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

Abstract

<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 ΔHSF1^186–201 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