Methionine Sulfoxide Reductase System Research Articles

Impact of Hydrogen Peroxide on the Activity, Structure, and Conformational Stability of the Oxidized Protein Repair Enzyme Methionine Sulfoxide Reductase A

The oxidized protein repair methionine sulfoxide reductase (Msr) system has been implicated in aging, in longevity, and in the protection against oxidative stress. This system is made of two different enzymes (MsrA and MsrB) that catalyze the reduction of the two diastereoisomers S- and R-methionine sulfoxide back to methionine within proteins, respectively. Due to its role in cellular protection against oxidative stress that is believed to originate from its reactive oxygen species scavenging ability in combination with exposed methionine at the surface of proteins, the susceptibility of MsrA to hydrogen-peroxide-mediated oxidative inactivation has been analyzed. This study is particularly relevant to the oxidized protein repair function of MsrA in both fighting against oxidized protein formation and being exposed to oxidative stress situations. The enzymatic properties of MsrA indeed rely on the activation of the catalytic cysteine to the thiolate anion form that is potentially susceptible to oxidation by hydrogen peroxide. The residual activity and the redox status of the catalytic cysteine were monitored before and after treatment. These experiments showed that the enzyme is only inactivated by high doses of hydrogen peroxide. Although no significant structural modification was detected by near- and far-UV circular dichroism, the conformational stability of oxidized MsrA was decreased as compared to that of native MsrA, making it more prone to degradation by the 20S proteasome. Decreased conformational stability of oxidized MsrA may therefore be considered as a key factor for determining its increased susceptibility to degradation by the proteasome, hence avoiding its intracellular accumulation upon oxidative stress.

Selenium and the Methionine Sulfoxide Reductase System

Selenium is a chemical element participating in the synthesis of selenocysteine residues that play a pivotal role in the enzymatic activity efficiency of selenoproteines. The methionine sulfoxide reductase (Msr) system that reduces methionine sulfoxide (MetO) to methionine comprises the selenoprotein MsrB (MsrB1) and the non-selenoprotein MsrA, which reduce the R- and the S- forms of MetO, respectively. The effects of a selenium deficient (SD) diet, which was administrated to wild type (WT) and MsrA knockout mice (MsrA-/-), on the expression and function of Msr-related proteins are examined and discussed. Additionally, new data about the levels of selenium in brain, liver, and kidneys of WT and MsrA-/- mice are presented and discussed.

Dietary selenium (Se) and copper (Cu) affect the activity and expression of the hepatic selenoprotein methionine sulfoxide reductase B (MsrB) in rats

As reported by Jenkinson et al. (J Nutr 1982) and Prohaska et al. (J Nutr Biochem 1992) Cu deficiency (CuD) decreases the activity and mRNA expression of the selenoprotein GPx. Because both Se and Cu are important in oxidative defense, we wanted to determine the effect of a combined deficiency on the methionine (Met) sulfoxide reductase system, which is comprised of MsrA and MsrB. MsrA reduces L‐methionine S‐sulfoxide to Met and MsrB (a selenoprotein) reduces L‐methionine R‐sulfoxide to Met. In a 2 x 2 experiment, male weanling Sprague‐Dawley rats were fed an amino acid‐based diet containing 0 or 0.2 µg Se (as selenite)/g and <1 or 6 µg Cu (as Cu carbonate)/g for 5 wk. Neither Se nor Cu had an effect on MsrA enzyme activity or mRNA expression. Se and Cu interacted to affect MsrB enzyme activity; both Se deficiency and CuD decreased the activity, however, the decrease by CuD was only significant in rats fed 0.2 µg Se. CuD resulted in downregulation of MsrB mRNA. CuD decreased GPx in Se adequate rats. The results show that Cu has a role in regulation of selenoproteins; whether this role is direct or indirect is not known.1Pooled SD. 2nmol/min/mg protein, N=4/group. 3fold change, N=7‐9/group. 4SD for delta Ct. 5U/mg protein, N=7‐8/group.

The Role of Methionine Oxidation/Reduction in the Regulation of Immune Response

Methionine oxidation by reactive oxygen species and reduction mediated by the methionine sulfoxide reductase (Msr) system may attenuate protein function in signal transduction pathways. This review will focus on two potential protein targets for methionine oxidation involved in signal transduction of the immune response: Ca(2+)/calmodulin-regulated phosphatase calcineurin (Cn) and inhibitor of kappa B-alpha (IkBα). The major known function of Cn is to regulate nuclear localization of the nuclear factor of activated T cells (NFAT), a family of transcription factors during immune stimulus. Like wise, IκBα inhibits the activity of nuclear factor kappa B (NFkB), which is known to regulate the transcription of various genes participating in immunological and oxidative stress response. Modification of Met (45) in IκBα enhances its resistance to protein-degredation; thereby, preventing NFkB from activating transcription in cells of the immune system. Similarly, the human Cn molecule contains several methionine residues that are either located next to a cysteine residue or a methionine residue. Accordingly, it is suggested that oxidation of a specific Cn-methionine may interfere with the proper NFAT nuclear-localization and transcriptional activation in T-cell. Thus, the roles of oxidized-methionine residues and their reduction, by the Msr system, are discussed as potential regulators of cellular immune response.

MsrA knockout mouse exhibits abnormal behavior and brain dopamine levels

Oxidative stress can cause methionine oxidation that has been implicated in various proteins malfunctions, if not adequately reduced by the methionine sulfoxide reductase system. Recent evidence has found oxidized methionine residues in neurodegenerative conditions. Previously, we have described elevated levels of brain pathologies and an abnormal walking pattern in the methionine sulfoxide reductase A knockout ( MsrA -/- ) mouse. Here we show that MsrA -/- mice have compromised complex task learning capabilities relative to wild-type mice. Likewise, MsrA -/- mice exhibit lower locomotor activity and altered gait that exacerbated with age. Furthermore, MsrA -/- mice were less responsive to amphetamine treatment. Consequently, brain dopamine levels were determined. Surprisingly, relative to wild-type mice, MsrA -/- brains contained significantly higher levels of dopamine up to 12 months of age, while lower levels of dopamine were observed at 16 months of age. Moreover, striatal regions of MsrA -/- mice showed an increase of dopamine release parallel to observed dopamine levels. Similarly, the expression pattern of tyrosine hydroxylase activating protein correlated with the age-dependent dopamine levels. Thus, it is suggested that dopamine regulation and signaling pathways are impaired in MsrA -/- mice, which may contribute to their abnormal behavior. These observations may be relevant to age-related neurological diseases associated with oxidative stress.

Methionine Sulfoxide Reductases and Virulence of Bacterial Pathogens

Oxidation of methionine (Met) residues in proteins by reactive oxygen species and reactive nitrogen intermediates results in altered protein structures, which subsequently affect their functions. Oxidized Met (Met-O) residues are reduced to Met by the methionine sulfoxide reductase (Msr) system, which includes mainly MsrA and MsrB. MsrA and MsrB show no sequence and structural identity with each other but both reduce methionine sulfoxides. MsrA is specific to the reduction of methionine-S-sulfoxide, whereas MsrB is specific to the reduction of methionine-R-sulfoxide. Genes encoding the enzymes MsrA and MsrB exist in most living organisms including bacteria. In recent times, absence of these enzymes has been implicated in the virulence of bacterial pathogens. In particular, pathogens deficient in Msr have been reported to have reduced ability to adhere with eukaryotic cells, to survive inside hosts and to resist in vitro oxidative stress. Bacterial proteins that are susceptible to Met oxidation, in the absence of Msr, have also been identified. This review discusses the current knowledge on the role of Msr in bacterial virulence.

Elevated levels of brain-pathologies associated with neurodegenerative diseases in the methionine sulfoxide reductase A knockout mouse

One of the posttranslational modifications to proteins is methionine oxidation, which is readily reversible by the methionine sulfoxide reductase (Msr) system. Thus, accumulation of faulty proteins due to a compromised Msr system may lead to the development of aging-associated diseases like neurodegenerative diseases. In particular, it was interesting to monitor the consequential effects of methionine oxidation in relation to markers that are associated with Alzheimer's disease as methionine oxidation was implied to play a role in beta-amyloid toxicity. In this study, a knockout mouse strain of the methionine sulfoxide reductase A gene (MsrA ( -/- )) caused an enhanced neurodegeneration in brain hippocampus relative to its wild-type control mouse brain. Additionally, a loss of astrocytes integrity, elevated levels of beta-amyloid deposition, and tau phosphorylation were dominant in various regions of the MsrA ( -/- ) hippocampus but not in the wild-type. Also, a comparison between cultured brain slices of the hippocampal region of both mouse strains showed more sensitivity of the MsrA ( -/- ) cultured cells to H(2)O(2) treatment. It is suggested that a deficiency in MsrA activity fosters oxidative-stress that is manifested by the accumulation of faulty proteins (via methionine oxidation), deposition of aggregated proteins, and premature brain cell death.

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.

Prolonged selenium deficient diet in MsrA knockout mice causes enhanced oxidative modification to proteins and affects the levels of antioxidant enzymes in a tissue-specific manner

The methionine sulfoxide reductase (Msr) system (comprised of MsrA and MsrB) is responsible for reducing methionine sulfoxide (MetO) to methionine. One major form of MsrB is a selenoprotein. Following prolonged selenium deficient diet (SD), through F2 generation, the MsrA − / −mice exhibited higher protein–MetO and carbonyl levels relative to their wild-type (WT) control in most organs. More specifically, the SD diet caused alteration in the expression and/or activities of certain antioxidants as follows: lowering the specific activity of MsrB in the MsrA − / −cerebellum in comparison to WT mice; lowering the activities of glutathione peroxidase (Gpx) and thioredoxin reductase (Trr) especially in brains of MsrA − / −mice; elevation of the cellular levels of selenoprotein P (SelP) in most tissues of the MsrA − / −relative to WT. Unexpectedly, the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) were mainly elevated in lungs and hearts of MsrA − / −mice. Moreover, the body weight of the MsrA − / −mice lagged behind the WT mice body weight up to 120 days of the SD diet. In summary, it is suggested that the lack of the MsrA gene in conjunction with prolonged SD diet causes decreased antioxidant capability and enhanced protein oxidation.

Protein-carbonyl accumulation in the non-replicative senescence of the methionine sulfoxide reductase A (msrA) knockout yeast strain

The major enzyme of the methionine sulfoxide reductase (Msr) system is MsrA. Senescing msrA knockout mother yeast cells accumulated significant amounts of protein-carbonyl both at 5 generation-old (young) and 21 generation-old (old) cultures, while the control mother cells showed significant levels of protein-carbonyl mainly in the old culture. The Msr activities of both yeast strains declined with age and exposure of cells to H(2)O(2) caused an accumulation of protein-carbonyl especially in the msrA knockout strain. It is suggested that a compromised MsrA activity may serve as a marker for non-replicative aging.

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.

Oxidized protein degradation and repair in ageing and oxidative stress

Cellular ageing is characterized by the accumulation of oxidatively modified proteins which may be due to increased protein damage and/or decreased elimination of oxidized protein. Since the proteasome is in charge of protein turnover and removal of oxidized protein, its fate during ageing and upon oxidative stress has received special attention, and evidence has been provided for an age-related impairment of proteasome function. However, proteins when oxidized at the level of sulfur-containing amino acids can also be repaired. Therefore, the fate of the methionine sulfoxide reductase system during ageing has also been addressed as well as its role in protection against oxidative stress.

Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H 2O 2-mediated oxidative stress

Proteins are modified by reactive oxygen species, and oxidation of specific amino acid residues can impair their biological functions, leading to an alteration in cellular homeostasis. Oxidized proteins can be eliminated through either degradation or repair. Repair is limited to the reversion of a few modifications such as the reduction of methionine oxidation by the methionine sulfoxide reductase (Msr) system. However, accumulation of oxidized proteins occurs during aging, replicative senescence, or neurological disorders or after an oxidative stress, while Msr activity is impaired. In order to more precisely analyze the relationship between oxidative stress, protein oxidative damage, and MsrA, we stably overexpressed MsrA full-length cDNA in SV40 T antigen-immortalized WI-38 human fibroblasts. We report here that MsrA-overexpressing cells are more resistant than control cells to hydrogen peroxide-induced oxidative stress, but not to ultraviolet A irradiation. This MsrA-mediated resistance is accompanied by a decrease in intracellular reactive oxygen species and is partially abolished when cells are cultivated at suboptimal concentration of methionine. These results indicate that MsrA may play an important role in cellular defenses against oxidative stress, by catalytic removal of oxidant through the reduction of methionine sulfoxide, and in protection against death by limiting, at least in part, the accumulation of oxidative damage to proteins.

Oxidized proteins and their biological impact

Aging is characterized by an increased accumulation of damaged macromolecules and oxidized protein build up is considered to be a hallmark of cellular aging. Advanced glycation end products (AGE) have been analyzed in aging human peripheral blood lymphocytes since such glycoxidative modifications have been reported to increase with age in a variety of cellular and tissular systems and are believed to contribute to the intracellular age‐related accumulation of damaged proteins, a process that has been associated with the cellular functional deficits that occur with age. The pattern of glycated protein has been studied using two dimensional gel electrophoresis followed by Western blotting with an anti‐AGE antibody. The protein silver stain and the immunoblot patterns were not superimposable indicating that glycoxidative modifications are targeting only a restricted set of proteins. Among these preferential protein targets, seven of them exhibited a significant age‐related increased immunoreactivity with the anti‐AGE antibody suggesting that the corresponding modified proteins might serve as biomarkers of aging lymphocytes [1]. Other age‐related protein modifications such as carbonyl formation and conjugation with the lipid peroxidation product 4‐hydroxy‐2‐nonenal are also currently studied. The age‐related accumulation of altered protein raises the problem of the efficacy of intracellular protein maintenance, in particular the protein degradation and the protein repair systems. Indeed, if these systems that take care of the removal or repair of damaged proteins are affected with aging, they would therefore directly contribute to the increased intracellular load of functionally impaired protein. Since cytosolic oxidized protein degradation and basal protein turnover have been shown to be mostly carried out by the proteasomal system, the fate of proteasome in aging has been addressed and a decline of proteasomal proteolytic activity has been reported [2].The impact of aging on human lymphocyte 26S proteasome has been recently investigated and age‐related alterations of proteasome structure and function have been evidenced. Indeed, we observed a decline of 26S proteasome specific activity which is correlated to an increasing yield of post‐translational modifications of proteasome subunits [3]. In fact, some proteasome subunits and particularly assembly and catalytic subunits are specifically modified with age. According to bidimensional westernblotting, some subunits were found either glycated, conjugated with the lipid peroxidation product 4‐hydroxy‐2‐nonenal or even ubiquitinated. In vitro treatment of the proteasome by glyoxal, that promotes the formation of Nɛ‐carboxymethyllysine adducts, or by the lipid peroxidation product 4‐hydroxy‐2‐nonenal was found to inactivate its peptidase activities although to different extent. In other studies aimed at monitoring the effect of oxidative stress on proteasome structure and function, we have shown on an in vivo rat model that coronary occlusion/reperfusion resulted in inactivation of the proteasome [4]. This inactivation is associated with selective modification by the lipid peroxidation product 4‐hydroxy‐2‐nonenal of three 20S proteasome α‐subunits. In contrast, the observed inhibition of proteasome upon exposure of human keratinocytes to UV stress was mainly due to the stress‐induced formation of endogeneous inhibitors, including certain oxidatively modified proteins [5]. In addition, the role of the peptide methionine sulfoxide reductase, one of the very few protein repair enzyme described, and its possible implication in the age‐related decline of protein maintenance has been investigated. The peptide methionine sulfoxide reductase system (Msr A and Msr B) catalyzes the reduction of methionine sulfoxide to methionine within proteins and its activity and the expression of Msr A have been shown to decline in different organs of aged rats [6] and more recently in senescent human fibroblasts. Moreover, we have recently shown that the peptide methionine sulfoxide reductase Msr A is present both in the cytosol and in the mitochondrial matrix, although under different isoforms [7]. In conclusion, during aging or in certain oxidative stress situations, impairment of both peptide methionine sulfoxide reductases and proteasome activity appears as a critical factor in the decreased efficacy of intracellular protein maintenance, contributing to the increased intracellular load of modified and functionally impaired proteins that may ultimately lead to a global deterioration of cellular homeostasis.

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.

Reduction of Sulindac to its active metabolite, sulindac sulfide: assay and role of the methionine sulfoxide reductase system

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