Abordagens moleculares e prote?micas para a caracteriza??o de leveduras resistentes ao mangan?s II.

Programa de P?s-Gradua??o em Ci?ncias Biol?gicas. N?cleo de Pesquisas em Ci?ncias Biol?gicas, Pr?-Reitoria de Pesquisa de P?s Gradua??o, Universidade Federal de Ouro Preto.A polui??o do meio ambiente pelo metal pesado mangan?s (Mn) tem aumentado, j? que ? um subproduto industrial, especialmente de minera??es. Estrat?gias biotecnol?gicas promissoras para descontamina??o t?m sido desenvolvidas. A biorremedia??o, ? uma delas, emprega microrganismos, por exemplo, leveduras. O objetivo principal deste estudo foi caracterizar isolados de leveduras quanto aos mecanismos de resist?ncia e capacidade de remo??o do ?on Mn2+ e descrever como estas respondem ao estresse ambiental alterando seu padr?o de express?o proteica. Tr?s leveduras foram isoladas de ?gua proveniente de uma mina de rejeito de minera??o, localizada no Quadril?tero Ferr?fero-MG e identificadas como Meyerozyma guilliermondii, Meyerozyma caribbica e Rhodotorula mucilaginosa utilizando an?lise bioqu?mica, por Auxanograma e filogen?tica, baseadas no 16S rDNA. A avalia??o da capacidade dos isolados removerem ?ons Mn+2 demonstrou que ambos os g?neros estudados s?o resistentes ao metal, sendo que a presen?a de excesso de Mn n?o interferiu negativamente no crescimento. As Meyerozyma sp. removeram 100% do Mn2+ por processo de biossor??o e as Rhodotorula sp. 10%, provavelmente por oxida??o. A menor capacidade de remo??o do ?ltimo g?nero em rela??o ao primeiro n?o o torna menos importante, uma vez que apresenta toler?ncia e capacidade de remo??o a concentra??es elevadas de Mn2+ quando comparado a outros. O primeiro relato do proteoma sol?vel total e diferencial induzidos por Mn+2 do g?nero Meyerozyma sp., bem como das intera??es prote?naprote?na foi realizado a partir da t?cnica shotgun/bottom-up, em que extratos prot?icos de M. guilliermondii contendo as fra??es sol?veis foram obtidos ap?s crescimento nas condi??es aus?ncia e presen?a de MnSO4 (0,91 mM). Os pept?deos tr?pticos foram analisados por cromatografia l?quida acoplada a espectrometria de massa (LC-MS/MS), seguida de an?lises de bioinform?tica. No proteoma um total de 1257 prote?nas foi identificado, sendo que a an?lise qualitativa demonstrou que dessas 117 eram exclusivas da condi??o aus?ncia e 69 expressas unicamente na presen?a de Mn2+ . A an?lise quantitativa apresentou 71 prote?nas upregulated induzidas pelo excesso de Mn2+, em que foram identificados sete enriquecimentos funcionais e 43 vias metab?licas. A maioria das prote?nas anotadas na condi??o presen?a de Mn2+ est? relacionada ? atividade de oxidoredutases, resposta ao estresse oxidativo, atividades metab?licas, reparo de DNA e remodela??o da express?o de genes. Diante do exposto ? poss?vel concluir que as tr?s esp?cies s?o tolerantes a alta concentra??o de Mn2+, a import?ncia compreens?o dos processos celulares e dos mecanismos regulat?rios moleculares, pois eles permitem o conhecimento dos mecanismos de defesa que minimizam o impacto do metal atrav?s da express?o de prote?nas antioxidantes, por exemplo, permitindo o ajuste na resposta de defesa associados ? toler?ncia do Mn2+ . Esse conhecimento permitir? estudos futuros e a explora??o do potencial biotecnol?gico em futuros processos de biorremedia??o.Pollution of the environment by heavy metal manganese (Mn) has increased as it is an industrial byproduct, especially of mining. Promising biotechnological strategies for decontamination have been developed. Bioremediation, one of them, employs microorganisms, for example, yeast. The main objective of this study was to characterize yeast isolates regarding the mechanisms of resistance and ability to remove the Mn 2 + ion and to describe how they respond to environmental stress by altering their protein expression pattern. Three yeasts were isolated from water from a mine tailings, located in the Quadril?tero Ferr?fero-MG and identified as Meyerozyma guilliermondii, Meyerozyma caribbica and Rhodotorula mucilaginosa using biochemical analysis, by Auxanograma and phylogenetic, based on 16S rDNA. The evaluation of the ability of the isolates to remove Mn+2 ions showed that both studied genera are resistant to metal, and the presence of excess Mn did not negatively affect growth. The Meyerozyma sp. 100% of the Mn+2 by biosorption process and the Rhodotorula sp. 10%, probably due to oxidation. The lower removal capacity of the latter genus in relation to the former does not make it less important, since it presents tolerance and the ability to remove at high concentrations of Mn+2 when compared to others. The first report of the Mn+2 induced total and differential soluble proteome of the genus Meyerozyma sp., As well as the protein-protein interactions was performed using the shotgun /bottom-up technique, in which protein extracts of M. guilliermondii containing the fractions were obtained after growth in the absence and presence of MnSO 4 (0.91 mM). Tryptic peptides were analyzed by liquid chromatography coupled to mass spectrometry (LC-MS/MS), followed by bioinformatics analyzes. In the proteome a total of 1257 proteins were identified, and the qualitative analysis demonstrated that of these 117 were exclusive to the absence condition and 69 expressed solely in the presence of Mn+2. The quantitative analysis showed 71 upregulated proteins induced by excess Mn+2, in which seven functional enrichments and 43 metabolic pathways were identified. Most of the proteins noted in the presence of Mn+2 are related to oxidoreductase activity, response to oxidative stress, metabolic activities, DNA repair and gene expression remodeling. In view of the above, it is possible to conclude that the three species are tolerant to high concentration of Mn+2, the importance of understanding cellular processes and molecular regulatory mechanisms, since they allow the knowledge of the defense mechanisms that minimize the impact of the metal through the expression of proteins antioxidants, for example, allowing adjustment in the defense response associated with Mn+2 tolerance. This knowledge will allow future studies and the exploration of the biotechnological potential in future bioremediation processes

Manganese alters expression of proteins involved in the oxidative stress of Meyerozyma guilliermondii.

Organisms, 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

Alterations in the proteomic composition of Serratia marcescens in response to manganese (II)

Abstract Background 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

Manganese alters expression of proteins involved in the oxidative stress of Meyerozyma guilliermondii.

Organisms, 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

Manganese (Mn2+) tolerance and biosorption by Meyerozyma guilliermondii and Meyerozyma caribbica strains.

Bioremediation 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

PPAR? is a major regulator of branched-chain amino acid blood levels and catabolism in white and brown adipose tissues.

Objective We investigated whether PPAR? modulates adipose tissue BCAA metabolism, and whether this mediates the attenuation of obesity-associated insulin resistance induced by pharmacological PPAR? activation. Methods Mice with adipocyte deletion of one or two PPAR? 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 PPAR? 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 ?-ketoacid dehydrogenase (BCKDH) complex subunits. Adipocyte deletion of two PPAR? copies induced lipodystrophy, severe glucose intolerance and markedly increased serum BCAA. Rosiglitazone abolished the increase in serum BCAA induced by adipocyte PPAR? 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 PPAR?, 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. PPAR? agonism, therefore, may improve whole-body and muscle insulin sensitivity by reducing blood BCAA, alleviating mTORC1-mediated inhibitory IRS1 phosphorylation