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Methionine oxidation and reduction is a common phenomenon occurring in biological systems under both physiological and oxidative-stress conditions. The levels of methionine sulfoxide (MetO) are dependent on the redox status in the cell or organ, and they are usually elevated under oxidative-stress conditions, aging, inflammation, and oxidative-stress related diseases. MetO modification of proteins may alter their function or cause the accumulation of toxic proteins in the cell/organ. Accordingly, the regulation of the level of MetO is mediated through the ubiquitous and evolutionary conserved methionine sulfoxide reductase (Msr) system and its associated redox molecules. Recent published research has provided new evidence for the involvement of free MetO or protein-bound MetO of specific proteins in several signal transduction pathways that are important for cellular function. In the current review, we will focus on the role of MetO in specific signal transduction pathways of various organisms, with relation to their physiological contexts, and discuss the contribution of the Msr system to the regulation of the observed MetO effect.

Methionine sulfoxide reductase enzymes are a protective system against biological oxidative stress in aerobic organisms. Modifications to this antioxidant system have been shown to impact the lifespan of several model system organisms. In humans, methionine oxidation of critical proteins and deficiencies in the methionine sulfoxide reductase system have been linked to age-related diseases, including cancer and neurodegenerative disease. Substrates for methionine sulfoxide reductases have been reviewed multiple times, and are still an active area of discovery. In contrast, less is known about the genetic regulation of methionine sulfoxide reductases. In this review, we discuss studies on the genetic regulation of the methionine sulfoxide reductase system with relevance to longevity and age-related diseases. A better understanding of genetic regulation for methionine sulfoxide reductases may lead to new therapeutic approaches for age-related diseases in the future.

Iron is an element with redox properties. It is active sites of many enzymes and plays an important role in various cellular and biological functions including ATP production and DNA synthesis. However, as a redox element, iron promotes free radical generation and lipid peroxidation, causing oxidative damage and cell death. Iron-mediated oxidation is a central player in ferroptosis, a type of cell death process that is different from apoptosis and necrosis. Thus, iron metabolism and homeostasis are sophisticatedly regulated. There has been exciting progress in understanding iron metabolism and regulation since hepcidin was recognized as the central regulator of iron homeostasis. Hepcidin mainly regulates the iron export function of the ferrous iron permease, ferroportin, which is the only known iron exporter expressed by mammalian cells. Particularly, epigenetic regulation has been a recent focus on iron homeostasis. Epigenetic phenomena have been demonstrated to modulate key proteins including hepcidin in iron metabolism. Here, we review the rapid progress in recent years in understanding molecular mechanisms of iron homeostasis with a focus on epigenetic regulation of hepcidin, ferritin, and ferroptosis. Interactions between methionine oxidation and iron is also discussed. Furthermore, many studies have suggested that the severity of neuronal damage after stroke is proportional to the magnitude of brain iron accumulation. Recent discoveries regarding iron metabolism in stroke is briefly discussed. Understanding the underlying mechanism in iron regulation could provide insight into the treatment of various intractable diseases including stroke.

Methionine sulfoxide (MetO) is an oxidative posttranslational modification that primarily occurs under oxidative stress conditions, leading to alteration of protein structure and function. This modification is regulated by MetO reduction through the evolutionarily conserved methionine sulfoxide reductase (Msr) system. The Msr type A enzyme (MsrA) plays an important role as a cellular antioxidant and promotes cell survival. The ubiquitin- (Ub) like neddylation pathway, which is controlled by the c-Jun activation domain-binding protein-1 (Jab1), also affects cell survival. Jab1 negatively regulates expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1B (P27) through binding and targeting P27 for ubiquitination and degradation. Here we report the finding that MsrA interacts with Jab1 and enhances Jab1&prime, s deneddylase activity (removal of Nedd8). In turn, an increase is observed in the level of deneddylated Cullin-1 (Cul-1, a component of E3 Ub ligase complexes). Furthermore, the action of MsrA increases the binding affinity of Jab1 to P27, while MsrA ablation causes a dramatic increase in P27 expression. Thus, an interaction between MsrA and Jab1 is proposed to have a positive effect on the function of Jab1 and to serve as a means to regulate cellular resistance to oxidative stress and to enhance cell survival.

Methionine sulfoxide reductase A (MsrA) is an enzyme involved in redox balance and signaling, and its aberrant activity is implicated in a number of diseases (for example, Alzheimer's disease and cancer). Since there is no simple small molecule tool to monitor MsrA activity in real time in vivo, we aimed at developing one. We have designed a BODIPY-based probe called (S)-Sulfox-1, which is equipped with a reactive sulfoxide moiety. Upon reduction with a model MsrA (E. coli), it exhibits a bathochromic shift in the fluorescence maximum. This feature was utilized for the real-time ratiometric fluorescent imaging of MsrA activity in E. coli cells. Significantly, our probe is capable of capturing natural variations of the enzyme activity in vivo.

Methionine sulfoxide reductases (MsrA and MsrB) protect the biological activity of proteins from oxidative modifications to methionine residues and are important for protecting against the pathological effects of neurodegenerative diseases. In the current study, we characterized the auditory phenotype of the MsrA knockout mouse. Young MsrA knockout mice showed small high-frequency threshold elevations for auditory brainstem response and distortion product otoacoustic emission compared to those of wild-type mice, which progressively worsened in older MsrA knockout mice. MsrA knockout mice showed an increased sensitivity to noise at young and older ages, suggesting that MsrA is part of a mechanism that protects the cochlea from acoustic damage. MsrA mRNA in the cochlea was increased following acoustic stimulation. Finally, expression of mRNA MsrB1 was compromised at 6 months old, but not in younger MsrA knockout mice (compared to controls). The identification of MsrA in the cochlea as a protective mediator from both early onset hearing loss and acoustic trauma expands our understanding of the pathways that may induce protection from acoustic trauma and foster further studies on how to prevent the damaging effect of noise exposure through Msr-based therapy.

Aims The enzyme catechol-O-methyl transferase (COMT) plays a primary role in the metabolism of catecholamine neurotransmitters and is implicated in the modulation of cognitive and emotional responses. The best-characterized single nucleotide polymorphism (SNP) of the COMT gene consists of a valine (Val)-to-methionine (Met) substitution at codon 108/158. The Met-containing variant confers a marked reduction in COMT catalytic activity. We recently showed that the activity of recombinant COMT is positively regulated by the enzyme Met sulfoxide reductase (MSR), which counters the oxidation of Met residues of proteins. The current study was designed to assess whether brain COMT activity may be correlated to MSR in an allele-dependent fashion.

Angiogenesis is regulated by hyperglycemic conditions, which can induce cellular stress responses, reactive oxygen species (ROS), and anti-oxidant defenses that modulate intracellular signaling to prevent oxidative damage. The RUNX2 DNA-binding transcription factor is activated by a glucose-mediated intracellular pathway, plays an important role in endothelial cell (EC) function and angiogenesis, and is a target of oxidative stress. RUNX2 DNA-binding and EC differentiation in response to glucose were conserved in ECs from different tissues and inhibited by hyperglycemia, which stimulated ROS production through the aldose reductase glucose-utilization pathway. Furthermore, the redox status of cysteine and methionine residues regulated RUNX2 DNA-binding and reversal of oxidative inhibition was consistent with an endogenous Methionine sulfoxide reductase-A (MsrA) activity. Low molecular weight MsrA substrates and sulfoxide scavengers were potent inhibitors of RUNX2 DNA binding in the absence of oxidative stress, but acted as antioxidants to increase DNA binding in the presence of oxidants. MsrA was associated with RUNX2:DNA complexes, as measured by a sensitive, quantitative DNA-binding ELISA. The related RUNX2 protein family member, RUNX1, which contains an identical DNA-binding domain, was a catalytic substrate of recombinant MsrA. These findings define novel redox pathways involving aldose reductase and MsrA that regulate RUNX2 transcription factor activity and biological function in ECs. Targeting of these pathways could result in more effective strategies to alleviate the vascular dysfunction associated with diabetes or cancer.

Membranous adenylyl cyclase 1 (AC1) is associated with memory and learning. AC1 is activated by the eukaryotic Ca(2+)-sensor calmodulin (CaM), which contains nine methionine residues (Met) important for CaM-target interactions. During ageing, Met residues are oxidized to (S)- and (R)-methionine sulfoxide (MetSO) by reactive oxygen species arising from an age-related oxidative stress. We examined how oxidation by H2O2 of Met in CaM regulates CaM activation of AC1. We employed a series of thirteen mutant CaM proteins never assessed before in a single study, where leucine is substituted for Met, in order to analyze the effects of oxidation of specific Met. CaM activation of AC1 is regulated by oxidation of all of the C-terminal Met in CaM, and by two N-terminal Met, M36 and M51. CaM with all Met oxidized is unable to activate AC1. Activity is fully restored by the combined catalytic activities of methionine sulfoxide reductases A and B (MsrA and B), which catalyze reduction of the (S)- and (R)-MetSO stereoisomers. A small change in secondary structure is observed in wild-type CaM upon oxidation of all nine Met, but no significant secondary structure changes occur in the mutant proteins when Met residues are oxidized by H2O2, suggesting that localized polarity, flexibility and structural changes promote the functional changes accompanying oxidation. The results signify that AC1 catalytic activity can be delicately adjusted by mediating CaM activation of AC1 by reversible Met oxidation in CaM. The results are important for memory, learning and possible therapeutic routes for regulating AC1.

Does oxytocin influence intimate partner violence (IPV)? Clues from prior research suggest that oxytocin increases prosocial behavior, but this effect is reversed among people with aggressive tendencies or in situations involving defensive aggression. Animal research also indicates that oxytocin plays a central role in defensive maternal aggression (i.e., protecting pups from intruders). Among highly aggressive people, a boost of oxytocin may cause them to use aggression toward close others as a means of maintaining their relationship. Adopting an interactionist approach, we predicted that oxytocin would increase IPV inclinations, but this effect would be limited to people high in trait physical aggression. In a double-blind, placebo-controlled, between-subject experiment, participants varying in trait physical aggression received either 24 international unit of oxytocin or a placebo. Following two provocation tasks, participants rated the probability that they would engage in various aggressive behaviors (e.g., slapping, throwing an object that could hurt) toward a romantic partner. Oxytocin increased IPV inclinations, but this effect was limited to participants prone to physical aggression. These data offer the first evidence that IPV inclinations have a biological basis in a combination of oxytocin and trait physical aggressiveness.

Methionine sulfoxide is a common posttranslational oxidative modification that can alter protein function. Vulnerability of specific proteins to methionine oxidation varies and depends on their structure. In the current study, detection of methionine sulfoxide in intact proteins is mediated by novel anti-methionine sulfoxide antibody that resulted in the identification of three major methionine sulfoxide-proteins in brain: bisphosphate aldolase A and C, α and β subunits of hemoglobin, and serum albumin. The locations of the methionine sulfoxide residues were determined by massspectrometry analyses. It is suggested that the in vivo methionine oxidation of these proteins represent early posttranslational oxidative modification of proteins in brain. Thus, elevated levels of methionine-sulfoxide in these proteins may serve as bio-markers for enhanced oxidative stress in brain, which may be associated with brain disorders and diseases.

The enzyme catechol-O-methyltransferase (COMT), which catalyses the degradation of dopamine and norepinephrine, is posited to participate in the pathophysiology of bipolar disorder (BD) and schizophrenia. In support of this notion, rich evidence has documented that the severity of various BD and schizophrenia symptoms is moderated by rs4680, a single nucleotide polymorphism of the COMT gene featuring a valine (Val)-to-methionine (Met) substitution that results in lower catalytic activity. Nevertheless, the specific relevance of COMT enzymatic activity in the pathophysiology of BD and schizophrenia dimensions remains elusive.We measured COMT catalytic activity in post-mortem prefrontal cortices, striata and cerebella of schizophrenia and BD patients, as well as non-affected controls. These values were then correlated with rs4680 genotypes and psychopathology scores in the last week of life.No direct correlation between COMT activity and rs4680 genotypes was found; however, the severity of manic symptoms was highly correlated with COMT activity in the striatum, irrespective of the diagnostic group.These results suggest that COMT striatal activity, but not rs4680 genotype, may serve as a biomarker for manic symptoms. Future studies are warranted to confirm these findings and assess the neurobiological links between COMT striatal activity and manic symptoms.

Accumulation of oxidized proteins, and especially β-amyloid (Aβ), is thought to be one of the common causes of Alzheimer's disease (AD). The current studies determine the effect of an in vivo methionine sulfoxidation of Aβ through ablation of the methionine sulfoxide reductase A (MsrA) in a mouse model of AD, a mouse that overexpresses amyloid precursor protein (APP) and Aβ in neurons. Lack of MsrA fosters the formation of methionine sulfoxide in proteins, and thus its ablation in the AD-mouse model will increase the formation of methionine sulfoxide in Aβ. Indeed, the novel MsrA-deficient APP mice ( APP+/ MsrAKO) exhibited higher levels of soluble Aβ in brain compared with APP+ mice. Furthermore, mitochondrial respiration and the activity of cytochrome c oxidase were compromised in the APP+/ MsrAKO compared with control mice. These results suggest that lower MsrA activity modifies Aβ solubility properties and causes mitochondrial dysfunction, and augmenting its activity may be beneficial in delaying AD progression.

The blind subterranean mole rat (Spalax ehrenbergi) exhibits a relatively long life span, which is attributed to an efficient antioxidant defense affording protection against accumulation of oxidative modifications of proteins. Methionine residues can be oxidized to methionine sulfoxide (MetO) and then enzymatically reduced by the methionine sulfoxide reductase (Msr) system. In the current study we have isolated the cDNA sequences of the Spalax Msr genes as well as 23 additional selenoproteins and monitored the activities of Msr enzymes in liver and brain of rat (Rattus norvegicus), Spalax galili, and Spalax judaei under normoxia, hypoxia, and hyperoxia. Under normoxia, the Msr activity was lower in S. galili in comparison to S. judaei and R. norvegicus especially in the brain. The pattern of Msr activity of the three species was similar throughout the tested conditions. However, exposure of the animals to hypoxia caused a significant enhancement of Msr activity, especially in S. galili. Hyperoxic exposure showed a highly significant induction of Msr activity compared with normoxic conditions for R. norvegicus and S. galili brain. It was concluded that among all species examined, S. galili appears to be more responsive to oxygen tension changes and that the Msr system is upregulated mainly by severe hypoxia.

Convergent evidence suggests that oxidative stress plays a central role in the pathology of Alzheimer's disease (AD). We asked if consequently, oxidation of methionine residues to methionine sulfoxide (MetO) was increased in plasma proteins of persons carrying familial AD (FAD) mutations.Plasma was collected from 31 persons from families harboring PSEN1 or APP mutations. Using Western blot analysis with a novel anti-MetO polyclonal antibody, MetO levels were measured and compared between FAD mutation carriers (MCs) and non-mutation carrying (NCs) kin.A MetO-positive 120-kDa gel band distinguished FAD MCs and NCs (mean 11.4 ± 2.8 vs. 4.0 ± 3.1, p = 0.02). In a subset of subjects for whom both measurements were available, MetO levels correlated well with plasma F2-isoprostane (r = 0.81, p0.001) and superoxide dismutase 1 (r = 0.52, p = 0.004) levels.Our data provide evidence for elevated MetO levels in persons carrying FAD mutations that correlate with other indices of oxidative stress and suggest that plasma oxidative stress markers may be useful for diagnosis of AD.

Self-assembly of amyloid β-protein (Aβ) into toxic oligomers and fibrillar polymers is believed to cause Alzheimer's disease (AD). In the AD brain, a high percentage of Aβ contains Met-sulfoxide at position 35, though the role this modification plays in AD is not clear. Oxidation of Met(35) to sulfoxide has been reported to decrease the extent of Aβ assembly and neurotoxicity, whereas surprisingly, oxidation of Met(35) to sulfone yields a toxicity similar to that of unoxidized Aβ. We hypothesized that the lower toxicity of Aβ-sulfoxide might result not only from structural alteration of the C-terminal region but also from activation of methionine-sulfoxide reductase (Msr), an important component of the cellular antioxidant system. Supporting this hypothesis, we found that the low toxicity of Aβ-sulfoxide correlated with induction of Msr activity. In agreement with these observations, in MsrA(-/-) mice the difference in toxicity between native Aβ and Aβ-sulfoxide was essentially eliminated. Subsequently, we found that treatment with N-acetyl-Met-sulfoxide could induce Msr activity and protect neuronal cells from Aβ toxicity. In addition, we measured Msr activity in a double-transgenic mouse model of AD and found that it was increased significantly relative to that of nontransgenic mice. Immunization with a novel Met-sulfoxide-rich antigen for 6 months led to antibody production, decreased Msr activity, and lowered hippocampal plaque burden. The data suggest an important neuroprotective role for the Msr system in the AD brain, which may lead to development of new therapeutic approaches for AD.

The abnormal regulation of dopamine (DA) has been associated with multiple neurodegenerative disease states (Bird and Iversen, 1974, Morgan et al., 1987), yet the role of DA reserve pool storage and mobilization in the pathophysiology of these conditions is now just being revealed. Many of these conditions, such as Parkinson’s disease, Huntington’s disease, and Lou Gehrig’s disease, have been associated with the increased production or reactive oxygen species, thereby enhancing the degree of cell oxidative stress in the brain (Patten et al., 2010, Cohen, 1983, Perez-Severiano et al., 2004). Interestingly, elevated DA levels have also been associated with enhanced oxidative stress (Oien et al., 2008b). Indeed, one model of oxidative stress, methionine sulfoxide reductase A knockout (MsrA−/−) mice, have been reported to have chronically high brain DA levels (Oien et al., 2008b). These mice lack the antioxidant enzyme MsrA, which is part of the Msr system. Methionine sulfoxide posttranslational modifications can be reversed by the Msr system, which consists of MsrA (reduces S methionine sulfoxide enantiomer) and MsrB (reduces R methionine sulfoxide enantiomer) (Moskovitz, 2005). The MsrA−/− mouse is hypersensitive to oxidative stress, accumulates higher levels of carbonylated protein, and expresses brain pathologies associated with neurodegenerative diseases (Moskovitz et al., 2001, Pal et al., 2007). Recent studies have shown that these mice have abnormally high DA levels in the brain at the ages of 6 and 12 months, compared to wild type (WT) control mice. Additionally, these high levels parallel an increased presynaptic DA release when stimulated in vitro without drug treatments. A possible mechanism for an increase in stimulated DA release in MsrA−\− mice involves the mobilization of reserve pool DA. In general, DA-containing vesicles are believed to be separated into three pools: the readily releasable pool (RRP), the recycling pool, and the reserve pool (Neves and Lagnado, 1999, Rizzoli and Betz, 2005). The RRP undergoes exocytosis upon mild stimulation and is replenished by the mobilization of the recycling pool vesicles. The reserve pool, mobilized upon prolonged periods of synaptic activity (Neves and Lagnado, 1999), is the largest pool consisting of 80–90% of the total vesicles (Rizzoli and Betz, 2005). Pharmacological manipulations, using a combination of alpha-methyl-p-tyrosine (aMPT) and either cocaine (COC) or amphetamine (AMPH) (Venton et al., 2006, Ortiz et al., 2010), have been used to quantitatively measure reserve pool dopamine. Other factors, such as calcium transport, may also influence the amplitude of stimulated dopamine release plots. Transient increases in intracellular calcium concentration trigger vesicular exocytosis (Nachshen and Sanchez-Armass, 1987, Kume-Kick and Rice, 1998) as well as the movement of RRP and reserve pool vesicles (Rose et al., 2002). Moreover, the increase in oxidative stress may result in calcium dysregulation. For example, the activity of calmodulin, a calcium regulatory protein that activates the plasma membrane calcium ATPase (PMCA), diminishes due to oxidative post-translational modifications as tissues age (Michaelis et al., 1996). The oxidation of specific methionines in calmodulin results in about a 50% reduction of PMCA activation (Bartlett et al., 2003), thereby impairing the ability of cells to clear calcium from the cell (Palacios et al., 2004). Oxidized calmodulin can accumulate in brain tissues as a result of low antioxidant levels and it is speculated that oxidation of methionines on calmodulin may be acting as a molecular switch in calcium regulation, oxidative stress, and DA release (Chen et al., 2001, Bigelow and Squier, 2005). To investigate possible mechanisms underlying elevated DA content and release found in MsrA−/− mice, fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used to measure the mobilization and efflux of reserve pool DA in striatal brain slices from MsrA−/− mice and WT control mice (Oien et al., 2008b). We hypothesized that the DA reserve pool is enhanced in MsrA−/− mice compared to WT control mice. In order to measure reserve pool DA, slices were pre-treated with αMPT and then treated with either AMPH, to measure the efflux of reserve pool DA, or with COC, to measure the stimulated release of mobilized DA reserve pool vesicles. Collectively, our results suggest that reserve pool DA is more abundant in the MsrA−/− striatum and that the number of vesicles is greater compared to WT controls.

The formation and accumulation of protein-carbonyl by reactive oxygen species may serve as a marker of oxidative stress, aging, and age-related diseases. Enzymatic reversal of the protein-carbonyl modification has not yet been detected. However, an enzymatic reversal of protein-methionine sulfoxide modification exists and is mediated by the methionine sulfoxide reductase (Msr) system. Methionine sulfoxide modifications to proteins may precede the formation of protein-carbonyl adducts because of consequent structural changes that increase the vulnerability of amino acid residues to carbonylation. Supportive evidence for this possibility arises from the elevated protein-carbonyl accumulations observed in organisms, such as yeast and mice, lacking the methionine sulfoxide reductase A (MsrA) enzyme. In addition, advanced age or enhanced oxidative-stress conditions foster the accumulations of protein-carbonyls. This review discusses the possible involvement of methionine sulfoxide formation in the occurrence of protein-carbonyl adducts and their relevance to the aging process and neurodegenerative diseases.

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.

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrPC) to a misfolded, pathogenic isoform (PrPSc). Prion inoculation experiments in mice expressing homologous PrPCmolecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrPC, our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

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.

It is commonly accepted that aging is associated with a decline in the antioxidant defense of the cell; accordingly, certain redox enzymes are used as markers of biological senescence. To further test and specify this general concept, we studied age-related changes in the enzymes of the methionine-centered redox cycle (MCRC) in four aero-digestive organs of rats. The levels of cytosolic thioredoxin (Trx), thioredoxin reductase (TrxR), and methionine sulfoxide reductase (Msr), all tended to decline with age. The enzymatic activities of MsrA and MsrB were significantly lower in the organs of aged animals. In general, the magnitude of this decline increased in the order: tongue < sternohyoid muscle < larynx < esophagus. The relative stability of MCRC in the old tongues might be part of the well-preserved oxidative metabolism as confirmed by the age-related increase in mitochondrial marker and muscle tissue in these tongues. In total, the results suggest that age-associated oxidative damage is organ-specific and could reflect differences in morphological composition of these tissues, and among them, relative content of striated muscles.

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.

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

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.

Previously we have shown that the binding complex formation of methionine sulfoxide reductase A (msrA) promoter and calcium phospholipid binding protein (CPBP) enhances msrA transcription and expression. The msrA promoter-CPBP-binding complex (PmsrA-CPBP) formation was similar in Deltatrx1, Deltatrx2, and Deltatrx3 yeast strains and their control, with or without exposure to H(2)O(2). In Deltatrx1/Deltatrx2 double mutant the PmsrA-CPBP was similar to its parent strain, following exposure to H(2)O(2) for 30 min. However, a late-onset loss of PmsrA-CPBP binding activity occurred following exposure to H(2)O(2) for 24 hours. Hence, it was inferred that both Trx1 and Trx2 are involved in the PmsrA-CPBP formation during prolonged oxidative stress conditions. In addition, the survival rate of the Deltatrx1Delta/trx2 double mutant was approximately 10% of its parent strain when exposed to H(2)O(2.) The MsrA activity was obliterated in Deltatrx1/Deltatrx2 and Deltatrx1 strains and remained intact in the Deltatrx2 and Deltatrx3 strains. The msrA mRNA level in Deltatrx1 was significantly reduced in comparison to that of its control, slightly reduced in Deltatrx2, and unchanged in Deltatrx3, respectively. It is suggested that under normal growth conditions Trx1 is essential for msrA transcription and activity. Moreover, following long-term oxidative stress conditions, Trx1 and Trx2 appear to promote PmsrA-CPBP-binding activity and cell survival.

The enzyme catechol-O-methyltransferase (COMT) plays a primary role in the metabolism of catecholamine neurotransmitters and is implicated in the modulation of cognitive and emotional responses. The best characterized single nucleotide polymorphism (SNP) of the COMT gene consists of a valine (Val)-to-methionine (Met) substitution at codon 108/158. The Met-containing variant confers a marked reduction in COMT catalytic activity. We recently showed that the activity of recombinant COMT is positively regulated by the enzyme Met sulphoxide reductase (MSR), which counters the oxidation of Met residues of proteins. The current study was designed to assess whether brain COMT activity may be correlated to MSR in an allele-dependent fashion.COMT and MSR activities were measured from post-mortem samples of prefrontal cortices, striata and cerebella of 32 subjects by using catechol and dabsyl-Met sulphoxide as substrates, respectively. Allelic discrimination of COMT Val(108/185) Met SNP was performed using the Taqman 5'nuclease assay.Our studies revealed that, in homozygous carriers of Met, but not Val alleles, the activity of COMT and MSR was significantly correlated throughout all tested brain regions.These results suggest that the reduced enzymatic activity of Met-containing COMT may be secondary to Met sulphoxidation and point to MSR as a key molecular determinant for the modulation of COMT activity.

One of the most oxidation-sensitive amino acids is methionine. Oxidation of methionine to methionine sulfoxide (MetO) could, on the one hand, be an important component of signal transduction pathways and on the other hand, may lower the cellular antioxidant capacity, alter protein function, interfere with signal transduction, and damage proteins. The latter changes could lead to the accumulation and malfunction of various proteins. As a result, enhanced development of certain diseases and signs of aging may occur. So far, two major enzymes that could reduce MetO in proteins have been described, denoted as MsrA and MsrB (Methionine sulfoxide reductases). In general, Msrs have been shown to be important in protecting cells from oxidative stress throughout many species from bacteria to mammals. In addition, the activities of certain enzymes could be restored or controlled following reduction of their MetO residues, through the Msr system. Of all Msrs, MsrA seems to be important in controlling MetO reduction in general and MsrB, thioredoxin reductase (Trr), and the adhesion capabilities of certain bacterial cells in particular. The recently discovered MsrB can reduce specifically the R-MetO enantiomer while MsrA can reduce specifically the S-MetO enantiomer. Another significant difference between MsrA and MsrB is that the latter's major form in mammalian cells is a selenoprotein. The current review will discuss the major characteristics of methionine sulfoxide reductases as physiological antioxidants, repair systems, and cellular regulating enzymes.

The biological function of cellular prion protein PrPc has not been established, despite in vitro studies suggesting antioxidant activity or link to signal transduction pathways. In this study, mice were exposed to hyperoxia to establish whether oxidative stress affected prion expression in vivo. C57Bl/6J mice aged 6, 18, and 24 months, maintained under normoxic conditions, exhibited age-related increases in PrPc in both cerebral microvessels and in microvessel-depleted brain homogenate. We demonstrate that PrPc is differentially affected by exposure to hyperoxia in vivo for 1 (24 h) or 2 (48 h) days, or for 1 day hyperoxia, followed by 1 day normoxia. Brain parenchymal cells from 6-month-old mice exposed to 1 day hyperoxia showed elevation of a glycosylated approximately 36 kDa form, whereas in 24-month-old mice cellular prion level was substantially reduced. Extending hyperoxia from 1 to 2 days resulted in significantly reduced PrPc level, regardless of age. Parenchymal PrPc is substantially elevated in 6-month-old mice, but declines in 18- and 24-month-old animals following 1 day hyperoxia. By contrast, PrPc content in cerebral microvessels from 6-month-old mice declined after a 2 day exposure to hyperoxia, while microvessels from 24-month-old brains showed elevated prion levels 24 h after hyperoxia. Moreover, unglycosylated 25-30 kDa PrPc, and a previously undescribed 50-64 kDa band containing at least some glycosylated protein, predominated in microvessels with lesser content of the glycosylated approximately 36 kDa form. Cellular content of these unglycosylated forms was correlated with age, while the response to hyperoxia was evident in both unglycosylated and glycosylated forms of the protein following 1 and 2 day exposures. The observed elevation of the 25-30 and 50-64 kDa bands of microvessel PrPc is not sustainable following 1 day hyperoxia, but returns to near normoxic levels within 24 h after hyperoxia. We also show in a knockout mouse for methionine sulfoxide reductase (MsrA), the enzyme responsible for reducing methionine sulfoxide back to methionine, and a regulator of cellular antioxidant defence, that following hyperoxia brain PrPc in the null mutant is elevated relative to PrPc content in the parent strain. Our results show up-regulated PrPc expression or reduced turnover in response to age-related, and hyperoxia-induced oxidative stress.

Actinobacillus actinomycetemcomitans is an oral pathogen that is a causative agent for periodontal disease as well as other non-oral infections. The chronic inflammation associated with periodontal diseases suggests that the bacterium must be able to neutralize oxygen intermediates to survive in the host tissues. Methionine sulfoxide reductase (MsrA) is an enzyme that has been demonstrated to have a role in protection against oxidative damage and has also been identified to be required for the proper expression or maintenance of functional adhesins on the surface of several pathogenic bacteria. The A. actinomycetemcomitans homologue of msrA has been isolated and a chromosomal insertion mutant constructed by allele replacement mutagenesis. Inactivation of the gene led to a complete loss of enzymic activity toward a synthetic substrate. However, the isogenic mutant was not more sensitive to oxidative stress or less adherent to epithelial cells as compared with the parent strain. These data suggest that this strain of A. actinomycetemcomitans has redundant systems that compensate for the MsrA activities ascribed for other organisms.

The mammalian methionine sulfoxide reductase B (MsrB) has been found to be a selenoprotein which can reduce R form of both free and protein-incorporated methionine sulfoxide to methionine. Together with MsrA, which reduces specifically the S form of methionine sulfoxide, the living cell can repair methionine-damaged proteins and salvage free methionine under oxidative stress conditions. Here, we report about the pivotal role of the selenocysteine residue in the protein putative active site by site-directed mutagenesis directed to the selenocysteine codon. Using the Escherichia coli SECIS (selenocysteine insertion sequence) element, needed for the recognition of the UGA codon as a selenocysteine codon in E. coli, we expressed the seleno-MsrB as a recombinant selenoprotein in E. coli. The recombinant seleno-MsrB has been shown to be much more active than the cysteine mutant, whereas the mutations to alanine and serine rendered the protein inactive. Although the yields of expression of the full-length N-terminus and C-terminus His-tagged seleno-MsrB were only 3% (of the total MsrB expressed), the C-terminus His-tagged protein enabled us to get a pure preparation of the seleno-MsrB. Using both recombinant selenoproteins, the N-terminus His-tagged and the C-terminus His-tagged proteins, we were able to determine the specific activities of the recombinant seleno-MsrB, which were found to be much higher than the cysteine mutant homologue. This finding confirmed our suggestion that the selenocysteine is essential for maintaining high reducing activity of MsrB. In addition, using radioactive selenium we were able to determine the in vivo presence of MsrB as a selenoprotein in mammalian cell cultures.

Previous studies have indicated that human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) are less active at blocking viral replication in HIV-1 infected peripheral blood monocytes/macrophages (M/M) than in HIV-1-infected T cells. We explored the hypothesis that oxidative modification and/or metabolism of the PIs in M/M might account for this reduced potency. We first tested the susceptibility of several PIs (kynostatin-272 [KNI-272], saquinavir, indinavir, ritonavir, or JE-2147) to oxidation after exposure to hydrogen peroxide (H2O2): only KNI-272 was highly susceptible to oxidation. Treatment of KNI-272 with low millimolar concentrations of H2O2resulted in mono-oxidation of the sulfur in theS-methyl cysteine (methioalanine) moiety, as determined by reversed-phase high-performance liquid chromatography and mass spectrometry (RP-HPLC/MS). Higher concentrations of H2O2led to an additional oxidation of the sulfur in the thioproline moiety of KNI-272. None of the PIs were metabolized or oxidized when added to T cells and cultured for up to 12 days. However, when KNI-272 was added to M/M, the concentration of the original KNI-272 steadily decreased with a corresponding increase in the production of three KNI-272 metabolites as identified by RP-HPLC/MS. The structures of these metabolites were different from those produced by H2O2treatment. The two major products of M/M metabolism of KNI-272 were identified as isomeric forms of KNI-272 oxidized solely on the thioproline ring. Both metabolites had reduced capacities to inhibit HIV-1 protease activity when tested in a standard HIV-1 protease assay. These studies demonstrate that antiviral compounds can be susceptible to oxidative modification in M/M and that this can affect their antiviral potency.

Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous single-nucleotide polymorphism (SNP) that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood post-translational mechanisms. One post-translational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, whereas brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined.

The roles of methionine residues in proteins have not been well defined, but a review of available studies leads to the conclusion that methionine, like cysteine, functions as an antioxidant and as a key component of a system for regulation of cellular metabolism. Methionine is readily oxidized to methionine sulfoxide by many reactive species. The oxidation of surface exposed methionines thus serves to protect other functionally essential residues from oxidative damage. Methionine sulfoxide reductases have the potential to reduce the residue back to methionine, increasing the scavenging efficiency of the system. Reversible covalent modification of amino acids in proteins provides the mechanistic basis for most systems of cellular regulation. Interconversion of methionine and methionine sulfoxide can function to regulate the biological activity of proteins, through alteration in catalytic efficiency and through modulation of the surface hydrophobicity of the protein.

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H2O2 resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H2O2 led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.

The yeast peptide-methionine sulfoxide reductase (MsrA) was overexpressed in aSaccharomyces cerevisiaenull mutant ofmsrAby using a high-copy plasmid harboring themsrAgene 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 themsrAmutant 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 themsrAmutant. 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 bovinemsrAand 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 themsrAgene is involved in the resistance of yeast and mammalian cells to oxidative stress.

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, Δ msrA::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.

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.