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Abstract
Accumulation of unfolded, misfolded and aggregated proteins in the endoplasmic reticulum (ER) causes ER stress. ER stress can result from physiological situations such as acute increases in secretory protein biosynthesis or pathological conditions that perturb ER homeostasis such as alterations in the ER redox state. Here we monitored ER redox together with transcriptional output of the Unfolded Protein Response (UPR) in INS-1 insulinoma cells stably expressing eroGFP (ER-redox-sensor) and mCherry protein driven by a GRP78 promoter (UPR-sensor). Live cell imaging, flow cytometry and biochemical characterization were used to examine these parameters in response to various conditions known to induce ER stress. As expected, treatment of the cells with the reducing agent dithiothreitol caused a decrease in the oxidation state of the ER accompanied by an increase in XBP-1 splicing. Unexpectedly however, other treatments including tunicamycin, thapsigargin, DL-homocysteine, elevated free fatty acids or high glucose had essentially no influence on the ER redox state, despite inducing ER stress. Comparable results were obtained with dispersed rat islet cells expressing eroGFP. Thus, unlike in yeast cells, ER stress in pancreatic β-cells is not associated with a more reducing ER environment.
Highlights
Biosynthesis, folding and maturation of membrane and secretory proteins occur in the endoplasmic reticulum (ER)
Cells respond to ER stress by activating the Unfolded Protein Response (UPR) that transiently inhibits translation to reduce the amount of new protein synthesis and enhances folding capacity by increasing ER chaperones and ER-associated degradation (ERAD) [4,5,6]
The UPR is activated by the coordinate action of three ER stress sensors that reside in the ER membrane, RNA-dependent protein kinase-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE-1), and activating transcription factor 6 (ATF6) [5,6,7]
Summary
Biosynthesis, folding and maturation of membrane and secretory proteins occur in the ER. Reducing agents disrupt the oxidizing ER environment critical for disulfide bond formation, which leads to accumulation of unfolded and misfolded proteins causing ER stress. Cells respond to ER stress by activating the Unfolded Protein Response (UPR) that transiently inhibits translation to reduce the amount of new protein synthesis and enhances folding capacity by increasing ER chaperones and ER-associated degradation (ERAD) [4,5,6]. Because quantification of misfolded or unfolded proteins directly is not possible ER stress is typically monitored by evaluating whether the UPR sensors are activated or by examining the levels of UPR response genes such as chaperone proteins.
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