The endoplasmic reticulum (ER) is an important organelle responsible for protein folding and transport. Protein folding stress in the ER can lead to the accumulation of unfolded or misfolded proteins, known as "ER stress", which activates the unfolded protein response (UPR). However, UPR can also be activated in the absence of ER stress. Deciphering the complex relationship between ER stress and UPR is crucial for understanding cellular stress responses in physiological and pathological conditions.

On June 11, 2024, a team led by Prof. WANG Likun from the Institute of Biophysics, Chinese Academy of Sciences, published a research paper in Cell Reports. The study developed a novel fluorescent reporting system that allows researchers to visualize the accumulation of unfolded proteins within the ER lumen in living cells in real-time. Through this system, researchers can better understand the intricate relationship between ER stress and UPR, revealing the dynamic changes in ER protein homeostasis under various physiological and pathological conditions.

The researchers fused the ER luminal domain of the ER stress signaling protein PERK (PERK(LD)) with green fluorescent protein (EGFP) and a peptide segment that tends to form hexamers (HOTag) to create a fluorescent reporting system. After binding with unfolded proteins, this system rapidly and reversibly forms fluorescent spots that can be visualized with confocal microscopy. This system exhibited high sensitivity and reversibility in HeLa and COS-7 cells, providing real-time insights into the dynamic changes of ER stress and UPR.

Using various techniques, including co-localization experiments and immunoprecipitation, the researchers validated the direct binding between the fluorescent reporting system and unfolded proteins. They demonstrated that the system could quantitatively detect the accumulation of unfolded proteins in the ER and displayed a dose-response relationship under different concentrations.

To further elucidate the relationship between ER stress and UPR, the researchers studied the differential UPR responses to the anti-cancer drug Bortezomib in different cell types, revealing the complexity of how Bortezomib regulates UPR and unfolded protein accumulation through different mechanisms in various cell types.

This study unveils the complex interactions between ER stress and UPR, providing a powerful tool for understanding how these processes operate in health and disease. This discovery offers a new perspective for developing novel therapeutic strategies, particularly in diseases involving ER stress, such as neurodegenerative diseases, diabetes, and cancer.

Fig. Researchers have developed a fluorescent reporting system to observe the accumulation of unfolded proteins within the endoplasmic reticulum (ER) of live cells. After binding with unfolded proteins, this system rapidly and reversibly forms fluorescent spots, allowing real-time visualization of the accumulation of unfolded proteins within the ER lumen, aiding in a better understanding of how ER stress is related to the unfolded protein response (UPR).

(Image by WANG Likun's group)

Article link: https://doi.org/10.1016/j.celrep.2024.114358

Contact: WANG Likun

Institute of Biophysics, Chinese Academy of Sciences

Beijing 100101, China

E-mail: wanglikun@ibp.ac.cn

（Reported by Prof. WANG Likun's group）