RESUMO
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.
Assuntos
Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Contração Miocárdica/efeitos dos fármacos , Peptídeos/farmacologia , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Animais , Anti-Hipertensivos/química , Sítios de Ligação , Quimotripsina/química , Quimotripsina/metabolismo , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiopatologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Masculino , Simulação de Acoplamento Molecular , NG-Nitroarginina Metil Éster/química , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Peptídeos/síntese química , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Tripsina/química , Tripsina/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/química , Vasodilatação/efeitos dos fármacosRESUMO
Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61â¯Å, respectively. The structures presented refinement parameters of Rworkâ¯=â¯14.52 % and Rfreeâ¯=â¯15.59 %; Rworkâ¯=â¯15.60 % and Rfreeâ¯=â¯18.78 %, and different surface area between the peptide and the enzyme of 1024â¯Å2 and 1070â¯Å2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358â¯Å between the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.
Assuntos
Oligopeptídeos/química , Sementes/química , Inibidor da Tripsina de Soja de Bowman-Birk/química , Tripsina/química , Vigna/embriologia , Linhagem Celular Tumoral , Cristalografia por Raios X , HumanosRESUMO
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.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Passiflora/química , Peptídeo Hidrolases/metabolismo , Extratos Vegetais/farmacologia , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Anticoagulantes/química , Estabilidade Enzimática , Frutas/química , Dados de Sequência Molecular , Extratos Vegetais/química , Inibidores de Proteases/química , Tempo de Protrombina , Inibidor da Tripsina de Soja de Bowman-Birk/química , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologiaRESUMO
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.
Assuntos
Endopeptidases/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/química , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Sequência de Aminoácidos , Animais , Bovinos , Quimotripsina/antagonistas & inibidores , Análise por Conglomerados , Desenho de Fármacos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Software , Propriedades de Superfície , Inibidores da Tripsina/químicaRESUMO
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.
Assuntos
Animais , Endopeptidases/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/química , Modelos Moleculares , Sequência de Aminoácidos , Bovinos , Análise por Conglomerados , Desenho de Fármacos , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Inibidores da Tripsina/química , Quimotripsina/antagonistas & inibidoresRESUMO
Several methods have been applied to study protein-protein interaction from structural and thermodynamic point of view. The present study reveals that atomic force microscopy (AFM), molecular modeling, and docking approaches represent alternative methods offering new strategy to investigate structural aspects in oligomerization process of proteinase inhibitors. The topography of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) was recorded by AFM and compared with computational rigid-bodies docking approaches. Multimeric states of BTCI identified from AFM analysis showed globular-ellipsoidal shapes. Monomers, dimers, trimers, and hexamers were the most prominent molecular arrays observed in AFM images as evaluated by molecular volume calculations and corroborated by in silico docking and theoretical approaches. We therefore propose that BTCI adopts stable and well-packed self-assembled states in monomer-dimer-trimer-hexamer equilibrium. Although there are no correlation between specificity and packing efficiency among proteinases and proteinase inhibitors, the AFM and docked BTCI analyses suggest that these assemblies may exist in situ to play their potential function in oligomerization process.
Assuntos
Microscopia de Força Atômica , Inibidor da Tripsina de Soja de Bowman-Birk/química , Inibidor da Tripsina de Soja de Bowman-Birk/ultraestrutura , Biologia Computacional , Dimerização , Estrutura Quaternária de Proteína , Reprodutibilidade dos Testes , Relação Estrutura-AtividadeRESUMO
Two isoforms of a protease inhibitor were isolated by ion-exchange chromatography of tepary bean (Phaseolus acutifolius G.) seed proteins. The main isoform was used to determine the amino acid sequence of the protein. It is an 80 amino acid residue protein with a molecular mass of 8765 Da, showing sequence homology with the Bowman-Birk family of protease inhibitors. Several regions with amino acid microheterogeneity were found, corroborating the possible presence of isoforms. Mass spectrometry analysis was carried out to confirm isoforms. The presence of dimer and trimer forms of the inhibitor was shown through electrophoresis and mass spectrometry. Another unusual characteristic for this inhibitor was its ability to bind metals. The presence of four sequential histidines at the N-terminal end of the protein could account for this binding. Mass spectrometry and atomic absorption spectroscopy support the presence of calcium in the native inhibitor.
Assuntos
Phaseolus/química , Inibidores de Proteases/química , Sementes/química , Sequência de Aminoácidos , Cromatografia por Troca Iônica , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Isoformas de Proteínas , Homologia de Sequência de Aminoácidos , Inibidor da Tripsina de Soja de Bowman-Birk/químicaRESUMO
A novel serine proteinase inhibitor, DgTI, was purified from Dioclea glabra seeds by acetone precipitation, and ion-exchange and reverse phase chromatography. The inhibitor belongs to the Bowman-Birk family, and its primary sequence, determined by Edman degradation and mass spectrometry, of 67 amino acids is: SSGPCCDRCRCTKSEPPQCQCQDVRLNSCHSACEACVCSHSMPGLCSCLDITHFCHEPCKSSGDDED++ +. Although two reactive sites were determined by susceptibility to trypsin (Lys(13) and His(40)), the inhibitory function was assigned only to the first site. The inhibitor forms a 1:1 complex with trypsin, and Ki is 0.5 x 10(-9) M. Elastase, chymotrypsin, kallikreins, factor Xa, thrombin, and plasmin were not inhibited. By its properties, DgTI is a Bowman-Birk inhibitor with structural and inhibitory properties between the class of Bowman-Birk type I (with a fully active second reactive site), and Bowman-Birk type II (devoid of second reactive site).
Assuntos
Proteínas de Plantas/química , Inibidores da Tripsina/química , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Sementes/química , Homologia de Sequência , Temperatura , Tripsina/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/químicaRESUMO
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.
Assuntos
Fabaceae/metabolismo , Plantas Medicinais , Sementes/química , Inibidor da Tripsina de Soja de Bowman-Birk/isolamento & purificação , Inibidores da Tripsina/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Anticoagulantes/farmacologia , Sítios de Ligação , Coagulação Sanguínea/efeitos dos fármacos , Bovinos , Quimotripsina/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Hidrólise , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Ratos/imunologia , Tripsina/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/química , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologiaRESUMO
TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds of Torresea acreana, one of the two known species of Torresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears as M(r) 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2, Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.