# Proteasome Inhibition Amplifies Endoplasmic Reticulum (ER) Stress Responses: Comparative Proteomics of Chinese Hamster Ovary Cell Lines

## TL;DR

This study explores how proteasome inhibition affects ER stress responses in different Chinese hamster ovary cell lines used for drug production.

## Contribution

The study reveals cell line-specific proteomic responses to proteasome and glycosylation inhibition, offering new insights into ER stress and protein quality control.

## Key findings

- Proteasome inhibition with MG132 intensified ER stress and altered ERAD in CHO cells.
- Combined treatment with tunicamycin and MG132 caused cell line-specific proteomic changes.
- CHO-K1 showed broad proteome shifts, while NISTCHO and CHO DP-12 upregulated specific stress-related proteins.

## Abstract

Chinese hamster ovary (CHO) cells are widely utilised in the biopharmaceutical industry to produce therapeutic proteins. Understanding the mechanisms of endoplasmic reticulum (ER) stress and its interplay with protein degradation pathways remains pivotal for improving production efficiency and product quality. In this study, we investigated the proteomic responses of CHO-K1 (non-producer), CHO DP-12 (IgG-producer), and NISTCHO (IgG-producer) cell lines under ER stress induced by a combination of the proteasome inhibitor MG132 and the glycosylation inhibitor tunicamycin. Viability, cell growth, and IgG titre were measured after 24 h, 48 h, and 72 h of treatment and the 48 h timepoint was used for the comparative analysis of the proteomic data across the three cell lines. Proteasome inhibition with MG132 intensified ER stress and altered ER-associated protein degradation (ERAD). Combined tunicamycin + MG132 treatment was associated with cell line-specific proteomic changes: NISTCHO upregulated ER translocation and glycoprotein quality control proteins (SSR4, SEC24C, UGGT1), CHO DP-12 activated redox/disulfide regulators (DNAJC10, CAPN1), while CHO-K1 showed broad proteome shifts, suggesting differences in baseline stress handling. These findings provide mechanistic insights into ER stress and protein quality control in CHO cells, offering a foundation for strategies to enhance cell line robustness and optimise biopharmaceutical production.