Real-Time Redox Measurements during Endoplasmic Reticulum Stress Reveal Interlinked Protein Folding Functions

Abstract

SUMMARY Disruption of protein folding in the endoplasmic reticulum (ER) causes unfolded proteins to accumulate, triggering the unfolded protein response (UPR). UPR outputs in turn decrease ER unfolded proteins to close a negative feedback loop. However, because it is infeasible to directly measure the concentration of unfolded proteins in vivo, cells are generically described as experiencing “ER stress” whenever the UPR is active. Because ER redox potential is optimized for oxidative protein folding, we reasoned that measureable redox changes should accompany unfolded protein accumulation. To test this concept, we employed fluorescent protein reporters to dynamically measure ER redox status and UPR activity in single cells. Using these tools, we show that diverse stressors, both experimental and physiological, compromise ER protein oxidation when UPR-imposed homeostatic control is lost. Using genetic analysis we uncovered redox heterogeneities in isogenic cell populations, and revealed functional interlinks between ER protein folding, modification, and quality control systems.

Autophagy deficiency in beta cells leads to compromised unfolded protein response and progression from obesity to diabetes in mice

“…Our data show that autophagy is necessary for UPR constitutively and also in response to ER stress in pancreatic beta cells. Probably because of insufficient UPR, autophagy-deficient beta cells were found to be susceptible to ER stressors in vitro and in vivo, which is consistent with previous reports suggesting that UPR is not only a marker of, but also an adaptation to, ER stress, and that insufficient UPR in the presence of ER stress renders cells prone to cell death [20]. Our data from Atg7 Δβ-cell mice are different from previous studies showing increased UPR in autophagy-deficient tissue [27,28], which may be attributable to peculiarities of pancreatic beta cells with respect to ER stress [29].…”

“…Susceptibility of autophagy-deficient beta cells to lipid injury in vitro Considering a previous paper showing that appropriate UPR is important for survival in the presence of ER stress [20], compromised UPR expression in Atg7-deficient beta cells may impair their adaptation to ER stress. To address this issue, we treated primary islet cells from Fig.…”

Firefly luciferase mutants as sensors of proteome stress

“…Therefore, HSF1 activity cannot be used to infer the changes in cellular proteostasis under these conditions. Protein misfolding in the ER results in an activation of the unfolded protein response (Walter and Ron, 2011) which can be semi-quantitatively measured by reporter based assays (Merksamer et al, 2008). The activation of heat shock response and unfolded protein response indicate that a response has been initiated but does not report whether homeostasis is restored.…”

Approaches to imaging unfolded secretory protein stress in living cells

“…Interestingly, FLIM successfully reported changes in the ER redox induced by DTT and thapsigargin, but not by other drugs that induce gross secretory protein misfolding, such as Tm [86]. The latter result contrasts with a previous study in yeast showing that both DTT and Tm induced changes in ER redox poise [61]. Changes in eroGFP signal in yeast appear to reflect accumulation of the ER-targeted reporter to the cytoplasm during prolonged stress as a result of poor secretory protein translocation [12].…”

“…Several fluorescent reporters have been developed to enable quantification of the transcriptional activity of the UPR following induction of misfolded protein stress in the ER. In yeast, these reporters consist of promoters containing UPR elements (UPRE) fused to FPs such as GFP or mCherry [61] ( Figure 2 ). Alternatively, FPs containing an appropriate ER retrieval motif (HDEL) can be chromosomally inserted to tag UPR targets, such as Kar2 [62, 63].…”