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

The intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding domain 1 (NBD1) of the cystic fibrosis 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 cystic fibrosis 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. This article is protected by copyright. All rights reserved

Analysis of distinct molecular assembly complexes of keratin K8 and K18 by hydrogen-deuterium exchange

Keratins are intermediate filament (IF) proteins that form complex filament systems in epithelial cells, thus serving as scaffolding elements and mechanical stress absorbers. The building blocks of keratin IFs are parallel coiled-coil dimers of two distinct sequence-related proteins distinguished as type I and type II keratins. To gain more insight into their structural dynamics, we resorted to hydrogen–deuterium exchange mass spectrometry of keratins K8 and K18, which are characteristic for simple epithelial cells. Using this powerful technique not employed with IFs before, we mapped patterns of protected versus unprotected regions in keratin complexes at various assembly levels. In particular, we localized protein segments exhibiting different hydrogen exchange patterns in tetramers versus filaments. We observed a general pattern of precisely positioned regions of stability intertwining with flexible regions, mostly represented by the non-α-helical segments. Notably, some regions within the coiled-coil domains are significantly more dynamic than others, while the IF-consensus motifs at the end domains of the central α-helical “rod” segment, which mediate the “head-to-tail” dimer–dimer interaction in the filament elongation process, become distinctly more protected upon formation of filaments. Moreover, to gain more insight into the dynamics of the individual keratins, we investigated the properties of homomeric preparations of K8 and K18. The physiological importance of keratins without a partner is encountered in both pathological and experimental situations when one of the two species is present in robust excess or completely absent, such as in gene-targeted mice

Influence of alder (Alnus glutinosa Gaerthn.) veneers on selected mechanical properties of layered pine (Pinus sylvestris L.) composites

Influence of alder (Alnus glutinosa Gaerthn.) veneers on selected mechanical properties of layered pine (Pinus sylvestris L.) composites. The aim of the study was to analyse the influence of using hardwood veneers in the base layer on selected mechanical properties of composites made of coniferous veneers dedicated for flooring applications. The modulus of elasticity and stiffness at three-point bending were determined in static, dynamic and fatigue tests. All tested mechanical properties of pine-alder composites showed, to a different extent, higher values than composites with a base layer made only of pine veneers.Wpływ zastosowania obłogu olchowego (Alnus glutinosa Gaerthn.) na wybrane właściwości mechaniczne kompozytów warstwowych z drewna sosnowego (Pinus sylvestris L.). Celem badań była analiza wpływu zastosowania fornirów z drewna liściastego w warstwie podbudowy na wybrane właściwości mechaniczne kompozytów z fornirów iglastych przeznaczonych do aplikacji na podłogach. Określono moduł sprężystości oraz sztywność przy zginaniu trzypunktowym w testach: statycznym, dynamicznym oraz zmęczeniowym. Wszystkie badane właściwości mechaniczne kompozytów sosnowoolchowych wykazały, w różnym stopniu, wyższe wartości od kompozytów o podstawie wykonanej jedynie z obłogów sosnowych

Discovery of novel potent ΔF508-CFTR correctors that target the nucleotide binding domain

International audienceThe 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