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Measuring signaling by the unfolded protein response
David J Cox1, Natalie Strudwick, Ahmed A Ali
1School of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom.
Methods in Enzymology
|February 19, 2011
Summary
The unfolded protein response (UPR) is crucial for understanding diseases and drug actions. This study details methods to experimentally activate and monitor the three UPR signaling pathways in yeast and mammalian cells.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The unfolded protein response (UPR) is a cellular stress response triggered by misfolded proteins in the endoplasmic reticulum (ER).
- UPR activation is implicated in various diseases, including cancer, metabolic disorders (type II diabetes, fatty liver), and neurodegenerative conditions (Alzheimer's disease).
- The UPR also responds to toxic chemicals and influences drug efficacy, highlighting its significance in toxicology and pharmacology.
Purpose of the Study:
- To provide a comprehensive overview of experimental methods for activating the UPR.
- To detail reliable assays for monitoring the activation status of the three main UPR signaling pathways (IRE1, PERK, ATF6).
- To equip researchers, especially newcomers, with the necessary protocols to assess UPR activation in yeast and mammalian cells.
Main Methods:
- Experimental activation of UPR in *Saccharomyces cerevisiae* and cultured mammalian cells.
- Monitoring IRE1 pathway activation via HAC1 mRNA splicing (yeast) and XBP1 processing (mammalian cells) using Northern blotting and RT-PCR.
- Assessing PERK pathway activation through eIF2α phosphorylation, CHOP induction (mRNA/protein), and ATF4 protein levels.
- Evaluating ATF6 pathway activation by detecting its processed 50 kDa fragment via Western blotting.
- Utilizing RT-PCR for UPR target gene expression and histological assays for tissue sections.
Main Results:
- Detailed protocols are presented for key assays measuring UPR activation across its three branches.
- Methods cover both yeast and mammalian systems, allowing for versatile application.
- The described repertoire of methods enables comprehensive assessment of UPR activation status.
Conclusions:
- This study provides a valuable resource of standardized methods for UPR research.
- The presented protocols facilitate the investigation of UPR's role in disease pathogenesis and drug response.
- The comprehensive approach empowers researchers to effectively study the unfolded protein response.
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