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Neil J. Bulleid - One of the best experts on this subject based on the ideXlab platform.
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protein secondary structure determines the temporal relationship between folding and disulfide formation
2019Co-Authors: Philip Robinson, Shingo Kanemura, Xiaofei Cao, Neil J. BulleidAbstract:Abstract How and when Disulfides form in proteins during their folding is a fundamental question in cell biology. Two models describe the relationship between disulfide formation and folding, the folded precursor model, in which formation of nascent structure occurs prior to the Disulfides and the quasi-stochastic model where Disulfides form prior to complete domain folding. Here we investigate oxidative folding within a cellular milieu of three structurally diverse substrates in order to understand the folding mechanisms required to achieve correct cysteine coupling. We use a eukaryotic translation system in which we can manipulate the redox conditions and produce stalled translation intermediates representative of different stages of translocation. We identify different disulfide bonded isomers by non-reducing SDS-PAGE. Using this approach, we determined whether each substrate followed a folding driven or disulfide driven mechanism. Our results demonstrate that the folding model is substrate-dependent with Disulfides forming prior to complete domain folding in a cysteine-rich domain lacking secondary structure, whereas disulfide formation was absent in proteins with defined structural elements. In addition, we demonstrate the presence and rearrangement of non-native Disulfides specifically in substrates following the quasi-stochastic model. These findings demonstrate why non-native Disulfides are prevented from forming in proteins with well-defined secondary structure. Significance statement A third of human proteins contain structural elements called disulfide bonds that are often crucial for stability and function. Disulfides form between cysteines in the specialised environment of the endoplasmic reticulum (ER), during the complex process of protein folding. Many proteins contain multiple cysteines that can potentially form correct or incorrect cysteine pairings. To investigate how correct disulfide pairs are formed in a biological context, we developed an experimental approach to assess disulfide formation and rearrangement as proteins enter the ER. We found that a disulfide-dense protein domain with atypical secondary structure undergoes disulfide orchestrated folding as it enters the ER and is prone to incorrect disulfide formation. In contrast, proteins with defined secondary structure form folding dependent, native Disulfides. These findings show how different mechanisms of disulfide formation can be rationalised from structural features of the folding domains.
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cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the er
2017Co-Authors: Greg J Poet, Philip Robinson, Ojore B V Oka, Marcel Van Lith, Zhenbo Cao, Marie Anne Pringle, Elias S J Arner, Neil J. BulleidAbstract:Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native Disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native Disulfides are reduced so that the correct or native Disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native Disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native Disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway. ![][1] Correcting non‐native Disulfides in secreted proteins, an unexpected role for the cytosol. [1]: /embed/graphic-1.gif
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thiol disulfide exchange between the pdi family of oxidoreductases negates the requirement for an oxidase or reductase for each enzyme
2015Co-Authors: Ojore B V Oka, Hui Ying Yeoh, Neil J. BulleidAbstract:The formation of Disulfides in proteins entering the secretory pathway is catalysed by the protein disulfide isomerase (PDI) family of enzymes. These enzymes catalyse the introduction, reduction and isomerization of Disulfides. To function continuously they require an oxidase to reform the disulfide at their active site. To determine how each family member can be recycled to catalyse disulfide exchange, we have studied whether Disulfides are transferred between individual PDI family members. We studied disulfide exchange either between purified proteins or by identifying mixed disulfide formation within cells grown in culture. We show that disulfide exchange occurs efficiently and reversibly between specific PDIs. These results have allowed us to define a hierarchy for members of the PDI family, in terms of ability to act as electron acceptors or donors during thiol-disulfide exchange reactions and indicate that there is no kinetic barrier to the exchange of Disulfides between several PDI proteins. Such promiscuous disulfide exchange negates the necessity for each enzyme to be oxidized by Ero1 (ER oxidoreductin 1) or reduced by a reductive system. The lack of kinetic separation of the oxidative and reductive pathways in mammalian cells contrasts sharply with the equivalent systems for native disulfide formation within the bacterial periplasm.
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inactivation of mammalian ero1α is catalysed by specific protein disulfide isomerases
2014Co-Authors: Colin Shepherd, Ojore B V Oka, Neil J. BulleidAbstract:Disulfide formation within the endoplasmic reticulum is a complex process requiring a disulfide exchange protein such as PDI (protein disulfide-isomerase) and a mechanism to form Disulfides de novo. In mammalian cells, the major pathway for de novo disulfide formation involves the enzyme Ero1α (endoplasmic reticulum oxidase 1α) which couples oxidation of thiols to the reduction of molecular oxygen to form hydrogen peroxide (H2O2). Ero1α activity is tightly regulated by a mechanism that requires the formation of regulatory Disulfides. These regulatory Disulfides are reduced to activate and reform to inactivate the enzyme. To investigate the mechanism of inactivation we analysed regulatory disulfide formation in the presence of various oxidants under controlled oxygen concentration. Neither molecular oxygen nor H2O2 was able to oxidize Ero1α efficiently to form the correct regulatory Disulfides. However, specific members of the PDI family, such as PDI or ERp46 (endoplasmic reticulum-resident protein 46), were able to catalyse this process. Further studies showed that both active sites of PDI contribute to the formation of regulatory Disulfides in Ero1α and that the PDI substrate-binding domain is crucial to allow electron transfer between the two enzymes. The results of the present study demonstrate a simple feedback mechanism of re-gulation of mammalian Ero1α involving its primary substrate.
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multiple ways to make Disulfides
2011Co-Authors: Neil J. Bulleid, Lars EllgaardAbstract:Our concept of how Disulfides form in proteins entering the secretory pathway has changed dramatically in recent years. The discovery of endoplasmic reticulum (ER) oxidoreductin 1 (ERO1) was followed by the demonstration that this enzyme couples oxygen reduction to de novo formation of Disulfides. However, mammals deficient in ERO1 survive and form Disulfides, which suggests the presence of alternative pathways. It has recently been shown that peroxiredoxin 4 is involved in peroxide removal and disulfide formation. Other less well-characterized pathways involving quiescin sulfhydryl oxidase, ER-localized protein disulfide isomerase peroxidases and vitamin K epoxide reductase might all contribute to disulfide formation. Here we discuss these various pathways for disulfide formation in the mammalian ER and highlight the central role played by glutathione in regulating this process.
Deborah Fass - One of the best experts on this subject based on the ideXlab platform.
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disulfide bonding in protein biophysics
2012Co-Authors: Deborah FassAbstract:It has been known for many decades that cell surface, soluble-secreted, and extracellular matrix proteins are generally rich in disulfide bonds, but only more recently has the functional diversity of disulfide bonding in extracellular proteins been appreciated. In addition to the classic mechanisms by which disulfide bonds enhance protein thermodynamic stability, Disulfides in certain configurations contribute particular mechanical properties to proteins that sense and respond to tensile forces. Disulfides may help warp protein folds for the evolution of new functions, or they may fasten aggregation-prone flaps of polypeptide to protein surfaces to prevent fibrilization or oligomerization. Disulfides can also be used to package and secure macromolecular cargo for intercellular transport. A series of case studies illustrating diverse biophysical roles of disulfide bonding are reviewed, with a focus on proteins functioning in the extracellular environment.
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oxidative activity of yeast ero1p on protein disulfide isomerase and related oxidoreductases of the endoplasmic reticulum
2010Co-Authors: Elvira Vitu, Carolyn S Sevier, Nimrod Heldman, Chris A Kaiser, Sunghwan Kim, Omer Lutzky, Moran Bentzur, Tamar Unger, Meital Yona, Deborah FassAbstract: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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the erv family of sulfhydryl oxidases
2008Co-Authors: Deborah FassAbstract:The Erv flavoenzymes contain a compact module that catalyzes the pairing of cysteine thiols into disulfide bonds. High-resolution structures of plant, animal, and fungal Erv enzymes that function in different contexts and intracellular compartments have been determined. Structural features can be correlated with biochemical properties, revealing how core sulfhydryl oxidase activity has been tailored to various functional niches. The introduction of Disulfides into cysteine-containing substrates by Erv sulfhydryl oxidases is compared with the mechanisms used by NADPH-driven disulfide reductases and thioredoxin-like oxidoreductases to reduce and transfer Disulfides, respectively.
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modulation of cellular disulfide bond formation and the er redox environment by feedback regulation of ero1
2007Co-Authors: Carolyn S Sevier, Hongjing Qu, Nimrod Heldman, Einav Gross, Deborah Fass, Chris A KaiserAbstract: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.
Michael J Davies - One of the best experts on this subject based on the ideXlab platform.
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characterization of disulfide cystine oxidation by hocl in a model peptide evidence for oxygen addition disulfide bond cleavage and adduct formation with thiols
2020Co-Authors: Maryam Karimi, Ben Crossett, Stuart J Cordwell, David I Pattison, Michael J DaviesAbstract:Abstract Disulfide bonds play a key role in stabilizing proteins by cross-linking secondary structures. Whilst many Disulfides are effectively unreactive, it is increasingly clear that some Disulfides are redox active, participate in enzymatic reactions and/or regulate protein function by allosteric mechanisms. Previously (Karimi et al., Sci. Rep. 2016, 6, 38752) we have shown that some Disulfides react rapidly with biological oxidants due to favourable interactions with available lone-pairs of electrons. Here we present data from kinetic, mechanistic and product studies for HOCl-mediated oxidation of a protected nine-amino acid model peptide containing a N- to C-terminal disulfide bond. This peptide reacts with HOCl with k2 1.8 × 106 M−1 s−1, similar to other highly-reactive disulfide-containing compounds. With low oxidant excesses, oxidation yields multiple oxidation products from the disulfide, with reaction predominating at the N-terminal Cys to give sulfenic, sulfinic and sulfonic acids, and disulfide bond cleavage. Limited oxidation occurs, with higher oxidant excesses, at Trp and His residues to give mono- and di- (for Trp) oxygenated products. Site-specific backbone cleavage also occurs between Arg and Trp, probably via initial side-chain modification. Treatment of the previously-oxidised peptide with thiols (GSH, N-Ac-Cys), results in adduction of the thiol to the oxidised peptide, with this occurring at the original disulfide bond. This gives an open-chain peptide, and a new mixed disulfide containing GSH or N-Ac-Cys as determined by mass spectrometry. Disulfide bond oxidation may therefore markedly alter the structure, activity and function of disulfide-containing proteins, and provides a potential mechanism for protein glutathionylation.
David Wilson - One of the best experts on this subject based on the ideXlab platform.
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disulfide arrangement and functional domains of beta 1 4 endoglucanse e5 from thermomonospora fusca
1993Co-Authors: Kathleen Mcginnis, David WilsonAbstract:Thermomonospora fusca cellulase E5 contains six cysteine residues. The number and location of the disulfide bonds and the effect of reduction of the Disulfides and modification of the resulting half-cystine residues on enzymatic activity were determined. No free sulfhydryl groups were found in E5. Reduction and subsequent labeling with iodoacetamide of E5 and of an enzymatically active 32-kDa proteolytic derivative of E5 (E5cd) showed that one of the three Disulfides is accessible to reduction under nondenatured conditions while the other two are not accessible. Full reduction of the Disulfides and complete carboxymethylation of the six cysteines decrease the specific activity of E5 on CMC by more than half, but reduction of only the exposed disulfide bond does not affect enzymatic activity or binding of E5 to cellulose. A 14-kDa proteolytic fragment of E5 containing 120 amino acids from the N-terminus of the protein was shown to bind to crystalline cellulose. This confirms earlier evidence that the cellulose binding domain of E5 is located at the N-terminus of the protein. This 14-kDa fragment contains the accessible disulfide bond involving Cys93 and Cys100. The location of the two disulfide bonds in the other fragment (E5cd) was determined by cleaving it with cyanogen bromide under conditions that left the disulfide bonds intact. The resulting peptides were separated under both nonreducing and reducing conditions using RP-HPLC. Amino acid analysis of peptide peaks indicated that one disulfide linkage in E5cd joins Cys138 to Cys143 while the other joins Cys166 to Cys406.
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disulfide arrangement and chemical modification of beta 1 4 endoglucanase e2 from thermomonospora fusca
1993Co-Authors: Kathleen Mcginnis, David WilsonAbstract:Thermomonospora fusca endoglucanase E2 contains six cysteine residues scattered along the protein sequence. Four of the cysteine residues were shown to participate in two disulfide bonds while the last two form a third disulfide bond. Neither full reduction of the Disulfides nor complete carboxymethylation of all six cysteines totally destroys enzymatic activity, but the activity of the reduced enzyme is much lower than the native enzyme and the iodoacetamide-modified enzyme has very low activity. Reduction of only the accessible Disulfides drastically decreases the enzyme's thermostability. One disulfide linkage joins Cys80 to Cys125, another joins Cys232 to Cys267, and the third joins Cys315 to Cys407. The first two bonds are similar to those in cellobiohydrolase II, which also belongs to cellulase family B (Rouvinen et al., 1990; Lao et al., 1991; Henrissat et al., 1989). Direct evidence for the involvement of carboxyl groups in catalysis by E2 was demonstrated by chemical modification with carbodiimide.
Hiram F Gilbert - One of the best experts on this subject based on the ideXlab platform.
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catalysis of thiol disulfide exchange glutaredoxin 1 and protein disulfide isomerase use different mechanisms to enhance oxidase and reductase activities
2005Co-Authors: Ruoyu Xiao, Arne Holmgren, Johanna Lundstromljung, Hiram F GilbertAbstract:Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative Disulfides in scrambled ribonuclease and protein-glutathione mixed Disulfides 30–180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.
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the contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum
2004Co-Authors: Ruoyu Xiao, Bonney Wilkinson, Arne Holmgren, Jakob R Winther, Johanna Lundstromljung, Anton Solovyov, Hiram F GilbertAbstract:In vitro, protein disulfide isomerase (Pdi1p) introduces Disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect Disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 ± 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.
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Protein disulfide isomerase.
2004Co-Authors: Bonney Wilkinson, Hiram F GilbertAbstract:During the maturation of extracellular proteins, disulfide bonds that chemically cross-link specific cysteines are often added to stabilize a protein or to join it covalently to other proteins. Disulfide formation, which requires a change in the covalent structure of the protein, occurs as the protein folds into its three-dimensional structure. In the eukaryotic endoplasmic reticulum and in the bacterial periplasm, an elaborate system of chaperones and folding catalysts ensure that Disulfides connect the proper cysteines and that the folding protein does not make improper interactions. This review focuses specifically on one of these folding assistants, protein disulfide isomerase (PDI), an enzyme that catalyzes disulfide formation and isomerization and a chaperone that inhibits aggregation.
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reduction reoxidation cycles contribute to catalysis of disulfide isomerization by protein disulfide isomerase
2003Co-Authors: Melissa Schwaller, Bonney Wilkinson, Hiram F GilbertAbstract:Abstract Protein-disulfide isomerase (PDI) catalyzes the formation and isomerization of Disulfides during oxidative protein folding. This process can be error-prone in its early stages, and any incorrect Disulfides that form must be rearranged to their native configuration. When the second cysteine (CGHC) in the PDI active site is mutated to Ser, the isomerase activity drops by 7–8-fold, and a covalent intermediate with the substrate accumulates. This led to the proposal that the second active site cysteine provides an escape mechanism, preventing PDI from becoming trapped with substrates that isomerize slowly (Walker, K. W., and Gilbert, H. F. (1997) J. Biol. Chem. 272, 8845–8848). Escape also reduces the substrate, and if it is invoked frequently, disulfide isomerization will involve cycles of reduction and reoxidation in preference to intramolecular isomerization of the PDI-bound substrate. Using a gel-shift assay that adds a polyethylene glycol-conjugated maleimide of 5 kDa for each sulfhydryl group, we find that PDI reduction and oxidation are kinetically competent and essential for isomerization. Oxidants inhibit isomerization and oxidize PDI when a redox buffer is not present to maintain the PDI redox state. Reductants also inhibit isomerization as they deplete oxidized PDI. These rapid cycles of PDI oxidation and reduction suggest that PDI catalyzes isomerization by trial and error, reducing Disulfides and oxidizing them in a different configuration. Disulfide reduction-reoxidation may set up critical folding intermediates for intramolecular isomerization, or it may serve as the only isomerization mechanism. In the absence of a redox buffer, these steady-state reduction-oxidation cycles can balance the redox state of PDI and support effective catalysis of disulfide isomerization.
