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Title: Using a Caesalpinia echinata Lam. protease inhibitor as a tool for studying the roles of neutrophil elastase, cathepsin G and proteinase 3 in pulmonary edema
Authors: Cruz-Silva, Ilana [UNIFESP]
Neuhof, Christiane
Gozzo, Andrezza Justino [UNIFESP]
Nunes, Viviane Abreu
Hirata, Izaura Yoshico [UNIFESP]
Sampaio, Misako Uemura
Figueiredo-Ribeiro, Rita de Cassia
Neuhof, Heinz
Araujo, Mariana da Silva [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
Univ Giessen
Universidade de São Paulo (USP)
Inst Bot
Keywords: Caesalpinia echinata Lam
Pulmonary edema
Protease inhibitors
Caesalpinia echinata elastase inhibitor
Issue Date: 1-Dec-2013
Publisher: Elsevier B.V.
Citation: Phytochemistry. Oxford: Pergamon-Elsevier B.V., v. 96, p. 235-243, 2013.
Abstract: Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. for this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEl is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K-i 3.7 mu M), leading to hydrolysis of specific synthetic substrates. in a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. in experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy. (C) 2013 Elsevier B.V. All rights reserved.
ISSN: 0031-9422
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