Alterations in mitochondrial and cytosolic methionine sulfoxide reductase activity during cardiac ischemia and reperfusion

During cardiac ischemia/reperfusion, proteins are targets of reactive oxygen species produced by the mitochondrial respiratory chain resulting in the accumulation of oxidatively modified protein. Sulfur-containing amino acids are among the most sensitive to oxidation. Certain cysteine and methionine oxidation products can be reversed back to their reduced form within proteins by specific repair enzymes. Oxidation of methionine in protein produces methionine-S-sulfoxide and methionine-R-sulfoxide that can be catalytically reduced by two stereospecific enzymes, methionine sulfoxide reductases A and B, respectively. Due to the importance of the methionine sulfoxide reductase system in the maintenance of protein structure and function during conditions of oxidative stress, the fate of this system during ischemia/reperfusion was investigated. Mitochondrial and cytosolic methionine sulfoxide reductase activities are decreased during ischemia and at early times of reperfusion, respectively. Partial recovery of enzyme activity was observed upon extended periods of reperfusion. Evidence indicates that loss in activity is not due to a decrease in the level of MsrA but may involve structural modification of the enzyme.     

Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals

Oxidation of proteins by reactive oxygen species is associated with aging, oxidative stress, and many diseases. Although free and protein-bound methionine residues are particularly sensitive to oxidation to methionine sulfoxide derivatives, these oxidations are readily repaired by the action of methionine sulfoxide reductase (MsrA). To gain a better understanding of the biological roles of MsrA in metabolism, we have created a strain of mouse that lacks the MsrA gene. Compared with the wild type, this mutant: (i) exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen); (ii) has a shorter lifespan under both normal and hyperoxic conditions; (iii) develops an atypical (tip-toe) walking pattern after 6 months of age; (iv) accumulates higher tissue levels of oxidized protein (carbonyl derivatives) under oxidative stress; and (v) is less able to up-regulate expression of thioredoxin reductase under oxidative stress. It thus seems that MsrA may play an important role in aging and neurological disorders.     

Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates

Inducible nitric oxide synthase (iNOS) plays an important role in host defense. Macrophages expressing iNOS release the reactive nitrogen intermediates (RNI) nitrite and S-nitrosoglutathione (GSNO), which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages. We sought to characterize the active intrabacterial forms of these RNI and their molecular targets. Peptide methionine sulfoxide reductase (MsrA; EC ) catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met). E. coli lacking MsrA were hypersensitive to killing not only by hydrogen peroxide, but also by nitrite and GSNO. The wild-type phenotype was restored by transformation with plasmids encoding msrA from E. coli or M. tuberculosis, but not by an enzymatically inactive mutant msrA, indicating that Met oxidation was involved in the death of these cells. It seemed paradoxical that nitrite and GSNO kill bacteria by oxidizing Met residues when these RNI cannot themselves oxidize Met. However, under anaerobic conditions, neither nitrite nor GSNO was bactericidal. Nitrite and GSNO can both give rise to NO, which may react with superoxide produced by bacteria during aerobic metabolism, forming peroxynitrite, a known oxidant of Met to Met-O. Thus, the findings are consistent with the hypotheses that nitrite and GSNO kill E. coli by intracellular conversion to peroxynitrite, that intracellular Met residues in proteins constitute a critical target for peroxynitrite, and that MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage.     

Thiol-disulfide exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase

Peptide methionine sulfoxide reductase (MsrA; EC ) reverses the inactivation of many proteins due to the oxidation of critical methionine residues by reducing methionine sulfoxide, Met(O), to methionine. MsrA activity is independent of bound metal and cofactors but does require reducing equivalents from either DTT or a thioredoxin-regenerating system. In an effort to understand these observations, the four cysteine residues of bovine MsrA were mutated to serine in a series of permutations. An analysis of the enzymatic activity of the variants and their free sulfhydryl states by mass spectrometry revealed that thiol-disulfide exchange occurs during catalysis. In particular, the strictly conserved Cys-72 was found to be essential for activity and could form disulfide bonds, only upon incubation with substrate, with either Cys-218 or Cys-227, located at the C terminus. The significantly decreased activity of the Cys-218 and Cys-227 variants in the presence of thioredoxin suggested that these residues shuttle reducing equivalents from thioredoxin to the active site. A reaction mechanism based on the known reactivities of thiols with sulfoxides and the available data for MsrA was formulated. In this scheme, Cys-72 acts as a nucleophile and attacks the sulfur atom of the sulfoxide moiety, leading to the formation of a covalent, tetracoordinate intermediate. Collapse of the intermediate is facilitated by proton transfer and the concomitant attack of Cys-218 on Cys-72, leading to the formation of a disulfide bond. The active site is returned to the reduced state for another round of catalysis by a series of thiol-disulfide exchange reactions via Cys-227, DTT, or thioredoxin.     

Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.     

Modulation of potassium channel function by methionine oxidation and reduction

Oxidation of amino acid residues in proteins can be caused by a variety of oxidizing agents normally produced by cells. The oxidation of methionine in proteins to methionine sulfoxide is implicated in aging as well as in pathological conditions, and it is a reversible reaction mediated by a ubiquitous enzyme, peptide methionine sulfoxide reductase. The reversibility of methionine oxidation suggests that it could act as a cellular regulatory mechanism although no such in vivo activity has been demonstrated. We show here that oxidation of a methionine residue in a voltage-dependent potassium channel modulates its inactivation. When this methionine residue is oxidized to methionine sulfoxide, the inactivation is disrupted, and it is reversed by coexpression with peptide methionine sulfoxide reductase. The results suggest that oxidation and reduction of methionine could play a dynamic role in the cellular signal transduction process in a variety of systems.     

The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo

A gene homologous to methionine sulfoxide reductase (msrA) was identified as the predicted ORF (cosmid 9379) in chromosome V of Saccharomyces cerevisiae encoding a protein of 184 amino acids. The corresponding protein has been expressed in Escherichia coli and purified to homogeneity. The recombinant yeast MsrA possessed the same substrate specificity as the other known MsrA enzymes from mammalian and bacterial cells. Interruption of the yeast gene resulted in a null mutant, ΔmsrA::URA3 strain, which totally lost its cellular MsrA activity and was shown to be more sensitive to oxidative stress in comparison to its wild-type parent strain. Furthermore, high levels of free and protein-bound methionine sulfoxide were detected in extracts of msrA mutant cells relative to their wild-type parent cells, under various oxidative stresses. These findings show that MsrA is responsible for the reduction of methionine sulfoxide in vivo as well as in vitro in eukaryotic cells. Also, the results support the proposition that MsrA possess an antioxidant function. The ability of MsrA to repair oxidative damage in vivo may be of singular importance if methionine residues serve as antioxidants.     

Methionine sulfoxide reductase A is important for lens cell viability and resistance to oxidative stress

Age-related cataract, an opacity of the eye lens, is the leading cause of visual impairment in the elderly, the etiology of which is related to oxidative stress damage. Oxidation of methionine to methionine sulfoxide is a major oxidative stress product that reaches levels as high as 60% in cataract while being essentially absent from clear lenses. Methionine oxidation results in loss of protein function that can be reversed through the action of methionine sulfoxide reductase A (MsrA), which is implicated in oxidative stress protection and is an essential regulator of longevity in species ranging from Escherichia coli to mice. To establish a role for MsrA in lens protection against oxidative stress, we have examined the levels and spatial expression patterns of MsrA in the human lens and have tested the ability of MsrA to protect lens cells directly against oxidative stress. In the present report, we establish that MsrA is present throughout the human lens, where it is likely to defend lens cells and their components against methionine oxidation. We demonstrate that overexpression of MsrA protects lens cells against oxidative stress damage, whereas silencing of the MsrA gene renders lens cells more sensitive to oxidative stress damage. We also provide evidence that MsrA is important for lens cell function in the absence of exogenous stress. Collectively, these data implicate MsrA as a key player in lens cell viability and resistance to oxidative stress, a major factor in the etiology of age-related cataract.     

Free methionine-(R)-sulfoxide reductase from Escherichia coli reveals a new GAF domain function

The reduction of methionine sulfoxide (MetO) is mediated by methionine sulfoxide reductases (Msr). The MsrA and MsrB families can reduce free MetO and MetO within a peptide or protein context. This process is stereospecific with the S- and R-forms of MetO repaired by MsrA and MsrB, respectively. Cell extracts from an MsrA(-)B(-) knockout of Escherichia coli have several remaining Msr activities. This study has identified an enzyme specific for the free form of Met-(R)-O, fRMsr, through proteomic analysis. The recombinant enzyme exhibits the same substrate specificity and is as active as MsrA family members. E. coli fRMsr is, however, 100- to 1,000-fold more active than non-selenocysteine-containing MsrB enzymes for free Met-(R)-O. The crystal structure of E. coli fRMsr was previously determined, but no known function was assigned. Thus, the function of this protein has now been determined. The structural similarity of the E. coli and yeast proteins suggests that most fRMsrs use three cysteine residues for catalysis and the formation of a disulfide bond to enclose a small active site cavity. This latter feature is most likely a key determinant of substrate specificity. Moreover, E. coli fRMsr is the first GAF domain family member to show enzymatic activity. Other GAF domain proteins substitute the Cys residues and others to specifically bind cyclic nucleotides, chromophores, and many other ligands for signal potentiation. Therefore, Met-(R)-O may represent a signaling molecule in response to oxidative stress and nutrients via the TOR pathway in some organisms.     

Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins.

An enzyme that reduces methionine sulfoxide [Met(O)] residues in proteins [peptide Met(O) reductase (MsrA), EC 1.8.4.6; originally identified in Escherichia coli] was purified from bovine liver, and the cDNA encoding this enzyme was cloned and sequenced. The mammalian homologue of E. coli msrA (also called pmsR) cDNA encodes a protein of 255 amino acids with a calculated molecular mass of 25,846 Da. This protein has 61% identity with the E. coli MsrA throughout a region encompassing a 199-amino acid overlap. The protein has been overexpressed in E. coli and purified to homogeneity. The mammalian recombinant MsrA can use as substrate, proteins containing Met(O) as well as other organic compounds that contain an alkyl sulfoxide group such as N-acetylMet(O), Met(O), and dimethyl sulfoxide. Northern analysis of rat tissue extracts showed that rat msrA mRNA is present in a variety of organs with the highest level found in kidney. This is consistent with the observation that kidney extracts also contained the highest level of enzyme activity.     

Selenoprotein R is a zinc-containing stereo-specific methionine sulfoxide reductase

Selenoprotein R (SelR) is a mammalian selenocysteine-containing protein with no known function. Here we report that cysteine homologs of SelR are present in all organisms except certain parasites and hyperthermophiles, and this pattern of occurrence closely matches that of only one protein, peptide methionine sulfoxide reductase (MsrA). Moreover, in several genomes, SelR and MsrA genes are fused or clustered, and their expression patterns suggest a role of both proteins in protection against oxidative stress. Consistent with these computational screens, growth of Saccharomyces cerevisiae SelR and MsrA mutant strains was inhibited, and the strain lacking both genes could not grow, in the presence of H2O2 and methionine sulfoxide. We found that the cysteine mutant of mouse SelR, as well as the Drosophila SelR homolog, contained zinc and reduced methionine-R-sulfoxide, but not methionine-S-sulfoxide, in in vitro assays, a function that is both distinct and complementary to the stereo-specific activity of MsrA. These findings identify a function of the conserved SelR enzyme family, define a pathway of methionine sulfoxide reduction, reveal a case of convergent evolution of similar function in structurally distinct enzymes, and suggest a previously uncharacterized redox regulatory role of selenium in mammals.     

Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress

The yeast peptide-methionine sulfoxide reductase (MsrA) was overexpressed in a Saccharomyces cerevisiae null mutant of msrA by using a high-copy plasmid harboring the msrA gene and its promoter. The resulting strain had about 25-fold higher MsrA activity than its parent strain. When exposed to either hydrogen peroxide, paraquat, or 2,2′-azobis-(2-amidinopropane) dihydrochloride treatment, the MsrA overexpressed strain grew better, had lower free and protein-bound methionine sulfoxide and had a better survival rate under these conditions than did the msrA mutant and its parent strain. Substitution of methionine with methionine sulfoxide in a medium lacking hydrogen peroxide had little effect on the growth pattern, which suggests that the oxidation of free methionine in the growth medium was not the main cause of growth inhibition of the msrA mutant. Ultraviolet A radiation did not result in obvious differences in survival rates among the three strains. An enhanced resistance to hydrogen peroxide treatment was shown in human T lymphocyte cells (Molt-4) that were stably transfected with the bovine msrA and exposed to hydrogen peroxide. The survival rate of the transfected strain was much better than its parent strain when grown in the presence of hydrogen peroxide. These results support the proposition that the msrA gene is involved in the resistance of yeast and mammalian cells to oxidative stress.     

Impairment of methionine sulfoxide reductase during UV irradiation and photoaging

During chronic UV irradiation, which is part of the skin aging process, proteins are damaged by reactive oxygen species resulting in the accumulation of oxidatively modified protein. UV irradiation generates irreversible oxidation of the side chains of certain amino acids resulting in the formation of carbonyl groups on proteins. Nevertheless, certain amino acid oxidation products such as methionine sulfoxide can be reversed back to their reduced form within proteins by specific repair enzymes, the methionine sulfoxide reductases A and B. Using quantitative confocal microscopy, the amount of methionine sulfoxide reductase A was found significantly lower in sun-exposed skin as compared to sun-protected skin. Due to the importance of the methionine sulfoxide reductase system in the maintenance of protein structure and function during aging and conditions of oxidative stress, the fate of this system was investigated after UVA irradiation of human normal keratinocytes. When keratinocytes are exposed to 15 J/cm(2) UVA, methionine sulfoxide reductase activity and content are decreased, indicating that the methionine sulfoxide reductase system is a sensitive target for UV-induced inactivation.     

Methionine oxidation contributes to bacterial killing by the myeloperoxidase system of neutrophils

Reactive oxygen intermediates generated by neutrophils kill bacteria and are implicated in inflammatory tissue injury, but precise molecular targets are undefined. We demonstrate that neutrophils use myeloperoxidase (MPO) to convert methionine residues of ingested Escherichia coli to methionine sulfoxide in high yield. Neutrophils deficient in individual components of the MPO system (MPO, H₂O₂, chloride) exhibited impaired bactericidal activity and impaired capacity to oxidize methionine. HOCl, the principal physiologic product of the MPO system, is a highly efficient oxidant for methionine, and its microbicidal effects were found to correspond linearly with oxidation of methionine residues in bacterial cytosolic and inner membrane proteins. In contrast, outer envelope proteins were initially oxidized without associated microbicidal effect. Disruption of bacterial methionine sulfoxide repair systems rendered E. coli more susceptible to killing by HOCl, whereas over-expression of a repair enzyme, methionine sulfoxide reductase A, rendered them resistant, suggesting a direct role for methionine oxidation in bactericidal activity. Prominent among oxidized bacterial proteins were those engaged in synthesis and translocation of peptides to the cell envelope, an essential physiological function. Moreover, HOCl impaired protein translocation early in the course of bacterial killing. Together, our findings indicate that MPO-mediated methionine oxidation contributes to bacterial killing by neutrophils. The findings further suggest that protein translocation to the cell envelope is one important pathway targeted for damage.     

Methionine sulfoxide reductase A down-regulation in human breast cancer cells results in a more aggressive phenotype

Breast cancer is one of the most frequent of human malignancies, and it is therefore fundamental to identify the underlying molecular mechanisms leading to cancer transformation. Among other causative agents in the development of breast cancers, an important role for reactive oxygen species (ROS) has emerged. However, most studies on the role of ROS in cancer have not reached specific conclusions, and many issues remain controversial. In the present study, we show that methionine sulfoxide reductase A (MsrA), which is known to protect proteins from oxidation and which acts as a ROS scavenger, is down-regulated in a number of breast cancers. Moreover, levels of MsrA correlate with advanced tumor grade. We therefore investigated the functional role of MsrA in breast cancer cells. Our data show that reduction of MsrA levels results in increased cell proliferation and extracellular matrix degradation, and consequently in a more aggressive cellular phenotype, both in vivo and in vitro. We also show that the underlying molecular mechanisms involve increased ROS levels, resulting in reduction of phosphatase and tensin homolog deleted on chromosome ten protein (PTEN), and activation of the phosphoinositide 3-kinase pathway. In addition, MsrA down-regulation results in up-regulation of VEGF, providing additional support for tumor growth in vivo.     

Oxidation of methionine residues in proteins of activated human neutrophils.

A simple assay for the detection of 35S-labeled methionine sulfoxide residues in proteins is described. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide, requires the prelabeling of cellular proteins with [35S]methionine. The assay was used to study the extent of methionine oxidation in newly synthesized proteins of both activated and quiescent human neutrophils. In cells undergoing a phorbol 12-myristate 13-acetate-induced respiratory burst, about 66% of all methionine residues in newly synthesized proteins were oxidized to the sulfoxide derivative, as compared with 9% in cells not treated with the phorbol ester. In contrast, quantitation of methionine sulfoxide content in the total cellular protein by means of amino acid analysis showed that only 22% of all methionine residues were oxidized in activated cells as compared with 9% in quiescent cells. It is proposed that methionine residues in nascent polypeptide chains are more susceptible to oxidation than those in completed proteins.     

Thionein can serve as a reducing agent for the methionine sulfoxide reductases

It has been generally accepted, primarily from studies on methionine sulfoxide reductase (Msr) A, that the biological reducing agent for the members of the Msr family is reduced thioredoxin (Trx), although high levels of DTT can be used as the reductant in vitro. Preliminary experiments using both human recombinant MsrB2 (hMsrB2) and MsrB3 (hMsrB3) showed that although DTT can function in vitro as the reducing agent, Trx works very poorly, prompting a more careful comparison of the ability of DTT and Trx to function as reducing agents with the various members of the Msr family. Escherichia coli MsrA and MsrB and bovine MsrA efficiently use either Trx or DTT as reducing agents. In contrast, hMsrB2 and hMsrB3 show <10% of the activity with Trx as compared with DTT, raising the possibility that, in animal cells, Trx may not be the direct hydrogen donor or that there may be a Trx-independent reducing system required for MsrB2 and MsrB3 activity. A heat-stable protein has been detected in bovine liver that, in the presence of EDTA, can support the Msr reaction in the absence of either Trx or DTT. This protein has been identified as a zinc-containing metallothionein (Zn-MT). The results indicate that thionein (T), which is formed when the zinc is removed from Zn-MT, can function as a reducing system for the Msr proteins because of its high content of cysteine residues and that Trx can reduce oxidized T.     

Enzymatic reduction of protein-bound methionine sulfoxide.

An enzyme that catalyzes the reduction of methionine sulfoxide residues in ribosomal protein L12 has been partially purified from Escherichia coli extracts. Methionine sulfoxide present in oxidize [Met]enkephalin is also reduced by the purified enzyme. The enzyme is different from a previously reported E. coli enzyme that catalyzes the reduction of methionine sulfoxide to methionine [Ejiri, S. I., Weissbach, H. & Brot, N. (1980) Anal. Biochem. 102, 393--398]. Extracts of rat tissues, Euglena gracilis, Tetrahymena pyriformis, HeLa cells, and spinach also can catalyze the reduction of methionine sulfoxide residues in protein.