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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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

On page 4 of this published article an error in Figure 2(a) has occurred. Accordingly, Figure 2(a) has been replaced with a corrected version of the figure as shown below. Figure 2 Phosphorylation of α-synuclein in MsrA−/− and wild-type (WT) brain extracts. (a) Tris-soluble and Urea-soluble brain extracts (40 μg protein) of both mouse types were prepared as described in Section 2. These extracts ...

Meconium ileus (MI), a life-threatening intestinal obstruction due to meconium with abnormal protein content, occurs in approximately 15 percent of neonates with cystic fibrosis (CF). Analysis of twins with CF demonstrates that MI is a highly heritable trait, indicating that genetic modifiers are largely responsible for this complication. Here, we performed regional family-based association analysis of a locus that had previously been linked to MI and found that SNP haplotypes 5′ to and within the MSRA gene were associated with MI (P = 1.99×10−5 to 1.08×10−6; Bonferroni P = 0.057 to 3.1×10−3). The haplotype with the lowest P value showed association with MI in an independent sample of 1,335 unrelated CF patients (OR = 0.72, 95% CI [0.53–0.98], P = 0.04). Intestinal obstruction at the time of weaning was decreased in CF mice with Msra null alleles compared to those with wild-type Msra resulting in significant improvement in survival (P = 1.2×10−4). Similar levels of goblet cell hyperplasia were observed in the ilea of the Cftr −/− and Cftr −/− Msra −/− mice. Modulation of MSRA, an antioxidant shown to preserve the activity of enzymes, may influence proteolysis in the developing intestine of the CF fetus, thereby altering the incidence of obstruction in the newborn period. Identification of MSRA as a modifier of MI provides new insight into the biologic mechanism of neonatal intestinal obstruction caused by loss of CFTR function., Author Summary Cystic fibrosis (CF) is a monogenic disease with considerable phenotypic variability. About 15% of newborns with CF suffer from an intestinal obstruction called meconium ileus (MI), and studies in CF twins have shown that modifier genes play a substantial role in the development of this complication. We used a family-based study design to enrich for genetic modifiers of MI and found that variations in the MSRA gene, represented by combinations of SNPs, or haplotypes, were protective against this manifestation of CF. We investigated association between one of the MSRA haplotypes and MI in an independent sample of CF patients and showed that it had a similar protective effect. Furthermore, CF mice lacking Msra expression had lower mortality due to intestinal obstruction at the time of transitioning to solid food and lived longer than CF mice with normal Msra, thus supporting the protective effect of the haplotype we observed in human CF subjects. The identification of modifiers of MI such as MSRA offers new insight into the mechanism of this life-threatening complication of CF.

Previously, we have showed that overexpression of methionine-oxidized α-synuclein in methionine sulfoxide reductase A (MsrA) null mutant yeast cells inhibits α-synuclein phosphorylation and increases protein fibrillation. The current studies show that ablation of mouse MsrA gene caused enhanced methionine oxidation of α-synuclein while reducing its own phophorylation levels, especially in the hydrophobic cell-extracted fraction. These data provide supportive evidence that a compromised MsrA function in mammalian brain may cause enhanced pathologies associated with altered α-synuclein oxidation and phosphorylation levels.

Previous research suggests that brain oxidative stress and altered rodent locomotor behavior are linked. We observed bio-behavioral changes in methionine sulfoxide reductase A knockout mice associated with abnormal dopamine signaling. Compromised ability of these knockout mice to reduce methionine sulfoxide enhances accumulation of sulfoxides in proteins. We examined the dopamine D(2)-receptor function and expression, which has an atypical arrangement and quantity of methionine residues. Indeed, protein expression levels of dopamine D(2)-receptor were higher in knockout mice compared with wild-type. However, the binding of dopamine D(2)-receptor agonist was compromised in the same fractions of knockout mice. Coupling efficiency of dopamine D(2)-receptors to G-proteins was also significantly reduced in knockout mice, supporting the compromised agonist binding. Furthermore, pre-synaptic dopamine release in knockout striatal sections was less responsive than control sections to dopamine D(2)-receptor ligands. Behaviorally, the locomotor activity of knockout mice was less responsive to the inhibitory effect of quinpirole than wild-type mice. Involvement of specific methionine residue oxidation in the dopamine D(2)-receptor third intracellular loop is suggested by in vitro studies. We conclude that ablation of methionine sulfoxide reductase can affect dopamine signaling through altering dopamine D(2)-receptor physiology and may be related to symptoms associated with neurological disorders and diseases.

Schistosomiasis, also known as Bilharzia, is an infectious disease caused by several species of Schistosoma. Twenty million individuals suffer severe symptoms and 200,000 people die annually from the disease. The host responds to the presence of S. mansoni by producing reactive oxygen species that cause oxidative stress. We hypothesized that schistosomes produce antioxidants in response to oxidative stress. A known antioxidant protein is methionine sulfoxide reductase (Msr). Methionine residues can be oxidized to methionine sulfoxide in the presence of oxidizing agents, and the process is readily reversed by the action of the Msr system. Two S. mansoni MsrB genes (MsrB1 and MsrB2) were cloned and the recombinant proteins were expressed in bacteria and purified. The S. mansoni MsrB proteins contained the common conserved catalytic-and zinc-coordinating cysteines. Analysis of the proteins showed that both proteins promote the reduction of both free methionine sulfoxide (Met[O]) and dabsyl-Met(O) to free methionine (Met) and dabsyl-Met, respectively, while exhibiting differences in their specific activities toward these substrates. Using real-time polymerase-chain reaction (RT-PCR), both proteins were found to be expressed in all stages of the parasite's life cycle, with the highest level of expression of both proteins in the egg stage. This is the first description of MsrB proteins from a parasite.

Oxidative stress is associated with the aging process, a risk factor for neurodegenerative diseases, and decreased by reduced energy intake. Oxidative modifications can affect protein function; the sulfur-containing amino acids, including methionine, are particularly susceptible to oxidation. A methionine sulfoxide can be enzymatically reduced by the methionine sulfoxide reductase (Msr) system. Previously, we have shown that MsrA(-/-) mice exhibit altered locomotor activity and brain dopamine levels as function of age. Previous studies have demonstrated that a caloric restriction enhances antioxidant defense and reduces the action of reactive oxygen species. Here we examine locomotor behavior and dopamine levels of MsrA(-/-) mice after caloric restriction starting at eight months of age and ending at 17 months. The MsrA(-/-) mice did not have any significant difference in spontaneous distance traveled when compared to controls at 17 months of age. In contrast, our previous report showed decreased locomotor activity in the MsrA(-/-) mice at 12 months of age and older when fed ad-libitum. After completion of the caloric restriction diet, dopamine levels were comparable to control mice. This differs from the abnormal dopamine levels previously observed in MsrA(-/-) mice fed ad-libitum. Thus, caloric restriction had a neutralization effect on MsrA ablation. In summary, it is suggested that caloric restriction alleviates abnormal locomotor activity and dopamine levels in the brain of the methionine sulfoxide reductase A knockout mouse.

Aggregated alpha-synuclein and the point mutations Ala30Pro and Ala53Thr of alpha-synuclein are associated with Parkinson's disease. The physiological roles of alpha-synuclein and methionine oxidation of the alpha-synuclein protein structure and function are not fully understood. Methionine sulfoxide reductase A (MsrA) reduces methionine sulfoxide residues and functions as an antioxidant. To monitor the effect of methionine oxidation to alpha-synuclein on basic cellular processes, alpha-synucleins were expressed in msrA null mutant and wild-type yeast cells. Protein degradation was inhibited in the alpha-synuclein-expressing msrA null mutant cells compared to alpha-synuclein-expressing wild-type cells. Increased inhibition of degradation and elevated accumulations of fibrillated proteins were observed in SynA30P-expressing msrA null mutant cells. Additionally, methionine oxidation inhibited alpha-synuclein phosphorylation in yeast cells and in vitro by casein kinase 2. Thus, a compromised MsrA function combined with alpha-synuclein overexpression may promote processes leading to synucleinopathies.

6 pags, 7 figs, Methionine sulfoxide (MetO) is a common posttranslational modification to proteins occurring in vivo. These modifications are prevalent when reactive oxygen species levels are increased. To enable the detection of MetO in pure and extracted proteins from various sources, we have developed novel antibodies that can recognize MetO-proteins. These antibodies are polyclonal antibodies raised against an oxidized methionine-rich zein protein (MetO-DZS18) that are shown to recognize methionine oxidation in pure proteins and mouse and yeast extracts. Furthermore, mouse serum albumin and immunoglobulin (IgG) were shown to accumulate MetO as function of age especially in serums of methionine sulfoxide reductase A knockout mice. Interestingly, high levels of methionine-oxidized IgG in serums of subjects diagnosed with Alzheimer's disease were detected by western blot analysis using these antibodies. It is suggested that anti-MetO-DZS18 antibodies can be applied in the identification of proteins that undergo methionine oxidation under oxidative stress, aging, or disease state conditions. © 2009 Elsevier Inc. All rights reserved., This study was partially supported by the National Institute of Aging Grant AG027363

Hypochlorous acid (HOCl) is produced by the neutrophil enzyme, myeloperoxidase, and reacts with amines to generate chloramines. These oxidants react readily with thiols and methionine and can affect cell-regulatory pathways. In the present study, we have investigated the ability of HOCl, glycine chloramine (Gly-Cl) and taurine chloramine (Tau-Cl) to oxidize IkappaBalpha, the inhibitor of NF-kappaB (nuclear factor kappaB), and to prevent activation of the NF-kappaB pathway in Jurkat cells. Glycine chloramine (Gly-Cl) and HOCl were permeable to the cells as determined by oxidation of intracellular GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase, whereas Tau-Cl showed no detectable cell permeability. Both Gly-Cl (20-200 muM) and HOCl (50 microM) caused oxidation of IkappaBalpha methionine, measured by a shift in electrophoretic mobility, when added to the cells in Hanks buffer. In contrast, a high concentration of Tau-Cl (1 mM) in Hanks buffer had no effect. However, Tau-Cl in full medium did modify IkappaBalpha. This we attribute to chlorine exchange with other amines in the medium to form more permeable chloramines. Oxidation by Gly-Cl prevented IkappaBalpha degradation in cells treated with TNFalpha (tumour necrosis factor alpha) and inhibited nuclear translocation of NF-kappaB. IkappaBalpha modification was reversed by methionine sulphoxide reductase, with both A and B forms required for complete reduction. Oxidized IkappaBalpha persisted intracellularly for up to 6 h. Reversion occurred in the presence of cycloheximide, but was prevented if thioredoxin reductase was inhibited, suggesting that it was due to endogenous methionine sulphoxide reductase activity. These results show that cell-permeable chloramines, either directly or when formed in medium, could regulate NF-kappaB activation via reversible IkappaBalpha oxidation.

Staphylococcus aureus contains three genes encoding MsrA-specific methionine sulfoxide reductase (Msr) activity (msrA1, msrA2 and msrA3) and an additional gene that encodes MsrB-specific Msr activity. Data presented here suggest that MsrA1 is the major contributor of the MsrA activity in S. aureus. In mutational analysis, while the total Msr activity in msrA2 mutant was comparable to that of the parent, Msr activity was significantly up-regulated in the msrA1 or msrA1 msrA2 double mutant. Assessment of substrate specificity together with increased reactivity of the cell-free protein extracts of the msrA1 mutants to anti-MsrB polyclonal antibodies in Western analysis provided evidence that increased Msr activity was due to elevated synthesis of MsrB in the MsrA1 mutants. Previously, it was reported that oxacillin treatment of S. aureus cells led to induced synthesis of MsrA1 and a mutation in msrA1 increased the susceptibility of the organism to H2O2. A mutation in the msrA2 gene, however, was not significant for the bacterial oxidative stress response. In complementation assays, while the msrA2 gene was unable to complement the msrA1 msrA2 double mutant for H2O2 resistance, the same gene restored H2O2 tolerance in the double mutant when placed under the control of the msrA1 promoter. However, msrA1 which was able to complement the oxidative stress response in msrA1 mutants could not restore the tolerance of the msrA1 msrA2 mutants to H2O2 when placed under the control of the msrA2 promoter. Additionally, although the oxacillin minimum inhibitory concentration of the msrA1 mutant was comparable to that of the wild-type parent, in shaking liquid culture, the msrA1 mutant responded more efficiently to sublethal doses of oxacillin. The data suggest complex regulation of Msr proteins and a more significant physiological role for msrA1/msrB in S. aureus.

Methionine sulfoxide reductase A (MsrA) maintains the function of many proteins by reversing oxidation of methionine residues. Lack of this repair mechanism very likely increases aging-related disease susceptibility. In Saccharomyces cerevisiae , disruption of the msrA gene increases free and protein-bound methionine sulfoxide and decreases cell viability. Although the underlying mechanisms in the induction of the msrA gene are still unknown, a transcriptional regulation may be involved. Hence, a search of nuclear proteins regulating the msrA gene is a major target of the experiments reported in this article. Using protein purification combined with MS, we discovered that calcium phospholipid-binding protein (CPBP), a homologue of elongation factor-1γ, is a component of a complex that binds to the msrA promoter. By measuring CPBP cooperative binding to the msrA promoter, we have mapped the CPBP binding site to a 39-bp sequence at the 3′ end of the promoter. In a mutant yeast strain lacking the CPBP-encoding gene, the ability to overexpress msrA mRNA and MsrA protein was impaired and MsrA catalytic activity was greatly reduced, suggesting that CPBP may enhance msrA gene expression.

Mammals contain two methionine sulfoxide (MetO) reductases, MsrA and MsrB, that catalyze the thioredoxin-dependent reduction of the S -MetO and R -MetO derivatives, respectively, to methionine. The major mammalian MsrB is a selenoprotein (except in the heart). Here, we show that there is a loss of MsrB activity in the MsrA – / – mouse that correlates with parallel losses in the levels of MsrB mRNA and MsrB protein, suggesting that MsrA might have a role in MsrB transcription. Moreover, mice that were grown on a selenium-deficient (SD) diet showed a substantial decrease in the levels of MsrB-catalytic activity, MsrB protein, and MsrB mRNA in liver and kidney tissues of both WT and MsrA – / – mouse strains. Whereas no significant protein-MetO could be detected in tissue proteins of young mature mice grown on a selenium-adequate diet, growth on the SD diet led to substantial accumulations of MetO in proteins and also of protein carbonyl derivatives in the liver, kidney, cerebrum, and cerebellum, respectively. In addition, accumulation of protein-MetO derivatives increased with age in tissues of mice fed with a selenium-adequate diet. It should be pointed out that even though the total Msr level is at least 2-fold higher in WT than in MsrA – / – mice, SD diet causes an equal elevation of protein-MetO (except in brain cerebellum) and carbonyl levels in both strains, suggesting involvement of other selenoproteins in regulation of the level of cellular protein-MetO accumulation. Furthermore, the development of the ``tip-toe'' walking behavior previously observed in the MsrA – / – mice occurred earlier when they were fed with the SD diet.

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.