Methionine sulfoxide reductase A (MsrA) protects cultured mouse embryonic stem cells from H2O2‐mediated oxidative stress

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. J. Cell. Biochem. 111: 94–103, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of a truncated form of methionine sulfoxide reductase a expressed in mouse embryonic stem cells

Background  

Methionine Sulfoxide Reductase A (MsrA), an enzyme in the Msr gene family, is important in the cellular anti-oxidative stress defense mechanism. It acts by reducing the oxidized methionine sulfoxide in proteins back to sulfide and by reducing the cellular level of reactive oxygen species. MsrA, the only enzyme in the Msr gene family that can reduce the S-form epimers of methionine sulfoxide, has been located in different cellular compartments including mitochondria, cytosol and nuclei of various cell lines.     

The mirrored methionine sulfoxide reductases of Neisseria gonorrhoeae pilB

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.     

A methionine sulfoxide reductase in Escherichia coli that reduces the R enantiomer of methionine sulfoxide

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.     

Increased catalytic efficiency following gene fusion of bifunctional methionine sulfoxide reductase enzymes from Shewanella oneidensis

Methionine sulfoxide reductase enzymes MsrA and MsrB have complementary stereospecificities that reduce the S and R stereoisomers of methionine sulfoxide (MetSO), respectively, and together function as critical antioxidant enzymes. In some pathogenic and metal-reducing bacteria, these genes are fused to form a bifunctional methionine sulfoxide reductase (i.e., MsrBA) enzyme. To investigate how gene fusion affects the substrate specificity and catalytic activities of Msr, we have cloned and expressed the MsrBA enzyme from Shewanella oneidensis, a metal-reducing bacterium and fish pathogen. For comparison, we also cloned and expressed the wild-type MsrA enzyme from S. oneidensis and a genetically engineered MsrB protein. MsrBA is able to completely reduce (i.e., repair) MetSO in the calcium regulatory protein calmodulin (CaM), while only partial repair is observed using both MsrA and MsrB enzymes together at 25 degrees C. A restoration of the normal protein fold is observed co-incident with the repair of MetSO in oxidized CaM (CaMox by MsrBA, as monitored by time-dependent increases in the anisotropy associated with the rigidly bound multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH). Underlying the efficient repair of MetSO in CaMox is the coordinate activity of the two catalytic domains in the MsrBA fusion protein, which results in a 1 order of magnitude rate enhancement in comparison to those of the individual MsrA or MsrB enzyme alone. The coordinate binding of both domains of MsrBA permits the full repair of all MetSO in CaMox. The common expression of Msr fusion proteins in bacterial pathogens is consistent with an important role for this enzyme activity in the maintenance of protein function necessary for bacterial survival under highly oxidizing conditions associated with pathogenesis or bioremediation.     

The Arabidopsis plastidic methionine sulfoxide reductase B proteins. Sequence and activity characteristics, comparison of the expression with plastidic methionine sulfoxide reductase A, and induction by photooxidative stress

Two types of methionine (Met) sulfoxide reductases (Msr) catalyze the reduction of Met sulfoxide (MetSO) back to Met. MsrA, well characterized in plants, exhibits an activity restricted to the Met-S-SO-enantiomer. Recently, a new type of Msr enzyme, called MsrB, has been identified in various organisms and shown to catalytically reduce the R-enantiomer of MetSO. In plants, very little information is available about MsrB and we focused our attention on Arabidopsis (Arabidopsis thaliana) MsrB proteins. Searching Arabidopsis genome databases, we have identified nine open reading frames encoding proteins closely related to MsrB proteins from bacteria and animal cells. We then analyzed the activity and abundance of the two chloroplastic MsrB proteins, MsrB1 and MsrB2. Both enzymes exhibit an absolute R-stereospecificity for MetSO and a higher catalytic efficiency when using protein-bound MetSO as a substrate than when using free MetSO. Interestingly, we observed that MsrB2 is reduced by thioredoxin, whereas MsrB1 is not. This feature of MsrB1 could result from the lack of the catalytical cysteine (Cys) corresponding to Cys-63 in Escherichia coli MsrB that is involved in the regeneration of Cys-117 through the formation of an intramolecular disulfide bridge followed by thioredoxin reduction. We investigated the abundance of plastidial MsrA and B in response to abiotic (water stress, photooxidative treatment) and biotic (rust fungus) stresses and we observed that MsrA and B protein levels increase in response to the photooxidative treatment. The possible role of plastidic MsrB in the tolerance to oxidative damage is discussed.     

Specific activity of methionine sulfoxide reductase in CD-1 mice is significantly affected by dietary selenium but not zinc

Reactive oxygen species-mediated oxidation of methionine residues in protein results in a racemic mixture of R and S forms of methionine sulfoxide (MetO). MetO is reduced back to methionine by the methionine sulfoxide reductases MsrA and MsrB. MsrA is specific toward the S form and MsrB is specific toward the R form of MetO. MsrB is a selenoprotein reported to contain zinc (Zn). To determine the effects of dietary selenium (Se) and Zn on Msr activity, CD-1 mice (N=16/group) were fed, in a 2×2 design, diets containing 0 or 0.2 μg Se/g and 3 or 15 ∥ Zn/g. As an oxidative stress, half of the mice received L-buthionine sulfoximine (BSO; ip; 2 mmol/kg, three times per week for the last 3 wk); the others received saline. After 9.5 wk, Msr (the combined specific activities of MsrA and MsrB) was measured in the brain, kidney, and liver. Se deficiency decreased (p<0.0001) Msr in all three tissues, but Zn had no direct effect. BSO treatment was expected to result in increased Msr activity; this was not seen. Additionally, we found that the ratio of MetO to methionine in liver protein was increased (indicative of oxidative damage) by Se deficiency. The results show that Se deficiency increases oxidation of methionyl residues in protein, that Se status affects Msr (most likely through effects on the selenoprotein MsrB), and that marginal Zn deficiency has little effect on Msr in liver and kidney. Finally, the results show that the oxidative effects of limited BSO treatment did not upregulate Msr activity.     

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.     

Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism

Almost all forms of reactive oxygen species (ROS) oxidize methionine residues of proteins to a mixture of the R- and S-isomers of methionine sulfoxide. Because organisms contain methionine sulfoxide reductases (Msr's) that can catalyze the thioredoxin-dependent reduction of the sulfoxides back to methionine, it was proposed that the cyclic oxidation/reduction of methionine residues might serve as antioxidants to scavenge ROS, and also to facilitate the regulation of critical enzyme activities. We summarize here results of studies showing that organisms possess two different forms of Msr – namely, MsrA that catalyzes reduction of the S-isomer and MsrB that catalyzes the reduction of the R-isomer. Deletion of the msrA gene in mice leads to increased sensitivity to oxidative stress and to a decrease (40%) in the maximum lifespan. This suggests that elimination of both Msr's would have more serious consequences.     

Functions and evolution of selenoprotein methionine sulfoxide reductases

Methionine sulfoxide reductases (Msrs) are thiol-dependent enzymes which catalyze conversion of methionine sulfoxide to methionine. Three Msr families, MsrA, MsrB, and fRMsr, are known. MsrA and MsrB are responsible for the reduction of methionine-S-sulfoxide and methionine-R-sulfoxide residues in proteins, respectively, whereas fRMsr reduces free methionine-R-sulfoxide. Besides acting on proteins, MsrA can additionally reduce free methionine-S-sulfoxide. Some MsrAs and MsrBs evolved to utilize catalytic selenocysteine. This includes MsrB1, which is a major MsrB in cytosol and nucleus in mammalian cells. Specialized machinery is used for insertion of selenocysteine into MsrB1 and other selenoproteins at in-frame UGA codons. Selenocysteine offers catalytic advantage to the protein repair function of Msrs, but also makes these proteins dependent on the supply of selenium and requires adjustments in their strategies for regeneration of active enzymes. Msrs have roles in protecting cellular proteins from oxidative stress and through this function they may regulate lifespan in several model organisms.     

The peptide methionine sulfoxide reductases, MsrA and MsrB (hCBS-1), are downregulated during replicative senescence of human WI-38 fibroblasts

In contrast to other oxidative modifications of amino acids, methionine sulfoxide can be enzymatically reduced back to methionine in proteins by the peptide methionine sulfoxide reductase system, composed of MsrA and MsrB. The expression of MsrA and one member of the MsrB family, hCBS-1, was analyzed during replicative senescence of WI-38 human fibroblasts. Gene expression decreased for both enzymes in senescent cells compared to young cells, and this decline was associated with an alteration in catalytic activity and the accumulation of oxidized proteins during senescence. These results suggest that downregulation of MsrA and hCBS-1 can alter the ability of senescent cells to cope with oxidative stress, hence contributing to the age-related accumulation of oxidative damage.     

Selenocompounds can serve as oxidoreductants with the methionine sulfoxide reductase enzymes

In a recent study on the reducing requirement for the methionine sulfoxide reductases (Msr) (Sagher, D., Brunell, D., Hejtmancik, J. F., Kantorow, M., Brot, N. & Weissbach, H. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 8656-8661), we have shown that thioredoxin, although an excellent reducing system for Escherichia coli MsrA and MsrB and bovine MsrA, is not an efficient reducing agent for either human MsrB2 (hMsrB2) or human MsrB3 (hMsrB3). In a search for another reducing agent for hMsrB2 and hMsrB3, it was recently found that thionein, the reduced, metal-free form of metallothionein, could function as a reducing system for hMsrB3, with weaker activity using hMsrB2. In the present study, we provide evidence that some selenium compounds are potent reducing agents for both hMsrB2 and hMsrB3.     

Methionine sulfoxide reductase from the hyperthermophilic archaeon Thermococcus kodakaraensis, an enzyme designed to function at suboptimal growth temperatures

Methionine sulfoxide reductase (Msr) catalyzes the thioredoxin-dependent reduction and repair of methionine sulfoxide (MetO). Although Msr genes are not present in most hyperthermophile genomes, an Msr homolog encoding an MsrA-MsrB fusion protein (MsrAB(Tk)) was present on the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. Recombinant proteins corresponding to MsrAB(Tk) and the individual domains (MsrA(Tk) and MsrB(Tk)) were produced, purified, and biochemically examined. MsrA(Tk) and MsrB(Tk) displayed strict substrate selectivity for Met-S-O and Met-R-O, respectively. MsrAB(Tk), and in particular the MsrB domain of this protein, displayed an intriguing behavior for an enzyme from a hyperthermophile. While MsrAB(Tk) was relatively stable at temperatures up to 80 degrees C (with a half-life of approximately 30 min at 80 degrees C), a 75% decrease in activity was observed after 2.5 min at 85 degrees C, the optimal growth temperature of this archaeon. Moreover, maximal levels of MsrB activity of MsrAB(Tk) were observed at the strikingly low temperature of 30 degrees C, which also was observed for MsrB(Tk). Consistent with the low-temperature-specific biochemical properties of MsrAB(Tk), the presence of the protein was greater in T. kodakaraensis cells grown at suboptimal temperatures (60 to 70 degrees C) and could not be detected at 80 to 90 degrees C. We found that the amount of intracellular MsrAB(Tk) protein increased with exposure to higher dissolved oxygen levels, but only at suboptimal growth temperatures. While measuring background rates of the Msr enzyme reactions, we observed significant levels of MetO reduction at high temperatures without enzyme. The occurrence of nonenzymatic MetO reduction at high temperatures may explain the specific absence of Msr homologs in most hyperthermophiles. Together with the fact that the presence of Msr in T. kodakaraensis is exceptional among the hyperthermophiles, the enzyme may represent a novel strategy for this organism to deal with low-temperature environments in which the dissolved oxygen concentrations increase.     

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.     

Characterization of the amino acids from Neisseria meningitidis methionine sulfoxide reductase B involved in the chemical catalysis and substrate specificity of the reductase step

Methionine sulfoxide reductases (Msrs) are antioxidant repair enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The Msr family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the S and R isomers of the sulfoxide function, respectively. Both classes of Msr share a similar three-step chemical mechanism involving first a reductase step that leads to the formation of a sulfenic acid intermediate. In this study, the invariant amino acids of Neisseria meningitidis MsrB involved in the reductase step catalysis and in substrate binding have been characterized by the structure-function relationship approach. Altogether the results show the following: 1) formation of the MsrB-substrate complex leads to an activation of the catalytic Cys-117 characterized by a decreased pKapp of approximately 2.7 pH units; 2) the catalytic active MsrB form is the Cys-117-/His-103+ species with a pKapp of 6.6 and 8.3, respectively; 3) His-103 and to a lesser extent His-100, Asn-119, and Thr-26 (via a water molecule) participate in the stabilization of the polarized form of the sulfoxide function and of the transition state; and 4) Trp-65 is essential for the catalytic efficiency of the reductase step by optimizing the position of the substrate in the active site. A scenario for the reductase step is proposed and discussed in comparison with that of MsrA.     

Alternative first exon splicing regulates subcellular distribution of methionine sulfoxide reductases

Background  

Methionine sulfoxide reduction is an important protein repair pathway that protects against oxidative stress, controls protein function and has a role in regulation of aging. There are two enzymes that reduce stereospecifically oxidized methionine residues: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In many organisms, these enzymes are targeted to various cellular compartments. In mammals, a single MsrA gene is known, however, its product is present in cytosol, nucleus, and mitochondria. In contrast, three mammalian MsrB genes have been identified whose products are located in different cellular compartments.     

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.     

Functional characterization of the Aspergillus nidulans methionine sulfoxide reductases (msrA and msrB)

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). 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. Here, we describe the homologs of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage.     

Studies on the reducing systems for plant and animal thioredoxin-independent methionine sulfoxide reductases B

Two distinct stereospecific methionine sulfoxide reductases (Msr), MsrA and MsrB reduce the oxidized methionine (Met), methionine sulfoxide [Met(O)], back to Met. In this report, we examined the reducing systems required for the activities of two chloroplastic MsrB enzymes (NtMsrB1 and NtMsrB2) from tobacco (Nicotiana tabacum). We found that NtMrsB1, but not NtMsrB2, could use dithiothreitol as an efficient hydrogen donor. In contrast Escherichia coli thioredoxin (Trx) could serve as a reducing agent for NtMsrB2, but not for NtMsrB1. Similar to previously reported human Trx-independent hMsrB2 and hMsrB3, NtMsrB1 could also use bovine liver thionein and selenocysteamine as reducing agents. Furthermore, the unique plant Trx-like protein CDSP32 was shown to reduce NtMsrB1, hMsrB2 and hMsrB3. All these tested Trx-independent MsrB enzymes lack an additional cysteine (resolving cysteine) that is capable of forming a disulfide bond on the enzyme during the catalytic reaction. Our results indicate that plant and animal MsrB enzymes lacking a resolving cysteine likely share a similar reaction mechanism.     

Methionine sulfoxide reductases: selenoprotein forms and roles in antioxidant protein repair in mammals

Msrs (methionine sulfoxide reductases), MsrA and MsrB, are repair enzymes that reduce methionine sulfoxide residues in oxidatively damaged proteins to methionine residues in a stereospecific manner. These enzymes protect cells from oxidative stress and have been implicated in delaying the aging process and progression of neurodegenerative diseases. In recent years, significant efforts have been made to explore the catalytic properties and physiological functions of these enzymes. In the current review, we present recent progress in this area, with the focus on mammalian MsrA and MsrBs including their roles in disease, evolution and function of selenoprotein forms of MsrA and MsrB, and the biochemistry of these enzymes.