The recombinant plant Bauhinia bauhinioides elastase inhibitor reduces rat thrombus without alterations in hemostatic parameters.

The anti-inflammatory effects of the plant protease inhibitor BbCI (Bauhinia bauhinioides cruzipain inhibitor), which blocks elastase, cathepsin G, and L, and proteinase 3 has been demonstrated. Here, we investigated the recombinant rBbCI-His(6) (containing a histidine tail) in an experimental venous thrombosis model of vena cava (VC) ligature in rats, comparing to heparin. We evaluate the effects of the inhibitors (native or recombinant) or heparin on the activated partial thromboplastin time (aPTT) and prothrombin time (PT) in human and rat plasmas. The rats undergoing treatment received a saline solution or increasing concentrations of rBbCI-His(6), heparin, or a mixture of both. After 4 h of ligature VC, thrombus, if present was removed and weighed. aPTT, PT, and cytokines were measured in blood collected by cardiac puncture. aPTT, PT, and bleeding time (BT) were also measured at the time of VC (vena cava) ligature. rBbCI-His(6) (0.45 or 1.40 mg/kg) does not alter aPTT, PT or BT. No differences in coagulation parameters were detected in rBbCI-His(6) treated rats at the time of VC ligature or when the thrombus was removed. There was a significant decrease in the weight of thrombus in the animals of the groups treated with the rBbCI-His(6) (1.40 mg/kg), with the rBbCI-His(6) mixture (1.40 mg/kg) + heparin (50 IU/kg) and heparin (100 IU/kg) in relation to control group (saline). The growth-related oncogene/keratinocyte chemoattractant (GRO/KC) serum levels in rats treated with rBbCI-His(6) (1.40 mg/kg) or heparin (200 IU/kg) were reduced. In the experimental model used, rBbCI-His(6) alone had an antithrombotic effect, not altering blood clotting or bleeding time.

Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S.

Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells.

Crataeva tapia bark lectin (CrataBL) is a chemoattractant for endothelial cells that targets heparan sulfate and promotes in vitro angiogenesis.

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent.

Plaquetas: papéis tradicionais e não tradicionais na hemostasia, na inflamação e no câncer / Platelets: traditional and nontraditional roles in hemostasis, inflammation and cancer

A principal e mais conhecida função plaquetária ainda está relacionada à parada de sangramento após um dano vascular. No entanto, plaquetas estão envolvidas em diversos processos, tais como iniciar e amplificar a inflamação, interagir com células da resposta imune, além de participar na progressão tumoral, angiogênese e metástase. Neste sentido, está claro que plaquetas apresentam funções no processo inflamatório e podem influenciar respostas imune, além de desordens plaquetárias autoimune erelacionadas a presença de auto-anticorpos após transfusões, comopor exemplo, na lesão pulmonar aguda associada à transfusão. Após muita especulação, recentes observações têm estabelecido novos paradigmas relacionando plaquetas à biologia molecular. Plaquetas humanas contêm fatores de spliceossomo, incluindo pequenos RNAs nucleares, proteínas de splicing e pre-mRNA endógenos. Outro ponto importante é o controle do número de plaquetas circulantes, resultado do equilíbrio entre a produção e destruição dessas células. Assim, é proposto um processo de morte programada da célula anucleada que determina seu tempo de vida. Esse processo é alvo de especulações desde a década de 60 e ainda permanece em discussão. A noção geral de que plaquetas funcionais são importantes para o sucesso de processos hematogênicos corroboram com inovações experimentais e também ligam a processos de interação plaquetas-células tumorais e seu microambiente que regula a progressão maligna. Plaquetas contribuem na sobrevivência e disseminação de células tumorais. Desta forma, discutimos aqui os mecanismos pelos quais as plaquetas atuam na imunidade, na inflamação e no câncer, uma vez que estas pequenas células são mais versáteis do que se pensava.

The principal and the most known function of platelets still remains stopping hemorrhage following vascular injury. However, platelets are involved in diverse processes such as triggering inflammation, participating in the immune response, besides tumor progression, angiogenesis, and metastasis. In this sense, it is becoming increasingly clear that platelets display inflammatory functions and can influence both innate and adaptive immune responses, such as autoimmune and alloimmune platelet disorders, and transfusion-related acute lung injury (TRALI). Despite much speculation recent observations have established new paradigms relevant to influence of platelets on molecular biology. Primary human platelets contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. Other point is, like all lineages of blood cells, the steady state number of mature platelets is the result of a balance between their production and destruction. Thus, it isproposed a programmed anuclear cell death delimits platelet lifespan is subject of speculation since the 1960s and has remainedelusive. The general notion that functional platelets are importantfor successful hematogenous tumor metastasis dates more than 4 decades and has been corroborated in numerous experimentalsettings. The dynamic crosstalk between tumors and their microenvironment is increasingly recognized as a key regulator ofmalignant progression. These contributions of platelets to tumorcell survival and spread suggest platelets as a new avenue forresearch. Here, we discuss the mechanisms by which plateletscontribute to immunity, inflammation, and cancer, since thesesmall cells are more versatile than we once thought.

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.

Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy.

A plant Kunitz-type inhibitor mimics bradykinin-induced cytosolic calcium increase and intestinal smooth muscle contraction.

BbKI is a kallikrein inhibitor with a reactive site sequence similar to that of kinins, the vasoactive peptides inserted in kininogen moieties. This structural similarity probably contributes to the strong interaction with plasma kallikrein, the enzyme that releases, from high-molecular weight kininogen (HMWK), the proinflammatory peptide bradykinin, which acts on B(2) receptors (B(2)R). BbKI was examined on smooth muscle contraction and Ca(2+) mobilization, in which the kallikrein-kinin system is involved. Contrary to expectations, BbKI (1.8 µm) increased [Ca(2+)](c) and contraction, as observed with BK (2.0 µm). Not blocked by B(1) receptors (B(1)R), the BbKI agonistic effect was blocked by the B(2)R antagonist, HOE-140 (6 µm), and the involvement of B(2)R was confirmed in B(2)R-knockout mice intestine. The same tissue response was obtained using a synthetic peptide derived from the BbKI reactive site structure, more resistant than BK to angiotensin I-converting enzyme (ACE) hydrolysis. Depending on the concentration, BbKI has a dual effect. At a low concentration, BbKI acts as a potent kallikrein inhibitor; however, due to the similarity to BK, in high concentrations, BbKI greatly increases Ca(2+) release from internal storages, as a consequence of its interaction with B(2)R. Therefore, the antagonistic and agonistic effects of BbKI may be considered in conditions of B(2)R involvement.

Effects of compounds from Passiflora edulis Sims f. flavicarpa juice on blood coagulation and on proteolytic enzymes.

Passion fruit (Passiflora edulis Sims f. flavicarpa) is popularly known for its sedative and calming properties and is consumed as a fresh fruit or as a juice. The clinical observation of blood incoagulability associated with excessive consumption of passion fruit juice, in a patient treated with warfarin, prompted the current study to investigate in vitro the presence of blood clotting inhibitors in Passiflora edulis Sims f. flavicarpa extract. After purification process, two compounds of distinct molecular weight and inhibitory action were better characterized. One is a trypsin inhibitor similar to inhibitors from Bowman-Birk family, named PeTI-I12, and other is a compound active in coagulation that prolongs aPTT and PT, but does not change TT. The aim of this study is to provide evidence that passion fruit extract's components play a role on hemostasis and therefore may be relevant in the handling of patients treated with anticoagulants or suffering hemorrhagic diseases.

Enterolobium contortisiliquum trypsin inhibitor (EcTI), a plant proteinase inhibitor, decreases in vitro cell adhesion and invasion by inhibition of Src protein-focal adhesion kinase (FAK) signaling pathways.

Tumor cell invasion is vital for cancer progression and metastasis. Adhesion, migration, and degradation of the extracellular matrix are important events involved in the establishment of cancer cells at a new site, and therefore molecular targets are sought to inhibit such processes. The effect of a plant proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on the adhesion, migration, and invasion of gastric cancer cells was the focus of this study. EcTI showed no effect on the proliferation of gastric cancer cells or fibroblasts but inhibited the adhesion, migration, and cell invasion of gastric cancer cells; however, EcTI had no effect upon the adhesion of fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular organization of molecules involved in the formation and maturation of invadopodia, such as integrin ß1, cortactin, neuronal Wiskott-Aldrich syndrome protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover, gastric cancer cells treated with EcTI presented a significant decrease in intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent cell signaling components. Together, these results indicate that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathways.

Structural and functional properties of kunitz proteinase inhibitors from leguminosae: a mini review.

Seed proteins that inhibit proteinases are classified in families based on amino acid sequence similarity, nature of reactive site and mechanism of action, and are used as tools for investigating proteinases in physiological and pathological events. More recently, the plant Kunitz family of inhibitors with two disulphide bridges was enlarged with members containing variable number of cysteine residues, ranging from no cysteine at all to more than four residues. The characteristic of these proteins, as well the interactions with their target proteinases, are briefly discussed.

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition.

Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH2 (RGE) and IVYYPDRGETGL-NH2 (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED(50) 0.16% and LD(50) 0.09%), this being even more effective than the native protein.

Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor.

Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7A resolution were obtained using hanging drop method by vapor diffusion at 18 degrees C. The refined structure shows the conservative beta-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function.

A Kunitz-type glycosylated elastase inhibitor with one disulfide bridge.

A glycosylated Bauhinia rufa elastase inhibitor (gBrEI) was purified and characterized using acetone precipitation, affinity chromatography on concanavalin A-Sepharose, ion-exchange chromatography on a HiTrap Q column, size exclusion chromatography on a Superdex 200 column and reverse-phase chromatography on a C18 column. gBrEI inhibited pancreatic porcine elastase with an equilibrium dissociation constant (K(i)) of 6.18 x 10(-8) M, but it did not inhibit human neutrophil elastase, bovine trypsin, human plasma kallikrein or porcine pancreatic kallikrein. On SDS-electrophoresis, gBrEI appeared as a single 20-kDa band, also after reduction. Schiff reagent staining indicated a carbohydrate portion in the protein, which was confirmed by mass spectrometry. The glycosylated site was Asn 38, and a carbohydrate portion of 1.17 kDa was identified. gBrEI was found to contain 144 amino acid residues, and a FASTA database analysis showed that it belongs to the plant Kunitz-type inhibitor family. Val66 was identified as reactive site P1 residue by comparison of conserved positions in the sequences. Since gBrEI harbors a single disulfide bridge, it may be considered a new type of Kunitz inhibitor, intermediate between the classical Kunitz inhibitors, which contain two disulfide bridges, and those from B. bauhinioides, which do not have such bridges.

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.