An association study on contrasting cystic fibrosis endophenotypes recognizes KRT8 but not KRT18 as a modifier of cystic fibrosis disease severity and CFTR mediated residual chloride secretion

<p>Abstract</p> <p>Background</p> <p>F508del-CFTR, the most frequent disease-causing mutation among Caucasian cystic fibrosis (CF) patients, has been characterised as a mutant defective in protein folding, processing and trafficking. We have investigated the two neighbouring cytokeratin genes <it>KRT8 </it>and <it>KRT18 </it>in a candidate gene approach to ask whether variants in <it>KRT8 </it>and/or <it>KRT18 </it>modify the impaired ion conductance known as the CF basic defect, and whether they are associated with correct trafficking of mutant CFTR and disease severity of CF.</p> <p>Methods</p> <p>We have selected contrasting F508del-<it>CFTR </it>homozygous patient subpopulations stratified for disease severity, comparing 13 concordant mildly affected sib pairs vs. 12 concordant severely affected sib pairs, or manifestation of the CF basic defect in intestinal epithelium, comparing 22 individuals who exhibit CFTR-mediated residual chloride secretion vs. 14 individuals who do not express any chloride secretion, for an association. The <it>KRT8</it>/<it>KRT18 </it>locus was initially interrogated with one informative microsatellite marker. Subsequently, a low density SNP map with four SNPs in KRT8 and two SNPs in KRT18, each selected for high polymorphism content, was used to localize the association signal.</p> <p>Results</p> <p><it>KRT8</it>, but not <it>KRT18</it>, showed an association with CF disease severity (P_best= 0.00131; P_corr= 0.0185) and CFTR mediated residual chloride secretion (P_best= 0.0004; P_corr= 0.0069). Two major four-marker-haplotypes spanning 13 kb including the entire <it>KRT8 </it>gene accounted for 90% of chromosomes, demonstrating strong linkage disequilibrium at that locus. Absence of chloride secretion was associated with the recessive haplotype 1122 at rs1907671, rs4300473, rs2035878 and rs2035875. The contrasting haplotype 2211 was dominant for the presence of CFTR mediated residual chloride secretion. In consistency, the <it>KRT8 </it>haplotype 2211 was associated with mild CF disease while 1122 was observed as risk haplotype. Analysis of microsatellite allele distributions on the SNP background suggests that the mild <it>KRT8 </it>haplotype 2211 is phylogenetically older than its severe counterpart.</p> <p>Conclusions</p> <p>The two opposing <it>KRT8 </it>alleles which have been identified as a benign and as a risk allele in this work are likely effective in the context of epithelial cell differentiation. As the mild <it>KRT8 </it>allele is associated with CFTR mediated residual chloride secretion among F508del-<it>CFTR </it>homozygotes, the KRT8/KRT18 heterodimeric intermediary filaments of the cytoskeleton apparently are an essential component for the proper targeting of CFTR to the apical membrane in epithelial cells.</p

DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription

<p>Abstract</p> <p>Background</p> <p>The clinical course of cystic fibrosis (CF) is characterized by recurrent pulmonary infections and chronic inflammation. We have recently shown that decreased methylation of the toll-like receptor-2 (TLR2) promoter leads to an apparent CF-related up-regulation of TLR2. This up-regulation could be responsible, in part, for the CF-associated enhanced proinflammatory responses to various bacterial products in epithelial cells. However, the molecular mechanisms underlying DNA hypomethylation-dependent enhancement of TLR2 expression in CF cells remain unknown.</p> <p>Results</p> <p>The present study indicates that there is a specific CpG region (CpG#18-20), adjacent to the SP1 binding site that is significantly hypomethylated in several CF epithelial cell lines. These CpGs encompass a minimal promoter region required for basal TLR2 expression, and suggests that CpG#18-20 methylation regulates TLR2 expression in epithelial cells. Furthermore, reporter gene analysis indicated that the SP1 binding site is involved in the methylation-dependent regulation of the TLR2 promoter. Inhibition of SP1 with mithramycin A decreased TLR2 expression in both CF and 5-azacytidine-treated non-CF epithelial cells. Moreover, even though SP1 binding was not affected by CpG methylation, SP1-dependent transcription was abolished by CpG methylation.</p> <p>Conclusion</p> <p>This report implicates SP1 as a critical component of DNA demethylation-dependent up-regulation of TLR2 expression in CF epithelial cells.</p

Chaperones rescue the energetic landscape of mutant CFTR at single molecule and in cell

Molecular chaperones are pivotal in folding and degradation of the cellular proteome but their impact on the conformational dynamics of near-native membrane proteins with disease relevance remains unknown. Here we report the effect of chaperone activity on the functional conformation of the temperature-sensitive mutant cystic fibrosis channel (Delta F508-CFTR) at the plasma membrane and after reconstitution into phospholipid bilayer. Thermally induced unfolding at 37 degrees C and concomitant functional inactivation of Delta F508-CFTR are partially suppressed by constitutive activity of Hsc70 and Hsp90 chaperone/co-chaperone at the plasma membrane and post-endoplasmic reticulum compartments in vivo, and at singlemolecule level in vitro, indicated by kinetic and thermodynamic remodeling of the mutant gating energetics toward its wild-type counterpart. Thus, molecular chaperones can contribute to functional maintenance of Delta F508-CFTR by reshaping the conformational energetics of its final fold, a mechanism with implication in the regulation of metastable ABC transporters and other plasma membrane proteins activity in health and diseases

Mild Electrical Stimulation with Heat Shock Ameliorates Insulin Resistance via Enhanced Insulin Signaling

Low-intensity electrical current (or mild electrical stimulation; MES) influences signal transduction and activates phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Because insulin resistance is characterized by a marked reduction in insulin-stimulated PI3K-mediated activation of Akt, we asked whether MES could increase Akt phosphorylation and ameliorate insulin resistance. In addition, it was also previously reported that heat shock protein 72 (Hsp72) alleviates hyperglycemia. Thus, we applied MES in combination with heat shock (HS) to in vitro and in vivo models of insulin resistance. Here we show that 10-min treatment with MES at 5 V (0.1 ms pulse duration) together with HS at 42°C increased the phosphorylation of insulin signaling molecules such as insulin receptor substrate (IRS) and Akt in HepG2 cells maintained in high-glucose medium. MES (12 V)+mild HS treatment of high fat-fed mice also increased the phosphorylation of insulin receptor β subunit (IRβ) and Akt in mice liver. In high fat-fed mice and db/db mice, MES+HS treatment for 10 min applied twice a week for 12–15 weeks significantly decreased fasting blood glucose and insulin levels and improved insulin sensitivity. The treated mice showed significantly lower weight of visceral and subcutaneous fat, a markedly improved fatty liver and decreased size of adipocytes. Our findings indicated that the combination of MES and HS alleviated insulin resistance and improved fat metabolism in diabetes mouse models, in part, by enhancing the insulin signaling pathway

The Interaction between the First Transmembrane Domain and the Thumb of ASIC1a Is Critical for Its N-Glycosylation and Trafficking

Acid-sensing ion channel-1a (ASIC1a), the primary proton receptor in the brain, contributes to multiple diseases including stroke, epilepsy and multiple sclerosis. Thus, a better understanding of its biogenesis will provide important insights into the regulation of ASIC1a in diseases. Interestingly, ASIC1a contains a large, yet well organized ectodomain, which suggests the hypothesis that correct formation of domain-domain interactions at the extracellular side is a key regulatory step for ASIC1a maturation and trafficking. We tested this hypothesis here by focusing on the interaction between the first transmembrane domain (TM1) and the thumb of ASIC1a, an interaction known to be critical in channel gating. We mutated Tyr71 and Trp287, two key residues involved in the TM1-thumb interaction in mouse ASIC1a, and found that both Y71G and W287G decreased synaptic targeting and surface expression of ASIC1a. These defects were likely due to altered folding; both mutants showed increased resistance to tryptic cleavage, suggesting a change in conformation. Moreover, both mutants lacked the maturation of N-linked glycans through mid to late Golgi. These data suggest that disrupting the interaction between TM1 and thumb alters ASIC1a folding, impedes its glycosylation and reduces its trafficking. Moreover, reducing the culture temperature, an approach commonly used to facilitate protein folding, increased ASIC1a glycosylation, surface expression, current density and slowed the rate of desensitization. These results suggest that correct folding of extracellular ectodomain plays a critical role in ASIC1a biogenesis and function

Serum and glucocorticoid-inducible kinase1 increases plasma membrane wt-CFTR in human airway epithelial cells by inhibiting its endocytic retrieval

Background: Chloride (Cl) secretion by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) located in the apical membrane of respiratory epithelial cells plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of pathogens. Previously, we and others have shown that the serum and glucocorticoid-inducible kinase-1 (SGK1) increases wild type CFTR (wt-CFTR) mediated Cl transport in Xenopus oocytes by increasing the amount of wt-CFTR protein in the plasma membrane. However, the effect of SGK1 on the membrane abundance of wt-CFTR in airway epithelial cells has not been examined, and the mechanism whereby SGK1 increases membrane wt-CFTR has also not been examined. Thus, the goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of wt-CFTR in human airway epithelial cells. Methods and Results: We report that elevated levels of SGK1, induced by dexamethasone, increase plasma membrane abundance of wt-CFTR. Reduction of SGK1 expression by siRNA (siSGK1) and inhibition of SGK1 activity by the SGK inhibitor GSK 650394 abrogated the ability of dexamethasone to increase plasma membrane wt-CFTR. Overexpression of a constitutively active SGK1 (SGK1-S422D) increased plasma membrane abundance of wt-CFTR. To understand the mechanism whereby SGK1 increased plasma membrane wt-CFTR, we examined the effects of siSGK1 and SGK1-S442D on the endocytic retrieval of wt-CFTR. While siSGK1 increased wt-CFTR endocytosis, SGK1-S442D inhibited CFTR endocytosis. Neither siSGK1 nor SGK1-S442D altered the recycling of endocytosed wt-CFTR back to the plasma membrane. By contrast, SGK1 increased the endocytosis of the epidermal growth factor receptor (EGFR). Conclusion: This study demonstrates for the first time that SGK1 selectively increases wt-CFTR in the plasma membrane of human airway epithelia cells by inhibiting its endocytic retrieval from the membrane. © 2014 Bomberger et al

The Hsc/Hsp70 Co-Chaperone Network Controls Antigen Aggregation and Presentation during Maturation of Professional Antigen Presenting Cells

The maturation of mouse macrophages and dendritic cells involves the transient deposition of ubiquitylated proteins in the form of dendritic cell aggresome-like induced structures (DALIS). Transient DALIS formation was used here as a paradigm to study how mammalian cells influence the formation and disassembly of protein aggregates through alterations of their proteostasis machinery. Co-chaperones that modulate the interplay of Hsc70 and Hsp70 with the ubiquitin-proteasome system (UPS) and the autophagosome-lysosome pathway emerged as key regulators of this process. The chaperone-associated ubiquitin ligase CHIP and the ubiquitin-domain protein BAG-1 are essential for DALIS formation in mouse macrophages and bone-marrow derived dendritic cells (BMDCs). CHIP also cooperates with BAG-3 and the autophagic ubiquitin adaptor p62 in the clearance of DALIS through chaperone-assisted selective autophagy (CASA). On the other hand, the co-chaperone HspBP1 inhibits the activity of CHIP and thereby attenuates antigen sequestration. Through a modulation of DALIS formation CHIP, BAG-1 and HspBP1 alter MHC class I mediated antigen presentation in mouse BMDCs. Our data show that the Hsc/Hsp70 co-chaperone network controls transient protein aggregation during maturation of professional antigen presenting cells and in this way regulates the immune response. Similar mechanisms may modulate the formation of aggresomes and aggresome-like induced structures (ALIS) in other mammalian cell types

A Chaperone Trap Contributes to the Onset of Cystic Fibrosis

Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a ‘chaperone trap’. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF