and in vitro
studies have suggested that enhancing ΔF508 CFTR activity by as little as 5% may diminish the symptoms of CF (2
). Therefore, a major thrust of CF research has been directed at identifying and characterizing small molecules that facilitate ΔF508 CFTR rescue from ERAD. Previously, we established that two of these correctors, corr-4a and VRT-325, stabilized the surface pool of ΔF508 CFTR by inhibiting endocytosis (20
). In the present study, we focused on the mechanism by which corr-4a rescues and stabilizes ΔF508 CFTR, and the functionality of the rescued protein in the presence and absence of corr-4a. We asked the following questions: (1
) Do low-temperature culture and corr-4a use the same mechanism to rescue ΔF508 CFTR? (2
) How long after delivery to the cell surface can ΔF508 CFTR be activated by physiological stimuli, such as FSK, in the presence or absence of corr-4a? (3
) Do cell surface ΔF508 CFTR levels correlate with physiological activity in the presence of corr-4a?
Although the effects of low temperature on ΔF508 CFTR have been established for quite some time (10
), the mechanism by which low temperature facilitates ΔF508 CFTR exit from the ER is not clear. The simplest interpretation is that, at lower temperature, the channel folds properly and is competent to exit the ER. However, if that is true, why is the majority of protein retained in the ER, and why is the function of the rescued cell surface ΔF508 CFTR compromised? One possibility is that only a small percentage of the protein is properly folded and rescued. However, if this were true, the functionality and stability of the rescued protein should be comparable to the wild type. Our results suggest that corr-4a stabilizes ΔF508 CFTR, yet the channel activity is still compromised. These results are consistent with those of Cui and coworkers (44
), who showed that ΔF508 plays a direct role in channel gating and residency time in the closed state. The other possibility is that some partially folded protein escapes ERAD, because the components or the pathways of the quality control machinery are altered. To support this possibility, our results indicate that both low-temperature culture and corr-4a increase the level of ΔF508 CFTR band B. Furthermore, both conditions alter the function of the ubiquitin-dependent degradative pathways. Using luciferase-based reporter constructs, we found that low-temperature culture inhibited both the E1–E3 cascade and the function of the proteasome. In contrast, corr-4a only inhibited the E1–E3 cascade. Therefore, it is likely that inhibition of the E1–E3 cascade by corr-4a is responsible for the stabilization ΔF508 CFTR at both the ER and the cell surface. Although we did not monitor the ubiquitination levels directly for WT or ΔF508 CFTR, there is also the possibility that corr-4a treatment interferes with the delivery of the ΔF508 CFTR to the degradative compartment as the result of inhibited ubiquitination. Interestingly, attempts by us and others to rescue ΔF508 CFTR using proteasome inhibitors have not been successful, although inhibition of the proteasome does stabilize the B band (4
) and cell surface CFTR in other models (31
). More recent results have indirectly suggested that selective inhibition of ERAD may rescue ΔF508 CFTR in CF tracheal epithelial cells transfected with ΔF508 CFTR (46
). Considering these data, and that CFTR has to be extracted from the ER membrane before degradation by the proteasome (4
), it is tempting to speculate that inhibition of ERAD steps before extraction, such as the E1–E3 ligase cascade, may indirectly facilitate protein escape from the ER.
Considering that corr-4a treatment had no effect on WT CFTR cell surface stability, these results complement a previous report demonstrating that low-temperature–rescued ΔF508 CFTR is still folded incorrectly at the cell surface and directed to the lysosome through a ubiquitin-dependent pathway, whereas the degradation of the WT protein from the cell surface is not ubiquitin dependent (47
). Although the studies by Sharma and coworkers (47
) show ubiquitin-dependent lysosomal degradation of ΔF508 CFTR, the studies by Gentzsch and coworkers (31
) indicate that the cell surface stability of ΔF508 CFTR increased after proteasome inhibition. Therefore, whether ΔF508 CFTR degradation from the cell surface is mediated by the lysosome, the proteasome, or both remains an open question. Importantly, both proposed pathways seem to require ubiquitin, and our data clearly demonstrate that corr-4a interferes with ubiquitination.
Regarding the cell surface stability of ΔF508 CFTR, it has been established that rescued ΔF508 CFTR is rapidly cleared from the cell surface at 37°C (20
). Results from Swiatecka-Urban (48
) and coworkers suggest that the surface half-life of ΔF508 CFTR in CFBE41o- cells is 1 hour, which agrees with our previous studies indicating that the half-life is 2 hours (20
). Chemical chaperones such as corr-4a seem to stabilize ΔF508 CFTR, but their mechanisms of actions are not clear. Our previous studies showed that the correctors, corr-4a and VRT-325, inhibit ΔF508 CFTR endocytosis and are specific for mutant CFTR (20
). In our studies using human airway epithelial cells, we did not observe a corr-4a effect on WT CFTR levels or function. However, Pedemonte and coworkers (15
) reported corr-4a effects in baby hamster kidney cells expressing WT CFTR. These results support the cell-specific differences in the processing of WT CFTR, as we described previously (49
The results presented here also agree with our previous findings indicating that corr-4a had no effect on the cell surface stability or endocytosis of WT CFTR or the transferrin receptor, because cell surface clearance of these proteins does not require ubiquitination (20
). We therefore hypothesize that corr-4a treatment may affect the cell surface clearance of all proteins that require ubiquitination. Considering that the optimal “corrector” for CF would be ΔF508 CFTR specific, our results also point to the importance of studies that investigate the complex mechanisms by which ΔF508 CFTR correctors work.
A recent study suggests that ΔF508 CFTR export requires a local folding environment that is sensitive to heat/stress-inducible factors found in some cell types, and these results suggest that low-temperature correction is necessary, but not sufficient, for ΔF508 export (50
). In this intriguing study, the authors proposed that folding and rescue efficiency of ΔF508 CFTR depend on the cell type, and that the chaperone environment is a critical component for successful ΔF508 CFTR folding at reduced temperatures. Moreover, different chaperone activities or balance of activities may be responsible for the folding of ΔF508 CFTR at reduced temperature, and these may be different from WT. As it relates to our study, it is possible that E1–E3 inhibition also alters the chaperone environment, possibly through inducing ER stress and activating the unfolded protein response (24
Importantly, corr-4a enhanced the responsiveness of ΔF508 CFTR to cAMP after a 6-hour treatment. Although this result represents a significant functional improvement over untreated controls, cell surface biotinylation studies also indicated that, although ΔF508 CFTR was also present at the membrane for a longer time period (12 h), it did not respond to cAMP. This observation suggests that protein stability and functional integrity are distinct features of rescued ΔF508 CFTR. Therefore, regarding endpoint results for experimental therapies, FSK-stimulated channel activity is a better readout than the presence of protein at the cell surface. Our results point to a clearly unmet need for additional measures (e.g., potentiators) beyond stabilizing the protein, measures that functionally correct channel activity to activate all rescued channels at maximum capacity, and ameliorate CF symptoms.
Based on previous results, it is clear that, in human airway epithelial cells, ΔF508 CFTR channel function is compromised at 37°C, but not at 27°C (20
). Furthermore, we have demonstrated that returning the cells to physiological conditions (37°C) after low-temperature rescue causes a rapid loss of functional activity (20
). This observation clearly differentiates the act of stabilizing the protein at the cell surface, or, for that matter, promoting ER exit, from correcting channel function. Our studies also establish that understanding how low-temperature rescue works is critical for developing therapies designed to promote rescue, protein stability, and channel activity of ΔF508 CFTR. It may well be that identifying a single compound that corrects all ΔF508 CFTR defects may prove to be difficult. In addition to examining the mechanism and the effects of low-temperature rescue and corr-4a on ΔF508 CFTR, our studies also establish a protocol for the evaluation of newly identified chemical chaperones.