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Öğe Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process(Biochemical and Biophysical Research Communications, 2014) Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çağlar; Koç, AhmetSuperoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1D and sod2D strains. However, ccs1D mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1D, sod2D, and ccs1D mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.Öğe Assessment of chronological lifespan dependent molecular damages in yeast lacking mitochondrial antioxidant genes(Biochemical and Biophysical Research Communications, 2010) Demir, Ayşe Banu; Koç, AhmetThe free radical theory of aging states that oxidative damage to biomolecules causes aging and that antioxidants neutralize free radicals and thus decelerate aging. Mitochondria produce most of the reactive oxygen species, but at the same time have many antioxidant enzymes providing protection from these oxidants. Expecting that cells without mitochondrial antioxidant genes would accumulate higher levels of oxidative damage and, therefore, will have a shorter lifespan, we analyzed oxidative damages to biomolecules in young and chronologically aged mutants lacking the mitochondrial antioxidant genes: GRX2, CCP1, SOD1, GLO4, TRR2, TRX3, CCS1, SOD2, GRX5, and PRX1. Among these mutants, ccp1D, trx3D, grx5D, prx1D, mutants were sensitive to diamide, and ccs1D and sod2D were sensitive to both diamide and menadione. Most of the mutants were less viable in stationary phase. Chronologically aged cells produced higher amount of superoxide radical and accumulated higher levels of oxidative damages. Even though our results support the findings that old cells harbor higher amount of molecular damages, no significant difference was observed between wild type and mutant cells in terms of their damage content.Öğe Boron stress activates the general amino acid control mechanism and ınhibits protein synthesis(PLoS ONE, 2011) Uluışık, İrem; Kaya, Alaattin; Dima, Fomenko; Karakaya, Hüseyin Çağlar; Brad, Carlson; Vadim, Gladyshev; Koç, AhmetBoron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2a in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2a in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance.Öğe Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast(Gene, 2014) Koç, Ahmet; Bozdağ, Gönensin O.; Kaya, Alaattin; Koç, Ahmet; Noll, Gündüla A.Nickel is an essential micronutrient due to its involvement in many enzymatic reactions as a cofactor. However, excess of this element is toxic to biological systems. Here, we constructed a cDNA library from Beta maritima and screened it in the yeast system to identify genes that confer resistance to toxic levels of nickel. A cDNA clone (NIC6), which encodes for a putative membrane protein with unknown function, was found to help yeast cells to tolerate toxic levels of nickel. A GFP fused form of Nic6 protein was localized to multivesicular structures in tobacco epidermal cells. Thus, our results suggest a possible role of Nic6 in nickel and intracellular ion homeostasis.Öğe Characterization of two genes encoding metal tolerance proteins from Beta vulgaris subspecies maritima that confers manganese tolerance in yeast(BioMetals, 2013) Bozdağ, Gönensin Ozan; Erbasol, Isil; Koç, Ahmet; Pedas, Pai; Karakaya, Hüseyin ÇağlarManganese (Mn2?) is an essential micronutrient in plants. However increased Mn2? levels are toxic to plant cells. Metal tolerance proteins (MTPs), member of cation diffusion facilitator protein (CDF) family, have important roles in metal homeostatis in different plant species and catalyse efflux of excess metal ions. In this study, we identified and characterized two MTP genes from Beta vulgaris spp. maritima (B. v. ssp. maritima). Overexpression of these two genes provided Mn tolerance in yeast cells. Sequence analyses displayed BmMTP10 and BmMTP11as members of the Mn-CDF family. Functional analyses of these proteins indicated that they are specific to Mn2? with a role in reducing excess cellular Mn2? levels when expressed in yeast. GFP-fusion constructs of both proteins localized to the Golgi apparatus as a punctuated pattern. Finally, Q-RT-PCR results showed that BmMTP10 expression was induced threefold in response to the excess Mn2? treatment. On the other hand BmMTP11 expression was not affected in response to excess Mn2? levels. Thus, our results suggest that the BmMTP10 and BmMTP11 proteins from B. v. ssp. maritima have non-redundant functions in terms of Mn2? detoxification with a similar in planta localization and function as the Arabidopsis Mn-CDF homolog AtMTP11 and this conservation shows the evolutionary importance of these vesicular proteins in heavy metal homeostatis among plant species.Öğe Checkpoint deficient rad53 11 yeast cannot accumulate dNTPs in response to DNA damage(Biochem Biophys Res Commun., 2007) Koç, Ahmet; Merrill, Gary F.Deoxyribonucleotide pools are maintained at levels that support efficient and yet accurate DNA replication and repair. Rad53 is part of a protein kinase regulatory cascade that, conceptually, promotes dNTP accumulation in four ways: (1) it activates the transcription of ribonucleotide reductase subunits by inhibiting the Crt1 repressor; (2) it plays a role in relocalization of ribonucleotide reductase subunits RNR2 and RNR4 from nucleus to cytoplasm; (3) it antagonizes the action of Sml1, a protein that binds and inhibits ribonucleotide reductase; and (4) it blocks cell-cycle progression in response to DNA damage, thus preventing dNTP consumption through replication forks. Although several lines of evidence support the above modes of Rad53 action, an effect of a rad53 mutation on dNTP levels has not been directly demonstrated. In fact, in a previous study, a rad53-11 mutation did not result in lower dNTP levels in asynchronous cells or in synchronized cells that entered the S-phase in the presence of the RNR inhibitor hydroxyurea. These anomalies prompted us to investigate whether the rad53-11 mutation affected dNTP levels in cells exposed to a DNA-damaging dose of ethylmethyl sulfonate (EMS). Although dNTP levels increased by 2- to 3-fold in EMS treated wild-type cells, rad53-11 cells showed no such change. Thus, the results indicate that Rad53 checkpoint function is not required for dNTP pool maintenance in normally growing cells, but is required for dNTP pool expansion in cells exposed to DNA-damaging agents. 2006 Elsevier Inc. All rights reserved.Öğe Compartmentalization and Regulation of Mitochondrial Function by Methionine Sulfoxide Reductases in Yeast(Biochemistry, 2010) Kaya, Alaattin; Koç, Ahmet; Lee, Byung Cheon; Fomenko, Dmitri E.; Rederstorff, Mathieu; Krol, Alain; Lescure, Alain; Gladyshev, Vadim N.Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine (S)-sulfoxide (Met-SO) and methionine (R)-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB, or both proteins had normal levels of mitochondria but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments.Öğe DELESYONU MAYADA YAŞAM SÜRESİNİ UZATAN MİTOKONDRİYEL METABOLİZMA GENLERİNDEN PPA2, AFG3 VE DSS1’E AİT MAYA MUTANTLARININ KARAKTERİZASYONU(2017) Koç, AhmetYaslanmanın moleku?ler mekanizmaları tam olarak bilinmemektedir. Mitokondriler hu?cresel metabolizma ve yaslanmada merkezi bir rol oynarlar. Bu çalısma kapsamında maya hu?creleri (S. cerevisiae) model olarak kullanılarak mitokondriyel metabolizma genlerinin yasam su?resi ile alakaları incelendi. Hayat su?resinde uzamaya neden olan u?ç adet delesyon mutantı tespit edildi (?ppa2, ?dss1 and ?afg3). Bu mutantların hayat su?relerinin uzama nedenini ortaya çıkarmak için mitokondriyel respirasyon hızları, mitokondriyel membran potensiyelleri, toplam ATP seviyeleri, reaktif oksijen tu?rlerinin seviyesi, mtDNA miktarlarının tespiti, mitokondrilerin miktar ve morfolojik olarak durumlarının tespiti, mitokondri ile çekirdek arasında gerçeklesen retrograt sinyal yolagının statu?su?, TCA döngu?su?nu?n statu?su?, global gen ifadelenme profilleri ve ilgili genlerin asırı ifade edilmelerinin hayat su?resine olan etkileri incelenmistir. Elde edilen verilere göre mutant hu?crelerin oksijenli solunum yapamadıkları, mitokondriyel respirasyon için oksijen kullanamadıklarından dolayı reaktif oksijen tu?rlerinin olusmadıgı, daha az ATP sentezledikleri, mtDNA miktarlarının az oldugu, yaslılık su?recinde sitozolik pH larını normal sınırlarda idame edebildikleri, mitokondri morfolojilerinin degistigi ve retrograt sinyal yolagını aktive ettikleri tespit edilmistir. Ayrıca mutant hu?crelerde kontrol hu?crelere kıyasla ifadelenme farklılıgı gösteren genler tespit edilerek mutantların yaslanma su?reci ile alakası olabilecek biyokimyasal yolaklar belirlenmistir. Sonuç olarak, serbest radikallerin azlıgı, yaslı hu?crelerdeki normal sitoplazmik pH degerleri ve genel olarak mitokondriyel disfonksiyona baglı olarak kompensasyon amaçlı aktive edilen retrograt sinyal iletim yolagı sonucu mutant hu?crelerin yabani tip kontrol hu?crelerine kıyasla daha avantajlı hale geldikleri ve bu nedenlerden dolayı daha uzun yasayabildikleri tespit edilmistir.Öğe Effect of Thioredoxin Deletion and p53 Cysteine Replacement on Human p53 Activity in Wild type and Thioredoxin Reductase Null Yeast(Biochemistry, 2009) Stoner, Chris; George, Perason; Koç, Ahmet; Jason, Merwin; Nathan, Lopez; Gary, MerrillReporter gene transactivation by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. To investigate the role of thioredoxin in controlling p53 activity, the level of reporter gene transactivation by p53 was determined in yeast lacking the TRX1 and TRX2 genes encoding cytosolic thioredoxin. Surprisingly, p53 activity was unimpaired in yeast lacking thioredoxin. Subsequent analyses showed that thioredoxin deletion suppressed the inhibitory effect of thioredoxin reductase deletion, suggesting that accumulation of oxidized thioredoxin in mutant yeast was necessary for p53 inhibition. Purified human thioredoxin and p53 interacted in vitro (Kd = 0.9 µM thioredoxin). To test the idea that dithio-disulfide exchange reactions between p53 and thioredoxin were responsible for p53 inhibition in mutant yeast, each p53 cysteine was changed to serine and the effect of the substitution on p53 activity in TRR1 and Δtrr1 yeast was determined. Substitutions at Zn-coordinating cysteines C176, C238 or C242 resulted in p53 inactivation. Unexpectedly, substitution at cysteine C275 also inactivated p53, which was the first evidence for a non-zinc-coordinating cysteine being essential for p53 function. Cysteine substitutions at six positions (C124, C135, C141, C182, C229 and C277) neither inactivated p53 nor relieved the requirement for thioredoxin reductase. Furthermore, no tested combination of these six cysteine substitutions relieved thioredoxin reductase dependence. The results suggested that p53 dependence on thioredoxin reductase either was indirect, perhaps mediated by an upstream activator of p53, or was due to oxidation of one or more of the four essential cysteines.Öğe Effects of deleting mitochondrial Antioxidant genes on life span(Ann N Y Acad Sci., 2007) Ünlü, Ercan Selçuk; Koç, AhmetReactive oxygen species (ROS) damage biomolecules, accelerate aging, and shorten life span, whereas antioxidant enzymes mitigate these effects. Because mitochondria are a primary site of ROS generation and also a primary target of ROS attack, they have become a major focus area of aging studies. Here, we employed yeast genetics to identify mitochondrial antioxidant genes that are important for replicative life span. In our studies, it was found that among the known mitochondrial antioxidant genes (TTR1, CCD1, SOD1, GLO4, TRR2, TRX3, CCS1, SOD2, GRX5, PRX1), deletion of only three genes, SOD1 (Cu, Zn superoxide dismutase), SOD2 (Manganese-containing superoxide dismutase), and CCS1 (Copper chaperone), shortened the life span enormously. The life span decreased 40% forΔsod1 mutant, 72% forΔsod2 mutant, and 50% forΔccs1 mutant. Deletion of the other genes had little or no effect on life span.Öğe Evidence for the presence of a second electron donor for cytoplasmic thioredoxins in the yeast s cerevisiae(T Journal Biol, 2006) Koç, Ahmet; Karakaya, Hüseyin Çağlar; Ünlü, Ercan SelçukIn yeast, the cytoplasmic thioredoxin system is composed of NADPH, thioredoxin reductase-1 (TRR1) and 2 thioredoxin genes (TRX1, TRX2). In this study, using yeast knockout mutants for TRR1, TRX1 and TRX2 genes, the role of the thioredoxin system in methionine sulfoxide reduction was investigated. Cells lacking both TRX1 and TRX2 genes simultaneously were not able to reduce methionine sulfoxides to methionine; however, mutants missing the TRR1 gene were able to reduce methionine sulfoxides to methionine, which showed that electrons could be transferred from NADPH to thioredoxins in the absence of TRR1. Similar results were observed for 3-phosphoadenosine 5-phosphosulfate reduction in the inorganic sulfate assimilation pathway. Results from both assays suggested that yeast cells have additional cytoplasmic thioredoxin reductase activity that could compensate for methionine sulfoxide reduction and sulfate assimilation in the absence of TRR1. This report also constitutes the first evidence that thioredoxins are the in vivo electron donors for methionine sulfoxide reductases in yeast.Öğe Functional characterization of new mutations in Wilson disease gene ATP7B using the yeast model(J Trace Elem Med Biol., 2015) Papur, Özlenen Şimşek; Koç, Ahmet; Terzioğlu, OrhanThe Wilson disease gene, a copper transporting ATPase (Atp7b), is responsible for the sequestration of Cu into secretory vesicles, and this function is exhibited by the orthologous Ccc2p in the yeast. In this study, we aimed to characterize clinically relevant new mutations of human ATP7B (p.T788I, p.V1036I and p.R1038G-fsX83)in yeastlacking the CCC2 gene. Expression of human wild type ATP7B gene in ccc2 mutant yeast restored the growth deficiency and copper transport activity; however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. Our data support that p.T788I, p.V1036I and p.R1038G-fsX83 mutations cause functional deficiency in ATP7B functions and suggest that these residues are important for normal ATP7B function.Öğe High copy overexpression screening reveals pdr5 as the main doxorubicin resistance gene in yeast(PLoS One, 2015) Demir, Ayşe Banu; Koç, AhmetDoxorubicin is one of the most potent anticancer drugs used in the treatment of various cancer types. The efficacy of doxorubicin is influenced by the drug resistance mechanisms and its cytotoxicity. In this study, we performed a high-copy screening analysis to find genes that play a role in doxorubicin resistance and found several genes (CUE5, AKL1, CAN1, YHR177W and PDR5) that provide resistance. Among these genes, overexpression of PDR5 provided a remarkable resistance, and deletion of it significantly rendered the tolerance level for the drug. Q-PCR analyses suggested that transcriptional regulation of these genes was not dependent on doxorubicin treatment. Additionally, we profiled the global expression pattern of cells in response to doxorubicin treatment and highlighted the genes and pathways that are important in doxorubicin tolerance/toxicity. Our results suggest that many efflux pumps and DNA metabolism genes are upregulated by the drug and required for doxorubicin tolerance.Öğe Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools(J Biol Chem, 2003) Koç, Ahmet; Wheeler, Linda J.; Mathews, Christopher K.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 G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 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-G1 levels. Accumulation of dNTPs at G1/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.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 Identification of respiratory chain gene mutations that shorten replicative life span in yeast(Experimental Gerontology, 2012) Hacıoğlu, Elise; Demir, Ayşe Banu; Koç, AhmetAging is the progressive accumulation of alterations in cells that elevates the risk of death. The mitochondrial theory of aging postulates that free radicals produced by the mitochondrial respiratory system contribute to the aging process. However, the roles of individual electron transfer chain (ETC) components in cellular aging have not been elucidated. In this study, we analyzed the replicative life span of 73 yeast deletion mutants lacking the genes of the mitochondrial electron transfer chain system, and found that nine of these mutants (Δnde1, Δtcm62, Δrip1, Δcyt1, Δqrc8, Δpet117, Δcox11, Δatp11, Δfmc1) had significantly shorter life spans. These mutants had lower rates of respiration and were slightly sensitive to exogenous administration of hydrogen peroxide. However, only two of them, Δnde1 and Δfmc1, produced higher amounts of intrinsic superoxide radicals in the presence of glucose compared to that of wild type cells. Interestingly, there were no significant alterations in the mitochondrial membrane potentials of these mutants. We speculate that the shorter life spans of ETC mutants result from multiple mechanisms including the low respiration rate and low energy production rather than just a ROS-dependent path.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 MsrB1 Methionine R sulfoxide Reductase 1 Knock out Mice ROLES OF MsrB1 IN REDOX REGULATION AND IDENTIFICATION OF A NOVEL SELENOPROTEIN FORM(Journal of Biological Chemistry, 2009) Dima, Fomenko; Sergey, Novoselov; Sathish, Natarajan; Koç, Ahmet; Brad, Carlson; Lee, TH; Hatfield, Dolph; Vadim, NebraskaProtein oxidation has been linked to accelerated aging and is a contributing factor to many diseases.Methionine residues are particularly susceptible to oxidation, but the resulting mixture of methionine R-sulfoxide (Met-RO) and methionine S-sulfoxide (Met-SO) can be repaired by thioredoxin-dependent enzymes MsrB and MsrA, respectively. Here, we describe a knock-out mouse deficient in selenoprotein MsrB1, the main mammalian MsrB located in the cytosol and nucleus. In these mice, in addition to the deletion of 14-kDa MsrB1, a 5-kDa selenoprotein form was specifically removed. Further studies revealed that the 5-kDa protein occurred in both mouse tissues and human HEK 293 cells; was down-regulated by MsrB1 small interfering RNA, selenium deficiency, and selenocysteine tRNA mutations; and was immunoprecipitated and recognized by MsrB1 antibodies. Specific labeling with 75Se and mass spectrometry analyses revealed that the 5-kDa selenoprotein corresponded to the C-terminal sequence of MsrB1. The MsrB1 knock-out mice lacked both 5- and 14-kDa MsrB1 forms and showed reduced MsrB activity, with the strongest effect seen in liver and kidney. In addition, MsrA activity was decreased by MsrB1 deficiency. Liver and kidney of the MsrB1 knock-out mice also showedincreasedlevels ofmalondialdehyde, protein carbonyls, protein methionine sulfoxide, and oxidized glutathione as well as reduced levels of free and protein thiols, whereas these parameters were little changed in other organs examined. Overall, this study established an important contribution of MsrB1 to the redox controlinmouseliver and kidney andidentified a novel form of this protein.Öğe Overexpression of yeast YLR177w gene provides tolerance to antifungal drug tioconazole(2022) Koç, Salman; Dündar, Muhammed; Koç, Ahmet; Isik, Ozlem AybukeAim: To find genes that provide resistance to antifungal drug tioconazole by a genomic DNA screening approach. Materials and Methods: Wild-type yeast (BY4741) cells were transformed with a high copy genomic DNA expression library and transformants that can grow on toxic levels of Tioconazole were determined. Library plasmids were isolated from the transformants and sequenced for the identification of potential tioconazole resistance gene(s). Results: Yeast YLR177w gene was isolated as a potential tioconazole resistance gene. Its overexpression led cells to tolerate normally toxic levels of tioconazole. Deletion of the YLR177w gene from the yeast genome made cells more vulnerable to the drug. Conclusion: Yeast YLR177w gene encodes a putative protein of unknown function. Our analyses suggest a potential function for the YLR177w gene for tioconazole resistance.
