Christian Appenzeller-Herzog - Academia.edu (original) (raw)

Papers by Christian Appenzeller-Herzog

International journal of cell biology, 2013

Peroxidases are enzymes that reduce hydroperoxide substrates. In many cases, hydroperoxide reduct... more Peroxidases are enzymes that reduce hydroperoxide substrates. In many cases, hydroperoxide reduction is coupled to the formation of a disulfide bond, which is transferred onto specific acceptor molecules, the so-called reducing substrates. As such, peroxidases control the spatiotemporal distribution of diffusible second messengers such as hydrogen peroxide (H2O2) and generate new disulfides. Members of two families of peroxidases, peroxiredoxins (Prxs) and glutathione peroxidases (GPxs), reside in different subcellular compartments or are secreted from cells. This review discusses the properties and physiological roles of PrxIV, GPx7, and GPx8 in the endoplasmic reticulum (ER) of higher eukaryotic cells where H2O2 and-possibly-lipid hydroperoxides are regularly produced. Different peroxide sources and reducing substrates for ER peroxidases are critically evaluated. Peroxidase-catalyzed detoxification of hydroperoxides coupled to the productive use of disulfides, for instance, in the...

Journal of Cell Science, 2011

The redox-active tripeptide glutathione is an endogenous reducing agent that is found in abundanc... more 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.

Antioxidants & Redox Signaling, 2012

The oxidative process of disulfide-bond formation is essential for the folding of most secretory ... more The oxidative process of disulfide-bond formation is essential for the folding of most secretory and membrane proteins in the endoplasmic reticulum (ER). It is driven by electron relay pathways that transfer two electrons derived from the fusion of two adjacent cysteinyl side chains onto various types of chemical oxidants. The conserved, ER-resident endoplasmic oxidoreductin 1 (Ero1) sulfhydryl oxidases that reduce molecular oxygen to generate an active-site disulfide represent one of these pathways. In mammals, two family members exist, Ero1α and Ero1β. The two mammalian Ero1 enzymes differ in transcriptional and post-translational regulation, tissue distribution, and catalytic turnover. A specific protein-protein interaction between either isoform and protein disulfide isomerase (PDI) facilitates the propagation of disulfides from Ero1 via PDI to nascent polypeptides, and inbuilt oxidative shutdown mechanisms in Ero1α and Ero1β prevent excessive oxidation of PDI. Besides disulfide-bond generation, Ero1α also regulates calcium release from the ER and the secretion of disulfide-linked oligomers through its reversible association with the chaperone ERp44. This review explores the functional repertoire and possible redundancy of mammalian Ero1 enzymes. Systematic analyses of different knockout mouse models will be the most promising strategy to shed new light on unique and tissue-specific roles of Ero1α and Ero1β. Moreover, in-depth characterization of the known physical interactions of Ero1 with peroxidases and PDI family members will help broaden our functional and mechanistic understanding of Ero1 enzymes.

Antioxidants & Redox Signaling, 2012

Redox Biology, 2016

Endoplasmic reticulum (ER) oxidoreductin 1α (Ero1α) is a disulfide producer in the ER of mammalia... more Endoplasmic reticulum (ER) oxidoreductin 1α (Ero1α) is a disulfide producer in the ER of mammalian cells. Besides four catalytic cysteines (Cys(94), Cys(99), Cys(394), Cys(397)), Ero1α harbors four regulatory cysteines (Cys(104), Cys(131), Cys(208), Cys(241)). These cysteines mediate the formation of inhibitory intramolecular disulfide bonds, which adapt the activation state of the enzyme to the redox environment in the ER through feedback signaling. Accordingly, disulfide production by Ero1α is accelerated by reducing conditions, which minimize the formation of inhibitory disulfides, or by mutations of regulatory cysteines. Here we report that reductive stimulation enhances Ero1α activity more potently than the mutation of cysteines. Specifically, mutation of Cys(208)/Cys(241) does not mechanistically mimic reductive stimulation, as it lowers the turnover rate of Ero1α in presence of a reducing agent. The Cys(208)/Cys(241) pair therefore fulfills a function during catalysis that reaches beyond negative regulation. In agreement, we identify a reciprocal crosstalk between the stabilities of the Cys(208)-Cys(241) disulfide and the inhibitory disulfide bonds involving Cys(104) and Cys(131), which also controls the recruitment of the H2O2 scavenger GPx8 to Ero1α. Two possible mechanisms by which thiol-disulfide exchange at the Cys(208)/Cys(241) pair stimulates the catalytic turnover under reducing conditions are discussed.

Free radical biology & medicine, Jan 2, 2015

Cellular metabolism is inherently linked to the production of oxidizing by-products, including re... more Cellular metabolism is inherently linked to the production of oxidizing by-products, including reactive oxygen species (ROS) hydrogen peroxide (H2O2). When present in excess, H2O2 can damage cellular biomolecules, but when produced in coordinated fashion, it typically serves as a mobile signaling messenger. It is therefore not surprising that cell health critically relies on both low-molecular-weight and enzymatic antioxidant components, which protect from ROS-mediated damage and shape the propagation and duration of ROS signals. This review focuses on H2O2-antioxidant cross talk in the endoplasmic reticulum (ER), which is intimately linked to the process of oxidative protein folding. ER-resident or ER-regulated sources of H2O2 and other ROS, which are subgrouped into constitutive and stimulated sources, are discussed and set into context with the diverse antioxidant mechanisms in the organelle. These include two types of peroxide-reducing enzymes, a high concentration of glutathion...

International journal of cell biology, 2014

GBM Annual Spring meeting Mosbach 2006, 2006

The Journal of biological chemistry, Jan 26, 2004

The recycling mannose lectin ERGIC-53 operates as a transport receptor by mediating efficient end... more 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...

Free Radical Biology and Medicine, 2015

Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated d... more Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O2) and releases hydrogen peroxide (H2O2), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys(208)-Cys(241) disulfide in hyperactive Ero1α (Ero1α-C104A/C131A) potentiates H2O2 production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the flavin cofactor where O2 is reduced to H2O2. Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys(208)/Cys(241)-dependent mixed-disulfide complex with Ero1α. The H2O2-detoxifying glutathione peroxidase 8 also binds to the Cys(208)/Cys(241) loop region. Supported by O2 diffusion simulations, these data describe the first enzymatically controlled O2 access into a flavoprotein active site, provide molecular-level understanding of Ero1α regulation and H2O2 production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity.

Redox Biology, 2013

Glutathione is present in millimolar concentrations in the cell, but its relative distribution am... more Glutathione is present in millimolar concentrations in the cell, but its relative distribution among cellular compartments remains elusive. We have chosen the endoplasmic reticulum (ER) as an example organelle to study compartment-specific glutathione levels. Using a glutaredoxin sensor (sCGrx1pER), which rapidly and specifically equilibrates with the reduced glutathione (GSH)-glutathione disulfide (GSSG) redox couple with known equilibrium constant, we showed that the [GSH]:[GSSG] ratio in the ER of intact HeLa cells is less than 7:1. Taking into consideration the previously determined value for [GSH](2):[GSSG] in the ER of 83 mM, this translates into a total glutathione concentration in the ER ([GStot]=[GSH]+2[GSSG]) of greater than 15 mM. Since the integrated, intracellular [GStot] was measured as ~7 mM, we conclude the existence of a [GStot] gradient across the ER membrane. A possible homeostatic mechanism by which cytosol-derived glutathione is trapped in the ER is discussed. We propose a high [GStot] as a distinguishing feature of the ER environment compared to the extracellular space.

Molecular Biology of the Cell, 2005

Some secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture... more Some secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture mechanism, but the signals required for the cargo-receptor interaction are largely unknown. Here, we describe a novel targeting motif that is composed of a high-mannose type oligosaccharide intimately associated with a surface-exposed peptide beta-hairpin loop. The motif accounts for lectin ERGIC-53-assisted ER-export of the lyososomal enzyme procathepsin Z. The second oligosaccharide chain of procathepsin Z exhibits no binding activity for ERGIC-53, illustrating the selective lectin properties of ERGIC-53. Our data suggest that the conformation-based motif is only present in fully folded procathepsin Z and that its recognition by ERGIC-53 reflects a quality control mechanism that acts complementary to the primary folding machinery in the ER. A similar oligosaccharide/beta-hairpin loop structure is present in cathepsin C, another cargo of ERGIC-53, suggesting the general nature of this ER-exit signal. To our knowledge this is the first documentation of an ER-exit signal in soluble cargo in conjunction with its decoding by a transport receptor.

Trends in Cell Biology, 2012

Many cellular processes including apoptosis, autophagy, translation, energy metabolism, and infla... more 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.

Frontiers in Genetics, 2013

Pathological endoplasmic reticulum (ER) stress is tightly linked to the accumulation of reactive ... more Pathological endoplasmic reticulum (ER) stress is tightly linked to the accumulation of reactive oxidants, which can be both upstream and downstream of ER stress. Accordingly, detrimental intracellular stress signals are amplified through establishment of a vicious cycle. An increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. Experimental monitoring of stress-induced, time-resolved changes in ER reduction-oxidation (redox) states is therefore important. Organelle-specific examination of redox changes has been facilitated by the advent of genetically encoded, fluorescent probes, which can be targeted to different subcellular locations by means of specific amino acid extensions. These probes include redox-sensitive green fluorescent proteins (roGFPs) and the yellow fluorescent protein-based redox biosensor HyPer. In the case of roGFPs, variants with known specificity toward defined redox couples are now available. Here, we review the experimental framework to measure ER redox changes using ER-targeted fluorescent biosensors. Advantages and drawbacks of plate-reader and microscopy-based measurements are discussed, and the power of these techniques demonstrated in the context of selected cell culture models for ER stress.

Journal of Cell Science, 2014

In many physiological contexts, intracellular reduction-oxidation (redox) conditions and the unfo... more 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 Ca(2+) 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.

The EMBO Journal, 2008

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powe... more 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 Ero1a, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1a in cells arises from the formation of a disulphide bond-identified by mass spectrometry-between the active-site Cys 94 (connected to Cys 99 in the active enzyme) and Cys 131 . Competition between substrate thiols and Cys 131 creates a feedback loop where activation of Ero1a is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1a-Cys131Ala or the isoform Ero1b, 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.

The EMBO Journal, 2010

The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cell... more 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 Ero1a 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 Ero1a, 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 disulphidemediated oxidation of PDIs constitutes an important element of ER redox homeostasis.

Proceedings of the National Academy of Sciences, 2009

The quality control system of the endoplasmic reticulum (ER) discriminates between native and non... more 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.

PLoS ONE, 2012

Background: Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key transcription factor regu... more 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 11b-hydroxysteroid dehydrogenase-1 (11b-HSD1).

International journal of cell biology, 2013

Peroxidases are enzymes that reduce hydroperoxide substrates. In many cases, hydroperoxide reduct... more Peroxidases are enzymes that reduce hydroperoxide substrates. In many cases, hydroperoxide reduction is coupled to the formation of a disulfide bond, which is transferred onto specific acceptor molecules, the so-called reducing substrates. As such, peroxidases control the spatiotemporal distribution of diffusible second messengers such as hydrogen peroxide (H2O2) and generate new disulfides. Members of two families of peroxidases, peroxiredoxins (Prxs) and glutathione peroxidases (GPxs), reside in different subcellular compartments or are secreted from cells. This review discusses the properties and physiological roles of PrxIV, GPx7, and GPx8 in the endoplasmic reticulum (ER) of higher eukaryotic cells where H2O2 and-possibly-lipid hydroperoxides are regularly produced. Different peroxide sources and reducing substrates for ER peroxidases are critically evaluated. Peroxidase-catalyzed detoxification of hydroperoxides coupled to the productive use of disulfides, for instance, in the...

Journal of Cell Science, 2011

The redox-active tripeptide glutathione is an endogenous reducing agent that is found in abundanc... more 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.

Antioxidants & Redox Signaling, 2012

The oxidative process of disulfide-bond formation is essential for the folding of most secretory ... more The oxidative process of disulfide-bond formation is essential for the folding of most secretory and membrane proteins in the endoplasmic reticulum (ER). It is driven by electron relay pathways that transfer two electrons derived from the fusion of two adjacent cysteinyl side chains onto various types of chemical oxidants. The conserved, ER-resident endoplasmic oxidoreductin 1 (Ero1) sulfhydryl oxidases that reduce molecular oxygen to generate an active-site disulfide represent one of these pathways. In mammals, two family members exist, Ero1α and Ero1β. The two mammalian Ero1 enzymes differ in transcriptional and post-translational regulation, tissue distribution, and catalytic turnover. A specific protein-protein interaction between either isoform and protein disulfide isomerase (PDI) facilitates the propagation of disulfides from Ero1 via PDI to nascent polypeptides, and inbuilt oxidative shutdown mechanisms in Ero1α and Ero1β prevent excessive oxidation of PDI. Besides disulfide-bond generation, Ero1α also regulates calcium release from the ER and the secretion of disulfide-linked oligomers through its reversible association with the chaperone ERp44. This review explores the functional repertoire and possible redundancy of mammalian Ero1 enzymes. Systematic analyses of different knockout mouse models will be the most promising strategy to shed new light on unique and tissue-specific roles of Ero1α and Ero1β. Moreover, in-depth characterization of the known physical interactions of Ero1 with peroxidases and PDI family members will help broaden our functional and mechanistic understanding of Ero1 enzymes.

Antioxidants & Redox Signaling, 2012

Redox Biology, 2016

Endoplasmic reticulum (ER) oxidoreductin 1α (Ero1α) is a disulfide producer in the ER of mammalia... more Endoplasmic reticulum (ER) oxidoreductin 1α (Ero1α) is a disulfide producer in the ER of mammalian cells. Besides four catalytic cysteines (Cys(94), Cys(99), Cys(394), Cys(397)), Ero1α harbors four regulatory cysteines (Cys(104), Cys(131), Cys(208), Cys(241)). These cysteines mediate the formation of inhibitory intramolecular disulfide bonds, which adapt the activation state of the enzyme to the redox environment in the ER through feedback signaling. Accordingly, disulfide production by Ero1α is accelerated by reducing conditions, which minimize the formation of inhibitory disulfides, or by mutations of regulatory cysteines. Here we report that reductive stimulation enhances Ero1α activity more potently than the mutation of cysteines. Specifically, mutation of Cys(208)/Cys(241) does not mechanistically mimic reductive stimulation, as it lowers the turnover rate of Ero1α in presence of a reducing agent. The Cys(208)/Cys(241) pair therefore fulfills a function during catalysis that reaches beyond negative regulation. In agreement, we identify a reciprocal crosstalk between the stabilities of the Cys(208)-Cys(241) disulfide and the inhibitory disulfide bonds involving Cys(104) and Cys(131), which also controls the recruitment of the H2O2 scavenger GPx8 to Ero1α. Two possible mechanisms by which thiol-disulfide exchange at the Cys(208)/Cys(241) pair stimulates the catalytic turnover under reducing conditions are discussed.

Free radical biology & medicine, Jan 2, 2015

Cellular metabolism is inherently linked to the production of oxidizing by-products, including re... more Cellular metabolism is inherently linked to the production of oxidizing by-products, including reactive oxygen species (ROS) hydrogen peroxide (H2O2). When present in excess, H2O2 can damage cellular biomolecules, but when produced in coordinated fashion, it typically serves as a mobile signaling messenger. It is therefore not surprising that cell health critically relies on both low-molecular-weight and enzymatic antioxidant components, which protect from ROS-mediated damage and shape the propagation and duration of ROS signals. This review focuses on H2O2-antioxidant cross talk in the endoplasmic reticulum (ER), which is intimately linked to the process of oxidative protein folding. ER-resident or ER-regulated sources of H2O2 and other ROS, which are subgrouped into constitutive and stimulated sources, are discussed and set into context with the diverse antioxidant mechanisms in the organelle. These include two types of peroxide-reducing enzymes, a high concentration of glutathion...

International journal of cell biology, 2014

GBM Annual Spring meeting Mosbach 2006, 2006

The Journal of biological chemistry, Jan 26, 2004

The recycling mannose lectin ERGIC-53 operates as a transport receptor by mediating efficient end... more 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...

Free Radical Biology and Medicine, 2015

Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated d... more Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O2) and releases hydrogen peroxide (H2O2), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys(208)-Cys(241) disulfide in hyperactive Ero1α (Ero1α-C104A/C131A) potentiates H2O2 production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the flavin cofactor where O2 is reduced to H2O2. Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys(208)/Cys(241)-dependent mixed-disulfide complex with Ero1α. The H2O2-detoxifying glutathione peroxidase 8 also binds to the Cys(208)/Cys(241) loop region. Supported by O2 diffusion simulations, these data describe the first enzymatically controlled O2 access into a flavoprotein active site, provide molecular-level understanding of Ero1α regulation and H2O2 production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity.

Redox Biology, 2013

Glutathione is present in millimolar concentrations in the cell, but its relative distribution am... more Glutathione is present in millimolar concentrations in the cell, but its relative distribution among cellular compartments remains elusive. We have chosen the endoplasmic reticulum (ER) as an example organelle to study compartment-specific glutathione levels. Using a glutaredoxin sensor (sCGrx1pER), which rapidly and specifically equilibrates with the reduced glutathione (GSH)-glutathione disulfide (GSSG) redox couple with known equilibrium constant, we showed that the [GSH]:[GSSG] ratio in the ER of intact HeLa cells is less than 7:1. Taking into consideration the previously determined value for [GSH](2):[GSSG] in the ER of 83 mM, this translates into a total glutathione concentration in the ER ([GStot]=[GSH]+2[GSSG]) of greater than 15 mM. Since the integrated, intracellular [GStot] was measured as ~7 mM, we conclude the existence of a [GStot] gradient across the ER membrane. A possible homeostatic mechanism by which cytosol-derived glutathione is trapped in the ER is discussed. We propose a high [GStot] as a distinguishing feature of the ER environment compared to the extracellular space.

Molecular Biology of the Cell, 2005

Some secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture... more Some secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture mechanism, but the signals required for the cargo-receptor interaction are largely unknown. Here, we describe a novel targeting motif that is composed of a high-mannose type oligosaccharide intimately associated with a surface-exposed peptide beta-hairpin loop. The motif accounts for lectin ERGIC-53-assisted ER-export of the lyososomal enzyme procathepsin Z. The second oligosaccharide chain of procathepsin Z exhibits no binding activity for ERGIC-53, illustrating the selective lectin properties of ERGIC-53. Our data suggest that the conformation-based motif is only present in fully folded procathepsin Z and that its recognition by ERGIC-53 reflects a quality control mechanism that acts complementary to the primary folding machinery in the ER. A similar oligosaccharide/beta-hairpin loop structure is present in cathepsin C, another cargo of ERGIC-53, suggesting the general nature of this ER-exit signal. To our knowledge this is the first documentation of an ER-exit signal in soluble cargo in conjunction with its decoding by a transport receptor.

Trends in Cell Biology, 2012

Many cellular processes including apoptosis, autophagy, translation, energy metabolism, and infla... more 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.

Frontiers in Genetics, 2013

Pathological endoplasmic reticulum (ER) stress is tightly linked to the accumulation of reactive ... more Pathological endoplasmic reticulum (ER) stress is tightly linked to the accumulation of reactive oxidants, which can be both upstream and downstream of ER stress. Accordingly, detrimental intracellular stress signals are amplified through establishment of a vicious cycle. An increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. Experimental monitoring of stress-induced, time-resolved changes in ER reduction-oxidation (redox) states is therefore important. Organelle-specific examination of redox changes has been facilitated by the advent of genetically encoded, fluorescent probes, which can be targeted to different subcellular locations by means of specific amino acid extensions. These probes include redox-sensitive green fluorescent proteins (roGFPs) and the yellow fluorescent protein-based redox biosensor HyPer. In the case of roGFPs, variants with known specificity toward defined redox couples are now available. Here, we review the experimental framework to measure ER redox changes using ER-targeted fluorescent biosensors. Advantages and drawbacks of plate-reader and microscopy-based measurements are discussed, and the power of these techniques demonstrated in the context of selected cell culture models for ER stress.

Journal of Cell Science, 2014

In many physiological contexts, intracellular reduction-oxidation (redox) conditions and the unfo... more 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 Ca(2+) 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.

The EMBO Journal, 2008

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powe... more 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 Ero1a, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1a in cells arises from the formation of a disulphide bond-identified by mass spectrometry-between the active-site Cys 94 (connected to Cys 99 in the active enzyme) and Cys 131 . Competition between substrate thiols and Cys 131 creates a feedback loop where activation of Ero1a is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1a-Cys131Ala or the isoform Ero1b, 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.

The EMBO Journal, 2010

The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cell... more 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 Ero1a 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 Ero1a, 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 disulphidemediated oxidation of PDIs constitutes an important element of ER redox homeostasis.

Proceedings of the National Academy of Sciences, 2009

The quality control system of the endoplasmic reticulum (ER) discriminates between native and non... more 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.

PLoS ONE, 2012

Background: Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key transcription factor regu... more 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 11b-hydroxysteroid dehydrogenase-1 (11b-HSD1).