Análises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensis
Autor(a) principal: | |
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Data de Publicação: | 2010 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFG |
Texto Completo: | http://repositorio.bc.ufg.br/tede/handle/tde/1282 |
Resumo: | The cell wall of pathogenic microbes acts as an initial barrier that is in contact with hostile environments. Besides functioning as a mechanical barrier, it harbours an immunogenic macromolecules arsenal. One of the ways that proteins can be associated to the cell wall, it is through GPI anchor. The hydrophobic C-terminal end of the β-1,3-glucanosyltransferase enzyme of the human pathogenic fungus Paracoccidioides brasiliensis is characteristic of GPI anchored proteins. The β-1,3-glucan assembling and rearrangement are essential since this molecule acts as a scaffold to support cell wall proteins and polysaccharides. In the thermodimorphic fungus P. brasiliensis, β-1,3-glucan is found predominantly in mycelium form and α-1,3-glucan is predominant in the yeast form. In this work, it was screened possible protein-protein interactions performed by β-1,3-glucanosyltransferase 1 of P. brasiliensis (PbGel1p). To obtain these results, a P. brasiliensis cDNA library was screened with PbGel1p using the Saccharomyces cerevisiae two hybrid system. In addition, pull-down assay was used as an in vitro complementary technique to isolate proteins that interact direct or indirectly with PbGel1p. It was screened 38 gene products using two hybrid system and it was identified 3 proteins using the pull-down assay associated with mass spectrometry. The PbGel1p role in the cell wall maintenance and remodeling was indicated through the analysis of screened interactions, like alpha-glucosides permease, acid phosphatase, GDSL lipase, septin, actin, tubulin, HSP90 and pyruvate kinase. Furthermore, nuclear localization of PbGel1p and its role in the locus-specific transcriptional silencing were suggested based on such interactions: Qde2 argonaute, transcription elongation factor spt6, others transcription factors and ATP-citrate synthase. Therefore, this study indicated, for the first time, that PbGel1p has multiple location and it participates either in roles classically described for glucanosyltransferases, as the cell wall remodeling, or in recently described functions for this family of proteins, as the locus-specific transcriptional silencing. |
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SOARES, Célia Maria de Almeidahttp://lattes.cnpq.br/8539946335852637http://lattes.cnpq.br/7291301552786046BAILÃO, Elisa Flávia Luiz Cardoso2014-07-29T15:16:35Z2010-05-032010-01-28BAILÃO, Elisa Flávia Luiz Cardoso. In vivo and in vitro analysis of Paracoccidioides brasiliensis Beta-1,3-glucanosyltransferase 1 intermolecular interactions. 2010. 107 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2010.http://repositorio.bc.ufg.br/tede/handle/tde/1282ark:/38995/001300000bwggThe cell wall of pathogenic microbes acts as an initial barrier that is in contact with hostile environments. Besides functioning as a mechanical barrier, it harbours an immunogenic macromolecules arsenal. One of the ways that proteins can be associated to the cell wall, it is through GPI anchor. The hydrophobic C-terminal end of the β-1,3-glucanosyltransferase enzyme of the human pathogenic fungus Paracoccidioides brasiliensis is characteristic of GPI anchored proteins. The β-1,3-glucan assembling and rearrangement are essential since this molecule acts as a scaffold to support cell wall proteins and polysaccharides. In the thermodimorphic fungus P. brasiliensis, β-1,3-glucan is found predominantly in mycelium form and α-1,3-glucan is predominant in the yeast form. In this work, it was screened possible protein-protein interactions performed by β-1,3-glucanosyltransferase 1 of P. brasiliensis (PbGel1p). To obtain these results, a P. brasiliensis cDNA library was screened with PbGel1p using the Saccharomyces cerevisiae two hybrid system. In addition, pull-down assay was used as an in vitro complementary technique to isolate proteins that interact direct or indirectly with PbGel1p. It was screened 38 gene products using two hybrid system and it was identified 3 proteins using the pull-down assay associated with mass spectrometry. The PbGel1p role in the cell wall maintenance and remodeling was indicated through the analysis of screened interactions, like alpha-glucosides permease, acid phosphatase, GDSL lipase, septin, actin, tubulin, HSP90 and pyruvate kinase. Furthermore, nuclear localization of PbGel1p and its role in the locus-specific transcriptional silencing were suggested based on such interactions: Qde2 argonaute, transcription elongation factor spt6, others transcription factors and ATP-citrate synthase. Therefore, this study indicated, for the first time, that PbGel1p has multiple location and it participates either in roles classically described for glucanosyltransferases, as the cell wall remodeling, or in recently described functions for this family of proteins, as the locus-specific transcriptional silencing.A parede celular de microrganismos patogênicos atua como uma barreira inicial no contato entre o parasito e o hospedeiro. Além de funcionar como uma barreira mecânica, ela abriga um arsenal de macromoléculas imunogênicas. Uma forma pela qual as proteínas estão associadas à parede celular é por meio de âncoras-GPI. A extremidade carboxila hidrofóbica da enzima β-1,3-glicanosiltransferase do fungo patogênico humano Paracoccidioides brasiliensis é característica de proteínas GPI-ancoradas. A montagem e o rearranjo de β-1,3- glicana são de fundamental importância porque esta molécula serve como um esqueleto sobre o qual outros polissacarídeos e proteínas da parede celular estão associados. No fungo termodimórfico P. brasiliensis, β-1,3-glicana é encontrada prioritariamente em micélio, sendo α-1,3-glicana predominante em levedura. Foram rastreadas neste trabalho possíveis interações proteína-proteína realizadas pela β-1,3-glicanosiltransferase 1 de P. brasiliensis (PbGel1p). Para isso utilizou-se a técnica de duplo-híbrido em Saccharomyces cerevisiae, rastreando-se uma biblioteca de cDNA do fungo P. brasiliensis com a enzima estudada. Adicionalmente, foi utilizado, como técnica complementar, o ensaio de pull-down, que isolou in vitro proteínas que interagem direta ou indiretamente com a PbGel1p. Foi possível rastrear 38 produtos gênicos através do sistema de duplo híbrido e isolar três proteínas pelo ensaio de pull-down, identificadas por espectrometria de massas. O papel da PbGel1p na manutenção e no remodelamento da parede celular do fungo foi indicado através da análise das interações rastreadas, como permease de α-glicosídeos, fosfatase ácida, lipase da família GDSL, septina, actina, tubulina, HSP90 e piruvato quinase. Além disso, foram sugeridos a localização da PbGel1p no núcleo das células do fungo e seu papel no silenciamento gênico mediado por alterações estruturais, por meio do rastreamento das seguintes proteínas ligantes a PbGel1p: argonauta Qde2, fator de alongamento transcricional spt6, outros fatores transcricionais e ATP-citrato sintase. Portanto, este estudo indicou, pela primeira vez, que PbGel1p tem localização múltipla e participa tanto de funções classicamente descritas para glicanosiltransferases, como o remodelamento da parede celular, quanto de funções recentemente descritas para essa família de proteínas, como o silenciamento transcricional sítio-específico.Made available in DSpace on 2014-07-29T15:16:35Z (GMT). No. of bitstreams: 1 Dissertacao FINAL_EF_corrigida_BC-UFG.pdf: 1485117 bytes, checksum: ecdc9729cc08da2832a6566c39052f65 (MD5) Previous issue date: 2010-01-28application/pdfhttp://repositorio.bc.ufg.br/TEDE/retrieve/3998/Dissertacao%20FINAL_EF_corrigida_BC-UFG.pdf.jpgporUniversidade Federal de GoiásMestrado em BiologiaUFGBRCiências BiolóicasGlicanosiltransferase, duplo híbrido e pull-downGlucanosyltransferase, two hybrid and pull-downCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARAnálises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensisIn vivo and in vitro analysis of Paracoccidioides brasiliensis Beta-1,3-glucanosyltransferase 1 intermolecular interactionsinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFGinstname:Universidade Federal de Goiás (UFG)instacron:UFGORIGINALDissertacao FINAL_EF_corrigida_BC-UFG.pdfapplication/pdf1485117http://repositorio.bc.ufg.br/tede/bitstreams/a6ba06e5-2b56-4551-addf-c3b8216d5b2f/downloadecdc9729cc08da2832a6566c39052f65MD51THUMBNAILDissertacao FINAL_EF_corrigida_BC-UFG.pdf.jpgDissertacao FINAL_EF_corrigida_BC-UFG.pdf.jpgGenerated Thumbnailimage/jpeg6106http://repositorio.bc.ufg.br/tede/bitstreams/3a7eb02a-ab81-436b-9130-8ae85599a745/download8efa727aedb87ad86602fff44bed6be1MD52tde/12822014-07-30 03:10:00.367open.accessoai:repositorio.bc.ufg.br:tde/1282http://repositorio.bc.ufg.br/tedeRepositório InstitucionalPUBhttp://repositorio.bc.ufg.br/oai/requesttasesdissertacoes.bc@ufg.bropendoar:2014-07-30T06:10Repositório Institucional da UFG - Universidade Federal de Goiás (UFG)false |
dc.title.por.fl_str_mv |
Análises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensis |
dc.title.alternative.eng.fl_str_mv |
In vivo and in vitro analysis of Paracoccidioides brasiliensis Beta-1,3-glucanosyltransferase 1 intermolecular interactions |
title |
Análises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensis |
spellingShingle |
Análises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensis BAILÃO, Elisa Flávia Luiz Cardoso Glicanosiltransferase, duplo híbrido e pull-down Glucanosyltransferase, two hybrid and pull-down CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
title_short |
Análises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensis |
title_full |
Análises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensis |
title_fullStr |
Análises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensis |
title_full_unstemmed |
Análises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensis |
title_sort |
Análises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensis |
author |
BAILÃO, Elisa Flávia Luiz Cardoso |
author_facet |
BAILÃO, Elisa Flávia Luiz Cardoso |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
SOARES, Célia Maria de Almeida |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/8539946335852637 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/7291301552786046 |
dc.contributor.author.fl_str_mv |
BAILÃO, Elisa Flávia Luiz Cardoso |
contributor_str_mv |
SOARES, Célia Maria de Almeida |
dc.subject.por.fl_str_mv |
Glicanosiltransferase, duplo híbrido e pull-down |
topic |
Glicanosiltransferase, duplo híbrido e pull-down Glucanosyltransferase, two hybrid and pull-down CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
dc.subject.eng.fl_str_mv |
Glucanosyltransferase, two hybrid and pull-down |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
description |
The cell wall of pathogenic microbes acts as an initial barrier that is in contact with hostile environments. Besides functioning as a mechanical barrier, it harbours an immunogenic macromolecules arsenal. One of the ways that proteins can be associated to the cell wall, it is through GPI anchor. The hydrophobic C-terminal end of the β-1,3-glucanosyltransferase enzyme of the human pathogenic fungus Paracoccidioides brasiliensis is characteristic of GPI anchored proteins. The β-1,3-glucan assembling and rearrangement are essential since this molecule acts as a scaffold to support cell wall proteins and polysaccharides. In the thermodimorphic fungus P. brasiliensis, β-1,3-glucan is found predominantly in mycelium form and α-1,3-glucan is predominant in the yeast form. In this work, it was screened possible protein-protein interactions performed by β-1,3-glucanosyltransferase 1 of P. brasiliensis (PbGel1p). To obtain these results, a P. brasiliensis cDNA library was screened with PbGel1p using the Saccharomyces cerevisiae two hybrid system. In addition, pull-down assay was used as an in vitro complementary technique to isolate proteins that interact direct or indirectly with PbGel1p. It was screened 38 gene products using two hybrid system and it was identified 3 proteins using the pull-down assay associated with mass spectrometry. The PbGel1p role in the cell wall maintenance and remodeling was indicated through the analysis of screened interactions, like alpha-glucosides permease, acid phosphatase, GDSL lipase, septin, actin, tubulin, HSP90 and pyruvate kinase. Furthermore, nuclear localization of PbGel1p and its role in the locus-specific transcriptional silencing were suggested based on such interactions: Qde2 argonaute, transcription elongation factor spt6, others transcription factors and ATP-citrate synthase. Therefore, this study indicated, for the first time, that PbGel1p has multiple location and it participates either in roles classically described for glucanosyltransferases, as the cell wall remodeling, or in recently described functions for this family of proteins, as the locus-specific transcriptional silencing. |
publishDate |
2010 |
dc.date.available.fl_str_mv |
2010-05-03 |
dc.date.issued.fl_str_mv |
2010-01-28 |
dc.date.accessioned.fl_str_mv |
2014-07-29T15:16:35Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
BAILÃO, Elisa Flávia Luiz Cardoso. In vivo and in vitro analysis of Paracoccidioides brasiliensis Beta-1,3-glucanosyltransferase 1 intermolecular interactions. 2010. 107 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2010. |
dc.identifier.uri.fl_str_mv |
http://repositorio.bc.ufg.br/tede/handle/tde/1282 |
dc.identifier.dark.fl_str_mv |
ark:/38995/001300000bwgg |
identifier_str_mv |
BAILÃO, Elisa Flávia Luiz Cardoso. In vivo and in vitro analysis of Paracoccidioides brasiliensis Beta-1,3-glucanosyltransferase 1 intermolecular interactions. 2010. 107 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2010. ark:/38995/001300000bwgg |
url |
http://repositorio.bc.ufg.br/tede/handle/tde/1282 |
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por |
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Universidade Federal de Goiás |
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Mestrado em Biologia |
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UFG |
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BR |
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Ciências Biolóicas |
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Universidade Federal de Goiás |
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