Methionine S-sulfoxide Research Articles

PpMYB105 inhibits chilling injury by regulating PpMsrA1 in peach fruit.

MeJA supplementation enhanced the chilling tolerance and gene expression of PpMsrA1. PpMYB105 protein positively regulated the PpMsrA1 promoter. PpMYB105 mediated the MeJA-boosted chilling tolerance by regulating PpMsrA1. Cold storage can maintain the quality of postharvest fruit. However, peaches easily suffer from chilling injury (CI) during cold storage, leading to economic loss. Results showed that methyl jasmonate (MeJA) supplementation reduced the CI severity, and enhanced the gene expression of methionine sulfoxide reductase A1 (PpMsrA1). It was found that MeJA application elevated the MsrA activity and methionine (Met) content, and reduced the methionine-S-sulfoxide (Met-S-SO) content and reactive oxygen species (ROS) production afterwards. Moreover, PpMYB105 could activate the transcription of PpMsrA1 by binding to the MYB binding element in its promoter. The gene expression of PpMYB105 was up-regulated by MeJA application. Overexpression of PpMYB105 in tomatoes enhanced the chilling tolerance and gene expression of SlMsrA1. Virus-induced gene silencing of PpMYB105 in peaches resulted in the increase in CI severity and the decrease in gene expression of PpMsrA1. Thus, PpMYB105 was involved in the MeJA-boosted chilling tolerance by regulating PpMsrA1.

Methionine sulfoxide reductase B5 plays a key role in preserving seed vigor and longevity in rice (Oryza sativa).

Oxidation of methionine leads to the formation of methionine S-sulfoxide and methionine R-sulfoxide, which can be reverted by two types of methionine sulfoxide reductase (MSR): MSRA and MSRB. Though the role of MSR enzymes has been elucidated in various physiological processes, the regulation and role of MSR in seeds remains poorly understood. In this study, through molecular, biochemical, and genetic studies using seed-specific overexpression and RNAi lines of OsMSRB5 in Oryza sativa, we demonstrate the role of OsMSRB5 in maintaining seed vigor and longevity. We show that an age-induced reduction in the vigor and viability of seeds is correlated with reduced MSR activity and increased methionine sulfoxide (MetSO) formation. OsMSRB5 expression increases during seed maturation and is predominantly localized to the embryo. Further analyses on transgenic lines reveal the role of OsMSRB5 in modulating reactive oxygen species (ROS) homeostasis to preserve seed vigor and longevity. We show that ascorbate peroxidase and PROTEIN l-ISOASPARTYL METHYLTRANSFERASE undergo MetSO modification in seeds that affects their functional competence. OsMSRB5 physically interacts with these proteins and reverts this modification to facilitate their functions and preserve seed vigor and longevity. Our results thus illustrate the role of OsMSRB5 in preserving seed vigor and longevity by modulating ROS homeostasis in seeds.

Implication of Staphylococcus aureus MsrB dimerization upon oxidation

Oxidative modification of protein structure has been shown to play a significant role in bacterial virulence and metabolism. The sulfur-containing residues are susceptible to oxidation and the enzymatic reversal of oxidized cysteine or methionine is detected in many organisms. Methionine sulfoxide reductases (Msr) are responsible for reducing oxidized methionine. The two different Msrs, MsrA and MsrB, reduce methionine R-sulfoxide and methionine S-sulfoxide, respectively through self-oxidation. This study elucidated the structure of MsrB from Staphylococcus aureus Mu50 and its changes upon oxidation. The active site shows two reduced cysteines in a close contact, implying disulfide bond would form without major structural rearrangement. When the protein is exposed to an oxidative condition, a dimeric state is observed. The dimerization of SAMsrB creates a valley structure for accepting peptidyl substrates. To the best of our knowledge, oxidation induced dimerization of SAMsrB would help to understand mechanism behind redox control that has not been well characterized.

Methionine Sulfoxide Reductase A (MsrA) and Its Function in Ubiquitin-Like Protein Modification in Archaea.

ABSTRACTMethionine sulfoxide reductase A (MsrA) is an antioxidant enzyme found in all domains of life that catalyzes the reduction of methionine-S-sulfoxide (MSO) to methionine in proteins and free amino acids. We demonstrate that archaeal MsrA has a ubiquitin-like (Ubl) protein modification activity that is distinct from its stereospecific reduction of MSO residues. MsrA catalyzes this Ubl modification activity, with the Ubl-activating E1 UbaA, in the presence of the mild oxidant dimethyl sulfoxide (DMSO) and in the absence of reductant. In contrast, the MSO reductase activity of MsrA is inhibited by DMSO and requires reductant. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis reveals that MsrA-dependent Ubl conjugates are associated with DNA replication, protein remodeling, and oxidative stress and include the Ubl-modified MsrA, Orc3 (Orc1/Cdc6), and Cdc48d (Cdc48/p97 AAA+ ATPase). Overall, we found archaeal MsrA to have opposing MSO reductase and Ubl modifying activities that are associated with oxidative stress responses and controlled by exposure to mild oxidant.

Methionine sulfoxide reductase A protects against lipopolysaccharide-induced septic shock via negative regulation of the proinflammatory responses

Methionine sulfoxide reductase A (MsrA) is a major antioxidant enzyme that specifically catalyzes the reduction of methionine S-sulfoxide. In this study, we used MsrA gene-knockout (MsrA–/–) mice and bone marrow-derived macrophages (BMDMs) to investigate the role of MsrA in the regulation of inflammatory responses induced by lipopolysaccharide (LPS). MsrA–/– mice were more susceptible to LPS-induced lethal shock than wild-type (MsrA+/+) mice. Serum levels of the proinflammatory cytokines IL-6 and TNF-α induced by LPS were higher in MsrA–/– than in MsrA+/+ mice. MsrA deficiency in the BMDMs also increased the LPS-induced cytotoxicity as well as TNF-α level. Basal and LPS-induced reactive oxygen species (ROS) levels were higher in MsrA–/– than in MsrA+/+ BMDMs. Phosphorylation levels of p38, JNK, and ERK were higher in MsrA–/– than in MsrA+/+ BMDMs in response to LPS, suggesting that MsrA deficiency increases MAPK activation. Furthermore, MsrA deficiency increased the expression and nuclear translocation of NF-κB and the expression of inducible nitric oxide synthase, a target gene of NF-κB, in response to LPS. Taken together, our results suggest that MsrA protects against LPS-induced septic shock, and negatively regulates proinflammatory responses via inhibition of the ROS–MAPK–NF-κB signaling pathways.

Methionine sulfoxide reductase A protects hepatocytes against acetaminophen-induced toxicity via regulation of thioredoxin reductase 1 expression

Thioredoxin reductase 1 (TXNRD1) is associated with susceptibility to acetaminophen (APAP)-induced liver damage. Methionine sulfoxide reductase A (MsrA) is an antioxidant and protein repair enzyme that specifically catalyzes the reduction of methionine S-sulfoxide residues. We have previously shown that MsrA deficiency exacerbates acute liver injury induced by APAP. In this study, we used primary hepatocytes to investigate the underlying mechanism of the protective effect of MsrA against APAP-induced hepatotoxicity. MsrA gene-deleted (MsrA–/–) hepatocytes showed higher susceptibility to APAP-induced cytotoxicity than wild-type (MsrA+/+) cells, consistent with our previous in vivo results. MsrA deficiency increased APAP-induced glutathione depletion and reactive oxygen species production. APAP treatment increased Nrf2 activation more profoundly in MsrA–/– than in MsrA+/+ hepatocytes. Basal TXNRD1 levels were significantly higher in MsrA–/– than in MsrA+/+ hepatocytes, while TXNRD1 depletion in both MsrA–/– and MsrA+/+ cells resulted in increased resistance to APAP-induced cytotoxicity. In addition, APAP treatment significantly increased TXNRD1 expression in MsrA–/– hepatocytes, while no significant change was observed in MsrA+/+ cells. Overexpression of MsrA reduced APAP-induced cytotoxicity and TXNRD1 expression levels in APAP-treated MsrA–/– hepatocytes. Collectively, our results suggest that MsrA protects hepatocytes from APAP-induced cytotoxicity through the modulation of TXNRD1 expression.

Methionine Sulfoxide Reductase A, B1 and B2 Are Likely to Be Involved in the Protection against Oxidative Stress in the Inner Ear

The methionine sulfoxide reductase (Msr) family of proteins is a class of repair enzymes that reduce methionine-S (MsrA) or methionine-R (MsrB) sulfoxide to methionine. Recent studies have reported that mutations in the MSRB3 gene cause autosomal recessive hearing loss in humans, and in mice MsrB3 deficiency leads to profound hearing loss due to hair cell apoptosis and stereocilia degeneration. However, apart from MsrB3, studies on Msr proteins in the inner ear have not yet been reported. In this study, we identified and characterized Msr expression in the cochlea and vestibule. First, we confirmed RNA expression levels of Msr family members in the cochlea and vestibule using reverse transcription PCR and detected Msr family members in both tissues. We also conducted immunohistochemical staining to localize Msr family members within the cochlea and vestibule. In the cochlea, MsrA was detected in supporting cells, spiral ligament, spiral limbus, Reissner's membrane and the spiral ganglion. MsrB1 was specifically expressed in hair cells and the spiral ganglion. MsrB2 was noted in the spiral ganglion, tectorial membrane and stria vascularis. In the vestibule, MsrA and MsrB1 were detected in hair cells and the vestibular ganglion, while MsrB2 was restricted to the vestibular ganglion. In this study, we identified distinct distributions of Msr family members in the organ of Corti and hypothesized that MsrA, MsrB1 and MsrB2 protect proteins in the organ of Corti from oxidative stress.

Characterization and Solution Structure of Mouse Myristoylated Methionine Sulfoxide Reductase A

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. The cytosolic form of the enzyme is myristoylated, but it is not known to translocate to membranes, and the function of myristoylation is not established. We compared the biochemical and biophysical properties of myristoylated and nonmyristoylated mouse methionine sulfoxide reductase A. These were almost identical for both forms of the enzyme, except that the myristoylated form reduced methionine sulfoxide in protein much faster than the nonmyristoylated form. We determined the solution structure of the myristoylated protein and found that the myristoyl group lies in a relatively surface exposed "myristoyl nest." We propose that this structure functions to enhance protein-protein interaction.

Structural Plasticity of the Thioredoxin Recognition Site of Yeast Methionine S-Sulfoxide Reductase Mxr1

The methionine S-sulfoxide reductase MsrA catalyzes the reduction of methionine sulfoxide, a ubiquitous reaction depending on the thioredoxin system. To investigate interactions between MsrA and thioredoxin (Trx), we determined the crystal structures of yeast MsrA/Mxr1 in their reduced, oxidized, and Trx2-complexed forms, at 2.03, 1.90, and 2.70 Å, respectively. Comparative structure analysis revealed significant conformational changes of the three loops, which form a plastic "cushion" to harbor the electron donor Trx2. The flexible C-terminal loop enabled Mxr1 to access the methionine sulfoxide on various protein substrates. Moreover, the plasticity of the Trx binding site on Mxr1 provides structural insights into the recognition of diverse substrates by a universal catalytic motif of Trx.

Compartmentalization and regulation of mitochondrial function by methionine sulfoxide reductases in yeast.

Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine (S)-sulfoxide (Met-SO) and methionine (R)-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB, or both proteins had normal levels of mitochondria but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments.

Functional Analysis of Free Methionine-R-sulfoxide Reductase from Saccharomyces cerevisiae

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 Cys(101) as catalytic and Cys(125) as resolving residues in yeast fRMsr. These residues as well as a third Cys, resolving Cys(91), 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.

MsrB1 (Methionine-R-sulfoxide Reductase 1) Knock-out Mice: ROLES OF MsrB1 IN REDOX REGULATION AND IDENTIFICATION OF A NOVEL SELENOPROTEIN FORM

Protein oxidation has been linked to accelerated aging and is a contributing factor to many diseases. Methionine residues are particularly susceptible to oxidation, but the resulting mixture of methionine R-sulfoxide (Met-RO) and methionine S-sulfoxide (Met-SO) can be repaired by thioredoxin-dependent enzymes MsrB and MsrA, respectively. Here, we describe a knock-out mouse deficient in selenoprotein MsrB1, the main mammalian MsrB located in the cytosol and nucleus. In these mice, in addition to the deletion of 14-kDa MsrB1, a 5-kDa selenoprotein form was specifically removed. Further studies revealed that the 5-kDa protein occurred in both mouse tissues and human HEK 293 cells; was down-regulated by MsrB1 small interfering RNA, selenium deficiency, and selenocysteine tRNA mutations; and was immunoprecipitated and recognized by MsrB1 antibodies. Specific labeling with (75)Se and mass spectrometry analyses revealed that the 5-kDa selenoprotein corresponded to the C-terminal sequence of MsrB1. The MsrB1 knock-out mice lacked both 5- and 14-kDa MsrB1 forms and showed reduced MsrB activity, with the strongest effect seen in liver and kidney. In addition, MsrA activity was decreased by MsrB1 deficiency. Liver and kidney of the MsrB1 knock-out mice also showed increased levels of malondialdehyde, protein carbonyls, protein methionine sulfoxide, and oxidized glutathione as well as reduced levels of free and protein thiols, whereas these parameters were little changed in other organs examined. Overall, this study established an important contribution of MsrB1 to the redox control in mouse liver and kidney and identified a novel form of this protein.

Functioning methionine sulfoxide reductases A and B are present in human epidermal melanocytes in the cytosol and in the nucleus

Oxidation of methionine residues by reactive oxygen (ROS) in protein structures leads to the formation of methionine sulfoxide which can consequently lead to a plethora of impaired functionality. The generation of methionine sulfoxide yields ultimately a diastereomeric mixture of the S and R sulfoxides. So far two distinct enzyme families have been identified. MSRA reduces methionine S-sulfoxide, while MSRB reduces the R-diastereomer. It has been shown that these enzymes are involved in regulation of protein function and in elimination of ROS via reversible methionine formation besides protein repair. Importantly, both enzymes require coupling to the NADPH/thioredoxin reductase/thioredoxin electron donor system. In this report, we show for the first time the expression and function of both sulfoxide reductases together with thioredoxin reductase in the cytosol as well as in the nucleus of epidermal melanocytes which are especially sensitive to ROS. Since this cell resides in the basal layer of the epidermis and its numbers and functions are reduced upon ageing and for instance also in depigmentation processes, we believe that this discovery adds an intricate repair mechanism to melanocyte homeostasis and survival.

Methionine Sulfoxide Reduction and Assimilation in Escherichia coli : New Role for the Biotin Sulfoxide Reductase BisC

Methionine ranks among the amino acids most sensitive to oxidation, which converts it to a racemic mixture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO). The methionine sulfoxide reductases MsrA and MsrB reduce free and protein-bound MetSO, MsrA being specific for Met-S-SO and MsrB for Met-R-SO. In the present study, we report that an Escherichia coli metB1 auxotroph lacking both msrA and msrB is still able to use either of the two MetSO enantiomers. This indicates that additional methionine sulfoxide reductase activities occur in E. coli. BisC, a poorly characterized biotin sulfoxide reductase, was identified as one of these new methionine sulfoxide reductases. BisC was purified and found to exhibit reductase activity with free Met-S-SO but not with free Met-R-SO as a substrate. Moreover, a metB1 msrA msrB bisC strain of E. coli was unable to use Met-S-SO for growth, but it retained the ability to use Met-R-SO. Mass spectrometric analyses indicated that BisC is unable to reduce protein-bound Met-S-SO. Hence, this study shows that BisC has an essential role in assimilation of oxidized methionines. Moreover, this work provides the first example of an enzyme that reduces free MetSO while having no activity on peptide-bound MetSO residues.

Reaction Mechanism, Evolutionary Analysis, and Role of Zinc in Drosophila Methionine-R-sulfoxide Reductase

Methionine residues in proteins are susceptible to oxidation, and the resulting methionine sulfoxides can be reduced back to methionines by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Herein, we have identified two MsrB families that differ by the presence of zinc. Evolutionary analyses suggested that the zinc-containing MsrB proteins are prototype enzymes and that the metal was lost in certain MsrB proteins later in evolution. Zinc-containing Drosophila MsrB was further characterized. The enzyme was found to employ a catalytic Cys(124) thiolate, which directly interacted with methionine sulfoxide, resulting in methionine and a Cys(124) sulfenic acid intermediate. A subsequent reaction of this intermediate with Cys(69) generated an intramolecular disulfide. Dithiothreitol could reduce either the sulfenic acid or the disulfide, but the disulfide was a preferred substrate for thioredoxin, a natural electron donor. Interestingly, the C69S mutant could complement MsrA/MsrB deficiency in yeast, and the corresponding natural form of mouse MsrB was active with thioredoxin. These data indicate that MsrB proteins employ alternative mechanisms for sulfenic acid reduction. Four other conserved cysteines in Drosophila MsrB (Cys(51), Cys(54), Cys(101), and Cys(104)) were found to coordinate structural zinc. Mutation of any one or a combination of these residues resulted in complete loss of metal and catalytic activity, demonstrating an essential role of zinc in Drosophila MsrB. In contrast, two conserved histidines were important for thioredoxin-dependent activity, but were not involved in zinc binding. A Drosophila MsrA gene was also cloned, and the recombinant enzyme was found to be metal-free and specific for methionine S-sulfoxide and to employ a similar sulfenic acid/disulfide mechanism.