RESUMO
Copper (Cu)-only superoxide dismutases (SOD) represent a newly characterized class of extracellular SODs important for virulence of several fungal pathogens. Previous studies of the Cu-only enzyme SOD5 from the opportunistic fungal pathogen Candida albicans have revealed that the active-site structure and Cu binding of SOD5 strongly deviate from those of Cu/Zn-SODs in its animal hosts, making Cu-only SODs a possible target for future antifungal drug design. C. albicans also expresses a Cu-only SOD4 that is highly similar in sequence to SOD5, but is poorly characterized. Here, we compared the biochemical, biophysical, and cell biological properties of C. albicans SOD4 and SOD5. Analyzing the recombinant proteins, we found that, similar to SOD5, Cu-only SOD4 can react with superoxide at rates approaching diffusion limits. Both SODs were monomeric and they exhibited similar binding affinities for their Cu cofactor. In C. albicans cultures, SOD4 and SOD5 were predominantly cell wall proteins. Despite these similarities, the SOD4 and SOD5 genes strongly differed in transcriptional regulation. SOD5 was predominantly induced during hyphal morphogenesis, together with a fungal burst in reactive oxygen species. Conversely, SOD4 expression was specifically up-regulated by iron (Fe) starvation and controlled by the Fe-responsive transcription factor SEF1. Interestingly, Candida tropicalis and the emerging fungal pathogen Candida auris contain a single SOD5-like SOD rather than a pair, and in both fungi, this SOD was induced by Fe starvation. This unexpected link between Fe homeostasis and extracellular Cu-SODs may help many fungi adapt to Fe-limited conditions of their hosts.
Assuntos
Candida/enzimologia , Candidíase/microbiologia , Ferro/metabolismo , Superóxido Dismutase/metabolismo , Candida/metabolismo , Candida albicans/enzimologia , Candida albicans/metabolismo , Candida tropicalis/enzimologia , Candida tropicalis/metabolismo , Cobre/metabolismo , Humanos , Modelos Moleculares , Espécies Reativas de Oxigênio/metabolismoRESUMO
Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.
Assuntos
Candida albicans/patogenicidade , Candidíase/metabolismo , Estresse Oxidativo/fisiologia , Simportadores de Próton-Fosfato/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Transporte Biológico/fisiologia , Drosophila , Proteínas Fúngicas/metabolismo , Humanos , Camundongos , Fosfatos/metabolismo , Transdução de Sinais/fisiologia , VirulênciaRESUMO
The copper-containing superoxide dismutases (SODs) represent a large family of enzymes that participate in the metabolism of reactive oxygen species by disproportionating superoxide anion radical to oxygen and hydrogen peroxide. Catalysis is driven by the redox-active copper ion, and in most cases, SODs also harbor a zinc at the active site that enhances copper catalysis and stabilizes the protein. Such bimetallic Cu,Zn-SODs are widespread, from the periplasm of bacteria to virtually every organelle in the human cell. However, a new class of copper-containing SODs has recently emerged that function without zinc. These copper-only enzymes serve as extracellular SODs in specific bacteria (i.e. Mycobacteria), throughout the fungal kingdom, and in the fungus-like oomycetes. The eukaryotic copper-only SODs are particularly unique in that they lack an electrostatic loop for substrate guidance and have an unusual open-access copper site, yet they can still react with superoxide at rates limited only by diffusion. Copper-only SOD sequences similar to those seen in fungi and oomycetes are also found in the animal kingdom, but rather than single-domain enzymes, they appear as tandem repeats in large polypeptides we refer to as CSRPs (copper-only SOD-repeat proteins). Here, we compare and contrast the Cu,Zn versus copper-only SODs and discuss the evolution of copper-only SOD protein domains in animals and fungi.
Assuntos
Cobre , Proteínas Fúngicas , Fungos/enzimologia , Metaloproteínas , Mycobacterium/enzimologia , Oomicetos/enzimologia , Proteínas Periplásmicas , Superóxido Dismutase , Zinco , Cobre/química , Cobre/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Metaloproteínas/química , Metaloproteínas/classificação , Metaloproteínas/metabolismo , Proteínas Periplásmicas/química , Proteínas Periplásmicas/classificação , Proteínas Periplásmicas/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/classificação , Superóxido Dismutase/metabolismo , Zinco/química , Zinco/metabolismoRESUMO
In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This "synthetic lethality" was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection.
Assuntos
ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Superóxido Dismutase/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Substituição de Aminoácidos , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Genes Fúngicos , Modelos Biológicos , Modelos Moleculares , Mutação , Estresse Oxidativo , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
Cu/Zn superoxide dismutase (SOD1) is an abundant enzyme that has been best studied as a regulator of antioxidant defense. Using the yeast Saccharomyces cerevisiae, we report that SOD1 transmits signals from oxygen and glucose to repress respiration. The mechanism involves SOD1-mediated stabilization of two casein kinase 1-gamma (CK1γ) homologs, Yck1p and Yck2p, required for respiratory repression. SOD1 binds a C-terminal degron we identified in Yck1p/Yck2p and promotes kinase stability by catalyzing superoxide conversion to peroxide. The effects of SOD1 on CK1γ stability are also observed with mammalian SOD1 and CK1γ and in a human cell line. Therefore, in a single circuit, oxygen, glucose, and reactive oxygen can repress respiration through SOD1/CK1γ signaling. Our data therefore may provide mechanistic insight into how rapidly proliferating cells and many cancers accomplish glucose-mediated repression of respiration in favor of aerobic glycolysis.
Assuntos
Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Caseína Quinase I/metabolismo , Linhagem Celular , Glucose/metabolismo , Glicólise , Humanos , Peróxido de Hidrogênio/metabolismo , Dados de Sequência Molecular , Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1 , Superóxidos/metabolismoRESUMO
SIGNIFICANCE: Antioxidant enzymes are thought to provide critical protection to cells against reactive oxygen species (ROS). However, many organisms can fully compensate for the loss of such enzymatic defenses by accumulating metabolites and Mn²âº, which can form catalytic Mn-antioxidants. Accumulated metabolites can direct reactivity of Mn²âº with superoxide and specifically shield proteins from oxidative damage. RECENT ADVANCES: There is mounting evidence that Mn-Pi (orthophosphate) complexes act as potent scavengers of superoxide in all three branches of life. Moreover, it is evident that Mn²âº in complexes with carbonates, peptides, nucleosides, and organic acids can also form catalytic Mn-antioxidants, pointing to diverse metabolic routes to oxidative stress resistance. CRITICAL ISSUES: What conditions favor utility of Mn-metabolites versus enzymatic means for removing ROS? Mn²âº-metabolite defenses are critical for preserving the activity of repair enzymes in Deinococcus radiodurans exposed to intense radiation stress, and in Lactobacillus plantarum, which lacks antioxidant enzymes. In other microorganisms, Mn-antioxidants can serve as an auxiliary protection when enzymatic antioxidants are insufficient or fail. These findings of a critical role of Mn-antioxidants in the survival of prokaryotes under oxidative stress parallel the trends developing for the simple eukaryote Saccharomyces cerevisiae. FUTURE DIRECTIONS: Phosphates, peptides and organic acids are just a snapshot of the types of anionic metabolites that promote such reactivity of Mn²âº. Their probable roles in pathogen defense against the host immune response and in ROS-mediated signaling pathways are also areas that are worthy of serious investigation. Moreover, it is clear that these protective chemical processes can be harnessed for practical purposes.
Assuntos
Manganês/metabolismo , Redes e Vias Metabólicas , Estresse Oxidativo , Antioxidantes/metabolismo , Bactérias/metabolismo , Manganês/química , Peptídeos/química , Peptídeos/metabolismo , Células Procarióticas/metabolismo , Tolerância a Radiação , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase/metabolismo , Leveduras/metabolismoRESUMO
The redox-active metal manganese plays a key role in cellular adaptation to oxidative stress. As a cofactor for manganese superoxide dismutase or through formation of non-proteinaceous manganese antioxidants, this metal can combat oxidative damage without deleterious side effects of Fenton chemistry. In either case, the antioxidant properties of manganese are vulnerable to iron. Cellular pools of iron can outcompete manganese for binding to manganese superoxide dismutase, and through Fenton chemistry, iron may counteract the benefits of non-proteinaceous manganese antioxidants. In this minireview, we highlight ways in which cells maximize the efficacy of manganese as an antioxidant in the midst of pro-oxidant iron.
Assuntos
Ferro/química , Manganês/química , Estresse Oxidativo , Antioxidantes/química , Bactérias/metabolismo , Bioquímica/métodos , Citosol/metabolismo , Escherichia coli/metabolismo , Humanos , Metais/química , Mitocôndrias/metabolismo , Modelos Biológicos , Oxidantes/química , Oxirredução , Superóxido Dismutase/químicaRESUMO
The copper chaperone for superoxide dismutase 1 (Ccs1) provides an important cellular function against oxidative stress. Ccs1 is present in the cytosol and in the intermembrane space (IMS) of mitochondria. Its import into the IMS depends on the Mia40/Erv1 disulfide relay system, although Ccs1 is, in contrast to typical substrates, a multidomain protein and lacks twin Cx(n)C motifs. We report on the molecular mechanism of the mitochondrial import of Saccharomyces cerevisiae Ccs1 as the first member of a novel class of unconventional substrates of the disulfide relay system. We show that the mitochondrial form of Ccs1 contains a stable disulfide bond between cysteine residues C27 and C64. In the absence of these cysteines, the levels of Ccs1 and Sod1 in mitochondria are strongly reduced. Furthermore, C64 of Ccs1 is required for formation of a Ccs1 disulfide intermediate with Mia40. We conclude that the Mia40/Erv1 disulfide relay system introduces a structural disulfide bond in Ccs1 between the cysteine residues C27 and C64, thereby promoting mitochondrial import of this unconventional substrate. Thus the disulfide relay system is able to form, in addition to double disulfide bonds in twin Cx(n)C motifs, single structural disulfide bonds in complex protein domains.
Assuntos
Cisteína , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Transporte Proteico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Cisteína/química , Cisteína/metabolismo , Citosol/metabolismo , Dissulfetos/metabolismo , Regulação Fúngica da Expressão Gênica , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Plasmídeos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Transdução GenéticaRESUMO
Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we present structures of the pathogenic SOD1 variants D124V and H80R, both of which demonstrate compromised zinc-binding sites. The disruption of the zinc-binding sites in H80R SOD1 leads to conformational changes in loop elements, permitting non-native SOD1-SOD1 interactions that mediate the assembly of these proteins into higher-order filamentous arrays. Analytical ultracentrifugation sedimentation velocity experiments indicate that these SOD1 variants are more prone to monomerization than the wild-type enzyme. Although D124V and H80R SOD1 proteins appear to have fully functional copper-binding sites, inductively coupled plasma mass spectrometery (ICP-MS) and anomalous scattering X-ray diffraction analyses reveal that zinc (not copper) occupies the copper-binding sites in these variants. The absence of copper in these proteins, together with the results of covalent thiol modification experiments in yeast strains with and without the gene encoding the copper chaperone for SOD1 (CCS), suggests that CCS may not fully act on newly translated forms of these polypeptides. Overall, these findings lend support to the hypothesis that immature mutant SOD1 species contribute to toxicity in SOD1-linked ALS.
Assuntos
Esclerose Lateral Amiotrófica , Cobre/metabolismo , Chaperonas Moleculares/metabolismo , Superóxido Dismutase , Zinco/metabolismo , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Animais , Sítios de Ligação/genética , Cristalografia por Raios X , Humanos , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/genética , Mutação , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Difração de Raios X , Raios XRESUMO
Heme biosynthesis consists of a series of eight enzymatic reactions that originate in mitochondria and continue in the cytosol before returning to mitochondria. Although these core enzymes are well studied, additional mitochondrial transporters and regulatory factors are predicted to be required. To discover such unknown components, we utilized a large-scale computational screen to identify mitochondrial proteins whose transcripts consistently coexpress with the core machinery of heme biosynthesis. We identified SLC25A39, SLC22A4, and TMEM14C, which are putative mitochondrial transporters, as well as C1orf69 and ISCA1, which are iron-sulfur cluster proteins. Targeted knockdowns of all five genes in zebrafish resulted in profound anemia without impacting erythroid lineage specification. Moreover, silencing of Slc25a39 in murine erythroleukemia cells impaired iron incorporation into protoporphyrin IX, and vertebrate Slc25a39 complemented an iron homeostasis defect in the orthologous yeast mtm1Delta deletion mutant. Our results advance the molecular understanding of heme biosynthesis and offer promising candidate genes for inherited anemias.
Assuntos
Heme/biossíntese , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Anemia/genética , Animais , Embrião não Mamífero/metabolismo , Técnicas de Silenciamento de Genes , Heme/metabolismo , Proteínas Ferro-Enxofre/genética , Camundongos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Família Multigênica , RNA Interferente Pequeno/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Since the discovery of SOD1 in 1969, there have been numerous achievements made in our understanding of the enzyme's biochemical reactivity and its role in oxidative stress protection and as a genetic determinant in amyotrophic lateral sclerosis. Many recent advances have also been made in understanding the "activation" of SOD1, i.e. the process by which an inert polypeptide is converted to a mature active enzyme through post-translational modifications. To date, two such activation pathways have been identified: one requiring the CCS copper chaperone and one that works independently of CCS to insert copper and activate SOD1 through oxidation of an intramolecular disulfide. Depending on an organism's lifestyle and complexity, different eukaryotes have evolved to favor one pathway over the other. Some organisms rely solely on CCS for activating SOD1, and others can only activate SOD1 independently of CCS, whereas the majority of eukaryotes appear to have evolved to use both pathways. In this minireview, we shall highlight recent advances made in understanding the mechanisms by which the CCS-dependent and CCS-independent pathways control the activity, structure, and intracellular localization of copper,zinc superoxide dismutase, with relevance to amyotrophic lateral sclerosis and an emphasis on evolutionary biology.
Assuntos
Mutação , Superóxido Dismutase/metabolismo , Animais , Dissulfetos/química , Evolução Molecular , Humanos , Mitocôndrias/enzimologia , Modelos Biológicos , Modelos Moleculares , Oxigênio/química , Plantas/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais , Superóxido Dismutase/química , Superóxido Dismutase/genéticaRESUMO
Superoxide dismutase 2 (SOD2) is one of the rare mitochondrial enzymes evolved to use manganese as a cofactor over the more abundant element iron. Although mitochondrial iron does not normally bind SOD2, iron will misincorporate into Saccharomyces cerevisiae Sod2p when cells are starved for manganese or when mitochondrial iron homeostasis is disrupted by mutations in yeast grx5, ssq1, and mtm1. We report here that such changes in mitochondrial manganese and iron similarly affect cofactor selection in a heterologously expressed Escherichia coli Mn-SOD, but not a highly homologous Fe-SOD. By x-ray absorption near edge structure and extended x-ray absorption fine structure analyses of isolated mitochondria, we find that misincorporation of iron into yeast Sod2p does not correlate with significant changes in the average oxidation state or coordination chemistry of bulk mitochondrial iron. Instead, small changes in mitochondrial iron are likely to promote iron-SOD2 interactions. Iron binds Sod2p in yeast mutants blocking late stages of iron-sulfur cluster biogenesis (grx5, ssq1, and atm1), but not in mutants defective in the upstream Isu proteins that serve as scaffolds for iron-sulfur biosynthesis. In fact, we observed a requirement for the Isu proteins in iron inactivation of yeast Sod2p. Sod2p activity was restored in mtm1 and grx5 mutants by depleting cells of Isu proteins or using a dominant negative Isu1p predicted to stabilize iron binding to Isu1p. In all cases where disruptions in iron homeostasis inactivated Sod2p, we observed an increase in mitochondrial Isu proteins. These studies indicate that the Isu proteins and the iron-sulfur pathway can donate iron to Sod2p.
Assuntos
Ferro/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Superóxido Dismutase/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Absorciometria de Fóton , Regulação Fúngica da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Immunoblotting , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Superóxido Dismutase/genéticaRESUMO
Over 100 mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Two pathogenic SOD1 mutations, His46Arg (H46R) and His48Gln (H48Q), affect residues that act as copper ligands in the wild type enzyme. Transgenic mice expressing a human SOD1 variant containing both mutations develop paralytic disease akin to ALS. Here we show that H46R/H48Q SOD1 possesses multiple characteristics that distinguish it from the wild type. These properties include the following: (1) an ablated copper-binding site, (2) a substantially weakened affinity for zinc, (3) a binding site for a calcium ion, (4) the ability to form stable heterocomplexes with the copper chaperone for SOD1 (CCS), and (5) compromised CCS-mediated oxidation of the intrasubunit disulfide bond in vivo. The results presented here, together with data on pathogenic SOD1 proteins coming from cell culture and transgenic mice, suggest that incomplete posttranslational modification of nascent SOD1 polypeptides via CCS may be a characteristic shared by familial ALS SOD1 mutants, leading to a population of destabilized, off-pathway folding intermediates that are toxic to motor neurons.
Assuntos
Substituição de Aminoácidos/genética , Variação Genética , Mutação , Superóxido Dismutase/química , Superóxido Dismutase/genética , Animais , Arginina/genética , Linhagem Celular , Cobre/química , Cristalografia por Raios X , Estabilidade Enzimática/genética , Glutamina/genética , Histidina/genética , Humanos , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Processamento de Proteína Pós-Traducional/genética , Eletricidade Estática , Superóxido Dismutase/metabolismo , Superóxido Dismutase/toxicidade , Superóxido Dismutase-1RESUMO
In various organisms, high intracellular manganese provides protection against oxidative damage through unknown pathways. Herein we use a genetic approach in Saccharomyces cerevisiae to analyze factors that promote manganese as an antioxidant in cells lacking Cu/Zn superoxide dismutase (sod1 Delta). Unlike certain bacterial systems, oxygen resistance in yeast correlates with high intracellular manganese without a lowering of iron. This manganese for antioxidant protection is provided by the Nramp transporters Smf1p and Smf2p, with Smf1p playing a major role. In fact, loss of manganese transport by Smf1p together with loss of the Pmr1p manganese pump is lethal to sod1 Delta cells despite normal manganese SOD2 activity. Manganese-phosphate complexes are excellent superoxide dismutase mimics in vitro, yet through genetic disruption of phosphate transport and storage, we observed no requirement for phosphate in manganese suppression of oxidative damage. If anything, elevated phosphate correlated with profound oxidative stress in sod1 Delta mutants. The efficacy of manganese as an antioxidant was drastically reduced in cells that hyperaccumulate phosphate without effects on Mn SOD activity. Non-SOD manganese can provide a critical backup for Cu/Zn SOD1, but only under appropriate physiologic conditions.
Assuntos
Sobrevivência Celular/fisiologia , Sequestradores de Radicais Livres/metabolismo , Estresse Oxidativo/fisiologia , Saccharomyces cerevisiae/fisiologia , Superóxido Dismutase/metabolismo , Aerobiose/fisiologia , ATPases Transportadoras de Cálcio/genética , Proteínas de Transporte de Cátions/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Compostos de Manganês/farmacologia , Chaperonas Moleculares , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
Copper,zinc superoxide dismutase (SOD1) in mammals is activated principally via a copper chaperone (CCS) and to a lesser degree by a CCS-independent pathway of unknown nature. In this study, we have characterized the requirement for CCS in activating SOD1 from Drosophila. A CCS-null mutant (Ccs(n)(29)(E)) of Drosophila was created and found to phenotypically resemble Drosophila SOD1-null mutants in terms of reduced adult life span, hypersensitivity to oxidative stress, and loss of cytosolic aconitase activity. However, the phenotypes of CCS-null flies were less severe, consistent with some CCS-independent activation of Drosophila SOD1 (dSOD1). Yet SOD1 activity was not detectable in Ccs(n)(29)(E) flies, due largely to a striking loss of SOD1 protein. In contrast, human SOD1 expressed in CCS-null flies is robustly active and rescues the deficits in adult life span and sensitivity to oxidative stress. The dependence of dSOD1 on CCS was also observed in a yeast expression system where the dSOD1 polypeptide exhibited unusual instability in CCS-null (ccs1Delta) yeast. The residual dSOD1 polypeptide in ccs1Delta yeast was nevertheless active, consistent with CCS-independent activation. Stability of dSOD1 in ccs1Delta cells was readily restored by expression of either yeast or Drosophila CCS, and this required copper insertion into the enzyme. The yeast expression system also revealed some species specificity for CCS. Yeast SOD1 exhibits preference for yeast CCS over Drosophila CCS, whereas dSOD1 is fully activated with either CCS molecule. Such variation in mechanisms of copper activation of SOD1 could reflect evolutionary responses to unique oxygen and/or copper environments faced by divergent species.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Evolução Molecular , Chaperonas Moleculares/metabolismo , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estabilidade Enzimática/genética , Expressão Gênica , Humanos , Longevidade/genética , Chaperonas Moleculares/genética , Estresse Oxidativo/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
The CCS copper chaperone is critical for maturation of Cu, Zn-superoxide dismutase (SOD1) through insertion of the copper co-factor and oxidization of an intra-subunit disulfide. The disulfide helps stabilize the SOD1 polypeptide, which can be particularly important in cases of amyotrophic lateral sclerosis (ALS) linked to misfolding of mutant SOD1. Surprisingly, however, over-expressed CCS was recently shown to greatly accelerate disease in a G93A SOD1 mouse model for ALS. Herein we show that disease in these G93A/CCS mice correlates with incomplete oxidation of the SOD1 disulfide. In the brain and spinal cord, CCS over-expression failed to enhance oxidation of the G93A SOD1 disulfide and if anything, effected some accumulation of disulfide-reduced SOD1. This effect was mirrored in culture with a C244,246S mutant of CCS that has the capacity to interact with SOD1 but can neither insert copper nor oxidize the disulfide. In spite of disulfide effects, there was no evidence for increased SOD1 aggregation. If anything, CCS over-expression prevented SOD1 misfolding in culture as monitored by detergent insolubility. This protection against SOD1 misfolding does not require SOD1 enzyme activation as the same effect was obtained with the C244,246S allele of CCS. In the G93A SOD1 mouse, CCS over-expression was likewise associated with a lack of obvious SOD1 misfolding marked by detergent insolubility. CCS over-expression accelerates SOD1-linked disease without the hallmarks of misfolding and aggregation seen in other mutant SOD1 models. These studies are the first to indicate biological effects of CCS in the absence of SOD1 enzymatic activation.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Chaperonas Moleculares/metabolismo , Superóxido Dismutase/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Humanos , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/genética , Mutação , Dobramento de Proteína , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
Mutations in Cu,Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (ALS). It has been proposed that neuronal cell death might occur due to inappropriately increased Cu interaction with mutant SOD1. Using Cu immobilized metal-affinity chromatography (IMAC), we showed that mutant SOD1 (A4V, G85R, and G93A) expressed in transfected COS7 cells, transgenic mouse spinal cord tissue, and transformed yeast possessed higher affinity for Cu than wild-type SOD1. Serine substitution for cysteine at the Cys111 residue in mutant SOD1 abolished the Cu interaction on IMAC. C111S substitution reversed the accelerated degradation of mutant SOD1 in transfected cells, suggesting that the Cys111 residue is critical for the stability of mutant SOD1. Aberrant Cu binding at the Cys111 residue may be a significant factor in altering mutant SOD1 behavior and may explain the benefit of controlling Cu access to mutant SOD1 in models of familial ALS.
Assuntos
Esclerose Lateral Amiotrófica/etiologia , Cobre/metabolismo , Cisteína/química , Medula Espinal/enzimologia , Superóxido Dismutase/metabolismo , Substituição de Aminoácidos , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Animais , Células COS , Chlorocebus aethiops , Cromatografia de Afinidade/métodos , Cobre/química , Cisteína/genética , Estabilidade Enzimática/genética , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Serina/química , Serina/genética , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase-1 , TransfecçãoRESUMO
Many metalloproteins have the capacity to bind diverse metals, but in living cells connect only with their cognate metal cofactor. In eukaryotes, this metal specificity can be achieved through metal-specific metallochaperone proteins. Herein, we describe a mechanism whereby Saccharomyces cerevisiae manganese superoxide dismutase (SOD2) preferentially binds manganese over iron based on the differential bioavailability of these ions within mitochondria. The bulk of mitochondrial iron is normally unavailable to SOD2, but when mitochondrial iron homeostasis is disrupted, for example, by mutations in S. cerevisiae mtm1, ssq1 and grx5, iron accumulates in a reactive form that potently competes with manganese for binding to SOD2, inactivating the enzyme. Studies in mtm1 mutants indicate that iron inactivation of SOD2 involves the Mrs3p/Mrs4p mitochondrial carriers and iron-binding frataxin (Yfh1p). A small pool of SOD2-reactive iron also exists under normal iron homeostasis conditions and binds SOD2 when mitochondrial manganese is low. The ability to control this reactive pool of iron is critical to maintaining SOD2 activity and has important potential implications for oxidative stress in disorders of iron overload.
Assuntos
Ferro/metabolismo , Manganês/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ativação Enzimática , Homeostase , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , FrataxinaRESUMO
Prolonged exposure to hyperoxia represents a serious danger to cells, yet little is known about the specific cellular factors that affect hyperoxia stress. By screening the yeast deletion library, we have identified genes that protect against high-O2 damage. Out of approx. 4800 mutants, 84 were identified as hyperoxia-sensitive, representing genes with diverse cellular functions, including transcription and translation, vacuole function, NADPH production, and superoxide detoxification. Superoxide plays a significant role, since the majority of hyperoxia-sensitive mutants displayed cross-sensitivity to superoxide-generating agents, and mutants with compromised SOD (superoxide dismutase) activity were particularly vulnerable to hyperoxia. By comparison, factors known to guard against H2O2 toxicity were poorly represented amongst hyperoxia-sensitive mutants. Although many cellular components are potential targets, our studies indicate that mitochondrial glutathione is particularly vulnerable to hyperoxia damage. During hyperoxia stress, mitochondrial glutathione is more susceptible to oxidation than cytosolic glutathione. Furthermore, two factors that help maintain mitochondrial GSH in the reduced form, namely the NADH kinase Pos5p and the mitochondrial glutathione reductase (Glr1p), are critical for hyperoxia resistance, whereas their cytosolic counterparts are not. Our findings are consistent with a model in which hyperoxia toxicity is manifested by superoxide-related damage and changes in the mitochondrial redox state.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glutationa/fisiologia , Mitocôndrias/fisiologia , Mutação , Oxirredução , Estresse Oxidativo , Paraquat/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Superóxidos/metabolismoRESUMO
Manganese is an essential, but potentially toxic, trace metal in biological systems. Overexposure to manganese is known to cause neurological deficits in humans, but the pathways that lead to manganese toxicity are largely unknown. We have employed the bakers' yeast Saccharomyces cerevisiae as a model system to identify genes that contribute to manganese-related damage. In a genetic screen for yeast manganese-resistance mutants, we identified S. cerevisiae MAM3 as a gene which, when deleted, would increase cellular tolerance to toxic levels of manganese and also increased the cell's resistance towards cobalt and zinc. By sequence analysis, Mam3p shares strong similarity with the mammalian ACDP (ancient conserved domain protein) family of polypeptides. Mutations in human ACDP1 have been associated with urofacial (Ochoa) syndrome. However, the functions of eukaryotic ACDPs remain unknown. We show here that S. cerevisiae MAM3 encodes an integral membrane protein of the yeast vacuole whose expression levels directly correlate with the degree of manganese toxicity. Surprisingly, Mam3p contributes to manganese toxicity without any obvious changes in vacuolar accumulation of metals. Furthermore, through genetic epistasis studies, we demonstrate that MAM3 operates independently of the well-established manganese-trafficking pathways in yeast, involving the manganese transporters Pmr1p, Smf2p and Pho84p. This is the first report of a eukaryotic ACDP family protein involved in metal homoeostasis.