Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis airway cells

Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4′-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties.

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Increased functional cell surface expression of CFTR and ΔF508-CFTR by the anthracycline doxorubicin

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.5.c1031 ◽

2001 ◽

Vol 280 (5) ◽

pp. C1031-C1037 ◽

Cited By ~ 22

Author(s):

Rangan Maitra ◽

Collin M. Shaw ◽

Bruce A. Stanton ◽

Joshua W. Hamilton

Keyword(s):

Cystic Fibrosis ◽

Cell Surface ◽

Protein Expression ◽

Surface Expression ◽

Chloride Secretion ◽

Cell Surface Expression ◽

Common Mutation ◽

Transmembrane Conductance Regulator ◽

Δf508 Cftr ◽

Cftr Protein

Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, ΔF508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, ΔF508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 μM) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased ΔF508-CFTR cell surface expression and ΔF508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF.

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Increased Cell Surface Expression Of Respiratory Syncytial Virus Receptor (Nucleolin) In Ciliated Columnar Airway Epithelial Cells From Children With Cystic Fibrosis And Asthma

10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a5486 ◽

2012 ◽

Author(s):

Richard G. Hegele ◽

Peter Mastrangelo ◽

Angela Fonceca ◽

Paul S. McNamara

Keyword(s):

Cystic Fibrosis ◽

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

Cell Surface ◽

Surface Expression ◽

Airway Epithelial Cells ◽

Cell Surface Expression ◽

Airway Epithelial ◽

Virus Receptor ◽

Syncytial Virus

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ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555218763310 ◽

2018 ◽

Vol 23 (8) ◽

pp. 823-831

Author(s):

Puay-Wah Phuan ◽

Guido Veit ◽

Joseph-Anthony Tan ◽

Ariel Roldan ◽

Walter E. Finkbeiner ◽

...

Keyword(s):

Cystic Fibrosis ◽

Cell Surface ◽

Nucleotide Binding ◽

Surface Expression ◽

Cell Surface Expression ◽

Wild Type ◽

Nucleotide Binding Domain ◽

Δf508 Cftr ◽

Nucleotide Binding Domain 1 ◽

Cftr Modulators

The most common cystic fibrosis–causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (∆F508). The ∆F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ∆F508-CFTR modulators that act on NBD1. A biochemical screen for ΔF508-NBD1 cell surface expression was done in Madin–Darby canine kidney cells expressing a chimeric reporter consisting of ΔF508-NBD1, the CD4 transmembrane domain, and an extracellular horseradish peroxidase (HRP) reporter. Using a luminescence readout of HRP activity, the screen was robust with a Z′ factor of 0.7. The screening of ~20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased ∆F508-NBD1 cell surface expression by up to 4-fold; for comparison, a 12-fold increased cell surface expression was found for a wild-type NBD1 chimera. While the compounds were inactive as correctors of full-length ΔF508-CFTR, several carboxamide-benzothiophenes had potentiator activity with low micromolar EC50. Interestingly, the potentiators did not activate G551D or wild-type CFTR. Our results provide a proof of concept for a cell-based NBD1 domain screen to identify ∆F508-CFTR modulators that target the NBD1 domain.

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A foldable CFTRΔF508 biogenic intermediate accumulates upon inhibition of the Hsc70–CHIP E3 ubiquitin ligase

The Journal of Cell Biology ◽

10.1083/jcb.200410065 ◽

2004 ◽

Vol 167 (6) ◽

pp. 1075-1085 ◽

Cited By ~ 118

Author(s):

J. Michael Younger ◽

Hong-Yu Ren ◽

Liling Chen ◽

Chun-Yang Fan ◽

Andrea Fields ◽

...

Keyword(s):

Cystic Fibrosis ◽

Cell Surface ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Surface Expression ◽

Cell Surface Expression ◽

Folding Defect ◽

Cellular Components ◽

Soluble State

CFTRΔF508 exhibits a correctable protein-folding defect that leads to its misfolding and premature degradation, which is the cause of cystic fibrosis (CF). Herein we report on the characterization of the CFTRΔF508 biogenic intermediate that is selected for proteasomal degradation and identification of cellular components that polyubiquitinate CFTRΔF508. Nonubiquitinated CFTRΔF508 accumulates in a kinetically trapped, but folding competent conformation, that is maintained in a soluble state by cytosolic Hsc70. Ubiquitination of Hsc70-bound CFTRΔF508 requires CHIP, a U box containing cytosolic cochaperone. CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a. Inactivation of the Hsc70–CHIP E3 leads CFTRΔF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface. Inhibition of CFTRΔF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.

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