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.

Heparin affects the interaction of kininogen on endothelial cells.

In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Zn(+2), HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PK binding to cell- or ECM-bound-HK and PK activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn(2+). Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment.

Bradykinin release and inactivation in brain of rats submitted to an experimental model of Alzheimer's disease.

The kallikrein-kinin system is involved in a variety of physiological and pathological processes. Components of this system, identified in rat and human brains, can be altered in neurodegenerative processes such as Alzheimer's disease. Here, we studied kinin release and its inactivation in rats submitted to chronic cerebroventricular infusion of beta-amyloid (Abeta) peptide. Neurodegeneration was confirmed by histological analysis of brain samples. In cerebrospinal fluid of animals infused with Abeta, bradykinin concentration was increased, as determined by radioimmunoassay. However, in the brain of Abeta group, we only detected the tripeptide Arg-Pro-Pro, purified by reversed-phase chromatography and characterized by liquid chromatography-electrospray ionization mass spectrometry. This fragment of bradykinin indicated the possible participation of kinin-processing enzymes in the brain such as a prolyl oligopeptidase.

Heparin modulation of human plasma kallikrein on different substrates and inhibitors.

The interplay of different proteases and glycosaminoglycans is able to modulate the activity of the enzymes and to affect their structures. Human plasma kallikrein (huPK) is a proteolytic enzyme involved in intrinsic blood clotting, the kallikrein-kinin system and fibrinolysis. We investigated the effect of heparin on the action, inhibition and secondary structure of huPK. The catalytic efficiency for the hydrolysis of substrates by huPK was determined by Michaelis-Menten kinetic plots: 5.12x10(4) M-1 s-1 for acetyl-Phe-Arg-p-nitroanilide, 1.40x10(5) M-1 s-1 for H-D-Pro-Phe-Arg-p-nitroanilide, 2.25x10(4) M-1 s-1 for Abz-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-EDDnp, 4.24x10(2)M-1 s-1 for factor XII and 5.58x10(2) M-1 s-1 for plasminogen. Heparin reduced the hydrolysis of synthetic substrates (by 2.0-fold), but enhanced factor XII and plasminogen hydrolysis (7.7- and 1.4-fold, respectively). The second-order rate constants for inhibition of huPK by antithrombin and C1-inhibitor were 2.40x10(2) M-1 s-1 and 1.70x10(4) M-1 s-1, respectively. Heparin improved the inhibition of huPK by these inhibitors (3.4- and 1.4-fold). Despite the fact that huPK was able to bind to a heparin-Sepharose matrix, its secondary structure was not modified by heparin, as monitored by circular dichroism. These actions may have a function in the control or maintenance of some pathophysiological processes in which huPK participates.

Vitamin E prevents cell death induced by mild oxidative stress in chicken skeletal muscle cells.

Apoptosis and necrosis are two forms of cell death that can occur in response to various agents and oxidative damage. In addition to necrosis, apoptosis contributes to muscle fiber loss in various muscular dystrophies as well participates in the exudative diathesis in chicken, pathology caused by dietary deficiency of vitamin E and selenium, which affects muscle tissue. We have used chicken skeletal muscle cells and bovine fibroblasts to study molecular events involved in the cell death induced by oxidative stress and apoptotic agents. The effect of vitamin E on cell death induced by oxidants was also investigated. Treatment of cells with anti-Fas antibody (50 to 400 ng/mL), staurosporine (0.1 to 100 microM) and TNF-alpha (10 and 50 ng/mL) resulted in a little loss of Trypan blue exclusion ability. Those stimuli conducted cells to apoptosis detected by an enhancement in caspase activity upon fluorogenic substrates but this activity was not fully blocked by the caspase inhibitor Z-VAD-fmk. Oxidative stress induced by menadione (10, 100 and 250 muM) promoted a significant reduction in cell viability (10%, 20% and 35% for fibroblasts; 20%, 30% and 75% for muscle cells, respectively) and caused an increase in caspase activity and DNA fragmentation. H2O2 also promoted apoptosis verified by caspase activation and DNA fragmentation, but in higher doses induced necrosis. Vitamin E protected cells from death induced by low doses of oxidants. Although it was ineffective in reducing caspase activity in fibroblasts, this vitamin diminished the enzyme activity in muscle cells. These data suggested that oxidative stress could activate apoptotic mechanisms; however the mode of cell death will depend on the intensity and duration of the stimulus, and on the antioxidant status of the cells.

A proteinase inhibitor from Caesalpinia echinata (pau-brasil) seeds for plasma kallikrein, plasmin and factor XIIa.

Caesalpinia echinata is a tree belonging to the Leguminosae family. The red color of the trunk, looking like burning wood ('brasa' in Portuguese), is the origin of the name Brazil. Seeds of leguminous plants contain high amounts of serine proteinase inhibitors that can affect different biological processes. Here we show that a protein isolated from seeds of C. echinata is able to inhibit enzymes that participate in blood coagulation and fibrinolysis. This inhibitor (CeKI) was purified to homogeneity by ion exchange and reversed-phase chromatography. SDS-PAGE indicated a single polypeptide chain with a molecular mass of 20 kDa. CeKI inhibits human plasma kallikrein ( K i =3.1 nM), plasmin ( K i =0.18 nM), factor XIIa ( K i =0.18 nM), trypsin ( K i =21.5 nM) and factor Xa ( K i =0.49 mM). CeKI inhibited kinin release from highmolecular- mass kininogen by kallikrein in vitro . The N-terminal sequence, determined by automatic Edman degradation, identified the inhibitor as a member of the Kunitz family. The secondary structure, determined by circular dichroism, is mainly a random coil followed by beta-sheet structure. The action of CeKI on enzymes of the blood-clotting intrinsic pathway was confirmed by prolongation of the activated partial thromboplastin time.