Methionine oxidation of carbohydrate-active enzymes during white-rot wood decay.

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.

The selenoprotein methionine sulfoxide reductase B1 (MSRB1).

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.

Biosensor-Linked Immunosorbent Assay for the Quantification of Methionine Oxidation in Target Proteins.

Methionine oxidation is involved in regulating the protein activity and often leads to protein malfunction. However, tools for quantitative analyses of protein-specific methionine oxidation are currently unavailable. In this work, we developed a biological sensor that quantifies oxidized methionine in the form of methionine-R-sulfoxide in target proteins. The biosensor "tpMetROG" consists of methionine sulfoxide reductase B (MsrB), circularly permuted yellow fluorescent protein (cpYFP), thioredoxin, and protein G. Protein G binds to the constant region of antibodies against target proteins, specifically capturing them. Then, MsrB reduces the oxidized methionine in these proteins, leading to cpYFP fluorescence changes. We assessed this biosensor for quantitative analysis of methionine-R-sulfoxide in various proteins, such as calmodulin, IDLO, LegP, Sacde, and actin. We further developed an immunosorbent assay using the biosensor to quantify methionine oxidation in specific proteins such as calmodulin in animal tissues. The biosensor-linked immunosorbent assay proves to be an indispensable tool for detecting methionine oxidation in a protein-specific manner. This is a versatile tool for studying the redox biology of methionine oxidation in proteins.

Distribution of methionine sulfoxide reductases in fungi and conservation of the free-methionine-R-sulfoxide reductase in multicellular eukaryotes.

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.

Development of a novel fluorescent biosensor for dynamic monitoring of metabolic methionine redox status in cells and tissues.

Aberrant production of reactive oxygen species (ROS) leads to tissue damage accumulation, which is associated with a myriad of human pathologies. Although several sensors have been developed for ROS quantification, their applications for ROS-related human physiologies and pathologies still remain problematic due to the unstable nature of ROS. Herein, we developed Trx1-cpYFP-fRMsr (TYfR), a genetically-encoded fluorescent biosensor with the remarkable specificity and sensitivity toward fMetRO (free Methionine-R-sulfoxide), allowing for dynamic quantification of physiological levels of fMetRO, a novel indicator of ROS and methionine redox status in vitro and in vivo. Moreover, using the sensor, we observed a significant fMetRO enrichment in serum from patients with acute coronary syndrome, one of the most severe cardiovascular diseases, which becomes more evident following percutaneous coronary intervention. Collectively, this study proposes that fMetRO is a novel biomarker of tissue damage accumulation in ROS-associated human pathologies, and that TYfR is a promising tool for quantifying fMetRO with potentials in versatile applications.

Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase.

In proteins, methionine (Met) can be oxidized into Met sulfoxide (MetO). The ubiquitous methionine sulfoxide reductases (Msr) A and B are thiol-oxidoreductases reducing MetO. Reversible Met oxidation has a wide range of consequences, from protection against oxidative stress to fine-tuned regulation of protein functions. Bacteria distinguish themselves by the production of molybdenum-containing enzymes reducing MetO, such as the periplasmic MsrP which protects proteins during acute oxidative stress. The versatile dimethyl sulfoxide (DMSO) reductases were shown to reduce the free amino acid MetO, but their ability to reduce MetO within proteins was never evaluated. Here, using model oxidized proteins and peptides, enzymatic and mass spectrometry approaches, we showed that the Rhodobacter sphaeroides periplasmic DorA-type DMSO reductase reduces protein bound MetO as efficiently as the free amino acid L-MetO and with catalytic values in the range of those described for the canonical Msrs. The identification of this fourth type of enzyme able to reduce MetO in proteins, conserved across proteobacteria and actinobacteria, suggests that organisms employ enzymatic systems yet undiscovered to regulate protein oxidation states.

Rhodobactersphaeroides methionine sulfoxide reductase P reduces R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates.

Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S-diastereomer and R-diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram-negative bacteria, was identified as a key actor for the reduction of oxidized periplasmic proteins. The haem-binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein-MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria Rhodobacter sphaeroides as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO is found in clusters, in amino acid sequences devoid of threonine and proline on the C-terminal side. Moreover, R. sphaeroides MsrP lacks stereospecificity as it can reduce both R- and S-diastereomers of MetO, similarly to its Escherichia coli homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.

Physiological Roles of Plant Methionine Sulfoxide Reductases in Redox Homeostasis and Signaling.

Oxidation of methionine (Met) leads to the formation of two S- and R-diastereoisomers of Met sulfoxide (MetO) that are reduced back to Met by methionine sulfoxide reductases (MSRs), A and B, respectively. Here, we review the current knowledge about the physiological functions of plant MSRs in relation with subcellular and tissue distribution, expression patterns, mutant phenotypes, and possible targets. The data gained from modified lines of plant models and crop species indicate that MSRs play protective roles upon abiotic and biotic environmental constraints. They also participate in the control of the ageing process, as shown in seeds subjected to adverse conditions. Significant advances were achieved towards understanding how MSRs could fulfil these functions via the identification of partners among Met-rich or MetO-containing proteins, notably by using redox proteomic approaches. In addition to a global protective role against oxidative damage in proteins, plant MSRs could specifically preserve the activity of stress responsive effectors such as glutathione-S-transferases and chaperones. Moreover, several lines of evidence indicate that MSRs fulfil key signaling roles via interplays with Ca2+- and phosphorylation-dependent cascades, thus transmitting ROS-related information in transduction pathways.

Monitoring of Methionine Sulfoxide Content and Methionine Sulfoxide Reductase Activity.

The sulfur-containing amino acid methionine (Met) plays critical roles in protein synthesis, methylation, and sulfur metabolism. Both in its free form and in the form of an amino acid residue, it can be oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Organisms evolved methionine sulfoxide reductases (MSRs) to reduce MetO to Met, with the MSRs type A (MSRA) and type B (MSRB) being specific for the S and R forms of MetO, respectively. In mammals, the selenoprotein MSRB1 plays an important protein repair function, and its expression is tightly regulated by dietary selenium. In this chapter, we describe a protocol for determining the concentration of protein-based Met-R-O and its analysis in HEK293 cells using a genetically encoded ratiometric fluorescent biosensor MetROx. We also describe the procedure for quantifying MSR activities in cell extracts using specific substrates and a reverse phase HPLC-based method.

Probing the Surface of a Laccase for Clues towards the Design of Chemo-Enzymatic Catalysts.

Systems featuring a multi-copper oxidase associated with transition-metal complexes can be used to perform oxidation reactions in mild conditions. Here, a strategy is presented for achieving a controlled orientation of a ruthenium-polypyridyl graft at the surface of a fungal laccase. Laccase variants are engineered with unique surface-accessible lysine residues. Distinct ruthenium-polypyridyl-modified laccases are obtained by the reductive alkylation of lysine residues precisely located relative to the T1 copper centre of the enzyme. In none of these hybrids does the presence of the graft compromise the catalytic efficiency of the enzyme on the substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Furthermore, the efficiency of the hybrids in olefin oxidation coupled to the light-driven reduction of O2 is highly dependent on the location of the graft at the enzyme surface. Simulated RuII -CuII electron coupling values and distances fit well the observed reactivity and could be used to guide future hybrid designs.

Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide.

In cells, physiological and pathophysiological conditions may lead to the formation of methionine sulfoxide (MetO). This oxidative modification of methionine exists in the form of two diastereomers, R and S, and may occur in both free amino acid and proteins. MetO is reduced back to methionine by methionine sulfoxide reductases (MSRs). Methionine oxidation was thought to be a nonspecific modification affecting protein functions and methionine availability. However, recent findings suggest that cyclic methionine oxidation and reduction is a posttranslational modification that actively regulates protein function akin to redox regulation by cysteine oxidation and phosphorylation. Methionine oxidation is thus an important mechanism that could play out in various physiological contexts. However, detecting MetO generation and MSR functions remains challenging because of the lack of tools and reagents to detect and quantify this protein modification. We recently developed two genetically encoded diasterospecific fluorescent sensors, MetSOx and MetROx, to dynamically monitor MetO in living cells. Here, we provide a detailed procedure for their use in bacterial and mammalian cells using fluorimetric and fluorescent imaging approaches. This method can be adapted to dynamically monitor methionine oxidation in various cell types and under various conditions.

Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors.

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly permuted yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, respectively named MetSOx and MetROx for their ability to detect S and R forms of MetO, were used for targeted analysis of protein oxidation, regulation and repair as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO and examining responses to physiological stimuli.

MsrB1 and MICALs regulate actin assembly and macrophage function via reversible stereoselective methionine oxidation.

Redox control of protein function involves oxidation and reduction of amino acid residues, but the mechanisms and regulators involved are insufficiently understood. Here, we report that in conjunction with Mical proteins, methionine-R-sulfoxide reductase B1 (MsrB1) regulates mammalian actin assembly via stereoselective methionine oxidation and reduction in a reversible, site-specific manner. Two methionine residues in actin are specifically converted to methionine-R-sulfoxide by Mical1 and Mical2 and reduced back to methionine by selenoprotein MsrB1, supporting actin disassembly and assembly, respectively. Macrophages utilize this redox control during cellular activation by stimulating MsrB1 expression and activity as a part of innate immunity. We identified the regulatory role of MsrB1 as a Mical antagonist in orchestrating actin dynamics and macrophage function. More generally, our study shows that proteins can be regulated by reversible site-specific methionine-R-sulfoxidation.

Diversity of plant methionine sulfoxide reductases B and evolution of a form specific for free methionine sulfoxide.

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed the MSRB activity toward free Met-R-O with kinetic parameters similar to those reported for fRMSR from Escherichia coli, an enzyme specific for free Met-R-O. Overexpression of GmMSRB2 or GmMSRB4 in the yeast cytosol supported the growth of the triple MSRA/MSRB/fRMSR (Δ3MSRs) mutant on MetO and protected cells against H2O2-induced stress. Taken together, our data reveal an unexpected diversity of MSRBs in plants and indicate that, in contrast to mammals that cannot reduce free Met-R-O and microorganisms that use fRMSR for this purpose, plants evolved MSRBs for the reduction of both free and protein-based MetO.

A drought-sensitive barley variety displays oxidative stress and strongly increased contents in low-molecular weight antioxidant compounds during water deficit compared to a tolerant variety.

Barley displays a great genetic diversity, constituting a valuable source to delineate the responses of contrasted genotypes to environmental constraints. Here, we investigated the level of oxidative stress and the participation of antioxidant systems in two barley genotypes: Express, a variety known to be sensitive to drought, and Saïda, an Algerian landrace selected for its tolerance to water deficit. Soil-grown 15-day-old plants were subjected to water deficit for 8 days and then rewatered. We observed that upon water stress Express exhibits compared to Saïda accelerated wilting and a higher level of oxidative stress evaluated by HPLC measurements of lipid peroxidation and by imaging techniques. In parallel, Express plants also display lower levels of catalase and superoxide dismutase activity. No great difference was observed regarding peroxiredoxins and methionine sulfoxide reductases, enzymes detoxifying peroxides and repairing oxidized proteins, respectively. In contrast, upon water stress and recovery, much higher contents and oxidation ratios of glutathione and ascorbate were measured in Express compared to Saïda. Express also shows during water deficit greater increases in the pools of lipophilic antioxidants like xantophyll carotenoids and α-tocopherol. Altogether, these data show that the differential behavior of the two genotypes involves distinct responses regarding antioxidant mechanisms. Indeed, the drought sensitivity of Express compared with Saïda is associated with oxidative damage and a lower enzymatic ROS-scavenging capacity, but in parallel with a much stronger enhancement of most mechanisms involving low-molecular weight antioxidant compounds.

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.

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.

Atypical thioredoxins in poplar: the glutathione-dependent thioredoxin-like 2.1 supports the activity of target enzymes possessing a single redox active cysteine.

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways.

Affinity chromatography: a valuable strategy to isolate substrates of methionine sulfoxide reductases?

Reactive oxygen species fulfill key roles in development and signaling, but lead at high concentration to damage in macromolecules. In proteins, methionine (Met) is particularly prone to oxidative modification and can be oxidized into Met sulfoxide (MetO). MetO reduction is catalyzed by specialized enzymes, termed methionine sulfoxide reductases (MSRs), involved in senescence and protection against diseases and environmental constraints. The precise physiological functions of MSRs remain often elusive because of very poor knowledge of their substrates. In this study, affinity chromatography was used to isolate partners of Arabidopsis thaliana plastidial methionine sulfoxide reductase B1 (MSRB1). Twenty-four proteins involved in photosynthesis, translation, and protection against oxidative stress, as well as in metabolism of sugars and amino acids, were identified. Statistical analysis shows that the abundance of MSRB1 partners in chromatography affinity samples is proportional to Met content. All proteins, for which structural modeling was feasible, display surface-exposed Met and are thus potentially susceptible to oxidation. Biochemical analyses demonstrated that H(2)O(2) treatment actually converts several MSRB1-interacting proteins into MSRB substrates. In consequence, we propose that affinity chromatography constitutes an efficient tool to isolate physiological targets of MSRs.