Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines

Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines

Author Ferreira, Rodrigo da Silva Autor UNIFESP Google Scholar
Zhou, Dongwen Google Scholar
Ferreira, Joana Gasperazzo Autor UNIFESP Google Scholar
Autor UNIFESP Google Scholar
Silva-Lucca, Rosemeire Aparecida Google Scholar
Mentele, Reinhard Google Scholar
Paredes-Gamero, Edgar Julian Autor UNIFESP Google Scholar
Bertolin, Thiago Carlos Autor UNIFESP Google Scholar
Santos Correia, Maria Tereza dos Google Scholar
Guedes Paiva, Patricia Maria Google Scholar
Gustchina, Alla Google Scholar
Wlodawer, Alexander Google Scholar
Oliva, Maria Luiza Vilela Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
NCI
Univ Estadual Oeste Parana
Max Planck Inst Biochem
Universidade Federal de Pernambuco (UFPE)
Abstract 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.
Language English
Sponsor U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences
Coordenação 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 Research
Grant number U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences: W-31-109-Eng-38
FAPESP: 09/53766-5
Date 2013-06-18
Published in Plos One. San Francisco: Public Library Science, v. 8, n. 6, 14 p., 2013.
ISSN 1932-6203 (Sherpa/Romeo, impact factor)
Publisher Public Library Science
Extent 14
Origin http://dx.doi.org/10.1371/journal.pone.0064426
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000320576400001
URI http://repositorio.unifesp.br/handle/11600/36427

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