Restriction lift date: 9999-12-31
Novel insights into the post-translational regulation of an ER localised ubiquitin conjugating enzyme
Loading...
Files
Date
2025
Authors
Algoufi, Noor Daifallah
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
Published Version
Abstract
Endoplasmic reticulum associated degradation (ERAD) of misfolded membrane proteins is critical for maintenance of cellular homeostasis. 'ER stress', caused by overload of misfolded proteins as commonly occurs in many physiological and disease states, activates the Unfolded Protein Response (UPR). This promotes restoration of homeostasis but also triggers apoptosis and other physiological responses. The ubiquitin conjugating enzyme Ube2J1 is a central component of the ERAD pathway. It is phosphorylated at serine 184 (S184) in response to p38 MAPKinase signalling, but apart from increasing its association with the E3 ligase cIAP1, the functional implications of this modification are not yet clear. Emerging evidence of Ube2J1 importance in cancer and virology show it as a key player in different pathways. We wondered whether additional, as yet undescribed modifications, might impact on its’ regulation and function.
Initially, this study investigated the regulation of Ube2J1 under various cellular signalling conditions, including those that can influence both the UPR and autophagy. Co-expression experiments in HEK293T cells revealed that Ube2J1 is modulated by multiple pathways including MAP kinases JNK and p38α, and also the ULK1 kinase that regulates autophagy. Notably, while JNK and ULK1 signalling both resulted in decreased Ube2J1 levels, ULK1 was linked to changes in electrophoretic mobility on SDS-PAGE, suggesting post-translational modifications. Equally, p38α and B-Raf signalling led to increased Ube2J1 levels, with modifications at the S184 phosphorylation site. Interestingly, a phospho-deficient S184A mutant still exhibited altered mobility, indicating the potential for additional regulatory modifications. These findings highlight the complex regulation of Ube2J1, suggesting its involvement in cellular processes beyond ERAD and autophagy.
Despite predictions from bioinformatics tools suggesting additional phosphorylation sites, previously only S184 had been experimentally validated. Through mutagenesis, we investigated the potential of S266 as a phosphorylation target but found no significant effects on the protein's electrophoretic mobility. Instead, we purified an S184-deficient form of Ube2J1 co-expressed with B-Raf-V600E and conducted phosphoproteomic analysis via mass spectrometry, identifying seven phosphorylation sites, including S266.
Finally, there is emerging evidence linking Ube2J1 to viral infections with elevated levels generally associated with increased infection rates. This is particularly the case for RNA viruses, where it may influence the expression of antiviral interferons. In this study, we utilized a BHK21 cell model to investigate the effects of Ube2J1 on Vesicular Stomatitis Virus (VSV) replication. Our findings demonstrate that overexpression of Ube2J1 significantly enhances viral titres, as evidenced by increased syncytial fusion areas when cells were co-transfected with the VSV-G protein and wild-type Ube2J1. This effect was abolished when Ube2J1 was replaced with catalytically inactive (C91S) or S184A phosphodeficient variants. Further investigations confirmed a role for the protein in augmenting VSV viral strength and correlated with an increase in the size and number of infection plaques. Given the growing evidence of Ube2J1's involvement in immune response and cancer signalling, our results provide valuable insights into its function in promoting syncytia formation, with implications for oncolytic virotherapy and viral pathogenesis.
This research not only expands our understanding of Ube2J1's function and involvement in a broad range of cellular processes, but it also raises the possibility that novel phosphorylation sites will allow for differential regulation of the enzyme. Our study will provide new avenues for studying the protein and exploring therapeutic targets in associated diseases.
Description
Keywords
ER stress
Citation
Algoufi, N. D. 2025. Novel insights into the post-translational regulation of an ER localised ubiquitin conjugating enzyme. PhD Thesis, University College Cork.
Link to publisher’s version
Copyright
© 2025, Noor Algoufi.