scholarly journals Balanced Ero1 activation and inactivation establishes ER redox homeostasis

2012 ◽  
Vol 196 (6) ◽  
pp. 713-725 ◽  
Author(s):  
Sunghwan Kim ◽  
Dionisia P. Sideris ◽  
Carolyn S. Sevier ◽  
Chris A. Kaiser
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Direct Reduction ◽  
Redox Homeostasis ◽  
Feedback Regulation ◽  
Redox Balance ◽  
Oxidative Protein Folding ◽  
Reduced And Oxidized Glutathione ◽  
Minimum Number ◽  
Substrate Proteins

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.

scholarly journals Identification of Redox Partners of the Thiol-Disulfide Oxidoreductase SdbA inStreptococcus gordonii

2019 ◽  
Vol 201 (10) ◽  
Author(s):  
Naif A. Jalal ◽  
Lauren Davey ◽  
Scott A. Halperin ◽  
Song F. Lee
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Extracellular Dna ◽  
Folding Pathway ◽  
Content Type ◽  
Cellular Processes ◽  
Oxidative Protein Folding ◽  
Redox Partners ◽  
Disulfide Oxidoreductase ◽  
Substrate Proteins

ABSTRACTWe previously identified a novel thiol-disulfide oxidoreductase, SdbA, inStreptococcus gordoniithat formed disulfide bonds in substrate proteins and played a role in multiple phenotypes. In this study, we used mutational, phenotypic, and biochemical approaches to identify and characterize the redox partners of SdbA. Unexpectedly, the results showed that SdbA has multiple redox partners, forming a complex oxidative protein-folding pathway. The primary redox partners of SdbA that maintain its active site in an oxidized state are a surface-exposed thioredoxin family lipoprotein called SdbB (Sgo_1171) and an integral membrane protein annotated as CcdA2. Inactivation ofsdbBandccdA2simultaneously, but not individually, recapitulated thesdbAmutant phenotype. ThesdbB-ccdA2mutant had defects in a range of cellular processes, including autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release. AtlS, the natural substrate of SdbA produced by thesdbB-ccdA2mutant lacked activity and an intramolecular disulfide bond. The redox state of SdbA in thesdbB-ccdA2mutant was found to be in a reduced form and was restored whensdbBandccdA2were knocked back into the mutant. In addition, we showed that SdbB formed a disulfide-linked complex with SdbA in the cell. Recombinant SdbB and CcdA2 exhibited oxidase activity and reoxidized reduced SdbAin vitro. Collectively, our results demonstrate thatS. gordoniiuses multiple redox partners for oxidative protein folding.IMPORTANCEStreptococcus gordoniiis a commensal bacterium of the human dental plaque. Previously, we identified an enzyme, SdbA, that forms disulfide bonds in substrate proteins and plays a role in a number of cellular processes inS. gordonii. Here, we identified the redox partners of SdbA. We showed that SdbA has multiple redox partners, SdbB and CcdA2, forming a complex oxidative protein-folding pathway. This pathway is essential for autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release inS. gordonii. These cellular processes are considered to be important for the success ofS. gordoniias a dental plaque organism. This is the first example of an oxidative protein-folding pathway in Gram-positive bacteria that consists of an enzyme that uses multiple redox partners to function.

The endoplasmic reticulum sulfhydryl oxidase Ero1β drives efficient oxidative protein folding with loose regulation

2011 ◽  
Vol 434 (1) ◽  
pp. 113-121 ◽  
Author(s):  
Lei Wang ◽  
Li Zhu ◽  
Chih-chen Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Disulfide Bonds ◽  
Critical Role ◽  
Feedback Regulation ◽  
Unfolded Protein ◽  
Oxidative Protein Folding ◽  
Genes Encoding ◽  
The Unfolded Protein Response ◽  
Protein Response

In eukaryotes, disulfide bonds are formed in the endoplasmic reticulum, facilitated by the Ero1 (endoplasmic reticulum oxidoreductin 1) oxidase/PDI (protein disulfide-isomerase) system. Mammals have two ERO1 genes, encoding Ero1α and Ero1β proteins. Ero1β is constitutively expressed in professional secretory tissues and induced during the unfolded protein response. In the present work, we show that recombinant human Ero1β is twice as active as Ero1α in enzymatic assays. Ero1β oxidizes PDI more efficiently than other PDI family members and drives oxidative protein folding preferentially via the active site in the a′ domain of PDI. Our results reveal that Ero1β oxidase activity is regulated by long-range disulfide bonds and that Cys130 plays a critical role in feedback regulation. Compared with Ero1α, however, Ero1β is loosely regulated, consistent with its role as a more active oxidase when massive oxidative power is required.

scholarly journals Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

2020 ◽  
Author(s):  
Reem Mousa ◽  
Taghreed Hidmi ◽  
Sergei Pomyalov ◽  
Shifra Lansky ◽  
Lareen Khouri ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Folding ◽  
Disulfide Bonds ◽  
Crystal Structure Analysis ◽  
Folding Rate ◽  
Oxidative Folding ◽  
Functional Studies ◽  
Oxidative Protein Folding ◽  
And Function

<p>The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. A well-known folding model for many disulfide-rich proteins is that of hirudin. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and folds through plagued pathway that involve highly heterogeneous intermediates and scrambled isomers. The formation of scrambled species is known to limit the rate and efficiency of <i>in vitro</i> oxidative folding of many proteins.</p><p>In the current manuscript we describe our recent work, intended to overcome the limitations of scrambled isomers formation during oxidative protein folding. In this research we deeply investigate the utility of introducing diselenide bridges at the three native disulfide crosslinks as well as at a non-native position on hirudin’s folding, structure and function. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the hirudin analogs, while reducing the complexity and heterogeneity of the process, and reducing the formation of scrambled isomers.</p><p>A parallel, equally important, objective of our study was to test if diselenide substitutions have structural and functional effects. Crystal structure analysis as well as functional studies indicated that diselenide crosslinks maintained the overall structure of the protein without causing major changes in function and structure. To substantiate these conclusions, we provide inhibition studies and high-resolution crystal structure of the wild-type hirudin and its seleno-analogs. </p>Taken together, we believe that the choice of hirudin as the model in this study has implications beyond its specific folding mechanism, and will serve as a useful methodology for the <i>in vitro</i> oxidative folding of many complex disulfide-rich proteins.

scholarly journals Endoplasmic reticulum oxidoreductin (ERO) provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics

2021 ◽  
Author(s):  
Jose Manuel Ugalde ◽  
Isabel Aller ◽  
Lika Kudrjasova ◽  
Romy Schmidt ◽  
Michelle Schloesser ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Redox Homeostasis ◽  
Midpoint Potential ◽  
Oxidative Protein Folding ◽  
Reductive Stress ◽  
Hypoxic Conditions ◽  
Glutathione Redox ◽  
Redox Dynamics

Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. While many key players involved in oxidative protein folding are known, our understanding of how redox homeostasis in the ER is maintained and how EROs, the Cys residues of nascent proteins, and the luminal glutathione redox buffer interact is limited. Here, we isolated viable ero1 ero2 double mutants largely deficient in ERO activity, which rendered the mutants highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER lumen in vivo, we expressed the glutathione redox potential (EGSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of -240 mV in the ER of Arabidopsis plants. We found EGSH values of -241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal EGSH was reduced further to -253 mV. Recovery to reductive ER stress, as induced by acute exposure to dithiothreitol, was delayed in ero1 ero2 mutants. The characteristic signature of EGSH dynamics in the ER lumen triggered by hypoxia was affected in the ero1 ero2 mutant reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis.

scholarly journals Structure of Ero1p, Source of Disulfide Bonds for Oxidative Protein Folding in the Cell

Cell ◽  
2004 ◽  
Vol 117 (5) ◽  
pp. 601-610 ◽  
Author(s):  
Einav Gross ◽  
David B Kastner ◽  
Chris A Kaiser ◽  
Deborah Fass
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Oxidative Protein Folding

scholarly journals Oxidative protein folding in eukaryotes

2004 ◽  
Vol 164 (3) ◽  
pp. 341-346 ◽  
Author(s):  
Benjamin P. Tu ◽  
Jonathan S. Weissman
Keyword(s):  
Protein Folding ◽  
Molecular Oxygen ◽  
Disulfide Bonds ◽  
Terminal Electron Acceptor ◽  
Oxidative Protein Folding ◽  
Disulfide Formation ◽  
Open Questions ◽  
The Many ◽  
Related Proteins ◽  
Protein Disulfide

The endoplasmic reticulum (ER) provides an environment that is highly optimized for oxidative protein folding. Rather than relying on small molecule oxidants like glutathione, it is now clear that disulfide formation is driven by a protein relay involving Ero1, a novel conserved FAD-dependent enzyme, and protein disulfide isomerase (PDI); Ero1 is oxidized by molecular oxygen and in turn acts as a specific oxidant of PDI, which then directly oxidizes disulfide bonds in folding proteins. While providing a robust driving force for disulfide formation, the use of molecular oxygen as the terminal electron acceptor can lead to oxidative stress through the production of reactive oxygen species and oxidized glutathione. How Ero1p distinguishes between the many different PDI-related proteins and how the cell minimizes the effects of oxidative damage from Ero1 remain important open questions.

scholarly journals Vitamin K epoxide reductase contributes to protein disulfide formation and redox homeostasis within the endoplasmic reticulum

2012 ◽  
Vol 23 (11) ◽  
pp. 2017-2027 ◽  
Author(s):  
Lori A. Rutkevich ◽  
David B. Williams
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Vitamin K ◽  
Redox Homeostasis ◽  
Human Hepatoma Cells ◽  
Vitamin K Epoxide Reductase ◽  
Oxidative Protein Folding ◽  
Disulfide Formation ◽  
Epoxide Reductase ◽  
Protein Disulfide

The transfer of oxidizing equivalents from the endoplasmic reticulum (ER) oxidoreductin (Ero1) oxidase to protein disulfide isomerase is an important pathway leading to disulfide formation in nascent proteins within the ER. However, Ero1-deficient mouse cells still support oxidative protein folding, which led to the discovery that peroxiredoxin IV (PRDX4) catalyzes a parallel oxidation pathway. To identify additional pathways, we used RNA interference in human hepatoma cells and evaluated the relative contributions to oxidative protein folding and ER redox homeostasis of Ero1, PRDX4, and the candidate oxidants quiescin-sulfhydryl oxidase 1 (QSOX1) and vitamin K epoxide reductase (VKOR). We show that Ero1 is primarily responsible for maintaining cell growth, protein secretion, and recovery from a reductive challenge. We further show by combined depletion with Ero1 that PRDX4 and, for the first time, VKOR contribute to ER oxidation and that depletion of all three activities results in cell death. Of importance, Ero1, PRDX4, or VKOR was individually capable of supporting cell viability, secretion, and recovery after reductive challenge in the near absence of the other two activities. In contrast, no involvement of QSOX1 in ER oxidative processes could be detected. These findings establish VKOR as a significant contributor to disulfide bond formation within the ER.

scholarly journals Oxidative Protein-Folding Systems in Plant Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
Author(s):  
Yayoi Onda
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
De Novo ◽  
Protein Body ◽  
Structural Diversity ◽  
Carrier Proteins ◽  
Exchange Reactions ◽  
Disulfide Exchange ◽  
Protein Storage ◽  
Oxidative Protein Folding

Plants are unique among eukaryotes in having evolved organelles: the protein storage vacuole, protein body, and chloroplast. Disulfide transfer pathways that function in the endoplasmic reticulum (ER) and chloroplasts of plants play critical roles in the development of protein storage organelles and the biogenesis of chloroplasts, respectively. Disulfide bond formation requires the cooperative function of disulfide-generating enzymes (e.g., ER oxidoreductase 1), which generate disulfide bonds de novo, and disulfide carrier proteins (e.g., protein disulfide isomerase), which transfer disulfides to substrates by means of thiol-disulfide exchange reactions. Selective molecular communication between disulfide-generating enzymes and disulfide carrier proteins, which reflects the molecular and structural diversity of disulfide carrier proteins, is key to the efficient transfer of disulfides to specific sets of substrates. This review focuses on recent advances in our understanding of the mechanisms and functions of the various disulfide transfer pathways involved in oxidative protein folding in the ER, chloroplasts, and mitochondria of plants.

scholarly journals Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

2020 ◽  
Author(s):  
Reem Mousa ◽  
Taghreed Hidmi ◽  
Sergei Pomyalov ◽  
Shifra Lansky ◽  
Lareen Khouri ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Folding ◽  
Disulfide Bonds ◽  
Crystal Structure Analysis ◽  
Folding Rate ◽  
Oxidative Folding ◽  
Functional Studies ◽  
Oxidative Protein Folding ◽  
And Function

<p>The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. A well-known folding model for many disulfide-rich proteins is that of hirudin. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and folds through plagued pathway that involve highly heterogeneous intermediates and scrambled isomers. The formation of scrambled species is known to limit the rate and efficiency of <i>in vitro</i> oxidative folding of many proteins.</p><p>In the current manuscript we describe our recent work, intended to overcome the limitations of scrambled isomers formation during oxidative protein folding. In this research we deeply investigate the utility of introducing diselenide bridges at the three native disulfide crosslinks as well as at a non-native position on hirudin’s folding, structure and function. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the hirudin analogs, while reducing the complexity and heterogeneity of the process, and reducing the formation of scrambled isomers.</p><p>A parallel, equally important, objective of our study was to test if diselenide substitutions have structural and functional effects. Crystal structure analysis as well as functional studies indicated that diselenide crosslinks maintained the overall structure of the protein without causing major changes in function and structure. To substantiate these conclusions, we provide inhibition studies and high-resolution crystal structure of the wild-type hirudin and its seleno-analogs. </p>Taken together, we believe that the choice of hirudin as the model in this study has implications beyond its specific folding mechanism, and will serve as a useful methodology for the <i>in vitro</i> oxidative folding of many complex disulfide-rich proteins.

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