Human heat shock protein 105/110 kDa (Hsp105/110) regulates biogenesis and quality control of misfolded cystic fibrosis transmembrane conductance regulator at multiple levels

Abstract

Heat shock protein 105/110-kDa (Hsp105/110), a member of the Hsp70 super family of molecular chaperones, serves as a nucleotide exchange factor for Hsc70, independently prevents the aggregation of misfolded proteins, and functionally relates to Hsp90. We investigated the roles of human Hsp105α, the constitutively expressed isoform, in the biogenesis and quality control of the cystic fibrosis transmembrane conductance regulator (CFTR). In the endoplasmic reticulum (ER), Hsp105 facilitates CFTR quality control at an early stage in its biosynthesis but promotes CFTR post-translational folding. Deletion of Phe-508 (ΔF508), the most prevalent mutation causing cystic fibrosis, interferes with de novo folding of CFTR, impairing its export from the ER and accelerating its clearance in the ER and post-Golgi compartments. We show that Hsp105 preferentially associates with and stabilizes ΔF508 CFTR at both levels. Introduction of the Hsp105 substrate binding domain potently increases the steady state level of ΔF508 CFTR by reducing its early-stage degradation. This in turn dramatically enhances ΔF508 CFTR cell surface functional expression in cystic fibrosis airway epithelial cells. Although other Hsc70 nucleotide exchange factors such as HspBP1 and BAG-2 inhibit CFTR post-translational degradation in the ER through cochaperone CHIP, Hsp105 has a primary role promoting CFTR quality control at an earlier stage. The Hsp105-mediated multilevel regulation of ΔF508 CFTR folding and quality control provides new opportunities to understand how chaperone machinery regulates the homeostasis and functional expression of misfolded proteins in the cell. Future studies in this direction will inform therapeutics development for cystic fibrosis and other protein misfolding diseases.

FIGURE 1.

Hsp105 knockdown increases the synthesis of wild-type and ΔF508 CFTR. A and B, HEK cells stably expressing a non-targeting shRNA (Cntrl) or an Hsp105 shRNA (105i) were transfected with wild-type (A) or ΔF508 (B) CFTR. Twenty-four hours post-transfection the cells were pulsed for 30 min and then chased for the indicated time periods. For all pulse-chase analyses, the levels of radiolabeled CFTR in band B or C were quantified by phosphorimaging and normalized to the level of CFTR band B at the 0-h chase in each set. The relative level of radiolabeled band B at the 0-h chase in each set was indicated below the autoradiograph. Shown are the means and S.E. of two independent experiments. C, the Cntrl and 105i cells were transfected with wild-type CFTR. Equal amounts of cell lysates were immunoblotted for the indicated chaperones and actin. For all figures, the steady state levels of CFTR or chaperones were quantified by densitometry, normalized to the levels of the actin loading control, and further normalized to the value of the Cntrl to facilitate comparison. The means and S.E. are shown. Unpaired, two-tailed t test was performed. Where indicated, * and ** denote p ≤ 0.05 and 0.01, respectively. n = 3.

FIGURE 2.

Hsp105 regulates CFTR quality control coincident with translation. A, the 105i cells were co-transfected with ΔF508 CFTR and increasing doses of an shRNA-refractory Hsp105 expression plasmid (r105) as indicated. HEK cells stably expressing a non-targeting shRNA (Cntrl) were co-transfected with ΔF508 CFTR and 0.5 μg of EGFP expression plasmid to serve as a control. Twenty-four hours after transfection the cells were lysed, and equal amounts of lysates were immunoblotted for CFTR, Hsp105, and calnexin as loading control. The levels of CFTR band B and Hsp105 were normalized to those of the Cntrl, and the horizontal line in the chart denotes the levels of Hsp105 and CFTR band B in the Cntrl cells. B, the 105i cells were co-transfected with ΔF508 CFTR together with EGFP (Cntrl) or r105. The transfected cells were subject to pulse-chase analysis. Shown are the means and S.E. of two independent experiments. C, the Cntrl and 105i cells were transfected with GST-EGFP fusion construct. After 24 h both cells were pulse-labeled for the indicated time periods. GST-EGFP fusion protein was recovered using glutathione-Sepharose. The levels of the pulse-labeled GST-EGFP were quantified by phosphorimaging. Linear regression was performed on the data to compare the initial rates of GST-EGFP synthesis in the two cell lines. D, the 105i cells were co-transfected with ΔF508 CFTR together with EGFP (Cntrl) or r105. The cells were pretreated with 200 μm ALLN or the DMSO control for 20 min before the 30-min pulse in the presence of ALLN or DMSO, respectively. CFTR was recovered by immunoprecipitation and separated by SDS-PAGE. Shown is the phosphorimage of the gel. The level of radioisotope incorporation into CFTR was quantified by phosphorimaging. To facilitate comparison, the relative levels of the radiolabeled CFTR band B in Cntrl and r105 in the absence of ALLN (lanes 1 and 2) were expressed as percentile value of the sum of the two, and the levels of the radiolabeled CFTR band B in the presence of ALLN (lanes 3 and 4) were normalized to the level of band B in Cntrl without ALLN (lane 1). The means and S.E. of three independent experiments are shown. ** indicates p ≤ 0.01.

FIGURE 3.

Hsp105 overexpression reduces CFTR synthesis but promotes its folding. A and B, HEK cells were co-transfected with wild-type (A) or ΔF508 (B) CFTR together with a control or Hsp105 (105) expression plasmid. Cells were lysed 24 h after transfection. Equal amounts of cell lysates were immunoblotted as indicated. Shown are the means and S.E. of three experiments. Unpaired, two-tailed t test was performed. * denotes p ≤ 0.05. C and D, HEK cells were co-transfected with wild-type (C) or ΔF508 (D) CFTR together with Cntrl or 105 expression plasmid. Twenty-four hours post-transfection, pulse-chase analyses were performed. The data were quantified as described in Fig. 1, A and B. Shown are the means and S.E. of 2 (C) and 3 (D) independent experiments, respectively. In D, unpaired, two-tailed t test was performed. ** denotes p ≤ 0.01.

FIGURE 4.

Hsp105 overexpression enhances the low temperature rescue of ΔF508 CFTR. HEK cells were co-transfected with ΔF508 CFTR together with a control or Hsp105 (105) expression plasmid. After 24 h, cells were shifted to 30 °C and incubated for additional 16 h. Cells were lysed, and equal amounts of cell lysates were immunoblotted as indicated. Shown are the means and S.E. of three experiments. Unpaired, two-tailed t test was performed. * denotes p ≤ 0.05.

FIGURE 5.

Hsp105 preferentially associates with misfolded ΔF508 CFTR in the ER. HEK cells were transfected with expression plasmids encoding wild-type (WT), ER-exit-code mutant DAA, or ΔF508 (ΔF) CFTR. After 16 h, 10 μg/ml brefeldin A (BFA) was added to the medium, and the cells were cultured for additional 24 h. The cells were lysed, and CFTR co-immunoprecipitation (IP) was performed. The precipitated proteins from equivalent amounts of cell lysates were immunoblotted as indicated. The mock-transfected cells were included as a control (mock) for nonspecific protein-protein interactions. The levels of associated Hsp105 were subtracted by that of the mock, normalized to the level of CFTR in band B, and then further normalized to the value of ΔF. The means and S.E. of two independent experiments are shown.

FIGURE 6.

Hsp105 preferentially associates with and stabilizes the rescued ΔF508 CFTR in post-Golgi compartments. A, HEK-ΔF (ΔF) or HEK-WT (WT) cells were cultured at 37 °C (37 °C) or were incubated at 30 °C for 21 h and then treated with 100 μg/ml cycloheximide (CHX) for an additional 12 h at 30 °C (30 °C). Cells were lysed, and CFTR co-immunoprecipitation was performed. The precipitated proteins from equivalent amounts of cell lysates were immunoblotted for the indicated proteins. HEK cells not expressing CFTR served as a control (mock) for nonspecific protein-protein interactions. B and C, the levels of the associated chaperones were subtracted by the levels of the mock and normalized to the total level of CFTR and then to the value of the ΔF at 37 °C. Shown are the means and S.E. of two independent experiments. D, HEK cells were co-transfected with ΔF508 CFTR together with a control (Cntrl) or Hsp105 (105) expression plasmid. After 24 h the cells were incubated at 37 °C in the presence of CHX for the indicated periods of time. Cells were lysed, and equal amounts of cell lysates were immunoblotted for CFTR and actin. The level of CFTR band C at each time point was normalized to the level of the actin loading control and then expressed as the percentage of the value at the 0 time point. The means and S.E. of two independent experiments are shown.

FIGURE 7.

Hsp105 SBD enhances ΔF508 CFTR expression by interfering with its early-stage ERAD. A, shown are the domain structure of human Hsp105α and the sequence alignment of a part of the ATPase domain between human Hsp105α and its yeast homologue Sse1. B, HEK cells were co-transfected with ΔF508 CFTR together with a plasmid expressing a control protein (Cntrl), wild-type Hsp105 (105), the Hsp105 G232D mutant (G-D), or the Hsp105 SBD. After 24 h, the cells were lysed, and equal amounts of the lysates were immunoblotted for the indicated proteins. The expressed Hsp105 SBD is labeled by an arrowhead. The steady state levels of CFTR and chaperones were quantified as described in Fig. 1C. Unpaired, two-tailed t test was performed. * and ** indicate p ≤ 0.05 and 0.01, respectively. n = 3. C, HEK cells were co-transfected with ΔF508 CFTR together with the Cntrl or SBD expression plasmid. Pulse-chase analyses were performed and quantified as described in Fig. 1B. n = 2. D, HEK cells were co-transfected with ΔF508 CFTR together with the Cntrl or SBD expression plasmid. The cells were subject to ALLN pulse analysis as described in Fig. 2D. The means and S.E. of three independent experiments are shown. Unpaired, two-tailed t test was performed. * denotes p ≤ 0.05.

FIGURE 8.

Hsp105 SBD enhances ΔF508 CFTR cell surface functional expression in IB3-1 CF airway epithelial cells. A, IB3-1 cells were transiently co-transfected with ΔF508 CFTR together with EGFP (GFP), Hsp105 (105), or SBD expression plasmid. Cells were either incubated at 37 °C for 24 h (37 °C) or further shifted to 30 °C and incubated for another 24 h (30 °C). Cells were lysed, and equal amounts of cell lysates were immunoblotted for the indicated proteins. Protein quantification was performed as described in Fig. 1C. The means and S.E. from three independent experiments are shown. Unpaired, two-tailed t test was performed. * and ** denote p ≤ 0.05 and 0.01, respectively. B and C, IB3-1 cells were co-transfected with ΔF508 CFTR together with GFP or SBD. IB3-1 cells transfected with equivalent amount of WT-CFTR were used as a positive control. More than 24 h later the cells were subjected to SPQ-based iodide efflux assay. Traces of the cAMP-induced dequenching and the iodide-mediated requenching of SPQ fluorescence are shown in B. The estimated initial rates of the SPQ dequenching are displayed in C. The means and S.E. of at least three different cell populations are shown. In C, unpaired, two-tailed t test was performed between GFP and SBD. * denotes p ≤ 0.05.

FIGURE 9.

Hsp105 regulates CFTR biogenesis and quality control at multiple levels. Shown is the current working model of the multilevel regulation of CFTR folding and quality control by Hsp105. JB12, DNAJB12.