HERC3 E3 ligase provides an ERAD branch eliminating select membrane proteins

Abstract

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, each with distinct substrate specificity, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. In this study, we have discovered a cytosolic Ub ligase called HERC3, which fulfills a distinct role in facilitating the ERAD of select polytopic membrane proteins. Using a series of multiplex knockdown/knockout experiments, we have demonstrated that HERC3 functions independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to facilitate the ubiquitination, retrotranslocation, and ERAD of misfolded CFTR. Furthermore, HERC3 collaborates with RNF5/185 to enhance the association of UBQLN proteins, thereby augmenting the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 specifically promotes the ERAD of CFTR, likely due to its ability to interact with the less hydrophobic membrane-spanning domains of CFTR. HERC3 may detect exposed transmembrane domains on the cytoplasmic surface of the ER, thereby facilitating the recruitment of UBQLN and subsequently accelerating the ERAD of select polytopic membrane proteins.

Small-molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states

“…Nonetheless, the modest changes to mNG-F508del stability and trafficking that we observed in RNF5 KO and RNF185/RNF5 KO K562 cells mirrors the modest changes in stability, trafficking, and ion transport previously observed in RNF5 knockdowns and RNF185/RNF5 knockouts in patient-derived bronchial epithelial cells ( Tomati et al. , 2015 ; Kamada et al. , 2023 ).…”

“…Therefore, we cannot rule-out that the more modest phenotype was due to residual expression. However, a recent manuscript observed a similarly modest change in transgenic CFTR-F508del half-life in clonal RNF185/RNF5 KO bronchial epithelial cell lines ( Kamada et al. , 2023 ).…”

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

“…∆F508-CFTR-HiBiT(CT), N1303K-CFTR-HiBiT(CT), Insig-1-HiBiT(CT), ∆Y490-ABCB1-HiBiT(CT), and ∆F508-CFTR-Nluc(Ex) were constructed previously [23][24][25]. pCold-GST-4xTR-TUBE (GST-TUBE) was constructed by in-fusion cloning (Takara Bio, Kusatsu, Japan) using the pCold-GST plasmid [25] and pRSET-4xGST-TR-TUBE (Addgene #110312) as templates.…”

“…All the cells used in this study were cultured in 5% CO 2 at 37 • C. 293MSR (Ther-moFisher Scientific, Cat# R79507) and 293MSR-RNF5/185 DKO [24] cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FBS). 293MSR or 293MSR-RNF5/185 DKO cells stably expressing HBH-∆F508-CFTR-3HA were grown in DMEM supplemented with 10% FBS, 0.5 mg/mL G418, and 5 µg/mL blasticidin S as previously [24].…”

“…In this study, we demonstrate that UBE3C promotes the ERAD of misfolded CFTR and ABCB1, even when both RNF185 and its functional ortholog RNF5 are absent. We employed a recently established HiBiT degradation assay [24], along with knockdown (KD) experiments, to illustrate that UBE3C KD delays the ERAD of misfolded ∆F508-CFTR, N1303K-CFTR, and ∆Y490-ABCB1. Importantly, these effects were also observed in cells in which both RNF5 and RNF185 had been ablated.…”