Análise de uma heme oxigenase funcional em Trypanosoma cruzi

Detalhes bibliográficos
Autor(a) principal: Souza, Cíntia Fernandes de
Data de Publicação: 2011
Tipo de documento: Tese
Idioma: por
Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
Texto Completo: http://www.bdtd.uerj.br/handle/1/16134
Resumo: Trypanosoma cruzi, the ethiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate host. These hematophagous insects ingest blood about 6 to 12 times its original weight, reaching in a single meal about 10 mM heme bound to hemoglobin. Heme (iron protoporphyrin IX) is an important molecule in metabolism of all living organisms. One important intracellular mechanism to control heme homeostasis is its enzymatic degradation by heme oxygenase (HO). HO catalyzes the degradation of heme to biliverdin (Bv), carbon monoxide and iron. HO is absent in T. cruzi genome, thus we have been investigating the presence of a functional HO in this parasite, since our previous results showed a presence of biliverdin in heme-treated epimastigotes. In the present work, we evaluated the effect of SnPPIX, a HO-1 inhibitor, CoPPIX, a HO inducer, and Bv upon T. cruzi epimastigotes proliferation. The addition of SnPPIX decreased the parasite proliferation in the absence or in the presence of heme. When Bv was added to the culture this effect was reversed; Bv increases the parasite proliferation in the presence of heme. On the other hand, CoPPIX did not interfered on proliferation. Furthermore, we showed through immunoblotting, using an anti-HO-1 monoclonal antibody, an increase in the protein expression in heme-treated epimastigotes. Differently of described HO-1 that has a mass molecular of a 32 kDa, we showed a 45 kDa protein, the only band recognize by the HO-1 antibody. HO-1 expression analysis in the presence of CoPPIX, SnPPIX and biliverdin, showed that only CoPPIX was able to modulate its expression level. Ultrastructural immunocytochemistry analysis suggests a higher expression of the enzyme in heme-treated epimastigotes, and that T. cruzi HO-1 might have a dual distribution, since the anti-HO-1 antibody labeled both cytosol and glycosomes. In order to investigate the T. cruzi HO-1 gene sequence, we isolated genomic DNA from T. cruzi for PCR amplification using primers designed as from the parasite genome. Unfortunately, the two pairs of the designed oligonucleotides tested were unable to amplify a sequence equivalent to a HO-1. In order to isolate the target protein, immunoprecipitation was performed and subsequently immunoblotted with anti-HO-1 antibody. The immunocomplex level was two-fold higher in heme-treated cells. Following the attempt to identify a HO-1 in T. cruzi, we used mass spectrometry from unidimensional electrophoresis. We showed a significant protein profile modification in heme-treated epimastigotes, but further experiments will be necessary, such as mass spectrometry from bidimensional electrophoresis, for identification of the target protein
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spelling Paes, Márcia Cristinahttp://lattes.cnpq.br/7463829190927034Coelho, Marsen Garcia Pintohttp://lattes.cnpq.br/4468352500732645Dutra, Patrícia Maria Lourençohttp://lattes.cnpq.br/1617851661398279Arruda, Maria Augusta Borges Cursino de Freitashttp://lattes.cnpq.br/0214033967250240Monteiro, Clarissa Menezes Mayahttp://lattes.cnpq.br/6910501689129124Atella, Georgia Correahttp://lattes.cnpq.br/9483943884586791http://lattes.cnpq.br/4603176980038483Souza, Cíntia Fernandes de2021-04-26T01:10:42Z2012-07-112011-02-21SOUZA, Cíntia Fernandes de. Análise de uma heme oxigenase funcional em Trypanosoma cruzi. 2011. 129 f. Tese (Doutorado em Biociências Nucleares; Ecologia) - Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2011.http://www.bdtd.uerj.br/handle/1/16134Trypanosoma cruzi, the ethiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate host. These hematophagous insects ingest blood about 6 to 12 times its original weight, reaching in a single meal about 10 mM heme bound to hemoglobin. Heme (iron protoporphyrin IX) is an important molecule in metabolism of all living organisms. One important intracellular mechanism to control heme homeostasis is its enzymatic degradation by heme oxygenase (HO). HO catalyzes the degradation of heme to biliverdin (Bv), carbon monoxide and iron. HO is absent in T. cruzi genome, thus we have been investigating the presence of a functional HO in this parasite, since our previous results showed a presence of biliverdin in heme-treated epimastigotes. In the present work, we evaluated the effect of SnPPIX, a HO-1 inhibitor, CoPPIX, a HO inducer, and Bv upon T. cruzi epimastigotes proliferation. The addition of SnPPIX decreased the parasite proliferation in the absence or in the presence of heme. When Bv was added to the culture this effect was reversed; Bv increases the parasite proliferation in the presence of heme. On the other hand, CoPPIX did not interfered on proliferation. Furthermore, we showed through immunoblotting, using an anti-HO-1 monoclonal antibody, an increase in the protein expression in heme-treated epimastigotes. Differently of described HO-1 that has a mass molecular of a 32 kDa, we showed a 45 kDa protein, the only band recognize by the HO-1 antibody. HO-1 expression analysis in the presence of CoPPIX, SnPPIX and biliverdin, showed that only CoPPIX was able to modulate its expression level. Ultrastructural immunocytochemistry analysis suggests a higher expression of the enzyme in heme-treated epimastigotes, and that T. cruzi HO-1 might have a dual distribution, since the anti-HO-1 antibody labeled both cytosol and glycosomes. In order to investigate the T. cruzi HO-1 gene sequence, we isolated genomic DNA from T. cruzi for PCR amplification using primers designed as from the parasite genome. Unfortunately, the two pairs of the designed oligonucleotides tested were unable to amplify a sequence equivalent to a HO-1. In order to isolate the target protein, immunoprecipitation was performed and subsequently immunoblotted with anti-HO-1 antibody. The immunocomplex level was two-fold higher in heme-treated cells. Following the attempt to identify a HO-1 in T. cruzi, we used mass spectrometry from unidimensional electrophoresis. We showed a significant protein profile modification in heme-treated epimastigotes, but further experiments will be necessary, such as mass spectrometry from bidimensional electrophoresis, for identification of the target proteinO Trypanosoma cruzi é o agente etiológico da doença de Chagas, transmitida através de insetos vetores triatomíneos durante a alimentação no hospedeiro vertebrado. Os triatomíneos ingerem numa única alimentação cerca de 10 mM de heme ligado à hemoglobina. O heme é uma importante molécula no metabolismo dos organismos. Um mecanismo intracelular importante no controle de sua homeostase é a degradação enzimática pela Heme Oxigenase (HO) formando biliverdina (Bv), monóxido de carbono e ferro. Como esta enzima não está presente no genoma de T. cruzi, esse trabalho tem por objetivo identificar uma atividade funcional de HO neste parasito, uma vez que dados do nosso laboratório mostram a presença de biliverdina nas incubações dessas células com heme. No presente trabalho testamos o efeito do SnPPIX (inibidor da HO-1), CoPPIX (indutor da HO-1) e Bv sobre a proliferação da forma epimastigota do parasito. A adição de SnPPIX diminuiu a proliferação do parasito na tanto na ausência quanto na presença de heme. Quando a Bv foi adicionada à cultura esse efeito foi revertido; a Bv aumenta a proliferação celular na presença de heme. Por outro lado, a adição de CoPPIX não interferiu na proliferação. Posteriormente, mostramos através da técnica de immunoblotting, utilizando anticorpo monoclonal contra a HO-1, um aumento da expressão de uma proteína em resposta ao heme. Diferentemente das HO-1 já descritas que possuem massa molecular de 32 kDa, a única banda reconhecida pelo anticorpo apresenta 45 kDa. Analisamos também a expressão da HO-1 na presença de CoPPIX, SnPPIX e biliverdina, e somente o CoPPIX foi capaz de modular os níveis de expressão da HO-1. A análise estrutural através da técnica de imunocitoquímica mostrou uma maior expressão da enzima na presença de heme, e que a HO-1 de T. cruzi pode ter mais de uma localização, apresentando marcação citoplasmática e glicossomal. A fim de investigar a sequência da HO-1 de T. cruzi, o DNA genômico foi extraído para amplificação por PCR do gene da HO-1 utilizando oligonucleotídeos desenhados no genoma de T. cruzi. Os dois pares de oligonucleotídeos utilizados nao foram capazes de amplificar uma sequência equivalente a uma HO. Em seguida, utilizamos a técnica de imunoprecipitação, seguida de immunoblotting, com anticorpo anti-HO-1, com objetivo de concentrar a proteína alvo, e observamos um aumento significativo do imunocomplexo nas células tratadas com heme 300 mM, cerca de 2 vezes em relação ao controle. Dando seguimento à tentativa de identificação da HO-1 de T. cruzi, utilizamos a técnica de espectrometria de massa a partir de eletroforese unidimensional, que mostrou uma grande alteração do perfil protéico na presença de heme, mas futuros experimentos são necessários, como eletroforese 2D, para a identificação da proteína alvoSubmitted by Boris INFORMAT (boris@uerj.br) on 2021-04-26T01:10:42Z No. of bitstreams: 1 Cintia.pdf: 9065013 bytes, checksum: d53d70aa42a5ddd6afed10ec21bcd275 (MD5)Made available in DSpace on 2021-04-26T01:10:42Z (GMT). No. of bitstreams: 1 Cintia.pdf: 9065013 bytes, checksum: d53d70aa42a5ddd6afed10ec21bcd275 (MD5) Previous issue date: 2011-02-21Fundação de Amparo à Pesquisa do Estado do Rio de Janeiroapplication/pdfporUniversidade do Estado do Rio de JaneiroPrograma de Pós-Graduação em BiociênciasUERJBRCentro Biomédico::Instituto de Biologia Roberto Alcantara GomesTrypanosoma cruziHemeHeme oxygenase-1Trypanosoma cruziHemeHeme oxigenase-1CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::METABOLISMO E BIOENERGETICAAnálise de uma heme oxigenase funcional em Trypanosoma cruziHeme oxygenase analysis in Trypanosoma cruziinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UERJinstname:Universidade do Estado do Rio de Janeiro (UERJ)instacron:UERJORIGINALCintia.pdfapplication/pdf9065013http://www.bdtd.uerj.br/bitstream/1/16134/1/Cintia.pdfd53d70aa42a5ddd6afed10ec21bcd275MD511/161342024-02-26 11:24:57.623oai:www.bdtd.uerj.br:1/16134Biblioteca Digital de Teses e Dissertaçõeshttp://www.bdtd.uerj.br/PUBhttps://www.bdtd.uerj.br:8443/oai/requestbdtd.suporte@uerj.bropendoar:29032024-02-26T14:24:57Biblioteca Digital de Teses e Dissertações da UERJ - Universidade do Estado do Rio de Janeiro (UERJ)false
dc.title.por.fl_str_mv Análise de uma heme oxigenase funcional em Trypanosoma cruzi
dc.title.alternative.eng.fl_str_mv Heme oxygenase analysis in Trypanosoma cruzi
title Análise de uma heme oxigenase funcional em Trypanosoma cruzi
spellingShingle Análise de uma heme oxigenase funcional em Trypanosoma cruzi
Souza, Cíntia Fernandes de
Trypanosoma cruzi
Heme
Heme oxygenase-1
Trypanosoma cruzi
Heme
Heme oxigenase-1
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::METABOLISMO E BIOENERGETICA
title_short Análise de uma heme oxigenase funcional em Trypanosoma cruzi
title_full Análise de uma heme oxigenase funcional em Trypanosoma cruzi
title_fullStr Análise de uma heme oxigenase funcional em Trypanosoma cruzi
title_full_unstemmed Análise de uma heme oxigenase funcional em Trypanosoma cruzi
title_sort Análise de uma heme oxigenase funcional em Trypanosoma cruzi
author Souza, Cíntia Fernandes de
author_facet Souza, Cíntia Fernandes de
author_role author
dc.contributor.advisor1.fl_str_mv Paes, Márcia Cristina
dc.contributor.advisor1Lattes.fl_str_mv http://lattes.cnpq.br/7463829190927034
dc.contributor.advisor-co1.fl_str_mv Coelho, Marsen Garcia Pinto
dc.contributor.advisor-co1Lattes.fl_str_mv http://lattes.cnpq.br/4468352500732645
dc.contributor.referee1.fl_str_mv Dutra, Patrícia Maria Lourenço
dc.contributor.referee1Lattes.fl_str_mv http://lattes.cnpq.br/1617851661398279
dc.contributor.referee2.fl_str_mv Arruda, Maria Augusta Borges Cursino de Freitas
dc.contributor.referee2Lattes.fl_str_mv http://lattes.cnpq.br/0214033967250240
dc.contributor.referee3.fl_str_mv Monteiro, Clarissa Menezes Maya
dc.contributor.referee3Lattes.fl_str_mv http://lattes.cnpq.br/6910501689129124
dc.contributor.referee4.fl_str_mv Atella, Georgia Correa
dc.contributor.referee4Lattes.fl_str_mv http://lattes.cnpq.br/9483943884586791
dc.contributor.authorLattes.fl_str_mv http://lattes.cnpq.br/4603176980038483
dc.contributor.author.fl_str_mv Souza, Cíntia Fernandes de
contributor_str_mv Paes, Márcia Cristina
Coelho, Marsen Garcia Pinto
Dutra, Patrícia Maria Lourenço
Arruda, Maria Augusta Borges Cursino de Freitas
Monteiro, Clarissa Menezes Maya
Atella, Georgia Correa
dc.subject.eng.fl_str_mv Trypanosoma cruzi
Heme
Heme oxygenase-1
topic Trypanosoma cruzi
Heme
Heme oxygenase-1
Trypanosoma cruzi
Heme
Heme oxigenase-1
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::METABOLISMO E BIOENERGETICA
dc.subject.por.fl_str_mv Trypanosoma cruzi
Heme
Heme oxigenase-1
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::METABOLISMO E BIOENERGETICA
description Trypanosoma cruzi, the ethiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate host. These hematophagous insects ingest blood about 6 to 12 times its original weight, reaching in a single meal about 10 mM heme bound to hemoglobin. Heme (iron protoporphyrin IX) is an important molecule in metabolism of all living organisms. One important intracellular mechanism to control heme homeostasis is its enzymatic degradation by heme oxygenase (HO). HO catalyzes the degradation of heme to biliverdin (Bv), carbon monoxide and iron. HO is absent in T. cruzi genome, thus we have been investigating the presence of a functional HO in this parasite, since our previous results showed a presence of biliverdin in heme-treated epimastigotes. In the present work, we evaluated the effect of SnPPIX, a HO-1 inhibitor, CoPPIX, a HO inducer, and Bv upon T. cruzi epimastigotes proliferation. The addition of SnPPIX decreased the parasite proliferation in the absence or in the presence of heme. When Bv was added to the culture this effect was reversed; Bv increases the parasite proliferation in the presence of heme. On the other hand, CoPPIX did not interfered on proliferation. Furthermore, we showed through immunoblotting, using an anti-HO-1 monoclonal antibody, an increase in the protein expression in heme-treated epimastigotes. Differently of described HO-1 that has a mass molecular of a 32 kDa, we showed a 45 kDa protein, the only band recognize by the HO-1 antibody. HO-1 expression analysis in the presence of CoPPIX, SnPPIX and biliverdin, showed that only CoPPIX was able to modulate its expression level. Ultrastructural immunocytochemistry analysis suggests a higher expression of the enzyme in heme-treated epimastigotes, and that T. cruzi HO-1 might have a dual distribution, since the anti-HO-1 antibody labeled both cytosol and glycosomes. In order to investigate the T. cruzi HO-1 gene sequence, we isolated genomic DNA from T. cruzi for PCR amplification using primers designed as from the parasite genome. Unfortunately, the two pairs of the designed oligonucleotides tested were unable to amplify a sequence equivalent to a HO-1. In order to isolate the target protein, immunoprecipitation was performed and subsequently immunoblotted with anti-HO-1 antibody. The immunocomplex level was two-fold higher in heme-treated cells. Following the attempt to identify a HO-1 in T. cruzi, we used mass spectrometry from unidimensional electrophoresis. We showed a significant protein profile modification in heme-treated epimastigotes, but further experiments will be necessary, such as mass spectrometry from bidimensional electrophoresis, for identification of the target protein
publishDate 2011
dc.date.issued.fl_str_mv 2011-02-21
dc.date.available.fl_str_mv 2012-07-11
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dc.identifier.citation.fl_str_mv SOUZA, Cíntia Fernandes de. Análise de uma heme oxigenase funcional em Trypanosoma cruzi. 2011. 129 f. Tese (Doutorado em Biociências Nucleares; Ecologia) - Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2011.
dc.identifier.uri.fl_str_mv http://www.bdtd.uerj.br/handle/1/16134
identifier_str_mv SOUZA, Cíntia Fernandes de. Análise de uma heme oxigenase funcional em Trypanosoma cruzi. 2011. 129 f. Tese (Doutorado em Biociências Nucleares; Ecologia) - Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2011.
url http://www.bdtd.uerj.br/handle/1/16134
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publisher.none.fl_str_mv Universidade do Estado do Rio de Janeiro
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