Recharging oxidative protein repair: Catalysis by methionine sulfoxide reductases towards their amino acid, protein, and model substrates

The sulfur-containing amino acid methionine (Met) in its free and amino acid residue forms can be readily oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Methionine sulfoxide reductases A (MSRA) and B (MSRB) reduce MetO back to Met in a stereospecific manner, acting on the S and R forms, respectively. A third MSR type, fRMSR, reduces the R form of free MetO. MSRA and MSRB are spread across the three domains of life, whereas fRMSR is restricted to bacteria and unicellular eukaryotes. These enzymes protect against abiotic and biotic stresses and regulate lifespan. MSRs are thiol oxidoreductases containing catalytic redox-active cysteine or selenocysteine residues, which become oxidized by the substrate, requiring regeneration for the next catalytic cycle. These enzymes can be classified according to the number of redox-active cysteines (selenocysteines) and the strategies to regenerate their active forms by thioredoxin and glutaredoxin systems. For each MSR type, we review catalytic parameters for the reduction of free MetO, low molecular weight MetO-containing compounds, and oxidized proteins. Analysis of these data reinforces the concept that MSRAs reduce various types of MetO-containing substrates with similar efficiency, whereas MSRBs are specialized for the reduction of MetO in proteins.     

Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins and protect cells from oxidative protein unfolding

Reduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis of MetO reduction in proteins, the physiological substrates of MSRA and MSRB. In this work we exploited an NADPH-dependent thioredoxin system and determined the kinetic parameters of yeast MSRA and MSRB using three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the K(m) lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereas MSRB was specialized for the reduction of MetO in proteins. Using oxidized glutathione S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins and that their activities increased with the unfolding state. Biochemical quantification and identification of MetO reduced in the substrates by mass spectrometry revealed that the increased activity was due to better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins.     

Regeneration Mechanisms of Arabidopsis thaliana Methionine Sulfoxide Reductases B by Glutaredoxins and Thioredoxins

Methionine oxidation leads to the formation of S- and R-diastereomers of methionine sulfoxide (MetSO), which are reduced back to methionine by methionine sulfoxide reductases (MSRs) A and B, respectively. MSRBs are classified in two groups depending on the conservation of one or two redox-active Cys; 2-Cys MSRBs possess a catalytic Cys-reducing MetSO and a resolving Cys, allowing regeneration by thioredoxins. The second type, 1-Cys MSRBs, possess only the catalytic Cys. The biochemical mechanisms involved in activity regeneration of 1-Cys MSRBs remain largely elusive. In the present work we used recombinant plastidial Arabidopsis thaliana MSRB1 and MSRB2 as models for 1-Cys and 2-Cys MSRBs, respectively, to delineate the Trx- and glutaredoxin-dependent reduction mechanisms. Activity assays carried out using a series of cysteine mutants and various reductants combined with measurements of free thiols under distinct oxidation conditions and mass spectrometry experiments show that the 2-Cys MSRB2 is reduced by Trx through a dithiol-disulfide exchange involving both redox-active Cys of the two partners. Regarding 1-Cys MSRB1, oxidation of the enzyme after substrate reduction leads to the formation of a stable sulfenic acid on the catalytic Cys, which is subsequently glutathionylated. The deglutathionylation of MSRB1 is achieved by both mono- and dithiol glutaredoxins and involves only their N-terminal conserved catalytic Cys. This study proposes a detailed mechanism of the regeneration of 1-Cys MSRB activity by glutaredoxins, which likely constitute physiological reductants for this type of MSR.Proteins are prone to oxidative modifications due to the action of reactive oxygen species. Methionine (Met), one of the most susceptible amino acids to oxidation (1), is converted into methionine sulfoxide (MetSO),³ resulting in altered conformation and activity for many proteins (1). Methionine sulfoxide reductases (MSRs), which catalyze the reduction of MetSO back to Met, are present in most living organisms. MSRA, the first MSR isolated (2), is specific of the MetSO S-diastereomer and participates in protection against oxidative stress (3). A second MSR type, MSRB, which catalytically reduces the MetSO R-diastereomer, was identified later (4). MSRA and MSRB are monomeric enzymes that display no sequence or structural homologies but share a similar three-step catalytic mechanism, (i) reduction of MetSO by MSR and formation of a sulfenic acid intermediate on the “catalytic” cysteine (Cys), (ii) formation of a disulfide bond between catalytic and “resolving” Cys and release of H₂O, and (iii) reduction of the disulfide bond by a reductant (5, 6). Thioredoxins (Trxs) have been proposed to be the biological reductant for MSRs (2, 7). Trxs are small and ubiquitous disulfide reductases with a WC(G/P)PC active site. They function as electron donors and play essential roles in many processes through control of protein conformation and activity by supplying the reducing power needed to reduce disulfide bonds in target proteins.Most MSRBs, named 2-Cys MSRBs, possess two conserved Cys and are actually reduced by Trxs (7, 8). However, in a second class of MSRBs, termed 1-Cys MSRBs and representing ∼40% of known MSRBs, the resolving Cys residue corresponding to Cys-63 in Escherichia coli is replaced by Thr or Ser (8, 9). Although some of these MSRBs possess another potential resolving Cys (9), most 1-Cys MSRBs do not have any additional Cys, indicating that an alternative mechanism, which does not involve the formation of an intramolecular disulfide reduction, is needed for their regeneration (7). Contrasting data concerning the role of Trxs in providing electrons to these MSRBs have been reported. Several studies showed that cytosolic Trx is not an efficient reductant for human 1-Cys MSRBs (10–12), whereas mitochondrial Trxs were recently reported to efficiently regenerate mitochondrial 1-Cys MSRBs (13). It has been proposed that regeneration of mammalian and plant 1-Cys MSRBs could involve direct reduction of the cysteine sulfenic acid form generated during catalysis (10, 13–15).Arabidopsis thaliana possesses two plastidial MSRBs referred to as MSRB1 and MSRB2 and related to 1-Cys and 2-Cys MSRB types, respectively. MSRB2 possesses two CXXC motifs potentially implicated in the coordination of a zinc atom, a Cys in position 187 corresponding to the catalytic Cys-117 of E. coli MSRB, a potential resolving Cys in position 134, and an additional Cys in position 115. MSRB1 also contains the four Cys residues potentially coordinating zinc, the potential catalytic Cys-186, and a Cys in position 116, whereas the potential resolving Cys is replaced by a Thr in position 132. Previously, we showed that various types of canonical Trxs are efficient electron suppliers to MSRB2, whereas MSRB1 can only be reduced by the peculiar Trx CDSP32 (chloroplastic drought-induced stress protein of 32 kDa) and by Grxs (15–17). Grxs are oxidoreductases of the Trx superfamily possessing either a monothiol CXXS or a dithiol CXXC active site and are generally reduced by glutathione (18). Grxs are able to reduce protein disulfides, but also glutathione-mixed disulfides, a reaction termed deglutathionylation, for which Trxs are not efficient catalysts (19, 20). Classical dithiol Grxs can reduce disulfide bonds using both active site Cys residues, as shown for E. coli ribonucleotide reductase, but can also reduce glutathione-mixed disulfides through a monothiol mechanism that requires only the N-terminal active site Cys (21). CXXS-type Grxs catalyze deglutathionylation either through a monothiol mechanism, as recently shown for chloroplastic GrxS12 (CSYS active site) (22), or through a dithiol mechanism as suggested for Grxs with a CGFS active site (20, 23).We reported recently the involvement of Grxs in the regeneration of MSRB activity (15). Nevertheless, the precise biochemical mechanism underlying regeneration by Grxs remains unknown. In this study we performed a detailed analysis of the roles of redox-active Cys in reductants (Trxs and Grxs) and in acceptors (plastidial Arabidopsis MSRBs). We provide evidence that reduction of MSRB2 by Trxs is achieved through a classical dithiol-disulfide exchange. The data on MSRB1 reveal that 1-Cys MSRBs are regenerated by Grxs through a glutathionylation step of the catalytic Cys.     

Involvement of thioredoxin y2 in the preservation of leaf methionine sulfoxide reductase capacity and growth under high light

Methionine (Met) in proteins can be oxidized to two diastereoisomers of methionine sulfoxide, Met‐S‐O and Met‐R‐O, which are reduced back to Met by two types of methionine sulfoxide reductases (MSRs), A and B, respectively. MSRs are generally supplied with reducing power by thioredoxins. Plants are characterized by a large number of thioredoxin isoforms, but those providing electrons to MSRs in vivo are not known. Three MSR isoforms, MSRA4, MSRB1 and MSRB2, are present in Arabidopsis thaliana chloroplasts. Under conditions of high light and long photoperiod, plants knockdown for each plastidial MSR type or for both display reduced growth. In contrast, overexpression of plastidial MSRBs is not associated with beneficial effects in terms of growth under high light. To identify the physiological reductants for plastidial MSRs, we analyzed a series of mutants deficient for thioredoxins f, m, x or y. We show that mutant lines lacking both thioredoxins y1 and y2 or only thioredoxin y2 specifically display a significantly reduced leaf MSR capacity (–25%) and growth characteristics under high light, related to those of plants lacking plastidial MSRs. We propose that thioredoxin y2 plays a physiological function in protein repair mechanisms as an electron donor to plastidial MSRs in photosynthetic organs.     

Different B-Type Methionine Sulfoxide Reductases in Chlamydomonas May Protect the Alga against High-Light, Sulfur-Depletion, or Oxidative Stress

The genome of unicellular green alga Chlamydomonas reinhardtii contains four genes encoding B-type methionine sulfoxide reductases, MSRBI.1, MSRB1.2, MSRB2.1, and MSRB2.2, with functions largely unknown. To understand the cell defense system mediated by the methionine suifoxide reductases in Chlamydomonas, we analyzed expression and physiological roles of the MSRBs under different abiotic stress conditions using immunoblotting and quantitative polymerase chain reaction （PCR） analyses. We showed that the MSRB2.2 protein was accumulated in cells treated with high light （1,300 μE-/m2 per s）, whereas MSRBI.1 was accumulated in the cells under 1 mmol/L H2O2 treatment or sulfur depletion. We observed that the cells with the MSRB2.2 knockdown and overexpression displayed increased and decreased sensitivity to high light, respectively, based on in situ chlorophyll a fluorescence measures. We also observed that the cells with the MSRBI.1 knockdown and overexpression displayed decreased and increased tolerance to sulfur-depletion and oxidative stresses, respectively, based on growth and H2- producing performance. The physiological implications revealed from the experimental data highlight the importance of MSRB2.2 and MSRBI.1 in protecting Chlamydomonas cells against adverse conditions such as high-light, sulfur-depletion, and oxidative stresses.     

Arabidopsis thaliana plastidial methionine sulfoxide reductases B,MSRBs, account for most leaf peptide MSR activity and are essential for growth under environmental constraints through a role in the preservation of photosystem antennae

Methionine oxidation to methionine sulfoxide (MetSO) is reversed by two types of methionine sulfoxide reductases (MSRs), A and B, specific to MetSO S‐ and R‐diastereomers, respectively. Two MSRB isoforms, MSRB1 and MSRB2, are present in chloroplasts of Arabidopsis thaliana. To assess their physiological role, we characterized Arabidopsis mutants knockout for the expression of MSRB1, MSRB2 or both genes. Measurements of MSR activity in leaf extracts revealed that the two plastidial MSRB enzymes account for the major part of leaf peptide MSR capacity. Under standard conditions of light and temperature, plants lacking one or both plastidial MSRBs do not exhibit any phenotype, regarding growth and development. In contrast, we observed that the concomitant absence of both proteins results in a reduced growth for plants cultivated under high light or low temperature. In contrast, double mutant lines restored for MSRB2 expression display no phenotype. Under environmental constraints, the MetSO level in leaf proteins is higher in plants lacking both plastidial MSRBs than in Wt plants. The absence of plastidial MSRBs is associated with an increased chlorophyll a/b ratio, a reduced content of Lhca1 and Lhcb1 proteins and an impaired photosynthetic performance. Finally, we show that MSRBs are able to use as substrates, oxidized cpSRP43 and cpSRP54, the two main components involved in the targeting of Lhc proteins to the thylakoids. We propose that plastidial MSRBs fulfil an essential function in maintaining vegetative growth of plants during environmental constraints, through a role in the preservation of photosynthetic antennae.     

Corynebacterium glutamicum Methionine Sulfoxide Reductase A Uses both Mycoredoxin and Thioredoxin for Regeneration and Oxidative Stress Resistance

Oxidation of methionine leads to the formation of the S and R diastereomers of methionine sulfoxide (MetO), which can be reversed by the actions of two structurally unrelated classes of methionine sulfoxide reductase (Msr), MsrA and MsrB, respectively. Although MsrAs have long been demonstrated in numerous bacteria, their physiological and biochemical functions remain largely unknown in Actinomycetes. Here, we report that a Corynebacterium glutamicum methionine sulfoxide reductase A (CgMsrA) that belongs to the 3-Cys family of MsrAs plays important roles in oxidative stress resistance. Deletion of the msrA gene in C. glutamicum resulted in decrease of cell viability, increase of ROS production, and increase of protein carbonylation levels under various stress conditions. The physiological roles of CgMsrA in resistance to oxidative stresses were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic-function (ECF) sigma factor SigH. Activity assays performed with various regeneration pathways showed that CgMsrA can reduce MetO via both the thioredoxin/thioredoxin reductase (Trx/TrxR) and mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) pathways. Site-directed mutagenesis confirmed that Cys56 is the peroxidatic cysteine that is oxidized to sulfenic acid, while Cys204 and Cys213 are the resolving Cys residues that form an intramolecular disulfide bond. Mrx1 reduces the sulfenic acid intermediate via the formation of an S-mycothiolated MsrA intermediate (MsrA-SSM) which is then recycled by mycoredoxin and the second molecule of mycothiol, similarly to the glutathione/glutaredoxin/glutathione reductase (GSH/Grx/GR) system. However, Trx reduces the Cys204-Cys213 disulfide bond in CgMsrA produced during MetO reduction via the formation of a transient intermolecular disulfide bond between Trx and CgMsrA. While both the Trx/TrxR and Mrx1/Mtr/MSH pathways are operative in reducing CgMsrA under stress conditions in vivo, the Trx/TrxR pathway alone is sufficient to reduce CgMsrA under normal conditions. Based on these results, a catalytic model for the reduction of CgMsrA by Mrx1 and Trx is proposed.     

Structural and Biochemical Characterization of Free Methionine-R-sulfoxide Reductase from Neisseria meningitidis

A new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys¹¹⁸ is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain.     

Methionine Sulfoxide Reductase B Displays a High Level of Flexibility

Methionine sulfoxide reductases (Msrs) are enzymes that catalyze the reduction of methionine sulfoxide back to methionine. In vivo, Msrs are essential in the protection of cells against oxidative damage to proteins and in the virulence of some bacteria. Two structurally unrelated classes of Msrs, named MsrA and MsrB, exist. MsrB are stereospecific to R epimer on the sulfur of sulfoxide. All MsrB share a common reductase step with the formation of a sulfenic acid intermediate. For the subclass of MsrB whose recycling process passes through the formation of an intradisulfide bond, the recycling reducer is thioredoxin. In the present study, X-ray structures of Neisseria meningitidis MsrB have been determined. The structures have a fold based on two β-sheets, similar to the fold already described for other MsrB, with the recycling Cys63 located in a position favorable for disulfide bond formation with the catalytic Cys117. X-ray structures of Xanthomonas campestris MsrB have also been determined. In the C117S MsrB structure with a bound substrate, the recycling Cys31 is far from Ser117, with Trp65 being essential in the reductase step located in between. This positioning prevents the formation of the Cys31-Cys117 disulfide bond. In the oxidized structure, a drastic conformational reorganization of the two β-sheets due to withdrawal of the Trp65 region from the active site, which remains compatible with an efficient thioredoxin-recycling process, is observed. The results highlight the remarkable structural malleability of the MsrB fold.     

A second human methionine sulfoxide reductase (hMSRB2) reducing methionine-R-sulfoxide displays a tissue expression pattern distinct from hMSRB1

Abstract

Peptide methionine sulfoxide reductases are important enzymes in the defense against cellular oxidative stress as they reduce methionine sulfoxide, the product of methionine oxidation by physiologically relevant reactive oxygen species. Two distinct enzyme classes, MSRA and MSRB, have evolved for selectively reducing the two epimers, methionine-S-sulfoxide and methionine-Rsulfoxide. A new human MSR enzyme (hMSRB2) specifically reducing methionine-R-sulfoxide, which showed a conversion rate for peptide-bound methionine-S-sulfoxide similar to hMSRB1, was characterized with respect to its tissue expression. As previously found for hMSRB1, expression of hMSRB2 mRNA was weak in brain, but strong in heart and skeletal muscle. In contrast to hMSRB1, its expression was high in smooth muscle-containing organs (digestive system, bladder), lung and aorta, while hMSRB1 displayed a higher expression than hMSRB2 in liver and kidney.     

Heterogeneity and function of mammalian MSRs: enzymes for repair, protection and regulation

Methionine sulfoxide, the physiologically relevant oxidation product of methionine, is enzymatically reduced by peptide methionine sulfoxide reductases (MSRs). Two distinct classes of these enzymes, MSRA and MSRB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Mammals typically possess only one gene encoding MSRA, but at least three genes encoding MSRBs. These MSRs show distinct tissue- and subcellular expression patterns and may play specific functional roles. Susceptibility of some ion channels to reversible methionine oxidation suggests that MSRs have a regulatory role in cellular excitability. Some--if not all--MSRs protect cells and organisms against a variety of oxidative stress episodes, including those by hypoxia and reperfusion, and play a modulatory role in lifespan determination. More MSR-dependent physiological phenomena await to be discovered.     

Structure-function relationship in an archaebacterial methionine sulphoxide reductase B

Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs.     

Functional Null Mutations of MSRB3 Encoding Methionine Sulfoxide Reductase Are Associated with Human Deafness DFNB74

The DFNB74 locus for autosomal-recessive, nonsyndromic deafness segregating in three families was previously mapped to a 5.36 Mb interval on chromosome 12q14.2-q15. Subsequently, we ascertained five additional consanguineous families in which deafness segregated with markers at this locus and refined the critical interval to 2.31 Mb. We then sequenced the protein-coding exons of 18 genes in this interval. The affected individuals of six apparently unrelated families were homozygous for the same transversion (c.265T>G) in MSRB3, which encodes a zinc-containing methionine sulfoxide reductase B3. c.265T>G results in a substitution of glycine for cysteine (p.Cys89Gly), and this substitution cosegregates with deafness in the six DFNB74 families. This cysteine residue of MSRB3 is conserved in orthologs from yeast to humans and is involved in binding structural zinc. In vitro, p.Cys89Gly abolished zinc binding and MSRB3 enzymatic activity, indicating that p.Cys89Gly is a loss-of-function allele. The affected individuals in two other families were homozygous for a transition mutation (c.55T>C), which results in a nonsense mutation (p.Arg19X) in alternatively spliced exon 3, encoding a mitochondrial localization signal. This finding suggests that DFNB74 deafness is due to a mitochondrial dysfunction. In a cohort of 1,040 individuals (aged 53–67 years) of European ancestry, we found no association between 17 tagSNPs for MSRB3 and age-related hearing loss. Mouse Msrb3 is expressed widely. In the inner ear, it is found in the sensory epithelium of the organ of Corti and vestibular end organs as well as in cells of the spiral ganglion. Taken together, MSRB3-catalyzed reduction of methionine sulfoxides to methionine is essential for hearing.     

A second human methionine sulfoxide reductase (hMSRB2) reducing methionine-R-sulfoxide displays a tissue expression pattern distinct from hMSRB1

Peptide methionine sulfoxide reductases are important enzymes in the defense against cellular oxidative stress as they reduce methionine sulfoxide, the product of methionine oxidation by physiologically relevant reactive oxygen species. Two distinct enzyme classes, MSRA and MSRB, have evolved for selectively reducing the two epimers, methionine-S-sulfoxide and methionine-R-sulfoxide. A new human MSR enzyme (hMSRB2) specifically reducing methionine-R-sulfoxide, which showed a conversion rate for peptide-bound methionine-S-sulfoxide similar to hMSRB1, was characterized with respect to its tissue expression. As previously found for hMSRB1, expression of hMSRB2 mRNA was weak in brain, but strong in heart and skeletal muscle. In contrast to hMSRB1, its expression was high in smooth muscle-containing organs (digestive system, bladder), lung and aorta, while hMSRB1 displayed a higher expression than hMSRB2 in liver and kidney.     

OsMSRA4.1 and OsMSRB1.1, two rice plastidial methionine sulfoxide reductases,are involved in abiotic stress responses

In proteins, methionine residues are especially sensitive to oxidation, leading to the formation of S- and R-methionine sulfoxide diastereoisomers, and these two methionine sulfoxides can be specifically reversed by two types of methionine sulfoxide reductases (MSRs), MSRA and MSRB. Previously, we have identified a gene encoding a putative MSR from NaCl-treated roots of Brazilian upland rice (Oryza sativa L. cv. IAPAR 9) via subtractive suppression hybridization (Wu et al. in Plant Sci 168:847–853, 2005). Blast database analysis indicated that at least four MSRA and three MSRB orthologs exist in rice, and two of them, OsMSRA4.1 and OsMSRB1.1, were selected for further functional analysis. Expression analysis showed that both OsMSRA4.1 and OsMSRB1.1 are constitutively expressed in all organs and can be induced by various stress conditions. Subcellular localization and in vitro activity assay revealed that both OsMSR proteins are targeted to the chloroplast and have MSR activity. Overexpression of either OsMSRA4.1 or OsMSRB1.1 in yeast enhanced cellular resistance to oxidative stress. In addition, OsMSRA4.1-overexpressing transgenic rice plants also showed enhanced viability under salt treatment. Our results provide genetic evidence of the involvement of OsMSRs in the plant stress responses. X. Guo and Y. Wu contributed equally to this work.     

Alkyl Hydroperoxide Reductase Repair by Helicobacter pylori Methionine Sulfoxide Reductase

Protein exposure to oxidants such as HOCl leads to formation of methionine sulfoxide (MetSO) residues, which can be repaired by methionine sulfoxide reductase (Msr). A Helicobacter pylori msr strain was more sensitive to HOCl-mediated killing than the parent. Because of its abundance in H. pylori and its high methionine content, alkyl hydroperoxide reductase C (AhpC) was hypothesized to be prone to methionine oxidation. AhpC was expressed as a recombinant protein in Escherichia coli. AhpC activity was abolished by HOCl, while all six methionine residues of the enzyme were fully to partially oxidized. Upon incubation with a Msr repair mixture, AhpC activity was restored to nonoxidized levels and the MetSO residues were repaired to methionine, albeit to different degrees. The two most highly oxidized and then Msr-repaired methionine residues in AhpC, Met₁₀₁ and Met₁₃₃, were replaced with isoleucine residues by site-directed mutagenesis, either individually or together. E. coli cells expressing variant versions were more sensitive to t-butyl hydroperoxide than cells expressing native protein, and purified AhpC variant proteins had 5% to 39% of the native enzyme activity. Variant proteins were still able to oligomerize like the native version, and circular dichroism (CD) spectra of variant proteins revealed no significant change in AhpC conformation, indicating that the loss of activity in these variants was not related to major structural alterations. Our results suggest that both Met₁₀₁ and Met₁₃₃ residues are important for AhpC catalytic activity and that their integrity relies on the presence of a functional Msr.     

Arrest defective 1 regulates the oxidative stress response in human cells and mice by acetylating methionine sulfoxide reductase A

Methionine sulfoxide reductase A (MSRA) protects proteins from oxidation, and also helps remove reactive oxygen species (ROS) by recovering antioxidant enzymes inactivated by oxidation. Although its functions have been investigated extensively, little is known about the mechanism by which MSRA is regulated. Arrest defective 1 (ARD1) is an enzyme that catalyzes not only N-terminal acetylation as a cotranslational modification but also lysine acetylation as a posttranslational modification. ARD1, which is expressed in most cell types, is believed to participate in diverse biological processes, but its roles are poorly understood. Given that MSRA was hunted in a yeast two-hybrid screen with ARD1 as the bait, we here investigated whether ARD1 is a novel regulator of MSRA. ARD1 was shown to interact with and acetylate MSRA in both cells and test tubes. It specifically acetylated the K49 residue of MSRA, and by doing so repressed the enzymatic function of MSRA. ARD1 increased cellular levels of ROS, carbonylated proteins and DNA breaks under oxidative stress. Moreover, it promoted cell death induced by pro-oxidants, which was attenuated in MSRA-deficient cells. When mice were exposed to hyperoxic conditions for 2 days, their livers and kidneys were injured and protein carbonylation was increased. The oxidative tissue injury was more severe in ARD1 transgenic mice than in their wild-type littermates. In conclusion, ARD1 has a crucial role in the cellular response to oxidative stress as a bona fide regulator of MSRA. ARD1 is a potential target for ameliorating oxidative injury or for potentiating ROS-producing anticancer agents.Aerobic respiration is essential for eukaryotic life because molecular oxygen participates in ATP production and various oxidative metabolic reactions.¹ When oxygen is used, reactive oxygen species (ROS) are inevitably generated and threaten life as harmful metabolites that damage macromolecules such as nucleic acids, lipids and proteins.^2,3 ROS also act as second messengers that promote cell proliferation or differentiation.^{4, 5, 6, 7} From a functional perspective, ROS act as a double-edged sword in determining cell fate, and the roles of ROS depend on cell contexts.⁸ A variety of cell metabolic reactions are regulated depending on the intracellular redox state, which reflects the balance between ROS-generating oxidases and ROS-scavenging antioxidants.⁹ Accordingly, knowledge about the redox-balancing mechanism will help us to better understand normal physiology and pathology.The sulfur atom of methionine is easily oxidized by ROS, with methionine being modified to methionine sulfoxide (MetO), which forms two enantiomers (S-sulfoxide and R-sulfoxide).¹⁰ When proteins are sulfoxidized at methionine residues, their functions become impaired or altered.¹¹ Therefore, MetO is not only a convincing biomarker for reflecting the extent of oxidative stress but also a pathogenic factor that contributes to oxidative stress-related diseases.¹² As MetO causes serious problems in life, the defense systems against MetO have been evolutionally conserved in prokaryotic and eukaryotic cells.¹³ One such system, methionine sulfoxide reductase (MSR), has a crucial role in preventing the accumulation of MetO, and includes two enzymes, methionine sulfoxide reductase A (MSRA) and MSRB, which reduce S-sulfoxide and R-sulfoxide, respectively.¹⁴Arrest defective 1 (ARD1) is an enzyme that catalyzes N-terminal acetylation of nascent peptides as a cotranslational modification and lysine acetylation as a posttranslational modification.¹⁵ In yeast and mammalian cells, ARD1 is known to have essential roles in cell growth and differentiation.^16,17 ARD1 has also been reported to control cell migration by acetylating myosin light chain kinase¹⁸ and to promote cancer growth by acetylating β-catenin or the androgen receptor.¹⁹ Considering that ARD1 is widely expressed in most mammalian cells,²⁰ it is expected that ARD1 has diverse functions beyond those mentioned above. To further understand the functions of ARD1, we sought novel targets of ARD1 using the yeast two-hybrid method and identified MSRA as an ARD1-interacting molecule. Furthermore, we tested the possibility that ARD1 determines cell fate under oxidative stress by regulating MSRA. This study may provide new insights into how MSRA is regulated and identifies ARD1 as a potential target for modulating the cellular response to oxidative stress.