13 research outputs found

    Quantitative Proteomic Profiling Reveals Differentially Regulated Proteins in Cystic Fibrosis Cells

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    The most prevalent cause of cystic fibrosis (CF) is the deletion of a phenylalanine residue at position 508 in CFTR (ΔF508-CFTR) protein. The mutated protein fails to fold properly, is retained in the endoplasmic reticulum via the action of molecular chaperones, and is tagged for degradation. In this study, the differences in protein expression levels in CF cell models were assessed using a systems biology approach aided by the sensitivity of MudPIT proteomics. Analysis of the differential proteome modulation without a priori hypotheses has the potential to identify markers that have not yet been documented. These may also serve as the basis for developing new diagnostic and treatment modalities for CF. Several novel differentially expressed proteins observed in our study are likely to play important roles in the pathogenesis of CF and may serve as a useful resource for the CF scientific community

    Quantitative Proteomic Profiling Reveals Differentially Regulated Proteins in Cystic Fibrosis Cells

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    The most prevalent cause of cystic fibrosis (CF) is the deletion of a phenylalanine residue at position 508 in CFTR (ΔF508-CFTR) protein. The mutated protein fails to fold properly, is retained in the endoplasmic reticulum via the action of molecular chaperones, and is tagged for degradation. In this study, the differences in protein expression levels in CF cell models were assessed using a systems biology approach aided by the sensitivity of MudPIT proteomics. Analysis of the differential proteome modulation without a priori hypotheses has the potential to identify markers that have not yet been documented. These may also serve as the basis for developing new diagnostic and treatment modalities for CF. Several novel differentially expressed proteins observed in our study are likely to play important roles in the pathogenesis of CF and may serve as a useful resource for the CF scientific community

    Stoichiometry of the WT and ΔF508 CFTR interaction with core chaperones.

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    <p>Table depicting the absolute amounts of CFTR, Hsp90 and Hsc70, expressed in pmol. Also shown are the molar ratios of chaperones to total ΔF508- or WT-CFTR. The fold change in the absolute amounts of CFTR, Hsp90 and Hsc70, expressed in pmol, relative to ΔF508-CFTR is shown in the final column.</p

    Quantification of CFTR, Hsc70 and Hsp90 in CFTR-containing complexes.

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    <p><b>A.</b> Absolute abundance (ng/µl) of Hsp90 calculated by <sup>15</sup>N protein labeling, AQUA labeling and Western blotting (WB). <b>B.</b> Absolute abundance (ng/µl) of Hsc70 calculated by <sup>15</sup>N protein labeling, AQUA labeling and Western blotting (WB). In all panels, data is shown as mean ± SD, n≥3.</p

    Minimal sequential ordering of intra- and inter-domain folding events responsible for CFTR folding and trafficking.

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    <p>Intra-domain folding of NBD1 is dictated by the Hsp90 system (step 1). A structural rearrangement occurs in response to the binding of cytoplasmic loop 4 (CL4) to the F508 containing hydrophobic pocket present WT NBD1 (step 2). The binding of CL4 provides a stabilizing effect on NBD1, releasing Hsp90 and promoting H8–H9 helix-coil transition. This H8–H9 transition would expose the NBD2-binding interface of NBD1 and allow NBD1 to ‘chaperone’ <i>in trans</i> the folding of NBD2 (step 3).</p

    Chemical inhibition of HSF1 synergizes with VX809 to improve F508del-CFTR function in patient-derived primary epithelium.

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    <p>(A) Short-circuit current analysis of human primary hBE cells (F508del/F508del, patient code CF006) treated with DMSO, 3 µM VX809, and 25 nM triptolide or a combination of VX809 and triptolide, for 96 h (daily dosing). The data is presented as fold change relative to the basal current seen with DMSO treatment, and shown as mean ± SD, <i>n</i>≥3 (replicated multiple times); * and # indicate <i>p</i><0.05 relative to DMSO or VX809, respectively. (B) Representative short-circuit current (I<sub>sc</sub>) traces for DMSO, VX809, triptolide, or triptolide + VX809 treatment of primary hBE cells from (A). (C) Quantitative analysis of organoid swelling (shown in D) that is indicative of CFTR function over the period of 60 min. Organoids were obtained from two distinct F508del/F508del CF patients (CF4, CF22), and treated with DMSO, 3 µM VX809, 25 nM triptolide, or a combination of VX809 and triptolide. Experiments were repeated once and results are shown as a mean ± SD, <i>n</i>≥2; * and # indicate <i>p</i><0.05 relative to DMSO or VX809, respectively. (D) Representative images of organoids derived from patients (CF4 and CF22) at T = 0 or after stimulus with Forskolin/Genistein at T = 60 min treated with the indicated compounds. Scale bar represents 110 µm. The underlying data used to make (A–C) in this figure can be found in the supplementary file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001998#pbio.1001998.s008" target="_blank">Data S1</a>.</p

    Q-state management of MSR to correct human disease.

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    <p>Illustrated is the activation state of the HSR in response to acute stress (red) or to the MSR (blue) seen in disease. Acute HSR activation, seen during acute stress insults, protects from and/or corrects misfolding and rapidly returns to basal levels, allowing normal biology to resume. In misfolding disease, chronic activation of the HSR alters the normal, physiologic Q-state (Q<sup>n</sup>) because of the continued expression of misfolded protein. Once chronically elevated (Q*), the folding environment becomes maladaptive as it fails to return to the Q<sup>n</sup> (light yellow area). Down-regulation of the MSR by siHSF1, sip23, or triptolide promotes a reduction of the Q*, which now falls within the proteostasis buffering capacity (green line), promoting a more normal cellular folding environment. This effect can be further improved (purple line) when combined with protein fold correctors (pharmacologic chaperones; PCs) which impart improved thermodynamic stability to the fold, or proteostasis regulators (PRs) that improve protein Q-state biology, improving function of disease-related misfolded protein and its proteome's associated environment, promoting abrogation of the chronic stress and improving health.</p

    Silencing of p23 improves F508del-CFTR function in CF by down-regulation of the MSR.

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    <p>Immunoblot of CFTR following sip23 treatment of F508del-expressing cells at 37°C (A) and 30°C (B) or WT-expressing cells (C). Histograms show quantification of CFTR band-B and C glycoforms and C/B ratios. Results are shown as a percent of the maximal signal for band-B glycoform and as fold change relative to control (set to 1) for the ratio C/B (mean ± SEM, n≥3). (D) qRT-PCR analysis of CFTR and p23 levels following p23 silencing in WT or F508del-expressing cells. Results represent a ratio of the indicated mRNA to GUS, and shown as the percent of siRNA control (mean ± SEM, n≥3). (E) Iodide efflux analysis of F508del-expressing cells in response to p23 siRNA or 30°C correction. Results are shown as a ratio of the efflux at stimulation (stim) to efflux at pre-stimulation (basal) (mean ± SD, n≥3). (F) Immunoblot of the indicated proteins in the cell lysate (input) or following CFTR IP (right panels) in response to sip23 treatment (CFTR−/− cells were used as a negative control for the CFTR IP). Histogram shows quantification of recovered Hsc/p90 (α and β) and Hsc/p70 by co-IP with CFTR. The data is shown as a ratio of the recovered chaperone to total CFTR and normalized to 1 for the control siRNA (mean ± SD, n = 3, replicated three times). For all data, * indicates <i>p</i><0.05 relative to control, and results were replicated at least once. The underlying data used to make (A–F) in this figure can be found in the supplementary file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001998#pbio.1001998.s008" target="_blank">Data S1</a>.</p
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