Methionine sulfoxide reductases protect Ffh from oxidative damages in Escherichia coli

In proteins, methionine residues are primary targets for oxidation. Methionine oxidation is reversed by methionine sulfoxide reductases A and B, a class of highly conserved enzymes. Ffh protein, a component of the ubiquitous signal recognition particle, contains a methionine-rich domain, interacting with a small 4.5S RNA. In vitro analyses reported here show that: (i) oxidized Ffh is unable to bind 4.5S RNA, (ii) oxidized Ffh contains methionine sulfoxide residues, (iii) oxidized Ffh is a substrate for MsrA and MsrB enzymes; and (iv) MsrA/B repairing activities allow oxidized Ffh to recover 4.5S RNA-binding abilities. In vivo analyses reveal that: (i) Ffh synthesized in the msrA msrB mutant contains methionine sulfoxide residues and is unstable, (ii) msrA msrB mutant requires high levels of Ffh synthesis for growth and (iii) msrA msrB mutation leads to defects in Ffh-dependent targeting of MalF. We conclude that MsrA and MsrB are required to repair Ffh oxidized by reactive oxygen species produced by aerobic metabolism, establishing an as-yet undescribed link between protein targeting and oxidation.     

New membrane-associated and soluble peptide methionine sulfoxide reductases in Escherichia coli

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.     

Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins and protect cells from oxidative protein unfolding

Reduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis of MetO reduction in proteins, the physiological substrates of MSRA and MSRB. In this work we exploited an NADPH-dependent thioredoxin system and determined the kinetic parameters of yeast MSRA and MSRB using three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the K(m) lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereas MSRB was specialized for the reduction of MetO in proteins. Using oxidized glutathione S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins and that their activities increased with the unfolding state. Biochemical quantification and identification of MetO reduced in the substrates by mass spectrometry revealed that the increased activity was due to better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins.     

Characterization of a novel methionine sulfoxide reductase A from tomato (Solanum lycopersicum ), and its protecting role in Escherichia coli

Methionine sulfoxide reductase A (MSRA) is a ubiquitous enzyme that has been demonstrated to reduce the S enantiomer of methionine sulfoxide (MetSO) to methionine (Met) and can protect cells against oxidative damage. In this study, we isolated a novel MSRA (SlMSRA2) from Micro-Tom (Solanum lycopersicum L. cv. Micro-Tom) and characterized it by subcloning the coding sequence into a pET expression system. Purified recombinant protein was assayed by HPLC after expression and refolding. This analysis revealed the absolute specificity for methionine-S-sulfoxide and the enzyme was able to convert both free and protein-bound MetSO to Met in the presence of DTT. In addition, the optimal pH, appropriate temperature, and Km and Kcat values for MSRA2 were observed as 8.5, 25oC, 352 ± 25 μM, and 0.066 ± 0.009 S(-1), respectively. Disk inhibition and growth rate assays indicated that SlMSRA2 may play an essential function in protecting E. coli against oxidative damage.     

Significance of Four Methionine Sulfoxide Reductases in Staphylococcus aureus

Staphylococcus aureus is a major human pathogen and emergence of antibiotic resistance in clinical staphylococcal isolates raises concerns about our ability to control these infections. Cell wall-active antibiotics cause elevated synthesis of methionine sulfoxide reductases (Msrs: MsrA1 and MsrB) in S. aureus. MsrA and MsrB enzymes reduce S-epimers and R-epimers of methionine sulfoxide, respectively, that are generated under oxidative stress. In the S. aureus chromosome, there are three msrA genes (msrA1, msrA2 and msrA3) and one msrB gene. To understand the precise physiological roles of Msr proteins in S. aureus, mutations in msrA1, msrA2 and msrA3 and msrB genes were created by site-directed mutagenesis. These mutants were combined to create a triple msrA (msrA1, msrA2 and msrA3) and a quadruple msrAB (msrA1, msrA2, msrA3, msrB) mutant. These mutants were used to determine the roles of Msr proteins in staphylococcal growth, antibiotic resistance, adherence to human lung epithelial cells, pigment production, and survival in mice relative to the wild-type strains. MsrA1-deficient strains were sensitive to oxidative stress conditions, less pigmented and less adherent to human lung epithelial cells, and showed reduced survival in mouse tissues. In contrast, MsrB-deficient strains were resistant to oxidants and were highly pigmented. Lack of MsrA2 and MsrA3 caused no apparent growth defect in S. aureus. In complementation experiments with the triple and quadruple mutants, it was MsrA1 and not MsrB that was determined to be critical for adherence and phagocytic resistance of S. aureus. Overall, the data suggests that MsrA1 may be an important virulence factor and MsrB probably plays a balancing act to counter the effect of MsrA1 in S. aureus.     

Important role for methionine sulfoxide reductase in the oxidative stress response of Xanthomonas campestris pv. phaseoli

A methionine sulfoxide reductase gene (msrA) from Xanthomonas campestris pv. phaseoli has unique expression patterns and physiological function. msrA expression is growth dependent and is highly induced by exposure to oxidants and N-ethylmaleimide in an OxyR- and OhrR-independent manner. An msrA mutant showed increased sensitivity to oxidants but only during stationary phase.     

The human IGF-I gene contains two cell type-specifically regulated promoters.

The human insulin-like growth factor-I (IGF-I) gene contains two alternative leader exons: exons 1 and 2. We have identified, by transient transfection experiments, the putative promoters P1 and P2 upstream of these leader exons. The promoter regions were cloned in front of the luciferase reporter gene and their promoter activities were measured in transfected SK-N-MC (human neuroepithelioma) and OVCAR-3 (human ovarian carcinoma) cells. Both of these cell lines express the IGF-I gene endogenously, resulting in normally sized IGF-I mRNAs of 7.6, 1.3 and 1.1 kb. In SK-N-MC cells, in which P1 is the most active IGF-I promoter, P2 displayed a three times lower promoter activity than P1. However, in OVCAR-3 cells, P2 is four times more active than P1, resulting in an overall 12-fold difference in the relative promoter activities of the two IGF-I gene promoters in these two cell types. This indicates that the IGF-I promoters show a cell type-specific expression pattern.     

Peptide Methionine Sulfoxide Reductase (MsrA) Is a Virulence Determinant in Mycoplasma genitalium

Mycoplasma genitalium is the smallest self-replicating microorganism and is implicated in human diseases, including urogenital and respiratory infections and arthritides. M. genitalium colonizes host cells primarily through adherence mechanisms mediated by a network of surface-associated membrane proteins, including adhesins and cytadherence-related proteins. In this paper, we show that cytadherence in M. genitalium is affected by an unrelated protein known as peptide methionine sulfoxide reductase (MsrA), an antioxidant repair enzyme that catalyzes the reduction of methionine sulfoxide [Met(O)] residues in proteins to methionine. An msrA disruption mutant of M. genitalium, constructed through homologous recombination, displayed markedly reduced adherence to sheep erythrocytes. In addition, the msrA mutant was incapable of growing in hamsters and exhibited hypersensitivity to hydrogen peroxide when compared to wild-type virulent M. genitalium. These results indicate that MsrA plays an important role in M. genitalium pathogenicity, possibly by protecting mycoplasma protein structures from oxidative damage or through alternate virulence-related pathways.     

A second human methionine sulfoxide reductase (hMSRB2) reducing methionine-R-sulfoxide displays a tissue expression pattern distinct from hMSRB1

Abstract

Peptide methionine sulfoxide reductases are important enzymes in the defense against cellular oxidative stress as they reduce methionine sulfoxide, the product of methionine oxidation by physiologically relevant reactive oxygen species. Two distinct enzyme classes, MSRA and MSRB, have evolved for selectively reducing the two epimers, methionine-S-sulfoxide and methionine-Rsulfoxide. A new human MSR enzyme (hMSRB2) specifically reducing methionine-R-sulfoxide, which showed a conversion rate for peptide-bound methionine-S-sulfoxide similar to hMSRB1, was characterized with respect to its tissue expression. As previously found for hMSRB1, expression of hMSRB2 mRNA was weak in brain, but strong in heart and skeletal muscle. In contrast to hMSRB1, its expression was high in smooth muscle-containing organs (digestive system, bladder), lung and aorta, while hMSRB1 displayed a higher expression than hMSRB2 in liver and kidney.     

A second human methionine sulfoxide reductase (hMSRB2) reducing methionine-R-sulfoxide displays a tissue expression pattern distinct from hMSRB1

Peptide methionine sulfoxide reductases are important enzymes in the defense against cellular oxidative stress as they reduce methionine sulfoxide, the product of methionine oxidation by physiologically relevant reactive oxygen species. Two distinct enzyme classes, MSRA and MSRB, have evolved for selectively reducing the two epimers, methionine-S-sulfoxide and methionine-R-sulfoxide. A new human MSR enzyme (hMSRB2) specifically reducing methionine-R-sulfoxide, which showed a conversion rate for peptide-bound methionine-S-sulfoxide similar to hMSRB1, was characterized with respect to its tissue expression. As previously found for hMSRB1, expression of hMSRB2 mRNA was weak in brain, but strong in heart and skeletal muscle. In contrast to hMSRB1, its expression was high in smooth muscle-containing organs (digestive system, bladder), lung and aorta, while hMSRB1 displayed a higher expression than hMSRB2 in liver and kidney.     

Methionine oxidation of Sup35 protein induces formation of the [PSI+] prion in a yeast peroxiredoxin mutant

The frequency with which the yeast [PSI(+)] prion form of Sup35 arises de novo is controlled by a number of genetic and environmental factors. We have previously shown that in cells lacking the antioxidant peroxiredoxin proteins Tsa1 and Tsa2, the frequency of de novo formation of [PSI(+)] is greatly elevated. We show here that Tsa1/Tsa2 also function to suppress the formation of the [PIN(+)] prion form of Rnq1. However, although oxidative stress increases the de novo formation of both [PIN(+)] and [PSI(+)], it does not overcome the requirement of cells being [PIN(+)] to form the [PSI(+)] prion. We use an anti-methionine sulfoxide antibody to show that methionine oxidation is elevated in Sup35 during oxidative stress conditions. Abrogating Sup35 methionine oxidation by overexpressing methionine sulfoxide reductase (MSRA) prevents [PSI(+)] formation, indicating that Sup35 oxidation may underlie the switch from a soluble to an aggregated form of Sup35. In contrast, we were unable to detect methionine oxidation of Rnq1, and MSRA overexpression did not affect [PIN(+)] formation in a tsa1 tsa2 mutant. The molecular basis of how yeast and mammalian prions form infectious amyloid-like structures de novo is poorly understood. Our data suggest a causal link between Sup35 protein oxidation and de novo [PSI(+)] prion formation.     

Heterogeneity and function of mammalian MSRs: enzymes for repair, protection and regulation

Methionine sulfoxide, the physiologically relevant oxidation product of methionine, is enzymatically reduced by peptide methionine sulfoxide reductases (MSRs). Two distinct classes of these enzymes, MSRA and MSRB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Mammals typically possess only one gene encoding MSRA, but at least three genes encoding MSRBs. These MSRs show distinct tissue- and subcellular expression patterns and may play specific functional roles. Susceptibility of some ion channels to reversible methionine oxidation suggests that MSRs have a regulatory role in cellular excitability. Some--if not all--MSRs protect cells and organisms against a variety of oxidative stress episodes, including those by hypoxia and reperfusion, and play a modulatory role in lifespan determination. More MSR-dependent physiological phenomena await to be discovered.     

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.     

Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase.

Oxidation of methionine residues to methionine sulfoxide can lead to inactivation of proteins. Methionine sulfoxide reductase (MsrA) has been known for a long time, and its repairing function well characterized. Here we identify a new methionine sulfoxide reductase, which we referred to as MsrB, the gene of which is present in genomes of eubacteria, archaebacteria, and eucaryotes. The msrA and msrB genes exhibit no sequence similarity and, in some genomes, are fused. The Escherichia coli MsrB protein (currently predicted to be encoded by an open reading frame of unknown function named yeaA) was used for genetic, enzymatic, and mass spectrometric investigations. Our in vivo study revealed that msrB is required for cadmium resistance of E. coli, a carcinogenic compound that induces oxidative stress. Our in vitro studies, showed that (i) MsrB and MsrA enzymes reduce free methionine sulfoxide with turn-over rates of 0.6 min(-1) and 20 min(-1), respectively, (ii) MsrA and MsrB act on oxidized calmodulin, each by repairing four to six of the eight methionine sulfoxide residues initially present, and (iii) simultaneous action of both MsrA and MsrB allowed full reduction of oxidized calmodulin. A possibility is that these two ubiquitous methionine sulfoxide reductases exhibit different substrate specificity.     

Diversity of Plant Methionine Sulfoxide Reductases B and Evolution of a Form Specific for Free Methionine Sulfoxide

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed the MSRB activity toward free Met-R-O with kinetic parameters similar to those reported for fRMSR from Escherichia coli, an enzyme specific for free Met-R-O. Overexpression of GmMSRB2 or GmMSRB4 in the yeast cytosol supported the growth of the triple MSRA/MSRB/fRMSR (Δ3MSRs) mutant on MetO and protected cells against H₂O₂-induced stress. Taken together, our data reveal an unexpected diversity of MSRBs in plants and indicate that, in contrast to mammals that cannot reduce free Met-R-O and microorganisms that use fRMSR for this purpose, plants evolved MSRBs for the reduction of both free and protein-based MetO.     

Methionine sulfoxide reductase A: Structure, function and role in ocular pathology

Methionine is a highly susceptible amino acid that can be oxidized to S and R diastereomeric forms of methionine sulfoxide by many of the reactive oxygen species generated in biological systems. Methionine sulfoxide reductases (Msrs) are thioredoxin-linked enzymes involved in the enzymatic conversion of methionine sulfoxide to methionine. Although MsrA and MsrB have the same function of methionine reduction, they differ in substrate specifi city, active site composition, subcellular localization, and evolution. MsrA has been localized in different ocular regions and is abundantly expressed in the retina and in retinal pigment epithelial (RPE) cells. MsrA protects cells from oxidative stress. Overexpression of MsrA increases resistance to cell death, while silencing or knocking down MsrA decreases cell survival; events that are mediated by mitochondria. MsrA participates in protein-protein interaction with several other cellular proteins. The interaction of MsrAwith α-crystallins is of utmost importance given the known functions of the latter in protein folding, neuroprotection, and cell survival. Oxidation of methionine residues in α-crystallins results in loss of chaperone function and possibly its antiapoptotic properties. Recent work from our laboratory has shown that MsrA is co-localized with αA and αB crystallins in the retinal samples of patients with age-related macular degen- eration. We have also found that chemically induced hypoxia regulates the expression of MsrA and MsrB2 in human RPE cells. Thus, MsrA is a critical enzyme that participates in cell and tissue protection, and its interaction with other proteins/growth factors may provide a target for therapeutic strategies to prevent degenerative diseases.     

Methionine sulfoxide reductases are essential for virulence of Salmonella typhimurium

Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H(2)O(2), and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H(2)O(2,) as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium.     

Alteration of DNA methylation status in K562 and MCF-7 cancer cell lines by nucleoside analogues

The effects of 2-chloro-2'-deoxyadenosine, beta-D-arabinofuranosyl-2-fluoroadenine, and 5-aza-2'-deoxycytidine on promoter methylation of the selected tumor suppressor genes (i.e., ERalpha, BRCA1, E-cadherin, PTEN, and APC) were estimated using methylation-sensitive restriction analysis (MSRA) in K562 cells (human erythroleukemic cell line) and MCF-7 cells (human breast cancer cell line). In both cell lines all tested drugs completely reduced methylation of PTEN and APC promoters. The results indicate that the tested nucleoside analogues, which are known inhibitors of DNA synthesis, also are implicated in indirect (or direct in the case of 5-aza-dCyd) regulation of post-replicative DNA modifications (i.e., DNA methylation).