Jackob Moskovitz

Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals

Oxidation of proteins by reactive oxygen species is associated with aging, oxidative stress, and many diseases. Although free and protein-bound methionine residues are particularly sensitive to oxidation to methionine sulfoxide derivatives, these oxidations are readily repaired by the action of methionine sulfoxide reductase (MsrA). To gain a better understanding of the biological roles of MsrA in metabolism, we have created a strain of mouse that lacks the MsrA gene. Compared with the wild type, this mutant: (i) exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen); (ii) has a shorter lifespan under both normal and hyperoxic conditions; (iii) develops an atypical (tip-toe) walking pattern after 6 months of age; (iv) accumulates higher tissue levels of oxidized protein (carbonyl derivatives) under oxidative stress; and (v) is less able to up-regulate expression of thioredoxin reductase under oxidative stress. It thus seems that MsrA may play an important role in aging and neurological disorders.

Methionine residues may protect proteins from critical oxidative damage.

Cysteine and methionine are the two sulfur-containing residues normally found in proteins. Cysteine residues function in the catalytic cycle of many enzymes, and they form disulfide bonds which contribute to protein structure. In contrast, the key functions of methionine residues are not known. We propose that methionine residues constitute an important antioxidant defense mechanism. A variety of oxidants react readily with methionine to form methionine sulfoxide, and surface exposed methionine residues create an extremely high concentration of reactant, providing for efficient scavenging of oxidants. The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system. Eight of the sixteen methionine residues could be oxidized with little effect on activity. The oxidizable methionine residues were found to be relatively surface exposed while the intact residues were generally buried within the core of the protein. Further, the susceptible residues were physically arranged in an array which guarded the entrance to the active site. Methionine sulfoxide can be reduced back to methionine by the enzyme methionine sulfoxide reductase, providing a catalytic amplification of the antioxidant potential of each methionine residue. Given the importance of oxidative stress during aging, the potential function of methionine residues as antioxidants during aging should be investigated experimentally.

Oxidation of Methionine in Proteins: Roles in Antioxidant Defense and Cellular Regulation

The roles of methionine residues in proteins have not been well defined, but a review of available studies leads to the conclusion that methionine, like cysteine, functions as an antioxidant and as a key component of a system for regulation of cellular metabolism. Methionine is readily oxidized to methionine sulfoxide by many reactive species. The oxidation of surface exposed methionines thus serves to protect other functionally essential residues from oxidative damage. Methionine sulfoxide reductases have the potential to reduce the residue back to methionine, increasing the scavenging efficiency of the system. Reversible covalent modification of amino acids in proteins provides the mechanistic basis for most systems of cellular regulation. Interconversion of methionine and methionine sulfoxide can function to regulate the biological activity of proteins, through alteration in catalytic efficiency and through modulation of the surface hydrophobicity of the protein.

Oxidation of Methionine Residues of Proteins: Biological Consequences

Most reactive oxygen species (ROS) can oxidize methionine (Met) residues of proteins to methionine sulfoxide (MetO). However, unlike the ROS-dependent oxidation of other amino acid residues of proteins (except cysteine residues), the oxidation of Met residues is readily reversed by the action of methionine sulfoxide reductase (Msr) that catalyzes the thioredoxin-dependent reduction of MetO residues of proteins back to Met. We summarize here results of studies showing that the cyclic interconversion of Met and MetO residues of proteins is involved in several different biological processes: (a) It is the basis of an important antioxidant mechanism for the scavenging of ROS. (b) It is likely involved in the regulation of enzyme activities. (c) It is involved in cell signaling. (d) It can target proteins for proteolytic degradation. Furthermore, a loss in the ability to catalyze the reduction of protein MetO to Met residues leads to a decrease in the maximum life span, whereas overexpression of this activity leads to an increase in the life span of animals. In addition, a decrease in Msr activities in brain tissues is associated with the development of Alzheimers disease.

Substrates of the Methionine Sulfoxide Reductase System and Their Physiological Relevance

Posttranslational modifications can change a proteins structure, function, and solubility. One specific modification caused by reactive oxygen species is the oxidation of the sulfur atom in the methionine (Met) side chain. This modified amino acid is denoted as methionine sulfoxide (MetO). MetOs in proteins are of considerable interest as they are involved in early posttranslational modification events. Thus, various organisms produce specific enzymes that can reverse these modifications. MetO reductases, known collectively as the methionine sulfoxide reductase (Msr) system, are the only known enzymes that can reduce MetOs. The current research field of Met redox cycles is consumed with elucidating its role in regulation, redox homeostasis, prevention of irreversible modifications, pathogenesis, and the aging process. Substrates of the Msr system can be loosely classified by the overall effect of the MetO on the protein. Regulated substrates utilize Met as a molecular switch to modulate activation; scavenging substrates use Mets to detoxify oxidants and protect important regions of the protein; and modified substrates are altered by Met oxidation resulting in various changes in their properties, including function, activity, structure, and degradation resistance.

Elevated levels of brain-pathologies associated with neurodegenerative diseases in the methionine sulfoxide reductase A knockout mouse

One of the posttranslational modifications to proteins is methionine oxidation, which is readily reversible by the methionine sulfoxide reductase (Msr) system. Thus, accumulation of faulty proteins due to a compromised Msr system may lead to the development of aging-associated diseases like neurodegenerative diseases. In particular, it was interesting to monitor the consequential effects of methionine oxidation in relation to markers that are associated with Alzheimer’s disease as methionine oxidation was implied to play a role in beta-amyloid toxicity. In this study, a knockout mouse strain of the methionine sulfoxide reductase A gene (MsrA−/−) caused an enhanced neurodegeneration in brain hippocampus relative to its wild-type control mouse brain. Additionally, a loss of astrocytes integrity, elevated levels of beta-amyloid deposition, and tau phosphorylation were dominant in various regions of the MsrA−/− hippocampus but not in the wild-type. Also, a comparison between cultured brain slices of the hippocampal region of both mouse strains showed more sensitivity of the MsrA−/− cultured cells to H2O2 treatment. It is suggested that a deficiency in MsrA activity fosters oxidative-stress that is manifested by the accumulation of faulty proteins (via methionine oxidation), deposition of aggregated proteins, and premature brain cell death.

HIV-2 protease is inactivated after oxidation at the dimer interface and activity can be partly restored with methionine sulphoxide reductase.

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H(2)O(2) resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H(2)O(2) led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.

Mouse methionine sulfoxide reductase B: effect of selenocysteine incorporation on its activity and expression of the seleno-containing enzyme in bacterial and mammalian cells

The mammalian methionine sulfoxide reductase B (MsrB) has been found to be a selenoprotein which can reduce R form of both free and protein-incorporated methionine sulfoxide to methionine. Together with MsrA, which reduces specifically the S form of methionine sulfoxide, the living cell can repair methionine-damaged proteins and salvage free methionine under oxidative stress conditions. Here, we report about the pivotal role of the selenocysteine residue in the protein putative active site by site-directed mutagenesis directed to the selenocysteine codon. Using the Escherichia coli SECIS (selenocysteine insertion sequence) element, needed for the recognition of the UGA codon as a selenocysteine codon in E. coli, we expressed the seleno-MsrB as a recombinant selenoprotein in E. coli. The recombinant seleno-MsrB has been shown to be much more active than the cysteine mutant, whereas the mutations to alanine and serine rendered the protein inactive. Although the yields of expression of the full-length N-terminus and C-terminus His-tagged seleno-MsrB were only 3% (of the total MsrB expressed), the C-terminus His-tagged protein enabled us to get a pure preparation of the seleno-MsrB. Using both recombinant selenoproteins, the N-terminus His-tagged and the C-terminus His-tagged proteins, we were able to determine the specific activities of the recombinant seleno-MsrB, which were found to be much higher than the cysteine mutant homologue. This finding confirmed our suggestion that the selenocysteine is essential for maintaining high reducing activity of MsrB. In addition, using radioactive selenium we were able to determine the in vivo presence of MsrB as a selenoprotein in mammalian cell cultures.

METHIONINE SULFOXIDE REDUCTASE IN ANTIOXIDANT DEFENSE

Publisher Summary Cells contain methionine sulfoxide reductases (MsrA) that catalyze the thioredoxin-dependent reduction of methionine sulfoxide [Met(O)] back to methionine (Met). The oxidation of methionine residues of some proteins may lead either to activation or to inactivation of their biological activities, whereas the oxidation of one or more methionine residues in other proteins may have-tittle or no effect on biological function. This has led to the suggestion that MsrA may have multiple biological functions: (1) it may serve to repair oxidative protein damage of some proteins, (2) it may play an important role in the regulation of enzyme activities by facilitating the interconversion of specific methionine residues of these proteins between oxidized and reduced forms, and (3) MsrA might also serve as an antioxidant enzyme to protect some enzymes from oxidative damage by various reactive oxygen species (ROS). The ability of MsrA to repair oxidative damage in vivo may be of singular importance if methionine residues serve as antioxidants. The chapter describes the expression and purification of recombinant eukaryotic MsrA.

Molecular characterization of a chromosomal locus in Staphylococcus aureus that contributes to oxidative defence and is highly induced by the cell-wall-active antibiotic oxacillin.

Previous studies employing two-dimensional gel electrophoresis and N-terminal protein sequencing have shown elevated synthesis of the enzyme methionine sulfoxide reductase (MsrA) in Staphylococcus aureus in response to cell-wall-active antibiotics. In the present study, the S. aureus msrA gene was cloned, overexpressed, purified as His-tagged MsrA and shown to have methionine sulfoxide reductase activity. The transcription of msrA was studied by assaying beta-galactosidase activity in an msrA promoter::lacZ fusion strain and by Northern blot analysis. Transcription of msrA was increased by oxacillin; but not by a variety of other stresses including H2O2. Northern blot analysis revealed that the size of the msrA transcript was 2.3 kb, considerably larger than the 531 nt msrA ORF. The msrA transcription start site was mapped 25 nt upstream of the msrA start codon. Computer analysis from database sequences indicated at least three additional ORFs downstream of msrA. The deduced amino acid sequences of two of these three ORFs showed significant sequence homologies to PilB, and enzyme IIA of the phosphotransferase system, respectively. The third ORF could not be identified by homology searches. Northern blot hybridization with probes specific to the msrA downstream region indicated that the S. aureus msrA was transcribed as part of a polycistronic message. Interestingly, purified S. aureus PilB was shown to possess approximately approximately 28-fold higher methionine sulfoxide reductase activity than the MsrA. An insertional knockout mutation in the first gene of this operon resulted in increased susceptibility of the mutant to H2O2 compared to the parent strain, but not to oxacillin.