Induction of HSP70 promotes ΔF508 CFTR trafficking

2001 ◽  
Vol 281 (1) ◽  
pp. L58-L68 ◽  
Author(s):  
Lee R. Choo-Kang ◽  
Pamela L. Zeitlin
Keyword(s):  
Heat Shock ◽  
Hsp70 Expression ◽  
Temperature Sensitive ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Genetic Techniques ◽  
Cystic Fibrosis Transmembrane ◽  
Dose Dependent Increase ◽  
Dose Dependent

The ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) is a temperature-sensitive trafficking mutant that is detected as an immature 160-kDa form (band B) in gel electrophoresis. The goal of this study was to test the hypothesis that HSP70, a member of the 70-kDa heat shock protein family, promotes ΔF508 CFTR processing to the mature 180-kDa form (band C). Both pharmacological and genetic techniques were used to induce HSP70. IB3-1 cells were treated with sodium 4-phenylbutyrate (4PBA) to promote maturation of ΔF508 CFTR to band C. A dose-dependent increase in band C and total cellular HSP70 was observed. Under these conditions, HSP70-CFTR complexes were increased and 70-kDa heat shock cognate protein-CFTR complexes were decreased. Increased ΔF508 CFTR maturation was also seen after transfection with an HSP70 expression plasmid and exposure to glutamine, an inducer of HSP70. With immunofluorescence techniques, the increased appearance of CFTR band C correlated with CFTR distribution beyond the perinuclear regions. These data suggest that induction of HSP70 promotes ΔF508 CFTR maturation and trafficking.

scholarly journals ΔF508 CFTR Pool in the Endoplasmic Reticulum Is Increased by Calnexin Overexpression

2004 ◽  
Vol 15 (2) ◽  
pp. 563-574 ◽  
Author(s):  
Tsukasa Okiyoneda ◽  
Kazutsune Harada ◽  
Motohiro Takeya ◽  
Kaori Yamahira ◽  
Ikuo Wada ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Endoplasmic Reticulum ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Cystic Fibrosis Transmembrane ◽  
Erad Pathway

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, ΔF508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of ΔF508 CFTR with calnexin, an ER chaperone, results in the ERAD of ΔF508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of ΔF508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the ΔF508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated ΔF508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of ΔF508 CFTR in the ER.

scholarly journals Correctors Promote Maturation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-processing Mutants by Binding to the Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33247-33251 ◽  
Author(s):  
Ying Wang ◽  
Tip W. Loo ◽  
M. Claire Bartlett ◽  
David M. Clarke
Keyword(s):  
Cystic Fibrosis ◽  
Channel Blocker ◽  
Cross Linking ◽  
Transmembrane Conductance Regulator ◽  
Mature Protein ◽  
Transmembrane Conductance ◽  
P Glycoprotein ◽  
Δf508 Cftr ◽  
Cross Linker ◽  
Cystic Fibrosis Transmembrane

The most common cause of cystic fibrosis (CF) is defective folding of a cystic fibrosis transmembrane conductance regulator (CFTR) mutant lacking Phe508 (ΔF508). The ΔF508 protein appears to be trapped in a prefolded state with incomplete packing of the transmembrane (TM) segments, a defect that can be repaired by expression in the presence of correctors such as corr-4a, VRT-325, and VRT-532. To determine whether the mechanism of correctors involves direct interactions with CFTR, our approach was to test whether correctors blocked disulfide cross-linking between cysteines introduced into the two halves of a Cys-less CFTR. Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 °C, we found that maturation could be restored if Val510 was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. The V510D mutation also promoted maturation of ΔF508 CFTR. The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Correctors and the CFTR channel blocker benzbromarone, but not P-glycoprotein substrates, inhibited cross-linking of mutant I340C(TM6)/S877C(TM7). These results suggest that corrector molecules such as corr-4a interact directly with CFTR.

scholarly journals Regulation of Ubiquitin-Proteasome System–mediated Degradation by Cytosolic Stress

2007 ◽  
Vol 18 (11) ◽  
pp. 4279-4291 ◽  
Author(s):  
Sean M. Kelly ◽  
Judy K. VanSlyke ◽  
Linda S. Musil
Keyword(s):  
Heat Shock ◽  
Secretory Pathway ◽  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Junction Protein ◽  
Ubiquitin Proteasome ◽  
Gap Junction Protein ◽  
Cystic Fibrosis Transmembrane

ER-associated, ubiquitin-proteasome system (UPS)-mediated degradation of the wild-type (WT) gap junction protein connexin32 (Cx32) is inhibited by mild forms of cytosolic stress at a step before its dislocation into the cytosol. We show that the same conditions (a 30-min, 42°C heat shock or oxidative stress induced by arsenite) also reduce the endoplasmic reticulum (ER)-associated turnover of disease-causing mutants of Cx32 and the cystic fibrosis transmembrane conductance regulator (CFTR), as well as that of WT CFTR and unassembled Ig light chain. Stress-stabilized WT Cx32 and CFTR, but not the mutant/unassembled proteins examined, could traverse the secretory pathway. Heat shock also slowed the otherwise rapid UPS-mediated turnover of the cytosolic proteins myoD and GFPu, but not the degradation of an ubiquitination-independent construct (GFP-ODC) closely related to the latter. Analysis of mutant Cx32 from cells exposed to proteasome inhibitors and/or cytosolic stress indicated that stress reduces degradation at the level of substrate polyubiquitination. These findings reveal a new link between the cytosolic stress-induced heat shock response, ER-associated degradation, and polyubiquitination. Stress-denatured proteins may titer a limiting component of the ubiquitination machinery away from pre-existing UPS substrates, thereby sparing the latter from degradation.

Dynasore inhibits removal of wild-type and ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) from the plasma membrane

2009 ◽  
Vol 421 (3) ◽  
pp. 377-385 ◽  
Author(s):  
Andrew Young ◽  
Martina Gentzsch ◽  
Cynthia Y. Abban ◽  
Ying Jia ◽  
Patricio I. Meneses ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Small Molecule ◽  
Short Exposure ◽  
Growth Media ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Cystic Fibrosis Transmembrane

Dynasore, a small molecule inhibitor of dynamin, was used to probe the role of dynamin in the endocytosis of wild-type and mutant CFTR (cystic fibrosis transmembrane conductance regulator). Internalization of both wild-type and ‘temperature-corrected’ ΔF508 CFTR was markedly inhibited by a short exposure to dynasore, implicating dynamin as a key element in the endocytic internalization of both wild-type and mutant CFTR. The inhibitory effect of dynasore was readily reversible upon washout of dynasore from the growth media. Corr-4 ({2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′]-bithiazolyl-2′-yl}-phenyl-methanonone), a pharmacological corrector of ΔF508 CFTR biosynthesis, caused a marked increase in the cell surface expression of mutant CFTR. Co-incubation of ΔF508 CFTR expressing cells with Corr-4 and dynasore caused a significantly greater level of cell surface CFTR than that observed in the presence of Corr-4 alone. These results argue that inhibiting the endocytic internalization of mutant CFTR provides a novel therapeutic target for augmenting the benefits of small molecule correctors of mutant CFTR biosynthesis.

Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive

Nature ◽  
1992 ◽  
Vol 358 (6389) ◽  
pp. 761-764 ◽  
Author(s):  
Gerene M. Denning ◽  
Matthew P. Anderson ◽  
Jane F. Amara ◽  
John Marshall ◽  
Alan E. Smith ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Temperature Sensitive ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

scholarly journals Structural comparative modeling of multi-domain ΔF508 CFTR

2021 ◽  
Author(s):  
Eli Fritz McDonald ◽  
Hope Woods ◽  
Shannon Smith ◽  
Minsoo Kim ◽  
Clara T. Schoeder ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Rational Design ◽  
Anion Channel ◽  
Comparative Modeling ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Δf508 Cftr ◽  
Domain Models ◽  
Cystic Fibrosis Transmembrane

Cystic Fibrosis (CF) is a common genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs - which increases the risk of infection and eventually causes death. CFTR is an ATP binding cassette (ABC) transporter composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 (ΔF508), making ΔF508 CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain ΔF508 CFTR structure has been determined and few studies have modeled ΔF508 using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a ΔF508 model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and ΔF508 CFTR structural models. These models demonstrate the destabilizing effects of ΔF508 on NBD1 and the NBD1/TMD interface in both the closed and open conformation of CFTR. Furthermore, we modeled ΔF508/R1070W and ΔF508 bound to a the CFTR corrector VX-809. Our models reveal the stabilizing effects of R1070W and VX-809 on multi-domain models of ΔF508 CFTR and pave the way for rational design of additional drugs that target ΔF508 CFTR for treatment of CF.

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.

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