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Herbert Weissbach - One of the best experts on this subject based on the ideXlab platform.

  • Methionine Sulfoxide Reductase A Mediates Dietary Restriction-Induced Lifespan Extension in Caenorhabditis elegans
    Journal of Aging Science, 2013
    Co-Authors: Justin Minnerly, Jiuli Zhang, Rebeca Aldunate, Herbert Weissbach
    Abstract:

    Background: Methionine Sulfoxide Reductase A (MsrA) is a well-studied antioxidant enzyme that has been found to be important for protecting cells against oxidative damage and regulating lifespan in several species. However, the role of MsrA in dietary restriction has not been examined. The authors evaluated the function of MsrA in dietary restriction-induced lifespan extension in Caenorhabditis elegans. Methods: C. elegans loss-of-function msra mutant animals and wild type control animals were subjected to two widely used dietary restriction treatments, solid dietary restriction (sDR) and dietary restriction by liquid bacteria (BDR). The survival of the animals was evaluated and the data was statistically analyzed. Results: The loss-of-function mutation of msra significantly suppressed the lifespan extension conferred by solid dietary restriction. By contrast, msra was dispensable for lifespan extension resulted from dietary restriction by diluted bacteria in liquid. Conclusion: msra-1 is a major factor in the sDR-induced lifespan extension. This result, coupled with the previous finding that MsrA mediates the effect of insulin-like signaling on lifespan extension, indicates an essential role of MsrA in the aging process in C. elegans.

  • Methionine Sulfoxide Reductase a msra protects cultured mouse embryonic stem cells from h2o2 mediated oxidative stress
    Journal of Cellular Biochemistry, 2010
    Co-Authors: Chi Zhang, Herbert Weissbach, Keith A. Webster, Xupei Huang, Sharon L. Lemanski, Mohan P Achary, Larry F. Lemanski
    Abstract:

    Methionine Sulfoxide Reductase A (MsrA), a member of the Msr gene family, can reduce Methionine Sulfoxide residues in proteins formed by oxidation of Methionine by reactive oxygen species (ROS). Msr is an important protein repair system which can also function to scavenge ROS. Our studies have confirmed the expression of MsrA in mouse embryonic stem cells (ESCs) in culture conditions. A cytosol-located and mitochondria-enriched expression pattern has been observed in these cells. To confirm the protective function of MsrA in ESCs against oxidative stress, a siRNA approach has been used to knockdown MsrA expression in ES cells which showed less resistance than control cells to hydrogen peroxide treatment. Overexpression of MsrA gene products in ES cells showed improved survivability of these cells to hydrogen peroxide treatment. Our results indicate that MsrA plays an important role in cellular defenses against oxidative stress in ESCs. Msr genes may provide a new target in stem cells to increase their survivability during the therapeutic applications.

  • A high-throughput screening compatible assay for activators and inhibitors of Methionine Sulfoxide Reductase A.
    Assay and Drug Development Technologies, 2010
    Co-Authors: David Brunell, Herbert Weissbach, Peter Hodder, Nathan Brot
    Abstract:

    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...

  • Methionine Sulfoxide Reductase a protects neuronal cells against brief hypoxia reoxygenation
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Olena Yermolaieva, Herbert Weissbach, Stefan H. Heinemann, Nathan Brot, Rong Xu, Carrie Schinstock, Toshinori Hoshi
    Abstract:

    Hypoxia/reoxygenation induces cellular injury by promoting oxidative stress. Reversible oxidation of Methionine in proteins involving the enzyme peptide Methionine Sulfoxide Reductase type A (MSRA) is postulated to serve a general antioxidant role. Therefore, we examined whether overexpression of MSRA protected cells from hypoxia/reoxygenation injury. Brief hypoxia increased the intracellular reactive oxygen species (ROS) level in PC12 cells and promoted apoptotic cell death. Adenovirus-mediated overexpression of MSRA significantly diminished the hypoxia-induced increase in ROS and facilitated cell survival. Measurements of the membrane potentials of intact mitochondria in PC12 cells and of isolated rat liver mitochondria showed that hypoxia induced depolarization of the mitochondrial membrane. The results demonstrate that MSRA plays a protective role against hypoxia/reoxygenation-induced cell injury and suggest the therapeutic potential of MSRA in ischemic heart and brain disease.

  • reduction of sulindac to its active metabolite sulindac sulfide assay and role of the Methionine Sulfoxide Reductase system
    Biochemical and Biophysical Research Communications, 2003
    Co-Authors: Frantzy Etienne, Nathan Brot, Lionel Resnick, Daphna Sagher, Herbert Weissbach
    Abstract:

    Sulindac is a known anti-inflammatory drug that functions by inhibition of cyclooxygenases 1 and 2 (COX). There has been recent interest in Sulindac and other non-steroidal anti-inflammatory drugs (NSAID) because of their anti-tumor activity against colorectal cancer. Studies with sulindac have indicated that it may also function as an anti-tumor agent by stimulating apoptosis. Sulindac is a pro-drug, containing a methyl Sulfoxide group, that must be reduced to sulindac sulfide to be active as a COX inhibitor. In the present studies we have developed a simple assay to measure sulindac reduction and tested sulindac as a substrate for 6 known members of the Methionine Sulfoxide Reductase (Msr) family that have been identified in Escherichia coli. Only MsrA and a membrane associated Msr can reduce sulindac to the active sulfide. The reduction of sulindac also has been demonstrated in extracts of calf liver, kidney, and brain. Sulindac Reductase activity is also present in mitochondria and microsomes.

Jackob Moskovitz - One of the best experts on this subject based on the ideXlab platform.

  • genetic regulation of longevity and age associated diseases through the Methionine Sulfoxide Reductase system
    Biochimica et Biophysica Acta, 2019
    Co-Authors: Derek B Oien, Jackob Moskovitz
    Abstract:

    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.

  • the functions of the mammalian Methionine Sulfoxide Reductase system and related diseases
    Antioxidants, 2018
    Co-Authors: Beichen Jiang, Jackob Moskovitz
    Abstract:

    This review article describes and discusses the current knowledge on the general role of the Methionine Sulfoxide Reductase (MSR) system and the particular role of MSR type A (MSRA) in mammals. A powerful tool to investigate the contribution of MSRA to molecular processes within a mammalian system/organism is the MSRA knockout. The deficiency of MSRA in this mouse model provides hints and evidence for this enzyme function in health and disease. Accordingly, the potential involvement of MSRA in the processes leading to neurodegenerative diseases, neurological disorders, cystic fibrosis, cancer, and hearing loss will be deliberated and evaluated.

  • a ratiometric fluorescent probe for imaging of the activity of Methionine Sulfoxide Reductase a in cells
    Angewandte Chemie, 2016
    Co-Authors: Nikolai Makukhin, Jackob Moskovitz, Vyacheslav Tretyachenko, Jiři Misek
    Abstract:

    Methionine Sulfoxide Reductase A (MsrA) is an enzyme involved in redox balance and signaling, and its aberrant activity is implicated in a number of diseases (for example, Alzheimer's disease and cancer). Since there is no simple small molecule tool to monitor MsrA activity in real time in vivo, we aimed at developing one. We have designed a BODIPY-based probe called (S)-Sulfox-1, which is equipped with a reactive Sulfoxide moiety. Upon reduction with a model MsrA (E. coli), it exhibits a bathochromic shift in the fluorescence maximum. This feature was utilized for the real-time ratiometric fluorescent imaging of MsrA activity in E. coli cells. Significantly, our probe is capable of capturing natural variations of the enzyme activity in vivo.

  • Induction of Methionine Sulfoxide Reductase activity by pergolide, pergolide Sulfoxide, and S-adenosyl-Methionine in neuronal cells
    Neuroscience Letters, 2012
    Co-Authors: Jade M. Franklin, Gonzalo A. Carrasco, Jackob Moskovitz
    Abstract:

    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.

  • Decreased Phosphorylation and Increased Methionine Oxidation of α-Synuclein in the Methionine Sulfoxide Reductase A Knockout Mouse
    Journal of Amino Acids, 2011
    Co-Authors: Derek B Oien, Gonzalo A. Carrasco, Jackob Moskovitz
    Abstract:

    Previously, we have showed that overexpression of Methionine-oxidized α-synuclein in Methionine Sulfoxide Reductase A (MsrA) null mutant yeast cells inhibits α-synuclein phosphorylation and increases protein fibrillation. The current studies show that ablation of mouse MsrA gene caused enhanced Methionine oxidation of α-synuclein while reducing its own phophorylation levels, especially in the hydrophobic cell-extracted fraction. These data provide supportive evidence that a compromised MsrA function in mammalian brain may cause enhanced pathologies associated with altered α-synuclein oxidation and phosphorylation levels.

Nathan Brot - One of the best experts on this subject based on the ideXlab platform.

  • A high-throughput screening compatible assay for activators and inhibitors of Methionine Sulfoxide Reductase A.
    Assay and Drug Development Technologies, 2010
    Co-Authors: David Brunell, Herbert Weissbach, Peter Hodder, Nathan Brot
    Abstract:

    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...

  • Methionine Sulfoxide Reductase a protects neuronal cells against brief hypoxia reoxygenation
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Olena Yermolaieva, Herbert Weissbach, Stefan H. Heinemann, Nathan Brot, Rong Xu, Carrie Schinstock, Toshinori Hoshi
    Abstract:

    Hypoxia/reoxygenation induces cellular injury by promoting oxidative stress. Reversible oxidation of Methionine in proteins involving the enzyme peptide Methionine Sulfoxide Reductase type A (MSRA) is postulated to serve a general antioxidant role. Therefore, we examined whether overexpression of MSRA protected cells from hypoxia/reoxygenation injury. Brief hypoxia increased the intracellular reactive oxygen species (ROS) level in PC12 cells and promoted apoptotic cell death. Adenovirus-mediated overexpression of MSRA significantly diminished the hypoxia-induced increase in ROS and facilitated cell survival. Measurements of the membrane potentials of intact mitochondria in PC12 cells and of isolated rat liver mitochondria showed that hypoxia induced depolarization of the mitochondrial membrane. The results demonstrate that MSRA plays a protective role against hypoxia/reoxygenation-induced cell injury and suggest the therapeutic potential of MSRA in ischemic heart and brain disease.

  • reduction of sulindac to its active metabolite sulindac sulfide assay and role of the Methionine Sulfoxide Reductase system
    Biochemical and Biophysical Research Communications, 2003
    Co-Authors: Frantzy Etienne, Nathan Brot, Lionel Resnick, Daphna Sagher, Herbert Weissbach
    Abstract:

    Sulindac is a known anti-inflammatory drug that functions by inhibition of cyclooxygenases 1 and 2 (COX). There has been recent interest in Sulindac and other non-steroidal anti-inflammatory drugs (NSAID) because of their anti-tumor activity against colorectal cancer. Studies with sulindac have indicated that it may also function as an anti-tumor agent by stimulating apoptosis. Sulindac is a pro-drug, containing a methyl Sulfoxide group, that must be reduced to sulindac sulfide to be active as a COX inhibitor. In the present studies we have developed a simple assay to measure sulindac reduction and tested sulindac as a substrate for 6 known members of the Methionine Sulfoxide Reductase (Msr) family that have been identified in Escherichia coli. Only MsrA and a membrane associated Msr can reduce sulindac to the active sulfide. The reduction of sulindac also has been demonstrated in extracts of calf liver, kidney, and brain. Sulindac Reductase activity is also present in mitochondria and microsomes.

  • a Methionine Sulfoxide Reductase in escherichia coli that reduces the r enantiomer of Methionine Sulfoxide
    Biochemical and Biophysical Research Communications, 2003
    Co-Authors: Frantzy Etienne, Nathan Brot, Daniel Spector, Herbert Weissbach
    Abstract:

    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.

  • high quality life extension by the enzyme peptide Methionine Sulfoxide Reductase
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Hongyu Ruan, Herbert Weissbach, Stefan H. Heinemann, Nathan Brot, Xiang Dong Tang, Mailei Chen, Meiling A Joiner, Linda E Iverson, Chunfang Wu, Toshinori Hoshi
    Abstract:

    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.

Rodney L Levine - One of the best experts on this subject based on the ideXlab platform.

  • Revision of JBC2012369116 A Low pKa Cysteine at the Active Site of Mouse Methionine Sulfoxide Reductase A
    2020
    Co-Authors: James M Gruschus, Barbara S Berlett, Nico Tjandra, Rodney L Levine
    Abstract:

    SUMMARY Methionine Sulfoxide Reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of Methionine and Methionine Sulfoxide. Recently it has also been shown to catalyze the reverse reaction, oxidizing Methionine residues to Methionine Sulfoxide. A cysteine at the enzyme’s active site is essential for both Reductase and oxidase activities. This cysteine has been reported to have a pK a of 9.5 in the absence of substrate, decreasing to 5.7 upon binding of substrate. Using 3 independent methods, we show that the pKa of the active site cysteine of mouse Methionine Sulfoxide Reductase is 7.2 even in the absence of substrate. The primary mechanism by which the pKa is lowered is hydrogen bonding of the active site Cys72 to protonated Glu115. The low pK a renders the active site cysteine susceptible to oxidation to sulfenic acid by micromolar concentrations of hydrogen peroxide. This characteristic supports a role for Methionine Sulfoxide Reductase in redox signaling.

  • Drosophila Methionine Sulfoxide Reductase A (MSRA) lacks Methionine oxidase activity.
    Free Radical Biology and Medicine, 2018
    Co-Authors: Sreya Tarafdar, Rodney L Levine
    Abstract:

    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.

  • myristoylated Methionine Sulfoxide Reductase a is a late endosomal protein
    Journal of Biological Chemistry, 2018
    Co-Authors: Rodney L Levine
    Abstract:

    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.

  • drosophila Methionine Sulfoxide Reductase a is not a Methionine oxidase
    Free Radical Biology and Medicine, 2017
    Co-Authors: Sreya Tarafdar, Nasser M Rusan, Rodney L Levine
    Abstract:

    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.

  • stard3 interacts with myristoylated Methionine Sulfoxide Reductase a
    Free Radical Biology and Medicine, 2016
    Co-Authors: Rodney L Levine
    Abstract:

    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.

Stefan H. Heinemann - One of the best experts on this subject based on the ideXlab platform.

  • Stereospecific electrophoretically mediated microanalysis assay for Methionine Sulfoxide Reductase enzymes.
    Analytical and Bioanalytical Chemistry, 2014
    Co-Authors: Rabab G. El-mergawy, Stefan H. Heinemann, Roland Schönherr, Gerhard K. E. Scriba
    Abstract:

    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.

  • Stereospecific micellar electrokinetic chromatography assay of Methionine Sulfoxide Reductase activity employing a multiple layer coated capillary
    Electrophoresis, 2013
    Co-Authors: Rabab G. El-mergawy, Stefan H. Heinemann, Roland Schönherr, Gerhard K. E. Scriba
    Abstract:

    : 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.

  • Treating Oxidative Neural Injury: Methionine Sulfoxide Reductase Therapy for Parkinson’s Disease
    Oxidative Neural Injury, 2009
    Co-Authors: Ramez Wassef, Stefan H. Heinemann, Toshinori Hoshi
    Abstract:

    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.

  • Methionine Sulfoxide Reductase a protects neuronal cells against brief hypoxia reoxygenation
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Olena Yermolaieva, Herbert Weissbach, Stefan H. Heinemann, Nathan Brot, Rong Xu, Carrie Schinstock, Toshinori Hoshi
    Abstract:

    Hypoxia/reoxygenation induces cellular injury by promoting oxidative stress. Reversible oxidation of Methionine in proteins involving the enzyme peptide Methionine Sulfoxide Reductase type A (MSRA) is postulated to serve a general antioxidant role. Therefore, we examined whether overexpression of MSRA protected cells from hypoxia/reoxygenation injury. Brief hypoxia increased the intracellular reactive oxygen species (ROS) level in PC12 cells and promoted apoptotic cell death. Adenovirus-mediated overexpression of MSRA significantly diminished the hypoxia-induced increase in ROS and facilitated cell survival. Measurements of the membrane potentials of intact mitochondria in PC12 cells and of isolated rat liver mitochondria showed that hypoxia induced depolarization of the mitochondrial membrane. The results demonstrate that MSRA plays a protective role against hypoxia/reoxygenation-induced cell injury and suggest the therapeutic potential of MSRA in ischemic heart and brain disease.

  • activity tissue distribution and site directed mutagenesis of a human peptide Methionine Sulfoxide Reductase of type b hcbs1
    FEBS Letters, 2002
    Co-Authors: Stephan Jung, Alfred Hansel, Toshinori Hoshi, Hubert Kasperczyk, Stefan H. Heinemann
    Abstract:

    Human CBS1 is a Methionine Sulfoxide Reductase of type B (MSRB) as it specifically reduced Met-R-SO in peptides with dithiothreitol or the thioredoxin system as reductants. Mutation C169S in the active site completely abolished enzymatic activity, while mutation W110A only reduced activity and C105S had no effect. Like human MSRA, hCBS1 showed in vivo reducing activity coexpressed with the Drosophila ShC/B potassium channel in oocytes, by accelerating the overall inactivation time course. hCBS1-encoding mRNA is most abundant in muscle tissues, especially in the heart and thereby shows an expression pattern different to the human MSRA.