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

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 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

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 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

Correlation of HLA-DR associated peptides and peptide clusters measured by MAPPs to the amount of protein present in subvisible particles.

<p>Linear regression analyses of the increase of the HLA-DR associated peptides and clusters as functions of the calculated amount of protein present in the subvisible particles. Left up: HLA-DR associated peptides of mAb1 vs protein amount in subvisible particles (r² = 0.994), left down: HLA-DR associated peptide clusters of mAb1 vs protein amount in subvisible particles (r² = 0.993), right up: HLA-DR associated peptides of mAb2 vs protein amount in subvisible particles (r² = 0.86), right down: HLA-DR associated peptide clusters of mAb2 vs protein amount in subvisible particles (r² = 0.943). HS: aggregates generated by heat and shake stress; FT: aggregates generated by freeze and thaw stress, S: aggregates generated by shear stress mAb1: monoclonal antibody 1, mAb2: monoclonal antibody 2, 1: stress level 1, 2: stress level 2. For further details, please, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086322#s4" target="_blank">Materials and Methods</a>.</p

Physicochemical characterization of stressed mAb materials.

<p>Representative MFI screenshots after freeze/thaw (FT) stress of mAb1 (A) unstressed, un; (B) stress level 1, sl1; (C) stress level 2, sl2 and mAb2 un, (E) sl1 and (F) sl2. (G) Particle Size distribution obtained by MFI of mAb1 (left) and mAb2 (right). For visualization the size binning 2–2.5, 2.5–5, 5–10 10–25, 25–50 and 50–400 µm was used. Representative images of individual particles formed by (H) heat/shake, HS; (I) freeze/thaw, FT and (J) shear stress, S. Polydispersity in % (PD%) revealed by DLS for (K) mAb1 and (L) mAb2.</p

Results from T-cell activation assay.

<p>T-cell responses determined by IFN-γ ELISpot from donors treated with mAb1, un, sl1 and sl2 material from (A) FT condition (8 donors) and from (B) HS condition (13 donors). The respective heat maps specify to which extent each single donor responded to each treatment. The horizontal line shows the geometric mean of the populations. ***: statistically significant (p<0.001); ns: statistically not significant. HS: aggregates generated by heat and shake stress, FT: aggregates generated by freeze and thaw stress, mAb1: monoclonal antibody 1, SI: Stimulation index, un: unstressed, sl1: stress level 1, sl2: stress level 2.</p