Hsp 70/Hsp 90 organizing protein as a nitrosylation target in cystic fibrosis therapy

Abstract

The endogenous signaling molecule S-nitrosoglutathione (GSNO) and other S-nitrosylating agents can cause full maturation of the abnormal gene product DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR). However, the molecular mechanism of action is not known. Here we show that Hsp70/Hsp90 organizing protein (Hop) is a critical target of GSNO, and its S-nitrosylation results in DeltaF508 CFTR maturation and cell surface expression. S-nitrosylation by GSNO inhibited the association of Hop with CFTR in the endoplasmic reticulum. This effect was necessary and sufficient to mediate GSNO-induced cell-surface expression of DeltaF508 CFTR. Hop knockdown using siRNA recapitulated the effect of GSNO on DeltaF508 CFTR maturation and expression. Moreover, GSNO acted additively with decreased temperature, which promoted mutant CFTR maturation through a Hop-independent mechanism. We conclude that GSNO corrects DeltaF508 CFTR trafficking by inhibiting Hop expression, and that combination therapies--using differing mechanisms of action--may have additive benefits in treating CF.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Combination treatment (low temperature and GSNO) synergistically up-regulates ΔF508 CFTR expression. CFBE41o− cells expressing ΔF508 CFTR were grown at 37 °C to 70% confluence, then incubated for an additional 48 h at 37 °C or 27 °C with GSNO (10 μM) present or absent (4 h). (A) Expression of the core glycosylated (band B) and mature, fully glycosylated (band C) forms of ΔF508 CFTR was detected by immunoblotting using mouse monoclonal anti-CFTR antibody (Chemicon). (B) Densitometric analysis revealed that the increases in the B and C bands were significant for both GSNO and 27 °C. Of note, combined treatment led to further full maturation of band B. [*P < 0.001 by two-way ANOVA for each experiment compared with no treatment/37 °C (C band) and both no treatment/GSNO and GSNO/27 °C (B band)]. Data represent the average fold change for three independent experiments.

Fig. 2.

GSNO cell-surface CFTR expression. (A and B) Cell-surface expression. CFBE41o− cells were treated with 5 or 10 μM GSNO (4 h). The glycosidic moieties of cell-surface membrane proteins were derivatized by exposing cells to sodium periodate and biotinylation using biotin-LC hydrazide (30 min at 20 °C). After IP and SDS/PAGE, biotinylated CFTR (cell surface C band only) was detected with streptavidin-conjugated HRP. The membrane was reprobed with anti-α-tubulin. (ANOVA: *P < 0.001; 5 and 10 μM compared with control). Data represent the average fold change for three experiments. (C and D) CFBE41o− cells were incubated with or without 5 or 10 μM GSNO (4 h), and cell membranes were isolated on a sucrose gradient before Western blotting for cell-surface CFTR (C band only; ANOVA: *P < 0.001, 5 and 10 μM compared with control). Data represent the average of three experiments. (E) Whole-cell extracts from CFBE41o− cells incubated with or without 10 μM GSNO (4 h) were Western blotted with anti-CFTR antibody A596 (50 μg protein/lane). Modification of core glycosylated (band B) and fully glycosylated mature (band C) forms of CFTR was confirmed by preincubation of cell lysates with Endo H or PNGase F respectively (2 h, 37 °C). (F) Human CF primary pseudostratified columnar epithelia at air-fluid interface were exposed to GSNO (100 μM every 6 h for 72 h to facilitate full-thickness penetration), membrane-permeable GSNO diethyl ester (60 μM every 12 h for 48 h), or vehicle alone (for each) before analysis in Using chambers (42). Relative to vehicle, forskolin-stimulated Cl− current was enhanced by GSNO diethyl ester (n = 4 donors, 2–3 wells each) but not by GSNO (n = 6 donors, three wells each). *P = 0.02.

Fig. 3.

GSNO, but not decreased temperature, decreases Hop expression to increase CFTR maturation. (A) CFPAC-1, CFBE41o−, and A549 cells incubated with or without 10 μM GSNO (4 h) were Western blotted using monoclonal anti-Hop antibody (50 μg of protein per lane). The blot was reprobed with anti-α-tubulin. (B) GSNO (4 h) decreased cytosolic Hop in CFBE41o− cells, as visualized by immunofluorescence. (C) Western blot for cells were incubated for 48 h at 37 °C or 27 °C before exposure to GSNO; GSNO, but not 27 °C, decreased Hop expression. (D) IB of Hop from CFBE41o− cells treated for 4 h with GSNO with or without pretreatment with actinomycin D or cycloheximide. (E) Hop Western blot from CFBE41o− cells treated with GSNO (10 μM, 4 h) after transfection with Hop siRNA or scrambled sequence DNA; Hsp 70 and Hsp 90 were Western blotted as controls. (F) Western blot of Hop and CFTR from CFBE41o− cells transfected with 50 nM of Hop siRNA (or control RNA) and analyzed after 48 h (50 μg protein per lane).

Fig. 4.

Hop S-nitrosylation decreases Hop expression and Hop-CFTR association. (A) Western blotting for calnexin and syntaxin 5 in the ER and Golgi fractions of CFBE41o− cells. (B) IP with anti-CFTR followed by Western blotting with anti-Hop revealed CFTR-associated Hop exclusively in the cytosol at baseline. Exposure to GSNO (10 μM) resulted in a translocation of CFTR-associated Hop to the ER at 2 h, with a subsequent loss of CFTR-associated Hop from all fractions thereafter. (C) At baseline, some Hop associated with the CFTR was S-nitrosylated (IP for CFTR followed by biotin switch, streptavidin isolation, and Western blot for Hop on each fraction). After 2 h GSNO treatment (10 μM), no CFTR-associated S-nitrosylated Hop was present. (D) In a time-course analysis, GSNO (10 μM) decreased CFTR-associated Hop (*P < 0.001 relative to baseline at 2 and 4 h, ANOVA). (E) Extracts from CFBE41o− cells treated with GSNO (10 μM, 2 h) underwent biotin switch followed by avidin affinity purification, proteolysis, and LC-MS. The relative intensity of a representative peptide from S-nitrosylated Hop (LAYINPDLALEEK) from treated and untreated cells is shown.

Fig. 5.

Hop degradation is prevented by C403S mutation and proteasomal inhibition. (A) CFBE41o− cells untransfected or overexpressing either WT Hop or C403S mutant Hop were treated for 2 h with 10 μM GSNO and Western blotted for Hop (Upper) and actin (representative of two experiments). Additionally, S-nitrosylated proteins from these samples underwent biotin substitution, followed by streptavidin isolation and Western blotting for Hop. C403S mutation inhibited Hop S-nitrosylation in Hop-overexpressing cells. (B) The proteasome inhibitor MG132 completely inhibited the ability of GSNO (10 μM, 2 h) to decrease Hop expression. (C) MG132 also preserved S-nitrosylated Hop (C) and Hop ubiquitination (D) in CFBE41o− cells treated with MG132 and/or GSNO. *P < 0.002; +P < 0.05.