Blood pressure-lowering effects of a Bowman-Birk inhibitor and its derived peptides in normotensive and hypertensive rats.

Bioactive plant peptides have received considerable interest as potential antihypertensive agents with potentially fewer side effects than antihypertensive drugs. Here, the blood pressure-lowering effects of the Bowman-Birk protease inhibitor, BTCI, and its derived peptides, PepChy and PepTry, were investigated using normotensive (Wistar-WR) and spontaneously hypertensive rats (SHR). BTCI inhibited the proteases trypsin and chymotrypsin, respectively, at 6 µM and 40 µM, a 10-fold greater inhibition than observed with PepTry (60 µM) and PepChy (400 µM). These molecules also inhibited angiotensin converting enzyme (ACE) with IC50 values of 54.6 ± 2.9; 24.7 ± 1.1; and 24.4 ± 1.1 µM, respectively, occluding its catalytic site, as indicated by molecular docking simulation, mainly for PepChy and PepTry. Gavage administration of BTCI and the peptides promoted a decrease of systolic and diastolic blood pressure and an increase of renal and aortic vascular conductance. These effects were more expressive in SHR than in WR. Additionally, BTCI, PepChy and PepTry promoted coronary vasodilation and negative inotropic effects in isolated perfused hearts. The nitric oxide synthase inhibitor blunted the BTCI and PepChy, with no cardiac effects on PepTry. The findings of this study indicate a therapeutic potential of BTCI and its related peptides in the treatment of hypertension.

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors.

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legume crop productivities. Protease inhibitors affect protein degradation and reduce the availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases and the growth and development of A. gemmatalis larvae exposed to soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan-Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmatalis was delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage wherein A. gemmatalis fed voraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline information about the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.

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.

Bowman-Birk inhibitors, proteasome peptidase activities and colorectal pre neoplasias induced by 1,2-dimethylhydrazine in Swiss mice.

Bowman-Birk inhibitors (BBIs) are protein molecules containing two inhibitory domains for enzymes similar to trypsin and chymotrypsin. Interest in these inhibitors arose from their properties against the cancer chemically induced by 1,2-dimethylhydrazine (DMH). In this study the effect of two BBI preparations (from Glycine max and Macrotyloma axillare) were evaluated for the prevention of colorectal neoplasia induced by intraperitoneal injections of DMH, given at a dose of 30 mg/kg, during 12 weeks. Mice treated with DMH presented histopathological alterations consistent with tumor development, augmented CD44 expression and increased proteasome peptidase activities. Lysosomal fractions, obtained from the intestines, were chromatographed in a Sepharose-BBI column and increased activity for trypsin and chymotrypsin-like proteases recovered from DMH-treated animals. In parallel, mice treated for eight weeks with BBIs showed a decrease in the chymotrypsin and trypsin-like proteasome activities compared to animals fed on normal diet. For the groups receiving simultaneous treatment with DMH and BBIs, dysplasic lesions were not observed and proteasome peptidase activities were similar to the control group after the 24th week. These results suggest that the mechanism by which BBIs could prevent the appearance of pre neoplastic lesions is associated with inhibition of both the lysosomal and proteasome-dependent proteolytic pathways.

Action of plant proteinase inhibitors on enzymes of physiopathological importance.

Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.

Action of plant proteinase inhibitors on enzymes of physiopathological importance

Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.

Obtidas de sementes leguminosas, várias proteínas inibem proteinases de origem animal, incluindo humanas, e podem ser consideradas para o desenvolvimento de compostos com atividade biológica. Inibidores da família Bowman-Birk e da família Kunitz vegetal tem sido caracterizados em relação a especificidade para proteinase, estrutura primária e sitio reativo. O nosso grupo majoritariamente vem estudando o gênero Bauhinia, principalmente as espécies bauhinioides, rufa, ungulatae variegata. Em algumas espécies, mais de um inibidor com propriedades diferentes foi caracterizado. Embora tais proteínas apresentem alta similaridade estrutural, diferem quanto à inibição de proteinases, e foram exploradas em estudos utilizando diversos modelos biológicos.

Proteinase inhibition using small Bowman-Birk-type structures.

Bowman-Birk inhibitors (BBIs) are cysteine-rich and highly cross-linked small proteins that function as specific pseudosubstrates for digestive proteinases. They typically display a "double-headed" structure containing an independent proteinase-binding loop that can bind and inhibit trypsin, chymotrypsin and elastase. In the present study, we used computational biology to study the structural characteristics and dynamics of the inhibition mechanism of the small BBI loop expressing a 35-amino acid polypeptide (ChyTB2 inhibitor) which has coding region for the mutated chymotrypsin-inhibitory site of the soybean BBI. We found that in the BBI-trypsin inhibition complex, the most important interactions are salt bridges and hydrogen bonds, whereas in the BBI-chymotrypsin inhibition complex, the most important interactions are hydrophobic. At the same time, ChyTB2 mutant structure maintained the individual functional domain structure and excellent binding/inhibiting capacities for trypsin and chymotrypsin at the same time. These results were confirmed by enzyme-linked immunosorbend assay experiments. The results showed that modeling combined with molecular dynamics is an efficient method to describe, predict and then obtain new proteinase inhibitors. For such study, however, it is necessary to start from the sequence and structure of the mutant interacting relatively strongly with both trypsin and chymotrypsin for designing the small BBI-type inhibitor against proteinases.

Proteinase inhibition using small Bowman-Birk-type structures

Bowman-Birk inhibitors (BBIs) are cysteine-rich and highly cross-linked small proteins that function as specific pseudosubstrates for digestive proteinases. They typically display a "double-headed" structure containing an independent proteinase-binding loop that can bind and inhibit trypsin, chymotrypsin and elastase. In the present study, we used computational biology to study the structural characteristics and dynamics of the inhibition mechanism of the small BBI loop expressing a 35-amino acid polypeptide (ChyTB2 inhibitor) which has coding region for the mutated chymotrypsin-inhibitory site of the soybean BBI. We found that in the BBI-trypsin inhibition complex, the most important interactions are salt bridges and hydrogen bonds, whereas in the BBI-chymotrypsin inhibition complex, the most important interactions are hydrophobic. At the same time, ChyTB2 mutant structure maintained the individual functional domain structure and excellent binding/inhibiting capacities for trypsin and chymotrypsin at the same time. These results were confirmed by enzyme-linked immunosorbend assay experiments. The results showed that modeling combined with molecular dynamics is an efficient method to describe, predict and then obtain new proteinase inhibitors. For such study, however, it is necessary to start from the sequence and structure of the mutant interacting relatively strongly with both trypsin and chymotrypsin for designing the small BBI-type inhibitor against proteinases.

Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis.

The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.

Relative importance of phytohemagglutinin (lectin) and trypsin-chymotrypsin inhibitor on bean (Phaseolus vulgaris L) protein absorption and utilization by the rat.

The main objective of this work was to perform a comparative study of the antinutritional and/or toxic properties of phytohemagglutinin and trypsin-chymotrypsin inhibitor extracted from the seed of a commercial cultivar of edible bean used in Brazil. Bean proteins were extracted in acidic salt solution and fractionated by dialysis and centrifugation, then freeze-dried. The total freeze-dried bean extract and the globulin or albumin protein fraction were resuspended in distilled water and heated (100 degrees C, 30 min) for inactivation of hemagglutinin. Diets were prepared with unheated bean protein fractions and heated ones (100% trypsin inhibitor activity, but 0% phytohemagglutinin activity). As a result, the inhibition of growth and poor dietary protein utilization were observed in rats fed diets containing unheated bean protein fractions, but not in rats fed diets containing heated fractions. It was thus assumed that phytohemagglutinin is the main antinutritional and toxic factor that in dry bean (Phaseolus) protein and that trypsin inhibitor (Bowman-Birk type) did not interfere with rat growth.

Purification and primary structure determination of a Bowman-Birk trypsin inhibitor from Torresea cearensis seeds.

A Bowman-Birk-type trypsin inhibitor (TcTI) was purified from seeds of Torresea cearensis, a Brazilian native tree of the Papilionoideae sub-family of Leguminosae. Three forms of the inhibitor were separated by anion exchange chromatography. The major form with 63 amino acids was entirely sequenced; it shows a high structural similarity to the Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of the inhibitor are a lysine residue at position 15 and a histidine at position 42 as identified by alignment to related inhibitors, direct chemical modification and specific enzymatic degradation. Immunoprecipitation with antibodies raised in rats is reduced significantly if TcTI is complexed with chymotrypsin and, to a lesser degree, if complexed with trypsin. TcTI forms a ternary complex with trypsin and chymotrypsin. The binary complexes with trypsin or chymotrypsin were isolated by gel filtration. Dissociation constants of the complexes with trypsin, plasmin, chymotrypsin, and factor XIIa are 1, 36, 50, 1450 nM, respectively; human plasma kallikrein, human factor Xa, porcine pancreatic kallikrein and bovine thrombin are not inhibited. TcTI prolongs blood clotting time of the contact phase activation pathway by inhibition of FXIIa.