disease. 78 differentially expressed spots were identified from 2D-PAGE proteome profiling yielding 28 identifications and postulating the existence of post-translation modifications (PTM). The ‘shotgun’ approach highlighted altered levels of proteins actively involved in CF: abnormal tissue/airway remodeling, protease/antiprotease imbalance, innate immune dysfunction, chronic inflammation, nutritional imbalance and Pseudomonas aeruginosa colonization. Members of the apolipoproteins family (VDBP, ApoA-I, and ApoB) presented gradually lower expression from non-CF to CF-carrier individuals and from those to CF patients, results validated by an independent assay. The multifunctional enzyme NDKB was identified only in the CF group and independently validated by WB. Its functions account for ion sensor in epithelial cells, pancreatic secretion, neutrophil-mediated inflammation and energy production, highlighting its physiological significance in the context of CF. Complementary proteomics-based approaches are reliable tools to reveal pathways and circulating proteins actively involved in a heterogeneous disease such as CF.

F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca2+-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be ‘restored’, i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue.

Objectives: The aim of this work was to establish protein profiles in serum and nasal epithelial cells of cystic fibrosis individuals in comparison with controls, asthma and chronic obstructive pulmonary disease patients for specific biomarker signatures identification. Design and methods: Protein extracts were analyzed by Surface Enhanced Laser Desorption/Ionization Time-Of-Flight Mass-Spectrometry (SELDI-TOF-MS). Results: The mass spectra revealed a set of peaks with differential expression in serum and nasal cells among the different groups studied, resulting into peak signatures representative/specific of each pathology. Logistic regressions were applied to those peaks; sensitivity, specificity, Youden's indexes and area under the curve (AUC) of the respective receiver operating characteristic (ROC) curves were compared. Discussion: Multivariate analysis demonstrated that combination of peaks has a better predictive value than the individual ones. These protein signatures may serve as diagnostic/prognostic markers for the studied diseases with common clinical features, or as follow-up assessment markers of therapeutic interventions.

Combined results from laser capture microdissection of mouse airway epithelial cells followed by high power (MALDI-FTICR) MS, and fluorescent two-dimensional gel elctrophoresis (2D-DIGE) of the whole lung, allowed us to identify proteins differentially expressed after naphthalene induced airway injury. Further, we discovered several novel aspects of Cystic Fibrosis (CF) lung pathology in an F508del-Cftr mouse model using this approach. The combined MALDI-FTICR-MS and 2D-DIGE data show that lung carbonyl reductase (CBR2), involved in prostaglandin metabolism, converting PGE2 to PGF2alpha, is localized to airway cells and is reduced 2-fold in mutant mice compared to normal, both before and after challenge. Further, we observe a downregulation of two key enzymes of retinoic acid metabolism after injury, which is more pronounced in CF mutant mice. These data show that state-of-the-art proteomics can be used to evaluate airway injury in small cell samples. Further, the results suggest the involvement of prostaglandin and retinoic acid metabolism in the abnormal responses of CF mutant mice to injury.

Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority of those correspond to ubiquitously expressed proteins. Consistent with the epithelial origin of this tissue, some of the identified proteins are epithelial markers (e.g. cytokeratins, palate lung and nasal epithelium clone protein (PLUNC), and squamous cell carcinoma antigen 1). Comparison of this protein profile with the one similarly obtained for CF nasal cells revealed a set of differentially expressed proteins. These included proteins related to chronic inflammation and some others involved in oxidative stress injury. Alterations were also observed in the levels of cytoskeleton proteins, being probably implicated with cytoskeleton organization changes described to occur in CF-airways. Lower levels were found for some mitochondrial proteins suggesting an altered mitochondrial metabolism in CF. Differential expression was also found for two more enzymes that have not been previously associated to CF. Further studies will clarify the involvement of such proteins in CF pathophysiology and whether they are targets for CF therapy.

Interest in the biochemical detection of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein followed soon after cloning of the gene and prediction of the protein structure. Ever since, antibodies (Abs) have been produced and used to detect CFTR in both heterologously and endogenously expressing cells and tissues. Although designed to be sensitive and specific, these Abs produce, in most cases, unsatisfactory results when used for the biochemical detection of CFTR either by Western blot or by immunoprecipitation. The lack of Abs that can reliably detect the CFTR protein is a major constraint to studies of CF. We compared 14 different Abs for their ability to detect CFTR in both stably transfected and endogenously expressing cell lines.

Numerous factors, other than mutations in the CFTR gene, affect the phenotypic variability of cystic fibrosis (CF). With a two-dimensional electrophoresis (2-DE) analysis of total protein expression profiles (proteomics) of CF versus non-CF cells it is possible to obtain an integrative picture of CF cellular alterations. Through this approach, proteins that interact differently with wild type- and mutant-CFTR can also be identified (interactomics). This can provide insight into CF pathophysiology as well as clues for novel therapeutic targets. Additionally, protein profiling can ultimately identify novel disease markers with the potential for a CF diagnosis not based on the analysis of CFTR gene.

A561E, a novel cystic fibrosis (CF) associated mutation in the first nucleotide binding domain of CFTR, is the second most common CF mutation in Portugal. Properties of the A561E-CFTR protein were studied by immunoblotting, pulse-chase, immunocytochemistry, and MQAE halide-efflux assay in stably transfected BHK cells. Altogether, results presented here suggest that A561E causes protein mislocalization in the endoplasmic reticulum where the mutant protein must be trapped by the quality control mechanism. We conclude that A561E originates a protein trafficking defect, thus belonging to class II of CFTR mutations. As it is the case for F508del-CFTR (the most common CF mutant), low temperature treatment partially rescues a functional A561E-CFTR channel, suggesting that substitution of glutamic acid for alanine at position 561 does not completely abolish CFTR function. Pharmacological strategies previously reported for treatment of CF patients with the F508del mutation could thus be also effective in CF patients bearing the A561E mutation.

We characterized the 3272-26A-->G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26A-->G/F508del genotype. Besides clinical and haplotype characterization of those patients, we report here results from CFTR transcript analysis in nasal brushings from all five patients. RT-PCR analysis supports alternative splicing in all patients and carriers, but not in controls. By sequencing, we determined that the alternative transcript includes 25 nucleotides from intron 17a, which predictively cause frameshift and a premature stop codon. The use of this alternative splice site causes a reduction in the levels of normal transcripts from the allele with this mutation and, most probably, of normal protein as well. By immunocytochemistry of both epithelial primary cell cultures and slices from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a high sodium absorption, characteristic of CF. Altogether, the results suggest that the main defect caused by the 3272-26A-->G mutation is a reduction in normal CFTR transcripts and protein and therefore this mutation should be included in class V, according to Zielenski and Tsui.

Attempts to promote normal processing and function of F508del-CFTR, the most common mutant in cystic fibrosis (CF), have been described as potential therapeutic strategies in the management of this disease. Here we described the proteomic approaches, namely two-dimensional electrophoresis (2DE), mass spectrometry (MS), and bioinformatics tools used in our recent studies to gain insight into the proteins potentially involved in low-temperature or mutagenic treatment-induced rescue process of F508del-CFTR. The proteins identified are part of the proteostasis network, such as the unfolded protein response (UPR) signaling pathways that may regulate the processing of CF transmembrane conductance regulator (CFTR) through the folding and trafficking progression. The complete characterization of these signaling pathways and their regulators in CF will certainly contribute to the development of novel therapeutic strategies against CF.

Proteomics was initially viewed as a promising new scientific discipline to study complex disorders such as polygenic, infectious and environment-related diseases. However, the first attempts to understand a monogenic disease such as cystic fibrosis (CF) by proteomics-based approaches have proved quite rewarding. In CF, the impairment of a unique protein, the CF transmembrane conductance regulator, does not completely explain the complex and variable CF clinical phenotype. The great advances in our knowledge about the molecular and cellular consequences of such impairment have not been sufficient to be translated into effective treatments, and CF patients are still dying due to chronic progressive lung dysfunction. The progression of proteomics application in CF will certainly unravel new proteins that could be useful as biomarkers either to elucidate CF basic mechanisms and to better monitor the disease progression, or to promote the development of novel therapeutic strategies against CF. This review will summarize the recent technological advances in proteomics and the first results of its application to address the most important issues in the CF field.

Genetically modified mouse strains are important research tools for the study of numerous human diseases. These models provide us with differentiated tissues, which are not often available from human sources. Furthermore, they allow for testing the effects of genetic manipulation and experimental therapeutics on physiology and pathology. Their importance relies on the assumption that biological processes in the mouse very closely resemble those in humans. Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population. CF is a monogenic disease whose phenotype variability is also attributed to genetic variation in other genes, the so-called modifier genes. Modulation of such modifier genes could be a therapeutic strategy to treat CF. CF mice models have been essential not only for understanding the disease better, but also for the discovery of modifier genes and testing of chemical compounds developed to repair the main protein dysfunction in CF, the CF transmembrane conductance regulator. Mice were also indispensable in gene therapy trials and for the study of CF and non-CF lung response to bacterial infections and inflammation challenges, although no spontaneous lung disease is developed in these mice. In this review, mouse models and their most important contribution to the understanding and management of CF will be presented and discussed.

A panel of 11 human cystic fibrosis transmembrane conductance regulator (hCFTR) antibodies were tested in ovine nasal, tracheal, and bronchial epithelial brushings. Two of these, G449 (polyclonal) and MATG1104 (monoclonal), recognized hCFTR but did not cross react with endogenous sheep CFTR. This specificity allows immunologic detection of hCFTR expressed in gene transfer studies in sheep against the background of endogenous ovine CFTR, thus enhancing the value of the sheep as a model animal in which to study CFTR gene transfer. Studies on mixed populations of human and sheep nasal epithelial cells showed that detection of hCFTR by these two antibodies was possible even at the lowest proportion of human cells (1:100). The hCFTR gene was delivered in vivo by local instillation using polyethylenimine-mediated gene transfer to the ventral surface of the ovine trachea and hCFTR mRNA and protein levels scored in a blinded fashion. Despite abundant hCFTR mRNA expression, the number of cells expressing hCFTR protein detectable by G449 was low (approximately 0.006-0.05%). Immunohistochemistry for hCFTR in animals treated by whole-lung aerosol demonstrated positive cells in sections of tracheal epithelium and in distal conducting airways. The strategic use of hCFTR-specific antibodies supports the utility of the normal sheep as a model for hCFTR gene transfer studies.

Studies on CFTR protein expression and localization in native tissues or in primary cultures of human epithelial cells are scarce due to the intrinsic instability of this protein, its low expression in most tissues and also to technical difficulties.However, such data are of the highest importance to understand the pathophysiology of CF. The purpose of this article is to outline several assays for the characterization of primary epithelial cultures and to review different CFTR immunostaining protocols.

For most expression studies focusing on the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, sensitive and specific antibodies (Abs) are critically needed. Several Abs have been produced commercially or by research laboratories for CFTR detection in both cell lines with heterologous or endogenous expression and native cells/tissues. Here, we review the applicability of most Abs currently in use in CF research for the biochemical and/or immunocytochemical detection of CFTR.

Detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein is usually a difficult task to accomplish due to the low levels of expression and high turnover that this membrane protein is submitted to in the cell. Common biochemical methods can be used for the detection of CFTR but several critical points must be taken into account. The scope of this article is to outline biochemical methods commonly used to assess CFTR expression, processing and membrane localization.

The autosomal recessive disease cystic fibrosis (CF, MIM 219700) is caused by a wide spectrum of mutations in the gene encoding for the CF transmembrane conductance regulator (CFTR, MIM 602421) protein, a cAMP regulated chloride (Cl−) channel located in the apical membrane of secretory epithelial cells. About 20% of the more than 1000 CFTR mutations reported so far are splicing mutations and generally they represent 15% of all point mutations.1 Although it is generally agreed that mutations at the conserved splice dinucleotide consensus sites GU (donor) and AG (acceptor) disrupt function of the resulting protein product, it is important to know exactly which alternatively spliced mRNA molecules will result in such situations. ### Key points

Estimates of the level of transcripts from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene required to develop a CF phenotype range from 4-20% of normal. Due to the importance of obtaining reliable data on this issue for therapeutic strategies, we developed a novel polymerase chain reaction-based method to quantify CFTR transcripts and applied it to the analysis of nasal epithelium RNA of five patients with CF and the 3272-26AG/F508del genotype. We calculated that 8.2 +/- 0.84% of the total CFTR RNA present in these five patients is normal full-length CFTR mRNA. We then demonstrated (in nasal samples from F508del carriers, n = 30) that the abundance of full-length F508del CFTR transcripts is reduced compared with wild-type transcripts, and estimated that the average ratio of F508del/wild-type transcripts is 0.87 +/- 0.06. To determine the amount of full-length transcripts relative to levels found in normal individuals, we corrected for the lower abundance of the F508del transcripts and calculated that the five patients with CF have, on average, 4.7 +/- 0.45% of the normal level of wild-type CFTR mRNA. Because these patients have mild CF compared with F508del homozygotes, this CFTR mRNA level appears to be sufficient to avoid the severe complications of the disease.

Editor—Cystic fibrosis (CF, MIM 219700) is a common, severe, autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene cloned in 1989.1-3The disease, characterised by chronic lung disease which is the main cause of morbidity and mortality, pancreatic dysfunction, raised electrolyte levels in sweat, and male infertility, is caused by altered chloride (Cl−) secretion across the apical membrane of epithelial cells.4 There is, however, substantial variability in the clinical manifestations affecting the various organs.4 5 One single mutation, F508del, generally associated with severe disease, accounts for about 70% of CF chromosomes world wide, although with a heterogeneous geographical distribution.5 Patients homozygous for the F508del mutation have the classical severe form of the disease which includes chronic mucous obstruction of the lung and conducting airways, followed by recurrent infections mostly by Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa), exocrine pancreatic insufficiency (PI), resulting in failure to gain weight and height, and raised levels of Cl−, sodium, and potassium in exocrine sweat.5 However, almost 1000 genetic alterations have been detected in the CFTR gene ( CFTR Mutation Database), most presumed to be disease causing mutations. About half of these are amino acid substitutions (missense mutations) and about 20% are splicing mutations. The remainder are nonsense, frameshift (including small deletions and insertions), and a small proportion of promoter mutations. The relationship between genotype, that is, the mutations in the CFTR gene, and the clinical phenotype of CF patients has been difficult to establish, in particular for lung disease. It was previously shown that the 3272-26A>G mutation leads to the creation of an alternative acceptor splice site competing with the normal one during RNA processing and resulting in the occurrence of an alternatively spliced mRNA with 25 extra nucleotides from …

Reliable methods for determining the localisation of mutant CFTR protein in native cells from CF individuals are necessary to allow the degree of mislocalisation of any genotype to be defined and to assess the effect of therapeutic agents on CFTR trafficking. Here, we present procedures for obtaining ciliated epithelial cells from CF patients by nasal brushing and a description of protocols for immunolocalisation of CFTR. The protocols are a consensus, following comparison of some aspects of methods currently used in the authors' laboratories.

The intracellular localization of cystic fibrosis transmembrane conductance regulator (CFTR) in native tissues is a major issue in the study of mutation, processing, and trafficking effects in CFTR and in the evaluation of therapeutic strategies in cystic fibrosis (CF). This work evaluated the applicability of ten different antibodies (Abs) under various fixation techniques for CFTR localization in fresh-brushed nasal epithelial cells collected from CF patients homozygous for F508del and control individuals. In parallel, the same Ab panel was also tested on BHK cell lines overexpressing wild-type or F508del CFTR. The Abs MATG1061, 169, Lis1, MP-CT1, CC24-R, MAB25031, and MAB1660 gave the best detection of CFTR in the apical region (AR) of nasal tall columnar epithelial (TCE) cells. The labeling pattern of these Abs was consistent with the postulated processing defect of F508del CFTR because only a minority of CF TCE cells present CFTR in the AR. In contrast, M3A7, MM13–4, and L12B4 weakly react with the AR and stain almost exclusively a cis-Golgi-like structure in the majority of CF and non-CF airway cells. In BHK cells, all the Abs enabled distinction between wild-type CFTR localization in cell membrane from F508del CFTR, which in these cells is exclusively located in the endoplasmic reticulum.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which has a major role as a chloride (Cl−) channel. Although perhaps all functions of CFTR are still not fully characterized, localization studies are necessary to understand the consequences of the more than 1000 mutations thus far identified. Our aim was to determine the histological localization of CFTR on respiratory and colon epithelia of human and murine origin with a panel of several antibodies produced against different CFTR epitopes, using an indirect immunofluorescence method. Our results on human tissues confirm the apical localization of CFTR in ciliated cells of the respiratory mucosa and show that in colon tissue CFTR is observed in both apical and basolateral membranes of epithelial cells from colon crypts. However, poor tissue preservation of colon biopsies after immunohistochemistry (IHC) raises doubts about the latter localization. Contrary to human, mouse colon epithelium (not biopsed) presents good tissue preservation and evidences many cylindrical surface cells with high apical expression of CFTR. For the antibodies's sensitivity, we demonstrate that MATG1061, 24-1, M3A7, and MPCT-1 give good results, allowing the histological localization of CFTR protein of both human and murine origin.

Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n = 12), in 42% of cells from F508del carriers (n = 20), and in 56% of cells from healthy individuals (n = 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p < 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation.

Attempts to promote normal processing and function of F508del-CFTR, the most common mutant in cystic fibrosis (CF), have been described as potential therapeutic strategies in the management of this disease. Here we described the proteomic approaches, namely two-dimensional electrophoresis (2DE), mass spectrometry (MS), and bioinformatics tools used in our recent studies to gain insight into the proteins potentially involved in low-temperature or mutagenic treatment-induced rescue process of F508del-CFTR. The proteins identified are part of the proteostasis network, such as the unfolded protein response (UPR) signaling pathways that may regulate the processing of CF transmembrane conductance regulator (CFTR) through the folding and trafficking progression. The complete characterization of these signaling pathways and their regulators in CF will certainly contribute to the development of novel therapeutic strategies against CF.