Inhibition of HDACs (Histone Deacetylases) Ameliorates High-Fat Diet-Induced Hypertension Through Restoration of the MsrA (Methionine Sulfoxide Reductase A)/Hydrogen Sulfide Axis.

[Figure: see text].

The Function of Selenium in Central Nervous System: Lessons from MsrB1 Knockout Mouse Models.

MsrB1 used to be named selenoprotein R, for it was first identified as a selenocysteine containing protein by searching for the selenocysteine insert sequence (SECIS) in the human genome. Later, it was found that MsrB1 is homologous to PilB in Neisseria gonorrhoeae, which is a methionine sulfoxide reductase (Msr), specifically reducing L-methionine sulfoxide (L-Met-O) in proteins. In humans and mice, four members constitute the Msr family, which are MsrA, MsrB1, MsrB2, and MsrB3. MsrA can reduce free or protein-containing L-Met-O (S), whereas MsrBs can only function on the L-Met-O (R) epimer in proteins. Though there are isomerases existent that could transfer L-Met-O (S) to L-Met-O (R) and vice-versa, the loss of Msr individually results in different phenotypes in mice models. These observations indicate that the function of one Msr cannot be totally complemented by another. Among the mammalian Msrs, MsrB1 is the only selenocysteine-containing protein, and we recently found that loss of MsrB1 perturbs the synaptic plasticity in mice, along with the astrogliosis in their brains. In this review, we summarized the effects resulting from Msr deficiency and the bioactivity of selenium in the central nervous system, especially those that we learned from the MsrB1 knockout mouse model. We hope it will be helpful in better understanding how the trace element selenium participates in the reduction of L-Met-O and becomes involved in neurobiology.

A Methionine Sulfoxide Reductase B Is Required for the Establishment of Astragalus sinicus-Mesorhizobium Symbiosis.

Methionine sulfoxide reductase B (MsrB) is involved in oxidative stress or defense responses in plants. However, little is known about its role in legume-rhizobium symbiosis. In this study, an MsrB gene was identified from Astragalus sinicus and its function in symbiosis was characterized. AsMsrB was induced under phosphorus starvation and displayed different expression patterns under symbiotic and nonsymbiotic conditions. Hydrogen peroxide or methyl viologen treatment enhanced the transcript level of AsMsrB in roots and nodules. Subcellular localization showed that AsMsrB was localized in the cytoplasm of onion epidermal cells and co-localized with rhizobia in nodules. Plants with AsMsrB-RNAi hairy roots exhibited significant decreases in nodule number, nodule nitrogenase activity and fresh weight of the aerial part, as well as an abnormal nodule and symbiosome development. Statistical analysis of infection events showed that plants with AsMsrB-RNAi hairy roots had significant decreases in the number of root hair curling events, infection threads and nodule primordia compared with the control. The content of hydrogen peroxide increased in AsMsrB-RNAi roots but decreased in AsMsrB overexpression roots at the early stage of infection. The transcriptome analysis showed synergistic modulations of the expression of genes involved in reactive oxygen species generation and scavenging, defense and pathogenesis and early nodulation. In addition, a candidate protein interacting with AsMsrB was identified and confirmed by bimolecular fluorescence complementation. Taken together, our results indicate that AsMsrB plays an essential role in nodule development and symbiotic nitrogen fixation by affecting the redox homeostasis in roots and nodules.

[Bacteria, model organisms to investigate the repair mechanisms of oxidized proteins]. / Les bactéries, organismes de choix pour comprendre les mécanismes de réparation des protéines oxydées.

TITLE: Les bactéries, organismes de choix pour comprendre les mécanismes de réparation des protéines oxydées. ABSTRACT: Dans le cadre de l'unité d'enseignement « Rédiger en sciences ¼ proposée par l'université d'Aix-Marseille, les étudiants du Master 2 de microbiologie se sont confrontés aux exigences de l'écriture scientifique. Quatre thématiques leur ont été proposées : les virus géants, les systèmes de sécrétion, la motilité bactérienne et la réparation des protéines oxydées. Après un travail préparatoire effectué avec l'équipe pédagogique et les auteurs des publications originales, les étudiants, organisés en groupes de trois ou quatre, ont rédigé une Nouvelle soulignant les résultats majeurs et l'originalité des quatre articles étudiés. Complété par un entretien avec les chercheurs auteurs de ces articles, l'ensemble offre un éclairage original sur la compréhension du vivant dans le domaine de la microbiologie.

Methionine sulfoxide reductase A of Salmonella Typhimurium interacts with several proteins and abets in its colonization in the chicken.

Defending phagocyte generated oxidants is the key for survival of Salmonella Typhimurium (S. Typhimurium) inside the host. Met residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO). Methionine sulfoxide reductase (Msr) can repair Met-SO to Met thus restoring the function(s) of Met-SO containing proteins. Using pull down method we have identified several MsrA interacting proteins in the S. Typhimurium, one of them was malate synthase (MS). MS is an enzyme of glyoxylate cycle. This cycle is essential for survival of S. Typhimurium inside the host under nutrient limiting conditions. By employing in vitro cross-linking and blot overlay techniques we showed that purified MsrA interacted with pure MS. Treatment of pure malate synthase with H2O2 resulted in reduction of MS activity. However, MsrA along with thioredoxin-thioredoxin reductase system partially restored the activity of oxidized MS. Our mass spectrometry data demonstrated H2O2 mediated oxidation and MsrA mediated repair of Met residues in MS. Further in comparison to S. Typhimurium, the msrA gene deletion (∆msrA) strain showed reduced (60%) malate synthase specific activity. Oral inoculation with wild type, ∆msrA and ∆ms strains of S. Typhimurium resulted in colonization of 100, 0 and 40% of the poultry respectively.

Methionine sulfoxide reductase A deficiency exacerbates acute liver injury induced by acetaminophen.

Acetaminophen (APAP) overdose induces acute liver injury via enhanced oxidative stress and glutathione (GSH) depletion. Methionine sulfoxide reductase A (MsrA) acts as a reactive oxygen species scavenger by catalyzing the cyclic reduction of methionine-S-sulfoxide. Herein, we investigated the protective role of MsrA against APAP-induced liver damage using MsrA gene-deleted mice (MsrA-/-). We found that MsrA-/- mice were more susceptible to APAP-induced acute liver injury than wild-type mice (MsrA+/+). The central lobule area of the MsrA-/- liver was more impaired with necrotic lesions. Serum alanine transaminase, aspartate transaminase, and lactate dehydrogenase levels were significantly higher in MsrA-/- than in MsrA+/+ mice after APAP challenge. Deletion of MsrA enhanced APAP-induced hepatic GSH depletion and oxidative stress, leading to increased susceptibility to APAP-induced liver injury in MsrA-deficient mice. APAP challenge increased Nrf2 activation more profoundly in MsrA-/- than in MsrA+/+ livers. Expression and nuclear accumulation of Nrf2 and its target gene expression were significantly elevated in MsrA-/- than in MsrA+/+ livers after APAP challenge. Taken together, our results demonstrate that MsrA protects the liver from APAP-induced toxicity. The data provided herein constitute the first in vivo evidence of the involvement of MsrA in hepatic function under APAP challenge.

Arabidopsis thaliana methionine sulfoxide reductase B8 influences stress-induced cell death and effector-triggered immunity.

KEY MESSAGE: Reactive oxygen species (ROS) oxidize methionine to methionine sulfoxide (MetSO) and thereby inactivate proteins. Methionine sulfoxide reductase (MSR) enzyme converts MetSO back to the reduced form and thereby detoxifies the effect of ROS. Our results show that Arabidopsis thaliana MSR enzyme coding gene MSRB8 is required for effector-triggered immunity and containment of stress-induced cell death in Arabidopsis. Plants activate pattern-triggered immunity (PTI), a basal defense, upon recognition of evolutionary conserved molecular patterns present in the pathogens. Pathogens release effector molecules to suppress PTI. Recognition of certain effector molecules activates a strong defense, known as effector-triggered immunity (ETI). ETI induces high-level accumulation of reactive oxygen species (ROS) and hypersensitive response (HR), a rapid programmed death of infected cells. ROS oxidize methionine to methionine sulfoxide (MetSO), rendering several proteins nonfunctional. The methionine sulfoxide reductase (MSR) enzyme converts MetSO back to the reduced form and thereby detoxifies the effect of ROS. Though a few plant MSR genes are known to provide tolerance against oxidative stress, their role in plant-pathogen interaction is not known. We report here that activation of cell death by avirulent pathogen or UV treatment induces expression of MSRB7 and MSRB8 genes. The T-DNA insertion mutant of MSRB8 exaggerates HR-associated and UV-induced cell death and accumulates a higher level of ROS than wild-type plants. The negative regulatory role of MSRB8 in HR is further supported by amiRNA and overexpression lines. Mutants and overexpression lines of MSRB8 are susceptible and resistant respectively, compared to the wild-type plants, against avirulent strains of Pseudomonas syringae pv. tomato DC3000 (Pst) carrying AvrRpt2, AvrB, or AvrPphB genes. However, the MSRB8 gene does not influence resistance against virulent Pst or P. syringae pv. maculicola (Psm) pathogens. Our results altogether suggest that MSRB8 function is required for ETI and containment of stress-induced cell death in Arabidopsis.

Methionine sulfoxide reductase B3 deficiency stimulates heme oxygenase-1 expression via ROS-dependent and Nrf2 activation pathways.

Methionine sulfoxide reductase B3 (MsrB3), which is primarily found in the endoplasmic reticulum (ER), is an important protein repair enzyme that stereospecifically reduces methionine-R-sulfoxide residues. We previously found that MsrB3 deficiency arrests the cell cycle at the G1/S stage through up-regulation of p21 and p27. In this study, we report a critical role of MsrB3 in gene expression of heme oxygenase-1 (HO-1), which has an anti-proliferative effect associated with p21 up-regulation. Depletion of MsrB3 elevated HO-1 expression in mammalian cells, whereas MsrB3 overexpression had no effect. MsrB3 deficiency increased cellular reactive oxygen species (ROS), particularly in the mitochondria. ER stress, which is associated with up-regulation of HO-1, was also induced by depletion of MsrB3. Treatment with N-acetylcysteine as an ROS scavenger reduced augmented HO-1 levels in MsrB3-depleted cells. MsrB3 deficiency activated Nrf2 transcription factor by enhancing its expression and nuclear import. The activation of Nrf2 induced by MsrB3 depletion was confirmed by increased expression levels of its other target genes, such as γ-glutamylcysteine ligase. Taken together, these data suggest that MsrB3 attenuates HO-1 induction by inhibiting ROS production, ER stress, and Nrf2 activation.

Methionine sulfoxide reductase A deficiency exacerbates progression of kidney fibrosis induced by unilateral ureteral obstruction.

Methionine sulfoxide reductase A (MsrA), which stereospecifically catalyzes the reduction of methionine-S-sulfoxide, is an important reactive oxygen species (ROS) scavenger. Tissue fibrosis is a maladaptive repair process following injury, associated with oxidative stress. In this study, we investigated the role of MsrA in unilateral ureteral obstruction (UUO)-induced kidney fibrosis and its underlying mechanisms by using MsrA gene-deleted mice (MsrA(-/-)). MsrA deletion increased collagen deposition in the interstitium and the expression of collagen III and α-smooth muscle actin in the UUO kidneys, indicating that MsrA deficiency exacerbated the progression of UUO-induced kidney fibrosis. UUO reduced the kidney expression of MsrA, MsrB1, and MsrB2, thereby decreasing MsrA and MsrB activity. UUO increased hydrogen peroxide and lipid peroxidation levels and the ratio of oxidized glutathione (GSSG) to total glutathione (GSH) in the kidneys. The UUO-induced elevations in the levels of these oxidative stress markers and leukocyte markers were much higher in the MsrA(-/-) than in the MsrA(+/+) kidneys, the latter suggesting that the exacerbated kidney fibrosis in MsrA(-/-) mice was associated with enhanced inflammatory responses. Collectively, our data suggest that MsrA plays a protective role in the progression of UUO-induced kidney fibrosis via suppression of fibrotic responses caused by oxidative stress and inflammation.

A protective role of methionine-R-sulfoxide reductase against cadmium in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe cells harboring the methionine- R-sulfoxide reductase (MsrB)-overexpressing recombinant plasmid pFMetSO exhibited better growth than vector control cells, when shifted into fresh medium containing cadmium chloride (abbreviated as Cd). Although both groups of cells contained enhanced reactive oxygen species (ROS) and nitric oxide (NO) levels in the presence of Cd, ROS and NO levels were significantly lower in the S. pombe cells harboring pFMetSO than in vector control cells. Conversely, the S. pombe cells harboring pFMetSO possessed higher total glutathione (GSH) levels and a greater reduced/oxidized GSH ratio than vector control cells under the same conditions.

Redox switch for actin.

Oxidation of actin methionine residues by the oxidation-reduction enzyme Mical is known to lead to actin filament depolymerization. SelR enzymes are now shown to reduce these oxidized actin methionines, revealing a regulated redox reaction mechanism through which cells control the assembly and disassembly of actin filaments.

SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics.

Actin's polymerization properties are markedly altered by oxidation of its conserved Met 44 residue. Mediating this effect is a specific oxidation-reduction (redox) enzyme, Mical, that works with Semaphorin repulsive guidance cues and selectively oxidizes Met 44. We now find that this actin-regulatory process is reversible. Employing a genetic approach, we identified a specific methionine sulfoxide reductase (MsrB) enzyme SelR that opposes Mical redox activity and Semaphorin-Plexin repulsion to direct multiple actin-dependent cellular behaviours in vivo. SelR specifically catalyses the reduction of the R isomer of methionine sulfoxide (methionine-R-sulfoxide) to methionine, and we found that SelR directly reduced Mical-oxidized actin, restoring its normal polymerization properties. These results indicate that Mical oxidizes actin stereospecifically to generate actin Met-44-R-sulfoxide (actin(Met(R)O-44)), and also implicate the interconversion of specific Met/Met(R)O residues as a precise means to modulate protein function. Our results therefore uncover a specific reversible redox actin regulatory system that controls cell and developmental biology.

Involvement of MsrB1 in the regulation of redox balance and inhibition of peroxynitrite-induced apoptosis in human lens epithelial cells.

Methionine sulfoxide reductases (Msrs) in lens cells are important for the maintenance of lens cell viability and resistance to oxidative stress damage. Peroxynitrite (ONOO(-)), as a strong oxidizing and nitrating agent, occurred in diabetic retinopathy patients and diabetic model animal. In an attempt to shed light on the roles of MsrB1, known as selenoprotein R, in protecting human lens epithelial (HLE) cells against peroxynitrite damage, and contribution of loss of its normal activity to cataract, the influences of MsrB1 gene silencing on peroxynitrite-induced apoptosis in HLE cells were studied. The results showed that both exogenous peroxynitrite and MsrB1 gene silencing by short interfering RNA (siRNA) independently resulted in oxidative stress, endoplasmic reticulum (ER) stress, activation of caspase-3 as well as an increase of apoptosis in HLE cells; moreover, when MsrB1-gene-silenced cells were exposed to 300 µM peroxynitrite, these indexes were further aggravated at the same conditions and DNA strand breaks occurred. The results demonstrate that in HLE cells MsrB1 may play important roles in regulating redox balance and mitigating ER stress as induced by oxidative stress under physiological conditions; MsrB1 may also protect HLE cells against peroxynitrite-induced apoptosis by inhibiting the activation of caspase-3 and oxidative damage of DNA under pathological conditions. Our results imply that loss of its normal activity is likely to contribute to cataract.

Methionine sulfoxide reductase B3 protects from endoplasmic reticulum stress in Drosophila and in mammalian cells.

Methionine sulfoxide reductase B3A (MsrB3A), which catalyzes the stereospecific reduction of methionine-R-sulfoxide to methionine, is localized to the endoplasmic reticulum (ER). Here, we report a critical role of the ER-targeted MsrB3 in protection against ER stress in Drosophila and in mammalian cells. Flies overexpressing human MsrB3A exhibited significantly increased resistance to ER stress induced by dithiothreitol. These flies also showed slightly enhanced resistance to tunicamycin-induced ER stress. In addition, overexpression of MsrB3A in mammalian cells increased resistance to dithiothreitol- and thapsigargin-induced ER stresses. However, MsrB3A overexpression had no effect on the resistance to tunicamycin-induced ER stress. Knockdown of MsrB3A in mammalian cells led to a significant decrease in the resistance to thapsigargin-induced ER stress, but had no effects on the resistance to either dithiothreitol- or tunicamycin-induced ER stress. Collectively, our data provide evidence that the ER-type of MsrB3 plays an important role in protection against ER stress, suggesting that MsrB3 may be involved in the regulation of ER homeostasis.

Myristoylated methionine sulfoxide reductase A protects the heart from ischemia-reperfusion injury.

Methionine sulfoxide reductase A (MsrA) catalytically scavenges reactive oxygen species and also repairs oxidized methionines in proteins. Increasing MsrA protects cells and organs from a variety of oxidative stresses while decreasing MsrA enhances damage, but the mechanisms of action have not been elucidated. A single gene encodes MsrA of which ∼25% is targeted to the mitochondria, a major site of reactive oxygen species production. The other ∼75% is targeted to the cytosol and is posttranslationally modified by myristoylation. To determine the relative importance of MsrA in each compartment in protecting against ischemia-reperfusion damage, we created a series of transgenic mice overexpressing MsrA targeted to the mitochondria or the cytosol. We used a Langendorff model of ischemia-reperfusion and assayed both the rate pressure product and infarct size following ischemia and reperfusion as measures of injury. While the mitochondrially targeted MsrA was expected to be protective, it was not. Notably, the cytosolic form was protective but only if myristoylated. The nonmyristoylated, cytosolic form offered no protection against injury. We conclude that cytosolic MsrA protects the heart from ischemia-reperfusion damage. The requirement for myristoylation suggests that MsrA must interact with a hydrophobic domain to provide protection.

The protein truncation caused by fusion of PEP-1 peptide and protective roles of transduced PEP-1-MsrA in skin cells.

PEP-1 peptide has been used for transduction of native protein into mammalian cells. This work describes the findings that the fusion of PEP-1 to target proteins led to protein truncation likely in a non-protein-specific manner. Approximately 75% of PEP-1-MsrA fusion protein was truncated in the N-terminal region of MsrA between Lys-27 and Val-28 during expression in Escherichia coli and purification. This large protein truncation was also observed in another PEP-1 fused protein, PEP-1- MsrB2, in the N-terminal region of MsrB2. The full-length PEP-1-MsrA protein was rapidly transduced into keratinocyte cells within 15 min. The transduced PEP-1-MsrA was functionally active and could protect skin cells against oxidative stress-and ultraviolet radiation-induced cell death. Collectively, our data demonstrated the protective roles of MsrA in skin cells and, moreover, may raise a concern of protein truncation caused by fusion of PEP-1 about the general use of this peptide for protein transduction.

Quantification of reserve pool dopamine in methionine sulfoxide reductase A null mice.

Methionine sulfoxide reductase A knockout (MsrA-/-) mice, which serve as a potential model for neurodegeneration, suffer from increased oxidative stress and have previously been found to have chronically elevated brain dopamine (DA) content levels relative to control mice. Additionally, these high levels parallel the increased presynaptic DA release. In this study, fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used to quantify striatal reserve pool DA in knockout mice and wild-type control mice. Reserve pool DA efflux, induced by amphetamine (AMPH), was measured in brain slices from knockout and wild type (WT) mice in the presence of α-methyl-p-tyrosine, a DA synthesis inhibitor. Additionally, the stimulated release of reserve pool DA, mobilized by cocaine (COC), was measured. Both efflux and stimulated release measurements were enhanced in slices from knockout mice, suggesting that these mice have greater reserve pool DA stores than wild-type and that these stores are effectively mobilized. Moreover, dopamine transporter (DAT) labeling data indicate that the difference in measured DA efflux was likely not caused by altered DAT protein expression. Additionally, slices from MsrA-/- and wild-type mice were equally responsive to increasing extracellular calcium concentrations, suggesting that potential differences in either calcium entry or intracellular calcium handling are not responsible for increased reserve pool DA release. Collectively, these results demonstrate that MsrA-/- knockout mice maintain a larger DA reserve pool than wild-type control mice, and that this pool is readily mobilized.

Role of methionine sulfoxide reductases A and B of Enterococcus faecalis in oxidative stress and virulence.

Methionine sulfoxide reductases A and B are antioxidant repair enzymes that reduce the S- and R-diastereomers of methionine sulfoxides back to methionine, respectively. Enterococcus faecalis, an important nosocomial pathogen, has one msrA gene and one msrB gene situated in different parts of the chromosome. Promoters have been mapped and mutants have been constructed in two E. faecalis strains (strains JH2-2 and V583) and characterized. For both backgrounds, the mutants are more sensitive than the wild-type parents to exposure to H2O2, and in combination the mutations seem to be additive. The virulence of the mutants has been analyzed in four different models. Survival of the mutants inside mouse peritoneal macrophages stimulated with recombinant gamma interferon plus lipopolysaccharide but not in naïve phagocytes is significantly affected. The msrA mutant is attenuated in the Galleria mellonella insect model. Deficiency in either Msr enzyme reduced the level of virulence in a systemic and urinary tract infection model. Virulence was reconstituted in the complemented strains. The combined results show that Msr repair enzymes are important for the oxidative stress response, macrophage survival, and persistent infection with E. faecalis.

Methionine sulfoxide reductase B1 (MsrB1) recovers TRPM6 channel activity during oxidative stress.

Mg(2+) is an essential ion for many cellular processes, including protein synthesis, nucleic acid stability, and numerous enzymatic reactions. Mg(2+) homeostasis in mammals depends on the equilibrium between intestinal absorption, renal excretion, and exchange with bone. The transient receptor potential melastatin type 6 (TRPM6) is an epithelial Mg(2+) channel, which is abundantly expressed in the luminal membrane of the renal and intestinal cells. It functions as the gatekeeper of transepithelial Mg(2+) transport. Remarkably, TRPM6 combines a Mg(2+)-permeable channel with an alpha-kinase domain. Here, by the Ras recruitment system, we identified methionine sulfoxide reductase B1 (MsrB1) as an interacting protein of the TRPM6 alpha-kinase domain. Importantly, MsrB1 and TRPM6 are both present in the renal Mg(2+)-transporting distal convoluted tubules. MsrB1 has no effect on TRPM6 channel activity in the normoxic conditions. However, hydrogen peroxide (H(2)O(2)) decreased TRPM6 channel activity. Co-expression of MsrB1 with TRPM6 attenuated the inhibitory effect of H(2)O(2) (TRPM6, 67 +/- 5% of control; TRPM6 + MsrB1, 81 +/- 5% of control). Cell surface biotinylation assays showed that H(2)O(2) treatment does not affect the expression of TRPM6 at the plasma membrane. Next, mutation of Met(1755) to Ala in TRPM6 reduced the inhibitory effect of H(2)O(2) on TRPM6 channel activity (TRPM6 M1755A: 84 +/- 10% of control), thereby mimicking the action of MsrB1. Thus, these data suggest that MsrB1 recovers TRPM6 channel activity by reducing the oxidation of Met(1755) and could, thereby, function as a modulator of TRPM6 during oxidative stress.