Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Outros Autores: | , , , , , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | http://repositorio.unifesp.br/handle/11600/36427 http://dx.doi.org/10.1371/journal.pone.0064426 |
Resumo: | A protein isolated from the bark of Crataeva tapia (CrataBL) is both a Kunitz-type plant protease inhibitor and a lectin. We have determined the amino acid sequence and three-dimensional structure of CrataBL, as well as characterized its selected biochemical and biological properties. We found two different isoforms of CrataBL isolated from the original source, differing in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser). CrataBL showed relatively weak inhibitory activity against trypsin (K-iapp = 43 mu M) and was more potent against Factor Xa (K-iapp = 8.6 mu M), but was not active against a number of other proteases. We have confirmed that CrataBL contains two glycosylation sites and forms a dimer at high concentration. the high-resolution crystal structures of two different crystal forms of isoform II verified the beta-trefoil fold of CrataBL and have shown the presence of dimers consisting of two almost identical molecules making extensive contacts (similar to 645 angstrom(2)). the structure differs from those of the most closely related proteins by the lack of the N-terminal beta-hairpin. in experiments aimed at investigating the biological properties of CrataBL, we have shown that addition of 40 mM of the protein for 48 h caused maximum growth inhibition in MTT assay (47% of DU145 cells and 43% of PC3 cells). the apoptosis of DU145 and PC3 cell lines was confirmed by flow cytometry using Annexin V/FITC and propidium iodide staining. Treatment with CrataBL resulted in the release of mitochondrial cytochrome c and in the activation of caspase-3 in DU145 and PC3 cells. |
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Ferreira, Rodrigo da Silva [UNIFESP]Zhou, DongwenFerreira, Joana Gasperazzo [UNIFESP]Silva, Mariana Cristina Cabral[UNIFESP]Silva-Lucca, Rosemeire AparecidaMentele, ReinhardParedes-Gamero, Edgar Julian [UNIFESP]Bertolin, Thiago Carlos [UNIFESP]Santos Correia, Maria Tereza dosGuedes Paiva, Patricia MariaGustchina, AllaWlodawer, AlexanderOliva, Maria Luiza Vilela [UNIFESP]Universidade Federal de São Paulo (UNIFESP)NCIUniv Estadual Oeste ParanaMax Planck Inst BiochemUniversidade Federal de Pernambuco (UFPE)2016-01-24T14:31:53Z2016-01-24T14:31:53Z2013-06-18Plos One. San Francisco: Public Library Science, v. 8, n. 6, 14 p., 2013.1932-6203http://repositorio.unifesp.br/handle/11600/36427http://dx.doi.org/10.1371/journal.pone.0064426WOS000320576400001.pdf10.1371/journal.pone.0064426WOS:000320576400001A protein isolated from the bark of Crataeva tapia (CrataBL) is both a Kunitz-type plant protease inhibitor and a lectin. We have determined the amino acid sequence and three-dimensional structure of CrataBL, as well as characterized its selected biochemical and biological properties. We found two different isoforms of CrataBL isolated from the original source, differing in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser). CrataBL showed relatively weak inhibitory activity against trypsin (K-iapp = 43 mu M) and was more potent against Factor Xa (K-iapp = 8.6 mu M), but was not active against a number of other proteases. We have confirmed that CrataBL contains two glycosylation sites and forms a dimer at high concentration. the high-resolution crystal structures of two different crystal forms of isoform II verified the beta-trefoil fold of CrataBL and have shown the presence of dimers consisting of two almost identical molecules making extensive contacts (similar to 645 angstrom(2)). the structure differs from those of the most closely related proteins by the lack of the N-terminal beta-hairpin. in experiments aimed at investigating the biological properties of CrataBL, we have shown that addition of 40 mM of the protein for 48 h caused maximum growth inhibition in MTT assay (47% of DU145 cells and 43% of PC3 cells). the apoptosis of DU145 and PC3 cell lines was confirmed by flow cytometry using Annexin V/FITC and propidium iodide staining. Treatment with CrataBL resulted in the release of mitochondrial cytochrome c and in the activation of caspase-3 in DU145 and PC3 cells.U.S. Department of Energy, Office of Science, Office of Basic Energy SciencesCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute, Center for Cancer ResearchUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilNCI, Macromol Crystallog Lab, Ctr Canc Res, Frederick, MD 21701 USAUniv Estadual Oeste Parana, Ctr Engn & Ciencias Exatas, Toledo, Parana, BrazilMax Planck Inst Biochem, Inst Clin Neuroimmunol LMU, Munich, GermanyMax Planck Inst Biochem, Dept Prot Analyt, Munich, GermanyUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniv Fed Pernambuco, Dept Bioquim, Recife, PE, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilU.S. Department of Energy, Office of Science, Office of Basic Energy Sciences: W-31-109-Eng-38FAPESP: 09/53766-5Web of Science14engPublic Library SciencePlos OneCrystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Linesinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESPORIGINALWOS000320576400001.pdfapplication/pdf2153530${dspace.ui.url}/bitstream/11600/36427/1/WOS000320576400001.pdf1883b5aa8da273ab9ed7517d98637404MD51open accessTEXTWOS000320576400001.pdf.txtWOS000320576400001.pdf.txtExtracted texttext/plain61856${dspace.ui.url}/bitstream/11600/36427/2/WOS000320576400001.pdf.txtb0ba893ae9571b6a30e292783ff85dceMD52open access11600/364272022-09-27 11:31:13.534open accessoai:repositorio.unifesp.br:11600/36427Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestopendoar:34652022-09-27T14:31:13Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.en.fl_str_mv |
Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines |
title |
Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines |
spellingShingle |
Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines Ferreira, Rodrigo da Silva [UNIFESP] |
title_short |
Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines |
title_full |
Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines |
title_fullStr |
Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines |
title_full_unstemmed |
Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines |
title_sort |
Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines |
author |
Ferreira, Rodrigo da Silva [UNIFESP] |
author_facet |
Ferreira, Rodrigo da Silva [UNIFESP] Zhou, Dongwen Ferreira, Joana Gasperazzo [UNIFESP] Silva, Mariana Cristina Cabral[UNIFESP] Silva-Lucca, Rosemeire Aparecida Mentele, Reinhard Paredes-Gamero, Edgar Julian [UNIFESP] Bertolin, Thiago Carlos [UNIFESP] Santos Correia, Maria Tereza dos Guedes Paiva, Patricia Maria Gustchina, Alla Wlodawer, Alexander Oliva, Maria Luiza Vilela [UNIFESP] |
author_role |
author |
author2 |
Zhou, Dongwen Ferreira, Joana Gasperazzo [UNIFESP] Silva, Mariana Cristina Cabral[UNIFESP] Silva-Lucca, Rosemeire Aparecida Mentele, Reinhard Paredes-Gamero, Edgar Julian [UNIFESP] Bertolin, Thiago Carlos [UNIFESP] Santos Correia, Maria Tereza dos Guedes Paiva, Patricia Maria Gustchina, Alla Wlodawer, Alexander Oliva, Maria Luiza Vilela [UNIFESP] |
author2_role |
author author author author author author author author author author author author |
dc.contributor.institution.none.fl_str_mv |
Universidade Federal de São Paulo (UNIFESP) NCI Univ Estadual Oeste Parana Max Planck Inst Biochem Universidade Federal de Pernambuco (UFPE) |
dc.contributor.author.fl_str_mv |
Ferreira, Rodrigo da Silva [UNIFESP] Zhou, Dongwen Ferreira, Joana Gasperazzo [UNIFESP] Silva, Mariana Cristina Cabral[UNIFESP] Silva-Lucca, Rosemeire Aparecida Mentele, Reinhard Paredes-Gamero, Edgar Julian [UNIFESP] Bertolin, Thiago Carlos [UNIFESP] Santos Correia, Maria Tereza dos Guedes Paiva, Patricia Maria Gustchina, Alla Wlodawer, Alexander Oliva, Maria Luiza Vilela [UNIFESP] |
description |
A protein isolated from the bark of Crataeva tapia (CrataBL) is both a Kunitz-type plant protease inhibitor and a lectin. We have determined the amino acid sequence and three-dimensional structure of CrataBL, as well as characterized its selected biochemical and biological properties. We found two different isoforms of CrataBL isolated from the original source, differing in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser). CrataBL showed relatively weak inhibitory activity against trypsin (K-iapp = 43 mu M) and was more potent against Factor Xa (K-iapp = 8.6 mu M), but was not active against a number of other proteases. We have confirmed that CrataBL contains two glycosylation sites and forms a dimer at high concentration. the high-resolution crystal structures of two different crystal forms of isoform II verified the beta-trefoil fold of CrataBL and have shown the presence of dimers consisting of two almost identical molecules making extensive contacts (similar to 645 angstrom(2)). the structure differs from those of the most closely related proteins by the lack of the N-terminal beta-hairpin. in experiments aimed at investigating the biological properties of CrataBL, we have shown that addition of 40 mM of the protein for 48 h caused maximum growth inhibition in MTT assay (47% of DU145 cells and 43% of PC3 cells). the apoptosis of DU145 and PC3 cell lines was confirmed by flow cytometry using Annexin V/FITC and propidium iodide staining. Treatment with CrataBL resulted in the release of mitochondrial cytochrome c and in the activation of caspase-3 in DU145 and PC3 cells. |
publishDate |
2013 |
dc.date.issued.fl_str_mv |
2013-06-18 |
dc.date.accessioned.fl_str_mv |
2016-01-24T14:31:53Z |
dc.date.available.fl_str_mv |
2016-01-24T14:31:53Z |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/article |
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article |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
Plos One. San Francisco: Public Library Science, v. 8, n. 6, 14 p., 2013. |
dc.identifier.uri.fl_str_mv |
http://repositorio.unifesp.br/handle/11600/36427 http://dx.doi.org/10.1371/journal.pone.0064426 |
dc.identifier.issn.none.fl_str_mv |
1932-6203 |
dc.identifier.file.none.fl_str_mv |
WOS000320576400001.pdf |
dc.identifier.doi.none.fl_str_mv |
10.1371/journal.pone.0064426 |
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WOS:000320576400001 |
identifier_str_mv |
Plos One. San Francisco: Public Library Science, v. 8, n. 6, 14 p., 2013. 1932-6203 WOS000320576400001.pdf 10.1371/journal.pone.0064426 WOS:000320576400001 |
url |
http://repositorio.unifesp.br/handle/11600/36427 http://dx.doi.org/10.1371/journal.pone.0064426 |
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Public Library Science |
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