Verónica Irazusta

Manganese is the link between frataxin and iron-sulfur deficiency in the yeast model of Friedreich ataxia.

Friedreich ataxia is a human neurodegenerative and myocardial disease caused by decreased expression of the mitochondrial protein frataxin. Proteomic analysis of the mutant yeast model of Friedreich ataxia presented in this paper reveals that these cells display increased amounts of proteins involved in antioxidant defenses, including manganese-superoxide dismutase. This enzyme shows, however, lower activity than that found in wild type cells. Our results indicate that this lack of activity is a consequence of cellular manganese deficiency, because in manganese-supplemented cultures, cell manganese content, and manganese-superoxide dismutase activity were restored. One of the hallmarks of Friedreich ataxia is the decreased activity of iron/sulfur-containing enzymes. The activities of four enzymes of this group (aconitase, glutamate synthase, succinate dehydrogenase, and isopropylmalate dehydratase) have been analyzed for the effects of manganese supplementation. Enzyme activities were recovered by manganese treatment, except for aconitase, for which, a specific interaction with frataxin has been demonstrated previously. Similar results were obtained when cells were grown in iron-limited media suggesting that manganese-superoxide dismutase deficiency is a consequence of iron overload. In conclusion, these data indicate that generalized deficiency of iron-sulfur protein activity could be a consequence of manganese-superoxide dismutase deficiency, and consequently, it opens new strategies for Friedreich ataxia treatment.

Major targets of iron-induced protein oxidative damage in frataxin-deficient yeasts are magnesium-binding proteins

Iron accumulation has been associated with several pathological conditions such as Friedreich ataxia. This human disorder is caused by decreased expression of frataxin. Iron-overload triggers oxidative stress, but the main targets of such stress are not known. In yeast cells lacking the frataxin ortholog YFH1, we have identified a set of 14 carbonylated proteins, which include mitochondrial ATP synthase, phosphoglycerate kinase, pyruvate kinase, and molecular chaperones. Interestingly, most of the target proteins are magnesium- and/or nucleotide-binding proteins. This key feature leads us to postulate that when iron accumulates, chelatable iron replaces magnesium at the corresponding metal-binding site, promoting selective damage to these proteins. Consistent with this hypothesis, in vitro experiments performed with pure pyruvate kinase and phosphoglycerate kinase showed that oxidation of these proteins can be prevented by magnesium and increased by the presence of ATP. Also, chelatable iron, which forms complexes with nucleotides, showed a sevenfold increase in Deltayfh1 cells. Moreover, lowering chelatable iron in Deltayfh1 cells by desferrioxamine prevented enzyme inactivation. As a general conclusion, we propose that magnesium bound to proteins is replaced by chelatable iron when this metal accumulates. This mechanism explains selective protein oxidation and provides clues for better understanding of iron-overloading pathologies.

Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage.

Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Deltayfh1 sensitivity to menadione. Finally, a Deltasod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.

Frataxin deficiency in neonatal rat ventricular myocytes targets mitochondria and lipid metabolism

Friedreich ataxia (FRDA) is a hereditary disease caused by deficient frataxin expression. This mitochondrial protein has been related to iron homeostasis, energy metabolism, and oxidative stress. Patients with FRDA experience neurologic alterations and cardiomyopathy, which is the leading cause of death. The specific effects of frataxin depletion on cardiomyocytes are poorly understood because no appropriate cardiac cellular model is available to researchers. To address this research need, we present a model based on primary cultures of neonatal rat ventricular myocytes (NRVMs) and short-hairpin RNA interference. Using this approach, frataxin was reduced down to 5 to 30% of control protein levels after 7 days of transduction. At this stage the activity and amount of the iron-sulfur protein aconitase, in vitro activities of several OXPHOS components, levels of iron-regulated mRNAs, and the ATP/ADP ratio were comparable to controls. However, NRVMs exhibited markers of oxidative stress and a disorganized mitochondrial network with enlarged mitochondria. Lipids, the main energy source of heart cells, also underwent a clear metabolic change, indicated by the increased presence of lipid droplets and induction of medium-chain acyl-CoA dehydrogenase. These results indicate that mitochondria and lipid metabolism are primary targets of frataxin deficiency in NRVMs. Therefore, they contribute to the understanding of cardiac-specific mechanisms occurring in FRDA and give clues for the design of cardiac-specific treatment strategies for FRDA.

Proteomic Strategies for the Analysis of Carbonyl Groups on Proteins

Oxidative stress is caused by an imbalance between formation and destruction of reactive oxygen species. Analysis of the reaction products of reactive oxygen species in biomolecules is an indirect way of determining the existence of oxidative stress. In this context, the formation of carbonyl groups in proteins has been one of the most studied oxidative stress markers because of its stability and easy detection. Various proteomic tools offer great potential for the discovery of new proteins susceptible to oxidative stress, determination of quantitative changes in the profile of these modifications under different biological conditions, and characterization of the type of modification it has suffered a particular protein. This paper reviews the different approaches used for the detection of protein carbonyls and the proteomic tools that can be used to identify them.

Disentangling metabolic pathways involved in copper resistance in Candida fukuyamaensis RCL-3 indigenous yeast.

Candida fukuyamaensis RCL‐3 yeast strain isolated from a copper filter plant is able to lower copper concentration in culture medium. In the present study, effect of copper in proteins expression and mechanisms involved in copper resistance were explored using comparative proteomics. Mono‐dimensional gel electrophoresis revealed differential band expressions between cells grown with or without copper. 2‐DE analysis of C. fukuyamaensis RCL‐3 revealed that copper exposure produced at least an over‐expression of 40 proteins. Sixteen proteins were identified and grouped in four categories according to their functions: glycolysis and ATP production, synthesis of proteins, oxidative stress response, and processing and transport of proteins. Integral membrane proteins and membrane‐associated proteins were analyzed, showing nine protein bands over‐expressed in Cu‐supplemented medium. Four proteins were identified, namely nucleoporin pom152, elongation factor 2, copper chaperone Sod1 Ccs1, and eiosome component Lsp1. The proteomic analysis performed allowed the identification of different metabolic pathways and certain proteins involved in metal input and storage related to cell ability to bioremediate copper. These proteins and mechanisms could be used for future applications of C. fukuyamaensis RCL‐3 in biotechnological processes such as remediation of heavy metals.

Copper Resistance and Oxidative Stress Response in Rhodotorula mucilaginosa RCL-11. Yeast Isolated from Contaminated Environments in Tucumán, Argentina

The threat of heavy metal pollution to public health and wildlife has led to a great interest in the development of effective technologies for heavy metal immobilization in a non-bioavailable form or their conversion into less toxic forms. Organisms subjected to metal exposure in their natural environments have developed resistance mechanisms such us dedicated components and sophisticated homeostasis. Rhodotorula mucilaginosa RCL-11, a pigmented yeast isolated from a filter plant of a copper mine in the province of Tucuman, Argentina, supports high concentrations of the heavy metal Cu(II). In order to understand the mechanism involved in resistance to copper in this yeast, a proteomic study was conducted. Identification of differentially expressed proteins was performed. The results obtained show that when R. mucilaginosa RCL-11 was exposed to 0.5 mM copper, differential proteins, involved in cell resistance mechanisms, were expressed. Moreover, copper overload augmented carotenoid biosynthesis in this yeast, modifying at the same time the relative proportion of the pigments produced. Inhibition of the synthesis pathway with diphenylamine suggests an inverse relationship between carotenoid and copper biosorption by R. mucilaginosa RCL-11. The increased activity of superoxide dismutase and catalase measured under inhibition of carotenoid biosynthesis could explain these observations. The change in the relative proportion of the carotenoids torularhodin, torulene, and beta-carotene, as well as the detection of gamma-carotene in the presence of Cu(II) allows to hypothesize that the carotenoids produced by R. mucilaginosa RCL-11 play different roles in the oxidative stress response of this yeast.

Proteomic and enzymatic response under Cr(VI) overload in yeast isolated from textile-dye industry effluent

Cyberlindnera jadinii M9 and Wickerhamomyces anomalus M10 isolated from textile-dye liquid effluents has shown capacity for chromium detoxification via Cr(VI) biological reduction. The aim of the study was to evaluate the effect of hexavalent chromium on synthesis of novel and/or specific proteins involved in chromium tolerance and reduction in response to chromium overload in two indigenous yeasts. A study was carried out following a proteomic approach with W. anomalus M10 and Cy. jadinii M9 strains. For this, proteins extracts belonging to total cell extracts, membranes and mitochondria were analyzed. When Cr(VI) was added to culture medium there was an over-synthesis of 39 proteins involved in different metabolic pathways. In both strains, chromium supplementation changed protein biosynthesis by upregulating proteins involved in stress response, methionine metabolism, energy production, protein degradation and novel oxide-reductase enzymes. Moreover, we observed that Cy. jadinii M9 and W. anomalus M10 displayed ability to activate superoxide dismutase, catalase and chromate reductase activity. Two enzymes from the total cell extracts, type II nitroreductase (Frm2) and flavoprotein wrbA (Ycp4), were identified as possibly responsible for inducing crude chromate-reductase activity in cytoplasm of W. anomalus M10 under chromium overload. In Cy.jadinii M9, mitochondrial Ferredoxine-NADP reductase (Yah1) and membrane FAD flavoprotein (Lpd1) were identified as probably involved in Cr(VI) reduction. To our knowledge, this is the first study proposing chromate reductase activity of these four enzymes in yeast and reporting a relationship between protein synthesis, enzymatic response and chromium biospeciation in Cy. jadinii and W. anomalus.

Bio-precipitates produced by two autochthonous boron tolerant Streptomyces strains

Boron is widespread in the environment. Although contaminated soils are hard to recover different strategies have been investigated in the recent years. Bioremediation is one of the most studied because it is eco-friendly and less costly than other techniques. The aim of this research was to evaluate whether two Streptomyces strains isolated from boron contaminated soils in Salta, Argentina, may help remove boron from such soils. For this, they were grown in different liquid media with two boric acid concentrations and their specific growth rate and specific boric acid consumption rate were determined. Both strains showed great capacity to remove boron from the media. Increasing boric acid concentrations affected negatively the specific growth rate, however the specific boric acid consumption rate was superior. Boron bio-precipitates were observed when the strains grew in the presence of boric acid, probably due to an adaptive response developed by the cells to the exposure, for which many proteins were differentially synthetized. This strategy to tolerate high concentrations of boron by immobilizing it in bio-precipitates has not been previously described, to the best of our knowledge, and may have a great potential application in remediating soils contaminated with boron compounds.

Biomineralización de cobre en Candida fukuyamaensis RCL-3

Candida fukuyamaensis RCL-3 yeast has the ability to decrease copper concentration in a culture medium. High copper concentrations change the cell color from white/cream to brown. The effect of color change ceases with the addition of KCN or when cells are grown in a culture medium without sulfate ions. These results could be associated with CuS bioaccumulation in the cell surface. This report revealed that mineralization would be a mechanism used by this yeast for copper bioremediation.