Tsukasa Okiyoneda

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR), whose folding, trafficking, and stability are tightly controlled by ubiquitination-dependent protein quality control (PQC) pathways. Although CFTR modulators have transformed CF therapy, their efficacy remains limited by persistent ubiquitination and degradation of rescued CFTR. This limitation is particularly evident in class I mutations, where premature termination codons (PTCs) reduce full-length CFTR protein production and no approved mutation-specific therapies are broadly available for canonical PTC variants. Recent advances highlight ubiquitination as a critical and druggable determinant of CFTR stability. The E3 ligase RFFL regulates peripheral CFTR PQC, restricting the stability of rescued CFTR at the plasma membrane (PM). Inhibition of RFFL, including via antisense oligonucleotides (ASO) and small molecules, enhances CFTR rescue and improves outcomes in combination with modulators and translational readthrough therapies. In parallel, deubiquitinase (DUB)-targeting chimeras (DUBTACs) have emerged as a novel modality to stabilize proteins by reversing ubiquitination. Here, we review current advances in targeting CFTR ubiquitination, with a focus on RFFL inhibition and DUBTAC-based strategies, and discuss their opportunities and translational limitations as components of next-generation CF therapies.

NRF1 is a key mediator of the proteasome recovery pathway, yet its regulation by ER-resident factors is not fully elucidated. Here, we demonstrate that selenoproteins SELS and SELK are critical regulators for NRF1 protein dynamics. SELS stabilizes NRF1, while SELK induces its insolubilization. Their deficiency leads to a hyper-accumulation and increased nuclear localization of NRF1 under proteasome inhibition condition. This results in an augmented transcriptional response of proteasome subunits. These results indicate that SELS and SELK cooperatively gate NRF1 activity by controlling its retrotranslocation and solubility, highlighting a novel layer of selenoprotein-mediated quality control in the proteostasis network.

Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the CFTR gene, which encodes a cAMP-regulated anion channel at the apical plasma membrane (PM) of epithelial cells. CFTR modulators have recently been approved as effective therapies for folding-defective mutations, including the most common variant, F508del. However, no clinically effective treatments are available for nonsense mutations such as G542X, the second most frequent CF-causing mutation. Translational readthrough-inducing drugs (TRIDs), such as G418, can suppress premature termination codons (PTCs) and partially restore full-length CFTR expression, but their therapeutic efficacy remains limited. Notably, combining TRIDs with CFTR modulators enhances functional rescue, suggesting that the restored full-length CFTR may be targeted by protein quality control (QC) pathways. Here, we investigated the QC mechanisms responsible for degrading TRID-induced full-length CFTR proteins harboring nonsense mutations. We identified the E3 ubiquitin ligases RNF5, RNF185, and RFFL as key regulators of CFTR turnover. Among these, RFFL played a particularly critical role in peripheral QC, targeting TRID-induced full-length CFTR for ubiquitination and degradation. Knockdown (KD) of RFFL markedly reduced CFTR ubiquitination, stabilized mature CFTR at the PM, and significantly enhanced functional rescue when TRIDs were combined with CFTR modulators. Enhanced CFTR channel activity confirmed that the stabilized proteins were functional. These findings indicate that RFFL-mediated degradation restricts the therapeutic benefit of TRID-based approaches. Targeting RFFL therefore represents a promising strategy to boost the efficacy of combination therapies involving TRIDs and CFTR modulators, offering new opportunities for the treatment of CF patients carrying nonsense CFTR mutations.

Abnormal proteins in the endoplasmic reticulum (ER) are eliminated via distinct ER-associated degradation (ERAD) pathways, each regulated by specific E3 ubiquitin ligases. While these pathways seem to work cooperatively to maintain ER proteostasis, their individual roles and potential compensatory mechanisms remain poorly defined in mammalian cells. In this study, we utilized multiple E3 ligase knockouts/knockdowns combined with a highly sensitive HiBiT-based ERAD assay to investigate pathway complementarity. We discovered that in the absence of RNF5/185 function, AMFR—a Hrd1 ortholog primarily involved in the ERAD-M branch—could partially compensate by facilitating degradation of mutant CFTR. In contrast, SYVN1, another Hrd1 ortholog, failed to show a similar effect. These findings reveal a novel bypass mechanism mediated by AMFR and demonstrate the functional flexibility of the ERAD network. Our results provide new insight into how E3 ligases maintain proteostasis under compromised conditions, advancing our understanding of ERAD pathway coordination in mammalian systems.

Protein ubiquitination extensively modulates protein functions and controls various biological processes, such as protein degradation, signal transduction, transcription, and DNA repair. Ubiquitination is a reversible post‐translational modification, and deubiquitinating enzymes cleave ubiquitin from proteins. Ubiquitin‐specific peptidase 46 (USP46), a deubiquitinase, is highly expressed in the brain and regulates neural functions. Deleting lysine 92 (ΔK92) in USP46 reduces murine depression‐like behavior in the tail suspension test. However, the molecular basis for USP46's role in regulating neural function has not yet been fully understood. Here we employed a proximity‐dependent biotinylation approach to characterize the USP46 protein interaction partners. Using homology‐independent targeted integration (HITI), a genome editing technique, we established knockin cell lines that stably express USP46 wildtype‐ or ΔK92‐biotin ligase fusion protein. We identified 286 candidate interaction partners, including well‐known binding partners of USP46. Although there were no obvious differences in the interactome of USP46 between wildtype and ΔK92, a gene ontology analysis revealed that centrosome‐related proteins were significantly enriched in the proximal proteins of USP46. Several centrosome‐related proteins were bound to USP46 in Neuro2a cells, but their protein expression levels were not affected in the brains of USP46‐deficient mice. These results uncover a potential relationship between USP46 and centrosome regulation independently of protein stabilization.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in CFTR gene expressing the anion selective channel CFTR located at the plasma membrane of different epithelial cells. The most commonly investigated variant causing CF is F508del. This mutation leads to structural defects in the CFTR protein, which are recognized by the endoplasmic reticulum (ER) quality control system. As a result, the protein is retained in the ER and degraded via the ubiquitin–proteasome pathway. Although blocking ubiquitination to stabilize the CFTR protein has long been considered a potential pharmacological approach in CF, progress in this area has been relatively slow. Currently, no compounds targeting this pathway have entered clinical trials for CF. On the other hand, the emergence of Orkambi initially, and notably the subsequent introduction of Trikafta/Kaftrio, have demonstrated the effectiveness of molecular chaperone-based therapies for patients carrying the F508del variant and even showed efficacy against other variants. These treatments directly target the CFTR variant protein without interfering with cell signaling pathways. This review discusses the limits and potential future of targeting protein ubiquitination in CF.