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Öğe Identification of a novel system for boron transport: atr1 ıs a main Boron exporter in yeast_(Molecular and Cellular Biology, 2009) Kaya, Alaattin; Karakaya, Hüseyin Çağlar; Fomenko, Dmitri E.; Gladyshev, Vadim N.; Koç, AhmetBoron 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.Öğe Methionine sulfoxide reductase regulation of yeast lifespan reveals reactive oxygen species dependent and independent components of aging(Proc Natl Acad Sci. USA., 2004) Koç, Ahmet; Gasch, Audrey P.; Rutherford, Julian C.; Kim, Hwa-Young; Gladyshev, Vadim N.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.Öğe Methionine sulfoxide reduction and the aging process(Ann N Y Acad Sci., 2007) Koç, Ahmet; Gladyshev, Vadim N.Aging has been described for multicellular and asymmetri-cally dividing organisms, but the mechanisms are poorly understood. Ox-idation of proteins is considered to be one of the major factors that leadsto aging. Oxidative damage to proteins results in the oxidation of certainamino acid residues, among which oxidation of sulfur-containing aminoacids, methionine and cysteine, is notable because of the susceptibility ofthese residues to damage, and occurrence of repair mechanisms. Methio-nine sulfoxide reductases, MsrA and MsrB, are thioredoxin-dependentoxidoreductases that reduce oxidized forms of methionine, methioninesulfoxides, in a stereospecific manner. These enzymes are present in allcell types and have shown to be regulating life spans in mammals, insects,and yeast. Here, their roles in modulating yeast life span are discussed.Öğe Reaction mechanism evolutionary analysis and role of zinc in Drosophila methionine R sulfoxide reductase(J Biol Chem, 2002) Kumar, R. Abhilash; Koç, Ahmet; Cerny, Ronald L.; Gladyshev, Vadim N.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. The enzyme was found to employ a catalytic Cys124 thiolate, which directly interacted with methionine sulfoxide, resulting in methionine and a Cys124 sulfenic acid intermediate. A subsequent reaction of this intermediate with Cys69 generated an intramolecular disulfide. Dithiothreitol could reduce either the sulfenic acid or the disulfide, but the disulfide was a preferred substrate for thioredoxin, a natural electron donor. Interestingly, the C69S mutant could complement MsrA/MsrB deficiency in yeast, and the corresponding natural form of mouse MsrB was active with thioredoxin. These data indicate that MsrB proteins employ alternative mechanisms for sulfenic acid reduction. Four other conserved cysteines in Drosophila MsrB (Cys51, Cys54, Cys101, and Cys104) 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.Öğe The roles of thiol oxidoreductases in yeast replicative aging(Mechanisms of Ageing and Development, 2010) Hacıoğlu, Elise; Esmer, Işıl; Fomenko, Dmitri E.; Gladyshev, Vadim N.; Koç, AhmetThiol-based redox reactions are involved in the regulation of a variety of biological functions, such as protection against oxidative stress, signal transduction and protein folding. Some proteins involved in redox regulation have been shown to modulate life span in organisms from yeast to mammals. To assess the role of thiol oxidoreductases in aging on a genome-wide scale, we analyzed the replicative life span of yeast cells lacking known and candidate thiol oxidoreductases. The data suggest the role of several pathways in controlling yeast replicative life span, including thioredoxin reduction, protein folding and degradation, peroxide reduction, PIP3 signaling, and ATP synthesis.Öğe Selenoprotein R is a zinc containing stereo specific methionine sulfoxide reductase(Proc Natl Acad Sci U S A., 2002) Kryukov, Gregory V.; Kumar, R. Abhilash; Koç, Ahmet; Sun, Zhaohui; Gladyshev, Vadim N.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.Öğe Thiol peroxidase deficiency leads to ıncreased mutational load and decreased fitness in saccharomyces cerevisiae(Genetics, 2014) Kaya, Alaattin; Lobanov, Alexei V.; Gerashchenko, Maxim V.; Koren, Amnon; Fomenko, Dmitri E.; Koç, Ahmet; Gladyshev, Vadim N.ABSTRACT Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (Δ8) is viable. In this study, we employed two independent Δ8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and Δ8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. Δ8 lines showed a significant increase in nonrecurrent point mutations and indels. The original Δ8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all Δ8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of Δ8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness.
