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

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

Methionine Sulfoxide Reductase A (MsrA) and Its Function in Ubiquitin-Like Protein Modification in Archaea.

Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme found in all domains of life that catalyzes the reduction of methionine-S-sulfoxide (MSO) to methionine in proteins and free amino acids. We demonstrate that archaeal MsrA has a ubiquitin-like (Ubl) protein modification activity that is distinct from its stereospecific reduction of MSO residues. MsrA catalyzes this Ubl modification activity, with the Ubl-activating E1 UbaA, in the presence of the mild oxidant dimethyl sulfoxide (DMSO) and in the absence of reductant. In contrast, the MSO reductase activity of MsrA is inhibited by DMSO and requires reductant. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis reveals that MsrA-dependent Ubl conjugates are associated with DNA replication, protein remodeling, and oxidative stress and include the Ubl-modified MsrA, Orc3 (Orc1/Cdc6), and Cdc48d (Cdc48/p97 AAA+ ATPase). Overall, we found archaeal MsrA to have opposing MSO reductase and Ubl modifying activities that are associated with oxidative stress responses and controlled by exposure to mild oxidant.IMPORTANCE Proteins that are damaged by oxidative stress are often targeted for proteolysis by the ubiquitin-proteasome system (UPS). The mechanisms that control this response are poorly understood, especially under conditions of mild oxidative stress when protein damage is modest. Here, we discovered a novel function of archaeal MsrA in guiding the Ubl modification of target proteins in the presence of mild oxidant. This newly reported activity of MsrA is distinct from its stereospecific reduction of methionine-S-sulfoxide to methionine residues. Our results are significant steps forward, first, in elucidating a protein factor that guides Ubl modification in archaea, and second, in providing an insight into oxidative stress responses that can trigger Ubl modification in a cell.

Hepatic overexpression of methionine sulfoxide reductase A reduces atherosclerosis in apolipoprotein E-deficient mice.

Methionine sulfoxide reductase A (MsrA), a specific enzyme that converts methionine-S-sulfoxide to methionine, plays an important role in the regulation of protein function and the maintenance of redox homeostasis. In this study, we examined the impact of hepatic MsrA overexpression on lipid metabolism and atherosclerosis in apoE-deficient (apoE(-/-)) mice. In vitro study showed that in HepG2 cells, lentivirus-mediated human MsrA (hMsrA) overexpression upregulated the expression levels of several key lipoprotein-metabolism-related genes such as liver X receptor α, scavenger receptor class B type I, and ABCA1. ApoE(-/-) mice were intravenously injected with lentivirus to achieve high-level hMsrA expression predominantly in the liver. We found that hepatic hMsrA expression significantly reduced plasma VLDL/LDL levels, improved plasma superoxide dismutase, and paraoxonase-1 activities, and decreased plasma serum amyloid A level in apoE(-/-) mice fed a Western diet, by significantly altering the expression of several genes in the liver involving cholesterol selective uptake, conversion and excretion into bile, TG biosynthesis, and inflammation. Moreover, overexpression of hMsrA resulted in reduced hepatic steatosis and aortic atherosclerosis. These results suggest that hepatic MsrA may be an effective therapeutic target for ameliorating dyslipidemia and reducing atherosclerosis-related cardiovascular diseases.

Regulation of expression of sodA and msrA genes of Corynebacterium glutamicum in response to oxidative and radiative stress.

Promoters of genes encoding superoxide dismutase (sodA) and peptide methionine sulfoxide reductase (msrA) from Cory-nebacterium glutamicum were cloned and sequenced. Promoter region analysis of sodA-msrA was unable to identify putative sites of fixed eventual regulators except for possible sites of fixed OxyR and integra-tion host factor. A study of the regulation of these genes was performed using the lacZ gene of Escherichia coli as a reporter placed under the control of sequences downstream of sodA and msrA. In silico analysis was used to identify regulators in the genome of C. glutamicum, which revealed the absence of homologs of soxRS and arcA and the presence of inactive oxyR and putative candidates of the homologs of ahpC, ohrR, integration host factor, furA, IdeR, diphtheria toxin repressor, and mntR.

Influence of DMF-induced oxidative stress on membrane and periplasmic proteins in Paracoccus sp. SKG.

The present study describes the N,N-dimethylformamide (DMF)-induced oxidative stress in Paracoccus sp. SKG. The oxidative stress was evaluated by analysing membrane and periplasmic proteins and K+ efflux, as well as by monitoring the activities of antioxidant enzymes like catalase, superoxide dismutase (SOD) and glutathione S-transferase (GST). The exposure of bacterial cells to a higher concentration of DMF resulted in the modification of membrane fatty acid composition which is accompanied by K+ efflux. Further, this oxidative stress resulted in increased periplasmic protein which can be attributed to the induction of GST and methionine sulphoxide reductase (Msr) enzymes under solvent stress. Paracoccus sp. SKG is tolerant to high concentrations of DMF up to 6% (v/v) and its toxic effects. DMF concentration-dependent induction of GST and Msr activities advocates the significant role of these enzymes in the bacterial defence system. The present study provides information which helps us to understand the ROS scavenging machinery in bacteria. The high tolerance of Paracoccus sp. SKG to DMF can be efficiently explored for various bioremediation and biotransformation applications.

Expression and biological properties of a novel methionine sulfoxide reductase A in tobacco (Nicotiana tabacum).

Methionine (Met) residues in proteins/peptides are extremely susceptible to oxidation mediated by reactive oxygen species, resulting in the formation of methionine sulfoxide, which could be inversely reduced back to Met by methionine sulfoxide reductase (MSR). In the present study, an A-type MSR gene, termed NtMSRA4, was isolated from tobacco (Nicotiana tabacum). Sequence analysis of NtMSRA4 amino acid sequence indicated that the gene, encoded a polypeptide with a molecular weight of 21 kDa, possessed the highly conserved motif, 'GCFWG' in the N-terminus and 'KGCNDPIRCY' motif in the C-terminus respectively. Substrate specific analysis revealed that recombinant NtMSRA4 protein could reduce specifically S-isomer of Dabsyl-MetSO to Dabsyl-Met in vitro using dithiothreitol as an electron donor. Enzymatic properties analysis showed that the temperature of 42 °C and pH 9.0 were optimum for NtMSRA4 activity. The K m and K cat values of NtMSRA4 were determined to be 40.04 µM and 0.048 S(-1) in the thioredoxin dependent reduction system. Overexpression of NtMSRA4 in E. coli cells enhanced resistance to H2O2 toxicity. Subcellular localization result showed that NtMSRA4 was located in the chloroplast. The expression level of NtMSRA4 was affected differently after exposure to various abiotic stresses.

Induction of methionine sulfoxide reductase activity by pergolide, pergolide sulfoxide, and S-adenosyl-methionine in neuronal cells.

The reduction of methionine sulfoxide in proteins is facilitated by the methionine sulfoxide reductase (Msr) system. The Msr reduction activity is important for protecting cells from oxidative stress related damages. Indeed, we have recently shown that treatment of cells with N-acetyl-methionine sulfoxide can increase Msr activity and protect neuronal cells from amyloid beta toxicity. Thus, in search of other similar Msr-inducing molecules, we examined the effects of pergolide, pergolide sulfoxide, and S-adenosyl-methionine on Msr activity in neuronal cells. Treatment of neuronal cells with a physiological range of pergolide and pergolide sulfoxide (0.5-1.0 µM) caused an increase of about 40% in total Msr activity compared with non-treated control cells. This increase in activity correlated with similar increases in methionine sulfoxide reductase A protein expression levels. Similarly, treatment of cells with S-adenosyl methionine also increased cellular Msr activity, which was milder compared to increases induced by pergolide and pergolide sulfoxide. We found that all the examined compounds are able to increase cellular Msr activity to levels comparable to N-acetyl-methionine sulfoxide treatment. Pergolide, pergolide sulfoxide, and S-adenosyl methionine can cross the blood-brain barrier. Therefore, we hypothesize that they can be useful in the treatment of symptoms/pathologies that are associated with reduced Msr activity.

Mulberry (Morus L.) methionine sulfoxide rreductase gene cloning, sequence analysis, and expression in plant development and stress response.

Methionine sulfoxide reductase plays a regulatory role in plant growth and development, especially in scavenging reactive oxygen species by restoration of the oxidation of methionine in protein. A full-length cDNA sequence encoding methionine sulfoxide reductase (MSR) from mulberry, which we designated MMSR, was cloned based on mulberry expressed sequence tags (ESTs). Sequence analysis showed that the MMSR is 810 bp long, encoding 194 amino acids with a predicted molecular weight of 21.6 kDa and an isoelectric point of 6.78. The expression level of the MMSR gene under conditions of drought and salt stresses was quantified by qRT-PCR. The results show that the expression level changed significantly under the stress conditions compared to the normal growth environment. It helps us to get a better understanding of the molecular basis for signal transduction mechanisms underlying the stress response in mulberry.

Oxidation of methionine 35 reduces toxicity of the amyloid beta-peptide(1-42) in neuroblastoma cells (IMR-32) via enzyme methionine sulfoxide reductase A expression and function.

The beta amyloid peptide (Abeta), the major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of free radicals that may lead to neurodegeneration. Our recent evidence suggest that the redox state of methionine residue in position 35 (Met-35) of Abeta has the ability to deeply modify peptide's neurotoxic actions. Reversible oxidation of methionine in proteins involving the enzyme methionine sulfoxide reductase type A (MsrA) is postulated to serve a general antioxidant role and a decrease in MsrA has been implicated in Alzheimer's disease. In rat neuroblastoma cells (IMR-32), we used Abeta(1-42), in which the Met-35 is present in the reduced state, with a modified peptide with oxidized Met-35 (Abeta(1-42)Met35(OX)), as well as an Abeta-derivative in which Met-35 is substituted with norleucine (Abeta(1-42)Nle35) to investigate the relationship between Met-35 redox state, expression and function of MsrA and reactive oxygen species (ROS) generation. The obtained results shown that MsrA activity, as well as mRNA levels, increase in IMR-32 cells treated with Abeta(1-42)Met35(OX), differently to that shown by the reduced derivative. The increase in MsrA function and expression was associated with a decline of ROS levels. None of these effects were observed when cells were exposed to Abeta containing oxidized Met35 (Abeta1-42)Met35(OX). Taken together, the results of the present study indicate that the differential toxicity of Abeta peptides containing reduced or oxidised Met-35 depends on the ability of the latter form to reduce ROS generation by enhancing MsrA gene expression and function and suggests the therapeutic potential of MsrA in Alzheimer's disease.

Overexpression of methionine-R-sulfoxide reductases has no influence on fruit fly aging.

Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine residues in proteins. This function implicated Msrs in antioxidant defense and the regulation of aging. There are two known Msr types in animals: MsrA specific for the reduction of methionine-S-sulfoxide, and MsrB that catalyzes the reduction of methionine-R-sulfoxide. In a previous study, overexpression of MsrA in the nervous system of Drosophila was found to extend lifespan by 70%. Overexpression of MsrA in yeast also extended lifespan, whereas MsrB overexpression did so only under calorie restriction conditions. The effect of MsrB overexpression on lifespan has not yet been characterized in animal model systems. Here, the GAL4-UAS binary system was used to drive overexpression of cytosolic Drosophila MsrB and mitochondrial mouse MsrB2 in whole body, fatbody, and the nervous system of flies. In contrast to MsrA, MsrB overexpression had no consistent effect on the lifespan of fruit flies on either corn meal or sugar yeast diets. Physical activity, fecundity, and stress resistance were also similar in MsrB-overexpressing and control flies. Thus, MsrA and MsrB, the two proteins with similar function in antioxidant protein repair, have different effects on aging in fruit flies.

Up-regulation of ADM and SEPX1 in the lymphoblastoid cells of patients in monozygotic twins discordant for schizophrenia.

The contribution of genetic factors to schizophrenia is well established and recent studies have indicated several strong candidate genes. However, the pathophysiology of schizophrenia has not been totally elucidated yet. To date, studies of monozygotic twins discordant for schizophrenia have provided insight into the pathophysiology of this illness; this type of study can exclude inter-individual variability and confounding factors such as effects of drugs. In this study we used DNA microarray analysis to examine the mRNA expression patterns in the lymphoblastoid (LB) cells derived from two pairs of monozygotic twins discordant for schizophrenia. From five independent replicates for each pair of twins, we selected five genes, which included adrenomedullin (ADM) and selenoprotein X1 (SEPX1), as significantly changed in both twins with schizophrenia. Interestingly, ADM was previously reported to be up-regulated in both the LB cells and plasma of schizophrenic patients, and SEPX1 was included in the list of genes up-regulated in the peripheral blood cells of schizophrenia patients by microarray analysis. Then, we performed a genetic association study of schizophrenia in the Japanese population and examined the copy number variations, but observed no association. These findings suggest the possible role of ADM and SEPX1 as biomarkers of schizophrenia. The results also support the usefulness of gene expression analysis in LB cells of monozygotic twins discordant for an illness.