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Atrial fibrillation (AF) is the most prevalent sustained tachyarrhythmia in patients with cardiovascular diseases. Recently, it has been discovered that oxidative stress is an important contributor to AF. Therefore, antioxidant therapies for AF have great potential for clinical applications. Methionine, a sulfur-containing amino acid residue other than cysteine, is recognized as a functional redox switch, which could be rescued from the reversible oxidation of methionine sulfoxide by methionine sulfoxide reductase A (MsrA). S -Methyl-L-cysteine (SMLC), a natural analogue of Met, which is abundantly found in garlic and cabbage, could substitute for Met oxidations and mediate MsrA to scavenge free radicals. However, whether SMLC alleviates AF is unclear. This study aims to clarify the effects of SMLC on AF and elucidate the underlying pharmacological and molecular mechanisms. In vivo , SMLC (70, 140 and 280 mg kg -1 day -1 ) was orally administered to mice for 4 weeks with angiotensin II (Ang II) by subcutaneous infusion using osmotic pumps to induce AF. Ang II significantly prompted high AF susceptibility and atrial remodeling characterized by oxidative stress, conductive dysfunction and fibrosis. SMLC played a remarkable protective role in Ang II-induced atrial remodeling dose-dependently. Moreover, RNA sequencing was performed on atrial tissues to identify the differentially expressed mRNA, which was to screen out MSRA, CAMK2 and MAPK signaling pathways. Western blots confirmed that Ang II-induced downregulation of MsrA and upregulation of oxidized CaMKII (ox-CaMKII) and p38 MAPK could be reversed in a concentration-dependent manner by SMLC. To investigate the underlying mechanisms, HL-1 cells (mouse atria-derived cardiomyocytes) treated with Ang II were used for an in vitro model. SMLC alleviated Ang II-induced cytotoxicity, mitochondrial damage and oxidative stress. Additionally, knockdown MsrA could attenuate the protective effects of SMLC, which were eliminated by the p38 MAPK inhibitor SB203580. In summary, the present study demonstrates that SMLC protects against atrial remodeling in AF by inhibiting oxidative stress through the mediation of the MsrA/p38 MAPK signaling pathway.

Citrus huanglongbing (HLB) has been causing enormous damage to the global citrus industry. As the main causal agent, 'Candidatus Liberibacter asiaticus' (CLas) delivers a set of effectors to modulate host responses, while the modes of action adopted remain largely unclear. Here, we demonstrated that CLIBASIA_00185 (CLas0185) could attenuate reactive oxygen species (ROS)-mediated cell death in Nicotiana benthamiana. Transgenic expression of CLas0185 in Citrus sinensis 'Wanjincheng' enhanced plant susceptibility to CLas. We found that methionine sulphoxide reductase B1 (CsMsrB1) was targeted by the effector, and its abundance was elevated in CLas0185-transgenic citrus plants. Their interaction promoted CLas proliferation. We then determined that CsMsrB1 sustained redox state and enzymatic activity of ascorbate peroxidase 1 (CsAPX1) under oxidative stress. The latter reduced H 2 O 2 accumulation and was associated with host susceptibility to CLas infection. Consistently, citrus plants expressing CLas0185 and CsMsrB1 conferred enhanced APX activity and decreased H 2 O 2 content. Taken together, these findings revealed how CLas0185 benefits CLas colonization by targeting CsMsrB1, which facilitated the antioxidant activity and depressed ROS during pathogen infection., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Background: Chondrocyte oxidative stress and apoptosis are critical factors contributing to the pathogenesis of osteoarthritis (OA). Methionine sulfoxide reductase B2 (MSRB2) is a mitochondrial protein that protects cells from oxidative stress and is involved in apoptosis. This study aimed to investigated the expression of MSRB2 in articular cartilage tissues and elucidated its effect on H 2 O 2 -stimulated chondrocytes., Methods: Human chondrocytes were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12. MSRB2 overexpression in chondrocytes was achieved by transfecting with an MSRB2 overexpression plasmid. Western blot, quantitative RT-PCR, Immunofluorescence staining, and TUNEL assay were employed in this study., Results: MSRB2 expression was found to be reduced in OA patients. Furthermore, overexpression of MSRB2 in H 2 O 2 -induced chondrocytes mitigated apoptosis and enhanced cell viability. Elevated MSRB2 expression diminished chondrocyte ROS contents, decreased cytochrome C (Cyc) in the cytoplasm, and regulated mitochondrial membrane potential to maintain mitochondrial homeostasis. Interestingly, knockdown of charged multivesicular body protein 5 (CHMP5) led to a decreased inMSRB2 expression in chondrocytes. Additionally, protein levels of CHMP5 and MSRB2 were reduced in H 2 O 2 -stimulated chondrocytes, and silencing CHMP5 reduced MSRB2 expression. Knockdown of CHMP5 increased cleaved caspase-3 expression in H 2 O 2 -induced chondrocytes and elevated TUNEL-positive chondrocytes., Conclusion: MSRB2 decreased in OA, and overexpression of MSRB2 alleviated oxidative stress and apoptosis of chondrocyte.

Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme that repairs the oxidation of methionine residues in proteins and free methionine in autism spectrum disorder (ASD). The present study aimed to assess the level of MsrA and neurotransmission enzymes in ASD individuals. Results confirmed that ASD associated with significant (P<0.05) reduction of MsrA and modulated mission enzymes. The role of MsrA as repair enzyme should be taken into account for study the activity of brain enzymes and proteins in ASD including ASMT that has a role in melatonin problems production in ASD due to higher AANAT level. The influence of MsrA also should be studied with MAT in mice to give more evidence.

Methionine sulfoxide reductase A (MsrA) has emerged as promising biocatalysts in the enantioselective kinetic resolution of racemic (rac) sulfoxides. In this study, we engineered robust MsrA variants through directed evolution, demonstrating substantial improvements of thermostability. Mechanism analysis reveals that the enhanced thermostability results from the strengthening of intracellular interactions and increase in molecular compactness. Moreover, these variants demonstrated concurrent improvements in catalytic activities, and notably, these enhancements in stability and activity collectively contributed to a significant improvement in enzyme substrate tolerance. We achieved kinetic resolution on a series of rac-sulfoxides with high enantioselectivity under initial substrate concentrations reaching up to 93.0 g/L, representing a great improvement in the aspect of the substrate concentration for biocatalytic preparation of chiral sulfoxide. Hence, the simultaneously improved thermostability, activity and substrate tolerance of MsrA represent an excellent biocatalyst for the green synthesis of optically pure sulfoxides., Competing Interests: Declaration of competing interest The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The reversible oxidation of methionine plays a crucial role in redox regulation of proteins. Methionine oxidation in proteins causes major structural modifications that can destabilize and abrogate their function. The highly conserved methionine sulfoxide reductases protect proteins from oxidative damage by reducing their oxidized methionines, thus restoring their stability and function. Deletion or mutation in conserved methionine sulfoxide reductases leads to aging and several human neurological disorders and also reduces yeast growth on nonfermentable carbon sources. Despite their importance in human health, limited information about their physiological substrates in humans and yeast is available. For the first time, we show that Mxr2 interacts in vivo with two core proteins of the cytoplasm to vacuole targeting (Cvt) autophagy pathway, Atg19, and Ape1 in Saccharomyces cerevisiae. Deletion of MXR2 induces instability and early turnover of immature Ape1 and Atg19 proteins and reduces the leucine aminopeptidase activity of Ape1 without affecting the maturation process of Ape1. Additonally, Mxr2 interacts with the immature Ape1, dependent on Met17 present within the propeptide of Ape1 as a single substitution mutation of Met17 to Leu abolishes this interaction. Importantly, Ape1 M17L mutant protein resists oxidative stress-induced degradation in WT and mxr2Δ cells. By identifying Atg19 and Ape1 as cytosolic substrates of Mxr2, our study maps the hitherto unexplored connection between Mxr2 and the Cvt autophagy pathway and sheds light on Mxr2-dependent oxidative regulation of the Cvt pathway., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Sulphur containing amino acids, methionine and cysteine are highly prone to oxidation. Reduction of oxidized methionine (Met-SO) residues to methionine (Met) by methionine sulfoxide reductases (Msrs) enhances the survival of bacterial pathogens under oxidative stress conditions. S. Typhimurium encodes two types (cytoplasmic and periplasmic) of Msrs. Periplasmic proteins, due to their location are highly vulnerable to host-generated oxidants. Therefore, the periplasmic Msr (MsrP) mediated repair (as compared to the cytoplasmic counterpart) might play a more imperative role in defending host-generated oxidants. Contrary to this, we show that in comparison to the ΔmsrP strain, the mutant strains in the cytoplasmic Msrs (ΔmsrA and ΔmsrAC strains) showed many folds more susceptibility to chloramine-T and neutrophils. Further ΔmsrA and ΔmsrAC strains accumulated higher levels of ROS and showed compromised fitness in mice spleen and liver. Our data suggest the pivotal role of cytoplasmic Msrs in oxidative stress survival of S. Typhimurium., Competing Interests: Declaration of competing interest Authors declare no competing interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: Chronic obstructive pulmonary disease (COPD) is multi-factorial disorder which results from environmental influences and genetic factors. We aimed to investigate whether methionine sulfoxide reductase A (MSRA) rs10903323 gene polymorphism is associated with COPD development and severity in Serbian adult population., Methods: The study included 155 patients with COPD and 134 healthy volunteers. Genotyping was determined performing home-made polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The difference between the inhibitory activities of normal and oxidized Alpha-1-Antitrypsin (A1AT) against elastase and trypsin was used for determination of Oxidized Alpha-1-Antitrypsin (OxyA1AT) (expressed as % and g/L). Functional activity of A1AT was presented as a specific inhibitor activity to elastase (SIA-Elastase, kU/g)., Results: Frequencies of the genotypes AA, AG and GG were 80.0%, 20.0%, 0% in COPD patients and 80.5%, 18.5% and 1.5% in the control group, and there was no significant difference in genotype or allele distributions between groups. Serum level of A1AT (g/L) and OxyA1AT was significantly higher in COPD patients than in the control group, but functional activity of A1AT (SIA-Elastase) was significantly lower in COPD patients than in the control group. In COPD group, increased level of OxyA1AT was present in G allele carriers who were smokers relative to G allele carriers who were not smokers. In the smoker group of patients with severe and very severe COPD (GOLD3+4), significant increase in OxyA1AT level was present in G allele carriers compared to AA homozygotes., Conclusion: These findings suggest that MSRA rs10903323 gene polymorphism is probably not a risk for COPD by itself but could represent a COPD modifier, since minor, G allele, is associated with an increased level of oxidized A1AT, indicating impaired ability of MSRA to repair oxidized A1AT in COPD-smokers, and in severe form of COPD., Competing Interests: Declaration of interest The authors declare that there are no conflicts of interest., (Copyright © 2021 Sociedade Portuguesa de Pneumologia. Published by Elsevier España, S.L.U. All rights reserved.)

Salmonella encounters but survives host inflammatory response. To defend host-generated oxidants, Salmonella encodes primary antioxidants and protein repair enzymes. Methionine (Met) residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO) which compromises protein functions and subsequently cellular survival. However, by reducing Met-SO to Met, methionine sulfoxide reductases (Msrs) enhance cellular survival under stress conditions. Salmonella encodes five Msrs which are specific for particular Met-SO (free/protein bound), and 'R'/'S' types. Earlier studies assessed the effect of deletions of one or two msrs on the stress physiology of S. Typhimurium. We generated a pan msr gene deletion (Δ5msr) strain in S. Typhimurium. The Δ5msr mutant strain shows an initial lag in in vitro growth. However, the Δ5msr mutant strain depicts very high sensitivity (p < 0.0001) to hypochlorous acid (HOCl), chloramine T (ChT) and superoxide-generating oxidant paraquat. Further, the Δ5msr mutant strain shows high levels of malondialdehyde (MDA), protein carbonyls, and protein aggregation. On the other side, the Δ5msr mutant strain exhibits lower levels of free amines. Further, the Δ5msr mutant strain is highly susceptible to neutrophils and shows defective fitness in the spleen and liver of mice. The results of the current study suggest that the deletions of all msrs render S. Typhimurium highly prone to oxidative stress and attenuate its virulence., (© 2023. The Author(s).)

Methionine restriction (MetR) extends lifespan in various organisms, but its mechanistic understanding remains incomplete. Whether MetR during a specific period of adulthood increases lifespan is not known. In Drosophila, MetR is reported to extend lifespan only when amino acid levels are low. Here, by using an exome-matched holidic medium, we show that decreasing Met levels to 10% extends Drosophila lifespan with or without decreasing total amino acid levels. MetR during the first four weeks of adult life only robustly extends lifespan. MetR in young flies induces the expression of many longevity-related genes, including Methionine sulfoxide reductase A (MsrA), which reduces oxidatively-damaged Met. MsrA induction is foxo-dependent and persists for two weeks after cessation of the MetR diet. Loss of MsrA attenuates lifespan extension by early-adulthood MetR. Our study highlights the age-dependency of the organismal response to specific nutrients and suggests that nutrient restriction during a particular period of life is sufficient for healthspan extension., (© 2023. The Author(s).)

Importance: Campylobacter concisus is an excellent model organism to study respiration diversity, including anaerobic respiration of physiologically relevant N-/S-oxides compounds, such as biotin sulfoxide, dimethyl sulfoxide, methionine sulfoxide (MetO), nicotinamide N-oxide, and trimethylamine N-oxide. All C. concisus strains harbor at least two, often three, and up to five genes encoding for putative periplasmic Mo/W-bisPGD-containing N-/S-oxide reductases. The respective role (substrate specificity) of each enzyme was studied using a mutagenesis approach. One of the N/SOR enzymes, annotated as "BisA", was found to be essential for anaerobic respiration of both N- and S-oxides. Additional phenotypes associated with disruption of the bisA gene included increased sensitivity toward oxidative stress and elongated cell morphology. Furthermore, a biochemical approach confirmed that BisA can repair protein-bound MetO residues. Hence, we propose that BisA plays a role as a periplasmic methionine sulfoxide reductase. This is the first report of a Mo/W-bisPGD-enzyme supporting both N- or S-oxide respiration and protein-bound MetO repair in a pathogen., Competing Interests: The authors declare no conflict of interest.

Trypanosoma cruzi is the causal agent of Chagas Disease and is a unicellular parasite that infects a wide variety of mammalian hosts. The parasite exhibits auxotrophy by L-Met; consequently, it must be acquired from the extracellular environment of the host, either mammalian or invertebrate. Methionine (Met) oxidation produces a racemic mixture (R and S forms) of methionine sulfoxide (MetSO). Reduction of L-MetSO (free or protein-bound) to L-Met is catalyzed by methionine sulfoxide reductases (MSRs). Bioinformatics analyses identified the coding sequence for a free-R-MSR (fRMSR) enzyme in the genome of T. cruzi Dm28c. Structurally, this enzyme is a modular protein with a putative N-terminal GAF domain linked to a C-terminal TIP41 motif. We performed detailed biochemical and kinetic characterization of the GAF domain of fRMSR in combination with mutant versions of specific cysteine residues, namely, Cys 12 , Cys 98 , Cys 108 , and Cys 132 . The isolated recombinant GAF domain and full-length fRMSR exhibited specific catalytic activity for the reduction of free L-Met(R)SO (non-protein bound), using tryparedoxins as reducing partners. We demonstrated that this process involves two Cys residues, Cys 98 and Cys 132 . Cys 132 is the essential catalytic residue on which a sulfenic acid intermediate is formed. Cys 98 is the resolutive Cys, which forms a disulfide bond with Cys 132 as a catalytic step. Overall, our results provide new insights into redox metabolism in T. cruzi, contributing to previous knowledge of L-Met metabolism in this parasite., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Methionine restriction (MR) extends lifespan in various model organisms, and understanding the molecular effectors of MR could expand the repertoire of tools targeting the aging process. Here, we address to what extent the biochemical pathway responsible for redox metabolism of methionine plays in regulating the effects of MR on lifespan and health span. Aerobic organisms have evolved methionine sulfoxide reductases to counter the oxidation of the thioether group contained in the essential amino acid methionine. Of these enzymes, methionine sulfoxide reductase A (MsrA) is ubiquitously expressed in mammalian tissues and has subcellular localization in both the cytosol and mitochondria. Loss of MsrA increases sensitivity to oxidative stress and has been associated with increased susceptibility to age-associated pathologies including metabolic dysfunction. We rationalized that limiting the available methionine with MR may place increased importance on methionine redox pathways, and that MsrA may be required to maintain available methionine for its critical uses in cellular homeostasis including protein synthesis, metabolism, and methylation. Using a genetic mutant mouse lacking MsrA, we tested the requirement for this enzyme in the effects of MR on longevity and markers of healthy aging late in life. When initiated in adulthood, we found that MR had minimal effects in males and females regardless of MsrA status. MR had minimal effect on lifespan with the exception of wild-type males where loss of MsrA slightly increased lifespan on MR. We also observed that MR drove an increase in body weight in wild-type mice only, but mice lacking MsrA tended to maintain more stable body weight throughout their lives. We also found that MR had greater benefit to males than females in terms of glucose metabolism and some functional health span assessments, but MsrA generally had minimal impact on these metrics. Frailty was also found to be unaffected by MR or MsrA in aged animals. We found that in general, MsrA was not required for the beneficial effects of MR on longevity and health span., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Methionine sulfoxide reductases (MSRs) are key enzymes in the cellular oxidative defense system. Reactive oxygen species oxidize methionine residues to methionine sulfoxide, and the methionine sulfoxide reductases catalyze their reduction back to methionine. We previously identified the cholesterol transport protein STARD3 as an in vivo binding partner of MSRA (methionine sulfoxide reductase A), an enzyme that reduces methionine-S-sulfoxide back to methionine. We hypothesized that STARD3 would also bind the cytotoxic cholesterol hydroperoxides and that its two methionine residues, Met307 and Met427, could be oxidized, thus detoxifying cholesterol hydroperoxide. We now show that in addition to binding MSRA, STARD3 binds all three MSRB (methionine sulfoxide reductase B), enzymes that reduce methionine-R-sulfoxide back to methionine. Using pure 5, 6, and 7 positional isomers of cholesterol hydroperoxide, we found that both Met307 and Met427 on STARD3 are oxidized by 6α-hydroperoxy-3β-hydroxycholest-4-ene (cholesterol-6α-hydroperoxide) and 7α-hydroperoxy-3β-hydroxycholest-5-ene (cholesterol-7α-hydroperoxide). MSRs reduce the methionine sulfoxide back to methionine, restoring the ability of STARD3 to bind cholesterol. Thus, the cyclic oxidation and reduction of methionine residues in STARD3 provides a catalytically efficient mechanism to detoxify cholesterol hydroperoxide during cholesterol transport, protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Methionine sulfoxide reductases are enzymes that reduce methionine oxidation in the cell. In mammals there are three B-type reductases that act on the R-diastereomer of methionine sulfoxide, and one A-type reductase (MSRA) that acts on the S-diastereomer. Unexpectedly, knocking out the four genes in the mouse protected from oxidative stresses such as ischemia-reperfusion injury and paraquat. To elucidate the mechanism by which lack of the reductases protects against oxidative stresses, we aimed to create a cell culture model with AML12 cells, a differentiated hepatocyte cell line. We employed CRISPR/Cas9 to create lines lacking the four individual reductases. All were viable and their susceptibility to oxidative stresses was the same as the parental strain. The triple knockout lacking all three methionine sulfoxide reductases B was also viable, but the quadruple knockout was lethal. We thus modeled the quadruple knockout mouse by creating an AML12 line lacking the three MSRB and heterozygous for the MSRA (Msrb3KO-Msra +/- ). We measured the effect of ischemia-reperfusion on the various AML12 cell lines, using a protocol that modeled the ischemic phase by glucose and oxygen deprivation for 36 h followed by return of glucose and oxygen for 3 h as the reperfusion phase. This stress killed ∼50% of the parental line, an effect we chose to facilitate detection of either protective or deleterious changes in the knockout lines. Unlike the protection afforded the mouse, the knockout lines produced by CRISPR/Cas9 did not differ from the parental line in their response to ischemia-reperfusion injury or paraquat poisoning. In the mouse, inter-organ communication may be essential for protection induced by lack of methionine sulfoxide reductases., Competing Interests: Declaration of competing interest None., (Published by Elsevier Inc.)

Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme that reduces the oxidized methionine (Met-O) in proteins to methionine (Met). Its pivotal role in the cellular processes has been well established by overexpressing, silencing, and knocking down MsrA or deleting the gene encoding MsrA in several species. We are specifically interested in understanding the role of secreted MsrA in bacterial pathogens. To elucidate this, we infected mouse bone marrow-derived macrophages (BMDMs) with recombinant Mycobacterium smegmatis strain (MSM), secreting a bacterial MsrA or M. smegmatis strain (MSC) carrying only the control vector. BMDMs infected with MSM induced higher levels of ROS and TNF-α than BMDMs infected with MSC. The increased ROS and TNF-α levels in MSM-infected BMDMs correlated with elevated necrotic cell death in this group. Further, RNA-seq transcriptome analysis of BMDMs infected with MSC and MSM revealed differential expression of protein and RNA coding genes, suggesting that bacterial-delivered MsrA could modulate the host cellular processes. Finally, KEGG pathway enrichment analysis identified the down-regulation of cancer-related signaling genes in MSM-infected cells, indicating that MsrA can potentially regulate the development and progression of cancer., Competing Interests: Declaration of competing interest “The authors declare no conflict of interest.”, (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Methionine is a sulfur-containing residue found in most proteins which are particularly susceptible to oxidation. Although methionine oxidation causes protein damage, it can in some cases activate protein function. Enzymatic systems reducing oxidized methionine have evolved in most bacterial species and methionine oxidation proves to be a reversible post-translational modification regulating protein activity. In this review, we inspect recent examples of methionine oxidation provoking protein loss and gain of function. We further speculate on the role of methionine oxidation as a multilayer endogenous antioxidant system and consider its potential consequences for bacterial virulence., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Among others, methionine residues are highly susceptible to host-generated oxidants. Repair of oxidized methionine (Met-SO) residues to methionine (Met) by methionine sulfoxide reductases (Msrs) play a chief role in stress survival of bacterial pathogens, including Salmonella Typhimurium. Periplasmic proteins, involved in many important cellular functions, are highly susceptible to host-generated oxidants. According to location in cell, two types of Msrs, cytoplasmic and periplasmic are present in S. Typhimurium. Owing to its localization, periplasmic Msr (MsrP) might play a crucial role in defending the host-generated oxidants. Here, we have assessed the role of MsrP in combating oxidative stress and colonization of S. Typhimurium. ΔmsrP (mutant strain) grew normally in in-vitro media. In comparison to S. Typhimurium (wild type), mutant strain showed mild hypersensitivity to HOCl and chloramine-T (ChT). Following exposure to HOCl, mutant strain showed almost similar protein carbonyl levels (a marker of protein oxidation) as compared to S. Typhimurium strain. Additionally, ΔmsrP strain showed higher susceptibility to neutrophils than the parent strain. Further, the mutant strain showed very mild defects in survival in mice spleen and liver as compared to wild-type strain. In a nutshell, our results indicate that MsrP plays only a secondary role in combating oxidative stress and colonization of S. Typhimurium., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Oxidation of methionine leads to the formation of methionine S-sulfoxide and methionine R-sulfoxide, which can be reverted by two types of methionine sulfoxide reductase (MSR): MSRA and MSRB. Though the role of MSR enzymes has been elucidated in various physiological processes, the regulation and role of MSR in seeds remains poorly understood. In this study, through molecular, biochemical, and genetic studies using seed-specific overexpression and RNAi lines of OsMSRB5 in Oryza sativa, we demonstrate the role of OsMSRB5 in maintaining seed vigor and longevity. We show that an age-induced reduction in the vigor and viability of seeds is correlated with reduced MSR activity and increased methionine sulfoxide (MetSO) formation. OsMSRB5 expression increases during seed maturation and is predominantly localized to the embryo. Further analyses on transgenic lines reveal the role of OsMSRB5 in modulating reactive oxygen species (ROS) homeostasis to preserve seed vigor and longevity. We show that ascorbate peroxidase and PROTEIN l-ISOASPARTYL METHYLTRANSFERASE undergo MetSO modification in seeds that affects their functional competence. OsMSRB5 physically interacts with these proteins and reverts this modification to facilitate their functions and preserve seed vigor and longevity. Our results thus illustrate the role of OsMSRB5 in preserving seed vigor and longevity by modulating ROS homeostasis in seeds., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Methionine (Met) can be oxidized to methionine sulfoxide (MetO), which exist as R- and S-diastereomers. Present in all three domains of life, methionine sulfoxide reductases (MSR) are the enzymes that reduce MetO back to Met. Most characterized among them are MSRA and MSRB, which are strictly stereospecific for the S- and R-diastereomers of MetO, respectively. While the majority of MSRs use a catalytic Cys to reduce their substrates, some employ selenocysteine. This is the case of mammalian MSRB1, which was initially discovered as selenoprotein SELR or SELX and later was found to exhibit an MSRB activity. Genomic analyses demonstrated its occurrence in most animal lineages, and biochemical and structural analyses uncovered its catalytic mechanism. The use of transgenic mice and mammalian cell culture revealed its physiological importance in the protection against oxidative stress, maintenance of neuronal cells, cognition, cancer cell proliferation, and the immune response. Coincident with the discovery of Met oxidizing MICAL enzymes, recent findings of MSRB1 regulating the innate immunity response through reversible stereospecific Met-R-oxidation of cytoskeletal actin opened up new avenues for biological importance of MSRB1 and its role in disease. In this review, we discuss the current state of research on MSRB1, compare it with other animal Msrs, and offer a perspective on further understanding of biological functions of this selenoprotein., (Copyright © 2022. Published by Elsevier Inc.)

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that often encounters hypoxic/anoxic environments within the host, which increases its tolerance to many conventional antibiotics. Toward identifying novel treatments, we explored the therapeutic potential of chlorate, a pro-drug that kills hypoxic/anoxic, antibiotic-tolerant P. aeruginosa populations. While chlorate itself is relatively nontoxic, it is enzymatically reduced to the toxic oxidizing agent, chlorite, by hypoxically induced nitrate reductase. To better assess chlorate's therapeutic potential, we investigated mechanisms of chlorate toxicity and resistance in P. aeruginosa. We used transposon mutagenesis to identify genes that alter P. aeruginosa fitness during chlorate treatment, finding that methionine sulfoxide reductases (Msr), which repair oxidized methionine residues, support survival during chlorate stress. Chlorate treatment leads to proteome-wide methionine oxidation, which is exacerbated in a ∆msrA∆msrB strain. In response to chlorate, P. aeruginosa upregulates proteins involved in a wide range of functions, including metabolism, DNA replication/repair, protein repair, transcription, and translation, and these newly synthesized proteins are particularly vulnerable to methionine oxidation. The addition of exogenous methionine partially rescues P. aeruginosa survival during chlorate treatment, suggesting that widespread methionine oxidation contributes to death. Finally, we found that mutations that decrease nitrate reductase activity are a common mechanism of chlorate resistance., (© 2022 John Wiley & Sons Ltd.)

Selenium incorporates into selenocysteine (Sec) which is a key component of selenoproteins implicated in antioxidant defense and redox homeostasis. Methionine sulfoxide reductases (Msr) play crucial roles in cellular defense against environmental stress. Whereas mammals have the MsrB selenoprotein form, unicellular organisms have MsrA. The Sec residue at the conserved catalytic sites of selenoprotein MsrA confers a metabolic advantage over the non-selenoprotein type MsrA. In the present study, the novel selenoprotein HpMsrA from Haematococcus pluvialis was cloned by the rapid amplification of cDNA ends and transformed into the model green alga Chlamydomonas reinhardtii. Alignment of homologs revealed the presence of the conserved catalytic domain GUFW and showed that the HpMsrA protein comprises Sec (U) at the N-terminus but no recycled Cys at the C-terminus. We studied the response of HpMsrA expression to selenite, high light intensity, hydrogen peroxide, cadmium nitrate, and glyphosate exposure via real-time quantitative PCR and enzyme activity analysis. The results demonstrated that HpMsrA protects cellular proteins against oxidative and environmental stressors. Compared with wild type C. reinhardtii, the transformant exhibited a superior antioxidant ability. The discoveries made herein shed light on the antioxidant physiology and environmental stress resistance mechanisms of the selenoproteins in microalgae. This information may aid in conducting environmental risk assessments of aquatic ecosystems involving microalgae known to respond rapidly and quantitatively to abiotic stress factors promoting excessive reactive oxygen species generation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Pod borer, Helicoverpa armigera, a polyphagus herbivore causes extensive economic losses to crops, including pigeonpea. Exploitation of pod borer resistance in wild relatives is pertinent due to the absence of resistance sources in cultivated pigeonpea and crossing-incompatibility with the resistant wild relatives. We present leads obtained in deeper understanding of pod borer resistance mechanism in Cajanus platycarpus, a pigeonpea wild relative. Surge in cellular ROS during herbivory leads to redox-PTMs (post translational modifications) of methionine-rich proteins including antioxidant enzymes, causing oxidative damage. Plants then officiate methionine sulfoxide reductases (MSRs), that maintain the redox status of methionine and hence homeostasis. We demonstrate functionality of MSRs (MSRA and MSRB) in the resistance response of the wild relative to pod borer. Among 5 MSRA and 3 MSRB genes, CpMSRA2 and CpMSRB1 were herbivore-responsive based on expression during herbivory. Clues about the stress-responsiveness were obtained upon analyses of cis-elements and co-expressing genes. Apparently, the wild relative followed a non-canonical mode of redox management, as divulged by antioxidant enzymes and the scavenging capacity. Differential lipid peroxidation as an early response provided evidences for an effective redox management in the wild relative. This is the first report signifying redox homeostasis in the resistance response towards herbivory., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Fusobacterium nucleatum, an anaerobic Gram-negative bacterium frequently found in the human oral cavity and some extra-oral sites, is implicated in several important diseases: periodontitis, adverse pregnancy outcomes, and colorectal cancer. To date, how this obligate anaerobe copes with oxidative stress and host immunity within multiple human tissues remains unknown. Here, we uncovered a critical role in this process of a multigene locus encoding a single, fused methionine sulfoxide reductase (MsrAB), a two-component signal transduction system (ModRS), and thioredoxin (Trx)- and cytochrome c (CcdA)-like proteins, which are induced when fusobacterial cells are exposed to hydrogen peroxide. Comparative transcriptome analysis revealed that the response regulator ModR regulates a large regulon that includes trx , ccdA , and many metabolic genes. Significantly, specific mutants of the msrAB locus, including msrAB , are sensitive to reactive oxygen species and defective in adherence/invasion of colorectal epithelial cells. Strikingly, the msrAB mutant is also defective in survival in macrophages, and it is severely attenuated in virulence in a mouse model of preterm birth, consistent with its failure to spread to the amniotic fluid and colonize the placenta. Clearly, the MsrAB system regulated by the two-component system ModRS represents a major oxidative stress defense pathway that protects fusobacteria against oxidative damage in immune cells and confers virulence by enabling attachment and invasion of multiple target tissues. IMPORTANCE F. nucleatum colonizes various human tissues, including oral cavity, placenta, and colon. How this obligate anaerobe withstands oxidative stress in host immune cells has not been described. We report here that F. nucleatum possesses a five-gene locus encoding a fused methionine sulfoxide reductase (MsrAB), a two-component signal transduction system (ModRS), and thioredoxin- and cytochrome c -like proteins. Regulated by ModRS, MsrAB is essential for resistance to reactive oxygen species, adherence/invasion of colorectal epithelial cells, and survival in macrophage. Unable to colonize placenta and spread to amniotic fluid, the msrAB mutant failed to induce preterm birth in a murine model.

Reactive oxygen species (ROS) can cause destructive damage to biological macromolecules and protein dysfunction in bacteria. Methionine sulfoxide reductase (Msr) with redox-active Cys and/or seleno-cysteine (Sec) residues can restore physiological functions of the proteome, which is essential for oxidative stress tolerance of the extremophile Deinococcus radiodurans. However, the underlying mechanism regulating MsrA enzyme activity in D. radiodurans under oxidative stress has remained elusive. Here, we identified the function of MsrA in response to oxidative stress. msrA expression in D. radiodurans was significantly upregulated under oxidative stress. The msrA mutant showed a deficiency in antioxidative capacity and an increased level of dabsyl-Met-S-SO, indicating increased sensitivity to oxidative stress. Moreover, msrA mRNA was posttranscriptionally regulated by a small RNA, DsrO. Analysis of the molecular interaction between DsrO and msrA mRNA demonstrated that DsrO increased the half-life of msrA mRNA and then upregulated MsrA enzyme activity under oxidative stress compared to the wild type. msrA expression was also transcriptionally regulated by the DNA-repairing regulator DrRRA, providing a connection for further analysis of protein restoration during DNA repair. Overall, our results provide direct evidence that DsrO and DrRRA regulate msrA expression at two levels to stabilize msrA mRNA and increase MsrA protein levels, revealing the protective roles of DsrO signaling in D. radiodurans against oxidative stress. IMPORTANCE The repair of oxidized proteins is an indispensable function allowing the extremophile D. radiodurans to grow in adverse environments. Msr proteins and various oxidoreductases can reduce oxidized Cys and Met amino acid residues of damaged proteins to recover protein function. Consequently, it is important to investigate the molecular mechanism maintaining the high reducing activity of MsrA protein in D. radiodurans during stresses. Here, we showed the protective roles of an sRNA, DsrO, in D. radiodurans against oxidative stress. DsrO interacts with msrA mRNA to improve msrA mRNA stability, and this increases the amount of MsrA protein. In addition, we also showed that DrRRA transcriptionally regulated msrA gene expression. Due to the importance of DrRRA in regulating DNA repair, this study provides a clue for further analysis of MsrA activity during DNA repair. This study indicates that protecting proteins from oxidation is an effective strategy for extremophiles to adapt to stress conditions.

Reactive oxygen species (ROS) are inevitably generated in aerobic organisms as by-products of common metabolism and as the result of defense and development. ROS readily oxidizes methionine (Met) residues of proteins to form Met-R-sulfoxide or Met-S-sulfoxide (MetSO), resulting in protein inactivation or malfunction. Although it is known that MetSO can be reverted to Met by methionine sulfoxide reductase (Msr), the mechanism how Msr interacts with its target proteins is poorly understood. In this study, two target proteins of tomato MsrB2 (SlMsrB2), catalase 2 (CAT2) and the Rubisco small subunit RBCS3B, were identified. Silencing of SlMsrB2 by RNA interference (RNAi) in tomato led to decreased drought tolerance, accompanied by increased ROS accumulation and chlorophyll degradation. By contrast, overexpression of SlMsrB2 in tomato significantly reduced ROS accumulation and enhanced drought tolerance. Protein interaction analysis showed that SlMsrB2 interacts with CAT2 and RBCS3B in vitro and in planta. Silencing of CAT2 by RNAi and RBCS3B by virus-induced gene silencing (VIGS) resulted in development of pale green leaves and enhanced ROS accumulation in tomato plants. These results demonstrate that SlMsrB2 functions in drought tolerance and promotes chlorophyll accumulation by modulating ROS accumulation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Methionine restriction (MR) extends lifespan and improves several markers of health in rodents. However, the proximate mechanisms of MR on these physiological benefits have not been fully elucidated. The essential amino acid methionine plays numerous biological roles and limiting its availability in the diet directly modulates methionine metabolism. There is growing evidence that redox regulation of methionine has regulatory control on some aspects of cellular function but interactions with MR remain largely unexplored. We tested the functional role of the ubiquitously expressed methionine repair enzyme methionine sulfoxide reductase A (MsrA) on the metabolic benefits of MR in mice. MsrA catalytically reduces both free and protein-bound oxidized methionine, thus playing a key role in its redox state. We tested the extent to which MsrA is required for metabolic effects of MR in adult mice using mice lacking MsrA. As expected, MR in control mice reduced body weight, altered body composition, and improved glucose metabolism. Interestingly, lack of MsrA did not impair the metabolic effects of MR on these outcomes. Moreover, females had blunted MR responses regardless of MsrA status compared to males. Overall, our data suggests that MsrA is not required for the metabolic benefits of MR in adult mice., (© 2022. The Author(s).)

Ethylene plays an important role in regulating fruit ripening by triggering dynamic changes in expression of ripening-associated genes, but the functions of many of these genes are still unknown. Here, a methionine sulfoxide reductase gene (AdMsrB1) was identified by transcriptomics-based analysis as the gene most responsive to ethylene treatment in ripening kiwifruit. The AdMsrB1 protein exhibits a stereospecific activity toward the oxidative stress-induced R enantiomer of methionine sulfoxide (MetSO), reducing it to methionine (Met). Stable overexpression of AdMsrB1 in kiwifruit significantly increased the content of free Met and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, and increased ethylene production. Dual-luciferase assays indicated that the AdMsrB1 promoter was not directly upregulated by ethylene treatment but was modulated by two ethylene-inducible NAM/ATAF/CUC transcription factors (AdNAC2 and AdNAC72) that bind directly to the AdMsrB1 promoter. Overexpression of AdNAC72 in kiwifruit not only enhanced AdMsrB1 expression, but also increased free Met and ACC content and ethylene production rates. This finding establishes an unexpected regulatory loop that enhances ethylene production and the concentration of its biosynthetic intermediates., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Two pepper methionine sulfoxide reductase B2 (CaMsrB2) gene expressing transgenic rice lines (L-8 and L-23) were interrogated with respect to their physiological and photochemical attributes along with control (WT, Ilmi) as a standard against varying levels of salt concentration which are 75 mM, 150 mM and 225 mM. Against various levels of salt (NaCl) concentration, recurring detrimental effects of extreme salt stress was observed and more pronounced in the wild type plants as compared to our transgenic lines. As the exacerbated effects of salinity is responsible for pushing the plants to their ecological tolerance, our transgenic lines performed well uplifted in different realms of physiology and photochemistry such as relative water content (RWC = 60-75%), stomatal conductance (g s = 70-190 mmolm -2 s -1 ), performance index (PI ABS  = 1.0-4.5), maximal photochemical yield of PSII (F V /F M  = 0.48-0.72) and chlorophyll content index (CCI = 5-7.2 au) in comparison to the control. Relative gene expression, ion analysis and antioxidants activity were analyzed in all treatments to ensure the hypothesis obtained from data of physiology and photochemistry. Photosynthetic apparatus is known to lose energy in various forms such as NPQ, DI O /CS, damages of reaction center (F V /F O ) which are the markers of poor health were clearly decreased in the L-23 line as compared to L-8 and WT. Present study revealed the protruding tolerance of L-23 and L-8 transgenic lines with L-23 line in the lead in comparison to control and L-8 transgenic lines., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

[Figure: see text].

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δ msr ) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δ msr strain does not exhibit increased sensitivity to H 2 O 2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δ msr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δ msr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δ msr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δ msr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.

Methionine sulfoxide reductase A (MsrA) is a widely expressed antioxidant enzyme that counteracts oxidative protein damage and contributes to protein regulation by reversing oxidation of protein methionine residues. In retinal pigment epithelial (RPE) cells in culture, MsrA overexpression increases phagocytic capacity by supporting mitochondrial ATP production. Here, we show elevated retinal protein carbonylation indicative of oxidation, decreased RPE mitochondrial membrane potential, and attenuated RPE phagocytosis in msra -/ - mice. Moreover, electroretinogram recordings reveal decreased light responses specifically of cone photoreceptors despite normal expression and localization of cone opsins. Impairment in msra -/ - cone-driven responses is similar from 6 weeks to 13 months of age. These functional changes match dramatic decreases in lectin-labeled cone sheaths and reduction in cone arrestin in msra -/ - mice. Strikingly, cone defects in light response and in lectin-labeled cone sheath are completely prevented by dark rearing. Together, our data show that msra -/ - mice provide a novel small animal model of preventable cone-specific photoreceptor dysfunction that may have future utility in analysis of cone dystrophy disease mechanisms and testing therapeutic approaches aiming to alleviate cone defects., (Copyright © 2021. Published by Elsevier B.V.)

Methionine, either as a free amino acid or included in proteins, can be oxidized into methionine sulfoxide (MetO), which exists as R and S diastereomers. Almost all characterized organisms possess thiol-oxidoreductases named methionine sulfoxide reductase (Msr) enzymes to reduce MetO back to Met. MsrA and MsrB reduce the S and R diastereomers of MetO, respectively, with strict stereospecificity and are found in almost all organisms. Another type of thiol-oxidoreductase, the free-methionine-R-sulfoxide reductase (fRMsr), identified so far in prokaryotes and a few unicellular eukaryotes, reduces the R MetO diastereomer of the free amino acid. Moreover, some bacteria possess molybdenum-containing enzymes that reduce MetO, either in the free or protein-bound forms. All these Msrs play important roles in the protection of organisms against oxidative stress. Fungi are heterotrophic eukaryotes that colonize all niches on Earth and play fundamental functions, in organic matter recycling, as symbionts, or as pathogens of numerous organisms. However, our knowledge on fungal Msrs is still limited. Here, we performed a survey of msr genes in almost 700 genomes across the fungal kingdom. We show that most fungi possess one gene coding for each type of methionine sulfoxide reductase: MsrA, MsrB, and fRMsr. However, several fungi living in anaerobic environments or as obligate intracellular parasites were devoid of msr genes. Sequence inspection and phylogenetic analyses allowed us to identify non-canonical sequences with potentially novel enzymatic properties. Finaly, we identified several ocurences of msr horizontal gene transfer from bacteria to fungi., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The oxidation of methionine (Met) by reactive oxygen species (ROS) causes detrimental effects on the protein functions. Methionine sulfoxide reductase (Msr) is the secondary antioxidant enzyme involved in protein repair, and is divided into two distinct classes, MsrA and MsrB, although the mechanisms underlying the transcriptional regulation of Msrs remain largely unknown. In this study, the full-length cDNAs encoding MsrA and three alternatively spliced isoforms of MsrB were isolated from the red flour beetle, Tribolium castaneum. Exposure of female adults to oxidative, heat and cold stresses induced expressions of both MsrA and MsrB. RNAi-mediated knockdown of MsrA and MsrB resulted in increased sensitivity of T. castaneum to paraquat-induced oxidative stress. Treatment with 20-hydroxyecdysone (20E) increased expression levels of both MsrA and MsrB. Knockdown of transcription factor forkhead box O (FOXO) decreased both MsrA and MsrB mRNA levels and abolished the induction of MsrA and MsrB by paraquat. Luciferase reporter assays revealed that FOXO directly activates the promoters of both MsrA and MsrB. Moreover, paraquat treatment induced expression of two ecdysone biosynthesis genes, Shade and Phantom, 20E upregulated exoression of FOXO, promoted FOXO nuclear translocation，and knockdown of FOXO abolished induction of MsrA and MsrB expression by 20E, suggesting that regulation of MsrA and MsrB by 20E was mediated by FOXO. Overall, these results provide important insights into the transcriptional regulation of insect Msrs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Salmonella Typhimurium survives and replicates inside the oxidative environment of phagocytic cells. Proteins, because of their composition and location, are the foremost targets of host inflammatory response. Among others, Met-residues are highly prone to oxidation. Methionine sulfoxide reductase (Msr), with the help of thioredoxin-thioredoxin reductase, can repair oxidized methionine (Met-SO) residues to Met. There are four methionine sulfoxide reductases localized in the cytosol of S. Typhimurium, MsrA, MsrB, MsrC and BisC. MsrA repairs both protein-bound and free 'S' Met-SO, MsrB repairs protein-bound 'R' Met-SO, MsrC repairs free 'R' Met-SO and BisC repairs free 'S' Met-SO. To assess the role(s) of various Msrs in Salmonella, few studies have been conducted by utilizing ΔmsrA, ΔmsrB, ΔmsrC, ΔmsrAΔmsrB, ΔmsrBΔmsrC and ΔbisC mutant strains of S. Typhimurium. Out of the above-mentioned mutants, ΔmsrA and ΔmsrC were found to play important role in the stress survival of this bacterium; however, the combined roles of these two genes have not been determined. In the current study, we have generated msrAmsrC double gene deletion strain (ΔmsrAΔmsrC) of S. Typhimurium and evaluated the effect of gene deletions on the survival of Salmonella against hypochlorite stress and intramacrophage replication. In in vitro growth curve analysis, ΔmsrAΔmsrC mutant strain showed a longer lag phase during the initial stages of the growth; however, it attained similar growth as the wild type strain of S. Typhimurium after 5 h. The ΔmsrAΔmsrC mutant strain has been highly (~ 3000 folds more) sensitive (p < 0.001) to hypochlorite stress. Further, ΔmsrA and ΔmsrAΔmsrC mutant strains showed more than 8 and 26 folds more susceptibility to poultry macrophages, respectively. Our data suggest that the deletion of both msrA and msrC genes severely affect the oxidative stress survival and intramacrophage proliferation of S. Typhimurium.

Background: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans., Methods: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization., Results: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm., Conclusions and General Significance: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Oxidative and endoplasmic reticulum (ER) stress are involved in mediating high-fat diet (HFD)-induced insulin resistance. As the ER-localized methionine sulfoxide reductase B3 (MsrB3) protects cells against oxidative and ER stress, we hypothesized that MsrB3 might be associated with HFD-induced insulin resistance. To test this hypothesis, we examined the effect of MsrB3 deficiency on HFD-induced insulin resistance using MsrB3 knockout (KO) mice. Mice were fed a control diet or HFD for 12 weeks and insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp. HFD consumption increased the body weight of both wild-type and MsrB3 KO mice, and no significant difference was observed between the genotypes. The HFD increased oxidative stress and induced insulin resistance in the skeletal muscle of wild-type mice, but did not affect either in MsrB3 KO mice. The unfolded protein response (UPR) was increased in MsrB3 KO mice upon consumption of HFD, but not in wild-type mice. Mitochondrial oxidative phosphorylation proteins and the levels of superoxide dismutase 2 and glutathione peroxidase 1 were increased in MsrB3 KO mice upon HFD consumption. The respiratory control ratio was reduced in wild-type mice consuming HFD but not in MsrB3 KO mice. The levels of calcium/calmodulin-dependent protein kinase kinase β, phosphorylated AMP-activated protein kinase, and peroxisome proliferator-activated receptor gamma coactivator 1α were increased in MsrB3 KO mice following HFD consumption. These results suggest that MsrB3 deficiency inhibits HFD-induced insulin resistance, and the increased mitochondrial biogenesis and antioxidant induction might be the mechanisms underlying this phenomenon., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The increase in reactive oxygen species (ROS) production during cryopreservation of semen, leads to oxidation of biomolecules affecting the functionality of spermatozoa. Methionine residues in proteins are highly prone to oxidation and get converted into methionine sulfoxide (MetO). Methionine sulfoxide reductase A (MsrA) can improve the functionality of spermatozoa by reducing the MetO to methionine restoring the lost functionality of the affected proteins., Objective: The expression of catalytically active recombinant MsrA (rMsrA)., Methods: The msrA gene was PCR amplified, cloned and sequenced. Further, the recombinant clone was used for protein expression and purification. The protein was getting precipitated during dialysis in Tris-buffer. Hence, the purified rMsrA was dialyzed at 4°C against the Tris-buffer pH 7.5 containing MgCl2, KCl, NaCl, urea and triton X-100. During dialysis, changes of buffer were done at every 12 h interval with stepwise reduction in the concentrations of NaCl, urea and triton X-100. The final dialysis was done with buffer containing 10 mM MgCl2, 30 mM KCl, and 150 mM NaCl, 25 mM Tris-HCl pH 7.5. The activity of the rMsrA was checked spectrophotometrically., Results: The protein BLAST of buffalo MsrA with bovine sequence showed 14 amino acid mismatches. The rMsrA has been purified under denaturing conditions as it was forming inclusion bodies consistently during protein expression. After renaturation, the purified 33 kDa rMsrA was catalytically active by biochemical assay., Conclusion: The rMsrA expressed in prokaryotic system is catalytically active and can be used for supplementation to semen extender to repair the oxidatively damaged seminal plasma proteins that occur during cryopreservation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Ear morphology is an important determinant of sheep breeds. It includes different variable traits such as ear size and erectness, suggesting a polygenic architecture. Here, we performed a comprehensive genome-wide analysis to identify regions under selection for ear morphology in 515 sheep from 17 breeds fixed for diverse ear phenotypes using 34k SNP genotyping data. GWASs for two ear type traits, size and erectness, revealed a single genome-wide significant association on ovine chromosome 3. The derived marker alleles were enriched in sheep with large and/or floppy ears. The GWAS signal harboured the MSRB3 gene encoding methionine sulphoxide reductase B3, which has already been found to be associated with different ear types in other species. We attempted whole-genome resequencing to identify causal variant(s) within a 1 Mb interval around MSRB3. This experiment excluded major copy number variants in the interval, but failed to identify a compelling candidate causal variant. Fine-mapping suggested that the causal variant for large floppy ears most likely resides in a 175 kb interval downstream of the MSRB3 coding region., (© 2020 Stichting International Foundation for Animal Genetics.)

The oxidation of free methionine (Met) and Met residues inside proteins leads to the formation of methionine sulfoxide (Met-O). The reduction of Met-O to Met is catalysed by a ubiquitous enzyme family: the methionine sulfoxide reductases (Msr). The importance of Msr systems in bacterial physiology and virulence has been reported in many species. Salmonella Typhimurium, a facultative intracellular pathogen, contains four cytoplasmic Msr. Recently, a periplasmic Msr enzyme (MsrP) has been identified in Escherichia coli. In the present study, the STM14&#95;4072 gene from Salmonella was shown to encode the MsrP protein (StMsrP). We describe the experimental procedure and precautions for the production of this molybdo-enzyme. StMsrP was also demonstrated to reduce free Met-O and to catalyse the complete repair of an oxidized protein. More importantly, this study provides for the first time access to the exhaustive list of the Msr systems of a pathogen, including four cytoplasmic enzymes (MsrA, MsrB, MsrC, BisC) and one periplasmic enzyme (MsrP)., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Classical homocystinuria is the most common cause of isolated homocystinuria. The variants of the CBS gene remain unidentified in Indian children with this disorder. Based on the hallmark clinical features, family history, and/or biochemical clues for classical homocystinuria, 16 children below the age of 18 years were evaluated by Sanger sequencing of the coding exons of CBS gene with flanking intronic regions. The common C677T variant of the MTHFR gene was also screened by restriction fragment length polymorphism. Fifteen children were clinically suspected of having classical homocystinuria and one asymptomatic child with positive family history. Only seven children had biochemical features of classical homocystinuria. Sanger sequencing of the CBS gene confirmed 15 different pathogenic or likely pathogenic variants in 14 cases. Of these, seven variants were novel (three frameshift deletions, two nonsense, one missense, one splice site variant) and were predicted to be deleterious by Mutation Taster software. Seven cases were homozygous, another six were compound heterozygous, and one case was single heterozygous in the study. None of the three most frequent mutations reported worldwide viz., I278T, G307S, and IVS 11-2A>C were found in our cohort. No variants were detected in the exons 2, 8, 12, and 14 as compared to reported literature. Eleven out of 15 variants were associated with the conserved catalytic domain of the CBS polypeptide. The MTHFR polymorphism C677T was observed in heterozygous state in six cases. Our study reports the detailed genotype and seven novel variants in the CBS gene, causing classical homocystinuria in Indian children. The genetic analysis will help to offer accurate genetic counseling, prenatal diagnosis, and development of mutation-based novel therapeutic strategies.

Background: Methionine is an amino acid susceptible to be oxidized to give a racemic mixture of R and S forms of methionine sulfoxide (MetSO). This posttranslational modification has been reported to occur in vivo under either normal or stress conditions. The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductases (MSRs), thiol-dependent enzymes present in almost all organisms. These enzymes can reduce specifically one or another of the isomers of MetSO (free and protein-bound). This redox modification could change the structure and function of many proteins, either concerned in redox or other metabolic pathways. The study of antioxidant systems in Trypanosoma cruzi has been mainly focused on the involvement of trypanothione, a specific redox component for these organisms. Though, little information is available concerning mechanisms for repairing oxidized methionine residues in proteins, which would be relevant for the survival of these pathogens in the different stages of their life cycle., Methods: We report an in vitro functional and in vivo cellular characterization of methionine sulfoxide reductase B (MSRB, specific for protein-bound MetSO R-enantiomer) from T. cruzi strain Dm28c., Results: MSRB exhibited both cytosolic and mitochondrial localization in epimastigote cells. From assays involving parasites overexpressing MSRB, we observed the contribution of this protein to increase the general resistance against oxidative damage, the infectivity of trypomastigote cells, and intracellular replication of the amastigote stage. Also, we report that epimastigotes overexpressing MSRB exhibit inhibition of the metacyclogenesis process; this suggesting the involvement of the proteins as negative modulators in this cellular differentiation., Conclusions and General Significance: This report contributes to novel insights concerning redox metabolism in T. cruzi. Results herein presented support the importance of enzymatic steps involved in the metabolism of L-Met and in repairing oxidized macromolecules in this parasite., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Clinical application of doxorubicin (Dox) is limited due to its serious side effects including nephrotoxicity, and kidney podocytes play important roles in renal diseases. MicroRNAs (miRNAs) are critical regulators associated with human diseases. The purpose of this study was to explore a novel target in adjusting Dox-induced renal podocyte injury. Through a double luciferase reporter gene experiment, it was found that miR-874-3p directly targeted methionine sulfoxide reductase B3 (MsrB3). During the tests of miR-874-3p inhibitor and MsrB3 siRNA in human podocytes or miR-874-3p antagomir in mice, we found that the expression levels of downstream oxidative stress and apoptosis-related proteins were regulated by miR-874-3p/MsrB3 signal to alleviate or aggravate renal podocyte injury. The data in the present work showed that miR-874-3p aggravated Dox-caused renal podocyte injury by promoting apoptosis and oxidative damage via inhibiting MsrB3. Therefore, miR-874-3p/MsrB3 should be considered as a new therapeutic target in controlling renal podocyte injury induced by Dox., Competing Interests: In this experiment, the authors asserted that they had no conflict of interest., (Copyright © 2020 Yan Dai et al.)

Peptide methionine sulfoxide reductases (Msrs) are enzymes that repair ROS-damage to sulfur-containing amino acids such as methionine, ensuring functional integrity of cellular proteins. Here we have shown that unlike the majority of pro- and eukaryotic Msrs, the peptide methionine sulfoxide reductase (MsrAB) from the human pathobiont Haemophilus influenzae (Hi) is required for the repair of hypochlorite damage to cell envelope proteins, but more importantly, we were able to demonstrate that MsrAB plays a role in modulating the host immune response to Hi infection. Loss of MsrAB resulted in >1000-fold increase in sensitivity of Hi to HOCl-mediated killing, and also reduced biofilm formation and in-biofilm survival. Expression of msrAB was also induced by hydrogen peroxide and paraquat, but a Hi2019 Δ msrAB strain was not susceptible to killing by these ROS in vitro. Hi2019 Δ msrAB fitness in infection models was low, with a 3-fold reduction in intracellular survival in bronchial epithelial cells, increased susceptibility to neutrophil killing, and a 10-fold reduction in survival in a mouse model of lung infection. Interestingly, infection with Hi2019 Δ msrAB led to specific changes in the antibacterial response of human host cells, with genes encoding antimicrobial peptides (BPI, CAMP) upregulated between 4 and 9 fold compared to infection with Hi2019 WT , and reduction in expression of two proteins with antiapoptotic functions (BIRC3, XIAP). Modulation of host immune responses is a novel role for an enzyme of this type and provides first insights into mechanisms by which MsrAB supports Hi survival in vivo.

Methionine sulfoxide reductase A (MsrA) is a ubiquitous antioxidant repair enzyme which specifically reduces the oxidized methionine (Met-O) in proteins to methionine (Met). Previous studies have shown that lack of or overexpression of MsrA in cells affects the function of proteins and can lead to altered cellular processes. Interestingly, some pathogenic bacteria secrete and/or carry MsrA on their surface, suggesting some key roles for this enzyme in the modulation of host cellular processes. Therefore, we investigated how exogenously added MsrA affects the ability of the host cells in combating infection by using an in vitroMycoplasma genitalium cytotoxicity model. HeLa cells pretreated with MsrA and infected with M. genitalium showed significantly lower necrosis (cytotoxicity) than untreated cells infected with M. genitalium. Intriguingly, necrotic cell death pathway specific real time RT-PCR revealed that M. genitalium infection upregulates the expression of the TNF gene in HeLa cells and that MsrA pretreatment of the cells downregulates its expression significantly. Consistent with this, enzyme linked immunosorbent assay (ELISA) results showed that HeLa cells pretreated with MsrA secreted reduced levels of TNF-α following M. genitalium infection. Also, our study demonstrates that MsrA treatment of cells affects the phosphorylation status of transcriptional regulators such as NF-кB, JNK and p53 that regulate different cytokines. Further, fluorescent microscopy showed the cellular uptake of exogenously added MsrA fused with red fluorescent protein (MsrA-RFP). Altogether, our results suggest that secreted MsrA may help pathogens to modulate host cellular processes., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

We report on the development of high-throughput fluorogenic assay that can streamline directed evolution of enantioselective sulfoxide reductases. As a model, methionine sulfoxide reductase A (MsrA) has been evolved to expand its limited substrate scope. The resulting mutant MsrA can resolve a range of new challenging racemic sulfoxides with high efficiency including the pharmaceutically relevant albendazole sulfoxide. The simplicity and the level of throughput make this method also suitable for the screening of metagenomic libraries in future for the discovery of new enzymes with similar reactivities.

High-density lipoprotein (HDL), a well-known atheroprotective factor, can be converted to proatherogenic particles in chronic inflammation. HDL-targeted therapeutic strategy for atherosclerotic cardiovascular disease (CVD) is currently under development. This study aims to assess the role of methionine sulfoxide reductase A (MsrA) in abnormal HDL and its related disorders in scavenger receptor class B type I deficient (SR-BI -/- ) mice. First, we demonstrated that MsrA overexpression attenuated ROS level and inflammation in HepG2 cells. For the in vivo study, SR-BI -/- mice were intravenously injected with lentivirus to achieve hepatic MsrA overexpression. High-level hepatic MsrA significantly reduced the plasma free cholesterol contents, improved HDL functional proteins apolipoprotein A-I (apoAI), apoE, paraoxonase1 (PON1), and lecithin:cholesterol acyltransferase (LCAT), while decreased the pro-inflammatory property of dysfunctional HDL, contributing to reduced atherosclerosis and hepatic steatosis in Western diet-fed mice. Furthermore, the study revealed that hepatic MsrA altered the expression of several genes controlling HDL biogenesis, cholesterol esterification, cholesterol uptake mediated by low-density lipoprotein receptor (LDLR) and biliary excretion, as well as suppressed nuclear factor κB (NF-κB) signaling pathway, which largely relied on liver X receptor alpha (LXRα)-upregulation. These results provide original evidence that MsrA may be a promising target for the therapy of dysfunctional HDL-related CVD., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology.)

Abscission is the terminal step of cytokinesis leading to the physical separation of the daughter cells. In response to the abnormal presence of lagging chromatin between dividing cells, an evolutionarily conserved abscission/NoCut checkpoint delays abscission and prevents formation of binucleated cells by stabilizing the cytokinetic intercellular bridge (ICB). How this bridge is stably maintained for hours while the checkpoint is activated is poorly understood and has been proposed to rely on F-actin in the bridge region. Here, we show that actin polymerization is indeed essential for stabilizing the ICB when lagging chromatin is present, but not in normal dividing cells. Mechanistically, we found that a cytosolic pool of human methionine sulfoxide reductase B2 (MsrB2) is strongly recruited at the midbody in response to the presence of lagging chromatin and functions within the ICB to promote actin polymerization there. Consistently, in MsrB2-depleted cells, F-actin levels are decreased in ICBs, and dividing cells with lagging chromatin become binucleated as a consequence of unstable bridges. We further demonstrate that MsrB2 selectively reduces oxidized actin monomers and thereby counteracts MICAL1, an enzyme known to depolymerize actin filaments by direct oxidation. Finally, MsrB2 colocalizes and genetically interacts with the checkpoint components Aurora B and ANCHR, and the abscission delay upon checkpoint activation by nuclear pore defects also depends on MsrB2. Altogether, this work reveals that actin reduction by MsrB2 is a key component of the abscission checkpoint that favors F-actin polymerization and limits tetraploidy, a starting point for tumorigenesis., Competing Interests: The authors declare no competing interest.