![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CELLULAR AND MOLECULAR
Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, Centre National de la Recherche Scientifique, Poitiers, France
Received November 22, 2007; accepted January 28, 2008.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). This retention generally causes the absence of the client protein at its target site, preventing its physiological function and leading to abnormal cell homeostasis and functions. Indeed, defective protein trafficking has been recognized as an important mechanism for a growing number of inherited human diseases (Aridor and Hannan, 2000, 2002). However, in many cases, the trafficking-defective mutant protein retains residual function if it can be rescued to its final destination (Dalemans et al., 1991; Burrows et al., 2000). These trafficking-defective proteins can be classified as class 2 mutations. For example, most patients suffering from cystic fibrosis (CF) have the F508del mutation (Fig. 1A, top), a class 2 mutation (Welsh and Smith, 1993), on at least one CFTR allele, generating misfolding and retention in the endoplasmic reticulum, followed by substantial degradation of the mutated protein via the ubiquitin-26S proteasome pathway (Cheng et al., 1990; Kartner et al., 1992). However, when the trafficking defect is corrected by incubation at low temperature, for example (Denning et al., 1992), CFTR, it displays an altered gating (Dalemans et al., 1991). Several other less frequent CF mutations such as G551D or G1349D are classified as class 3 because the corresponding proteins, although correctly located at the plasma membrane, have a dysfunctional regulation (see for a recent review, MacDonald et al., 2007). The G622D-CFTR mutant, in which the pathophysiology is unclear, has been provisionally classified as a class 3 missense mutation after its identification in CF patients (http://www.genet.sickkids.on.ca/cftr) (Vankeerberghen et al., 1998). Gly622 is located within the N terminus of the R domain (Fig. 1A, bottom). The G622D mutant forms a cAMP-regulated chloride channel with significantly lower Po than the wild-type channels (Vankeerberghen et al., 1998).
|
Rescue of misfolded trafficking-defective mutant proteins by pharmacological chaperones emerged some years ago with the finding that ligands (agonists and antagonists) increased the efficiency of receptor maturation and restored function of these proteins (Ulloa-Aguirre et al., 2004; Robert et al., 2005; Gong et al., 2006). However, in most cases, the mechanism of pharmacological rescue is not clearly understood. In particular, numerous efforts aimed at developing small molecule pharmacotherapy for CF focused on identifying compounds that can either stabilize the tertiary structure of the F508del-CFTR protein or modify the interactions of the mutant protein with ER chaperones. By preventing these interactions, the newly synthesized, misfolded but functional F508del-CFTR protein might escape recognition by mechanisms responsible for its retention and its ultimate degradation (Powell and Zeitlin, 2002). A limited number of molecules have been shown to restore partial function in F508del-CFTR mutant cells, including 4-phenylbutyrate (4-PBA; Buphenyl) (Rubenstein et al., 1997; Rubenstein and Zeitlin, 2000), curcumin (Egan et al., 2002, 2004; Norez et al., 2006a), CFTRcor-325 (Wang et al., 2006), and the 
1,2-glucosidase inhibitor miglustat (Norez et al., 2006b).
In previous studies, we identified benzo[c]quinolizinium (MPB) derivatives (several chemical structures are shown in Fig. 1B together with two phenanthrene derivatives) as activators of wild-type CFTR, and G551D, G1349D, and F508del mutants (Becq et al., 1999; Dormer et al., 2001a; Marivingt-Mounir et al., 2004; Melin et al., 2004). Two of them, MPB-07 and MPB-91, have also been described as correctors of the F508del-CFTR trafficking (Dormer et al., 2001a,b) and as inhibitors of the first cytoplasmic-domain degradation of F508del-CFTR (Stratford et al., 2003).
In the present work, we performed a comparative analysis of the effect of several chemically diverse MPB and phenanthrene derivatives on two CFTR mutants, F508del and G622D. We identified the Gly622 amino acid as part of the putative site interfering with the correcting effect of MPB and showed that the rescue of F508del-CFTR by MPB is due to prevention of proteasomal degradation.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
), and COS-7 cells stably transfected with green fluorescent protein (GFP)-tagged CFTR vectors containing either wild-type (wt)-, F508del-, or G622D-CFTR. The F508del and G662D mutations were created and introduced into pS65T/enhanced GFP-C1/wt-CFTR constructs as described previously (Melin et al., 2004). The culture conditions are as described previously (Jefferson et al., 1990; Melin et al., 2004; Norez et al., 2006a).
Immunoprecipitation and Western Blot Analysis. For lysates, total protein was quantified using the Bradford (Bradford, 1976) protein assay reagent (Bio-Rad S.A, Marnes-la-Coquette, France), and 50 µg of protein was loaded onto an SDS-polyacrylamide gel electrophoresis apparatus. For immunoprecipitation, CF15 cell lysates were incubated with monoclonal anti-CFTR antibody (2 µg, IgG2a clone 24-1; R&D Systems, Minneapolis, MN). Immunoblots were probed with monoclonal mouse anti-GFP antibody (2 µl/ml; Sigma-Aldrich, St. Louis, MO), polyclonal rabbit anti-calnexin (CNX) antibody (2 µl/ml, SPA-860; Assay Designs, Ann Arbor, MI), polyclonal rabbit anti-heat shock protein (HSP)70 antibody (1:80,000, SPA-757; Assay Designs), or polyclonal rabbit anti-HSP90 antibody (1:250, SPS-771; Assay Designs). The protein levels were quantified by densitometry and expressed as a percentage of controls. Other details appear elsewhere (Norez et al., 2006a).
Immunofluorescence Study. CF15 cells were incubated with monoclonal anti-human CFTR antibody (1:100, IgG2a clone 24-1; R&D Systems) overnight at 4°C. Cells were then incubated with the FluoProbes 488 (1:400; FluoProbes, Interchim, France) secondary antibody. Nuclei were stained in red with TO-PRO-3 iodide (Invitrogen, Carlsbad, CA) for 15 min at room temperature (1:1000 in Tris-buffered saline). Fluorescence was examined with a spectral confocal station FV 1000 installed on an inverted microscope IX-81 (Olympus, Tokyo, Japan). For more details, see Norez et al., 2006a.
Functional Analysis of CFTR Channel Activity. The wt-, F508del-, and G622D-CFTR Cl– channel activities were assayed by measuring the rate of iodide (125I) efflux from CF15 cells and COS-7 cells as described previously (Melin et al., 2004; Norez et al., 2006a). Time-dependent rates of 125I efflux were calculated from the following: ln (125I t1/125It2)/(t1 – t2), where 125It is the intracellular 125I at time t, and t1 and t2 successive time points. Curves were constructed by plotting rates of 125I versus time. All comparisons were based on maximal values for the time-dependent rates (k = peak rates, min–1), excluding the points used to establish the baseline (k peak-k basal, min–1) (for other details, see Norez et al., 2006a.)
Proteasome Activity Assay. Proteasome enzymatic activity was measured as described by Canu et al. (2000) and per the manufacturer's protocol (20S Proteasome Activity Assay Kit; Millipore Bioscience Research Reagents, Temecula, CA). In brief, this assay is based on the detection of the fluorophore 7-amino-4-methylcoumarin (AMC) after cleavage from the labeled substrate LLVY-AMC by the proteasome machinery. The free AMC fluorescence was quantified using a 380/460-nm filter set. The intracellular proteasome activity was detected in cells after2hof treatment with several correctors, whereas testing the inhibition of purified proteasome enzyme by these correctors was performed after incubation of the 20S proteasome-positive control for 30 min according to the assay instructions.
Statistics. Results are expressed as the mean ± S.E.M. of n observations. Sets of data were compared with a Student's t test. Differences were considered statistically significant when p < 0.05. Significant differences were *, p < 0.05, **, p < 0.01, and ***, p < 0.001. All statistical tests were performed using Prism 4.0 software for Windows (GraphPad Software Inc., San Diego, CA).
Chemicals. The following MPB derivatives have been prepared in our laboratory as described previously (Marivingt-Mounir et al., 2004): MPB-07, MPB-80, MPB-91, MPB-104, MPB-89, and MPB-05. All other chemicals were obtained from Sigma-Aldrich, with the exception of miglustat (obtained from Toronto Research Chemicals, Toronto, ON, Canada), forskolin (Fsk), and genistein (Gst) (both obtained from PKC Pharmaceuticals, Woburn, MA). TS-TM calix[4]arene was provided by Dr. R. Bridges (University of Chicago, Chicago, IL).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Norez et al., 2004). In this assay, a rapid change in the rate of iodide efflux on stimulation was indicative of the presence of active CFTR at the plasma membrane. The transport activity of CFTR was stimulated by a cocktail of Fsk (10 µM) and Gst (30 µM) (Norez et al., 2006a). Figure 2A shows an example of CFTR-dependent iodide efflux in F508del-, G622D-, and wt-CFTR-expressing COS-7 cells. As expected, in F508del-CFTR-expressing cells, Fsk + Gst could not stimulate iodide efflux due to trafficking deficiency (Fig. 2A, black circles), as in mock-transfected cells (data not shown). For G622D-CFTR-expressing cells (Fig. 2A, black triangles), Fsk + Gst stimulated the iodide efflux (kpeak – kbasal = 0.046 ± 0.020 min–1) to a level 
3-fold lower than that of wt-CFTR cells (Fig. 2A, black squares; kpeak – kbasal = 0.155 ± 0.009 min–1). All of the effluxes stimulated by Fsk + Gst were inhibited by the CFTR inhibitor CFTRinh-172 (Ma et al., 2002) (data not shown). These results suggest that, although functional (in agreement with Vankeerberghen et al., 1998), G622D-expressing COS-7 cells have a reduced transport activity compared with wt-CFTR cells. This difference in activity can be explained by a dysfunctional activity, as reported previously (Vankeerberghen et al., 1998). An alternative explanation would be that the trafficking of G622D is abnormal. To test this hypothesis, we explored the effect of several trafficking correctors; MPB derivatives (Dormer et al., 2001a,b) and miglustat (Norez et al., 2006b). All cells maintained at 37°C were incubated for 2 h with culture medium supplemented with 100 µM of the given chemical compound. After the incubation period, CFTR-dependent effluxes were stimulated by the Fsk + Gst cocktail. Figure 2B presents typical iodide efflux responses, showing that in G622D-CFTR cells incubated with miglustat (black triangles), Fsk + Gst increased (2-fold) iodide efflux compared to untreated cells (Fig. 2B, open squares; p < 0.001). However, with G622D-CFTR cells incubated with MPB-104 (Fig. 2B, black circles), the Fsk + Gst-stimulated iodide efflux was not different from control. Normalized iodide effluxes for numerous experiments are presented in Fig. 2C. Therefore, these results show a partial trafficking defect of G622D-CFTR that can be reversed by miglustat as with the F508del mutant. We also observed that miglustat by itself is not a direct activator of CFTR, because replacement of Fsk + Gst by miglustat failed to stimulate CFTR-dependent efflux (data not shown). Altogether, our observations indicate successful functional rescue of F508del-CFTR by MPB-91 and MPB-07 and also by MPB-104 and MPB-80 to a similar level than cells corrected by miglustat (Fig. 2C, open bars). It is surprising to note that the incubation of cells with any of the MPB correctors did not rescue G622D-CFTR (Fig. 2C, gray bars).
|
If G622D is a partial trafficking-deficient mutant, as our functional data suggest, then we should observe less band C forms of the mutant compared to wt-CFTR, indicating a reduced pool of mature proteins. Thus, we performed biochemical assays to compare the G622D-CFTR protein expression in the presence or absence of these compounds. Anti-GFP immunoblotting from COS-7 cells stably expressing GFP-tagged wt-CFTR was used as a control to reveal the two characteristic bands of the CFTR protein (Fig. 2D, lane 1). Band C corresponds to the mature, fully glycosylated CFTR that underwent Golgi complex, and band B corresponds to the immature, core-glycosylated CFTR in the ER (Cheng et al., 1990). Anti-GFP immunoblotting in COS-7 cells expressing G622D-CFTR (Fig. 2D, lane 2) shows a reduced amount of band C form but a much higher quantity of band B form for G622D versus wt proteins, confirming the partial trafficking defect of G622D. In COS-7 cells expressing G622D-CFTR incubated for 2 h with 100 µM miglustat, anti-GFP immunoblotting revealed an increased band C intensity (Fig. 2D, lane 4). In contrast, a treatment by MPB-104 did not affect the molecular forms of the proteins (Fig. 2D, lane 3). Increasing the band C level by the CFTR corrector miglustat is in good agreement with our functional results. However, and contrary to F508del-CFTR rescued by MPB derivatives (Dormer et al., 2001a), the abnormal processing of G622D-CFTR cannot be rescued by MPB. Again, this is in good agreement with our functional data.
Effect of MPB Correctors on the Endogenous F508del-CFTR Trafficking. Because heterologous expression of F508del-CFTR can disturb the ER quality control (ERQC), we extended our study to evaluate the mechanism of action of MPB correctors on endogenous F508del-CFTR in the human airway epithelial CF15 cells. We first localized F508del-CFTR in CF15 cells before and after treatment with MPB. We found clear restriction of F508del proteins around the nucleus in untreated CF15 cells (Fig. 3A, a–d). After 2 h of treatment at 37°C with 100 µM MPB-91 (Fig. 3A, e–h) or MPB-104 (Fig. 3A, i–l), the cellular distribution of F508del-CFTR changed, with a new location at the plasma membrane and throughout the cells. The right panel (Fig. 3A, d, h, and l) is a merge of fluorescent images (Fig. 3A, b, f, and j) with the corresponding light micrograph (Fig. 3A, c, g, and k) showing the plasma membrane and cytosol location of F508del-CFTR proteins after MPB treatment in comparison to untreated cells.
|
30 and 50% of the miglustat correction, respectively) (see chemical structures in Fig. 1B). No rescue was obtained with either phenanthrene or 9-hydroxyphenanthrene (Fig. 3B), two MPB-related compounds lacking the quaternary ammonium (Fig. 1B). Thus, the minimal chemical structure for trafficking correction corresponds to the MPB-89 compound. We measured increasing potency with the hydroxyl-substituted derivative MPB-05 and with the group of substituted hydroxybenzo-[c]quinolizinium chloride MPB-07, MPB-80, MPB-91, and MPB-104 (Fig. 3B). To ascertain the identity of rescued F508del-CFTR, the pharmacological profile of inhibition was performed for CF15 cells corrected by those compounds. Figure 3C represents the pharmacological profile of inhibition obtained in CF15 cells treated for 2 h with 100 µM MPB-104. The efflux stimulated by Fsk + Gst was inhibited by diphenylamide-2-carboxylic acid (DPC), CFTRinh-172, and glibenclamide, but not by calixarene or DIDS as expected for CFTR (Norez et al., 2006a). Similar results were obtained after correction by MPB-07, MPB-80, and MPB-91 (data not shown). We then addressed several questions regarding the correcting effect of MPB. First, could an increase of the duration of incubation with the drug improve the iodide efflux response? It is interestingly to note that, as shown Fig. 3D, the response increased as a function of the duration of treatment to a maximum of 0.170 ± 0.005 min–1 obtained after 4 h of MPB-104 treatment. This response then declined, although there was still detectable rescue after 6 h (Fig. 3D) but not after 12 h (data not shown). In a separate series of experiments, we incubated CF15 cells for 2 h at 37°C with MPB-104 and then washed out the drug and analyzed the Fsk + Gst iodide efflux response every 2 h for 12 h. Figure 3E shows that rescue of F508del-CFTR function was maximal during the first 2 h after compound washout, and then it progressively declined between 4 and 12 h. Taken together, these data demonstrated reversible rescue of endogenous F508del-CFTR function with MPB in a time-dependent manner. These results also show that MPB is able to rescue endogenous F508del-CFTR with the following order of potency: MPB-104 =–91 = –07 =–80 >–05>–89, whereas phenanthrene and 9-hydroxyphenantrene are not active.
Interactions between F508del-CFTR and Molecular Chaperones in MPB-Corrected CF15 Cells. To study a potential effect of MPB on molecular chaperones, we performed coimmunoprecipitation experiments using CF15 cells. We first immunoprecipitated F508del-CFTR complexes using polyclonal anti-CFTR antibody followed by anti-calnexin Western blotting (Fig. 4A). Two conditions were used as positive controls: CF15 lysate (Fig. 4A, lane 1) and untreated CF15 cells immunoprecipitation (Fig. 4A, lane 3). Nonimmune mouse IgG (Fig. 4A, lane 2) was used as a negative control. In untreated CF15 cells (Fig. 4A, lane 3), we detected the calnexin band complexed with F508del-CFTR. This interaction was not affected when cells were incubated at 27°C (Fig. 4A, lane 4) but was prevented by the corrector miglustat (Fig. 4A, lane 5). Immunoblotting with CF15 cells corrected either by MPB-104, -91, -80, or -07 detected a similar intensity band (Fig. 4A, lanes 6–9) of calnexin as in the untreated CF15 cells (Fig. 4A, lane 3). Densitometry analysis of several independent experiments confirmed these results (Fig. 4, right panels). In a similar method, we evaluated the effect of MPB derivatives on the interaction between F508del-CFTR and the molecular HSP70 and HSP90. Histograms show an absence of effect of MPB-104, -91, -80, or -07 on the F508del-CFTR/HSP70 (Fig. 4B) or F508del-CFTR/HSP90 complexes (Fig. 4C). To verify that variations in the CFTR expression level did not affect the amount of chaperone that was pulled down, we compared the mRNA quantity of CFTR by the quantitative reverse transcription-polymerase chain reaction technique. Whatever the MPB treatment, we observed no variation of CFTR mRNA (data not shown). Taken together, these results show that the rescue of F508del-CFTR by MPB correctors cannot be explained by perturbation of the physical interaction between the mutant channel and the molecular chaperones CNX, HSP70, or HSP90.
|
To further study the rescue pathway of F508del-CFTR in MPB-treated cells, we investigated the effect of MPB-104 in competition with several inhibitors of the biosynthetic pathway and of the ER quality machinery (see the schematic drawing in Fig. 5) using the iodide efflux technique. We observed inhibition of the effect of MPB-104 and miglustat by BFA, a vesicular ER/Golgi-intermediate compartment traffic inhibitor (denoted BFA in Fig. 5A). This result indicates that F508del-CFTR proteins follow a conventional trafficking pathway when rescued by MPB-104 and miglustat. The sarcoendoplasmic reticulum calcium ATPase pump inhibitor TG is a partial corrector of F508del-CFTR (Fig. 5B, hatched bar), as shown previously (Egan et al., 2002; Norez et al., 2006a), and is known to alter the F508del-CFTR/calnexin interaction (Norez et al., 2006a). In the presence of TG, we observed a very significant (p < 0.001) potentiation of the F508del-CFTR rescue by MPB-104 (Fig. 5B, black bars) and a significant potentiation (p < 0.05) of the F508del-CFTR rescue by miglustat (Fig. 5B, open bars). The HSP90 inhibitor geldanamycin has been shown to stabilize F508del-CFTR by preventing the interaction between the chaperone and the mutant protein (Fuller and Cuthbert, 2000). We showed that MPB compounds do not inhibit the interaction between HSP90 and F508del-CFTR in CF15 cells (Fig. 4C). We measured the iodide efflux in CF15 cells incubated with MPB-104 and geldanamycin (Fig. 5C, black bars) and observed a significant potentiation (p < 0.001) of the Fsk + Gst response. When cells were treated with miglustat and geldanamycin (Fig. 5C, white bars), the Fsk + Gst response was also potentiated compared to cells treated only with miglustat, but the level of potentiation for miglustat/geldanamycin was smaller (p < 0.05) than for MPB-104/geldanamycin. Moreover, we observed only a small increase (p < 0.05) of the Fsk + Gst response after treating cells with MPB-104 in the presence of MG132, a proteasome inhibitor (Fig. 5D, black bars). When cells were treated with miglustat and MG132, the Fsk + Gst response was also potentiated compared to cells treated only with miglustat (Fig. 5D, white bars), but the level of potentiation was increased (p < 0.01) for miglustat/MG132 compared to MPB-104/MG132. MG132 by itself was not able to rescue F508del-CFTR function (Fig. 5D, hatched bar). Similar results were obtained with other MPB compounds (data not shown). These results suggest that the mechanism of correction of MPB and miglustat are different.
|
Effect of MPB Correctors on the Degradation Machinery. In a previous study, Stratford et al. (2003) suggested that MPB-07 and MPB-91 are inhibitors of the first cytoplasmic domain degradation of F508del-CFTR using an in vitro biochemical assay. To determine whether this mechanism of action takes place in CF15 cells, we evaluated the effect of correctors on the proteasome activity of CF15 cells. The proteasome activity of CF15 cells corrected by MPB-104, -07, -91, or -80 was significantly reduced compared to uncorrected and miglustat-corrected cells (Fig. 6A). In CF15 cells, we observed approximately 30% inhibition after MPB treatment in comparison to MG132 and lactacystin, two known proteasome inhibitors (91 and 79% inhibition, respectively). On the contrary, we did not detect inhibition of the purified 20S proteasome activity by any of the MPB correctors (Fig. 6B), whereas lactacystin and MG132 caused 97 and 91% inhibition, respectively. Miglustat has no effect on the proteasome activity in CF15 cells (Fig. 6A) or on the purified 20S proteasome activity (Fig. 6B). The inhibition of the cellular proteasome by MPB correctors was not due to a toxic effect because no cellular toxicity was observed (data not shown) for any of the MPB compounds [toxicity evaluated on CF15 cells with the 3-(4,5-dimethyl(thiazol-2-yl)-2,5-diphenyltetrazolium bromide test as described previously (Norez et al., 2006a)].
|
The results presented above show that after treatment of CF cells by MPB correctors, the cell proteasome activity was reduced. Because this effect could be attributed to a nonspecific action of MPB, we also measured the proteasome activity in mock COS-7 cells (Fig. 7A) or COS-7 cells stably expressing F508del-CFTR (Fig. 7B) or G622D-CFTR (Fig. 7C). In mock-transfected COS-7 cells, the overall proteasome activity was fully inhibited by MG132 and lactacystin. On the contrary, with MPB derivatives, phenanthrene or the vehicle (dimethyl sulfoxide), inhibition could not be detected (Fig. 7A). In sharp contrast, in COS-7 cells expressing F508del-CFTR, we obtained 50% inhibition of the proteasome activity by MPB treatment (100 µM, 2 h; Fig. 7B). Interestingly, we did not observe inhibition by phenanthrene, a non-F508del-CFTR corrector, and noted only 25% of proteasome inhibition in the presence of MPB-89, a partial F508del-CFTR corrector (see Fig. 3B). Therefore, these experiments revealed that inhibition of the cellular degradation machinery by MPB correctors is not only CFTR-dependent, but it also follows the same structure-activity relationship as demonstrated for functional correction, i.e., MPB-104-91-07-80 > 89 >> phenanthrene. Finally, because we showed that MPB correctors are not effective on G622D-CFTR, we asked whether this mutation could also affect the MPB-mediated inhibition of the degradation machinery. To study this theory, the proteasome activity was also measured in COS-7 cells expressing G622D proteins. Our results show that neither MPB derivatives nor phenanthrene were able to inhibit the cell proteasome activity (Fig. 7C). These results are undistinguishable from those obtained in mock COS-7 cells (Fig. 7A). Altogether, these results demonstrate that MPB correctors inhibit the cell proteasome activity in a CFTR-dependent manner and suggest that the mutation G622D prevents this inhibition.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Structural Determinants of MPB Compounds. In a previous study analyzing the effect of CFTR channel activators, we reported a structure-activity relationship of MPB compounds (Marivingt-Mounir et al., 2004), and we demonstrated that some of these derivatives were able to stimulate the channel activity of wt-, G551D-, G1349D-, G551D/G1349D-, and F508del-CFTR (Becq et al., 1999; Dormer et al., 2001a; Marivingt-Mounir et al., 2004; Melin et al., 2004). We determined that presence of the hydroxyl group associated with a chlorine atom, and that an alkyl chain in the MPB skeleton was more effective at activating CFTR (Marivingt-Mounir et al., 2004). For example, the product MPB-104 seems to be 100 times more potent than the parental compound MPB-07. In the present study, we analyzed the structure-activity relationship of several MPB and phenanthrene derivatives for their ability to rescue the abnormal trafficking of mutant CFTR proteins. The present results indicate that F508del-CFTR-dependent iodide efflux was rescued by MPB (in CF15 and COS-7 cells) to the same level as cells corrected by miglustat. In addition, a modest rescue was found with MPB-89 and MPB-05 (30–50% of the miglustat correction). No rescue of mutant CFTR was found with phenanthrene and 9-hydroxyphenanthrene, arguing that the quaternary ammonium is important for F508del-CFTR rescue. Thus, we hypothesize that the minimal conformation to partially rescue F508del trafficking corresponds to the structure of MPB-89, i.e., to the MPB skeleton (see structure in Fig. 1B). To increase its efficacy on F508del-CFTR rescue, further chemical substituents (Fig. 8A) are required, such as the 6-hydroxyl (MPB-05, -07, -80, -91, and -104). In contrast, adding an alkyl chain (as in MPB-91 and MPB-104) has no apparent effect on the rescue efficacy (for example, MPB-80 and MPB-91 have similar efficacy), whereas this greatly increases the activation efficacy of MPB-91 or MPB-104 as an activator of CFTR-chloride channels (Marivingt-Mounir et al., 2004).
|
Although several CFTR potentiators and/or activators have been reported in the literature, MPB compounds seem to be distinguished by their ability to activate several forms of CFTR (Becq et al., 1999; Dormer et al., 2001a; Marivingt-Mounir et al., 2004; Melin et al., 2004) and to rescue the defective trafficking of endogenous F508del-CFTR in human airway epithelial cells (this study; Dormer et al., 2001a,b). Because a significant number of human diseases involve defective traffic of misfolded glycoproteins, albeit partially functional (Aridor and Hannan, 2000, 2002; Ulloa-Aguirre et al., 2004), the use of new MPB congeners tailored to rescue class 2 CF mutation might prove to be beneficial for the management of CF and other diseases.
Mechanism of F508del-CFTR Rescue by MPB Correctors. Some of the pharmacological chaperones described so far, including miglustat (Fig. 8B), alter the recognition of the mutant protein by calnexin (Robert et al., 2005; Gong et al., 2006; Norez et al., 2006a,b). In all of these cases, the complex pharmacological chaperone/traffic-deficient protein seemed to be stabilized in an intermediate state in its folding path that more closely resembled the native state of the wild-type protein (Bernier et al., 2004). Thus, until now, preventing the calnexin interaction with the mutant protein in the ER has been regarded as a major mechanism of rescue. In this study, we show dramatic action of MPB on the release of F508del-CFTR from the ER by a calnexin-independent effect. Interaction between HSP90 and F508del-CFTR was unaffected by MPB, arguing against an effect of MPB derivatives on ERQC but more likely on endoplasmic reticulum-associated degradation (ERAD) machinery. Furthermore, Stratford et al. (2003) provide evidence that MPB-07 and MPB-91 protect a proteolytic cleavage site by direct binding to the first cytoplasmic domain of F508del-CFTR. MPB derivatives probably affect the trafficking of F508del-CFTR by interfering with CFTR architecture from the cytosol. Thus, we hypothesize that within the CFTR structure, occupation of a drug binding site by MPB may stabilize the conformation of F508del-CFTR protein, allowing the MPB/F508del-CFTR complex to escape the proteasome-degradation system reaching the plasma membrane (Fig. 8C).
ERAD has important consequences for protein folding, transport, and degradation (Meusser et al., 2005), and it is a central element of the secretory pathway. A direct inhibition of the proteasome with lactacystin and MG132 did not promote F508del-CFTR to the cell surface (Jensen et al., 1995; Ward et al., 1995). We also found that MG132 was unable to rescue F508del-CFTR in CF15 cells after incubation and iodide efflux assay. It is interesting to note that deoxyspergualin, which competitively inhibits peptide binding to HSP70, leads to a restoration of CFTR function in cells expressing F508del-CFTR (Jiang et al., 1998). Moreover, Rubenstein and Zeitlin (2000) demonstrated that decreased heat shock cognate 70 (HSC70) expression after 4-PBA treatment leads to improved F508del-CFTR trafficking. A recent study reported that a proteasome inhibition with bortezomid, which induces HSP70 and down-regulates valosin-containing protein expression, partially rescued mature F508del-CFTR in IB3-1 cells and human CF cells (Vij et al., 2006). These results are consistent with our data, demonstrating that MPB correctors prevent the recognition of F508del-CFTR by the proteasome machinery and allow restoration of F508del-CFTR to the cell surface via a BFA-sensitive pathway (Fig. 8C). Therefore, this study and others (Jiang et al., 1998; Rubenstein and Zeitlin, 2000; Vij et al., 2006) provide evidence that ERAD is a potential target to rescue defective F508del-CFTR trafficking.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ER, endoplasmic reticulum; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; 4-PBA, 4-phenylbutyrate; miglustat, N-butyl-deoxynojirimycin; MPB, benzo[c]quinolizinium; MPB-07, 10-chloro-6-hydroxychlorobenzo[c]quinolizinium chloride; MPB-91, 5-butyl-10-chloro-6-hydroxybenzo[c]quinolizinium chloride; GFP, green fluorescent protein; wt, wild-type; CNX, calnexin; HSP, heat shock protein; BFA, brefeldin A; TG, thapsigargin; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; AMC, 7-amino-4-methylcoumarin; MPB-80, 10-fluoro-6-hydroxybenzo[c]quinolizinium chloride; MPB-104, 5-butyl-7-chloro-6-hydroxybenzo[c]quinolizinium chloride; MPB-89, benzo[c]quinolizinium chloride; MPB-05, 6-hydroxybenzo[c]quinolizinium chloride; TS-TM calyx[4]arene, 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene; Fsk, forskolin; Gst, genistein; CFTRinh-172, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone; ERQC, ER quality control; DPC, diphenylamide-2-carboxylic acid; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; ERAD, endoplasmic reticulum-associated degradation; LLVY, Leu-Leu-Val-Tyr.
Address correspondence to: Dr. Caroline Norez, Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, Centre National de la Recherche Scientifique, 86022 Poitiers, France. E-mail: caroline.norez{at}univ-poitiers.fr
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aridor M and Hannan LA (2000) Traffic jam: a compendium of human diseases that affect intracellular transport processes. Traffic 1: 836–851.[CrossRef][Medline]
Aridor M and Hannan LA (2002) Traffic jams II: an update of diseases of intracellular transport. Traffic 3: 781–790.[CrossRef][Medline]
Becq F, Mettey Y, Gray MA, Galietta LJ, Dormer RL, Merten M, Metaye T, Chappe V, Marvingt-Mounir C, Zegarra-Moran O, et al. (1999) Development of substituted benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel. J Biol Chem 274: 27415–27425.
Bernier V, Bichet DG, and Bouvier M (2004) Pharmacological chaperone action on G-protein-coupled receptors. Curr Opin Pharmacol 4: 528–533.[CrossRef][Medline]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.[CrossRef][Medline]
Burrows JA, Willis LK, and Perlmutter DH (2000) Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: a potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency. Proc Natl Acad Sci U S A 97: 1796–1801.
Canu N, Barbato C, Ciotti MT, Serafino A, Dus L, and Calissano P (2000) Proteasome involvement and accumulation of ubiquitinated proteins in cerebellar granule neurons undergoing apoptosis. J Neurosci 20: 589–599.
Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O'Riordan CR, and Smith AE (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63: 827–834.[CrossRef][Medline]
Dalemans W, Barbry P, Champigny G, Jallat S, Dott K, Dreyer D, Crystal RG, Pavirani A, Lecocq JP, and Lazdunski M (1991) Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation. Nature 354: 526–528.[CrossRef][Medline]
Denning GM, Anderson MP, Amara JF, Marshall J, Smith AE, and Welsh MJ (1992) Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358: 761–764.[CrossRef][Medline]
Dormer RL, Derand R, McNeilly CM, Mettey Y, Bulteau-Pignoux L, Metaye T, Vierfond JM, Gray MA, Galietta LJ, Morris MR, et al. (2001a) Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells. J Cell Sci 114: 4073–4081.[Medline]
Dormer RL, McNeilly CM, Morris MR, Pereira MM, Doull IJ, Becq F, Mettey Y, Vierfond JM, and McPherson MA (2001b) Localisation of wild-type and DeltaF508-CFTR in nasal epithelial cells. Pflugers Arch 443 (Suppl 1): S117–S120.[CrossRef][Medline]
Egan ME, Glockner-Pagel J, Ambrose C, Cahill PA, Pappoe L, Balamuth N, Cho E, Canny S, Wagner CA, Geibel J, et al. (2002) Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells. Nat Med 8: 485–492.[CrossRef][Medline]
Egan ME, Pearson M, Weiner SA, Rajendran V, Rubin D, Glockner-Pagel J, Canny S, Du K, Lukacs GL, and Caplan MJ (2004) Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects. Science 304: 600–602.
Ellgaard L and Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4: 181–191.[CrossRef][Medline]
Fuller W and Cuthbert AW (2000) Post-translational disruption of the delta F508 cystic fibrosis transmembrane conductance regulator (CFTR)-molecular chaperone complex with geldanamycin stabilizes delta F508 CFTR in the rabbit reticulocyte lysate. J Biol Chem 275: 37462–37468.
Gong Q, Jones MA, and Zhou Z (2006) Mechanisms of pharmacological rescue of trafficking-defective hERG mutant channels in human long QT syndrome. J Biol Chem 281: 4069–4074.
Jefferson DM, Valentich JD, Marini FC, Grubman SA, Iannuzzi MC, Dorkin HL, Li M, Klinger KW, and Welsh MJ (1990) Expression of normal and cystic fibrosis phenotypes by continuous airway epithelial cell lines. Am J Physiol 259: 496–505.
Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, and Riordan JR (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83: 129–135.[CrossRef][Medline]
Jiang C, Fang SL, Xiao YF, O'Connor SP, Nadler SG, Lee DW, Jefferson DM, Kaplan JM, Smith AE, and Cheng SH (1998) Partial restoration of cAMP-stimulated CFTR chloride channel activity in DeltaF508 cells by deoxyspergualin. Am J Physiol Cell Physiol 275: C171–C178.
Kartner N, Augustinas O, Jensen TJ, Naismith AL, and Riordan JR (1992) Mislocalization of delta F508 CFTR in cystic fibrosis sweat gland. Nat Genet 1: 321–327.[CrossRef][Medline]
Ma T, Thiagarajah JR, Yang H, Sonawane ND, Folli C, Galietta LJ, and Verkman AS (2002) Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J Clin Invest 110: 1651–1658.[CrossRef][Medline]
MacDonald KD, McKenzie KR, and Zeitlin PL (2007) Cystic fibrosis transmembrane regulator protein mutations: "class" opportunity for novel drug innovation. Paediatr Drugs 9: 1–10.[Medline]
Marivingt-Mounir C, Norez C, Derand R, Bulteau-Pignoux L, Nguyen-Huy D, Viossat B, Morgant G, Becq F, Vierfond JM, and Mettey Y (2004) Synthesis, SAR, crystal structure, and biological evaluation of benzoquinoliziniums as activators of wild-type and mutant cystic fibrosis transmembrane conductance regulator channels. J Med Chem 47: 962–972.[CrossRef][Medline]
Melin P, Thoreau V, Norez C, Bilan F, Kitzis A, and Becq F (2004) The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein. Biochem Pharmacol 67: 2187–2196.[CrossRef][Medline]
Meusser B, Hirsch C, Jarosch E, and Sommer T (2005) ERAD: the long road to destruction. Nat Cell Biol 7: 766–772.[CrossRef][Medline]
Norez C, Heda GD, Jensen T, Kogan I, Hughes LK, Auzanneau C, Derand R, Bulteaux-Pignoux L, Li C, Ramjeesingh M, et al. (2004) Determination of CFTR chloride channel activity and pharmacology using radiotracer flux methods. J Cyst Fibros 3 (Suppl 2): 119–121.[Medline]
Norez C, Antigny F, Becq F, and Vandebrouck C (2006a) Maintaining low Ca2+ level in the endoplasmic reticulum restores abnormal endogenous F508del-CFTR trafficking in airway epithelial cells. Traffic 7: 562–573.[CrossRef][Medline]
Norez C, Noel S, Wilke M, Bijvelds M, Jorna H, Melin P, DeJonge H, and Becq F (2006b) Rescue of functional delF508-CFTR channels in cystic fibrosis epithelial cells by the alpha-glucosidase inhibitor miglustat. FEBS Lett 580: 2081–2086.[CrossRef][Medline]
Powell K and Zeitlin PL (2002) Therapeutic approaches to repair defects in deltaF508 CFTR folding and cellular targeting. Adv Drug Deliv Rev 54: 1395–1408.[CrossRef][Medline]
Robert J, Auzan C, Ventura MA, and Clauser E (2005) Mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin V1b/V3 receptor by a pharmacological chaperone. J Biol Chem 280: 42198–42206.
Rubenstein RC, Egan ME, and Zeitlin PL (1997) In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR. J Clin Invest 100: 2457–2465.[Medline]
Rubenstein RC and Zeitlin PL (2000) Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR. Am J Physiol Cell Physiol 278: C259–C267.
Stratford FL, Pereira MM, Becq F, McPherson MA, and Dormer RL (2003) Benzo-(c)quinolizinium drugs inhibit degradation of Delta F508-CFTR cytoplasmic domain. Biochem Biophys Res Commun 300: 524–530.[CrossRef][Medline]
Ulloa-Aguirre A, Janovick JA, Brothers SP, and Conn PM (2004) Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease. Traffic 5: 821–837.[CrossRef][Medline]
Vankeerberghen A, Wei L, Jaspers M, Cassiman JJ, Nilius B, and Cuppens H (1998) Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator. Hum Mol Genet 7: 1761–1769.
Vij N, Fang S, and Zeitlin PL (2006) Selective inhibition of endoplasmic reticulum-associated degradation rescues DeltaF508-cystic fibrosis transmembrane regulator and suppresses interleukin-8 levels: therapeutic implications. J Biol Chem 281: 17369–17378.
Wang Y, Bartlett MC, Loo TW, and Clarke DM (2006) Specific rescue of cystic fibrosis transmembrane conductance regulator processing mutants using pharmacological chaperones. Mol Pharmacol 70: 297–302.
Ward CL, Omura S, and Kopito RR (1995) Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83: 121–127.[CrossRef][Medline]
Welsh MJ and Smith AE (1993) Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73: 1251–1254.[CrossRef][Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
