Browsing by Author "Ruas, France Anne Dias"
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Item Abordagens moleculares e proteômicas para a caracterização de leveduras resistentes ao manganês II.(2019) Ruas, France Anne Dias; Cota, Renata Guerra de Sá; Cota, Renata Guerra de Sá; Santiago, Aníbal da Fonseca; Silva, Breno de Mello; Rúbio, Karina Taciana Santos; Borges, Marcia HelenaA poluição do meio ambiente pelo metal pesado manganês (Mn) tem aumentado, já que é um subproduto industrial, especialmente de minerações. Estratégias biotecnológicas promissoras para descontaminação têm sido desenvolvidas. A biorremediação, é uma delas, emprega microrganismos, por exemplo, leveduras. O objetivo principal deste estudo foi caracterizar isolados de leveduras quanto aos mecanismos de resistência e capacidade de remoção do íon Mn2+ e descrever como estas respondem ao estresse ambiental alterando seu padrão de expressão proteica. Três leveduras foram isoladas de água proveniente de uma mina de rejeito de mineração, localizada no Quadrilátero Ferrífero-MG e identificadas como Meyerozyma guilliermondii, Meyerozyma caribbica e Rhodotorula mucilaginosa utilizando análise bioquímica, por Auxanograma e filogenética, baseadas no 16S rDNA. A avaliação da capacidade dos isolados removerem íons Mn+2 demonstrou que ambos os gêneros estudados são resistentes ao metal, sendo que a presença de excesso de Mn não interferiu negativamente no crescimento. As Meyerozyma sp. removeram 100% do Mn2+ por processo de biossorção e as Rhodotorula sp. 10%, provavelmente por oxidação. A menor capacidade de remoção do último gênero em relação ao primeiro não o torna menos importante, uma vez que apresenta tolerância e capacidade de remoção a concentrações elevadas de Mn2+ quando comparado a outros. O primeiro relato do proteoma solúvel total e diferencial induzidos por Mn+2 do gênero Meyerozyma sp., bem como das interações proteínaproteína foi realizado a partir da técnica shotgun/bottom-up, em que extratos protéicos de M. guilliermondii contendo as frações solúveis foram obtidos após crescimento nas condições ausência e presença de MnSO4 (0,91 mM). Os peptídeos trípticos foram analisados por cromatografia líquida acoplada a espectrometria de massa (LC-MS/MS), seguida de análises de bioinformática. No proteoma um total de 1257 proteínas foi identificado, sendo que a análise qualitativa demonstrou que dessas 117 eram exclusivas da condição ausência e 69 expressas unicamente na presença de Mn2+ . A análise quantitativa apresentou 71 proteínas upregulated induzidas pelo excesso de Mn2+, em que foram identificados sete enriquecimentos funcionais e 43 vias metabólicas. A maioria das proteínas anotadas na condição presença de Mn2+ está relacionada à atividade de oxidoredutases, resposta ao estresse oxidativo, atividades metabólicas, reparo de DNA e remodelação da expressão de genes. Diante do exposto é possível concluir que as três espécies são tolerantes a alta concentração de Mn2+, a importância compreensão dos processos celulares e dos mecanismos regulatórios moleculares, pois eles permitem o conhecimento dos mecanismos de defesa que minimizam o impacto do metal através da expressão de proteínas antioxidantes, por exemplo, permitindo o ajuste na resposta de defesa associados à tolerância do Mn2+ . Esse conhecimento permitirá estudos futuros e a exploração do potencial biotecnológico em futuros processos de biorremediação.Item Alterations in the proteomic composition of Serratia marcescens in response to manganese (II).(2018) Queiroz, Pollyana Santos; Ruas, France Anne Dias; Barboza, Natália Rocha; Borges, William de Castro; Cota, Renata Guerra de SáBackground: Proteomics is an important tool for the investigation of dynamic physiological responses of microbes under heavy metal stress. To gain insight into how bacteria respond to manganese (II) and identify the proteins involved in Mn (II) oxidation, the shotgun proteomics approach was applied to a potential Mn (II)-oxidizing Serratia marcescens strain cultivated in the absence and presence of Mn (II). Results: The LG1 strain, which grew equally well in the two conditions, was found to express a set of proteins related to cellular processes vital for survival, as well as proteins involved in adaptation and tolerance to Mn (II). The multicopper oxidase CueO was identified, indicating its probable participation in the Mn (II) bio-oxidation; however, its expression was not modulated by the presence of Mn (II). A set of proteins related to cell and metabolic processes vital to the cells were downregulated in the presence of Mn (II), while cell membrane-related proteins involved in the maintenance of cell integrity and survival under stress were upregulated under this condition. Conclusions: These findings indicate that the LG1 strain may be applied successfully in the bioremediation of Mn (II), and the shotgun approach provides an efficient means for obtaining the total proteome of this species.Item In silico prediction of protein-protein interaction network induced by manganese II in Meyerozyma guilliermondii.(2020) Ruas, France Anne Dias; Cota, Renata Guerra de SáRecently, there has been an increasing interest in the use of yeast to produce biosorbent materials, because yeast is economical to use, adaptable to a variety of conditions, and amenable to morphological manipulations to yield better raw biomaterials. Previous studies from our laboratory have shown that Meyerozyma guilliermondii, a nonpathogenic haploid yeast (ascomycete), exhibits excellent biosorption capacity for Mn2+, as demonstrated by kinetic analyses. Shotgun/bottom-up analyses of soluble fractions revealed a total of 1257 identified molecules, with 117 proteins expressed in the absence of Mn2+ and 69 expressed only in the presence of Mn2+. In this article, we describe the first in silico prediction and screening of protein–protein interactions (PPIs) in M. guilliermondii using experimental data from shotgun/bottom-up analyses. We also present the categorization of biological processes (BPs), molecular functions (MFs), and metabolic pathways of 71 proteins upregulated in the M. guilliermondii proteome in response to stress caused by an excess of Mn2+ ions. Most of the annotated proteins were related to oxidation–reduction processes, metabolism, and response to oxidative stress. We identified seven functional enrichments and 42 metabolic pathways; most proteins belonged to pathways related to metabolic pathways (19 proteins) followed by the biosynthesis of secondary metabolites (10 proteins) in the presence of Mn2+. Using our data, it is possible to infer that defense mechanisms minimize the impact of Mn2+ via the expression of antioxidant proteins, thus allowing adjustment during the defense response. Previous studies have not considered protein interactions in this genus in a manner that permits comparisons. Consequently, the findings of the current study are innovative, highly relevant, and provide a description of interactive complexes and networks that yield insight into the cellular processes of M. guilliermondii. Collectively, our data will allow researchers to explore the biotechnological potential of M. guilliermondii in future bioremediation processes.Item Manganese (Mn2+) tolerance and biosorption by Meyerozyma guilliermondii and Meyerozyma caribbica strains.(2018) Amorim, Soraya Sander; Ruas, France Anne Dias; Barboza, Natália Rocha; Neves, Viviano Gomes de Oliveira; Leão, Versiane Albis; Cota, Renata Guerra de SáBioremediation of manganese (Mn) is notoriously difficult to achieve because of the high stability of Mn2+ in aqueous solutions. Regarding the biotechnology strategies for removal of Mn from water, the use of bioremediation by bacteria and fungi is well known, but little is known about how yeasts can participate in this process. Hence, the study’s aim was to isolate yeasts with the ability to remove Mn2+, also elucidate the mechanism related to Mn bioremediation. Two kinds of yeast organisms were isolated from Brazilian mining water and identified as Meyerozyma guilliermondii and Meyerozyma caribbica by biochemical and phylogenetic analyses. Both isolates survived and their colonies grew in up to 32 mM of Mn2+, and they were able to remove 100% of Mn2+ from the culture medium in small-scale batch experiments conducted overall 1-week period. It was observed that for both isolates the removal of Mn is independent of pH. Analysis by SEM/EDX revealed that the Mn was adsorbed by the cell walls of M. guilliermondii and M. caribbica in the biosorption assays in the biological removal of bound Mn2+. These results demonstrated that both yeasts, with living and dead biomass, have an excellent Mn2+ ion biosorption capacity, as demonstrated by kinetic equations, in which M. caribbica showed a higher velocity when compared to M. guilliermondii. (respectively 1.088 and 0.324 mgMn day−1). Taken together, our results showed that these two yeasts isolates have potential roles in developing new biotechnology applications related to Mn bioremediation from waters contaminated with this persistent ion.Item Manganese alters expression of proteins involved in the oxidative stress of Meyerozyma guilliermondii.(2019) Ruas, France Anne Dias; Barboza, Natália Rocha; Borges, William de Castro; Cota, Renata Guerra de SáOrganisms, in general, respond to environmental stress by altering their pattern of protein expression (proteome), as an alternative to growing in stressful conditions. A strain of Meyerozyma guilliermondii resistant to manganese was isolated from a sample of water collected from mine drainage in southeastern Minas Gerais (Brazil), and demonstrated manganese detoxification capacity. Protein extracts containing the soluble fractions were obtained after growth of the strain in the absence and presence of MnSO4. Tryptic peptides recovered from samples were analyzed by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Shotgun/bottom-up analyses of the soluble fractions revealed a total of 1257 identified molecules. Treatment with Mn did not affect the growth of yeast but induced changes in the protein profile, with 117 proteins expressed in the absence of Mn and 69 expressed only in its presence. Most of these are annotated as related to DNA repair, oxidoreductase activity, and remodeling of gene expression. This is the first proteomic report of M. guilliermondii, with promising characteristics for Mn bioremediation, and the first of the genus Meyerozyma. This proteomic characterization may help in the understanding of molecular regulatory mechanisms associated with tolerance to excess Mn, and the potential use of biomass in bioremediation processes. Significance: Environmental pollution by heavy metals such as manganese (Mn2+) has increased as it is a by-product of the mining industry and a potential environmental contaminant. Many studies have explored the use of bacteria for manganese bioremediation, but yeasts have emerged as a promising alternative, displaying faster growth and greater removal efficiency. Previous works of our laboratory showed that Meyerozyma guilliermondii, a non-pathogenic haploid yeast (ascomycete), has excellent removal and accumulation capacity of Mn2+, potentially useful in bioremediation. Nowadays efforts have been devoted to understanding the physiology of metal hyperaccumulation to gain insights into the molecular basis of hyperaccumulation. To obtain a comprehensive understanding of the molecular mechanism of Mn2+ hyperaccumulation in M. guilliermondii, proteomic approaches were employed yielding the first compositional proteomic map of total soluble proteins and their differential expression in the presence of Mn2+. We believe our findings are of biotechnological interest concerning the utilization of M. guilliermondii for bioremediation purposes.Item Manganese alters expression of proteins involved in the oxidative stress of Meyerozyma guilliermondii.(2019) Ruas, France Anne Dias; Barboza, Natália Rocha; Borges, William de Castro; Cota, Renata Guerra de SáOrganisms, in general, respond to environmental stress by altering their pattern of protein expression (proteome), as an alternative to growing in stressful conditions. A strain of Meyerozyma guilliermondii resistant to manganese was isolated from a sample of water collected from mine drainage in southeastern Minas Gerais (Brazil), and demonstrated manganese detoxification capacity. Protein extracts containing the soluble fractions were obtained after growth of the strain in the absence and presence of MnSO4. Tryptic peptides recovered from samples were analyzed by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Shotgun/bottomup analyses of the soluble fractions revealed a total of 1257 identified molecules. Treatment with Mn did not affect the growth of yeast but induced changes in the protein profile, with 117 proteins expressed in the absence of Mn and 69 expressed only in its presence. Most of these are annotated as related to DNA repair, oxidoreductase activity, and remodeling of gene expression. This is the first proteomic report of M. guilliermondii, with promising characteristics for Mn bioremediation, and the first of the genus Meyerozyma. This proteomic characterization may help in the understanding of molecular regulatory mechanisms associated with tolerance to excess Mn, and the potential use of biomass in bioremediation processes. Significance: Environmental pollution by heavy metals such as manganese (Mn2+) has increased as it is a byproduct of the mining industry and a potential environmental contaminant. Many studies have explored the use of bacteria for manganese bioremediation, but yeasts have emerged as a promising alternative, displaying faster growth and greater removal efficiency. Previous works of our laboratory showed that Meyerozyma guilliermondii, a non-pathogenic haploid yeast (ascomycete), has excellent removal and accumulation capacity of Mn2+, potentially useful in bioremediation. Nowadays efforts have been devoted to understanding the physiology of metal hyperaccumulation to gain insights into the molecular basis of hyperaccumulation. To obtain a comprehensive understanding of the molecular mechanism of Mn2+ hyperaccumulation in M. guilliermondii, proteomic approaches were employed yielding the first compositional proteomic map of total soluble proteins and their differential expression in the presence of Mn2+. We believe our findings are of biotechnological interest concerning the utilization of M. guilliermondii for bioremediation purposes.Item PPARγ is a major regulator of branched-chain amino acid blood levels and catabolism in white and brown adipose tissues.(2018) Blanchard, Pierre-Gilles; Moreira, Rafael Junges; Castro, Érique de; Caron, Alexandre; Côté, Marie; Andrade, Maynara Lucca; Silva, Tiago Eugênio Oliveira da; Silva, Milene Ortiz; Peixoto, Álbert Souza; Ruas, France Anne Dias; Gélinas, Yves; Cota, Renata Guerra de Sá; Deshaies, Yves; Festuccia, William Tadeu LaraObjective We investigated whether PPARγ modulates adipose tissue BCAA metabolism, and whether this mediates the attenuation of obesity-associated insulin resistance induced by pharmacological PPARγ activation. Methods Mice with adipocyte deletion of one or two PPARγ copies fed a chow diet and rats fed either chow, or high fat (HF) or HF supplemented with BCAA (HF/BCAA) diets treated with rosiglitazone (30 or 15 mg/kg/day, 14 days) were evaluated for glucose and BCAA homeostasis. Results Adipocyte deletion of one PPARγ copy increased mice serum BCAA and reduced inguinal white (iWAT) and brown (BAT) adipose tissue BCAA incorporation into triacylglycerol, as well as mRNA levels of branched-chain aminotransferase (BCAT)2 and branched-chain α-ketoacid dehydrogenase (BCKDH) complex subunits. Adipocyte deletion of two PPARγ copies induced lipodystrophy, severe glucose intolerance and markedly increased serum BCAA. Rosiglitazone abolished the increase in serum BCAA induced by adipocyte PPARγ deletion. In rats, HF increased serum BCAA, such levels being further increased by BCAA supplementation. Rosiglitazone, independently of diet, lowered serum BCAA and upregulated iWAT and BAT BCAT and BCKDH activities. This was associated with a reduction in mTORC1-dependent inhibitory serine phosphorylation of IRS1 in skeletal muscle and whole-body insulin resistance evaluated by HOMA-IR. Conclusions PPARγ, through the regulation of both BAT and iWAT BCAA catabolism in lipoeutrophic mice and muscle insulin responsiveness and proteolysis in lipodystrophic mice, is a major determinant of circulating BCAA levels. PPARγ agonism, therefore, may improve whole-body and muscle insulin sensitivity by reducing blood BCAA, alleviating mTORC1-mediated inhibitory IRS1 phosphorylation.Item Rhodotorula mucilaginosa isolated from the manganese mine water in Minas Gerais, Brazil : potential employment for bioremediation of contaminated water.(2020) Ruas, France Anne Dias; Amorim, Soraya Sander; Leão, Versiane Albis; Cota, Renata Guerra de SáManganese (Mn) is an essential heavy metal for living organisms. However, they tend to accumulate the excess of this element causing health problems. The increase of the environmental contamination by this heavy metalis mainly due to mining practices. This work aimed to isolate yeast from mining waters to evaluate its ability of removing Mn2+ ions for the development of further biotechnological and bioremediation applications. The growing interest of this study is because it could be effective in the biological treatment of contaminated water, which remains a major challenge for industry and environment. Yeasts were initially isolated from mining water and grown in YPD medium containing 1 to 54 mM Mn2+. Subsequently, the isolates were characterized biochemically and phylogenetically. Then, we evaluate the ability of the isolates of removing Mn2+ ion by SEM scanning electron microscopy coupled with SEM/EDX. Our results showed yeast growth up to 32 mM. There was no pH increase along the tests, suggesting a biological Mn2+ removal. Taken together, the morphological changes in the colony and the darkening of the culture medium suggest the yeast’s ability of oxidizing Mn2+. Five isolates remove these ions considerably, identified as Rhodotorula mucilaginosa. SEM/EDX analysis shows the ability to oxidize and adsorb Mn. The data obtained in this work allows us to conclude that R. mucilaginosa has an important role in the biogeochemical cycle of manganese and present potential biotechnological applications for bioremediation of water contaminated with Mn2+ ions.