Disclosing the involvement of proteases in an eczema murine animal model: Perspectives for protease inhibitor-based therapies.

Eczema is a skin condition characterized by itchy and inflammatory patches. The accumulation of neutrophils and the imbalance between enzymes and their inhibitors appears to be related to this condition. We proposed a neutrophil elastase (NE)-based eczema model in mice in order to verify histopathological features as well as the expression and activity of proteases and inhibitors. Mice skins were topically administered with human NE (0-2 pmol/cm2) for 24-168 h. It was observed thickening of epidermis, parakeratosis, spongiosis and leukocyte infiltration. Also, NE-treated skins presented high activity of epidermal kallikreins 5 and 7, and cathepsin B on synthetic substrates, and expression evaluated by RT-qPCR. The proteolytic activity was inhibited by soybean trypsin inhibitor, CA074 and Caesalpinia echinata kallikrein inhibitor (CeKI). The topic application of CeKI reversed eczema phenotype in NE-treated skins. Elafin expression was shown to be increased in NE-treated skins. These results suggest that the NE may trigger morphological and biochemical changes in skin similar to those observed in eczematous diseases. In addition to the establishment of this in vivo model, this work opens perspectives for the use of protease inhibitor-based drugs for the management of this skin condition.

Bioengineering of an elastase inhibitor from Caesalpinia echinata (Brazil wood) seeds.

Protease inhibitors have been widely used in several therapeutic applications such as in the treatment of bleeding disorders, hypertension, cancer and pulmonary diseases. In a previous work, we demonstrated that a Kunitz-type serine protease inhibitor isolated from the seeds of Caesalpinia echinata (CeEI) exhibits pharmacological potential in lung inflammatory diseases in which neutrophil elastase plays a crucial role. However, an important challenge in the use of natural products is to ensure a commercially viable production. In this work, we report the cloning, expression and purification of two recombinant CeEI isoinhibitors with 700 base pairs encoding two proteins with 181 amino acid residues (rCeEI-4 and rCeEI-5). After the expression, each yielding 22 mg/L of active protein, both isoinhibitors presented a molecular mass of about 23.0 kDa, evaluated by SDS-PAGE. The inhibition constants for human neutrophil elastase (HNE) were 0.67 nM (rCeEI-4) and 0.57 nM (rCeEI-5), i.e., similar to the native inhibitor (1.90 nM). Furthermore, rCeEI-4 was used as a template to design smaller functional peptides flanking the inhibitor reactive site: rCeEI-36, delimited between the amino acid residues N36 and S88 containing a disulfide bond in the reactive-site loop, and rCeEI-46, delimited between S46 and L75 without the disulfide bond. The yields were 18 mg/L (rCeEI-36) and 12 mg/L (rCeEI-46). Both peptides inhibit HNE in the nanomolar range (Ki 0.30 ± 0.01 and 8.80 ± 0.23, respectively). Considering their size and the inhibitory efficiency, these peptides may be considered in strategies for the development of drugs targeting pulmonary disorders where elastase is involved.

Protease Inhibitors Extracted from Caesalpinia echinata Lam. Affect Kinin Release during Lung Inflammation.

Inflammation is an essential process in many pulmonary diseases in which kinins are generated by protease action on kininogen, a phenomenon that is blocked by protease inhibitors. We evaluated kinin release in an in vivo lung inflammation model in rats, in the presence or absence of CeKI (C. echinata kallikrein inhibitor), a plasma kallikrein, cathepsin G, and proteinase-3 inhibitor, and rCeEI (recombinant C. echinata elastase inhibitor), which inhibits these proteases and also neutrophil elastase. Wistar rats were intravenously treated with buffer (negative control) or inhibitors and, subsequently, lipopolysaccharide was injected into their lungs. Blood, bronchoalveolar lavage fluid (BALF), and lung tissue were collected. In plasma, kinin release was higher in the LPS-treated animals in comparison to CeKI or rCeEI groups. rCeEI-treated animals presented less kinin than CeKI-treated group. Our data suggest that kinins play a pivotal role in lung inflammation and may be generated by different enzymes; however, neutrophil elastase seems to be the most important in the lung tissue context. These results open perspectives for a better understanding of biological process where neutrophil enzymes participate and indicate these plant inhibitors and their recombinant correlates for therapeutic trials involving pulmonary diseases.

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.

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, CeEI 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 µM), 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.

Biochemical aspects of a serine protease from Caesalpinia echinata Lam. (Brazilwood) seeds: a potential tool to access the mobilization of seed storage proteins.

Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP) hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (K(m) 55.7 µM) in an optimum pH of 7.1, and this activity is effectively retained until 50 °C. CeSP remained stable in the presence of kosmotropic anions (PO(4) (3-), SO(4) (2-), and CH(3)COO(-)) or chaotropic cations (K(+) and Na(+)). It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins.

Estudo da interação de proteínas dos sistema calicreína-cininas com glicosaminoglicanos / Study of the interaction of Kallikrein-Kinin system proteins with glycosaminoglycans

A calicreína plasmática humana (huPK) é uma serinoendopeptidas envolvida em muitos processos biológicos, entre eles o sistema calicreína-cinina. Em relação a esse sistema, tem sido mostrado que a procalicreína (PK), quando ligada à membrana de células endoteliais por meio do HK, é ativada à calicreína, resultando na liberação de bradicinina (BK), um potente mediador da resposta inflamatória. A liberação de BK pode ser influenciada por muitos compostos e objetivo desse trabalho foi investigar o efeito de glicosaminoglicanos na hidrólise do HK pela huPK in vitro, na conformação do HK e da huPK, na ligação do HK da PK à superfície e matriz extracelular de células endoteliais e em um modelo c inflamação in vivo. Para isso, huPK foi incubada com HK, na ausência e presença de diferentes glicosaminoglicanos. A liberação de BK, em diferentes tempos, foi medida por radioimunoensaio e as constantes catalíticas foram calculadas. Dermatam sulfato de atum e bovino reduziram a eficiência catalítica da huPK sobre o HK (controle = 4.1 x 104 M-¹ s-¹) em 80 por cento e 68 por cento, respectivamente. Então, o efeito do dermatam sulfato bovino (DSb) na resposta inflamatória foi estudado num modelo de edema de pata de rato induzido por carragenina. O volume da pata do rato foi medido com o auxílio de um pletismômetro, de 1 a 4 horas após administração da carragenina. O DSb reduziu significativamente (p< 0.05) a resposta inflamatória na primeira e segunda hora de medida, em 24 por cento e 28 por cento, respectivamente. A interação da huPK com um glicosaminoglicano foi verificada por cromatografia de afinidade em Heparina-Sepharose. A huPK apresentou uma alta afinidade pela heparina, já que foi eluída da coluna com NaCl 0,5 M. A influência dos glicosaminoglicanos na estrutura secundária da huPK e do HK foi analisada por dicroísmo circular. Apenas os condroitim 4- e 6-sulfato modificaram essas estruturas...

Inibidores de proteases encontrados em sementes de Caesalpinia echinata (paubrasil): isolamento e caracterização do inibidor de tripsina

Caesalpinia echinata, o pau-brasil, é uma árvore pertencente à família das Leguminosas, sub-família Caesalpinoidae. Como já foram encontrados inibidores de proteases em sementes de outras Leguminosas, o objetivo do trabalho é purificar e caracterizar o inibidor de tripsina extraído das sementes de C. echinata. Após extração salina e precipitação por acetona, os inibidores foram purificados por cromatografia de troca iônica e filtração em gel, apresentando massas moleculares de 19,5 e 10 kDa e constante de inibição da ordem de nM.