Methods for Monitoring Endoplasmic Reticulum Stress and the Unfolded Protein Response

Afshin Samali,Una Fitzgerald,Sanjeev Gupta,Shane Deegan

doi:10.1155/2010/830307

Abstract

The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

Highlights

The endoplasmic reticulum (ER) is the cellular site for Ca2+ storage and for synthesis, folding, and maturation of most secreted and transmembrane proteins
The ER luminal domain of PKRlike ER kinase (PERK), Inositol-requiring enzyme 1 (IRE1), and activated transcription factor 6 (ATF6) interacts with the ER chaperone GRP78; upon accumulation of unfolded proteins, GRP78 dissociates from these molecules, leading to their activation [3]
Instead we suggest that detection of downstream protein targets of ER stress such as CAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), HERP, X box-binding protein 1 (XBP1), GRP78, and Activating transcription factor 4 ATF6 (ATF4) be a more robust approach for detecting activation of the Unfolded Protein Response (UPR)

Summary

Introduction

The endoplasmic reticulum (ER) is the cellular site for Ca2+ storage and for synthesis, folding, and maturation of most secreted and transmembrane proteins. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR) [1] This concerted and complex cellular response is mediated initially by three molecules, PKRlike ER kinase (PERK), activated transcription factor 6 (ATF6), and Inositol-requiring enzyme 1 (IRE1) [2]. Further ER luminal domain of ATF6 is disulfide bonded and ER stress-induced reduction plays important role in both translocation to Golgi body and subsequent recognition by the site-1 and site-2 proteases (S1P and S2P) [10] These differences may explain the different kinetics in the activation of IRE1, PERK, and ATF6 to various ER stress inducers. This is because some assays are inappropriate, problematic, or may not work at all in particular cells, tissues, or model systems

Experimental Approaches for the Detection of ER Stress

Concluding Remarks