The Experts below are selected from a list of 105 Experts worldwide ranked by ideXlab platform
Bob B. Buchanan - One of the best experts on this subject based on the ideXlab platform.
-
A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Yves Balmer, William H. Vensel, William J. Hurkman, Wanda Manieri, Peter Schürmann, Bob B. BuchananAbstract:†‡ A growing number of processes throughout biology are regulated by redox via thiol– Disulfide exchange. This mechanism is particularly widespread in plants, where almost 200 Proteins have been linked to thioredoxin (Trx), a widely distributed small regulatory Disulfide Protein. The current study extends regulation by Trx to amyloplasts, organelles prevalent in heterotrophic plant tissues that, among other biosynthetic activities, catalyze the synthesis and storage of copious amounts of starch. Using proteomics and immunological methods, we identified the components of the ferredoxinTrx system (ferredoxin, ferredoxin–Trx reductase, and Trx), originally described for chloroplasts, in amyloplasts isolated from wheat starchy endosperm. Ferredoxin is reduced not by light, as in chloroplasts, but by metabolically generated NADPH via ferredoxin–NADP reductase. However, once reduced, ferredoxin appears to act as established for chloroplasts, i.e., via ferredoxin– Trx reductase and a Trx (m-type). A proteomics approach in combination with affinity chromatography and a fluorescent thiol probe led to the identification of 42 potential Trx target Proteins, 13 not previously recognized, including a major membrane transporter (Brittle-1 or ADP-glucose transporter). The Proteins function in a range of processes in addition to starch metabolism: biosynthesis of lipids, amino acids, and nucleotides; Protein folding; and several miscellaneous reactions. The results suggest a mechanism whereby light is initially recognized as a thiol signal in chloroplasts, then as a sugar during transit to the sink, where it is converted again to a thiol signal. In this way, amyloplast reactions in the grain can be coordinated with photosynthesis taking place in leaves. redox regulation target Proteins ferredoxin–thioredoxin reductase
-
Redox Regulation: A Broadening Horizon
Annual Review of Plant Biology, 2005Co-Authors: Bob B. Buchanan, Yves BalmerAbstract:Initially discovered in the context of photosynthesis, regulation by change in the redox state of thiol groups (S−S ↔ 2SH) is now known to occur throughout biology. Several systems, each linking a hydrogen donor to an intermediary Disulfide Protein, act to effect changes that alter the activity of target Proteins: the ferredoxin/thioredoxina small Protein, reduced enzymatically by NADPH or ferredoxin, that is active in thiol/Disulfide exchange and results in regulation or substrate conversion system, comprised of reduced ferredoxin, a thioredoxin, and the enzyme, ferredoxin-thioredoxin reductase; the NADP/thioredoxin system, including NADPH, a thioredoxin, and NADP-thioredoxin reductase; and the glutathione/glutaredoxina small Protein, reduced by glutathione, that is active in thiol/Disulfide exchange and results in regulation or substrate conversion system, composed of reduced glutathione and a glutaredoxin. A related Disulfide Protein, Protein Disulfide isomerase (PDI) acts in Protein assembly. Regulati...
-
Thioredoxin-linked mitigation of allergic responses to wheat (food allergyyallergen Disulfideyallergenic gliadins)
1997Co-Authors: Bob B. Buchanan, Rosa Lozano, M. Momma, L. FrickAbstract:Thioredoxin,aubiquitous12-kDaregulatory Disulfide Protein, was found to reduce Disulfide bonds of allergens (convert SOS to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strengthwasdeterminedbyskintestswithacaninemodelfor food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the Protein fractions extracted from wheat flour with the indi- catedsolventwasalsoassessed:thegliadins(ethanol)werethe strongest allergens, followed by glutenins (acetic acid), albu- mins (water), and globulins (salt water). Of the gliadins, the aand bfractions were most potent, followed by the gand v types. Thioredoxin mitigated the allergenicity associated with themajorProteinfractions—i.e,thegliadins(includingthe a, b, and g types) and the glutenins—but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP- thioredoxinreductaseorchemicallybydithiothreitol;reduced glutathione was without significant effect. As in previous studies,thioredoxinwasparticularlyeffectiveinthereduction of intramolecular (intrachain) Disulfide bonds. The present results demonstrate that the reduction of these Disulfide bondsisaccompaniedbyastatisticallysignificantdecreasein allergenicity of the active Proteins. This decrease occurs alongside the changes identified previously—i.e., increased susceptibilitytoproteolysisandheat,andalteredbiochemical activity. The findings open the door to the testing of the thioredoxinsystemintheproductionofhypoallergenic,more- digestible foods.
-
Thioredoxin-linked mitigation of allergic responses to wheat
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Bob B. Buchanan, Karoly Kobrehel, Rosa Lozano, M. Momma, C. Adamidi, Richard W. Ermel, Oscar L. FrickAbstract:Thioredoxin, a ubiquitous 12-kDa regulatory Disulfide Protein, was found to reduce Disulfide bonds of allergens (convert S—S to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the Protein fractions extracted from wheat flour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the α and β fractions were most potent, followed by the γ and ω types. Thioredoxin mitigated the allergenicity associated with the major Protein fractions—i.e, the gliadins (including the α, β, and γ types) and the glutenins—but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP–thioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) Disulfide bonds. The present results demonstrate that the reduction of these Disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active Proteins. This decrease occurs alongside the changes identified previously—i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, more-digestible foods.
-
The Ferredoxin-Thioredoxin System: Update on its Role in the Regulation of Oxygenic Photosynthesis
Advances in Molecular and Cell Biology, 1994Co-Authors: Bob B. BuchananAbstract:Publisher Summary The chapter describes the regulatory function of the ferredoxin–thioredoxin system in the carbon reactions of oxygenic photosynthesis process. In an outgrowth of the chloroplast research, thioredoxin has been found to function as a signal in seed germination. Here, thioredoxin acts by regulating (by reduction) the activity of a Disulfide Protein that inhibits α-amylase, and thereby controls the breakdown (mobilization) of starch. Such a mechanism contrasts to that for the activation of chloroplast enzymes in which thioredoxin acts by reducing defined Disulfide groups of the target enzymes independently of specific inhibitor Proteins. The function of thioredoxin in enzyme regulation complements its role in reducing storage Proteins, thereby, leading to their mobilization and use by the developing seedling.
Craig Harris - One of the best experts on this subject based on the ideXlab platform.
-
Differential antioxidant enzyme activities and glutathione content between rat and rabbit conceptuses.
Free Radical Biology and Medicine, 2001Co-Authors: Jason M. Hansen, Hyung Suk Choe, Edward W. Carney, Craig HarrisAbstract:Abstract Redox status regulates numerous cellular processes like transcription factor activation and binding, Protein folding, and calcium sequestration. Because the most abundant reducing equivalent in the cell is glutathione (GSH), it could play a role for teratogens that cause oxidative stress and disrupt pathways involved in differentiation and proliferation. Investigation of the redox status of two species that have demonstrated differential sensitivity to teratogens represents a novel approach for determining the role of redox alteration in teratogenesis. Furthermore, examining specific regions of the embryo may also help to explain why certain tissues are uniquely sensitive, while others are resistant to oxidative insult. In the presented study, New Zealand White rabbit (GD 12) and Sprague Dawley rat embryos (GD 13) were removed from the uterus on days of similar development. Each embryo was dissected into three portions—the limbs, the head, and the trunk. Samples were placed in the appropriate buffers for the measurement of both direct and indirect redox status contributors—GSH, cysteine, thioredoxin, glutathione Disulfide, Protein-glutathione mixed Disulfides, superoxide dismutase, glutathione peroxidase, and glutathione Disulfide reductase. Species comparison of whole embryos indicated that the rabbit embryo possesses a higher redox potential (more oxidative) than the rat embryo. Findings, in general, show that the rabbit may be more sensitive to redox-altering teratogens because it is inherently more pro-oxidizing and may be more easily perturbed resulting in misregulation of cellular processes. Differences were most apparent in the limb as compared to the embryonic head and trunk, where the rabbit limb has a significantly more pro-oxidizing redox environment than the rat limb. Species comparisons like these may help in the understanding of how redox shifts affect cellular processes and would contribute to regulation of biochemical and molecular events that may be associated with mechanisms of teratogenesis. These may contribute to a more complete rationale for choosing a species for study and provide a better correlation with human developmental toxicants.
-
Diamide-induced alterations of intracellular thiol status and the regulation of glucose metabolism in the developing rat conceptus in vitro.
Teratology, 1995Co-Authors: Roongrudee Hiranruengchok, Craig HarrisAbstract:Direct oxidation of embryonic reduced glutathione (GSH) by a thiol oxidant, diamide, has been demonstrated to result in in- creased glutathione Disulfide (GSSG) and Protein- glutathione mixed Disulfide (Protein-S-SG) forma- tion, which is accompanied by embryotoxicity and reductions in amniotic fluid volume. The altered functions of critical Proteins or enzymes caused by the formation of Protein-S-SG perturb cellular me- tabolism and may be involved in the embryotoxicity produced by GSH oxidation. The present study in- vestigates changes in the metabolism of glucose through glycolysis and the pentose phosphate shunt pathways (PPP) and their related enzymes under the oxidative conditions produced by dia- mide exposure in organogenesis-stage rat con- ceptus (gestational day 10) in vitro. The metabo- lism of glucose via the PPP, measured as amounts of C02 production from D-( 1 -'4C)-glucose, was significantly increased in the conceptus exposed to 100-500 pM diamide to levels 2.5-3-fold those of controls. It was found that these substantial in- creases in the PPP activity did not correlate well with a moderate activation of glucose 6-phosphate dehydrogenase (G6PD) activity, the key enzyme in the PPP pathway. Changes in glycolysis due to di- amide treatment were also determined by mea- surements of lactate production from D-(U-''c)- glucose. Production of lactate by the conceptus exposed to 250-500 pM diamide for 60 min was reduced (to approximately 54% of control values) concomitantly with a significant inhibition of the glycolytic enzymes, glyceraldehyde 3-phosphate dehydrogenase (GPD) and phosphofructokinase (PFK), indicating an overall decrease in glycolysis. Diamide was found to produce a differential effect on the enzymatic activities determined in this study, with greater degrees of inhibition seen in the tissue supernatants from the visceral yolk sac (VYS) com- pared to those from the embryo. Activities of GPD and PFK were decreased to approximately 22% and 43% control values, respectively, when deter- mined in the supernatants from the VYS of the con- ceptus exposed to 500 pM diamide for 60 min. In addition, more than 90% of the GPD activity in the VYS, but not the embryo, was rapidly inhibited by the thiol alkylating agent N-ethylmaleimide (NEM, 100 pM) within 15 min of the exposure. In contrast to diamide and NEM, no alterations in lactate pro- duction were seen in the conceptus treated with the GSH depletor L-buthionine-S,R-sulfoximine (1 mM) for 5 hr in the culture media. Further experiments demonstrated that the activity of the GPD, inhibited by a 30-min incubation with 500 pM diamide, can be reversed after removal of diamide and that this effect was potentiated by subsequent treatment with dithiothreitol (30 mM), a thiol reducing agent. These results indicated the involvement of thiol/di- sulfide status in regulation of the metabolism of glucose in the developing conceptus and support the hypothesis that GSH oxidation and Protein- S-SG formation could be a critical event associ- ated with mechanisms of embryotoxicity elicited by oxidative stress. It was suggested in this study that, under these experimental conditions, embryotoxi- city induced by diamide is primarily mediated via altered VYS functions, including disrupted energy production (glycolysis). o 1995 WiIey-Liss, Inc.
Lisandra L. Martin - One of the best experts on this subject based on the ideXlab platform.
-
Probing the stability of the Disulfide radical intermediate of thioredoxin using direct electrochemistry
Letters in Peptide Science, 2020Co-Authors: Daniel L. Johnson, Steven W. Polyak, John C. Wallace, Lisandra L. MartinAbstract:The original publication can be found at www.springerlink.comThioredoxin, a redox active Disulfide Protein, has been specifically immobilized at a modified gold electrode. The thioredoxin is uniquely oriented relative to the electrode surface via a histidine tag thereby enabling the redox mechanism of Protein to be examined. When scanning the applied potential in the negative direction (cathodic), two one-electron reduction waves can be observed. The first of these redox waves occurs at −90 mV and is electrochemically reversible at all scan rates whereas the second wave occurs at −433 mV is irreversible. These two processes are interpreted as the initial reduction of the Disulfide form of the Protein to a stable (reversible) semi-reduced radical anion intermediate, followed by an electrochemically irreversible process to form a fully reduced thioredoxin. These electron transfer characteristics suggest that a radical intermediate retaining the sulfur-sulfur bond is thermodynamically stable but the addition of a second electron results in bond scission.Daniel L. Johnson, Steven W. Polyak, John C. Wallace and Lisandra L. Marti
-
Probing the stability of the Disulfide radical intermediate of thioredoxin using direct electrochemistry
Letters in Peptide Science, 2003Co-Authors: Daniel L. Johnson, Steven W. Polyak, John C. Wallace, Lisandra L. MartinAbstract:Thioredoxin, a redox active Disulfide Protein, has been specifically immobilized at a modified gold electrode. The thioredoxin is uniquely oriented relative to the electrode surface via a histidine tag thereby enabling the redox mechanism of Protein to be examined. When scanning the applied potential in the negative direction (cathodic), two one-electron reduction waves can be observed. The first of these redox waves occurs at −90 mV and is electrochemically reversible at all scan rates whereas the second wave occurs at −433 mV is irreversible. These two processes are interpreted as the initial reduction of the Disulfide form of the Protein to a stable (reversible) semi-reduced radical anion intermediate, followed by an electrochemically irreversible process to form a fully reduced thioredoxin. These electron transfer characteristics suggest that a radical intermediate retaining the sulfur-sulfur bond is thermodynamically stable but the addition of a second electron results in bond scission.
Masaaki Hirose - One of the best experts on this subject based on the ideXlab platform.
-
Conformational state of ovalbumin at acidic pH as evaluated by a novel approach utilizing intrachain sulfhydryl-mixed Disulfide exchange reactions.
Biochemistry, 1998Co-Authors: Eizo Tatsumi, Daisuke Yoshimatsu, Masaaki HiroseAbstract:: Ovalbumin contains four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) and one cystine Disulfide (Cys73-Cys120). A highly reactive aromatic Disulfide, 2,2'-dipyridyl Disulfide, reacts specifically with Cys367 of ovalbumin at pH 2.2 generating a mixed Disulfide Protein derivative [Tatsumi, E., and Hirose, M. (1997) J. Biochem. 122, 300-308]. The mode of conformational fluctuation in ovalbumin was investigated at pH 2.2 using the mixed Disulfide derivatives of the cystine-intact and cystine-reduced Protein forms. In the presence of a high concentration of urea, both the mixed Disulfide derivatives underwent rapid cysteine sulfhydryl/mixed Disulfide exchanges, thereby releasing the quantitative amount of 2-thiopyridone. A peptide mapping analysis for Disulfide-forming cysteines revealed that this release was mostly accounted for by the nucleophile attack on the Cys367-mixed Disulfide by the nearest cysteine residue in the primary structure, Cys382. At the acidic pH, the exchange reaction was practically restricted to the cysteine sulfhydryl/mixed Disulfide exchanges; no other exchange reaction, such as the cysteine sulfhydryl/cystine Disulfide exchange reaction, was detected. In the absence of urea, the cystine-reduced form, but not the cystine-intact form, underwent significant sulfhydryl/mixed Disulfide exchange reactions at a physiological temperature, as determined by the release of 2-thiopyridone. A kinetic analysis for the generation of Disulfide-forming cysteines with Cys367 at 37 degreesC revealed that the rate for the intrachain exchange reaction was quite different for the five cysteine sulfhydryls. The effective concentrations of the five cysteine sulfhydryls relative to the Cys367-mixed Disulfide were determined by using three related model reactions: the obtained values were 11.4, 4.6, 15.2, 5.9, and 8.9 microM for Cys11, Cys30, Cys73, Cys120, and Cys382, respectively. Implications of the effective concentrations for the conformational state of acidic ovalbumin are discussed.
-
Highly Ordered Molten Globule—Like State of Ovalbumin at Acidic pH: Native-Like Fragmentation by Protease and Selective Modification of Cys367 with Dithiodipyridine
Journal of Biochemistry, 1997Co-Authors: Eizo Tatsumi, Masaaki HiroseAbstract:: Structural characteristics of ovalbumin at acidic pH were investigated by a variety of analytical approaches. At pH 2.2, the Protein appeared to assume a partially denatured, molten globule-like conformation as evaluated by the binding of a hydrophobic probe, anilino-1-naphthalene-8-sulfonate. The Protein was, however, resistant to proteolysis with pepsin under conditions in which the urea-denatured form was extensively hydrolyzed. Furthermore, under more drastic proteolytic conditions, the acid ovalbumin was specifically proteolyzed at the N-terminal site of Ala351, which is located in close proximity to the canonical serpin cleavage site Ala352-Ser353 that is known to be the cleavage site at neutral pH with subtilisin and elastase in native ovalbumin. Among the four cysteine residues (Cys11, Cys30, Cys367, and Cys382), which are all known to be buried in the native ovalbumin molecule, only Cys367 was specifically modified with 2,2'-dithiodipyridine, generating a mixed-Disulfide Protein derivative. Upon incubation of the derivative with a high concentration of L-cysteine, the thiopyridine mixed Disulfide did not undergo any bimolecular exchange reaction with the thiol in the absence of an added denaturant, indicating that the mixed Disulfide group is inaccessible. The far-UV CD spectra indicated that the native secondary structure is retained in either the modified or non-modified Protein; but as evaluated by the near-UV CD spectra, the asymmetric nature of aromatic side chains in the non-modified ovalbumin and of the mixed-Disulfide group in the modified Protein was almost lost at pH 2.2. These results are consistent with a highly ordered molten globule-like state for OVA at pH 2.2, in which side chains, but not the backbone chain, significantly fluctuate.
Yves Balmer - One of the best experts on this subject based on the ideXlab platform.
-
A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Yves Balmer, William H. Vensel, William J. Hurkman, Wanda Manieri, Peter Schürmann, Bob B. BuchananAbstract:†‡ A growing number of processes throughout biology are regulated by redox via thiol– Disulfide exchange. This mechanism is particularly widespread in plants, where almost 200 Proteins have been linked to thioredoxin (Trx), a widely distributed small regulatory Disulfide Protein. The current study extends regulation by Trx to amyloplasts, organelles prevalent in heterotrophic plant tissues that, among other biosynthetic activities, catalyze the synthesis and storage of copious amounts of starch. Using proteomics and immunological methods, we identified the components of the ferredoxinTrx system (ferredoxin, ferredoxin–Trx reductase, and Trx), originally described for chloroplasts, in amyloplasts isolated from wheat starchy endosperm. Ferredoxin is reduced not by light, as in chloroplasts, but by metabolically generated NADPH via ferredoxin–NADP reductase. However, once reduced, ferredoxin appears to act as established for chloroplasts, i.e., via ferredoxin– Trx reductase and a Trx (m-type). A proteomics approach in combination with affinity chromatography and a fluorescent thiol probe led to the identification of 42 potential Trx target Proteins, 13 not previously recognized, including a major membrane transporter (Brittle-1 or ADP-glucose transporter). The Proteins function in a range of processes in addition to starch metabolism: biosynthesis of lipids, amino acids, and nucleotides; Protein folding; and several miscellaneous reactions. The results suggest a mechanism whereby light is initially recognized as a thiol signal in chloroplasts, then as a sugar during transit to the sink, where it is converted again to a thiol signal. In this way, amyloplast reactions in the grain can be coordinated with photosynthesis taking place in leaves. redox regulation target Proteins ferredoxin–thioredoxin reductase
-
Redox Regulation: A Broadening Horizon
Annual Review of Plant Biology, 2005Co-Authors: Bob B. Buchanan, Yves BalmerAbstract:Initially discovered in the context of photosynthesis, regulation by change in the redox state of thiol groups (S−S ↔ 2SH) is now known to occur throughout biology. Several systems, each linking a hydrogen donor to an intermediary Disulfide Protein, act to effect changes that alter the activity of target Proteins: the ferredoxin/thioredoxina small Protein, reduced enzymatically by NADPH or ferredoxin, that is active in thiol/Disulfide exchange and results in regulation or substrate conversion system, comprised of reduced ferredoxin, a thioredoxin, and the enzyme, ferredoxin-thioredoxin reductase; the NADP/thioredoxin system, including NADPH, a thioredoxin, and NADP-thioredoxin reductase; and the glutathione/glutaredoxina small Protein, reduced by glutathione, that is active in thiol/Disulfide exchange and results in regulation or substrate conversion system, composed of reduced glutathione and a glutaredoxin. A related Disulfide Protein, Protein Disulfide isomerase (PDI) acts in Protein assembly. Regulati...
-
Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Yves Balmer, William H. Vensel, Charlene K. Tanaka, William J. Hurkman, Eric Gelhaye, Nicolas Rouhier, Jean-pierre Jacquot, Wanda Manieri, Peter Schürmann, Michel DrouxAbstract:Mitochondria contain thioredoxin (Trx), a regulatory Disulfide Protein, and an associated flavoenzyme, NADP/Trx reductase, which provide a link to NADPH in the organelle. Unlike animal and yeast counterparts, the function of Trx in plant mitochondria is largely unknown. Accordingly, we have applied recently devised proteomic approaches to identify soluble Trx-linked Proteins in mitochondria isolated from photosynthetic (pea and spinach leaves) and heterotrophic (potato tubers) sources. Application of the mitochondrial extracts to mutant Trx affinity columns in conjunction with proteomics led to the identification of 50 potential Trx-linked Proteins functional in 12 processes: photorespiration, citric acid cycle and associated reactions, lipid metabolism, electron transport, ATP synthesis/transformation, membrane transport, translation, Protein assembly/folding, nitrogen metabolism, sulfur metabolism, hormone synthesis, and stress-related reactions. Almost all of these targets were also identified by a fluorescent gel electrophoresis procedure in which reduction by Trx can be observed directly. In some cases, the processes targeted by Trx depended on the source of the mitochondria. The results support the view that Trx acts as a sensor and enables mitochondria to adjust key reactions in accord with prevailing redox state. These and earlier findings further suggest that, by sensing redox in chloroplasts and mitochondria, Trx enables the two organelles of photosynthetic tissues to communicate by means of a network of transportable metabolites such as dihydroxyacetone phosphate, malate, and glycolate. In this way, light absorbed and processed by means of chlorophyll can be perceived and function in regulating fundamental mitochondrial processes akin to its mode of action in chloroplasts.