ERβ Binds N-CoR in the Presence of Estrogens via an LXXLL-like Motif in the N-CoR C-terminus

Nuclear receptors (NRs) usually bind the corepressors N-CoR and SMRT in the absence of ligand or in the presence of antagonists. Agonist binding leads to corepressor release and recruitment of coactivators. Here, we report that estrogen receptor β (ERβ) binds N-CoR and SMRT in the presence of agonists, but not antagonists, in vitro and in vivo. This ligand preference differs from that of ERα interactions with corepressors, which are inhibited by estradiol, and resembles that of ERβ interactions with coactivators. ERβ /N-CoR interactions involve ERβ AF-2, which also mediates coactivator recognition. Moreover, ERβ recognizes a sequence (PLTIRML) in the N-CoR C-terminus that resembles coactivator LXXLL motifs. Inhibition of histone deacetylase activity specifically potentiates ERβ LBD activity, suggesting that corepressors restrict the activity of AF-2. We conclude that the ER isoforms show completely distinct modes of interaction with a physiologically important corepressor and discuss our results in terms of ER isoform specificity in vivo

Reduced PDZ Interactions of Rescued ΔF508CFTR Increases Its Cell Surface Mobility*

Deletion of phenylalanine 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). Though several maneuvers can rescue endoplasmic reticulum-retained ΔF508CFTR and promote its trafficking to the plasma membrane, rescued ΔF508CFTR remains susceptible to quality control mechanisms that lead to accelerated endocytosis, ubiquitination, and lysosomal degradation. To investigate the role of scaffold protein interactions in rescued ΔF508CFTR surface instability, the plasma membrane mobility of ΔF508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, ΔF508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Knock-down of the plasma membrane quality control proteins CHIP and Hsc70 partially restored ΔF508CFTR-scaffold association. Quantitative comparisons of CFTR cell surface diffusion and endocytosis kinetics suggested an association between reduced scaffold binding and CFTR internalization. Our surface diffusion measurements in live cells indicate defective scaffold interactions of rescued ΔF508CFTR at the cell surface, which may contribute to its defective peripheral processing

Correction: X-Box Binding Protein 1 (XBP1s) Is a Critical Determinant of Pseudomonas aeruginosa Homoserine Lactone-Mediated Apoptosis.

[This corrects the article DOI: 10.1371/journal.ppat.1003576.]

Inhibitors of Pseudomonas homoserine lactones

Pseudomonas aeruginosa infections are commonly associated with cystic fibrosis, pneumonias, neutropenia and burns. The P. aeruginosa quorum sensing molecule N-(3-oxo-dodecanoyl) homoserine lactone (C12) cause multiple deleterious host responses, including repression of NF-κB transcriptional activity and apoptosis. Inhibition of C12-mediated host responses is predicted to reduce P. aeruginosa virulence. We report here a novel, host-targeted approach for potential adjunctive anti-Pseudomonal therapy based on inhibition of C12-mediated host responses. A high-throughput screen was developed to identify C12 inhibitors that restore NF-κB activity in C12-treated, lipopolysaccharide (LPS)-stimulated cells. Triazolo[4,3-a]quinolines with nanomolar potency were identified as C12-inhibitors that restore NF-κB-dependent luciferase expression in LPS- and TNF-stimulated cell lines. In primary macrophages and fibroblasts, triazolo[4,3-a]quinolines inhibited C12 action to restore cytokine secretion in LPS-stimulated cells. Serendipitously, in the absence of an inflammatory stimulus, triazolo[4,3-a]quinolines prevented C12-mediated responses, including cytotoxicity, elevation of cytoplasmic calcium, and p38 MAPK phosphorylation. In vivo efficacy was demonstrated in a murine model of dermal inflammation involving intradermalC12 administration. The discovery of triazolo[4,3-a]quinolines provides a pharmacological tool to investigate C12-mediated host responses, and a potential host-targeted anti-Pseudomonal therapy

XBP1-independent cellular responses to C12.

<p>A. Immunoblot analysis of phosphorylated eIF2α (p-eIF2α, <i>left</i>) and phosphorylated p38 (p-p38, <i>right</i>) levels in wt, <i>Ire1α</i>^−/− and <i>Xbp1^−/−</i> MEFs. Cells were treated with 25 µM C12 for 45 minutes. B. (<i>top</i>) C12 treatment (25 µM, 45 min) of <i>Perk^−/−</i> MEFs results in phosphorylation of eIF2α (<i>bottom</i>) Control experiments confirm that ER stress (brefeldin A (BFA), 2 hours) in wt MEFs, but not <i>Perk^−/−</i> MEFs, produces p-eIF2α. C. C12 treatment increases c-Jun levels in wt MEFs but not in <i>Ire1α</i>^−/−, and <i>Xbp1^−/−</i> MEFs. Cells were treated with 25 µM C12 for 90 minutes (<i>bottom</i>) and c-Jun levels were characterized by image analysis of immunostained cells (green). Images of DAPI staining are also shown (magenta). D. Quantitative analysis of normalized c-Jun levels in wt, <i>Ire1α</i>^−/−, and <i>Xbp1^−/−</i> MEFs. Scale bar in panel C refers to all images.</p

Deletion of <i>Ire1α</i> prevents C12 cytotoxicity.

<p>A. Structure of the <i>Pseudomonas aeruginosa N</i>-(3-oxo-dodecanoyl) homoserine lactone (C12). B. (<i>left</i>) Cell density of wt (wild-type; <i>first panels</i>), <i>Perk^−/−</i> (<i>third panels</i>) and <i>Ire1α^−/−</i> (<i>fourth panels</i>) MEFs under control conditions (<i>top</i>) and after C12 challenge (25 µM, 4 hours, <i>bottom</i>) assessed by calcein AM labelling. Caspase inhibition prevents C12 cytotoxicity as assessed by calcein AM staining of z-VAD-fmk (z-VAD) treated wt MEFs cells (<i>second panels</i>). (<i>right</i>) RT-PCR (<i>top</i>) and western blot (<i>bottom</i>) analysis of <i>Perk^−/−</i> and <i>Ire1α^−/−</i> MEFs. C. Quantitative analysis of change in MEF cell density after C12 challenge in wt, <i>Perk^−/−</i> and <i>Ire1α^−/−</i> MEFs and in z-VAD-fmk-treated wt MEFs (<i>grey bar</i>). D. Normalized caspase 3/7 activity in wt, <i>Perk^−/−</i> and <i>Ire1α^−/−</i> MEFs in control conditions (−) and after C12 treatment (+; 25 µM, 4 hours). E. Dose response of normalized caspase 3/7 activity in wt MEFs treated with C12 for 4 hours. Statistical analysis was by t-test with reference to data from wt MEFs (C) or control experiments (E); * p<0.005, ** p<0.0001.</p

Correction: X-Box Binding Protein 1 (XBP1s) Is a Critical Determinant of <i>Pseudomonas aeruginosa</i> Homoserine Lactone-Mediated Apoptosis

Correction: X-Box Binding Protein 1 (XBP1s) Is a Critical Determinant of <i>Pseudomonas aeruginosa</i> Homoserine Lactone-Mediated Apoptosi