HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.

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, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.

Identification of α-Tocopherol succinate as an RFFL-substrate interaction inhibitor inducing peripheral CFTR stabilization and apoptosis.

The E3 ubiquitin ligase RFFL is an apoptotic inhibitor highly expressed in cancers and its knockdown suppresses cancer cell growth and sensitizes to chemotherapy. RFFL also participates in peripheral protein quality control which removes the functional cell surface ΔF508-CFTR channel and reduces the efficacy of pharmaceutical therapy for cystic fibrosis (CF). Although RFFL inhibitors have therapeutic potential for both cancer and CF, they remain undiscovered. Here, a chemical array screening has identified α-tocopherol succinate (αTOS) as an RFFL ligand. NMR analysis revealed that αTOS directly binds to RFFL's substrate-binding region without affecting the E3 enzymatic activity. Consequently, αTOS inhibits the RFFL-substrate interaction, ΔF508-CFTR ubiquitination and elimination from the plasma membrane of epithelial cells, resulting in the increased functional CFTR channel. Among the α-tocopherol (αTOL) analogs we tested, only αTOS inhibited the RFFL-substrate interaction and increased the cell surface ΔF508-CFTR, depending on RFFL expression. Similarly, the unique proapoptotic effect of αTOS was dependent on RFFL expression. Thus, unlike other αTOL analogs, αTOS acts as an RFFL protein-protein interaction inhibitor which may explain its unique biological properties among αTOL analogs. Moreover, αTOS may act as a CFTR stabilizer, a novel class of drugs that extend cell surface ΔF508-CFTR lifetime.