Deborah Penque

The human DnaJ homologue (Hdj)-1/heat-shock protein (Hsp) 40 co-chaperone is required for the in vivo stabilization of the cystic fibrosis transmembrane conductance regulator by Hsp70

The CFTR (cystic fibrosis transmembrane conductance regulator) gene, defective in cystic fibrosis, codes for a polytopic apical membrane protein functioning as a chloride channel. Wild-type (wt) CFTR matures inefficiently and CFTR with a deletion of Phe-508 (F508del), the most frequent mutation, is substantially retained as a core-glycosylated intermediate in the endoplasmic reticulum (ER), probably due to misfolding that is recognized by the cellular quality control machinery involving molecular chaperones. Here, we overexpressed the heat-shock protein (Hsp) 70 chaperone in vivo and observed no changes in degradation rate of the core-glycosylated form, nor in the efficiency of its conversion into the fully glycosylated form, for either wt- or F508del-CFTR, contrary to previous in vitro studies on the affect of heat-shock cognate (Hsc) 70 on part of the first nucleotide-binding domain of CFTR. Co-transfection of Hsp70 with its co-chaperone human DnaJ homologue (Hdj)-1/Hsp40, however, stabilizes the immature form of wt-CFTR, but not of F508del-CFTR, suggesting that these chaperones act on a wt-specific conformation. As the efficiency of conversion into the fully glycosylated form is not increased under Hsp70/Hdj-1 overexpression, the lack of these two chaperones does not seem to be critical for CFTR maturation and ER retention. The effects of 4-phenylbutyrate and deoxyspergualin, described previously to interfere with Hsp70 binding, were also tested upon CFTR degradation and processing. The sole effect observed was destabilization of F508del-CFTR.

Cystic fibrosis F508del patients have apically localized CFTR in a reduced number of airway cells.

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.

Two-dimensional gel electrophoresis and mass spectrometry for biomarker discovery

The proteome project, initiated in 1995, was made possible by 2‐DE combined with MS. The project main objective was and remains the identification of all proteins expressed by a cell, tissue or organism in a given time and condition. Following this objective, the global profiling of proteins in health versus pathological state by the 2‐DE/MS‐based proteomic approach has contributed to the elucidation of the basic mechanisms of disease by discovering candidate disease biomarkers and disease targets for new drug development. This review will briefly summarize the historical evolution of 2‐DE up to today, and review 2‐DE/MS technology and its specific methods of study of immunoresponse (immunoproteomics), PTM of proteins, complex protein–protein interactions (interactome), the proteome of cell membrane and intracellular proteome turnover in disease biomarker discovery.

Cystic fibrosis patients with the 3272‐26A→G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane

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. Hum Mutat 14:133–144, 1999.

CFTR Localization in Native Airway Cells and Cell Lines Expressing Wild-type or F508del-CFTR by a Panel of Different Antibodies

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.

Applicability of Different Antibodies for the Immunohistochemical Localization of CFTR In Respiratory and Intestinal Tissues of Human and Murine Origin

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 antibodiess 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.

Cystic fibrosis patients with the 3272-26A>G splicing mutation have milder disease than F508del homozygotes: a large European study

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 …

Low temperature restoring effect on F508del-CFTR misprocessing: A proteomic approach

To gain insight into the proteins potentially involved in the low temperature-induced F508del-CFTR rescue process, we have explored by two-dimensional electrophoresis (2DE) the proteome of BHK cell lines expressing wt or F508del-CFTR, grown at 37 degrees C or 26 degrees C/24h or 26 degrees C/48h followed by 3h of metabolic labelling with [(35)S]-methionine. A set of 139 protein spots (yielding 125 mass spectrometry identifications) was identified as differentially expressed (p ANOVA<0.05) among the six phenotypic groups analysed. The data analysis suggests that the unfolded protein response (UPR) induction and some cell-metabolism repression are the major cold-shock responses that may generate a favourable cellular environment to promote F508del-CFTR rescue. Down-regulation of proteasome regulatory PA28 and/or COP9 signalosome subunit, both involved in CFTR degradation, could also be a relevant cold-shock-induced condition for F508de-CFTR rescue. Moreover, cold-shock may promote the reestablishment of some proteostasis imbalance associated with over-expression of F508del-CFTR. In BHK-F508del cells, the deregulation of RACK1, a protein described to be important for stable expression of CFTR in the plasma membrane, is partially repaired after low temperature treatment. Together these findings give new insights about F508del-CFTR rescue by low temperature treatment and the proteins involved could ultimately constitute potential therapeutic targets in CF disease.

SELDI-TOF biomarker signatures for cystic fibrosis, asthma and chronic obstructive pulmonary disease

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, Youdens 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.

Proteomic Analysis of Naphthalene-Induced Airway Epithelial Injury and Repair in a Cystic Fibrosis Mouse Model

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.