Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas ma...Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.展开更多
Thioredoxin (Trx) is a small ubiquitous dithiol protein which together with the FADcontaining enzyme thioredoxin reductase (TR) and NADPH (the Trx system) is a hydrogen donor for ribonucleotide reductase essential for...Thioredoxin (Trx) is a small ubiquitous dithiol protein which together with the FADcontaining enzyme thioredoxin reductase (TR) and NADPH (the Trx system) is a hydrogen donor for ribonucleotide reductase essential for DNA synthesis and a general protein disulfide reductase involved in redox regulation. Selenite, selenodiglutathione (GS-Se-SG) and selenocystine are efficiently reduced by thioredoxins and also directly by NADPH and mammalian TR but not by the E. coli enzyme. Incubation of selenite or GS-Se-SG with the Trx system or with mammalian TR results in a rapid formation of selenide, which by redox cycling with oxygen may cause a large non-stoichiometric oxidation of NADPH. Selenocystine is efficiently reduced into two molecules of the selenol amino acid selenocysteine by mammalian TR with a Km-value (6μmol·L-1 ) and a high turnover number (kcat, 3200 min-1) almost identical to the natural substrate Trx-S2. TR also directly reduces lipid hydroperoxides and this peroxidase reaction is strongly stimulated by the presence of catalytic amounts of free selenocysteine. Glutaredoxin (Grx) which catalyzes GSH dependent disulfide reduction also via a redox-active disulfide and Trx are both efficient electron donors to the hut-nan plasrna glutathione peroxidase providing a mechanism by which human plasma glutathione peroxidase may reduce hydroperoxides in an environment almost free from glutathione. Selenate is reduced by Grx and Trx in the presence of GSH. The DNA-binding of the transcription factor AP-1 is strongly inhibited by GS-Se-SG and selenite. Furtherrnore, selenide formed by TR-mediated reduction of selenite and GS-Se-SG inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron. Mammalian TR with two subunits of 57 kDa has recently been cloned and shown to be homologous to glutathione reductase. The rat enzyme contains a selenocysteine residue in a unique Cterminal position and a conserved SEClS sequence directing insertion of the selenocysteine. The discovery of selenocysteine in mammalian TR may explain the broad substrate specificity of the enzyme and the requirement of seleflium for cell proliferation展开更多
Glutaredoxins （GRXs） are ubiquitous oxidoreductases that play a crucial role in response to oxidative stress by reducing disulfides in various organisms. In planta, three different GRX classes have been identified a...Glutaredoxins （GRXs） are ubiquitous oxidoreductases that play a crucial role in response to oxidative stress by reducing disulfides in various organisms. In planta, three different GRX classes have been identified according to their active site motifs. CPYC and CGFS classes are found in all organisms, whereas the CC-type class is specific for higher land plants. Recently, two Arabidopsis CC-type GRXs, ROXY1 and ROXY2, were shown to exert crucial functions in petal and anther initiation and differentiation. To analyze the function of CC-type GRXs in the distantly related monocots, we isolated and characterized OsROXY1 and OsROXY2-two rice homologs of ROXY1. Both genes are expressed in vegetative and reproductive stages. Although rice flower morphology is distinct from eudicots, OsROXY1/2 floral expression patterns are similar to their Arabidopsis counterparts ROXY1/2. Complementation experiments demonstrate that OsROXY1 and OsROXY2 can fully rescue the roxyl floral mutant phenotype. Overexpression of OsROXY1, OsROXY2, and ROXY1 in Arabidopsis causes similar vegetative and reproductive plant developmental defects. ROXY1 and its rice homologs thus exert a conserved function during eudicot and monocot flower development. Strikingly, overexpression of these CC-type GRXs also leads to an increased accumulation of hydrogen peroxide levels and hyper-susceptibility to infection from the necrotrophic pathogen Botrytis cinerea, revealing the importance of balanced redox processes in flower organ develop- ment and pathogen defence.展开更多
Glutaredoxins are small heat-stable oxidoreductases that transfer electrons from glutathione （GSH） to oxi- dized cysteine residues, thereby contributing to protein integrity and regulation. In Arabidopsis thaliana, ...Glutaredoxins are small heat-stable oxidoreductases that transfer electrons from glutathione （GSH） to oxi- dized cysteine residues, thereby contributing to protein integrity and regulation. In Arabidopsis thaliana, floral glutare- doxins ROXY1 and ROXY2 and pathogen-induced ROXY19/GRX480 interact with bZIP transcription factors of the TGACG （TGA） motif-binding family. ROXY1, ROXY2, and TGA factors PERIANTHIA, TGA9, and TGA10 play essential roles in floral development. In contrast, ectopically expressed ROXY19/GRX480 negatively regulates expression of jasmonic acid （JA）/ ethylene （ET）-induced defense genes through an unknown mechanism that requires clade II transcription factors TGA2, TGA5, and/or TGA6. Here, we report that at least 17 of the 21 land plant-specific glutaredoxins encoded in the Arabidopsis genome interact with TGA2 in a yeast-two-hybrid system. To investigate their capacity to interfere with the expression of JA/ET-induced genes, we developed a transient expression system. Activation of the ORA59 （OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF-domain protein 59） promoter by transcription factor EIN3 （ETHYLENE INSENSITVE 3） was sup- pressed by co-expressed ROXY19/GRX480. Suppression depended on the L＊＊LL motif in the C-terminus of ROXY19/ GRX480. This putative protein interaction domain was recently described as being essential for the TGA/ROXY interaction. Ten of the 17 tested ROXY proteins suppressed ORA59 promoter activity, which correlated with the presence of the C-terminal ALWL motif, which is essential for ROXY1 function in flower development. ROXY19/GRX480-mediated repres- sion depended on the GSH binding site, suggesting that redox modification of either TGA factors or as yet unknown target proteins is important for the suppression of ORA59 promoter activity.展开更多
A functional relationship between monothiol glutaredoxins and BolAs has been unraveled by genomic analyses and in several high-throughput studies. Phylogenetic analyses coupled to transient expression of green fluo- r...A functional relationship between monothiol glutaredoxins and BolAs has been unraveled by genomic analyses and in several high-throughput studies. Phylogenetic analyses coupled to transient expression of green fluo- rescent protein （GFP） fusions indicated that, in addition to the sulfurtransferase SufE1, which contains a C-terminal BolA domain, three BolA isoforms exist in Arabidopsis thaliana, BolA1 being plastidial, BolA2 nucleo-cytoplasmic, and BolA4 dual-targeted to mitochondria and plastids. Binary yeast two-hybrid experiments demonstrated that all BolAs and SufE 1, via its BolA domain, can interact with all monothiol glutaredoxins. Most interactions between protein couples of the same subcellular compartment have been confirmed by bimolecular fluorescence complementation. In vitro experiments indicated that monothiol glutaredoxins could regulate the redox state of BolA2 and SufE1, both proteins possessing a single conserved reactive cysteine. Indeed, a glutathionylated form of SufE1 lost its capacity to activate the cysteine desuifurase, Nfs2, but it is reactivated by plastidial glutaredoxins. Besides, a monomeric glutathionyiated form and a dimeric disulfide-bridged form of BolA2 can be preferentially reduced by the nucleo-cytoplasmic GrxS17. These results indicate that the glutaredoxin-BolA interaction occurs in several subcellular compartments and suggest that a redox regulation mechanism, disconnected from their capacity to form iron-sulfur cluster-bridged heterodimers, may be physiologically relevant for BolA2 and SufE1.展开更多
Holo glutaredoxin(Grx)is a homo-dimer that bridges a[2Fe-2S]cluster with two glutathione(GSH)ligands.In this study,both monothiol and dithiol holo Grxs are found capable of transferring their iron-sulfur(FeS)cluster t...Holo glutaredoxin(Grx)is a homo-dimer that bridges a[2Fe-2S]cluster with two glutathione(GSH)ligands.In this study,both monothiol and dithiol holo Grxs are found capable of transferring their iron-sulfur(FeS)cluster to an apo ferredoxin(Fdx)through direct interaction,regardless of FeS cluster stability in holo Grxs.The ligand GSH molecules in holo Grxs are unstable and can be exchanged with free GSH,which inhibits the FeS cluster transfer from holo Grxs to apo Fdx.This phenomenon suggests a novel role of GSH in FeS cluster trafficking.展开更多
Glutaredoxins(GRXs)play very important roles in maintaining intracellular redox homeostasis.In the present study,the full-length cDNA sequence encoding GRX2,named OfurGRX2(GenBank accession no.GU393246),was obtained f...Glutaredoxins(GRXs)play very important roles in maintaining intracellular redox homeostasis.In the present study,the full-length cDNA sequence encoding GRX2,named OfurGRX2(GenBank accession no.GU393246),was obtained from Ostrinia furnacalis,using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends.Sequence analysis revealed that the open reading frame of OfurGRX2 consists of 351 nucleotides encoding 116 amino acid residues with a predicted molecular weight of 12.6 kDa.Homolog research revealed that OfurGRX2 shares a common active site,CPYC/CPFC,with other insect counterparts.Expression profiles revealed that OfurGRX2 is a ubiquitous gene expressed in insect heads,fat bodies,epidermises,mid guts and muscles.The OfurGRX2 transcript peaked in 36-h larvae of 4th instars,and then suddenly declined in the molting stage.Hormone treatment experiments revealed that 20-hydroxyecodyson(20e)significantly induces the expression of the OfurGRX2 transcript,whereas juvenile hormone(JH)counteracts 20e effects.Adverse stress factors(including starvation,ultraviolet light,mechanical injury,Escherichia coli exposure,and high and low temperatures)dramatically induced OfurGRXGRX2 transcript expression,which confirmed for the first time that GRX2 play important roles in insecta during exposure to adverse environments.展开更多
The active center of human glutaredoxin(hGrx1)shares a common thioredoxin fold and specific affinity for substrate glutathione (GSH)with natural glutathione peroxidase(GPx).hGrx1 was redesigned to introduce the cataly...The active center of human glutaredoxin(hGrx1)shares a common thioredoxin fold and specific affinity for substrate glutathione (GSH)with natural glutathione peroxidase(GPx).hGrx1 was redesigned to introduce the catalytic selenocysteine residue to imi- tate the function of antioxidant selenoenzyme GPx in vivo.The human hGrx1 scaffold is a good candidate for potential medical application compared with other animal-originated protein scaffolds.Two consecutive rare codons(AGG-AGG)in the open reading frame of hGrx1 mRNA encoding Arg26-Arg27 residues can reduce seleno-hGrx1 expression level significantly in the Cys auxotrophic Escherichia coli strain BL21cysE51.Therefore,we optimized the rare codons,which resulted in a remarkable in- crease of the expression level in the Cys auxotrophic cells,which may be sufficient for future medical production.The engineered artificial selenoenzyme displays high GPx catalytic activity,rivaling that of some natural GPx proteins.Kinetic analysis of the engineered seleno-hGrx1 showed a typical ping-pong kinetic mechanism;its catalytic properties are similar to those of some nat- urally occurring GPx proteins.展开更多
基金Supported by The Swedish Research Council Medicine,No.3529The Swedish Cancer Society,No.961The Wallenberg Foundation
文摘Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.
文摘Thioredoxin (Trx) is a small ubiquitous dithiol protein which together with the FADcontaining enzyme thioredoxin reductase (TR) and NADPH (the Trx system) is a hydrogen donor for ribonucleotide reductase essential for DNA synthesis and a general protein disulfide reductase involved in redox regulation. Selenite, selenodiglutathione (GS-Se-SG) and selenocystine are efficiently reduced by thioredoxins and also directly by NADPH and mammalian TR but not by the E. coli enzyme. Incubation of selenite or GS-Se-SG with the Trx system or with mammalian TR results in a rapid formation of selenide, which by redox cycling with oxygen may cause a large non-stoichiometric oxidation of NADPH. Selenocystine is efficiently reduced into two molecules of the selenol amino acid selenocysteine by mammalian TR with a Km-value (6μmol·L-1 ) and a high turnover number (kcat, 3200 min-1) almost identical to the natural substrate Trx-S2. TR also directly reduces lipid hydroperoxides and this peroxidase reaction is strongly stimulated by the presence of catalytic amounts of free selenocysteine. Glutaredoxin (Grx) which catalyzes GSH dependent disulfide reduction also via a redox-active disulfide and Trx are both efficient electron donors to the hut-nan plasrna glutathione peroxidase providing a mechanism by which human plasma glutathione peroxidase may reduce hydroperoxides in an environment almost free from glutathione. Selenate is reduced by Grx and Trx in the presence of GSH. The DNA-binding of the transcription factor AP-1 is strongly inhibited by GS-Se-SG and selenite. Furtherrnore, selenide formed by TR-mediated reduction of selenite and GS-Se-SG inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron. Mammalian TR with two subunits of 57 kDa has recently been cloned and shown to be homologous to glutathione reductase. The rat enzyme contains a selenocysteine residue in a unique Cterminal position and a conserved SEClS sequence directing insertion of the selenocysteine. The discovery of selenocysteine in mammalian TR may explain the broad substrate specificity of the enzyme and the requirement of seleflium for cell proliferation
文摘Glutaredoxins （GRXs） are ubiquitous oxidoreductases that play a crucial role in response to oxidative stress by reducing disulfides in various organisms. In planta, three different GRX classes have been identified according to their active site motifs. CPYC and CGFS classes are found in all organisms, whereas the CC-type class is specific for higher land plants. Recently, two Arabidopsis CC-type GRXs, ROXY1 and ROXY2, were shown to exert crucial functions in petal and anther initiation and differentiation. To analyze the function of CC-type GRXs in the distantly related monocots, we isolated and characterized OsROXY1 and OsROXY2-two rice homologs of ROXY1. Both genes are expressed in vegetative and reproductive stages. Although rice flower morphology is distinct from eudicots, OsROXY1/2 floral expression patterns are similar to their Arabidopsis counterparts ROXY1/2. Complementation experiments demonstrate that OsROXY1 and OsROXY2 can fully rescue the roxyl floral mutant phenotype. Overexpression of OsROXY1, OsROXY2, and ROXY1 in Arabidopsis causes similar vegetative and reproductive plant developmental defects. ROXY1 and its rice homologs thus exert a conserved function during eudicot and monocot flower development. Strikingly, overexpression of these CC-type GRXs also leads to an increased accumulation of hydrogen peroxide levels and hyper-susceptibility to infection from the necrotrophic pathogen Botrytis cinerea, revealing the importance of balanced redox processes in flower organ develop- ment and pathogen defence.
文摘Glutaredoxins are small heat-stable oxidoreductases that transfer electrons from glutathione （GSH） to oxi- dized cysteine residues, thereby contributing to protein integrity and regulation. In Arabidopsis thaliana, floral glutare- doxins ROXY1 and ROXY2 and pathogen-induced ROXY19/GRX480 interact with bZIP transcription factors of the TGACG （TGA） motif-binding family. ROXY1, ROXY2, and TGA factors PERIANTHIA, TGA9, and TGA10 play essential roles in floral development. In contrast, ectopically expressed ROXY19/GRX480 negatively regulates expression of jasmonic acid （JA）/ ethylene （ET）-induced defense genes through an unknown mechanism that requires clade II transcription factors TGA2, TGA5, and/or TGA6. Here, we report that at least 17 of the 21 land plant-specific glutaredoxins encoded in the Arabidopsis genome interact with TGA2 in a yeast-two-hybrid system. To investigate their capacity to interfere with the expression of JA/ET-induced genes, we developed a transient expression system. Activation of the ORA59 （OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF-domain protein 59） promoter by transcription factor EIN3 （ETHYLENE INSENSITVE 3） was sup- pressed by co-expressed ROXY19/GRX480. Suppression depended on the L＊＊LL motif in the C-terminus of ROXY19/ GRX480. This putative protein interaction domain was recently described as being essential for the TGA/ROXY interaction. Ten of the 17 tested ROXY proteins suppressed ORA59 promoter activity, which correlated with the presence of the C-terminal ALWL motif, which is essential for ROXY1 function in flower development. ROXY19/GRX480-mediated repres- sion depended on the GSH binding site, suggesting that redox modification of either TGA factors or as yet unknown target proteins is important for the suppression of ORA59 promoter activity.
文摘A functional relationship between monothiol glutaredoxins and BolAs has been unraveled by genomic analyses and in several high-throughput studies. Phylogenetic analyses coupled to transient expression of green fluo- rescent protein （GFP） fusions indicated that, in addition to the sulfurtransferase SufE1, which contains a C-terminal BolA domain, three BolA isoforms exist in Arabidopsis thaliana, BolA1 being plastidial, BolA2 nucleo-cytoplasmic, and BolA4 dual-targeted to mitochondria and plastids. Binary yeast two-hybrid experiments demonstrated that all BolAs and SufE 1, via its BolA domain, can interact with all monothiol glutaredoxins. Most interactions between protein couples of the same subcellular compartment have been confirmed by bimolecular fluorescence complementation. In vitro experiments indicated that monothiol glutaredoxins could regulate the redox state of BolA2 and SufE1, both proteins possessing a single conserved reactive cysteine. Indeed, a glutathionylated form of SufE1 lost its capacity to activate the cysteine desuifurase, Nfs2, but it is reactivated by plastidial glutaredoxins. Besides, a monomeric glutathionyiated form and a dimeric disulfide-bridged form of BolA2 can be preferentially reduced by the nucleo-cytoplasmic GrxS17. These results indicate that the glutaredoxin-BolA interaction occurs in several subcellular compartments and suggest that a redox regulation mechanism, disconnected from their capacity to form iron-sulfur cluster-bridged heterodimers, may be physiologically relevant for BolA2 and SufE1.
基金supported by grants from Minis-try of Science and Technology of China(Nos.2012CB910703 and 2006DFA31210)National Natural Science Foundation of China(Grant No.30570353)to BXthe ANR Grant(No.2010BLAN1616)to NR and JPJ.
文摘Holo glutaredoxin(Grx)is a homo-dimer that bridges a[2Fe-2S]cluster with two glutathione(GSH)ligands.In this study,both monothiol and dithiol holo Grxs are found capable of transferring their iron-sulfur(FeS)cluster to an apo ferredoxin(Fdx)through direct interaction,regardless of FeS cluster stability in holo Grxs.The ligand GSH molecules in holo Grxs are unstable and can be exchanged with free GSH,which inhibits the FeS cluster transfer from holo Grxs to apo Fdx.This phenomenon suggests a novel role of GSH in FeS cluster trafficking.
基金supported by a National Corn Industry System Grant(NYCYTX-02)by the Natural Science Foundation of Henan Province(No.2011B180021).
文摘Glutaredoxins(GRXs)play very important roles in maintaining intracellular redox homeostasis.In the present study,the full-length cDNA sequence encoding GRX2,named OfurGRX2(GenBank accession no.GU393246),was obtained from Ostrinia furnacalis,using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends.Sequence analysis revealed that the open reading frame of OfurGRX2 consists of 351 nucleotides encoding 116 amino acid residues with a predicted molecular weight of 12.6 kDa.Homolog research revealed that OfurGRX2 shares a common active site,CPYC/CPFC,with other insect counterparts.Expression profiles revealed that OfurGRX2 is a ubiquitous gene expressed in insect heads,fat bodies,epidermises,mid guts and muscles.The OfurGRX2 transcript peaked in 36-h larvae of 4th instars,and then suddenly declined in the molting stage.Hormone treatment experiments revealed that 20-hydroxyecodyson(20e)significantly induces the expression of the OfurGRX2 transcript,whereas juvenile hormone(JH)counteracts 20e effects.Adverse stress factors(including starvation,ultraviolet light,mechanical injury,Escherichia coli exposure,and high and low temperatures)dramatically induced OfurGRXGRX2 transcript expression,which confirmed for the first time that GRX2 play important roles in insecta during exposure to adverse environments.
基金supported by the National Natural Science Foundation of China(91027023,20874036,20921003,and 21004028)the Natural Science Foundation for the Youth(21004028)+2 种基金the Natural Science Foundation of China for Outstanding Younger Scientist(20725415)the 111 pro-ject(B06009)the National Basic Research Program(2007CB808006)
文摘The active center of human glutaredoxin(hGrx1)shares a common thioredoxin fold and specific affinity for substrate glutathione (GSH)with natural glutathione peroxidase(GPx).hGrx1 was redesigned to introduce the catalytic selenocysteine residue to imi- tate the function of antioxidant selenoenzyme GPx in vivo.The human hGrx1 scaffold is a good candidate for potential medical application compared with other animal-originated protein scaffolds.Two consecutive rare codons(AGG-AGG)in the open reading frame of hGrx1 mRNA encoding Arg26-Arg27 residues can reduce seleno-hGrx1 expression level significantly in the Cys auxotrophic Escherichia coli strain BL21cysE51.Therefore,we optimized the rare codons,which resulted in a remarkable in- crease of the expression level in the Cys auxotrophic cells,which may be sufficient for future medical production.The engineered artificial selenoenzyme displays high GPx catalytic activity,rivaling that of some natural GPx proteins.Kinetic analysis of the engineered seleno-hGrx1 showed a typical ping-pong kinetic mechanism;its catalytic properties are similar to those of some nat- urally occurring GPx proteins.
基金Supported by Postdoctoral Researcher of Heilongjiang Postdoctoral Management Office(No.LBH-Q16225)Qiqihar Medical University(No.QY2016LX-01 and QY2016B-36)~~
