Biology subjects

Okiyoneda, T.

Publications and source records attributed to Okiyoneda, T..

3 recordsLinked to original sources

bioRxiv2025

AMFR provides an ERAD bypass mechanism to maintain proteostasis under canonical E3 ligase deficiency

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.

cell biology↗

bioRxiv2024

Ca2+-driven PDIA6 phase separation to ensure proinsulin quality control

The endoplasmic reticulum (ER) plays key roles in protein quality control1,2 and dynamic Ca2+ storage3,4 in eukaryotic cells. However, the protein homeostasis (proteostasis) system that regulates these ER functions is still incompletely characterised. Previous study revealed the importance of oligomerization in the function PDIA1, an ER-resident disulfide isomerase and molecular chaperone, regulates oligomeric states in accordance with client folding5. This result suggests that at least some of the 20 members of other PDI family may undergo regulated self-assembly in order to optimally function. Here, we show that Ca2+ triggers the phase separation of PDIA6 into liquid-like condensates. In contrast to the condensation mechanism observed for proteins containing low-complexity domains, our results indicate that transient but specific electrostatic interactions occur between the first and the third folded thioredoxin-like domains of PDIA6. We further show that the Ca2+-driven condensation of PDIA6 recruits PDIA3 and proinsulin, thus increasing their local concentrations. This process results in the 30-fold enhancement of proinsulin folding and in the inhibition of proinsulin aggregation. Our findings shed light on a condensation-driven Ca2+-mediated proteostasis cascade in the ER by revealing how the efficiency of the protein folding process can be enhanced within quality control granules.

biochemistry↗

bioRxiv2023

HERC3 E3 ligase provides an ERAD branch eliminating select membrane proteins

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.

cell biology↗