Publications by authors named "Carolyn S Sevier"

26 Publications

  • Page 1 of 1

The asymmetric Pitx2 gene regulates gut muscular-lacteal development and protects against fatty liver disease.

Cell Rep 2021 Nov;37(8):110030

Department of Molecular Medicine, College of Veterinary Medicine, Cornell, Ithaca, NY 14853, USA. Electronic address:

Intestinal lacteals are essential lymphatic channels for absorption and transport of dietary lipids and drive the pathogenesis of debilitating metabolic diseases. However, organ-specific mechanisms linking lymphatic dysfunction to disease etiology remain largely unknown. In this study, we uncover an intestinal lymphatic program that is linked to the left-right (LR) asymmetric transcription factor Pitx2. We show that deletion of the asymmetric Pitx2 enhancer ASE alters normal lacteal development through the lacteal-associated contractile smooth muscle lineage. ASE deletion leads to abnormal muscle morphogenesis induced by oxidative stress, resulting in impaired lacteal extension and defective lymphatic system-dependent lipid transport. Surprisingly, activation of lymphatic system-independent trafficking directs dietary lipids from the gut directly to the liver, causing diet-induced fatty liver disease. Our study reveals the molecular mechanism linking gut lymphatic function to the earliest symmetry-breaking Pitx2 and highlights the important relationship between intestinal lymphangiogenesis and the gut-liver axis.
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http://dx.doi.org/10.1016/j.celrep.2021.110030DOI Listing
November 2021

Pathways for Sensing and Responding to Hydrogen Peroxide at the Endoplasmic Reticulum.

Cells 2020 10 18;9(10). Epub 2020 Oct 18.

Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA.

The endoplasmic reticulum (ER) has emerged as a source of hydrogen peroxide (HO) and a hub for peroxide-based signaling events. Here we outline cellular sources of ER-localized peroxide, including sources within and near the ER. Focusing on three ER-localized proteins-the molecular chaperone BiP, the transmembrane stress-sensor IRE1, and the calcium pump SERCA2-we discuss how post-translational modification of protein cysteines by HO can alter ER activities. We review how changed activities for these three proteins upon oxidation can modulate signaling events, and also how cysteine oxidation can serve to limit the cellular damage that is most often associated with elevated peroxide levels.
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http://dx.doi.org/10.3390/cells9102314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603117PMC
October 2020

Activity of the yeast cytoplasmic Hsp70 nucleotide-exchange factor Fes1 is regulated by reversible methionine oxidation.

J Biol Chem 2020 01 5;295(2):552-569. Epub 2019 Dec 5.

Department of Molecular Medicine, Cornell University, Ithaca, New York 14853. Electronic address:

Cells employ a vast network of regulatory pathways to manage intracellular levels of reactive oxygen species (ROS). An effectual means used by cells to control these regulatory systems are sulfur-based redox switches, which consist of protein cysteine or methionine residues that become transiently oxidized when intracellular ROS levels increase. Here, we describe a methionine-based oxidation event involving the yeast cytoplasmic Hsp70 co-chaperone Fes1. We show that Fes1 undergoes reversible methionine oxidation during excessively-oxidizing cellular conditions, and we map the site of this oxidation to a cluster of three methionine residues in the Fes1 core domain. Making use of recombinant proteins and a variety of assays, we establish that oxidation inhibits Fes1 activity and, correspondingly, alters Hsp70 activity. Moreover, we demonstrate and in cells that Fes1 oxidation is reversible and is regulated by the cytoplasmic methionine sulfoxide reductase Mxr1 (MsrA) and a previously unidentified cytoplasmic pool of the reductase Mxr2 (MsrB). We speculate that inactivation of Fes1 activity during excessively-oxidizing conditions may help maintain protein-folding homeostasis in a suboptimal cellular folding environment. The characterization of Fes1 oxidation during cellular stress provides a new perspective as to how the activities of the cytoplasmic Hsp70 chaperones may be attuned by fluctuations in cellular ROS levels and provides further insight into how cells use methionine-based redox switches to sense and respond to oxidative stress.
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http://dx.doi.org/10.1074/jbc.RA119.010125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956543PMC
January 2020

Working Together: Redox Signaling between the Endoplasmic Reticulum and Mitochondria.

Chem Res Toxicol 2019 03 5;32(3):342-344. Epub 2019 Feb 5.

Department of Molecular Medicine , Cornell University , Ithaca , New York 14853 , United States.

The concept that reactive oxygen species (ROS) are primarily toxic, mitochondria-generated molecules has persisted for decades. Here we highlight the emerging complexity for ROS-based events, emphasizing the evolving importance of the endoplasmic reticulum as a source and platform for redox signaling.
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http://dx.doi.org/10.1021/acs.chemrestox.8b00379DOI Listing
March 2019

Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant.

Biochemistry 2018 02 1;57(7):1073-1086. Epub 2018 Feb 1.

Department of Molecular Medicine, Cornell University , Ithaca, New York 14853, United States.

The ATPase domain of members of the 70 kDa heat shock protein (Hsp70) family shows a high degree of sequence, structural, and functional homology across species. A broadly conserved residue within the Hsp70 ATPase domain that captured our attention is an unpaired cysteine, positioned proximal to the site of nucleotide binding. Prior studies of several Hsp70 family members show this cysteine is not required for Hsp70 ATPase activity, yet select amino acid replacements of the cysteine can dramatically alter ATP hydrolysis. Moreover, post-translational modification of the cysteine has been reported to limit ATP hydrolysis for several Hsp70s. To better understand the underlying mechanism for how perturbation of this noncatalytic residue modulates Hsp70 function, we determined the structure for a cysteine-to-tryptophan mutation in the constitutively expressed, mammalian Hsp70 family member Hsc70. Our work reveals that the steric hindrance produced by a cysteine-to-tryptophan mutation disrupts the hydrogen-bond network within the active site, resulting in a loss of proper catalytic magnesium coordination. We propose that a similarly altered active site is likely observed upon post-translational oxidation. We speculate that the subtle changes we detect in the hydrogen-bonding network may relate to the previously reported observation that cysteine oxidation can influence Hsp70 interdomain communication.
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http://dx.doi.org/10.1021/acs.biochem.7b01005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839340PMC
February 2018

How Are Proteins Reduced in the Endoplasmic Reticulum?

Trends Biochem Sci 2018 01 15;43(1):32-43. Epub 2017 Nov 15.

Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. Electronic address:

The reversal of thiol oxidation in proteins within the endoplasmic reticulum (ER) is crucial for protein folding, degradation, chaperone function, and the ER stress response. Our understanding of this process is generally poor but progress has been made. Enzymes performing the initial reduction of client proteins, as well as the ultimate electron donor in the pathway, have been identified. Most recently, a role for the cytosol in ER protein reduction has been revealed. Nevertheless, how reducing equivalents are transferred from the cytosol to the ER lumen remains an open question. We review here why proteins are reduced in the ER, discuss recent data on catalysis of steps in the pathway, and consider the implications for redox homeostasis within the early secretory pathway.
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http://dx.doi.org/10.1016/j.tibs.2017.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751730PMC
January 2018

An unexpected role for the yeast nucleotide exchange factor Sil1 as a reductant acting on the molecular chaperone BiP.

Elife 2017 03 3;6. Epub 2017 Mar 3.

Department of Molecular Medicine, Cornell University, Ithaca, United States.

Unfavorable redox conditions in the endoplasmic reticulum (ER) can decrease the capacity for protein secretion, altering vital cell functions. While systems to manage reductive stress are well-established, how cells cope with an overly oxidizing ER remains largely undefined. In previous work (Wang et al., 2014), we demonstrated that the chaperone BiP is a sensor of overly oxidizing ER conditions. We showed that modification of a conserved BiP cysteine during stress beneficially alters BiP chaperone activity to cope with suboptimal folding conditions. How this cysteine is reduced to reestablish 'normal' BiP activity post-oxidative stress has remained unknown. Here we demonstrate that BiP's nucleotide exchange factor - Sil1 - can reverse BiP cysteine oxidation. This previously unexpected reductant capacity for yeast Sil1 has potential implications for the human ataxia Marinesco-Sjögren syndrome, where it is interesting to speculate that a disruption in ER redox-signaling (due to genetic defects in ) may influence disease pathology.
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http://dx.doi.org/10.7554/eLife.24141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358974PMC
March 2017

A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities.

J Mol Biol 2016 10 16;428(20):4168-4184. Epub 2016 Aug 16.

Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA. Electronic address:

Among the amino acids, cysteine stands apart based on its highly reactive sulfur group. In general, cysteine is underrepresented in proteins. Yet, when present, the features of cysteine often afford unique function. We have shown previously that a cysteine within the ATPase domain of yeast BiP (Kar2) serves as a sensor of the endoplasmic reticulum (ER) redox environment [1, 2]. Under conditions of increased oxidant (oxidative stress), this cysteine becomes oxidized, changing Kar2 from an ATP-dependent foldase to an ATP-independent holdase. We were struck by the high degree of conservation for this cysteine between BiP orthologs, and we sought to determine how cysteine substitution impacts Kar2 function. We observed that no single amino acid replacement is capable of recreating the range of functions that can be achieved by wild-type Kar2 with its cysteine in either unmodified or oxidized states. However, we were able to generate mutants that could selectively replicate the distinct activities exhibited by either unmodified or oxidized Kar2. We found that the ATPase activity displayed by unmodified Kar2 is fully maintained when Cys63 is replaced with Ala or Val. Conversely, we demonstrate that several amino acid substitutions (including His, Phe, Pro, Trp, and Tyr) support an enhanced viability during oxidative stress associated with oxidized Kar2, although these alleles are compromised as an ATPase. We reveal that the range of activity demonstrated by wild-type Kar2 can be replicated by co-expression of Kar2 mutants that mimic either the unmodified or oxidized Kar2 state, allowing for growth during standard and oxidative stress conditions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5048563PMC
http://dx.doi.org/10.1016/j.jmb.2016.08.011DOI Listing
October 2016

Formation and Reversibility of BiP Protein Cysteine Oxidation Facilitate Cell Survival during and post Oxidative Stress.

J Biol Chem 2016 Apr 10;291(14):7541-57. Epub 2016 Feb 10.

From the Department of Molecular Medicine, Cornell University, Ithaca, New York 14853

Redox fluctuations within cells can be detrimental to cell function. To gain insight into how cells normally buffer against redox changes to maintain cell function, we have focused on elucidating the signaling pathways that serve to sense and respond to oxidative redox stress within the endoplasmic reticulum (ER) using yeast as a model system. Previously, we have shown that a cysteine in the molecular chaperone BiP, a Hsp70 molecular chaperone within the ER, is susceptible to oxidation by peroxide during ER-derived oxidative stress, forming a sulfenic acid (-SOH) moiety. Here, we demonstrate that this same conserved BiP cysteine is susceptible also to glutathione modification (-SSG). Glutathionylated BiP is detected both as a consequence of enhanced levels of cellular peroxide and also as a by-product of increased levels of oxidized glutathione (GSSG). Similar to sulfenylation, we observe glutathionylation decouples BiP ATPase and peptide binding activities, turning BiP from an ATP-dependent foldase into an ATP-independent holdase. We show glutathionylation enhances cell proliferation during oxidative stress, which we suggest relates to modified BiP's increased ability to limit polypeptide aggregation. We propose the susceptibility of BiP to modification with glutathione may serve also to prevent irreversible oxidation of BiP by peroxide.
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http://dx.doi.org/10.1074/jbc.M115.694810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817183PMC
April 2016

Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress.

Elife 2014 Jul 22;3:e03496. Epub 2014 Jul 22.

Department of Molecular Medicine, Cornell University, Ithaca, United States

Oxidative protein folding in the endoplasmic reticulum (ER) has emerged as a potentially significant source of cellular reactive oxygen species (ROS). Recent studies suggest that levels of ROS generated as a byproduct of oxidative folding rival those produced by mitochondrial respiration. Mechanisms that protect cells against oxidant accumulation within the ER have begun to be elucidated yet many questions still remain regarding how cells prevent oxidant-induced damage from ER folding events. Here we report a new role for a central well-characterized player in ER homeostasis as a direct sensor of ER redox imbalance. Specifically we show that a conserved cysteine in the lumenal chaperone BiP is susceptible to oxidation by peroxide, and we demonstrate that oxidation of this conserved cysteine disrupts BiP's ATPase cycle. We propose that alteration of BiP activity upon oxidation helps cells cope with disruption to oxidative folding within the ER during oxidative stress.
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http://dx.doi.org/10.7554/eLife.03496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132286PMC
July 2014

Balanced Ero1 activation and inactivation establishes ER redox homeostasis.

J Cell Biol 2012 Mar 12;196(6):713-25. Epub 2012 Mar 12.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The endoplasmic reticulum (ER) provides an environment optimized for oxidative protein folding through the action of Ero1p, which generates disulfide bonds, and Pdi1p, which receives disulfide bonds from Ero1p and transfers them to substrate proteins. Feedback regulation of Ero1p through reduction and oxidation of regulatory bonds within Ero1p is essential for maintaining the proper redox balance in the ER. In this paper, we show that Pdi1p is the key regulator of Ero1p activity. Reduced Pdi1p resulted in the activation of Ero1p by direct reduction of Ero1p regulatory bonds. Conversely, upon depletion of thiol substrates and accumulation of oxidized Pdi1p, Ero1p was inactivated by both autonomous oxidation and Pdi1p-mediated oxidation of Ero1p regulatory bonds. Pdi1p responded to the availability of free thiols and the relative levels of reduced and oxidized glutathione in the ER to control Ero1p activity and ensure that cells generate the minimum number of disulfide bonds needed for efficient oxidative protein folding.
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http://dx.doi.org/10.1083/jcb.201110090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308690PMC
March 2012

Erv2 and quiescin sulfhydryl oxidases: Erv-domain enzymes associated with the secretory pathway.

Authors:
Carolyn S Sevier

Antioxid Redox Signal 2012 Apr 11;16(8):800-8. Epub 2012 Jan 11.

Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA.

Significance: Members of the Erv/ALR/QSOX protein family contain an Erv sequence module and catalyze protein disulfide bond formation. Erv enzymes impact protein function within and outside cells that affects both normal and malignant cell growth. This protein family is named for its founding members: Erv1 (essential for respiratory and vegetative growth 1) and ALR (augmenter of liver regeneration), homologous mitochondrial proteins from yeast and mammals, respectively, and QSOX (quiescin sulfhydryl oxidase), an oxidase secreted from quiescent cells. This review will focus on a subset of Erv proteins that are localized within the secretory pathway: Erv2-like proteins, proteins present in the endoplasmic reticulum of fungi, and QSOX proteins, proteins localized within the secretory pathway and extracellular space and present in most eukaryotes, but not fungi.

Recent Advances: A wealth of structural and biochemical data has been obtained for Erv2 and QSOX proteins. These data have identified a generally conserved catalytic mechanism and structure for the Erv2 and QSOX proteins with unique features for each enzyme.

Critical Issues: Many fundamental questions remain about the activity for these proteins in living cells including the partners, pathways, and locations utilized by these enzymes in vivo.

Future Directions: A more comprehensive understanding of the cellular roles for Erv2 and QSOX enzymes will require identification of their partners and substrates. Also, determining when Erv2 and QSOX function during growth and development, and how changes in levels of active Erv2 and QSOX impact cell function, is necessary to facilitate a better understanding of these intriguing enzymes.
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http://dx.doi.org/10.1089/ars.2011.4450DOI Listing
April 2012

Steps in reductive activation of the disulfide-generating enzyme Ero1p.

Protein Sci 2010 Oct;19(10):1863-76

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.

Ero1p is the primary catalyst of disulfide bond formation in the yeast endoplasmic reticulum (ER). Ero1p contains a pair of essential disulfide bonds that participate directly in the electron transfer pathway from substrate thiol groups to oxygen. Remarkably, elimination of certain other Ero1p disulfides by mutation enhances enzyme activity. In particular, the C150A/C295A Ero1p mutant exhibits increased thiol oxidation in vitro and in vivo and interferes with redox homeostasis in yeast cells by hyperoxidizing the ER. Inhibitory disulfides of Ero1p are thus important for enzyme regulation. To visualize the differences between de-regulated and wild-type Ero1p, we determined the crystal structure of Ero1p C150A/C295A. The structure revealed local changes compared to the wild-type enzyme around the sites of mutation, but no conformational transitions within 25 A of the active site were observed. To determine how the C150--C295 disulfide nonetheless participates in redox regulation of Ero1p, we analyzed using mass spectrometry the changes in Ero1p disulfide connectivity as a function of time after encounter with reducing substrates. We found that the C150--C295 disulfide sets a physiologically appropriate threshold for enzyme activation by guarding a key neighboring disulfide from reduction. This study illustrates the diverse and interconnected roles that disulfides can play in redox regulation of protein activity.
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http://dx.doi.org/10.1002/pro.473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998722PMC
October 2010

Oxidative activity of yeast Ero1p on protein disulfide isomerase and related oxidoreductases of the endoplasmic reticulum.

J Biol Chem 2010 Jun 26;285(24):18155-65. Epub 2010 Mar 26.

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.

The sulfhydryl oxidase Ero1 oxidizes protein disulfide isomerase (PDI), which in turn catalyzes disulfide formation in proteins folding in the endoplasmic reticulum (ER). The extent to which other members of the PDI family are oxidized by Ero1 and thus contribute to net disulfide formation in the ER has been an open question. The yeast ER contains four PDI family proteins with at least one potential redox-active cysteine pair. We monitored the direct oxidation of each redox-active site in these proteins by yeast Ero1p in vitro. In this study, we found that the Pdi1p amino-terminal domain was oxidized most rapidly compared with the other oxidoreductase active sites tested, including the Pdi1p carboxyl-terminal domain. This observation is consistent with experiments conducted in yeast cells. In particular, the amino-terminal domain of Pdi1p preferentially formed mixed disulfides with Ero1p in vivo, and we observed synthetic lethality between a temperature-sensitive Ero1p variant and mutant Pdi1p lacking the amino-terminal active-site disulfide. Thus, the amino-terminal domain of yeast Pdi1p is on a preferred pathway for oxidizing the ER thiol pool. Overall, our results provide a rank order for the tendency of yeast ER oxidoreductases to acquire disulfides from Ero1p.
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http://dx.doi.org/10.1074/jbc.M109.064931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881739PMC
June 2010

New insights into oxidative folding.

Authors:
Carolyn S Sevier

J Cell Biol 2010 Mar;188(6):757-8

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The oxidoreductase ERO1 (endoplasmic reticulum [ER] oxidoreductin 1) is thought to be crucial for disulfide bond formation in the ER. In this issue, Zito et al. (2010. J. Cell Biol. doi:10.1083/jcb.200911086) examine the division of labor between the two mammalian isoforms of ERO1 (ERO1-alpha and -beta) in oxidative folding. Their analysis reveals a selective role for ERO1-beta in insulin production and a surprisingly minor contribution for either ERO1 isoform on immunoglobulin folding and secretion.
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http://dx.doi.org/10.1083/jcb.201002114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845070PMC
March 2010

The genetic landscape of a cell.

Science 2010 Jan;327(5964):425-31

Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for approximately 75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.
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http://dx.doi.org/10.1126/science.1180823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600254PMC
January 2010

Yeast Mpd1p reveals the structural diversity of the protein disulfide isomerase family.

J Mol Biol 2008 Dec 27;384(3):631-40. Epub 2008 Sep 27.

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.

Oxidoreductases belonging to the protein disulfide isomerase (PDI) family promote proper disulfide bond formation in substrate proteins in the endoplasmic reticulum. In plants and metazoans, new family members continue to be identified and assigned to various functional niches. PDI-like proteins typically contain tandem thioredoxin-fold domains. The limited information available suggested that the relative orientations of these domains may be quite uniform across the family, and structural models based on this assumption are appearing. However, the X-ray crystal structure of the yeast PDI family protein Mpd1p, described here, demonstrates the radically different domain orientations and surface properties achievable with multiple copies of the thioredoxin fold. A comparison of Mpd1p with yeast Pdi1p expands our perspective on the contexts in which redox-active motifs are presented in the PDI family.
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http://dx.doi.org/10.1016/j.jmb.2008.09.052DOI Listing
December 2008

Ero1 and redox homeostasis in the endoplasmic reticulum.

Biochim Biophys Acta 2008 Apr 23;1783(4):549-56. Epub 2007 Dec 23.

Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA.

Living cells must be able to respond to physiological and environmental fluctuations that threaten cell function and viability. A cellular event prone to disruption by a wide variety of internal and external perturbations is protein folding. To ensure protein folding can proceed under a range of conditions, the cell has evolved transcriptional, translational, and posttranslational signaling pathways to maintain folding homeostasis during cell stress. This review will focus on oxidative protein folding in the endoplasmic reticulum (ER) and will discuss the features of the main facilitator of biosynthetic disulfide bond formation, Ero1. Ero1 plays an essential role in setting the redox potential in the ER and regulation of Ero1 activity is central to maintain redox homeostasis and proper ER folding activity.
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http://dx.doi.org/10.1016/j.bbamcr.2007.12.011DOI Listing
April 2008

Modulation of cellular disulfide-bond formation and the ER redox environment by feedback regulation of Ero1.

Cell 2007 Apr;129(2):333-44

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Introduction of disulfide bonds into proteins entering the secretory pathway is catalyzed by Ero1p, which generates disulfide bonds de novo, and Pdi1p, which transfers disulfides to substrate proteins. A sufficiently oxidizing environment must be maintained in the endoplasmic reticulum (ER) to allow for disulfide formation, but a pool of reduced thiols is needed for isomerization of incorrectly paired disulfides. We have found that hyperoxidation of the ER is prevented by attenuation of Ero1p activity through noncatalytic cysteine pairs. Deregulated Ero1p mutants lacking certain cysteines show increased enzyme activity, a decreased lag phase in kinetic assays, and growth defects in vivo. We hypothesize that noncatalytic cysteine pairs in Ero1p sense the level of potential substrates in the ER and correspondingly modulate Ero1p activity as part of a homeostatic regulatory system governing the thiol-disulfide balance in the ER.
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http://dx.doi.org/10.1016/j.cell.2007.02.039DOI Listing
April 2007

Conservation and diversity of the cellular disulfide bond formation pathways.

Antioxid Redox Signal 2006 May-Jun;8(5-6):797-811

Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, USA.

Two pathways for the formation of biosynthetic protein disulfide bonds have been characterized in the endoplasmic reticulum (ER) of eukaryotes. In the major pathway, the membrane-associated flavoprotein Ero1 generates disulfide bonds for transfer to protein disulfide isomerase (PDI), which is responsible for directly introducing disulfide bonds into secretory proteins. In a minor fungal-specific protein oxidation pathway, the membrane-associated flavoprotein Erv2 can catalyze disulfide bond formation via the transfer of oxidizing equivalents to PDI. Genomic sequencing has revealed an abundance of enzymes sharing homology with Ero1, Erv2, or PDI. Herein the authors discuss the functional, mechanistic, and potential structural similarities between these homologs and the core enzymes of the characterized ER oxidation pathways. In addition they speculate about the possible differences between these enzymes that may explain why the cell contains multiple proteins dedicated to a single process. Finally, the eukaryotic ER protein oxidation and reduction pathways are compared to the corresponding prokaryotic periplasmic pathways, to highlight the functional, mechanistic, and structural similarities that exist between the pathways in these two kingdoms despite very low primary sequence homology between the protein and small molecule components.
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http://dx.doi.org/10.1089/ars.2006.8.797DOI Listing
March 2007

Disulfide transfer between two conserved cysteine pairs imparts selectivity to protein oxidation by Ero1.

Mol Biol Cell 2006 May 22;17(5):2256-66. Epub 2006 Feb 22.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The membrane-associated flavoprotein Ero1p promotes disulfide bond formation in the endoplasmic reticulum (ER) by selectively oxidizing the soluble oxidoreductase protein disulfide isomerase (Pdi1p), which in turn can directly oxidize secretory proteins. Two redox-active disulfide bonds are essential for Ero1p oxidase activity: Cys100-Cys105 and Cys352-Cys355. Genetic and structural data indicate a disulfide bond is transferred from Cys100-Cys105 directly to Pdi1p, whereas a Cys352-Cys355 disulfide bond is used to reoxidize the reduced Cys100-Cys105 pair through an internal thiol-transfer reaction. Electron transfer from Cys352-Cys355 to molecular oxygen, by way of a flavin cofactor, maintains Cys352-Cys355 in an oxidized form. Herein, we identify a mixed disulfide species that confirms the Ero1p intercysteine thiol-transfer relay in vivo and identify Cys105 and Cys352 as the cysteines that mediate thiol-disulfide exchange. Moreover, we describe Ero1p mutants that have the surprising ability to oxidize substrates in the absence of Cys100-Cys105. We show the oxidase activity of these mutants results from structural changes in Ero1p that allow substrates increased access to Cys352-Cys355, which are normally buried beneath the protein surface. The altered activity of these Ero1p mutants toward selected substrates leads us to propose the catalytic mechanism involving transfer between cysteine pairs evolved to impart substrate specificity to Ero1p.
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http://dx.doi.org/10.1091/mbc.e05-05-0417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1446090PMC
May 2006

Generating disulfides enzymatically: reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p.

Proc Natl Acad Sci U S A 2006 Jan 3;103(2):299-304. Epub 2006 Jan 3.

Department of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

Ero1p is a key enzyme in the disulfide bond formation pathway in eukaryotic cells in both aerobic and anaerobic environments. It was previously demonstrated that Ero1p can transfer electrons from thiol substrates to molecular oxygen. However, the fate of electrons under anaerobic conditions and the final fate of electrons under aerobic conditions remained obscure. To address these fundamental issues in the Ero1p mechanism, we studied the transfer of electrons from recombinant yeast Ero1p to various electron acceptors. Under aerobic conditions, reduction of molecular oxygen by Ero1p yielded stoichiometric hydrogen peroxide. Remarkably, we found that reduced Ero1p can transfer electrons to a variety of small and macromolecular electron acceptors in addition to molecular oxygen. In particular, Ero1p can catalyze reduction of exogenous FAD in solution. Free FAD is not required for the catalysis of dithiol oxidation by Ero1p, but it is sufficient to drive disulfide bond formation under anaerobic conditions. These findings provide insight into mechanisms for regenerating oxidized Ero1p and maintaining disulfide bond formation under anaerobic conditions in the endoplasmic reticulum.
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http://dx.doi.org/10.1073/pnas.0506448103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1326156PMC
January 2006

Structural determinants of substrate access to the disulfide oxidase Erv2p.

J Mol Biol 2005 Dec 10;354(4):952-66. Epub 2005 Oct 10.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Erv2p is a small, dimeric FAD-dependent sulfhydryl oxidase that generates disulfide bonds in the lumen of the endoplasmic reticulum. Mutagenic and structural studies suggest that Erv2p uses an internal thiol-transfer relay between the FAD-proximal active site cysteine pair (Cys121-Cys124) and a second cysteine pair (Cys176-Cys178) located in a flexible, substrate-accessible C-terminal tail of the adjacent dimer subunit. Here, we demonstrate that Cys176 and Cys178 are the only amino acids in the tail region required for disulfide transfer and that their relative positioning within the tail peptide is important for activity. However, intragenic suppressor mutations could be isolated that bypass the requirement for Cys176 and Cys178. These mutants were found to disrupt Erv2p dimerization and to increase the activity of Erv2p for thiol substrates such as glutathione. We propose that the two Erv2p subunits act together to direct the disulfide transfer to specific substrates. One subunit provides the catalytic domain composed of the active site cysteine residues and the FAD cofactor, while the second subunit appears to have two functions: it facilitates disulfide transfer to substrates via the tail cysteine residues, while simultaneously shielding the active site cysteine residues from non-specific reactions.
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http://dx.doi.org/10.1016/j.jmb.2005.09.076DOI Listing
December 2005

The prokaryotic enzyme DsbB may share key structural features with eukaryotic disulfide bond forming oxidoreductases.

Protein Sci 2005 Jun;14(6):1630-42

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Three different classes of thiol-oxidoreductases that facilitate the formation of protein disulfide bonds have been identified. They are the Ero1 and SOX/ALR family members in eukaryotic cells, and the DsbB family members in prokaryotic cells. These enzymes transfer oxidizing potential to the proteins PDI or DsbA, which are responsible for directly introducing disulfide bonds into substrate proteins during oxidative protein folding in eukaryotes and prokaryotes, respectively. A comparison of the recent X-ray crystal structure of Ero1 with the previously solved structure of the SOX/ALR family member Erv2 reveals that, despite a lack of primary sequence homology between Ero1 and Erv2, the core catalytic domains of these two proteins share a remarkable structural similarity. Our search of the DsbB protein sequence for features found in the Ero1 and Erv2 structures leads us to propose that, in a fascinating example of structural convergence, the catalytic core of this integral membrane protein may resemble the soluble catalytic domain of Ero1 and Erv2. Our analysis of DsbB also identified two new groups of DsbB proteins that, based on sequence homology, may also possess a catalytic core similar in structure to the catalytic domains of Ero1 and Erv2.
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http://dx.doi.org/10.1110/ps.051355705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2253379PMC
June 2005

Formation and transfer of disulphide bonds in living cells.

Nat Rev Mol Cell Biol 2002 Nov;3(11):836-47

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Protein disulphide bonds are formed in the endoplasmic reticulum of eukaryotic cells and the periplasmic space of prokaryotic cells. The main pathways that catalyse the formation of protein disulphide bonds in prokaryotes and eukaryotes are remarkably similar, and they share several mechanistic features. The recent identification of new redox-active proteins in humans and yeast that mechanistically parallel the more established redox-active enzymes indicates that there might be further uncharacterized redox pathways throughout the cell.
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http://dx.doi.org/10.1038/nrm954DOI Listing
November 2002

A new FAD-binding fold and intersubunit disulfide shuttle in the thiol oxidase Erv2p.

Nat Struct Biol 2002 Jan;9(1):61-7

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.

Erv2p is an FAD-dependent sulfhydryl oxidase that can promote disulfide bond formation during protein biosynthesis in the yeast endoplasmic reticulum. The structure of Erv2p, determined by X-ray crystallography to 1.5 A resolution, reveals a helix-rich dimer with no global resemblance to other known FAD-binding proteins or thiol oxidoreductases. Two pairs of cysteine residues are required for Erv2p activity. The first (Cys-Gly-Glu-Cys) is adjacent to the isoalloxazine ring of the FAD. The second (Cys-Gly-Cys) is part of a flexible C-terminal segment that can swing into the vicinity of the first cysteine pair in the opposite subunit of the dimer and may shuttle electrons between substrate protein dithiols and the FAD-proximal disulfide.
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http://dx.doi.org/10.1038/nsb740DOI Listing
January 2002
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