13 research outputs found

    HSF1 silencing increases F508del folding, trafficking, and function and improves the phenotype of other misfolding diseases.

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    <p>(A) Immunoblot of indicated proteins following siHSF-1. (B) Short-circuit current (<i>I</i><sub>sc</sub>) analysis of CFTR in polarized CFBE cells expressing WT- or F508del-CFTR treated with the indicated siRNA or temperature corrected at 30°C. Channel activity was determined in response to forskolin and genistein (positive deflection) or with CF specific inhibitor 172 (Inh172) (negative deflection). The ** indicates p<0.05 and * indicates p<0.1 relative to control siRNA or DMSO for VX809 (<i>n</i>≥4). (C) Immunoblot of the indicated proteins in WT- and Z-AAT expressing IB3 cells (<i>n</i> = 3). (D) Immunoblot of the indicated proteins in Z-AAT expressing IB3 cells in response to siHSF1 treatment. Shown for the AAT are the immature (I), mature (M), and secreted (S) forms (<i>n</i> = 3). (E) Immunoblot of the indicated proteins from primary fibroblasts derived from WT- or mutant I1061T-NPC1 patients (<i>n</i> = 3). (F) Immunoblot of the digestion pattern before and after endo-H digestion of WT- or mutant I1061T-NPC1 stably expressed in Hela cells and treated with the indicated siRNA (<i>n</i> = 3). Immunoblot shows NPC1 endo-H resistant band (R) and sensitive band (S). Quantification represents total I1061T NPC1 (R+S) in control or siHSF1-treated cells, shown as percentage of control. Percentage of endo-H sensitive band (S) or resistant band (R) is shown in gray and black color, respectively (<i>n</i>≥3, * indicates <i>p</i><0.05). (G) Immunoblots of indicated proteins and quantification of HSF1 and HSF1-P on brain homogenates obtained from WT or AD mice of approximately 4 months (4 m; <i>n</i> = 3 for WT, <i>n</i> = 3 for AD), 9 months (9 m; <i>n</i> = 2 for WT, <i>n</i> = 2 for AD), and 16 months of age (16 m; <i>n</i> = 3 for WT, <i>n</i> = 3 for AD). Results were normalized to tubulin loading control and shown as fold change relative to WT-3m set to 1 (* indicates <i>p</i><0.05 relative to age-matched WT mice). (H) Immunoblot for APP (110 kDa), Aβ<sub>42</sub> toxic species (monomer, 4 kDa, and multimers from 6–12 kDa), and actin control on particulate fractions of brain homogenates obtained from WT (on the right) or AD mice. Controls shown include 0.5 µg recombinant Aβ<sub>42</sub>; control and Alzheimer disease samples from human brain homogenates. The underlying data used to make (B), (F) and (G) 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

    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

    Expression of F508del induces chronic proteotoxic stress that affects cellular protein folding.

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    <p>(A) Immunoblots of WT-CFTR, F508del-CFTR, and HSF1-P during HS time course (42°C for a total of 60 min). (B) Quantification of total CFTR during HS, relative to pre-HS (T = 0) (<i>n</i> = 4). (C) Immunoblots of indicated proteins in CFBE41o- cellular lysates following the co-expression of F508del-CFTR with the constitutively active ΔHSF1<sup>186–201</sup> or control empty plasmid. (D) Immunoblots of HSF1-P and I-Hsp70 in WT or F508del expressing cells (<i>n</i> = 4). (E) Quantification of the expression of HSR markers in WT-CFTR, F508del-CFTR at 37°C or 30°C, and CFTR null (CFTR−/−) expressing cells. (F) Quantification of HFS-1 trimer levels in WT-, F508del- and CFTR null–CFBE cells. (E,F) Results are shown as percentage of that seen in WT-expressing cells, as a mean ± standard error of the mean (SEM), n≥3. qRT-PCR of I-Hsp70 (HspA1A, or HspA6), I-Hsp90 (Hsp90α), and I-Hsp40 (DNAJB1) in (G,J) or of cancer-related HSF1 responsive genes (CKS2, LY6K, EIF4A2) shown in (J) from mRNA isolated from WT-, F508del- and CFTR null–CFBE cells (G) or from mRNA obtained from hBE primary cells obtained from homozygous patients for WT- or F508del-CFTR (J). qRT-PCR data was normalized to the housekeeping gene beta-glucuronidase (GUS). Results are shown as percentage of WT-expressing cells set to a 100 (mean ± standard deviation [SD] or SEM, n≥3, and * indicates <i>p</i><0.05 relative to WT). Immunoblot (H) and quantification (I) of CFTR, HSF1-P, and I-Hsp70 from hBE primary cell lysates of WT or F508del patients. Data is shown as the relative protein expression normalized to actin (mean ± SD, n≥2). (K) Quantification of I-Hsp70 and I-Hsp40 protein level in F508del-expressing cells at 37°C (MSR) or following acute HS (shown as a percentage of the level seen in WT-expressing cells; mean ± SD, n≥2). (L) Firefly luciferase (FLuc) activity in WT- and F508del-CFTR expressing cells following siCFTR silencing. Results represent normalized specific activity of FLuc (luminescence/relative FLuc expression) for each condition. Data is shown as percentage of WT-CFTR expressing cells, mean ± SEM, n≥3, and *, # indicate <i>p</i><0.05 relative to WT and F508del (0 nM siCFTR), respectively. (M) (1) Diagram showing the proteostatic environment of WT folding, where substrates are properly managed by the physiological Q-state (yellow cloud). (2) Representation of the transient stress level observed during acute stress responses (dotted red line). (3) The MSR state induced in misfolding diseases (abnormal Q-state, gray cloud) that results in a continuous elevated (subacute) stress affecting global folding and cellular function. All experimental data was repeated at least once. The underlying data used to make (B), (E–G), and (I–L) 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

    Chemical inhibition of HSF1 improves F508del-CFTR function.

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    <p>Immunoblot and quantification of CFTR following treatment of F508del-CFTR–expressing CFBE cells with increasing concentration of triptolide (Trip.) alone (A) or in combination with the CF corrector VX809 (D). Results are expressed as percentage of maximum signal of CFTR band-B (set at 100%), and shown as mean ± SEM, <i>n</i>≥3; * represents <i>p</i><0.05 relative to DMSO. (C) Immunoblot and quantitative analysis of CFTR following a daily chronic dosing regimen (96 h) of 12 nM triptolide in F508del-CFTR expressing cells. Results are expressed as fold change relative to DMSO, and shown as mean ± SD, <i>n</i> = 2; * represents <i>p</i><0.05 relative to DMSO. YFP-quenching curves of F508del-CFTR expressing CFBE-YFP cells treated with the indicated compounds for 24 h alone (B) or in combination (E) (mean ± SD, <i>n</i>≥3). All results were repeated at least once. The underlying data used to make (A–E) 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

    p23 Hsp90 co-chaperone is a modulator of the MSR.

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    <p>(A) Quantification of HSF1-P after 1 h HS in F508del-expressing cells treated with control or p23 siRNA. Data is shown as percentage of control (mean ± SEM, n = 4). (B) Immunoblot of the indicated proteins following p23 silencing in F508del-CFTR expressing cells. Histograms represent quantification of the indicated proteins upon sip23, relative to levels seen with control siRNA, which is set to 100%. (C) qRT-PCR analysis of I-Hsp70 in F508del-expressing cells following sip23. The data represent a ratio of I-Hsp70 to the housekeeping gene (GUS) and are shown as percentage of control siRNA. All results are shown (B,C) as a mean ± SEM, n≥3, and * indicates <i>p</i><0.05 relative to control. (D) Quantification of HSF-1-P and I-Hsp70 protein levels in WT- and F508del- expressing cells after control or sip23 treatment. The data is shown as a percentage of F508del-expressing cells, and # represents <i>p</i><0.05 relative to F508del (mean ± SEM, n≥3). (E) Quantitative analysis of FLuc activity in WT- and F508del-CFTR expressing cells treated with control or sip23. The data represents normalized specific activity of FLuc (luminescence/relative FLuc expression) for each condition (mean ± SEM, n≥3). All results were replicated at least once. The symbols * and # represent <i>p</i><0.05 relative to WT and F508del respectively. The underlying data used to make (A–E) 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

    Canonical PRC2 function is essential for mammary gland development and affects chromatin compaction in mammary organoids

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    <div><p>Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using <i>in vitro</i> and <i>in vivo</i> studies, we show that deletion of <i>Suz12</i>, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with <i>Suz12</i> loss <i>in vivo</i>, we have explored gene expression and chromatin structure in wild-type and <i>Suz12</i>-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type–specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states.</p></div

    Silencing of HSF1 improves F508del folding and its cell surface stability.

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    <p>(A) Total protein synthesis on S<sup>35</sup> labeled samples from WT- or F508del-CFTR expressing cells treated with control or siHSF1. Results are shown as percent of WT for total protein normalized by number of cells in each condition (mean ± SEM, <i>n</i> = 3; * indicates <i>p</i><0.05 relative to WT). (B) Pulse-chase of F508del-CFTR in response to siHSF1 treatment. Numbers show percent of F508del relative to time 0 (T = 0; <i>n</i> = 2). (C) F508del-CFTR immunoblots and quantification in control or siHSF1 transfected cells before (T = 0) and after cycloheximide (CHX; 50 µM) chase for the indicated time (h). Results are shown as percent of band-B or band-C at T = 0 (mean ± SD, <i>n</i>≥4). (D) CFTR immunoblots and quantification in WT or F508del corrected at 30°C or by siHSF1 before (T = 0) and after brefeldin A treatment (BFA; 5 µg/ml) for the indicated time (h). The data is presented as a fraction of maximal band-C set to 1 at 1 h post-BFA treatment and represents the mean ± SD, <i>n</i>≥4. (E) Representative immunoblots from three experimental replicates of CFTR—WT, F508del at 37°C, F508del at 30°C, or F508del siHSF1—before and after proteolysis digestion with increasing concentration of trypsin (mg/ml). Upper blots show digestion pattern of CFTR NBD1 domain (18D1 antibody), and lower blots show digestion pattern of CFTR NBD2 domain (M3A7 antibody) (* indicates stable core fragment previously described) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001998#pbio.1001998-Hoelen1" target="_blank">[75]</a>. (F) Quantification of FLuc activity in WT- and F508del-CFTR expressing cells treated with the indicated siRNA. The data represents specific FLuc activity (luminescence/relative FLuc expression) for each condition. Results are shown as a mean ± SEM, <i>n</i>≥3; * and # indicate <i>p</i><0.05 relative to WT- and F508del-CFTR, respectively. The underlying data used to make (A), (C), (D) and (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

    Suz12 is essential for MaSC and progenitor cell activity and integrity of the PRC2 complex.

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    <p>(A) Western blot analysis of PRC2 proteins and histone marks in basal/MaSC-enriched (Basal, CD29<sup>hi</sup>CD24<sup>+</sup>), committed luminal progenitor (CD29<sup>lo</sup>CD24<sup>+</sup>CD14<sup>+</sup>), and mature luminal cells (CD29<sup>lo</sup>CD24<sup>+</sup>CD14<sup>−</sup>) populations sorted from 10 week old C57BL/6 mice. Molecular mass in KDa of the protein ladder are shown on the left-hand side. (B) Representative images of Giemsa-stained colonies (left) and quantification of colonies (right) grown from sorted epithelial populations from R26creERT2<sup>KI/+</sup>Suz12<sup>f/f</sup> mice and littermate controls in the presence or absence of 4OHT. Basal, luminal progenitor, and mature luminal cell populations (defined in A) were sorted from R26creERT2<sup>KI/+</sup>Suz12<sup>f/f</sup> mice and control genotypes and 100 cells plated onto irradiated 3T3s. Colony formation was assessed 1 week following addition of 4OHT to induce Suz12 deletion. Mean ± S.E.M. (<i>n</i> = 3–5 independent mice per genotype performed in duplicate). ** <i>P</i> < 0.01 for KI/+ f/f compared with all other genotypes (one-way ANOVA for multiple comparisons). Scale bars: 2 mm. (C) Western blot analysis of protein expression in MECs from R26creERT2<sup>KI/+</sup>Suz12<sup>f/f</sup> mice and the indicated control genotypes following addition of 4OHT to induce <i>Suz12</i> deletion on day 2. Cells were cultured for 1 week prior to preparation of protein lysates. Molecular mass in KDa of the protein ladder are shown on the left. Individual quantitative observations can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004986#pbio.2004986.s013" target="_blank">S6 Data</a>. 4OHT, 4-hyrdoxytamoxifen; Ezh2, Enhancer of Zeste homolog 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H3K27me1, histone 3 lysine 27 monomethylation; H3K27me2, histone 3 lysine 27 dimethylation; H3K27me3, histone 3 lysine 27 trimethylation; MaSC, mammary stem cell; MEC, mammary epithelial cell; PRC2, Polycomb repressive complex 2; Suz12, Suppressor of Zeste 12 protein homolog.</p
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