Christian Appenzeller-Herzog

Bidirectional crosstalk between endoplasmic reticulum stress and mTOR signaling

Many cellular processes including apoptosis, autophagy, translation, energy metabolism, and inflammation are controlled by the mammalian target of rapamycin (mTOR) kinase and the endoplasmic reticulum (ER) stress pathway, also known as the unfolded protein response (UPR). Although both of these signaling nodes have attracted wide attention in fundamental cell biology and drug discovery, crosstalk between the two pathways has emerged only very recently. mTOR complex 1 (mTORC1) operates both upstream and downstream of ER stress signals, which can either enhance or antagonize the anabolic output of mTORC1. Upon prolonged ER stress, mTORC1 contributes to apoptotic signaling by suppressing the survival kinase Akt through feedback inhibition. Likewise, chronic ER stress obstructs activation of Akt by mTOR complex 2. This review surveys our knowledge of mTOR-ER stress intersections and highlights potential therapeutic implications.

A novel disulphide switch mechanism in Ero1α balances ER oxidation in human cells

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox‐driven shutdown mechanism of Ero1α, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1α in cells arises from the formation of a disulphide bond—identified by mass spectrometry—between the active‐site Cys94 (connected to Cys99 in the active enzyme) and Cys131. Competition between substrate thiols and Cys131 creates a feedback loop where activation of Ero1α is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1α‐Cys131Ala or the isoform Ero1β, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.

Glutathione- and non-glutathione-based oxidant control in the endoplasmic reticulum

The redox-active tripeptide glutathione is an endogenous reducing agent that is found in abundance and throughout the cell. In the endoplasmic reticulum (ER), the ratio of glutathione to glutathione disulfide is lower compared with non-secretory organelles. This relatively oxidizing thiol-disulfide milieu is essential for the oxidative folding of nascent proteins in the ER and, at least in part, maintained by the activity of ER-resident endoplasmic oxidoreductin 1 (Ero1) enzymes that oxidize cysteine side chains at the expense of molecular oxygen. Glutathione disulfide and hydrogen peroxide formed as a consequence of Ero1 activity are widely considered as being inoperative and potentially dangerous by-products of oxidative protein folding in the ER. In contrast to this common view, this Commentary highlights the importance of glutathione- and non glutathione-based homeostatic redox control mechanisms in the ER. Stability in the thiol–disulfide system that prominently includes the protein disulfide isomerases is ensured by the contribution of tightly regulated Ero1 activity, ER-resident peroxidases and the glutathione–glutathione-disulfide redox pair that acts as a potent housekeeper of redox balance. Accordingly, the widely held concept that Ero1-mediated over-oxidation in the ER constitutes a common cause of cellular demise is critically re-evaluated.

Disulphide production by Ero1α–PDI relay is rapid and effectively regulated

The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cells are incompletely understood. Here, we show that after reductive challenge the ER steady‐state disulphide content is restored on a time scale of seconds. Both the oxidase Ero1α and the oxidoreductase protein disulphide isomerase (PDI) strongly contribute to the rapid recovery kinetics, but experiments in ERO1‐deficient cells indicate the existence of parallel pathways for disulphide generation. We find PDI to be the main substrate of Ero1α, and mixed‐disulphide complexes of Ero1 primarily form with PDI, to a lesser extent with the PDI‐family members ERp57 and ERp72, but are not detectable with another homologue TMX3. We also show for the first time that the oxidation level of PDIs and glutathione is precisely regulated. Apparently, this is achieved neither through ER import of thiols nor by transport of disulphides to the Golgi apparatus. Instead, our data suggest that a dynamic equilibrium between Ero1‐ and glutathione disulphide‐mediated oxidation of PDIs constitutes an important element of ER redox homeostasis.

pH-induced Conversion of the Transport Lectin ERGIC-53 Triggers Glycoprotein Release

The recycling mannose lectin ERGIC-53 operates as a transport receptor by mediating efficient endoplasmic reticulum (ER) export of some secretory glycoproteins. Binding of cargo to ERGIC-53 in the ER requires Ca2+. Cargo release occurs in the ERGIC, but the molecular mechanism is unknown. Here we report efficient binding of purified ERGIC-53 to immobilized mannose at pH 7.4, the pH of the ER, but not at slightly lower pH. pH sensitivity of the lectin was more prominent when Ca2+ concentrations were low. A conserved histidine in the center of the carbohydrate recognition domain was required for lectin activity suggesting it may serve as a molecular pH/Ca2+ sensor. Acidification of cells inhibited the association of ERGIC-53 with the known cargo cathepsin Z-related protein and dissociation of this glycoprotein in the ERGIC was impaired by organelle neutralization that did not impair the transport of a control protein. The results elucidate the molecular mechanism underlying reversible lectin/cargo interaction and establish the ERGIC as the earliest low pH site of the secretory pathway.

Endoplasmic reticulum : reduced and oxidized glutathione revisited

Summary The reducing power of glutathione, expressed by its reduction potential EGSH, is an accepted measure for redox conditions in a given cell compartment. In the endoplasmic reticulum (ER), EGSH is less reducing than elsewhere in the cell. However, attempts to determine EGSH(ER) have been inconsistent and based on ineligible assumptions. Using a codon-optimized and evidently glutathione-specific glutaredoxin-coupled redox-sensitive green fluorescent protein (roGFP) variant, we determined EGSH(ER) in HeLa cells as −208±4 mV (at pH 7.0). At variance with existing models, this is not oxidizing enough to maintain the known redox state of protein disulfide isomerase family enzymes. Live-cell microscopy confirmed ER hypo-oxidation upon inhibition of ER Ca2+ import. Conversely, stressing the ER with a glycosylation inhibitor did not lead to more reducing conditions, as reported for yeast. These results, which for the first time establish the oxidative capacity of glutathione in the ER, illustrate a context-dependent interplay between ER stress and EGSH(ER). The reported development of ER-localized EGSH sensors will enable more targeted in vivo redox analyses in ER-related disorders.

GPx8 peroxidase prevents leakage of H2O2 from the endoplasmic reticulum.

Unbalanced endoplasmic reticulum (ER) homeostasis (ER stress) leads to increased generation of reactive oxygen species (ROS). Disulfide-bond formation in the ER by Ero1 family oxidases produces hydrogen peroxide (H2O2) and thereby constitutes one potential source of ER-stress-induced ROS. However, we demonstrate that Ero1α-derived H2O2 is rapidly cleared by glutathione peroxidase (GPx) 8. In 293 cells, GPx8 and reduced/activated forms of Ero1α co-reside in the rough ER subdomain. Loss of GPx8 causes ER stress, leakage of Ero1α-derived H2O2 to the cytosol, and cell death. In contrast, peroxiredoxin (Prx) IV, another H2O2-detoxifying rough ER enzyme, does not protect from Ero1α-mediated toxicity, as is currently proposed. Only when Ero1α-catalyzed H2O2 production is artificially maximized can PrxIV participate in its reduction. We conclude that the peroxidase activity of the described Ero1α-GPx8 complex prevents diffusion of Ero1α-derived H2O2 within and out of the rough ER. Along with the induction of GPX8 in ER-stressed cells, these findings question a ubiquitous role of Ero1α as a producer of cytoplasmic ROS under ER stress.

Redox controls UPR to control redox

ABSTRACT In many physiological contexts, intracellular reduction–oxidation (redox) conditions and the unfolded protein response (UPR) are important for the control of cell life and death decisions. UPR is triggered by the disruption of endoplasmic reticulum (ER) homeostasis, also known as ER stress. Depending on the duration and severity of the disruption, this leads to cell adaptation or demise. In this Commentary, we review reductive and oxidative activation mechanisms of the UPR, which include direct interactions of dedicated protein disulfide isomerases with ER stress sensors, protein S-nitrosylation and ER Ca2+ efflux that is promoted by reactive oxygen species. Furthermore, we discuss how cellular oxidant and antioxidant capacities are extensively remodeled downstream of UPR signals. Aside from activation of NADPH oxidases, mitogen-activated protein kinases and transcriptional antioxidant responses, such remodeling prominently relies on ER–mitochondrial crosstalk. Specific redox cues therefore operate both as triggers and effectors of ER stress, thus enabling amplification loops. We propose that redox-based amplification loops critically contribute to the switch from adaptive to fatal UPR.

Suppression of the Nrf2-Dependent Antioxidant Response by Glucocorticoids and 11β-HSD1-Mediated Glucocorticoid Activation in Hepatic Cells

Background Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key transcription factor regulating a plethora of detoxifying enzymes and antioxidant genes involved in drug metabolism and defence against oxidative stress. The glucocorticoid receptor (GR) is a ligand-induced transcription factor involved in the regulation of energy supply for metabolic needs to cope with various stressors. GR activity is controlled by glucocorticoids, which are synthesized in the adrenal glands and regenerated mainly in the liver from inactive cortisone by 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1). Methods and Principal Findings Using transfected HEK-293 cells and hepatic H4IIE cells we show that glucocorticoids, activated by 11β-HSD1 and acting through GR, suppress the Nrf2-dependent antioxidant response. The expression of the marker genes NQO1, HMOX1 and GST2A was suppressed upon treatment of 11β-HSD1 expressing cells with cortisone, an effect that was reversed by 11β-HSD1 inhibitors. Furthermore, our results demonstrate that elevated glucocorticoids lowered the ability of cells to detoxify H2O2. Moreover, a comparison of gene expression in male and female rats revealed an opposite sexual dimorphism with an inverse relationship between 11β-HSD1 and Nrf2 target gene expression. Conclusions The results demonstrate a suppression of the cellular antioxidant defence capacity by glucocorticoids and suggest that elevated 11β-HSD1 activity may lead to impaired Nrf2-dependent antioxidant response. The gender-specific differences in hepatic expression levels of 11β-HSD1 and Nrf2 target genes and the impact of pharmacological inhibition of 11β-HSD1 on improving cellular capacity to cope with oxidative stress warrants further studies in vivo.

A luminal flavoprotein in endoplasmic reticulum-associated degradation

The quality control system of the endoplasmic reticulum (ER) discriminates between native and nonnative proteins. The latter are degraded by the ER-associated degradation (ERAD) pathway. Whereas many cytosolic and membrane components of this system are known, only few luminal players have been identified. In this study, we characterize ERFAD (ER flavoprotein associated with degradation), an ER luminal flavoprotein that functions in ERAD. Upon knockdown of ERFAD, the degradation of the ERAD model substrate ribophorin 332 is delayed, and the overall level of polyubiquitinated cellular proteins is decreased. We also identify the ERAD components SEL1L, OS-9 and ERdj5, a known reductase of ERAD substrates, as interaction partners of ERFAD. Our data show that ERFAD facilitates the dislocation of certain ERAD substrates to the cytosol, and we discuss the findings in relation to a potential redox function of the protein.