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Vadim N. Gladyshev - One of the best experts on this subject based on the ideXlab platform.
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an nmr based biosensor to measure stereo specific Methionine sulfoxide reductase msr activities in vitro and in vivo
Chemistry: A European Journal, 2020Co-Authors: Carolina Sanchezlopez, Natalia Labadie, Franco Biglione, Bruno Manta, Reeba Susan Jacob, Salim Abdelilahseyfried, Philipp Selenko, Verónica A. Lombardo, Vadim N. Gladyshev, Andres BinolfiAbstract:Oxidation of protein Methionines to Methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to Methionine by two families of conserved Methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S - or R -diastereoisomers of Methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments, including cell lysates and live zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways.
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an nmr based biosensor to measure stereospecific Methionine sulfoxide reductase activities in vitro and in vivo
Chemistry: A European Journal, 2020Co-Authors: Magda Carolina Sanchezlopez, Natalia Labadie, Franco Biglione, Bruno Manta, Reeba Susan Jacob, Salim Abdelilahseyfried, Verónica A. Lombardo, Vadim N. Gladyshev, Philipp SelenkoAbstract:Oxidation of protein Methionines to Methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to Methionine by two families of conserved Methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of Methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments, including cell lysates and live zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways.
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an nmr based biosensor to measure stereo specific Methionine sulfoxide reductase msr activities in vitro and in vivo
bioRxiv, 2020Co-Authors: Magda Carolina Sanchezlopez, Natalia Labadie, Franco Biglione, Bruno Manta, Reeba Susan Jacob, Salim Abdelilahseyfried, Philipp Selenko, Verónica A. Lombardo, Vadim N. Gladyshev, Andres BinolfiAbstract:Oxidation of protein Methionines to Methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to Methionine by two families of conserved Methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of Methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments that range from native cell lysates to zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways. Our approach further extends high-resolution in-cell NMR measurements of exogenously delivered biomolecules to an entire multicellular organism.
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functions and evolution of selenoprotein Methionine sulfoxide reductases
Biochimica et Biophysica Acta, 2009Co-Authors: Alexander Dikiy, Vadim N. GladyshevAbstract:Methionine sulfoxide reductases (Msrs) are thiol-dependent enzymes which catalyze conversion of Methionine sulfoxide to Methionine. Three Msr families, MsrA, MsrB, and fRMsr, are known. MsrA and MsrB are responsible for the reduction of Methionine-S-sulfoxide and MethionineR-sulfoxide residues in proteins, respectively, whereas fRMsr reduces free Methionine-R-sulfoxide. Besides acting on proteins, MsrA can additionally reduce free Methionine-S-sulfoxide. Some MsrAs and MsrBs evolved to utilize catalytic selenocysteine. This includes MsrB1, which is a major MsrB in cytosol and nucleus in mammalian cells. Specialized machinery is used for insertion of selenocysteine into MsrB1 and other selenoproteins at in-frame UGA codons. Selenocysteine offers catalytic advantage to the protein repair function of Msrs, but also makes these proteins dependent on the supply of selenium and requires adjustments in their strategies for regeneration of active enzymes. Msrs have roles in protecting cellular proteins from oxidative stress and through this function they may regulate lifespan in several model organisms.
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functional analysis of free Methionine r sulfoxide reductase from saccharomyces cerevisiae
Journal of Biological Chemistry, 2009Co-Authors: Dung Tien Le, Stefano M Marino, Yan Zhang, Dmitri E Fomenko, Alaattin Kaya, Elise Hacioglu, Geun Hee Kwak, Vadim N. GladyshevAbstract:Methionine sulfoxide reductases (Msrs) are oxidoreductases that catalyze thiol-dependent reduction of oxidized Methionines. MsrA and MsrB are the best known Msrs that repair Methionine-S-sulfoxide (Met-S-SO) and Methionine-R-sulfoxide (Met-R-SO) residues in proteins, respectively. In addition, an Escherichia coli enzyme specific for free Met-R-SO, designated fRMsr, was recently discovered. In this work, we carried out comparative genomic and experimental analyses to examine occurrence, evolution, and function of fRMsr. This protein is present in single copies and two mutually exclusive subtypes in about half of prokaryotes and unicellular eukaryotes but is missing in higher plants and animals. A Saccharomyces cerevisiae fRMsr homolog was found to reduce free Met-R-SO but not free Met-S-SO or dabsyl-Met-R-SO. fRMsr was responsible for growth of yeast cells on Met-R-SO, and the double fRMsr/MsrA mutant could not grow on a mixture of Methionine sulfoxides. However, in the presence of Methionine, even the triple fRMsr/MsrA/MsrB mutant was viable. In addition, fRMsr deletion strain showed an increased sensitivity to oxidative stress and a decreased life span, whereas overexpression of fRMsr conferred higher resistance to oxidants. Molecular modeling and cysteine residue targeting by thioredoxin pointed to Cys101 as catalytic and Cys125 as resolving residues in yeast fRMsr. These residues as well as a third Cys, resolving Cys91, clustered in the structure, and each was required for the catalytic activity of the enzyme. The data show that fRMsr is the main enzyme responsible for the reduction of free Met-R-SO in S. cerevisiae.
Cecilia Sundby - One of the best experts on this subject based on the ideXlab platform.
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Conserved Methionines in chloroplasts.
Biochimica et biophysica acta, 2005Co-Authors: Cecilia Sundby, Niklas Gustavsson, Ulrika Harndahl, Emma Åhrman, Denis J MurphyAbstract:Heat shock proteins counteract heat and oxidative stress. In chloroplasts, a small heat shock protein (Hsp21) contains a set of conserved Methionines, which date back to early in the emergence of terrestrial plants. Methionines M49, M52, M55, M59, M62, M67 are located on one side of an amphipathic helix, which may fold back over two other conserved Methionines (M97 and M101), to form a binding groove lined with Methionines, for sequence-independent recognition of peptides with an overall hydrophobic character. The sHsps protect other proteins from aggregation by binding to their hydrophobic surfaces, which become exposed under stress. Data are presented showing that keeping the conserved Methionines in Hsp21 in a reduced form is a prerequisite to maintain such binding. The chloroplast generates reactive oxygen species under both stress and unstressed conditions, but this organelle is also a highly reducing cellular compartment. Chloroplasts contain a specialized isoform of the enzyme, peptide Methionine sulfoxide reductase, the expression of which is light-induced. Recombinant proteins were used to measure that this reductase can restore Hsp21 Methionines after sulfoxidation. This paper also describes how Methionine sulfoxidation-reduction can be directly assessed by mass spectrometry, how Methionine-to-leucine substitution affects Hsp21, and discusses the possible role for an Hsp21 Methionine sulfoxidation-reduction cycle in quenching reactive oxygen species.
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a peptide Methionine sulfoxide reductase highly expressed in photosynthetic tissue in arabidopsis thaliana can protect the chaperone like activity of a chloroplast localized small heat shock protein
Plant Journal, 2002Co-Authors: Niklas Gustavsson, Bas P A Kokke, Ulrika Harndahl, Maria Silow, Ulrike Bechtold, Zaruhi Poghosyan, Wilbert C Boelens, Denis J Murphy, Cecilia SundbyAbstract:The oxidation of Methionine residues in proteins to Methionine sulfoxides occurs frequently and protein repair by reduction of the Methionine sulfoxides is mediated by an enzyme, peptide Methionine sulfoxide reductase (PMSR, EC 1.8.4.6), universally present in the genomes of all so far sequenced organisms. Recently, five PMSR-like genes were identified in Arabidopsis thaliana, including one plastidic isoform, chloroplast localised plastidial peptide Methionine sulfoxide reductase (pPMSR) that was chloroplast-localized and highly expressed in actively photosynthesizing tissue (Sadanandom A et al., 2000). However, no endogenous substrate to the pPMSR was identified. Here we report that a set of highly conserved Methionine residues in Hsp21, a chloroplast-localized small heat shock protein, can become sulfoxidized and thereafter reduced back to Methionines by this pPMSR. The pPMSR activity was evaluated using recombinantly expressed pPMSR and Hsp21 from Arabidopsis thaliana and a direct detection of Methionine sulfoxides in Hsp21 by mass spectrometry. The pPMSR-catalyzed reduction of Hsp21 Methionine sulfoxides occurred on a minute time-scale, was ultimately DTT-dependent and led to recovery of Hsp21 conformation and chaperone-like activity, both of which are lost upon Methionine sulfoxidation (Harndahl et al., 2001). These data indicate that one important function of pPMSR may be to prevent inactivation of Hsp21 by Methionine sulfoxidation, since small heat shock proteins are crucial for cellular resistance to oxidative stress. (Less)
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The chaperone-like activity of a small heat shock protein is lost after sulfoxidation of conserved Methionines in a surface-exposed amphipathic alpha-helix.
Biochimica et biophysica acta, 2001Co-Authors: Ulrika Harndahl, Kristina Berggren, N Gustavsson, Sara Linse, Bas P A Kokke, Wilbert C Boelens, Folke Tjerneld, Cecilia SundbyAbstract:The small heat shock proteins (sHsps) possess a chaperone-like activity which prevents aggregation of other proteins during transient heat or oxidative stress. The sHsps bind, onto their surface, molten globule forms of other proteins, thereby keeping them in a refolding competent state. In Hsp21, a chloroplast-located sHsp in all higher plants, there is a highly conserved region forming an amphipathic alpha-helix with several Methionines on the hydrophobic side according to secondary structure prediction. This paper describes how sulfoxidation of the Methionines in this amphipathic alpha-helix caused conformational changes and a reduction in the Hsp21 oligomer size, and a complete loss of the chaperone-like activity. Concomitantly, there was a loss of an outer-surface located alpha-helix as determined by limited proteolysis and circular dichroism spectroscopy. The present data indicate that the Methionine-rich amphipathic alpha-helix, a motif of unknown physiological significance which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone-like activity of Hsp21 very dependent on the chloroplast redox state.
Ulrika Harndahl - One of the best experts on this subject based on the ideXlab platform.
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Conserved Methionines in chloroplasts.
Biochimica et biophysica acta, 2005Co-Authors: Cecilia Sundby, Niklas Gustavsson, Ulrika Harndahl, Emma Åhrman, Denis J MurphyAbstract:Heat shock proteins counteract heat and oxidative stress. In chloroplasts, a small heat shock protein (Hsp21) contains a set of conserved Methionines, which date back to early in the emergence of terrestrial plants. Methionines M49, M52, M55, M59, M62, M67 are located on one side of an amphipathic helix, which may fold back over two other conserved Methionines (M97 and M101), to form a binding groove lined with Methionines, for sequence-independent recognition of peptides with an overall hydrophobic character. The sHsps protect other proteins from aggregation by binding to their hydrophobic surfaces, which become exposed under stress. Data are presented showing that keeping the conserved Methionines in Hsp21 in a reduced form is a prerequisite to maintain such binding. The chloroplast generates reactive oxygen species under both stress and unstressed conditions, but this organelle is also a highly reducing cellular compartment. Chloroplasts contain a specialized isoform of the enzyme, peptide Methionine sulfoxide reductase, the expression of which is light-induced. Recombinant proteins were used to measure that this reductase can restore Hsp21 Methionines after sulfoxidation. This paper also describes how Methionine sulfoxidation-reduction can be directly assessed by mass spectrometry, how Methionine-to-leucine substitution affects Hsp21, and discusses the possible role for an Hsp21 Methionine sulfoxidation-reduction cycle in quenching reactive oxygen species.
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a peptide Methionine sulfoxide reductase highly expressed in photosynthetic tissue in arabidopsis thaliana can protect the chaperone like activity of a chloroplast localized small heat shock protein
Plant Journal, 2002Co-Authors: Niklas Gustavsson, Bas P A Kokke, Ulrika Harndahl, Maria Silow, Ulrike Bechtold, Zaruhi Poghosyan, Wilbert C Boelens, Denis J Murphy, Cecilia SundbyAbstract:The oxidation of Methionine residues in proteins to Methionine sulfoxides occurs frequently and protein repair by reduction of the Methionine sulfoxides is mediated by an enzyme, peptide Methionine sulfoxide reductase (PMSR, EC 1.8.4.6), universally present in the genomes of all so far sequenced organisms. Recently, five PMSR-like genes were identified in Arabidopsis thaliana, including one plastidic isoform, chloroplast localised plastidial peptide Methionine sulfoxide reductase (pPMSR) that was chloroplast-localized and highly expressed in actively photosynthesizing tissue (Sadanandom A et al., 2000). However, no endogenous substrate to the pPMSR was identified. Here we report that a set of highly conserved Methionine residues in Hsp21, a chloroplast-localized small heat shock protein, can become sulfoxidized and thereafter reduced back to Methionines by this pPMSR. The pPMSR activity was evaluated using recombinantly expressed pPMSR and Hsp21 from Arabidopsis thaliana and a direct detection of Methionine sulfoxides in Hsp21 by mass spectrometry. The pPMSR-catalyzed reduction of Hsp21 Methionine sulfoxides occurred on a minute time-scale, was ultimately DTT-dependent and led to recovery of Hsp21 conformation and chaperone-like activity, both of which are lost upon Methionine sulfoxidation (Harndahl et al., 2001). These data indicate that one important function of pPMSR may be to prevent inactivation of Hsp21 by Methionine sulfoxidation, since small heat shock proteins are crucial for cellular resistance to oxidative stress. (Less)
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The chaperone-like activity of a small heat shock protein is lost after sulfoxidation of conserved Methionines in a surface-exposed amphipathic alpha-helix.
Biochimica et biophysica acta, 2001Co-Authors: Ulrika Harndahl, Kristina Berggren, N Gustavsson, Sara Linse, Bas P A Kokke, Wilbert C Boelens, Folke Tjerneld, Cecilia SundbyAbstract:The small heat shock proteins (sHsps) possess a chaperone-like activity which prevents aggregation of other proteins during transient heat or oxidative stress. The sHsps bind, onto their surface, molten globule forms of other proteins, thereby keeping them in a refolding competent state. In Hsp21, a chloroplast-located sHsp in all higher plants, there is a highly conserved region forming an amphipathic alpha-helix with several Methionines on the hydrophobic side according to secondary structure prediction. This paper describes how sulfoxidation of the Methionines in this amphipathic alpha-helix caused conformational changes and a reduction in the Hsp21 oligomer size, and a complete loss of the chaperone-like activity. Concomitantly, there was a loss of an outer-surface located alpha-helix as determined by limited proteolysis and circular dichroism spectroscopy. The present data indicate that the Methionine-rich amphipathic alpha-helix, a motif of unknown physiological significance which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone-like activity of Hsp21 very dependent on the chloroplast redox state.
Aiko Amagai - One of the best experts on this subject based on the ideXlab platform.
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The ethylene action in the development of cellular slime molds: an analogy to higher plants
Protoplasma, 1992Co-Authors: Aiko Amagai, Y. MaedaAbstract:The cellular slime mold Dictyostelium mucoroides -7 (Dm 7) and its mutant (MF 1) exhibit sexual or asexual development depending upon culture conditions. During the sexual cycle macrocyst formation occurs, whereas sorocarps containing spores and stalk cells are asexually formed. As previously reported, the macrocyst formation is marked by the emergence of true zygotes, and is induced by a potent plant hormone, ethylene. The concentration of ethylene required for macrocyst induction was determined to establish the similarity of ethylene action between this organism and higher plants. Macrocysts are induced by low (1 μl/l) exogenous concentrations of ethylene. Higher concentrations (10–1,000 ul/l) also gave essentially the same inductive activity. Ethionine, an analogue of Methionine, was found to inhibit zygote formation during sexual development through its interference with ethylene production by Dm 7 and MF 1 cells. In fact, the inhibitory effect of ethionine was mostly nullified by the application of ethylene, S-adenosyl-L-Methionine, or 1-aminocyclopropane-1-carboxylic acid. Taken together these results suggest that both the effective concentration of ethylene and the pathway of ethylene biosynthesis in D. mucoroides may be similar to those in higher plants. Ethylene was also found to be produced in various species and strains of cellular slime molds, even during the asexual process. The possible functions of ethylene in the asexual development are discussed in relation to cell aggregation and differentiation.
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Induction of Heterothallic and Homothallic Zygotes in Dictyostelium discoideum by Ethylene
Development Growth & Differentiation, 1992Co-Authors: Aiko AmagaiAbstract:Dictyostelium mucoroides-7 (Dm7) and a mutant (MF1) derived from it exhibit homothallic macrocyst formation in the sexual process. As previously shown, the zygote formation during macrocyst formation is induced by a potent plant hormone, ethylene. The present work was undertaken to know if ethylene is also involved in heterothallic and homothallic macrocyst formation in D. discoideum. In heterothallic macrocyst formation between NC4 and V12M2 cells, ethionine, an analogue of Methionine, inhibits macrocyst formation through arresting specifically the acquisition process of fusion competence. Such an inhibitory effect of ethionine was almost completely cancelled by co-application of ACC (1-aminocyclopropane-1-carboxylic acid), the immediate precursor of ethylene. Essentially the same effects of ethionine and ACC were also noticed on homothallic macrocyst formation in D. discoideum AC4. Thus it seems most likely that ethylene is required for the acquisition of fusion competence during macrocyst formation, and that in a variety of strains examined there is a common mechanism regulated by ethylene, beyond the difference of sexual modes.
Masatoshi Mita - One of the best experts on this subject based on the ideXlab platform.
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Involvement of cyclic adenosine 3',5'-monophosphate in methylation during 1-methyladenine production by starfish ovarian follicle cells.
General and Comparative Endocrinology, 1992Co-Authors: Masatoshi MitaAbstract:Resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeAde) produced by ovarian follicle cells under the influence of a gonad-stimulating substance (GSS). With respect to 1-MeAde production, the effect of GSS on follicle cells results in the receptor-mediated formation of cyclic AMP (cAMP). It has also been reported that methylation is involved in 1-MeAde production by GSS. This study was undertaken to determine whether cAMP is the agent responsible for mediating methylation in 1-MeAde biosynthesis by isolated follicle cells of the starfish Asterina pectinifera. Methionine and selenoMethionine enhanced 1-MeAde production by GSS in follicle cells. These stimulatory effects were dependent on the GSS concentration. Production of 1-MeAde by GSS was inhibited by ethionine and selenoethionine, competitive inhibitors of Methionine. Like GSS, 1-MeAde production induced by concanavalin A, trypsin, and 3-isobutyl-1-methylxanthine (IBMX), which stimulated cAMP accumulation in follicle cells, was influenced by Methionine and its related compounds. In contrast, although 1-methyladenosine (1-MeAde-R) induced 1-MeAde production by follicle cells without increasing cAMP levels, Methionine and its related compounds had no effect. Use of [methyl-14C]Methionine showed that a radiolabel was incorporated into 1-MeAde during incubation with GSS and IBMX, but not with 1-MeAde-R. These results strongly suggest that cAMP plays an important role in the process of methylation during 1-MeAde biosynthesis induced by GSS.
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Methylation during 1-methyladenine production by starfish ovarian follicle cells
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1991Co-Authors: Masatoshi MitaAbstract:Abstract 1. 1. Under the influence of gonad-stimulating substance (GSS), ovarian follicle cells of the starfish, Asterina pectinifera , produce 1-methyladenine (1-MeAde) for resumption of meiosis in oocytes. As a methyl donor, Methionine increased GSS-induced 1-MeAde production in follicle cells, but 5-methyltetrahydrofolate or homarine had no effect. 2. 2. 1-Methyladenine production by GSS in follicle cells was enhanced by l -Methionine, d -Methionine and seleno- dl -Methionine. Without GSS, however, these chemicals alone did not induce 1-MeAde production. 3. 3. Both l - and d -ethionine, competitive inhibitors of Methionine, reduced 1-MeAde levels produced by follicle cells in the presence of GSS. Seleno- dl -ethionine also inhibited 1-MeAde production. l -Methionine, d -Methionine and seleno- dl -Methionine abolished the ethionine-induced inhibition of 1-MeAde production.
