Abstract
The peripheral protein quality control (periQC) system eliminates the conformationally defective cystic fibrosis transmembrane conductance regulator (CFTR), including ∆F508-CFTR, from the plasma membrane (PM) and limits the efficacy of pharmacological therapy for cystic fibrosis (CF). The ubiquitin (Ub) ligase RFFL is responsible for the chaperone-independent ubiquitination and lysosomal degradation of CFTR in the periQC. Here, we report that the Ub ligase RNF34 participates in the CFTR periQC in parallel to RFFL. An in vitro study reveals that RNF34 directly recognizes the CFTR NBD1 and selectively promotes the ubiquitination of unfolded proteins. RNF34 was localized in the cytoplasm and endosomes, where RFFL was equally colocalized. RNF34 ablation increased the PM density as well as the mature form of ∆F508-CFTR rescued at low temperatures. RFFL ablation, with the exception of RNF34 ablation, increased the functional PM expression of ∆F508-CFTR upon a triple combination of CFTR modulators (Trikafta) treatment by inhibiting the K63-linked polyubiquitination. Interestingly, simultaneous ablation of RNF34 and RFFL dramatically increased the functional PM ∆F508-CFTR by inhibiting the ubiquitination in the post-Golgi compartments. The CFTR-NLuc assay demonstrates that simultaneous ablation of RNF34 and RFFL dramatically inhibits the degradation of mature ∆F508-CFTR after Trikafta treatment. Therefore, these results suggest that RNF34 plays a crucial role in the CFTR periQC, especially when there is insufficient RFFL. We propose that simultaneous inhibition of RFFL and RNF34 may improve the efficacy of CFTR modulators.
Highlights
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel expressed on the plasma membrane (PM) of epithelial cells (Riordan, 2008), where its mutation gives rise to cystic fibrosis (CF), a recessive genetic disorder (Guggino, 1999)
Understanding that the periQC system limits the efficacy of CFTR modulators (Okiyoneda et al, 2018); we evaluated the KD of RFFL and RNF34 on PM levels of ΔF508-CFTR upon treatment with the triple combination of CFTR modulators named Trikafta (VX-661/ VX-445/VX-770)
The halide-sensitive YFP quenching assay revealed that the RFFL/RNF34 double KD significantly increased the functional ΔF508-CFTR channel induced by Trikafta (Figures 3D,E)
Summary
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel expressed on the plasma membrane (PM) of epithelial cells (Riordan, 2008), where its mutation gives rise to cystic fibrosis (CF), a recessive genetic disorder (Guggino, 1999). RFFL, an FYVE-like domain-containing Ub ligase, directly interacts with mature ΔF508CFTR in the post-Golgi compartments, including endosomes and PM through its disordered regions located in the N-terminal region (Okiyoneda et al, 2018) Both CHIP and RFFL selectively bind and facilitate ubiquitination of the conformationally defective CFTR, resulting in rapid endocytosis and lysosomal degradation. RNF34mCherry and RFFL-mCherry were partially colocalized with ΔF508CFTR-GFP, and low-temperature incubation increased the colocalization measured by the Pearson’s correlation coefficient (PCC) (Figures 2B,C) These results suggest that RNF34 may participate in the CFTR periQC in addition to RFFL. The RFFL/RNF34 double KD further increased both immature and mature forms and PM levels of ΔF508-CFTR compared to every single KD (Figures 2D,F) These results suggest that RNF34 participates in the periQC in parallel to RFFL, limiting the PM levels of lowtemperature rescued ΔF508-CFTR
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