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Rodney L. Levine - One of the best experts on this subject based on the ideXlab platform.
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Loss of Methionine Sulfoxide reductases increases resistance to oxidative stress
Free radical biology & medicine, 2019Co-Authors: Lo Lai, Sreya Tarafdar, Junhui Sun, Chengyu Liu, Elizabeth Murphy, Geumsoo Kim, Rodney L. LevineAbstract:Oxidation of Methionine residues to Methionine Sulfoxide scavenges reactive species, thus protecting against oxidative stress. Reduction of the Sulfoxide back to Methionine by Methionine Sulfoxide reductases creates a cycle with catalytic efficiency. Protection by the Methionine Sulfoxide reductases is well documented in cultured cells, from microorganisms to mammals. However, knocking out one or two of the 4 mammalian reductases had little effect in mice that were not stressed. We hypothesized that the minimal effect is due to redundancy provided by the 4 reductases. We tested the hypothesis by creating a transgenic mouse line lacking all 4 reductases and predicted that this mouse would be exceptionally sensitive to oxidative stress. The mutant mice were phenotypically normal at birth, exhibited normal post-natal growth, and were fertile. Surprisingly, rather than being more sensitive to oxidative stress, they were more resistant to both cardiac ischemia-reperfusion injury and to parenteral paraquat, a redox-cycling agent. Resistance was not a result of hormetic induction of the antioxidant transcription factor Nrf2 nor activation of Akt. The mechanism of protection may be novel.
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Drosophila Methionine Sulfoxide reductase A (MSRA) lacks Methionine oxidase activity.
Free Radical Biology and Medicine, 2018Co-Authors: Sreya Tarafdar, Rodney L. LevineAbstract:Abstract Mouse, human, and E. coli Methionine Sulfoxide reductase A (MSRA) stereospecifically catalyze both the reduction of S-Methionine Sulfoxide to Methionine and the oxidation of Methionine to S-Methionine Sulfoxide. Calmodulin has 9 Methionine residues, but only Met77 is oxidized by MSRA, and this is completely reversed when MSRA operates in the reductase direction. Given the powerful genetic tools available for Drosophila, we selected this model organism to identify the in vivo calmodulin targets regulated by redox modulation of Met77. The active site sequences of mammalian and Drosophila MSRA are identical, and both contain two cysteine residues in their carboxy terminal domains. We produced recombinant Drosophila MSRA and studied its biochemical and biophysical properties. The enzyme is active as a Methionine Sulfoxide reductase, but it cannot function as a Methionine oxidase. The first step in the mammalian oxidase reaction is formation of a sulfenic acid at the active site, and the second step is the reaction of the sulfenic acid with a carboxy terminal domain cysteine to form a disulfide bond. The third step regenerates the active site through a disulfide exchange reaction with a second carboxy terminal domain cysteine. Drosophila MSRA carries out the first and second steps, but it cannot regenerate the active site in the third step. Thus, unlike the E. coli and mammalian enzymes, Drosophila MSRA catalyzes only the reduction of Methionine Sulfoxide and not the oxidation of Methionine.
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myristoylated Methionine Sulfoxide reductase a is a late endosomal protein
Journal of Biological Chemistry, 2018Co-Authors: Rodney L. LevineAbstract:Methionine residues in proteins provide antioxidant defense by reacting with oxidizing species, which oxidize Methionine to Methionine Sulfoxide. Reduction of the Sulfoxide back to Methionine is catalyzed by Methionine Sulfoxide reductases, essential for protection against oxidative stress. The nonmyristoylated form of Methionine Sulfoxide reductase A (MSRA) is present in mitochondria, whereas the myristoylated form has been previously reported to be cytosolic. Despite the importance of MSRA in antioxidant defense, its in vivo binding partners and substrates have not been identified. Starting with a protein array, and followed by immunoprecipitation experiments, colocalization studies, and subcellular fractionation, we identified the late endosomal protein, StAR-related lipid transfer domain-containing 3 (STARD3), as a binding partner of myristoylated MSRA, but not of nonmyristoylated MSRA. STARD3 is known to have both membrane-binding and cytosolic domains that are important in STARD3-mediated transport of cholesterol from the endoplasmic reticulum to the endosome. We found that the STARD3 cytosolic domain localizes MSRA to the late endosome. We propose that the previous conclusion that myristoylated MSRA is strictly a cytosolic protein is artifactual and likely due to vigorous overexpression of MSRA. We conclude that myristoylated MSRA is a late endosomal protein that may play a role in lipid metabolism or may protect endosomal proteins from oxidative damage.
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drosophila Methionine Sulfoxide reductase a is not a Methionine oxidase
Free Radical Biology and Medicine, 2017Co-Authors: Sreya Tarafdar, Nasser M. Rusan, Rodney L. LevineAbstract:Methionine Sulfoxide reductase A (MsrA) stereospecifically catalyzes the reduction of S-Methionine Sulfoxide to Methionine and is important in defense against oxidative stress. Recently, we reported that mammalian Methionine Sulfoxide reductase A stereospecifically and selectively oxidizes Met77 in calcium-bound calmodulin and can fully reduce it as well. The control mechanism that prevents futile cycling is hypothesized to be through interaction with a postulated regulatory protein. Thus, cyclic oxidation and reduction of Methionines in proteins by MsrA could function as a redox-based mechanism of cellular regulation. Our aim in this study was to elucidate the physiological significance of Methionine Sulfoxide reductase A mediated reversible oxidation of calmodulin Met77 in Drosophila. However, we found that Drosophila MsrA, unlike its mammalian counterpart, is not a Methionine oxidase. This led us to explore the mechanistic details of the enzyme. Using a double alkylation approach with HPLC-mass spectrometric sequencing, we found that the active site cysteine residue in Drosophila MsrA becomes locked in a disulfide bond with the terminal cysteine residue of the protein and thus cannot mediate oxidation. A mutant Drosophila MsrA lacking the two C-terminal cysteine residues also lacked oxidase activity, despite not being able to form a disulfide bond with the active site cysteine.
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stard3 interacts with myristoylated Methionine Sulfoxide reductase a
Free Radical Biology and Medicine, 2016Co-Authors: Rodney L. LevineAbstract:Methionine Sulfoxide reductase A (MsrA) is a component of the oxidative defense system that reduces Methionine Sulfoxide (MetO) in proteins back to Methionine. Although many proteins with MetO are in vitro substrates of MsrA, no in vivo substrates have been confidently identified. In vitro, MsrA can also function as an oxidase that converts Methionine to Methionine Sulfoxide, but it is also not known if it does so in vivo. The cytosolic form of MsrA is myristoylated, and overexpression of the myristoylated form protects the heart from ischemia-reperfusion injury while overexpression of the non-myristoylated form does not. Previous efforts in our laboratory to identify proteins that interact in vivo with MsrA had not been successful. We now report use of a human protein microarray to identify a protein that binds to MsrA. The ProtoArray Human Protein Microarray v5.0 contains 9,483 human proteins from multiple protein classes along with 2,016 controls. Each protein was expressed as an N-terminal GST- tagged protein and printed in duplicate on the nitrocellulose-coated slide glass. We incubated the slide with biotinylated human MsrA and interrogated the array with streptavidin tagged with Alexa Fluor 647. STARD3 emerged as an interacting protein with a high probability score. We co-expressed MsrA and STARD3 in HEK-293 cells, immunoprecipitated from a homogenate with anti-STARD3, and confirmed that MsrA was co-immunoprecipitated. When non-myristoylated MsrA was co-expressed, no interaction with STARD3 was detected. STARD3 contains the star-related lipid transfer (START) domain, and proteins with that domain have been implicated in lipid and steroid metabolism. STARD3 is known to bind cholesterol and transport it to the late endosome. We conclude that it also binds to myristoylated MsrA, and we are now investigating the physiological significance of the interaction.
Jackob Moskovitz - One of the best experts on this subject based on the ideXlab platform.
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genetic regulation of longevity and age associated diseases through the Methionine Sulfoxide reductase system
Biochimica et Biophysica Acta, 2019Co-Authors: Derek B Oien, Jackob MoskovitzAbstract:Abstract Methionine Sulfoxide reductase enzymes are a protective system against biological oxidative stress in aerobic organisms. Modifications to this antioxidant system have been shown to impact the lifespan of several model system organisms. In humans, Methionine oxidation of critical proteins and deficiencies in the Methionine Sulfoxide reductase system have been linked to age-related diseases, including cancer and neurodegenerative disease. Substrates for Methionine Sulfoxide reductases have been reviewed multiple times, and are still an active area of discovery. In contrast, less is known about the genetic regulation of Methionine Sulfoxide reductases. In this review, we discuss studies on the genetic regulation of the Methionine Sulfoxide reductase system with relevance to longevity and age-related diseases. A better understanding of genetic regulation for Methionine Sulfoxide reductases may lead to new therapeutic approaches for age-related diseases in the future.
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Detection and localization of Methionine Sulfoxide residues of specific proteins in brain tissue.
Protein and peptide letters, 2013Co-Authors: Jackob MoskovitzAbstract:Methionine Sulfoxide is a common posttranslational oxidative modification that can alter protein function. Vul- nerability of specific proteins to Methionine oxidation varies and depends on their structure. In the current study, detection of Methionine Sulfoxide in intact proteins is mediated by novel anti-Methionine Sulfoxide antibody that resulted in the identification of three major Methionine Sulfoxide-proteins in brain: bisphosphate aldolase A and C, � andsubunits of hemoglobin, and serum albumin. The locations of the Methionine Sulfoxide residues were determined by mass- spectrometry analyses. It is suggested that the in vivo Methionine oxidation of these proteins represent early posttransla- tional oxidative modification of proteins in brain. Thus, elevated levels of Methionine-Sulfoxide in these proteins may serve as bio-markers for enhanced oxidative stress in brain, which may be associated with brain disorders and diseases.
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Induction of Methionine Sulfoxide reductase activity by pergolide, pergolide Sulfoxide, and S-adenosyl-Methionine in neuronal cells
Neuroscience Letters, 2012Co-Authors: Jade M. Franklin, Gonzalo A. Carrasco, Jackob MoskovitzAbstract:Abstract The reduction of Methionine Sulfoxide in proteins is facilitated by the Methionine Sulfoxide reductase (Msr) system. The Msr reduction activity is important for protecting cells from oxidative stress related damages. Indeed, we have recently shown that treatment of cells with N -acetyl-Methionine Sulfoxide can increase Msr activity and protect neuronal cells from amyloid beta toxicity. Thus, in search of other similar Msr-inducing molecules, we examined the effects of pergolide, pergolide Sulfoxide, and S -adenosyl-Methionine on Msr activity in neuronal cells. Treatment of neuronal cells with a physiological range of pergolide and pergolide Sulfoxide (0.5–1.0 μM) caused an increase of about 40% in total Msr activity compared with non-treated control cells. This increase in activity correlated with similar increases in Methionine Sulfoxide reductase A protein expression levels. Similarly, treatment of cells with S -adenosyl Methionine also increased cellular Msr activity, which was milder compared to increases induced by pergolide and pergolide Sulfoxide. We found that all the examined compounds are able to increase cellular Msr activity to levels comparable to N -acetyl-Methionine Sulfoxide treatment. Pergolide, pergolide Sulfoxide, and S -adenosyl Methionine can cross the blood–brain barrier. Therefore, we hypothesize that they can be useful in the treatment of symptoms/pathologies that are associated with reduced Msr activity.
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Methionine Sulfoxide reductases and Methionine Sulfoxide in the subterranean mole rat (Spalax): characterization of expression under various oxygen conditions.
Comparative biochemistry and physiology. Part A Molecular & integrative physiology, 2011Co-Authors: Jackob Moskovitz, Assaf Malik, Alvaro G. Hernandez, Mark Band, Aaron AviviAbstract:The blind subterranean mole rat (Spalax ehrenbergi) exhibits a relatively long life span, which is attributed to an efficient antioxidant defense affording protection against accumulation of oxidative modifications of proteins. Methionine residues can be oxidized to Methionine Sulfoxide (MetO) and then enzymatically reduced by the Methionine Sulfoxide reductase (Msr) system. In the current study we have isolated the cDNA sequences of the Spalax Msr genes as well as 23 additional selenoproteins and monitored the activities of Msr enzymes in liver and brain of rat (Rattus norvegicus), Spalax galili, and Spalax judaei under normoxia, hypoxia, and hyperoxia. Under normoxia, the Msr activity was lower in S. galili in comparison to S. judaei and R. norvegicus especially in the brain. The pattern of Msr activity of the three species was similar throughout the tested conditions. However, exposure of the animals to hypoxia caused a significant enhancement of Msr activity, especially in S. galili. Hyperoxic exposure showed a highly significant induction of Msr activity compared with normoxic conditions for R. norvegicus and S. galili brain. It was concluded that among all species examined, S. galili appears to be more responsive to oxygen tension changes and that the Msr system is upregulated mainly by severe hypoxia.
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Protein carbonyl and the Methionine Sulfoxide reductase system.
Antioxidants & redox signaling, 2010Co-Authors: Jackob Moskovitz, Derek B OienAbstract:The formation and accumulation of protein-carbonyl by reactive oxygen species may serve as a marker of oxidative stress, aging, and age-related diseases. Enzymatic reversal of the protein-carbonyl modification has not yet been detected. However, an enzymatic reversal of protein-Methionine Sulfoxide modification exists and is mediated by the Methionine Sulfoxide reductase (Msr) system. Methionine Sulfoxide modifications to proteins may precede the formation of protein-carbonyl adducts because of consequent structural changes that increase the vulnerability of amino acid residues to carbonylation. Supportive evidence for this possibility arises from the elevated protein-carbonyl accumulations observed in organisms, such as yeast and mice, lacking the Methionine Sulfoxide reductase A (MsrA) enzyme. In addition, advanced age or enhanced oxidative-stress conditions foster the accumulations of protein-carbonyls. This review discusses the possible involvement of Methionine Sulfoxide formation in the occurrence of protein-carbonyl adducts and their relevance to the aging process and neurodegenerative diseases.
Derek B Oien - One of the best experts on this subject based on the ideXlab platform.
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genetic regulation of longevity and age associated diseases through the Methionine Sulfoxide reductase system
Biochimica et Biophysica Acta, 2019Co-Authors: Derek B Oien, Jackob MoskovitzAbstract:Abstract Methionine Sulfoxide reductase enzymes are a protective system against biological oxidative stress in aerobic organisms. Modifications to this antioxidant system have been shown to impact the lifespan of several model system organisms. In humans, Methionine oxidation of critical proteins and deficiencies in the Methionine Sulfoxide reductase system have been linked to age-related diseases, including cancer and neurodegenerative disease. Substrates for Methionine Sulfoxide reductases have been reviewed multiple times, and are still an active area of discovery. In contrast, less is known about the genetic regulation of Methionine Sulfoxide reductases. In this review, we discuss studies on the genetic regulation of the Methionine Sulfoxide reductase system with relevance to longevity and age-related diseases. A better understanding of genetic regulation for Methionine Sulfoxide reductases may lead to new therapeutic approaches for age-related diseases in the future.
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Protein carbonyl and the Methionine Sulfoxide reductase system.
Antioxidants & redox signaling, 2010Co-Authors: Jackob Moskovitz, Derek B OienAbstract:The formation and accumulation of protein-carbonyl by reactive oxygen species may serve as a marker of oxidative stress, aging, and age-related diseases. Enzymatic reversal of the protein-carbonyl modification has not yet been detected. However, an enzymatic reversal of protein-Methionine Sulfoxide modification exists and is mediated by the Methionine Sulfoxide reductase (Msr) system. Methionine Sulfoxide modifications to proteins may precede the formation of protein-carbonyl adducts because of consequent structural changes that increase the vulnerability of amino acid residues to carbonylation. Supportive evidence for this possibility arises from the elevated protein-carbonyl accumulations observed in organisms, such as yeast and mice, lacking the Methionine Sulfoxide reductase A (MsrA) enzyme. In addition, advanced age or enhanced oxidative-stress conditions foster the accumulations of protein-carbonyls. This review discusses the possible involvement of Methionine Sulfoxide formation in the occurrence of protein-carbonyl adducts and their relevance to the aging process and neurodegenerative diseases.
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Selenium and the Methionine Sulfoxide reductase system.
Molecules, 2009Co-Authors: Derek B Oien, Jackob MoskovitzAbstract:Selenium is a chemical element participating in the synthesis of selenocysteine residues that play a pivotal role in the enzymatic activity efficiency of selenoproteines. The Methionine Sulfoxide reductase (Msr) system that reduces Methionine Sulfoxide (MetO) to Methionine comprises the selenoprotein MsrB (MsrB1) and the non-selenoprotein MsrA, which reduce the R- and the S- forms of MetO, respectively. The effects of a selenium deficient (SD) diet, which was administrated to wild type (WT) and MsrA knockout mice (MsrA-/-), on the expression and function of Msr-related proteins are examined and discussed. Additionally, new data about the levels of selenium in brain, liver, and kidneys of WT and MsrA-/- mice are presented and discussed.
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Ablation of the mammalian Methionine Sulfoxide reductase A affects the expression level of cysteine deoxygenase.
Biochemical and Biophysical Research Communications, 2006Co-Authors: Derek B Oien, Jackob MoskovitzAbstract:Abstract Methionine Sulfoxide reductases (Msrs) are able to reduce Methionine Sulfoxide to Methionine both in proteins and free amino acids. By their action it is possible to regulate the function of specific proteins and the cellular antioxidant defense against oxidative damage. Similarly, cysteine deoxygenase (CDO) may be involved in the regulation of protein function and antioxidant defense mechanisms by its ability to oxidized cysteine residues. The two enzymes’ involvement in sulfur amino-acids metabolism seems to be connected. Lack of Methionine Sulfoxide reductase A (MsrA) in liver of MsrA−/− led to a significant drop in the cellular level of thiol groups and lowered the CDO level of expression. Moreover, following selenium deficient diet (applied to decrease the expression levels of selenoproteins like MsrB), the latter effect was maintained while the basal levels of thiol decreased in both mouse strains. We suggest that both enzymes are working in coordination to balance cellular antioxidant defense.
Herbert Weissbach - One of the best experts on this subject based on the ideXlab platform.
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Identification of activators of Methionine Sulfoxide reductases A and B.
Biochemical and Biophysical Research Communications, 2015Co-Authors: Predrag Cudic, Neelambari Joshi, Daphna Sagher, Brandon T. Williams, Maciej Stawikowski, Herbert WeissbachAbstract:The Methionine Sulfoxide reductase (Msr) family of enzymes has been shown to protect cells against oxidative damage. The two major Msr enzymes, MsrA and MsrB, can repair oxidative damage to proteins due to reactive oxygen species, by reducing the Methionine Sulfoxide in proteins back to Methionine. A role of MsrA in animal aging was first demonstrated in Drosophila melanogaster where transgenic flies over-expressing recombinant bovine MsrA had a markedly extended life span. Subsequently, MsrA was also shown to be involved in the life span extension in Caenorhabditis elegans. These results supported other studies that indicated up-regulation, or activation, of the normal cellular protective mechanisms that cells use to defend against oxidative damage could be an approach to treat age related diseases and slow the aging process. In this study we have identified, for the first time, compounds structurally related to the natural products fusaricidins that markedly activate recombinant bovine and human MsrA and human MsrB.
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A high-throughput screening compatible assay for activators and inhibitors of Methionine Sulfoxide reductase A.
Assay and Drug Development Technologies, 2010Co-Authors: David Brunell, Peter Hodder, Herbert Weissbach, Nathan BrotAbstract:Abstract The Methionine Sulfoxide reductase (Msr) system has been shown to play an important role in protecting cells against oxidative damage. This family of enzymes can repair damage to proteins resulting from the oxidation of Methionine residues to Methionine Sulfoxide, caused by reactive oxygen species. Previous genetic studies in animals have shown that increased levels of Methionine Sulfoxide reductase enzyme A (MsrA), an important member of the Msr family, can protect cells against oxidative damage and increase life span. A high-throughput screening (HTS) compatible assay has been developed to search for both activators and inhibitors of MsrA. The assay involves a coupled reaction in which the oxidation of NADPH is measured by either spectrophotometric or fluorometric analysis. Previous studies had shown that MsrA has a broad substrate specificity and can reduce a variety of methyl Sulfoxide compounds, including dimethylSulfoxide (DMSO). Since the chemicals in the screening library are dissolved in...
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New membrane-associated and soluble peptide Methionine Sulfoxide reductases in Escherichia coli.
Biochemical and biophysical research communications, 2003Co-Authors: Daniel Spector, Nathan Brot, Frantzy Etienne, Herbert WeissbachAbstract:Abstract It is known that reactive oxygen species can oxidize Methionine residues in proteins in a non-stereospecific manner, and cells have mechanisms to reverse this damage. MsrA and MsrB are members of the Methionine Sulfoxide family of enzymes that specifically reduce the S and R forms, respectively, of Methionine Sulfoxide in proteins. However, in Escherichia coli the level of MsrB activity is very low which suggested that there may be other enzymes capable of reducing the R epimer of Methionine Sulfoxide in proteins. Employing a msrA/B double mutant, a new peptide Methionine Sulfoxide reductase activity has been found associated with membrane vesicles from E. coli . Both the R and S forms of N -acetylMethionine Sulfoxide, d -ala-met(o)-enkephalin and Methionine Sulfoxide, are reduced by this membrane associated activity. The reaction requires NADPH and may explain, in part, how the R form of Methionine Sulfoxide in proteins is reduced in E. coli . In addition, a new soluble Msr activity was also detected in the soluble extracts of the double mutant that specifically reduces the S epimer of met(o) in proteins.
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a Methionine Sulfoxide reductase in escherichia coli that reduces the r enantiomer of Methionine Sulfoxide
Biochemical and Biophysical Research Communications, 2003Co-Authors: Frantzy Etienne, Nathan Brot, Daniel Spector, Herbert WeissbachAbstract:It is known that Escherichia coli Methionine mutants can grow on both enantiomers of Methionine Sulfoxide (met(o)), i.e., met-R-(o) or met-S-(o), indicating the presence of enzymes in E. coli that can reduce each of these enantiomers to Methionine (met). Previous studies have identified two members of the Methionine Sulfoxide reductase (Msr) family of enzymes, MsrA and fSMsr, that could reduce free met-S-(o), but the reduction of free met-R-(o) to met has not been elucidated. One possible candidate is MsrB which is known to reduce met-R-(o) in proteins to met. However, free met-R-(o) is a very poor substrate for MsrB and the level of MsrB activity in E. coli extracts is very low. A new member of the Msr family (fRMsr) has been identified in E. coli extracts that reduces free met-R-(o) to met. Partial purification of FRMsr has been obtained using extracts from an MsrA/MsrB double mutant of E. coli.
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The mirrored Methionine Sulfoxide reductases of Neisseria gonorrhoeae pilB
Nature Structural & Molecular Biology, 2002Co-Authors: W. Todd Lowther, Nathan Brot, Frantzy Etienne, Herbert Weissbach, Brian W. MatthewsAbstract:Methionine Sulfoxide reductases (Msr) protect against oxidative damage that can contribute to cell death. The tandem Msr domains (MsrA and MsrB) of the pilB protein from Neisseria gonorrhoeae each reduce different epimeric forms of Methionine Sulfoxide. The overall fold of the MsrB domain revealed by the 1.85 A crystal structure shows no resemblance to the previously determined MsrA structures from other organisms. Despite the lack of homology, the active sites show approximate mirror symmetry. In each case, conserved amino acid motifs mediate the stereo-specific recognition and reduction of the substrate. Unlike the MsrA domain, the MsrB domain activates the cysteine or selenocysteine nucleophile through a unique Cys-Arg-Asp/Glu catalytic triad. The collapse of the reaction intermediate most likely results in the formation of a sulfenic or selenenic acid moiety. Regeneration of the active site occurs through a series of thiol-disulfide exchange steps involving another active site Cys residue and thioredoxin. These observations have broad implications for modular catalysis, antibiotic drug design and continuing longevity studies in mammals.
Stefan H. Heinemann - One of the best experts on this subject based on the ideXlab platform.
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Stereospecific electrophoretically mediated microanalysis assay for Methionine Sulfoxide reductase enzymes.
Analytical and Bioanalytical Chemistry, 2014Co-Authors: Rabab G. El-mergawy, Stefan H. Heinemann, Roland Schönherr, Gerhard K. E. ScribaAbstract:An electrophoretically mediated microanalysis assay (EMMA) for the determination of the stereoselective reduction of l-Methionine Sulfoxide diastereomers by Methionine Sulfoxide reductase enzymes was developed using fluorenylmethyloxycarbonyl (Fmoc)-l-Methionine Sulfoxide as substrate. The separation of the diastereomers of Fmoc-l-Methionine Sulfoxide and the product Fmoc-l-Methionine was achieved in a successive multiple ionic-polymer layer-coated capillary using a 50 mM Tris buffer, pH 8.0, containing 30 mM sodium dodecyl sulfate as background electrolyte and an applied voltage of 25 kV. 4-Aminobenzoic acid was employed as internal standard. An injection sequence of incubation buffer, enzyme, substrate, enzyme, and incubation buffer was selected. The assay was optimized with regard to mixing time and mixing voltage and subsequently applied for the analysis of stereoselective reduction of Fmoc-l-Methionine-(S)-Sulfoxide by human Methionine Sulfoxide reductase A and of the Fmoc-l-Methionine-(R)-Sulfoxide by human Methionine Sulfoxide reductase B. The Michaelis–Menten constant, Km, and the maximum velocity, vmax, were determined. Essentially identical data were determined by the electrophoretically mediated microanalysis assay and the analysis of the samples by CE upon offline incubation. Furthermore, it was shown for the first time that Fmoc-Methionine-(R)-Sulfoxide is a substrate of human Methionine Sulfoxide reductase B.
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Stereospecific micellar electrokinetic chromatography assay of Methionine Sulfoxide reductase activity employing a multiple layer coated capillary
Electrophoresis, 2013Co-Authors: Rabab G. El-mergawy, Stefan H. Heinemann, Roland Schönherr, Gerhard K. E. ScribaAbstract:: A micellar electrokinetic chromatography method for the analysis of the l-Methionine Sulfoxide diastereomers employing a successive multiple ionic-polymer layer coated fused-silica capillary was developed and validated in order to investigate the stereospecificity of Methionine Sulfoxide reductases. The capillary coating consisted of a first layer of hexadimethrine and a second layer of dextran sulfate providing a stable strong cathodic EOF and consequently highly repeatable analyte migration times. The Methionine Sulfoxide diastereomers, Methionine as product as well as β-alanine as internal standard were derivatized by dabsyl chloride and separated using a 35 mM sodium phosphate buffer, pH 8.0, containing 25 mM SDS as BGE and a separation voltage of 25 kV. The method was validated in the range of 0.15-2.0 mM with respect to linearity and precision. The LODs of the analytes ranged between 0.04 and 0.10 mM. The assay was subsequently applied to determine the stereospecificity of Methionine Sulfoxide reductases as well as the enzyme kinetics of human Methionine Sulfoxide reductase A. Monitoring the decrease of the l-Methionine-(S)-Sulfoxide Km = 411.8 ± 33.8 μM and Vmax = 307.5 ± 10.8 μM/min were determined.
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Treating Oxidative Neural Injury: Methionine Sulfoxide Reductase Therapy for Parkinson’s Disease
Oxidative Neural Injury, 2009Co-Authors: Ramez Wassef, Stefan H. Heinemann, Toshinori HoshiAbstract:Parkinson’s disease is a common neurodegenerative disease that is characterized by loss of dopaminergic neurons in the substantia nigra and impaired motor function. The disease is multifactorial but oxidative injury is associated with the pathology and contributes to neuronal injury. Fibrillations of α-synuclein are present in pathological lesions in this disease. Oxidation of the sulfur moieties of Methionine residues on α-synuclein can contribute to α-synuclein fibrillation. An anti-oxidant enzyme Methionine Sulfoxide reductase can reverse the Methionine oxidation on α-synuclein and act as a sink scavenging reactive oxygen species. Thus, boosting Methionine Sulfoxide reductase activity may prevent oxidative injury in dopaminergic neurons that contributes to neurodegeneration and impaired neuronal function in Parkinson’s disease. One promising approach to augmenting Methionine Sulfoxide reductase activity in neurons is to provide a naturally occurring substrate for Methionine Sulfoxide reductase A, S-methyl-l-cysteine. Recent work in our lab with this compound supports the promise of this substance in preventing or delaying motor dysfunction in multiple model systems of Parkinson’s disease.
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mitochondrial targeting of the human peptide Methionine Sulfoxide reductase msra an enzyme involved in the repair of oxidized proteins
The FASEB Journal, 2002Co-Authors: Alfred Hansel, Toshinori Hoshi, Lioba Kuschel, Solveig Hehl, Cornelius Lemke, Hans-jurgen Agricola, Stefan H. HeinemannAbstract:SPECIFIC AIMSThe enzyme peptide Methionine Sulfoxide reductase (MSRA) catalyzes the reduction of free and protein-bound Methionine Sulfoxide to Methionine and is a repair mechanism for oxidatively ...
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high quality life extension by the enzyme peptide Methionine Sulfoxide reductase
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Hongyu Ruan, Stefan H. Heinemann, Nathan Brot, Xiang Dong Tang, Mailei Chen, Mei-ling A. Joiner, Linda E Iverson, Chunfang Wu, Herbert Weissbach, Toshinori HoshiAbstract:Cumulative oxidative damages to cell constituents are considered to contribute to aging and age-related diseases. The enzyme peptide Methionine Sulfoxide reductase A (MSRA) catalyzes the repair of oxidized Methionine in proteins by reducing Methionine Sulfoxide back to Methionine. However, whether MSRA plays a role in the aging process is poorly understood. Here we report that overexpression of the msrA gene predominantly in the nervous system markedly extends the lifespan of the fruit fly Drosophila. The MSRA transgenic animals are more resistant to paraquat-induced oxidative stress, and the onset of senescence-induced decline in the general activity level and reproductive capacity is delayed markedly. The results suggest that oxidative damage is an important determinant of lifespan, and MSRA may be important in increasing the lifespan in other organisms including humans.