UBE3C Facilitates the ER-Associated and Peripheral Degradation of Misfolded CFTR

Abstract

The ubiquitin E3 ligase UBE3C promotes the proteasomal degradation of cytosolic proteins and endoplasmic reticulum (ER) membrane proteins. UBE3C is proposed to function downstream of the RNF185/MBRL ER-associated degradation (ERAD) branch, contributing to the ERAD of select membrane proteins. Here, we report that UBE3C facilitates the ERAD of misfolded CFTR, even in the absence of both RNF185 and its functional ortholog RNF5 (RNF5/185). Unlike RNF5/185, UBE3C had a limited impact on the ubiquitination of misfolded CFTR. UBE3C knockdown (KD) resulted in an additional increase in the functional ∆F508-CFTR channels on the plasma membrane when combined with the RNF5/185 ablation, particularly in the presence of clinically used CFTR modulators. Interestingly, although UBE3C KD failed to attenuate the ERAD of insig-1, it reduced the ERAD of misfolded ∆Y490-ABCB1 and increased cell surface expression. UBE3C KD also stabilized the mature form of ∆F508-CFTR and increased the cell surface level of T70-CFTR, a class VI CFTR mutant. These results suggest that UBE3C plays a vital role in the ERAD of misfolded CFTR and ABCB1, even within the RNF5/185-independent ERAD pathway, and it may also be involved in maintaining the peripheral quality control of CFTR.

Ligand-based virtual-screening identified a novel CFTR ligand which improves the defective cell surface expression of misfolded ABC transporters

“…Treatment with FR3 increased the steady-state level of ΔF508-CFTR-NLuc upon TEZ/ELX/IVA treatment (Figure 3A). The CHX chase revealed a continuous reduction in the NLuc signal, with a half-life of approximately 100 min, consistent with the reported half-life of mature ΔF508-CFTR (Taniguchi et al, 2022;Kamada et al, 2023) (Figures 3B,C). Interestingly, FR3 treatment, in a concentration-dependent manner, weakly but significantly reduced the degradation of mature Frontiers in Pharmacology frontiersin.org ΔF508-CFTR induced by TEZ/ELX/IVA treatment, thereby extending its half-life (Figures 3B-D).…”

“…The CFTR expressions in CFBE cells were induced by treating them with 1 μg/mL dox for 4 days. BEAS-2B cells stably expressing CFTR variants-HiBiT, ∆Y490-ABCB1-HiBiT, or G601S-hERG-HiBiT were established by lentivirus transduction as previously ( Taniguchi et al, 2022 ) and were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FBS and 10 μg/mL blasticidin S. The HiBiT tag was introduced to the extracellular region of CFTR (4 th extracellular loop), ABCB1 (1 st extracellular loop) ( Kamada et al, 2023 ) or hERG (1 st extracellular loop). The hERG-HiBiT was produced by replacing the extracellular HA tag ( Apaja et al, 2013 ) with the HiBiT tag by PCR-based mutagenesis.…”