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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    Journal of Controlled Release 01/2014; 178(1). DOI:10.1016/j.jconrel.2014.01.007 · 7.71 Impact Factor
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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.
    PLoS ONE 01/2014; 9(1):e85183. DOI:10.1371/journal.pone.0085183 · 3.23 Impact Factor
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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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