Keratin K18 Increases Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Surface Expression by Binding to Its C-terminal Hydrophobic Patch

Duan, Yuanyuan; Sun, Ying; Zhang, Fan; Zhang, Wei Kevin; Wang, Dong; Wang, Yan; Cao, Xu; Hu, Wenbao; Xie, Changyan; Cuppoletti, John; Magin, Thomas M.; Wang, Haixia; Wu, Zhenguo; Li, Ning; Huang, Pingbo

doi:10.1074/jbc.m112.403584

Cited by 23 publications

(22 citation statements)

References 52 publications

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“…Since DRA is a central intestinal Cl Ϫ /HCO 3 Ϫ transporter, the nearly complete loss of DRA in the K8 Ϫ/Ϫ colon described here, together with the upregulation and mistargeting of AE1/2 (39), explains the decreased Cl Ϫ uptake. No major differences in mRNA levels for PAT-1 or CFTR were observed; however, a role for K8/K18 in CFTR function cannot be ruled out, since K18 was recently found to bind CFTR and regulate CFTR localization to the membrane (8). Indeed, in K18 Ϫ/Ϫ (8) and K8 Ϫ/Ϫ (2) small intestine villi (but not crypts), CFTR surface expression was reduced.…”

Section: Discussionmentioning

confidence: 92%

Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon

Asghar

Priyamvada

Nyström

et al. 2016

American Journal of Physiology-Gastrointestinal and Liver Physi

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Asghar MN, Priyamvada S, Nyström JH, Anbazhagan AN, Dudeja PK, Toivola DM. Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon. Am J Physiol Gastrointest Liver Physiol 310: G1147-G1154, 2016. First published April 28, 2016 doi:10.1152 doi:10. /ajpgi.00354.2015 are intermediate filament proteins important in protection from stress. The roles of keratins in the intestine are not clear, but K8 knockout (K8 Ϫ/Ϫ ) mice develop a Th2-type colonic inflammation, epithelial hyperproliferation, and mild diarrhea caused by a keratin level-dependent decrease in short-circuit current and net sodium and chloride absorption in the distal colon. The lack of K8 leads to mistargeting or altered levels of membrane proteins in colonocytes; however, the main transporter responsible for the keratin-related ion transport defect is unknown. We here analyzed protein and mRNA levels of candidate ion transporters CFTR, PAT-1, NHE-3, and DRA in ileum, cecum, and proximal and distal colon. Although no differences were observed for CFTR, PAT-1, or NHE-3, DRA mRNA levels were decreased by three-to fourfold and DRA protein was almost entirely lost in K8 Ϫ/Ϫ cecum and proximal and distal colon compared with K8 ϩ/ϩ , whereas the levels in ileum were normal. In K8 ϩ/Ϫ mice, DRA mRNA levels were unaltered, while decreased DRA protein levels were detected in the proximal colon. Immunofluorescence staining confirmed the loss of DRA in K8 Ϫ/Ϫ distal colon, while K8 ϩ/Ϫ displayed a similar but more patchy apical DRA distribution compared with K8 ϩ/ϩ . DRA was similarly decreased when K8 was knocked down in Caco-2 cells, confirming that K8 levels modulate DRA levels in an inflammationindependent manner. Taken together, the loss of DRA in the K8

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Section: Discussionmentioning

confidence: 92%

Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon

Asghar

Priyamvada

Nyström

et al. 2016

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…The IFs: K8 and keratin 18 (K18) prevent the delivery of functional ΔF508‐CFTR to the plasma membrane . K18 reportedly binds to the CFTR C‐terminus, regulating its function . We recently demonstrated that K8, but not K18, physically binds to the NBD1 of both wt and ΔF508 CFTR, with a greater tendency to target the unfolded destabilized conformation caused by F508 deletion.…”

Section: Introductionmentioning

confidence: 99%

New insights into interactions between the nucleotide‐binding domain of CFTR and keratin 8

et al. 2017

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The intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding domain 1 (NBD1) of the cystic fibrosis (CF) transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508-CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for CF patients bearing the ΔF508 mutation. Here, we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on recombinant wild-type (wt) NBD1 and ΔF508-NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt-NBD1 and ΔF508-NBD1. Finally, we performed HDX-MS analysis of the NBD1 molecules and full-length K8, revealing hydrogen-bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508-NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1.

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“…Other molecules such as curcumin or resveratrol derived from plants may act by modifying keratin 18 (K18) network (Hamdaoui et al, ; Lipecka et al, ). K18 heterodimerizes with keratin 8 (K8) and further evidence suggests that both proteins play a role in the trafficking of CFTR/ΔF508‐CFTR (Colas et al, ; Duan et al, ). Accordingly, we have reported that a decrease in K8 expression leads to functional correction of ΔF508‐CFTR (Colas et al, ).…”

Section: Introductionmentioning

confidence: 99%

Discovery of novel potent ΔF 508‐ CFTR correctors that target the nucleotide binding domain

Odolczyk¹,

Fritsch

Norez

et al. 2013

EMBO Mol Med

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The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508-CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508-NBD1 and housekeeping proteins prevents ΔF508-CFTR delivery to the plasma membrane. Based on this assumption we applied structure-based virtual screening to identify new low-molecular-weight compounds that should bind to ΔF508-NBD1 and act as protein–protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico-selected compounds induced functional expression of ΔF508-CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ΔF508-CFTR mice. The proposed compounds disrupt keratin8-ΔF508-CFTR interaction in ΔF508-CFTR HeLa cells. Structural analysis of ΔF508-NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ΔF508-CFTR trafficking defect known to date.

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Keratin K18 Increases Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Surface Expression by Binding to Its C-terminal Hydrophobic Patch

Cited by 23 publications

References 52 publications

Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon

Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon

New insights into interactions between the nucleotide‐binding domain of CFTR and keratin 8

Discovery of novel potent ΔF 508‐ CFTR correctors that target the nucleotide binding domain

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