Cystic Fibrosis Transmembrane Regulator Missing the First Four Transmembrane Segments Increases Wild Type and ΔF508 Processing

Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2008; 283(32):21926-33. DOI: 10.1074/jbc.M709156200
Source: PubMed


We previously generated an adenoassociated viral gene therapy vector, rAAV-Delta264 cystic fibrosis transmembrane conductance regulator (CFTR), missing the first four transmembrane domains of CFTR. When infected into monkey lungs, Delta264 CFTR increased the levels of endogenous wild type CFTR protein. To understand this process, we transfected Delta264 CFTR plasmid cDNA into COS7 cells, and we noted that protein expression from the truncation mutant is barely detectable when compared with wild type or DeltaF508 CFTR. Delta264 CFTR protein expression increases dramatically when cells are treated with proteasome inhibitors. Cycloheximide experiments show that Delta264 CFTR is degraded faster than DeltaF508 CFTR. VCP and HDAC6, two proteins involved in retrograde translocation from endoplasmic reticulum to cytosol for proteasomal and aggresomal degradation, coimmunoprecipitate with Delta264 CFTR. In cotransfection studies in COS7 cells and in transfection of Delta264 CFTR into cells stably expressing wild type and DeltaF508 CFTR, Delta264 CFTR increases wild type CFTR protein and increases levels of maturation of immature band B to mature band C of DeltaF508 CFTR. Thus the adenoassociated viral vector, rAAV-Delta264 CFTR, is a highly promising cystic fibrosis gene therapy vector because it increases the amount of mature band C protein both from wild type and DeltaF508 CFTR and associates with key elements in quality control mechanism of CFTR.

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    • "4B and S8). B band but not C band was detected in CFBE41o-cells expressing only endogenous ΔF508 CFTR, which is indicative of immature ΔF508 CFTR residing primarily in the endoplasmic reticulum [40]. However, a fully glycosylated C band was detected when these cells were treated with PEI-MPP carrying wild-type CFTR plasmid DNA (Fig. 4C). "
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    ABSTRACT: Inhaled gene carriers must penetrate the highly viscoelastic and adhesive mucus barrier in the airway in order to overcome rapid mucociliary clearance and reach the underlying epithelium; however, even the most widely used viral gene carriers are unable to efficiently do so. We developed two polymeric gene carriers that compact plasmid DNA into small and highly stable nanoparticles with dense polyethylene glycol (PEG) surface coatings. These highly compacted, densely PEG-coated DNA nanoparticles rapidly penetrate human cystic fibrosis (CF) mucus ex vivo and mouse airway mucus ex situ. Intranasal administration of the mucus penetrating DNA nanoparticles greatly enhanced particle distribution, retention and gene transfer in the mouse lung airways compared to conventional gene carriers. Successful delivery of a full-length plasmid encoding the cystic fibrosis transmembrane conductance regulator protein was achieved in mouse lungs and airway cells, including a primary culture of mucus-covered human airway epithelium grown at air-liquid interface, without causing acute inflammation or toxicity. Highly compacted mucus penetrating DNA nanoparticles hold promise for lung gene therapy.
    Full-text · Article · Jan 2014 · Journal of Controlled Release
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    • "African green monkey kidney cells (Cos7) were maintained in Dulbecco’s modified Eagle’s medium-high glucose 1x (DMEM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum as described previously [14]). "
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    ABSTRACT: Cystic fibrosis is caused by more than 1000 mutations, the most common being the ΔF508 mutation. These mutations have been divided into five classes [1], with ΔF508 CFTR in class II. Here we have studied the class V mutation A455E. We report that the mature and immature bands of A455E are rapidly degraded primarily by proteasomes; the short protein half-life of this mutant therefore resembles that of ΔF508 CFTR. A455E could be rescued by treatment of the cells with proteasome inhibitors. Furthermore, co-transfection of A455E with the truncation mutant Δ264 CFTR also rescued the mature C band, indicating that A455E can be rescued by transcomplementation. We found that Δ264 CFTR bound to A455E, forming a bimolecular complex. Treatment with the compound correctors C3 and C4 also rescued A455E. These results are significant because they show that although ΔF508 belongs to a different class than A455E, it can be rescued by the same strategies, offering therapeutic promise to patients with Class V mutations.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "For instance, compounds have been identified that rescue ΔF508-CFTR mutation via interactions with the TMDs (Loo et al., 2011). ΔF508 and other mutant CFTRs were also partially rescued by transcomplementation, in which co-expression of parts of CFTR were able to improve trafficking of CF-mutant CFTR from the ER (Cormet-Boyaka et al., 2004; Cebotaru et al., 2008). Insights into the rescue of ΔF508-CFTR also come from the yeast homologous ABC exporter, Yor1p (Pagant et al., 2007, 2008). "
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    ABSTRACT: Cystic fibrosis is a lethal genetic disease caused by lack of functional cystic fibrosis transmembrane conductance regulator (CFTR) proteins at the apical surface of secretory epithelia. CFTR is a multidomain protein, containing five domains, and its functional structure is attained in a hierarchical folding process. Most CF-causing mutations in CFTR, including the most common mutation, a deletion of phenylalanine at position 508 (ΔF508), are unable to properly fold into this functional native three dimensional structure. Currently, no high-resolution structural information about full length CFTR exists. However, insight has been gained through examining homologous ABC transporter structures, molecular modeling, and high-resolution structures of individual, isolated CFTR domains. Taken together, these studies indicate that the prevalent ΔF508 mutation disrupts two essential steps during the development of the native structure: folding of the first nucleotide binding domain (NBD1) and its later association with the fourth intracellular loop (ICL4) in the second transmembrane domain (TMD2). Therapeutics to rescue ΔF508 and other mutants in CFTR can be targeted to correct defects that occur during the complex folding process. This article reviews the structural relationships between CFTR and ABC transporters and current knowledge about how CFTR attains its structure-with a focus on how this process is altered by CF-causing mutations in a manner targetable by therapeutics.
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