Methionine Sulfoxide Research Articles

Monitoring of peroxide in gamma irradiated PE/EVOH/PE multilayer film using methionine probe

Methionine oxidation is used as a proxy to model the gamma radiation induced changes in single-use bags. These changes can be the formation of acids, radicals and hydroperoxides due to the irradiation of the multilayer film the single use bag is made from. Biopharmaceutical multilayer polyethylene bags irradiated from 0 to 260 kGy and aged from 0 to 36 months were filled with a methionine solution to follow the oxidation of methionine. Methionine sulfoxide was measured using high performance liquid chromatography after 21 days of storage. The maximum methionine sulfoxide concentration detected after 21 days of storage was analyzed through a design of experiments. The results to the design of experiments were considered to obtain the desirability domain for the optimization of the conditions of use for the single-use bags, limiting the oxidation of methionine as well as the release of reactive species thereof.

Tomato methionine sulfoxide reductase B2 functions in drought tolerance by promoting ROS scavenging and chlorophyll accumulation through interaction with Catalase 2 and RBCS3B

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.

Photo- and Radiation-Induced One-Electron Oxidation of Methionine in Various Structural Environments Studied by Time-Resolved Techniques.

Oxidation of methionine (Met) is an important reaction that plays a key role in protein modifications during oxidative stress and aging. The first steps of Met oxidation involve the creation of very reactive and short-lived transients. Application of complementary time-resolved radiation and photochemical techniques (pulse radiolysis and laser flash photolysis together with time-resolved CIDNP and ESR techniques) allowed comparing in detail the one-electron oxidation mechanisms initiated either by ●OH radicals and other one-electron oxidants or the excited triplet state of the sensitizers e.g., 4-,3-carboxybenzophenones. The main purpose of this review is to present various factors that influence the character of the forming intermediates. They are divided into two parts: those inextricably related to the structures of molecules containing Met and those related to external factors. The former include (i) the protection of terminal amine and carboxyl groups, (ii) the location of Met in the peptide molecule, (iii) the character of neighboring amino acid other than Met, (iv) the character of the peptide chain (open vs cyclic), (v) the number of Met residues in peptide and protein, and (vi) the optical isomerism of Met residues. External factors include the type of the oxidant, pH, and concentration of Met-containing compounds in the reaction environment. Particular attention is given to the neighboring group participation, which is an essential parameter controlling one-electron oxidation of Met. Mechanistic aspects of oxidation processes by various one-electron oxidants in various structural and pH environments are summarized and discussed. The importance of these studies for understanding oxidation of Met in real biological systems is also addressed.

Interaction of wheat methionine sulfoxide reductase TaMSRB5.2 with glutathione S-transferase TaGSTF3-A contributes to seedling osmotic stress resistance

Reactive oxygen species (ROS) produced due to drought stress can lead to the oxidation of methionine in plant cells; this can then be reduced by methionine sulfoxide reductases (MSRs). To the best of our knowledge, no information has been reported to date on the function and substrates of wheat cytoplasmic TaMSRB5.2, which is induced by drought mainly both in the leaves and roots. In the present study, its constitutive overexpression in wheat (TaOE) resulted in a notable increase in MSR activity, and enhanced the tolerance of seedlings, particularly to osmotic stress, but also to salt stress. The effects of constitutively active TaMSRB5.2 in wheat included a decrease in ROS accumulation, a heightening of endogenous abscisic acid (ABA) levels, an increase in the sensitivity of stomatal guard cells to exogenous ABA, and an enhancement of intracellular proline content. Wheat glutathione S-transferase F3-A (TaGSTF3-A), a potential interaction substrate of TaMSRB5.2, was identified by screening a wheat cDNA library, and further confirmed by means of yeast two-hybrid, split-luciferase complementation and co-immunoprecipitation assays. Under osmotic stress, TaGSTF3-A expression, overall GST enzyme activity and the ratio of GSH/GSSG in the TaOE lines increased, compared to the wild-type strain. L-buthionine sulfoximine, a GST inhibitor, was found to partially impair the TaMSRB5.2-induced response to osmotic stress. TaMSRB5.2 and TaGSTF3-A were observed to be expressed in the leaves, their proteins localized within the cytoplasm, and both acted in abiotic stress response, according to the present and previous studies. In conclusion, TaMSRB5.2 participates in osmotic response in wheat, partially via TaGSTF3-A.

Multi attribute method implementation using a High Resolution Mass Spectrometry platform: From sample preparation to batch analysis.

Quality control of biopharmaceuticals such as monoclonal antibodies (mAbs) has been evolving and becoming more challenging as the requirements of the regulatory agencies increase due to the demanding complexity of products under evaluation. Mass Spectrometry (MS)-based methods such as the multi-attribute method (MAM) are being explored to achieve a deeper understanding of the attributes critical for the safety, efficacy, and quality of these products. MAM uses high mass accuracy/high-resolution MS data that enables the direct and simultaneous monitoring of relevant product quality attributes (PQAs, in particular, chemical modifications) in a single workflow, replacing several orthogonal methods, reducing time and costs associated with these assays. Here we describe a MAM implementation process using a QTOF high resolution platform. Method implementation was accomplished using NIST (National Institute for Standards and Technology) mAb reference material and an in-process mAb sample. PQAs as glycosylation profiles, methionine oxidation, tryptophan dioxidation, asparagine deamidation, pyro-Glu at N-terminal and glycation were monitored. Focusing on applications that require batch analysis and high-throughput, sample preparation and LC-MS parameters troubleshooting are discussed. This MAM workflow was successfully explored as reference analytical tool for comprehensive characterization of a downstream processing (DSP) polishing platform and for a comparability study following technology transfer between different laboratories.

Mechanistic understanding of metal-catalyzed oxidation of polysorbate 80 and monoclonal antibody in biotherapeutic formulations

Surfactants are commonly used in biotherapeutic formulations to prevent the formation of aggregates and protect proteins from denaturation. Among them polysorbates are the most widely used. However, they are known to be prone to degradation, mainly via enzymatic hydrolysis and oxidation.In this study, the impact of different conditions and factors on the oxidation of polysorbate 80 (PS80) and of a monoclonal antibody (mAb) was evaluated. In particular, the role of different formulation components (e.g., mAb concentration, pH, buffer, surfactant grade, chelators) was investigated in the presence of iron as transition metal contaminant.The results of our studies demonstrated that PS80 oxidation was accelerated even in the presence of iron levels as low as 20 ppb. In addition, the results showed that the oxidation of a specific solvent-exposed mAb methionine increased with PS80 oxidation, in particular under accelerated stress conditions and that the oxidation phenomenon was hindered in absence of iron or after addition of EDTA. Our results showed that PS80 “all oleate” (PS80-AO) was more sensitive to oxidative degradation than PS80 “multi-compendial” (PS80-MC). Contrary to acetate and citrate buffers, the results showed that the kinetics of PS80 oxidation was pH-dependent in presence of histidine buffer. It was also demonstrated that, when increasing its concentration, the mAb exhibited a protective effect against metal catalyzed PS80 and methionine oxidation.Our systematic studies on the role of the formulation components and potential contaminants (i.e., iron) demonstrated the complexity of the oxidative mechanism and the importance of different competitive systems, including pro-oxidant factors (e.g., iron, pH, PS80 quality) and antioxidant factors (e.g., protein concentration, EDTA, citrate) that may occur in biologic formulations containing PS80.

Escherichia coli alanyl-tRNA synthetase maintains proofreading activity and translational accuracy under oxidative stress

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in Escherichia coli AlaRS, unlike in other aaRSs, this does not affect proofreading activity against the noncognate substrates serine and glycine and only results in a 1.6-fold decrease in efficiency of cognate Ala-tRNAAla formation. Mass spectrometry analysis of oxidized AlaRS revealed that the critical proofreading residue in the editing site, Cys666, and three methionine residues (M217 in the active site, M658 in the editing site, and M785 in the C-Ala domain) were modified to cysteine sulfenic acid and methionine sulfoxide, respectively. Alanine scanning mutagenesis showed that none of the identified residues were solely responsible for the change in cognate tRNAAla aminoacylation observed under oxidative stress, suggesting that these residues may act as reactive oxygen species “sinks” to protect catalytically critical sites from oxidative damage. Combined, our results indicate that E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.

Chemical Group-Based Metabolome Analysis Identifies Candidate Plasma Biomarkers Associated With Residual Feed Intake in Beef Steers

We applied chemical group-based metabolomics to identify blood metabolic signatures associated with residual feed intake in beef cattle. A group of 56 crossbred growing beef steers (average BW = 261.3 ± 18.5 kg) were adapted to a high-forage total mixed ration in a confinement dry lot equipped with GrowSafe intake nodes for period of 49 d to determine their residual feed intake classification (RFI). After RFI determination, weekly blood samples were collected three times from beef steers with the lowest RFI [most efficient (HFE);n= 8] and highest RFI and least-efficient [least efficient (LFE);n= 8]. Plasma was prepared by centrifugation and composited for each steer. Metabolome analysis was conducted using a chemical isotope labeling (CIL)/liquid chromatography–mass spectrometry, which permitted the analysis of metabolites containing amine/phenol-, carboxylic acid-, and carbonyl-chemical groups, which are metabolites associated with metabolisms of amino acids, fatty acids, and carbohydrates, respectively. A total number of 495 amine/phenol-containing metabolites were detected and identified; pathway analysis of all these metabolites showed that arginine biosynthesis and histidine metabolism were enriched (P< 0.10) in HFE, relative to LFE steers. Biomarker analyses of the amine/phenol-metabolites identified methionine, 5-aminopentanoic acid, 2-aminohexanedioic acid, and 4-chlorolysine as candidate biomarkers of RFI [false discovery rate ≤ 0.05; Area Under the Curve (AUC) > 0.90]. A total of 118 and 330 metabolites containing carbonyl- and carboxylic acid-chemical groups, respectively were detected and identified; no metabolic pathways associated with these metabolites were altered and only one candidate biomarker (methionine sulfoxide) was identified. These results identified five candidate metabolite biomarkers of RFI in beef cattle which are mostly associated with amino acid metabolism. Further validation using a larger cohort of beef cattle of different genetic pedigree is required to confirm these findings.

Betaine protects rats against ischemia/reperfusion injury-induced brain damage.

Brain ischemia and reperfusion (I/R) injury may lead to a poor prognosis for ischemic stroke, which could be alleviated by antioxidants with diminished oxidative stress. Betaine is a natural nutrient found in beetroot and seafood to improve cognitive performance in the elderly. The present study investigated whether betaine could protect the brain from I/R injury. Results showed that betaine treatment could reduce H2O2-induced cell death in the PC12 cell line. Pretreatment with betaine reduced the brain infarct volume and neuronal apoptosis in a rat I/R injury model induced by 2-h middle cerebral artery occlusion (MCAO). Biochemical analyses indicated that betaine treatment decreased proinflammatory cytokine production and reduced oxidative stress after I/R injury. Betaine increased the expression of antioxidative enzymes, such as glutathione peroxidase 4 (Gpx4) and superoxide dismutase 1 (Sod1), and antioxidative nonenzymatic genes, such as 3-mercaptopyruvate sulfurtransferase (Mpst), methionine sulfoxide reductases b1 (Msrb1), and Msrb2. These data suggest that betaine exerts a neuroprotective effect in I/R injury through enzymatic and nonenzymatic antioxidative systems and anti-inflammatory mechanisms.NEW & NOTEWORTHY These data suggested that betaine exerted a neuroprotective effect in I/R injury through enzymatic and nonenzymatic antioxidative systems.

γ-Aminobutyric acid boosts chilling tolerance by promoting the methionine sulfoxide reductase-thioredoxin reductase system in peach fruit

γ-Aminobutyric acid boosts chilling tolerance by promoting the methionine sulfoxide reductase-thioredoxin reductase system in peach fruit

Detecting Differences in Prion Protein Conformation by Quantifying Methionine Oxidation.

A prion’s pathogenic character is enciphered in its conformation, which also defines the chemical environments of its amino acids. Differences in chemical environments influence the reactivity of amino acid side chains, in a conformation-dependent manner. Chemical oxidation of susceptible methionines would identify those methionines on the surface of a prion, which would reveal conformation-dependent information. We identified a set of methionine-containing peptides derived from the tryptic, chymotryptic, or tryptic/chymotryptic digestion of recombinant prion protein and the Sc237 strain of hamster-adapted scrapie. We developed a multiple reaction monitoring-based method of quantifying the extent of the methionine oxidation in those peptides. This approach can be used to define a prion’s conformation and to distinguish among prion strains, which is an important component of food safety.

Design, Synthesis, and Evaluation of Probes for Spatially Resolved Imaging of Enantioselective Sulfoxide Reductases.

A novel concept of nonhydrolytic enzyme sensing based on aggregation-induced emission is described. As a proof of principle, fluorogenic probes for methionine sulfoxide reductases have been developed. Changes in the polarity and electronic nature upon reduction of sulfoxide to sulfide are translated to the aggregation potential of the probe. The new probes enable sensitive and highly spatially resolved imaging of the enzymatic activity.

A peptide-crosslinking approach identifies HSPA8 and PFKL as selective interactors of an actin-derived peptide containing reduced and oxidized methionine.

The oxidation of methionine to methionine sulfoxide occurs under conditions of cellular oxidative stress, and modulates the function of a diverse array of proteins. Enzymatic systems that install and reverse the methionine sulfoxide modifications have been characterized, however, little is known about potential readers of this oxidative modification. Here, we apply a peptide-crosslinking approach to identify proteins that are able to differentially interact with reduced and oxidized methionine-containing peptides. Specifically, we generated a photo-crosslinking peptide derived from actin, which contains two sites of methionine oxidation, M44 and M47. Our proteomic studies identified heat shock proteins, including HSPA8, as selective for the reduced methionine-containing peptide, whereas the phosphofructokinase isoform, PFKL, preferentially interacts with the oxidized form. We then demonstrate that the favored interaction of PFKL with oxidized methionine is also observed in the full-length actin protein, suggesting a role of methionine oxidation in regulating the actin-PFKL interaction in cells. Our studies demonstrate the potential to identify proteins that can differentiate between reduced and oxidized methionine and thereby mediate downstream protein functions under conditions of oxidative stress. Furthermore, given that numerous sites of methionine oxidation have now been identified, these studies set the stage to identify putative readers of methionine oxidation on other protein targets.

Plasma advanced glycation end products and the subsequent risk of microvascular complications in type 1 diabetes in the DCCT/EDIC

IntroductionTo assess impact of glycemic control on plasma protein-bound advanced glycation end products (pAGEs) and their association with subsequent microvascular disease.Research design and methodsEleven pAGEs were measured by liquid chromatography-mass...

Methionine oxidation of CLK4 promotes the metabolic switch and redox homeostasis in esophageal carcinoma via inhibiting MITF selective autophagy.

BackgroundMetabolic reprogramming and redox homeostasis contribute to esophageal squamous cell carcinoma (ESCC). CDC‐like kinase 4 (CLK4) is a dual‐specificity kinase that can phosphorylate substrates’ tyrosine or serine/threonine residue. However, the role and mechanism of CLK4 in ESCC remain unknown.MethodsCLK4 expression was analysed using publicly available datasets and confirmed in ESCC tissues and cell lines. The biological roles of CLK4 were studied with gain and loss‐of‐function experiments. Mass spectrometry was employed to examine the effects of CLK4 on metabolic profiling. In vitro kinase assay, co‐immunoprecipitation, glutathione S‐transferase pulldown, chromatin immunoprecipitation and luciferase reporter were used to elucidate the relationship among CLK4, microphthalmia‐associated transcription factor (MITF), COP1 and ZRANB1.ResultsCLK4 down‐regulation was observed in ESCC cell lines and clinical samples and associated with the methylation of its promoter. Low levels of CLK4 promoted ESCC development by affecting the purine synthesis pathway and nicotinamide adenine dinucleotide phosphate (NADPH)/nicotinamide adenine dinucleotide phosphate (NADP+) ratio. Interestingly, CLK4 inhibited ESCC development by blocking MITF‐enhanced de novo purine synthesis and redox balance. Mechanistically, wild type CLK4 (WT‐CLK4) but not kinase‐dead CLK4‐K189R mutant phosphorylated MITF at Y360. This modification promoted its interaction with E3 ligase COP1 and its K63‐linked ubiquitination at K308/K372, leading to sequestosome 1 recognition and autophagic degradation. However, the deubiquitinase ZRANB1 rescued MITF ubiquitination and degradation. In turn, MITF bound to E‐ rather than M‐boxes in CLK4 promoter and transcriptionally down‐regulated its expression in ESCC. Clinically, the negative correlations were observed between CLK4, MITF, and purine metabolic markers, which predicts a poor clinical outcome of ESCC patients. Notably, CLK4 itself was a redox‐sensitive kinase, and its methionine oxidation at M307 impaired kinase activity, enhanced mitochondria length and inhibited lipid peroxidation, contributing to ESCC.ConclusionsOur data highlight the potential role of CLK4 in modulating redox status and nucleotide metabolism, suggesting potential therapeutic targets in ESCC treatment.

Chalcogen-based ratiometric reversible BODIPY redox sensors for the determination of enantioselective methionine sulfoxide reductase activity.

Many serious diseases are associated with degenerative changes caused by oxidative stress triggered by elevated concentrations of reactive oxygen species (ROS) in cells. Therefore, the development of suitable probes for monitoring such processes is of great importance. Here, we introduce a series of sulfur- and selenium-substituted BODIPY derivatives as reversible redox sensors for ROS and enzymatic redox processes. Significant differences in emission maxima and fluorescence quantum yields between the reduced and oxidized forms make them excellent ratiometric turn-on/off probes. Installation of polar sulfonate groups improved their aqueous solubility while retaining their sensing properties, which allowed the probes to monitor the enzymatic activity of enantioselective methionine sulfoxide reductase.

“Comparative Assessment of Cytoplasmic (MsrA and MsrC) and Periplasmic (MsrP) Methionine Sulfoxide Reductases in Stress Survival and Virulence of S. Typhimurium”

“Comparative Assessment of Cytoplasmic (MsrA and MsrC) and Periplasmic (MsrP) Methionine Sulfoxide Reductases in Stress Survival and Virulence of S. Typhimurium”

Quantifying methionine sulfoxide in therapeutic protein formulation excipients as sensitive oxidation marker

Methionine is a common excipient used in therapeutic protein liquid formulations as stabilizer and antioxidant. The oxidation of methionine to methionine sulfoxide can be regarded as a sensitive marker of oxidative stress for drug product storage conditions. In this study, a sensitive HPLC method for the quantification of methionine sulfoxide in formulated protein product was developed and qualified according to regulatory requirements using a SIELC® Primesep 100 column with UV detection. The separation involves a mixed-mode mechanism including reversed phase and cationic exchange modalities. The operating range of the method was established between 1 µM and 35 µM of methionine sulfoxide. In this testing range, the method was shown to be linear (R2 > 0.99), accurate (Recovery 92.9 – 103.6%, average recovery = 99.8 ± 1.4%) and precise (intermediate precision at LoQ, CV = 2.9%). The developed test system was successfully applied to study the effects of temperature and storage conditions on methionine sulfoxide formation in complex therapeutic antibody formulations.

Chemical Stability of Proteins in Foods: Oxidation and the Maillard Reaction.

Protein is a major nutrient present in foods along with carbohydrates and lipids. Food proteins undergo a wide range of modifications during food production, processing, and storage. In this review, we discuss two major reactions, oxidation and the Maillard reaction, involved in chemical modifications of food proteins. Protein oxidation in foods is initiated by metal-, enzyme-, or light-induced processes. Food protein oxidation results in the loss of thiol groups and the formation of protein carbonyls and specific oxidation products of cysteine, tyrosine, tryptophan, phenylalanine, and methionine residues, such as disulfides, dityrosine, kynurenine, m-tyrosine, and methionine sulfoxide. The Maillard reaction involves the reaction of nucleophilic amino acid residues with reducing sugars, which yields numerous heterogeneous compounds such as α-dicarbonyls, furans, Strecker aldehydes, advanced glycation end-products, and melanoidins. Both protein oxidation and the Maillard reaction result in the loss of essential amino acids but may positively or negatively impact food structure and flavor.

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.