Methionine Residues In Proteins Research Articles

On the electrochemical oxidation of methionine residues of proteins

The oxidation of methionine side chain residues in proteins provides a great diversity of possible oxidation mechanisms dictated, among others, by the oxidizing species, solvent properties, and protein structure. The oxidation behavior of a series of short acetylated synthetic peptides, Ac-GMG, Ac-GGMGG and Ac-GGGMGGG, was investigated by differential pulse and square wave voltammetry, in a wide pH range, at glassy carbon electrode. It was found that always the first oxidation step represents the one-electron oxidation of thioether moiety with the formation of a radical cation. Following this, depending on the experimental conditions and side chain position of methionine, the radical is stabilized by the nucleophilic attack of water or by catalytic support of the neighboring carbonyl and amide groups in an intermediate structure, finally converted in methionine sulfoxide which can be further oxidized, at more positive potential, into methionine sulfone. For Ac-GGGMGGG, at pH 8.0, the amino and amide groups are active involved in the oxidation process and the electrode reaction takes place with proton transfer

An Intramolecular Charge Transfer-Förster Resonance Energy Transfer Integrated Unimolecular Platform for Two-Photon Ratiometric Fluorescence Sensing of Methionine Sulfoxide Reductases in Live-Neurons and Mouse Brain Tissues.

Oxidative stress in organisms is a factor leading to a series of diseases including tumors and neurological disorders, while methionine sulfoxide reductases (Msrs) may provide an antioxidant and self-repair mechanism through redox cycles of methionine residues in proteins. Thus, it is important to understand the crucial role of Msrs in maintaining the redox homeostasis. However, it remains a great challenge for real-time and quantitative monitoring of Msrs in live systems due to the lack of appropriate sensing tools. Herein, a novel unimolecular platform integrating the intramolecular charge transfer (ICT) and Förster resonance energy transfer (FRET) dual mechanisms was successfully developed. By employing the highly specific Msrs-catalyzed reduction from the electron-withdrawing sulfoxide moiety in the probe to an electron-donating sulfide group, a synergistic ICT-FRET activation process was achieved, leading to a ratiometric fluorescence response toward Msrs with high selectivity, sensitivity, and accuracy. Moreover, benefiting from the favorable features, including mitochondria-targeting, near-infrared two-photon excitation, low cytotoxicity, good stability, and biocompatibility, the probe was successfully used for monitoring mitochondrial Msrs levels in live-neurons, and a positively correlated up-regulation of endogenous Msrs levels under O2•- stimulation was observed for the first time, confirming a Msrs-involved adaptive antioxidant mechanism in neurons. Furthermore, two-photon microscopic imaging of various regions in Alzheimer's disease (AD) mice brains revealed a down-regulated Msrs levels compared with that in normal brains, especially in the cornuammonis of the hippocampus region, which may in turn lead to an aggravation of AD pathogenesis due to the weakened antioxidant and self-repair capability of neurons.

Staphylococcus aureus Peptide Methionine Sulfoxide Reductases Protect from Human Whole-Blood Killing.

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 (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr 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 Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr 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.

Role of methionine sulfoxide reductase in Alzheimer's disease

Cellular oxidative damage has been implicated as one of the factors in Alzheimer's disease (AD) pathogenesis. One of the enzyme systems preventing protein oxidation in mammalian cells is the methionine sulfoxide reductase (MSR) system; MSR system is comprised of MSRA and MSRB, which reduce oxidized methionine residues (Met(O)) present in proteins back to methionine. Further, the MSR system scavenges reactive oxygen species (ROS), by permitting methionine residues in proteins to function as catalytic antioxidants. There is growing evidence from many studies on the affect of altered MSRA levels on AD progression in different animal models and, also that oxidized Met 35 will promote aggregation of toxic forms of Aß. Our research aims to further understand the role of MSRA and Met35 in the pathogenic processes of AD. For our studies we have used, AD transgenic mice, J20 APP-Tg that overexpresses Aß to analyze MSR levels and, variants of commercially available Aß (1-42) to assess the effect of methionine oxidation. Our preliminary results indicate that the level of MSRA was decreased in the transgenic mice as compared to the wild type mice. We have also observed in Aß peptide aggregation experiments, oxidized Met35 Aβformed high molecular weight fibrillar aggregates compared to the WT- Aβ and, if Met is replaced with norleucine (Nle) only monomers were formed. These results suggest that MSRA plays a role in Aß aggregation and these studies provide a potential therapeutic target for AD.

Lack of the antioxidant enzyme methionine sulfoxide reductase A in mice impairs RPE phagocytosis and causes photoreceptor cone dysfunction

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.

On the functionality of a methionine sulfoxide reductase B from Trypanosoma cruzi

BackgroundMethionine 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. MethodsWe 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. ResultsMSRB 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 significanceThis 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.

Molecular Expression of Bioactive Recombinant Methionine Sulfoxide Reductase A (MsrA).

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. The expression of catalytically active recombinant MsrA (rMsrA). 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. 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. 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.

Repurposing the Pummerer Rearrangement: Determination of Methionine Sulfoxides in Peptides

The reversible oxidation of methionine residues in proteins has emerged as a biologically important post‐translational modification. However, detection and quantitation of methionine sulfoxide in proteins is difficult. Our aim is to develop a method for specifically derivatizing methionine sulfoxide residues. We report a Pummerer rearrangement of methionine sulfoxide treated sequentially with trimethylsilyl chloride and then 2‐mercaptoimidazole or pyridine‐2‐thiol to produce a dithioacetal product. This derivative is stable to standard mass spectrometry conditions, and its formation identified oxidized methionine residues. The scope and requirements of dithioacetal formation are reported for methionine sulfoxide and model substrates. The reaction intermediates have been investigated by computational techniques and by 13C NMR spectroscopy. These provide evidence for an α‐chlorinated intermediate. The derivatization allows for detection and quantitation of methionine sulfoxide in proteins by mass spectrometry and potentially by immunochemical methods.

Methionine sulfoxide reductase reverses oxidation of the nucleotide exchange factor Fes1 to regulate cytoplasmic Hsp70 chaperone cycle

Methionine residues in proteins are targets of oxidation by reactive oxygen species (ROS). Selective oxidation of methionine to methionine sulfoxide (MetO) can allow for reversible, beneficial modification of proteins to limit cell damage during conditions of excess ROS. Methionine sulfoxide reductases (Msrs) are conserved enzymes that reduce MetO residues in proteins, serving to both limit the over‐oxidation of protein methionines, and to remove MetO adducts when stress subsides. Although loss of Msr function has been implicated in neurodegenerative disorders and aging, it is unclear how Msr activities influence these disease pathologies because the vast majority of their physiological substrates remain unknown.We have identified the cytoplasmic Hsp70 nucleotide exchange factor Fes1 as an Msr substrate in yeast. Under oxidative stress, Fes1 is oxidized at a cluster of N‐terminal methionine residues. Current data show that this Msr‐controlled modification reversibly regulates interaction between Fes1 and Hsp70, thereby modulating chaperone nucleotide exchange, ATP hydrolysis, and peptide binding and release. We suggest that these altered Hsp70 activities, as a consequence of Fes1 modification, may help cells cope with elevated ROS by limiting peptide aggregation and/or activating cellular stress response pathways, whereas sustained modification of Fes1 in the absence of Msr activity is detrimental to proteostasis under non‐stress conditions. Moreover, our studies with Fes1 uncovered that the two yeast Msr proteins (Mxr1 and Mxr2) are expressed in the cytoplasm, in contrast to prior studies of Mxr2 suggesting sole mitochondrial localization. We show that Mxr1 and Mxr2 co‐operate to regulate the redox state of Fes1 both in vivo and in vitro. Together, these studies provide a new perspective on how activities of the well‐studied Hsp70 family can be modulated by ROS, and further define how methionine modification is used as a means for cells to sense and respond to oxidative stress.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abscisic acid-dependent nitric oxide pathway and abscisic acid-independent nitric oxide routes differently modulate NaCl stress induction of the gene expression of methionine sulfoxide reductase A and B in rice roots

The methionine residues of proteins are the preferred targets of oxidation by reactive oxygen species resulting in the formation of methionine sulfoxide (MetSO), which impairs protein function. Methionine sulfoxide reductase A and B (MSR) catalyze the reduction of the MetSO S and R epimers back to Met residues, respectively. The roles of abscisic acid (ABA) and nitric oxide (NO) on the transcript levels of methionine sulfoxide reductase (MSR; EC 1.8.4.6) in the roots of 2-d-old etiolated rice (Oryza sativa L.) seedlings exposed to NaCl were examined. The OsMSR transcript levels increased upon exposure to NaCl, which increased as the NaCl concentrations increased. Fluridone (Flu) pretreatment inhibited the increases in ABA and NO contents and the OsMSRA4, OsMSRA5, OsMSRB1.1, OsMSRB3 and OsMSRB5 transcripts by NaCl, while ABA application reversed the effects of Flu. Flu did not affect the OsMSRA2 and OsMSRB1.2 transcripts. The application of the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), to NaCl-treated roots inhibited the increase in all of the OsMSRs transcripts with the exception of OsMSRB1.2. Treatment with the NO donor sodium nitroprusside (SNP) increased all the OsMSRs transcripts. The inhibitory effect of Flu on the increase of the OsMSRA4, OsMSRA5, OsMSR1.1, OsMSRB 3, and OsMSRB5 transcripts in the NaCl-treated roots was reversed by SNP. cPTIO inhibited the expression of all the OsMSR genes. The OsMSRA2.1 and OsMSRB1.2 transcripts can be increased by SNP. The Flu-inhibited internal ABA contents cannot be recovered by treatment with cPTIO or SNP. In addition, NaCl-induced NO production can be divided into ABA-dependent and ABA-independent routes. Therefore, the ABA-dependent NO route regulated the expression of OsMSRA4, OsMSRA5, OsMSRB1.1, OsMSRB 3, and OsMSRB5 in the NaCl-treated rice roots, while the ABA-independent NO pathway modulated OsMSRA2.1, and the ABA-independent and NO-independent pathway modulated OsMSRB1.2 expression in response to NaCl treatment.

Mitigation of Oxidation in Therapeutic Antibody Formulations: a Biochemical Efficacy and Safety Evaluation of N-Acetyl-Tryptophan and L-Methionine.

Biotherapeutics can be susceptible to oxidation during manufacturing and storage. Free L-methionine is known to protect methionine residues in proteins from oxidation. Similarly, free tryptophan and other indole derivatives have been shown to protect tryptophan residues from oxidation. N-acetyl-DL-tryptophan was previously identified as a potentially superior antioxidant to tryptophan as it has a lower oxidation potential and produces less peroxide upon light exposure. This study sought to confirm the antioxidant efficacy and safety of N-acetyl-DL-tryptophan and L-methionine as formulation components for biotherapeutic drugs. Antibodies were subjected to AAPH and light exposure in the presence of N-acetyl-DL-tryptophan and L-methionine. Oxidation in relevant CDR and Fc residues was quantified by peptide map. In silico, in vitro, and in vivo studies were performed to evaluate the safety of N-acetyl-DL-tryptophan and L-methionine. Peptide mapping demonstrated that N-acetyl-DL-tryptophan was effective at protecting tryptophans from AAPH stress, and that the combination of N-acetyl-DL-tryptophan and L-methionine protected both tryptophan and methionine from AAPH stress. The safety assessment suggested an acceptable safety profile for both excipients. N-acetyl-tryptophan and L-methionine effectively reduce the oxidation of susceptible tryptophan and methionine residues in antibodies and are safe for use in parenteral biotherapeutic formulations.

Myristoylated methionine sulfoxide reductase A is a late endosomal protein

Methionine residues in proteins provide antioxidant defense by reacting with oxidizing species, which oxidize methionine to methionine sulfoxide. Reduction of the sulfoxide back to methionine is catalyzed by methionine sulfoxide reductases, essential for protection against oxidative stress. The nonmyristoylated form of methionine sulfoxide reductase A (MSRA) is present in mitochondria, whereas the myristoylated form has been previously reported to be cytosolic. Despite the importance of MSRA in antioxidant defense, its in vivo binding partners and substrates have not been identified. Starting with a protein array, and followed by immunoprecipitation experiments, colocalization studies, and subcellular fractionation, we identified the late endosomal protein, StAR-related lipid transfer domain-containing 3 (STARD3), as a binding partner of myristoylated MSRA, but not of nonmyristoylated MSRA. STARD3 is known to have both membrane-binding and cytosolic domains that are important in STARD3-mediated transport of cholesterol from the endoplasmic reticulum to the endosome. We found that the STARD3 cytosolic domain localizes MSRA to the late endosome. We propose that the previous conclusion that myristoylated MSRA is strictly a cytosolic protein is artifactual and likely due to vigorous overexpression of MSRA. We conclude that myristoylated MSRA is a late endosomal protein that may play a role in lipid metabolism or may protect endosomal proteins from oxidative damage.

Methionine sulfoxide reductase B3 requires resolving cysteine residues for full activity and can act as a stereospecific methionine oxidase.

The oxidation of methionine residues in proteins occurs during oxidative stress and can lead to an alteration in protein function. The enzyme methionine sulfoxide reductase (Msr) reverses this modification. Here, we characterise the mammalian enzyme Msr B3. There are two splice variants of this enzyme that differ only in their N-terminal signal sequence, which directs the protein to either the endoplasmic reticulum (ER) or mitochondria. We demonstrate here that the enzyme can complement a bacterial strain, which is dependent on methionine sulfoxide reduction for growth, that the purified recombinant protein is enzymatically active showing stereospecificity towards R-methionine sulfoxide, and identify the active site and two resolving cysteine residues. The enzyme is efficiently recycled by thioredoxin only in the presence of both resolving cysteine residues. These results show that for this isoform of Msrs, the reduction cycle most likely proceeds through a three-step process. This involves an initial sulfenylation of the active site thiol followed by the formation of an intrachain disulfide with a resolving thiol group and completed by the reduction of this disulfide by a thioredoxin-like protein to regenerate the active site thiol. Interestingly, the enzyme can also act as an oxidase catalysing the stereospecific formation of R-methionine sulfoxide. This result has important implications for the role of this enzyme in the reversible modification of ER and mitochondrial proteins.

Modeling the Oxidation of Methionine Residues by Peroxides in Proteins

We report the use of molecular modeling to predict the oxidation propensity of methionine residues in proteins. Oxidation of methionine to the sulfoxide form is one of the major degradation pathways for therapeutic proteins. Oxidation can occur during production, formulation, or storage of pharmaceuticals and it often reduces or eliminates biological activity. We use a molecular model based on atomistic simulations called 2-shell water coordination number to predict the oxidation rates for several model proteins and therapeutic candidates. In addition, we implement models that are based on static and simulation average of the solvent-accessible area (SAA) for either the side chain or the sulfur atom in the methionine residue. We then compare the results from the different models against the experimentally measured relative rates of methionine oxidation. We find that both the 2-shell model and the simulation-averaged SAA models are accurate in predicting the oxidation propensity of methionine residues for the proteins tested. We also find the appropriate parameter ranges where the models are most accurate. These models have significant predictive power and can be used to enable further protein engineering or to guide formulation approaches in stabilizing the unstable methionine residues.

Symposium 6: Trace Elements S6-1 - Selenium and Methionine Sulfoxide Reduction

Selenium is an essential trace element because it is present in proteins in the form of selenocysteine residue. Functionally characterized selenoproteins are oxidoreductases. Selenoprotein methionine-R-sulfoxide reductase B1 (MsrB1) is a repair enzyme that reduces ROS-oxidized methionine residues in proteins. Here, we explored a possibility that reversible methionine oxidation is also a mechanism that regulates protein function. We found that MsrB1, together with Mical proteins, regulated mammalian actin assembly via stereospecific methionine oxidation and reduction in a reversible, site-specific manner. Two methionine residues in actin were specifically converted to methionine-R-sulfoxide by Mical1 and Mical2 and reduced back to methionine by MsrB1, supporting actin disassembly and assembly, respectively. Macrophages utilized this redox control during cellular activation by stimulating MsrB1 expression and activity. Thus, we identified the regulatory role of MsrB1 as a Mical antagonist in orchestrating actin dynamics and macrophage function. More generally, our study showed that proteins can be regulated by reversible site-specific methionine-R-sulfoxidation and that selenium is involved in this regulation by being a catalytic component of MsrB1.

Abstract 2917: The role of Methionine sulfoxide reductase A in metastasis of breast cancer.

Abstract The role of oxidative stress in several human diseases is very well established, but the effect of oxidative stress on cancer cells, especially resulting from mitochondrial dysfunction, appears to be quite complex. Warburg first showed that cancer cells have a defect in mitochondrial respiration which explains why these cells have a greater dependency on glycolysis for energy production than normal cells, even in the presence of adequate oxygen. Cancer cells have been reported to be more sensitive to reactive oxygen species (ROS) and are known to have lower levels of several antioxidant enzymes, which are thought to function as tumor suppressors. In support of this are recent findings that indicate increased levels of ROS make cancer cells more aggressive and promote metastasis. However, in contrast there are other studies that indicate elevated levels of antioxidant enzymes drive tumor cells to metastasize and also prolong tumor cell survival. Our goal is to clarify the role of oxidative stress and ROS on cancer cell viability and aggressiveness. The focus will be on an important protein repair and catalytic antioxidant enzyme system, methionine sulfoxide reductase (Msr). The Msr system in mammalian cells (comprised of MsrA, MsrB1, MsrB2, MsrB3) reduces the oxidized methionine residues (Met(O)) in proteins back to methionine. Furthermore, the Msr system plays an important role in scavenging ROS by permitting methionine residues in proteins to function as catalytic antioxidants. MsrA has been studied in most detail. This enzyme plays an important role in protection against increased oxidative stress in a variety of organisms and can affect the life span of yeast and animals. The objective of our study was to determine whether the expression of MsrA is altered during tumor progression and to identify the molecular targets that regulate MsrA. Our preliminary data show that MsrA is downregulated in several types of breast cancer. We have used two lines of human mammary epithelial 21T series that represent different stages of tumor progression from a single patient and compared with metastatic human breast cancer cell line MDA-MB-231. Our data indicates that non-metastatic 21PT has higher protein levels of MsrA compared to both metastatic 21MT-1 and MDA-MB-231 cell lines as evidenced by western blots. MsrA protein levels of nonmetastatic 21PT are much lower than normal epithelial cells. Levels of MsrA activity were higher in nonmetastatic 21PT than metastatic 21MT-1 as determined by enzymatic activity assays. Enzyme activity of MsrA is nearly two fold higher in nonmetastatic 21PT than the MsrA levels in metastatic MDA-MB-231 cells. Our preliminary results also indicate upregulation of oncogenic (c-Myc) protein levels in metastatic lines suggesting an inverse correlation with MsrA. It is therefore likely that MsrA may be an important antioxidant enzyme in breast cancer cells whose regulation drives tumor progression. Citation Format: Shailaja Kesaraju, Xiaoping Wu, Diane Baronas-Lowell, Herbert Weissbach. The role of Methionine sulfoxide reductase A in metastasis of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2917. doi:10.1158/1538-7445.AM2013-2917

Wanted and wanting: Antibody against methionine sulfoxide

Methionine residues in protein can be oxidized by reactive oxygen or nitrogen species to generate methionine sulfoxide. This covalent modification has been implicated in processes ranging from normal cell signaling to neurodegenerative diseases. A general method for detecting methionine sulfoxide in proteins would be of great value in studying these processes, but development of a chemical or immunochemical technique has been elusive. Recently, an antiserum raised against an oxidized corn protein, DZS18, was reported to be specific for methionine sulfoxide in proteins (Arch. Biochem. Biophys. 485:35–40; 2009). However, data included in that report indicate that the antiserum is not specific. Utilizing well-characterized native and methionine-oxidized glutamine synthetase and aprotinin, we confirm that the antiserum does not possess specificity for methionine sulfoxide.

Characterization and Solution Structure of Mouse Myristoylated Methionine Sulfoxide Reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. The cytosolic form of the enzyme is myristoylated, but it is not known to translocate to membranes, and the function of myristoylation is not established. We compared the biochemical and biophysical properties of myristoylated and nonmyristoylated mouse methionine sulfoxide reductase A. These were almost identical for both forms of the enzyme, except that the myristoylated form reduced methionine sulfoxide in protein much faster than the nonmyristoylated form. We determined the solution structure of the myristoylated protein and found that the myristoyl group lies in a relatively surface exposed "myristoyl nest." We propose that this structure functions to enhance protein-protein interaction.

Structural and biochemical analysis of mammalian methionine sulfoxide reductase B2

Methionine sulfoxide reductases are antioxidant enzymes that repair oxidatively damaged methionine residues in proteins. Mammals have three members of the methionine-R-sulfoxide reductase family, including cytosolic MsrB1, mitochondrial MsrB2, and endoplasmic reticulum MsrB3. Here, we report the solution structure of reduced Mus musculus MsrB2 using high resolution nuclear magnetic resonance (NMR) spectroscopy. MsrB2 is a β-strand rich globular protein consisting of eight antiparallel β-strands and three N-terminal α-helical segments. The latter secondary structure elements represent the main structural difference between mammalian MsrB2 and MsrB1. Structural comparison of mammalian and bacterial MsrB structures indicates that the general topology of this MsrB family is maintained and that MsrB2 more resembles bacterial MsrBs than MsrB1. Structural and biochemical analysis supports the catalytic mechanism of MsrB2 that, in contrast to MsrB1, does not involve a resolving cysteine (Cys). pH dependence of catalytically relevant residues in MsrB2 was accessed by NMR spectroscopy and the pK(a) of the catalytic Cys162 was determined to be 8.3. In addition, the pH-dependence of MsrB2 activity showed a maximum at pH 9.0, suggesting that deprotonation of the catalytic Cys is a critical step for the reaction. Further mobility analysis showed a well-structured N-terminal region, which contrasted with the high flexibility of this region in MsrB1. Our study highlights important structural and functional aspects of mammalian MsrB2 and provides a unifying picture for structure-function relationships within the MsrB protein family.

Anoxia, acidosis, and intergenic interactions selectively regulate methionine sulfoxide reductase transcriptions in mouse embryonic stem cells

Methionine sulfoxide reductases (Msr) belong to a gene family that contains one MsrA and three MsrBs (MsrB1, MsrB2, and MsrB3). We have identified all four of the genes that are expressed in mouse embryonic stem cell cultures. The vital cellular functions of the Msr family of genes are to protect cells from oxidative damage by enzymatically reducing the oxidized sulfide groups of methionine residues in proteins from the sulfoxide form (--SO) back to sulfide thus restoring normal protein functions as well as reducing intracellular reactive oxygen species (ROS). We have performed studies on the Msr family genes to examine the regulation of gene expression. Our studies using real-time RT-PCR and Western blotting have shown that expression levels of the four Msr family genes are under differential regulation by anoxia/reoxygenation treatment, acidic culture conditions and interactions between MsrA and MsrB. Results from these in vitro experiments suggest that although these genes function as a whole in oxidative stress protection, each one of the Msr genes could be responsive to environmental stimulants differently at the tissue level.