A chaperone trap contributes to the onset of cystic fibrosis

Abstract

Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a 'chaperone trap'. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF.

Figure 1. Quantification of CFTR, Hsc70 and Hsp90 in CFTR-containing complexes.

A. Absolute abundance (ng/µl) of Hsp90 calculated by ¹⁵N protein labeling, AQUA labeling and Western blotting (WB). 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.

Figure 2. Quantification of WT and ΔF508 CFTR interactions with core chaperones.

A. 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. 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.

Figure 3. Quantification of ΔF508-CFTR interaction with core chaperones following temperature shift

. A. 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. 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. C. 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.

Figure 4. Structural mapping of the Interaction of NBD1 with Hsp90 using cross-linking.

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

Figure 5. Minimal sequential ordering of intra- and inter-domain folding events responsible for CFTR folding and trafficking.

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’ in trans the folding of NBD2 (step 3).