ORCID
- Esen Efeoglu: 0000-0002-3380-0499
Abstract
Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER-stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC-MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO-DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N-glycan biosynthesis, and ubiquitin-mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER-stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.
Keywords
Chinese hamster ovary (CHO) cells, K-ε-GG peptides, LC-MS/MS analysis, endoplasmic reticulum stress, proteasome inhibition, ubiquitination
DOI Link
Publication Date
2024-11-01
Publication Title
Biotechnology Journal
Volume
19
Issue
11
ISSN
1860-6768
Deposit Date
2025-07-18
Funding
This research was funded by a Science Foundation Ireland Frontiers for the Future Award (grant no. 19/FFP/6759). The Orbitrap Fusion Tribrid mass spectrometer was funded by a Science Foundation Ireland Infrastructure Award to Dublin City University (SFI 16/RI/3701). Graphical abstract figure was created with BioRender.com.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Additional Links
Recommended Citation
Selvaprakash, Karuppuchamy; Sideri, Christiana‐Kondylo; Henry, Michael; Efeoglu, Esen; Ryan, David; and Meleady, Paula, "Characterization of the Ubiquitin‐Modified Proteome of Recombinant Chinese Hamster Ovary Cells in Response to Endoplasmic Reticulum Stress" (2024). Research Outputs: 2025-Present. 10.
https://arrow.tudublin.ie/schfsehro/10