Quantitative Proteomic Profiling Reveals Differentially Regulated Proteins in Cystic Fibrosis Cells

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

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 ΔF508 CFTR interaction with core chaperones at physiological and corrective temperatures.

<p>Table depicting the absolute amounts of CFTR, Hsp90, Hsc70 and Hsp40, expressed in pmol. Also shown are the molar ratios of chaperones to total ΔF508-CFTR at both 37°C and 30°C. The fold change in the absolute amounts of CFTR, Hsp90 and Hsc70, expressed in pmol, relative to ΔF508-CFTR at 37°C is shown in the final column.</p

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

<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

Structural mapping of the Interaction of NBD1 with Hsp90 using cross-linking.

<p><b>A</b>. Ribbon diagram of NBD1 depicting Hsp90 interacting peptides. <b>B–C</b> Ribbon diagram of ΔF508-NBD1 (<b>B</b>) and WT-NBD1 (<b>C</b>) with associated Hsp90 interacting peptides shown as electrostatic map. <b>D–E.</b> Ribbon diagram of Hsp90 with associated ΔF508-NBD1 (<b>D</b>) and WT-NBD1 (<b>E</b>) interacting peptides shown as electrostatic map. Data shown is conserved peptides from 3 independent experiments.</p

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

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

Quantification of WT and ΔF508 CFTR interactions with core chaperones.

<p><b>A.</b> The absolute levels of CFTR, Hsp90 and Hsc70, expressed in pmol, in CFTR-containing complexes were determined using the absolute quantification strategy from HEK293 ΔF508-CFTR (white) and WT-CFTR (black) producing cells. <b>B.</b> Immunoblot and densitometric analysis for CFTR, Hsp90, Hsc/p70 and Hsp40 from CFTR-containing immunoprecipitates. A representative blot is shown. In the densitometric analysis, the relative protein amount is shown in arbitrary units (a.u.). In all panels, data is shown as mean ± SD, n = 3 and asterisks represent p value <0.05 as determined by two-tailed t-test using the WT sample as the reference.</p

Quantification of ΔF508-CFTR interaction with core chaperones following temperature shift

<p>. <b>A.</b> Western blot analysis of HEK293 cells stably expressing ΔF508-CFTR cultured at 37°C or 30°C in the presence of 50 μM cyclohexamide (CHX) or vehicle control for the indicated time. <b>B.</b> Absolute quantification of ΔF508 CFTR and interacting chaperones at 37°C (black) or 30°C for 16 h (white). Absolute protein abundance of CFTR, Hsp90, Hsc70, and Hsp40 in CFTR-containing complexes is shown and expressed in pmols. <b>C.</b> Immunoblot and densitometric analysis for CFTR, Hsp90, Hsc/p70 and Hsp40 in CFTR-containing immunoprecipitates. In the densitometric analysis, the relative protein amount is shown in arbitrary units (a.u.). In all panels, data is shown as mean ± SD, n = 3 and asterisks represent p value <0.05 as determined by two-tailed t-test using the ΔF508-CFTR at 37°C sample as the reference.</p

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

<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

Q-state management of MSR to correct human disease.

<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 (Qⁿ) because of the continued expression of misfolded protein. Once chronically elevated (Q*), the folding environment becomes maladaptive as it fails to return to the Qⁿ (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