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1.
Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP‐activated Cl channel, is extensively expressed in the epithelial cells of various tissues and organs. Accumulating evidence indicates that aberrant expression or mutation of CFTR is related to carcinoma development. Malignant gliomas are the most common and aggressive intracranial tumours; however, the role of CFTR in the development of malignant gliomas is unclear. Here, we report that CFTR is expressed in malignant glioma cell lines. Suppression of CFTR channel function or knockdown of CFTR suppresses glioma cell viability whereas overexpression of CFTR promotes it. Additionally, overexpression of CFTR suppresses apoptosis and promotes glioma progression in both subcutaneous and orthotopic xenograft models. Cystic fibrosis transmembrane conductance regulator activates Akt/Bcl2 pathway, and suppression of PI3K/Akt pathway abolishes CFTR overexpression–induced up‐regulation of Bcl2 (MK‐2206 and LY294002) and cell viability (MK‐2206). More importantly, the protein expression level of CFTR is significantly increased in glioblastoma patient samples. Altogether, our study has revealed a mechanism by which CFTR promotes glioma progression via up‐regulation of Akt/Bcl2‐mediated anti‐apoptotic pathway, which warrants future studies into the potential of using CFTR as a therapeutic target for glioma treatment.  相似文献   

2.
Curcumin has been reported to correct cystic fibrosis caused by the DeltaF508 mutation of the cystic fibrosis transmembrane regulator (CFTR) but its mechanistic action remains unclear. We have recently demonstrated that the ER chaperone calreticulin (CRT) negatively regulates the CFTR cell surface expression and activity. Thus, we aimed at determining whether CRT mediates the effect of curcumin on CFTR. We show here that the treatment with curcumin of Chinese hamster ovary cells suppressed CRT expression and increased wild-type CFTR but did not affect DeltaF508 CFTR expression. However, we determined that although curcumin did not augment DeltaF508 CFTR expression, it enhanced the functional competence of DeltaF508 CFTR induced by 26 degrees C incubation. Knock down of CRT by siRNA at low-temperature had a similar effect. Our findings suggest that the positive effect of curcumin on CFTR expression is mediated through the down-regulation of CRT, a negative regulator of CFTR.  相似文献   

3.
Cystic fibrosis (CF) is caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. We ascertained five patients with a novel complex CFTR allele, with two mutations, H939R and H949L, inherited in cis in the same exon of CFTR gene, and one different mutation per patient inherited in trans in a wide population of 289 Caucasian CF subjects from South Italy. The genotype-phenotype relationship in patients bearing this complex allele was investigated. The two associated mutations were related to classical severe CF phenotypes.  相似文献   

4.
The most common cystic fibrosis (CF)‐causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of Phe508 (ΔF508) in the first of two nucleotide‐binding domains (NBDs). Nucleotide binding and hydrolysis at the NBDs and phosphorylation of the regulatory (R) region are required for gating of CFTR chloride channel activity. We report NMR studies of wild‐type and ΔF508 murine CFTR NBD1 with the C‐terminal regulatory extension (RE), which contains residues of the R region. Interactions of the wild‐type NBD1 core with the phosphoregulatory regions, the regulatory insertion (RI) and RE, are disrupted upon phosphorylation, exposing a potential binding site for the first coupling helix of the N‐terminal intracellular domain (ICD). Phosphorylation of ΔF508 NBD1 does not as effectively disrupt interactions with the phosphoregulatory regions, which, along with other structural differences, leads to decreased binding of the first coupling helix. These results provide a structural basis by which phosphorylation of CFTR may affect the channel gating of full‐length CFTR and expand our understanding of the molecular basis of the ΔF508 defect.  相似文献   

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目的研究肠道组织CFTR基因表达与分泌性腹泻发生的关系。方法选取KM小鼠24只,雌雄各半,随机分为3组(每组8只):对照组经小鼠腹腔注射0.2 mL生理盐水,实验组小鼠经腹腔注射LPS[6 mg/(kg·bw)]分别作用1 h、8 h,于注射后通过小鼠精神状态、肠道组织形态学判定分泌性腹泻模型的建立,利用荧光定量PCR法检测各段肠道组织CFTR基因的表达。结果 LPS成功诱导小鼠发生了分泌性腹泻;CFTR基因在小鼠十二指肠、空肠、回肠和结肠组织中均有不同的表达丰度,以结肠最高,但各段肠道间差异不显著;与对照组相比,LPS上调了十二指肠、空肠和回肠CFTR基因的转录,下调了结肠CFTR基因的转录。结论提示肠道组织CFTR基因转录水平的上调与LPS诱导分泌性腹泻的发生密切相关,且在各肠段发挥的作用不同,其中空肠在氯离子(Cl-)分泌中发挥主要作用,结肠的作用最弱。  相似文献   

8.
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.  相似文献   

9.
Structural differences have been reported in the glycosylation patterns of cystic fibrosis glycoproteins. Although the gene mutated in cystic fibrosis (CFTR) has been cloned and characterized as a chloride channel, its relationship to the highly viscous mucus and structural glycoprotein and mucin abnormalities in cystic fibrosis still remains to be defined. We have evaluated O-glycan biosynthesis in CHO and BHK cells that express CFTR and F508 CFTR as in vitro models, and utilized the cftr knockout mouse as an in vivo model of CFTR dysfunction. Activities of glycosyltransferases and sulfotransferases synthesizing mucin type O-glycan chains were determined in these models. Differences in transferase activity levels were found between tissues and cell types and during mouse development. No specific patterns of activities were associated with the lack of CFTR or with F508CFTR expression. This suggests that it is not the presence or absence of normal CFTR, or the presence of mutant CFTR alone, but rather cell specific additional factors or pathophysiological consequences that determine the changes in mucin glycosylation in cystic fibrosis.  相似文献   

10.
Cystic fibrosis, the most common autosomal recessive disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated chloride and bicarbonate channel that regulates ion and water transport in secretory epithelia. Although all mutations lead to the lack or reduction in channel function, the mechanisms through which this occurs are diverse – ranging from lack of full-length mRNA, reduced mRNA levels, impaired folding and trafficking, targeting to degradation, decreased gating or conductance, and reduced protein levels to decreased half-life at the plasma membrane. Here, we review the different molecular mechanisms that cause cystic fibrosis and detail how these differences identify theratypes that can inform the use of directed therapies aiming at correcting the basic defect. In summary, we travel through CFTR life cycle from the gene to function, identifying what can go wrong and what can be targeted in terms of the different types of therapeutic approaches.  相似文献   

11.
The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl- channel regulated by protein phosphorylation. To identify cAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA). Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe(3+)-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full-length CFTR.  相似文献   

12.
Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis, a hereditary lethal disease. CFTR is a chloride channel expressed in the apical membrane of epithelia. It is activated by cAMP dependent phosphorylation and gated by the binding of ATP. The impaired chloride transport of some types of cystic fibrosis mutations could be pharmacologically solved by the use of chemical compounds called potentiators. Here it is undertaken the construction of a model of the CFTR activation pathways, and the possible modification produced by a potentiator application. The model yields a novel mechanism for the potentiator action, describing the activatory and inhibitory activities on two different positions in the CFTR activation pathway.  相似文献   

13.

Aims

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP regulated chloride channel expressed in the apical plasma membrane of pancreatic duct cells where it plays an important role in fluid secretion. The purpose of this study was to elucidate the role of the CFTR chloride channel on ion and fluid secretion from the guinea-pig pancreas by manipulating the expression of CFTR by RNA interference or by luminal application of a CFTR selective activator, MPB91, in isolated cultured pancreatic ducts.

Materials and methods

Using cDNA isolated from the guinea-pig small intestine, fragments of the CFTR gene were generated by polymerase chain reaction and directly sequenced. Two different RNA duplexes for small interference RNA (siRNA) were designed from the sequence obtained. Fluid secretion from the isolated guinea-pig pancreatic ducts was measured using video-microscopy. The amount of CFTR chloride channel or AQP1 water channel expressed in pancreatic ducts was examined by immunoblotting with antibodies against CFTR or AQP1, respectively.

Results

Guinea-pig CFTR consists of 1481 amino acid residues. An additional glutamine residue was found to be inserted between amino acid residues 403 and 404 of human CFTR. Forskolin-stimulated fluid secretion from intact pancreatic ducts was significantly higher in the presence of MPB91 compared to fluid secretion in the absence of MPB91. Both basal and forskolin-stimulated fluid secretion in pancreatic ducts transfected with CFTR specific siRNAs were reduced by ∼50% compared to fluid secretion from ducts transfected with scrambled negative control dsRNAs. The amount of CFTR and AQP1 proteins was reduced to 34% and 45% of control, respectively.

Conclusions

The activity of the CFTR chloride channel or the amount of CFTR protein expressed determines the rate of fluid secretion from the isolated guinea-pig pancreatic ducts.  相似文献   

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Altered terminal glycosylation, with increased fucosylation and decreased sialylation is a hallmark of the cystic fibrosis (CF) glycosylation phenotype. Oligosaccharides purified from the surface membrane glycoconjugates of CF airway epithelial cells have the Lewis x, selectin ligand in terminal positions. This review is focused on the investigations of the glycoconjugates of the CF airway epithelial cell surface. Two of the major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins which recognize fucose in -1,3 linkage and asialoglycoconjugates. Therefore, consideration has been given to the possibility that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to both the chronic infection and the robust, but ineffective, inflammatory response in the CF lung. Since the glycosylation phenotype of CF airway epithelial cells have been modulated by the expression of wtCFTR, the hypotheses which have been proposed to relate altered function of CFTR to the regulation of the glycosyltransferases are discussed. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as new approaches to the therapy of CF.  相似文献   

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Cystic fibrosis (CF) is a fatal disease affecting the lungs and digestive system by impairment of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). While over 1000 mutations in CFTR have been associated with CF, the majority of cases are linked to the deletion of phenylalanine 508 (ΔF508). F508 is located in the first nucleotide binding domain (NBD1) of CFTR. This mutation is sufficient to impair the trafficking of CFTR to the plasma membrane and, thus, its function. As an ABC transporter, recent structural data from the family provide a framework on which to consider the effect of the ΔF508 mutation on CFTR. There are fifty-seven known structures of ABC transporters and domains thereof. Only six of these structures are of the intact transporters. In addition, modern bioinformatic tools provide a wealth of sequence and structural information on the family. We will review the structural information from the RCSB structure repository and sequence databases of the ABC transporters. The available structural information was used to construct a model for CFTR based on the ABC transporter homologue, Sav1866, and provide a context for understanding the molecular pathology of Cystic Fibrosis.  相似文献   

18.
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-dependent anion channel. Disease-causing mutations can affect channel biogenesis, trafficking or function, and result in reduced ion transport at the apical surface of many tissues. The most common CFTR mutation is a deletion of phenylalanine at position 508 (ΔF508), which results in a misfolded protein that is prematurely targeted for degradation. This article focuses on how proteomic approaches have been utilized to explore the mechanisms of premature proteolysis in CF. Additionally, we emphasize the potential for proteomic-based technologies in expanding our understanding of CF pathophysiology and therapeutic approaches.  相似文献   

19.
Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl channel function is required for activating amiloride-sensitive epithelial Na+ channels (ENaC) in salt-absorbing human sweat duct. It is unclear whether ENaC channel function is also required for CFTR activation. The dysfunctional ENaC mutations in type-1 pseudohypoaldosteronism (PHA-1) provided a good opportunity to study this phenomenon of ion channel interaction between CFTR and ENaC. The PHA-1 ducts completely lacked spontaneous ENaC conductance (gENaC). In contrast, the normal ducts showed large spontaneous gENaC (46 ± 10 ms, mean ± SE). After permeabilization of the basolateral membrane with α-toxin, cAMP + ATP activation of CFTR Cl conductance (gCFTR) or alkalinization of cytosolic pH (6.8 to 8.5) stimulated gENaC of normal but not PHA-1 ducts. In contrast, both spontaneous gCFTR in intact ducts and (cAMP + ATP)-activated gCFTR of permeabilized ducts appeared to be similar in normal and PHA-1 subjects. Lack of gENaC completely blocked salt absorption and caused dramatic reversal of skin potentials associated with pilocarpine-induced sweat secretion from significantly negative in normal subjects (−13 ± 7.0 mV) to significantly positive (+22 ± 11.0 mV) in PHA-1 patients. We conclude that virtual lack of ENaC in PHA-1 ducts had little effect on CFTR activity and that the positive skin potentials could potentially serve as a diagnostic tool to identify type-1 pseudohypoaldosteronism. An erratum to this article is available at .  相似文献   

20.
Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer‐related deaths among women. New biomarkers with definite diagnostic and prognostic efficacy are urgently needed. Here, we showed that the promoter of the cystic fibrosis transmembrane conductance regulator (CFTR) was hypermethylated in breast cancer. The messenger RNA level of CFTR was downregulated in breast cancer. Notably, all 19 breast cancer patients with hypermethylated CFTR were diagnosed with invasive carcinoma. Moreover, CFTR was upregulated in decitabine (10 μM) treated breast cancer cells. Overexpression of CFTR inhibited cell growth whereas knockdown of CFTR promoted cell invasion. In the tissue array analysis, the CFTR protein level decreased significantly in breast cancer and low CFTR protein level correlated with poor survival with a P‐value of 0.034. Thus, promoter hypermethylation of the CFTR gene might be a novel diagnostic marker of breast cancer.  相似文献   

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