scholarly journals Distinct proteostasis states drive pharmacologic chaperone susceptibility for Cystic Fibrosis Transmembrane Conductance Regulator misfolding mutants

2021 ◽  
Author(s):  
Eli Fritz McDonald ◽  
Carleen Mae P. Sabusap ◽  
Minsoo Kim ◽  
Lars Plate
Keyword(s):  
Cystic Fibrosis ◽  
Protein Misfolding ◽  
Clinical Success ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Pharmacological Chaperone ◽  
Pharmacological Chaperones ◽  
Misfolding Diseases ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane

ABSTRACTPharmacological chaperones represent a class of therapeutic compounds for treating protein misfolding diseases. One of the most prominent examples is the FDA-approved pharmacological chaperone lumacaftor (VX-809), which has transformed cystic fibrosis (CF) therapy. CF is a fatal disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). VX-809 corrects folding of F508del CFTR, the most common patient mutation, yet F508del exhibits only mild VX-809 response. In contrast, rarer mutations P67L and L206W are hyper-responsive to VX-809, while G85E is non-responsive. Despite the clinical success of VX-809, the mechanistic origin for the distinct susceptibility of mutants remains unclear. Here, we use interactomics to characterize the impact of VX-809 on proteostasis interactions of P67L and L206W and compare these to F508del and G85E. We determine hyper-responsive mutations P67L and L206W exhibit decreased interactions with proteasomal, and autophagy degradation machinery compared to F508del and G85E. We then show inhibiting the proteasome attenuates P67L and L206W VX-809 response, and inhibiting the lysosome attenuates F508del VX-809 response. Our data suggests a previously unidentified but required role for protein degradation in VX-809 correction. Furthermore, we present an approach for identifying proteostasis characteristics of mutant-specific therapeutic response to pharmacological chaperones.

scholarly journals Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane

2003 ◽  
Vol 374 (3) ◽  
pp. 793-797 ◽  
Author(s):  
Mohabir RAMJEESINGH ◽  
Jackie F. KIDD ◽  
Ling Jun HUAN ◽  
Yanchun WANG ◽  
Christine E. BEAR
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Biology ◽  
Plasma Membranes ◽  
Chinese Hamster Ovary Cells ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane

CFTR (cystic fibrosis transmembrane conductance regulator) mediates chloride conduction across the apical membrane of epithelia, and mutations in CFTR lead to defective epithelial fluid transport. Recently, there has been considerable interest in determining the quaternary structure of CFTR at the cell surface, as such information is a key to understand the molecular basis for pathogenesis in patients harbouring disease-causing mutations. In our previous work [Ramjeesingh, Li, Kogan, Wang, Huan and Bear (2001) Biochemistry 40, 10700–10706], we showed that monomeric CFTR is the minimal functional form of the protein, yet when expressed in Sf 9 cells using the baculovirus system, it also exists as dimers. The purpose of the present study was to determine if dimeric CFTR exists at the surface of mammalian cells, and particularly in epithelial cells. CFTR solubilized from membranes prepared from Chinese-hamster ovary cells stably expressing CFTR and from T84 epithelial cells migrates as predicted for monomeric, dimeric and larger complexes when subjected to sizing by gel filtration and analysis by non-dissociative electrophoresis. Purification of plasma membranes led to the enrichment of CFTR dimers and this structure exists as the complex glycosylated form of the protein, supporting the concept that dimeric CFTR is physiologically relevant. Consistent with its localization in plasma membranes, dimeric CFTR was labelled by surface biotinylation. Furthermore, dimeric CFTR was captured at the apical surface of intact epithelial cells by application of a membrane-impermeable chemical cross-linker. Therefore it follows from the present study that CFTR dimers exist at the surface of epithelial cells. Further studies are necessary to understand the impact of dimerization on the cell biology of wild-type and mutant CFTR proteins.

scholarly journals Cystic Fibrosis–Related Diabetes: Pathophysiology and Therapeutic Challenges

2019 ◽  
Vol 12 ◽  
pp. 117955141985177 ◽  
Author(s):  
Ryan Kelsey ◽  
Fiona N Manderson Koivula ◽  
Neville H McClenaghan ◽  
Catriona Kelly
Keyword(s):  
Cystic Fibrosis ◽  
Bacterial Infections ◽  
Respiratory Health ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Current Review ◽  
Co Morbidity ◽  
Endocrine Pancreatic Function ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane

Cystic fibrosis–related diabetes (CFRD) is among the most common extrapulmonary co-morbidity associated with cystic fibrosis (CF), affecting an estimated 50% of adults with the condition. Cystic fibrosis is prevalent in 1 in every 2500 Caucasian live births and is caused by a mutation in the cystic fibrosis transmembrane conductance regulator ( CFTR) gene. Mutated CFTR leads to dehydrated epithelial surfaces and a build-up of mucus in a variety of tissues including the lungs and pancreas. The leading cause of mortality in CF is repeated respiratory bacterial infections, which prompts a decline in lung function. Co-morbid diabetes promotes bacterial colonisation of the airways and exacerbates the deterioration in respiratory health. Cystic fibrosis–related diabetes is associated with a 6-fold higher mortality rate compared with those with CF alone. The management of CFRD adds a further burden for the patient and creates new therapeutic challenges for the clinical team. Several proposed hypotheses on how CFRD develops have emerged, including exocrine-driven fibrosis and destruction of the entire pancreas and contrasting theories on the direct or indirect impact of CFTR mutation on islet function. The current review outlines recent data on the impact of CFTR on endocrine pancreatic function and discusses the use of conventional diabetic therapies and new CFTR-correcting drugs on the treatment of CFRD.

scholarly journals Differential thermostability and response to cystic fibrosis transmembrane conductance regulator potentiators of human and mouse F508del-CFTR

2019 ◽  
Vol 317 (1) ◽  
pp. L71-L86 ◽  
Author(s):  
Samuel J. Bose ◽  
Marcel J. C. Bijvelds ◽  
Yiting Wang ◽  
Jia Liu ◽  
Zhiwei Cai ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Membrane Expression ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane ◽  
Human And Mouse

Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl− channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl− channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl− channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl− channels.

scholarly journals Enhanced cell-surface stability of rescued ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) by pharmacological chaperones

2008 ◽  
Vol 410 (3) ◽  
pp. 555-564 ◽  
Author(s):  
Karoly Varga ◽  
Rebecca F. Goldstein ◽  
Asta Jurkuvenaite ◽  
Lan Chen ◽  
Sadis Matalon ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Half Life ◽  
Permissive Temperature ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Surface Stability ◽  
Pharmacological Chaperones ◽  
Δf508 Cftr ◽  
Cystic Fibrosis Transmembrane

Misfolded proteins destined for the cell surface are recognized and degraded by the ERAD [ER (endoplasmic reticulum) associated degradation] pathway. TS (temperature-sensitive) mutants at the permissive temperature escape ERAD and reach the cell surface. In this present paper, we examined a TS mutant of the CFTR [CF (cystic fibrosis) transmembrane conductance regulator], CFTR ΔF508, and analysed its cell-surface trafficking after rescue [rΔF508 (rescued ΔF508) CFTR]. We show that rΔF508 CFTR endocytosis is 6-fold more rapid (∼30% per 2.5 min) than WT (wild-type, ∼5% per 2.5 min) CFTR at 37 °C in polarized airway epithelial cells (CFBE41o−). We also investigated rΔF508 CFTR endocytosis under two further conditions: in culture at the permissive temperature (27 °C) and following treatment with pharmacological chaperones. At low temperature, rΔF508 CFTR endocytosis slowed to WT rates (20% per 10 min), indicating that the cell-surface trafficking defect of rΔF508 CFTR is TS. Furthermore, rΔF508 CFTR is stabilized at the lower temperature; its half-life increases from <2 h at 37 °C to >8 h at 27 °C. Pharmacological chaperone treatment at 37 °C corrected the rΔF508 CFTR internalization defect, slowing endocytosis from ∼30% per 2.5 min to ∼5% per 2.5 min, and doubled ΔF508 surface half-life from 2 to 4 h. These effects are ΔF508 CFTR-specific, as pharmacological chaperones did not affect WT CFTR or transferrin receptor internalization rates. The results indicate that small molecular correctors may reproduce the effect of incubation at the permissive temperature, not only by rescuing ΔF508 CFTR from ERAD, but also by enhancing its cell-surface stability.

scholarly journals Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier–dependent pathway

2013 ◽  
Vol 24 (2) ◽  
pp. 74-84 ◽  
Author(s):  
Annette Ahner ◽  
Xiaoyan Gong ◽  
Bela Z. Schmidt ◽  
Kathryn W. Peters ◽  
Wael M. Rabeh ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Airway Epithelial Cells ◽  
Small Heat Shock Proteins ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Shock Proteins ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane

Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27’s ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4’s impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin–proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein.

scholarly journals Cystic Fibrosis Transmembrane Conductance Regulator Folding Mutations Reveal Differences in Corrector Efficacy Linked to Increases in Immature Cystic Fibrosis Transmembrane Conductance Regulator Expression

2021 ◽  
Vol 12 ◽  
Author(s):  
Kathryn W. Peters ◽  
Xiaoyan Gong ◽  
Raymond A. Frizzell
Keyword(s):  
Cystic Fibrosis ◽  
Control Level ◽  
Epithelial Cell Line ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Band Ratio ◽  
Empty Vector ◽  
Induced Folding ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane

Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin–proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B.Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio.Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level.Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

scholarly journals Divergent signaling via SUMO modification: potential for CFTR modulation

2016 ◽  
Vol 310 (3) ◽  
pp. C175-C180 ◽  
Author(s):  
Annette Ahner ◽  
Xiaoyan Gong ◽  
Raymond A. Frizzell
Keyword(s):  
Cystic Fibrosis ◽  
Protein Misfolding ◽  
Small Heat Shock Protein ◽  
Short Review ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Sumo Modification ◽  
Anion Conductance ◽  
Apical Membranes ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) is generally responsible for the cAMP/PKA regulated anion conductance at the apical membranes of secretory epithelial cells. Mutations in CFTR underlie cystic fibrosis (CF), in which the most common variant, F508del, causes protein misfolding and its proteasome-mediated degradation. A new pathway that contributes to mutant CFTR degradation is mediated by the small heat shock protein, Hsp27, which cooperates with Ubc9, the E2 enzyme for SUMOylation, to selectively conjugate mutant CFTR with SUMO-2/3. This SUMO paralog can form polychains, which are recognized by the ubiquitin E3 enzyme, RNF4, leading to CFTR ubiquitylation and recognition by the proteasome. We found also that F508del CFTR could be modified by SUMO-1, a paralog that does not support SUMO polychain formation. The use of different SUMO paralogs to modify and target a single substrate for divergent purposes is not uncommon. In this short review we discuss the possibility that conjugation with SUMO-1 could protect mutant CFTR from disposal by RNF4 and similar ubiquitin ligases. We hypothesize that such a pathway could contribute to therapeutic efforts to stabilize immature mutant CFTR and thereby enhance the action of therapeutics that correct CFTR trafficking to the apical membranes.

scholarly journals Role of transmembrane spanning domain 1 in cystic fibrosis transmembrane conductance regulator folding

2021 ◽  
Author(s):  
Anna E. Patrick ◽  
Linda Millen ◽  
Philip J. Thomas
Keyword(s):  
Cystic Fibrosis ◽  
Transient Stability ◽  
Protein Quality ◽  
Regulatory Region ◽  
Common Mutation ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane

AbstractCystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein that disrupt its folding pathway. The most common mutation causing CF is a deletion of phenylalanine at position 508 (ΔF508). CFTR contains five domains that each form cotranslational structures that interact with other domains as they are produced and folded. CFTR is comprised of two transmembrane spanning domains (TMDs), two nucleotide binding domains (NBDs) and a unique regulatory region (R). The first domain translated, TMD1, forms interdomain interactions with the other domains in CFTR. In TMD1, long intracellular loops extend into the cytoplasm and interact with both NBDs via coupling helices and with TMD2 via transmembrane spans (TMs). We examined mutations in TMD1 to determine the impact on individual domain and multidomain constructs. We found that mutations in a TM span or in the cytosolic ICLs interfere with specific steps in the hierarchical folding of CFTR. TM1 CF-causing mutants, G85E and G91R, directly affect TMD1, whereas most ICL1 and ICL2 mutant effects become apparent in the presence of TMD2. A single mutant in ICL2 worsened CFTR trafficking in the presence of NBD2, supporting its role in the ICL2-NBD2 interface. Mutation of hydrophobic residues in ICL coupling helices tended to increased levels of pre-TMD2 biogenic intermediates but caused ER accumulation in the presence of TMD2. This suggests a tradeoff between transient stability during translation and final structure. NBD2 increased the efficiency of mutant trafficking from the ER, consistent with stabilization of the full-length constructs. While the G85E and G91R mutants in TM1 have immediately detectable effects, most of the studied mutant effects and the ΔF508 mutant are apparent after production of TMD2, supporting this intermediate as a major point of recognition by protein quality control.

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