Selenoprotein R (SelR) is a mammalian selenocysteine-containing protein with no known function. Here we report that cysteine homologs of SelR are present in all organisms except certain parasites and hyperthermophiles, and this pattern of occurrence closely matches that of only one protein, peptide methionine sulfoxide reductase (MsrA). Moreover, in several genomes, SelR and MsrA genes are fused or clustered, and their expression patterns suggest a role of both proteins in protection against oxidative stress. Consistent with these computational screens, growth of Saccharomyces cerevisiae SelR and MsrA mutant strains was inhibited, and the strain lacking both genes could not grow, in the presence of H2O2 and methionine sulfoxide. We found that the cysteine mutant of mouse SelR, as well as the Drosophila SelR homolog, contained zinc and reduced methionine-R-sulfoxide, but not methionine-S-sulfoxide, in in vitro assays, a function that is both distinct and complementary to the stereo-specific activity of MsrA. These findings identify a function of the conserved SelR enzyme family, define a pathway of methionine sulfoxide reduction, reveal a case of convergent evolution of similar function in structurally distinct enzymes, and suggest a previously uncharacterized redox regulatory role of selenium in mammals.

Aging is thought to be caused by the accumulation of damage, primarily from oxidative modifications of cellular components by reactive oxygen species (ROS). Here we used yeast methionine sulfoxide reductases MsrA and MsrB to address this hypothesis. In the presence of oxygen, these antioxidants could increase yeast lifespan and did so independent of the lifespan extension offered by caloric restriction. However, under ROS-deficient, strictly anaerobic conditions, yeast lifespan was shorter, not affected by MsrA or MsrB, and further reduced by caloric restriction. In addition, we identified changes in the global gene expression associated with aging in yeast, and they did not include oxidative stress genes. Our findings suggest how the interplay between ROS, antioxidants, and efficiency of energy production regulates the lifespan. The data also suggest a model wherein factors implicated in aging (for example, ROS) may influence the lifespan yet not be the cause of aging.A ging is typically viewed as the accumulation of damage that results in death. This damage is thought to be caused by reactive oxygen species (ROS). Indeed, ROS can damage biomolecules, accelerate aging, and shorten lifespan (1-3), whereas antioxidant enzymes alleviate these effects, providing support for a free radical theory of aging (4). The contrasting effects of ROS and antioxidants, as well as correlation between accumulation of oxidative damage in cellular components and the lifespan of an organism, have been the main arguments in favor of this theory.Proteins become oxidatively damaged by oxidation of their amino acid residues and cofactors. Methionine residues in proteins are particularly susceptible to oxidation by ROS, resulting in methionine-S-sulfoxides (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO) (5). Oxidized methionines can be repaired by antioxidant enzymes Met-S-SO reductase (MsrA) and Met-R-SO reductase (MsrB) (6). Recent studies revealed that methionine sulfoxide reduction provides lifespan extension in animals: deletion of the MsrA gene in mice reduced lifespan by Ϸ40% (7), whereas MsrA overexpression, predominantly in the nervous system, extended fruit fly lifespan by Ϸ70% (8). The correlation between methionine sulfoxide reductase activity and lifespan has been attributed to antioxidant function of MsrA. The role of MsrB in lifespan regulation has not been previously addressed.Most known modulators of the rate of aging are conserved across species, suggesting that common, conserved processes regulate and cause aging in diverse organisms. For example, caloric restriction (CR) is a dietary regimen that is known to extend lifespan in organisms from yeast to mammals (3, 9, 10). Saccharomyces cerevisiae has been extensively used as a model organism in studies on the mechanisms of aging (9, 10). Yeast express methionine sulfoxide reductases and can grow both aerobically and anaerobically, and in the present study, we used these features to examine the casual role of ROS-dependent processes in aging. Materials and MethodsYeas...

The relationship between dNTP levels and DNA synthesis was investigated using ␣ factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G 1 /S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G 1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37°C before being released to 25°C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G 1 levels. Accumulation of dNTPs at G 1 /S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25°C were able to replicate when subsequently switched to 37°C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/ Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold. Hydroxyurea (HU)1 is a potent inhibitor of the enzyme ribonucleotide reductase (RNR) and inhibits DNA replication in a wide variety of cells, including Saccharomyces cerevisiae (1). The simplest explanation for HU inhibition of DNA synthesis is that it starves the DNA polymerase at the replication forks for dNTPs. HU treatment has been shown to reduce the purine dNTP pools in a variety of mammalian cells (2-7); however, conflicting data exist concerning its modulation of pyrimidine dNTP pool levels. Furthermore, even for purine dNTPs, HU has only rarely been shown to cause a complete depletion of the dGTP or dATP pools (2-4). More commonly, HU results in only partial depletion of the purine dNTP pools (5-7). The complicated, often reciprocal, changes in individual dNTP pools that occur in HU-treated mammalian cells may be due to the compensatory activities of deoxyribonucleotide salvage pathways in higher eukaryotes. Budding yeast offers a simpler system in which to study the mechanism by which HU affects replication. Yeast possess no deoxyribonucleoside kinase activities, and thus deoxyribonucleotide synthesis is entirely dependent on ribonucleotide reductase. Also, yeast can easily be synchronized in G 1 using mating pheromone, and the availability of several temperature-sensitive cdc mutations allows cell cycle progression to be reversibly halted at specific points throughout the cell cycle (8). Reciprocal switch experiments in yeast have ordered the execution point of several cdc genes with respect to the HU-sensitive step during the cell cycle (9). Furthermore, mutational screens in yeast have identified regulatory proteins, such as Mec1 and Rad53, that are necessary for proper execution of the HU-induced replication arrest checkpoint (10). Despite the gene...

Methionine residues in proteins are susceptible to oxidation, and the resulting methionine sulfoxides can be reduced back to methionines by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Herein, we have identified two MsrB families that differ by the presence of zinc. Evolutionary analyses suggested that the zinc-containing MsrB proteins are prototype enzymes and that the metal was lost in certain MsrB proteins later in evolution. Zinc-containing Drosophila MsrB was further characterized. , and Cys 104 ) were found to coordinate structural zinc. Mutation of any one or a combination of these residues resulted in complete loss of metal and catalytic activity, demonstrating an essential role of zinc in Drosophila MsrB. In contrast, two conserved histidines were important for thioredoxin-dependent activity, but were not involved in zinc binding. A Drosophila MsrA gene was also cloned, and the recombinant enzyme was found to be metal-free and specific for methionine S-sulfoxide and to employ a similar sulfenic acid/disulfide mechanism.The side chains of the sulfur-containing amino acid residues (cysteine and methionine) are susceptible to oxidation by reactive oxygen species (1). Such modifications may change protein function, modulate its activity, or result in a signaling event. Cellular thiol-dependent antioxidant systems, primarily thioredoxin and glutathione systems, are involved in maintaining the reduced state of cysteines and methionines.The product of methionine oxidation is a diastereomeric mixture of methionine S-sulfoxide and methionine R-sulfoxide (2, 3). One of the enzymes that can reduce methionine sulfoxides, methionine-sulfoxide reductase (MsrA), has been known for several decades, but further studies revealed that its activity is restricted to methionine S-sulfoxides (3-7). Recent identification of a new methionine-sulfoxide reductase (MsrB; also called SelR) with specificity for methionine R-sulfoxides provided an explanation of how cells cope with racemic methionine oxidation (8 -10).Although MsrA has been well characterized structurally and functionally (4), little information on MsrB is available. Interesting aspects of MsrB function include the presence of zinc in mammalian and fruit fly MsrB proteins and the presence of selenocysteine in selenoprotein R, which is one of the mammalian MsrB proteins (8). In functionally characterized selenoproteins, selenium is located at enzyme active sites and is involved in redox reactions (11, 12), suggesting that selenocysteine in selenoprotein R and corresponding cysteines in other MsrB homologs are directly involved in catalysis (8).The reaction mechanism of MsrA has been characterized in great detail. The enzyme employs a reactive Cys thiolate that attacks a sulfoxide (13,14), with the formation of methionine and a Cys-based sulfenic acid intermediate (15). The shortlived sulfenic acid is then reduced by cysteines located in the C-terminal portion of the enzyme through a thiol/disulfide exchange mechanism (13, 15). T...

Boron is an essential element for plants and probably essential for human and animal health. Boron has a broad range of physiological effects on biological systems at low concentrations, whereas it is toxic to at high concentrations. Eventhough there are many studies on boron's biological effects and toxicity, more information is needed to understand the mechanisms of its action. The aim of the current work is to review boron's function, transport and toxicity in different biological systems.

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H 2 O 2 response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H 2 O 2 . Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H 2 O 2 . The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H 2 O 2 and other cellular proteins plays a secondary role.

Methionine sulfoxide reductases (Msrs) are oxidoreductases that catalyze thiol-dependent reduction of oxidized methionines. MsrA and MsrB are the best known Msrs that repair methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO) residues in proteins, respectively. In addition, an Escherichia coli enzyme specific for free Met-R-SO, designated fRMsr, was recently discovered. In this work, we carried out comparative genomic and experimental analyses to examine occurrence, evolution, and function of fRMsr. This protein is present in single copies and two mutually exclusive subtypes in about half of prokaryotes and unicellular eukaryotes but is missing in higher plants and animals. A Saccharomyces cerevisiae fRMsr homolog was found to reduce free Met-R-SO but not free Met-S-SO or dabsyl-Met-R-SO. fRMsr was responsible for growth of yeast cells on Met-R-SO, and the double fRMsr/MsrA mutant could not grow on a mixture of methionine sulfoxides. However, in the presence of methionine, even the triple fRMsr/ MsrA/MsrB mutant was viable. In addition, fRMsr deletion strain showed an increased sensitivity to oxidative stress and a decreased life span, whereas overexpression of fRMsr conferred higher resistance to oxidants. Molecular modeling and cysteine residue targeting by thioredoxin pointed to Cys 101 as catalytic and Cys 125 as resolving residues in yeast fRMsr. These residues as well as a third Cys, resolving Cys 91 , clustered in the structure, and each was required for the catalytic activity of the enzyme. The data show that fRMsr is the main enzyme responsible for the reduction of free Met-R-SO in S. cerevisiae.

Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1⌬ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1⌬ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1⌬ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.Boron has been proposed as an important micronutrient in plants and animals. Studies have shown the presence of several genes associated with boron transport and tolerance in plants (18,25,27); however, boron transport mechanisms in other organisms, including animals, remain unclear. In plants, boron functions as a cross-linker for rhammogalacturanon II in the cell membrane (9, 14, 21) and also as a structural component in cytoskeleton assembly (1). Arabidopsis thaliana BOR1 was the first gene shown to play a role in boron tolerance (28). Homologs of BOR1 were found in many organisms, including yeasts, plants, and mammals (22,25,29). A high level of boron leads to degradation of its own exporter, BOR1, in A. thaliana (27), and A. thaliana BOR1 cannot be used to produce genetically modified plants that grow in soil with high boron levels. However, transgenic plants expressing BOR4, one of six paralogs of BOR1, showed high tolerance to toxic levels of boron (18). Multicopy expression of BOT1, a BOR1 ortholog, provided boron tolerance to barley (25).The yeast Saccharomyces cerevisiae has been used as a model organism for characterization of plant boron tolerance genes (19,20,25,26,29). While 10 mM boric acid is lethal to Arabidopsis (18), yeast can grow in the presence of 80 mM boron and is considered a boron-tolerant organism (19,20). Yeast Bor1 was characterized in detail (10). This protein is localized to the plasma membrane and functions as a boric acid exporter (26). The bor1⌬ yeast strain overaccumulates boron (20,28), and cells that overexpress BOR1 have less intracellular boron and show resistance to boron treatment (20). In addition to Bor1, two other proteins, Dur3 and Fps1, have been implicated in boron tolerance in yeast, but their functions are not clear (20). Dur3 is a plasma membrane transporter that plays a ...