The Ubiquitin Ligase RNF34 Participates in the Peripheral Quality Control of CFTR (RNF34 Role in CFTR PeriQC)

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.

AlphaLISA 112.5 nM His6-sumo-NBD1-∆F1S denatured at 44°C for 5 min was incubated with either 75 nM GST-RFFL or 75 nM GST-RNF34 for 2 hours at room temperature in 384 well plates (Perkin Elmer). Then, Ni Donor beads (Perkin Elmer) and Glutathione alphaLISA acceptor beads (Perkin Elmer) were added and incubated for 1 hour in the dark according to the manufacturer's instructions. The direct interaction was detected using the EnSpire Alpha plate reader (Perkin Elmer).

Establishment of RNF34 KO cells by CRISPR/CAS9
The RNF34

Measurement of PM expression of CFTR
PM density of ∆F508-CFTR-HRP was measured as done previously (Phuan et al., 2014;Veit et al., 2014). For low-temperature rescues, CFBE and BEAS-2B cells were incubated at 26°C and 30°C, respectively, for 2 days followed by a 1 h incubation at 37°C to induce unfolding. PM density of ∆F508-CFTR-HiBiT was measured in 96 well plates using the Nano Glo HiBiT Extracellular system (Promega). For the Trikafta rescue, cells were incubated with 3 µM VX-661, 3 µM VX-445 and 1 µM VX-770 at 37°C for 24
The luminescent signal was measured using the Luminoskan and Varioskan Flash microplate reader (ThermoFisher).

Western Blotting
Cells were solubilized in a RIPA buffer supplemented with 1 mM PMSF, 5 µg/ml leupeptin and pepstatin A) where the cell lysates were analyzed by a Western blot as done previously (Okiyoneda et al., 2010).

Halide-sensitive YFP quenching assay
The ΔF508-CFTR function assay by halide-sensitive YFP fluorescence quenching was

mRNA isolation and q-PCR analysis
The mRNA isolation and q-PCR was performed as described previously (Okiyoneda et al., 2018). The following primers were used; RFFL FW primer 5'-

Statistical analysis
For quantification, data from more than two technical repeats for each independent experiments were used where the data is expressed as means ± SE. Statistical significance was assessed by either a two-tailed paired Student's t-test or a one-way ANOVA using GraphPad Prism 8 (GraphPad Software). The RNF34 mRNA expression in differentiated human bronchial epithelial cells (HBE) isolated from three donors with CFTR WT/WT and nine patients with CFTR ΔF508/ΔF508 genotype was measured by q-PCR. The HBE cells were differentiated on filter supports under air-liquid interface culture conditions for ≥ 4 weeks. Error bars and horizontal lines show SE and means of all data, respectively.