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Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman–Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts the quality of seed proteins. As an alternative approach, we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild-type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays, and qRT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss-of-function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed-specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities. [ABSTRACT FROM AUTHOR]

The article shows that proteolysis (enzymatic hydrolysis of proteins) is a fundamental biological reaction that underlies such biological processes as digestion, reutilization of tissue proteins, immunity, neuroendocrine regulation, inflammation and dystrophy, and in some cases, as defense against pathogens. The most important regulators of proteolysis are inhibitors of proteolytic enzymes, which make them effective factors influencing the state of a significant number of biological processes. There are 2 inhibitors of proteolytic enzymes in soybeans: Kunitz inhibitor (KI), which inhibits trypsin, and Bowman-Birk inhibitor (BBI), which inhibits trypsin, chymotrypsin, elastase, cathepsins, and microbial proteases. Unlike the KI inhibitor, the BBI inhibitor is thermostable and remains in soy products (meal, cake) after defatting. Therefore, it is important to use soy products with a minimum content of BBI in feeding animals. Goal of work. To determine the content of BBI in promising soybean varieties of Odesa selection. Materials and methods. 12 soybean varieties of the Odesa selection were studied (Euridika, Orpheus, Aurora, Pivdenna zorya, Ariadna, Phoenix, Vasylkivska, Taverna, Serenada, Odesitka, Zmina, Zvytyaga). After grinding the beans and defatting the flour with acetone, the inhibitor was extracted with acetate buffer (0.05 M, pH 4.5) and the extract was mixed with three volumes of acetone. After keeping the mixture at a temperature of -16°C for 2 days, the precipitate was centrifuged and dissolved in 0.5% NaHCO3. The content of BBI was determined by the effect on chymotrypsin, the activity of which was assessed by the rate of casein hydrolysis by Kunitz-Levytskyi method. Results. It was established that the content of BBI in the investigated soybean varieties varies from 93.6 ?g/g (Euridika) to 368.8 ?g/g (Serenada). The soybean varieties Taverna (323.8 ?g/g), Vasylkivska (290.9 ?g/g), as well as Zmina (282.0 ?g/g) and Ariadna (263.0 ?g/g) are characterized by a high content of BBI. Conclusion. New soybean varieties of the Odesa selection contain a small amount of BBI and can be used in animal feeding after conventional heat treatment without additional BBI removal.

Abstract Background Bowman-Birk inhibitors (BBI) are a family of serine-type protease inhibitors that modulate endogenous plant proteolytic activities during different phases of development. They also inhibit exogenous proteases as a component of plant defense mechanisms, and their overexpression can confer resistance to phytophagous herbivores and multiple fungal and bacterial pathogens. Dicot BBIs are multifunctional, with a “double-headed” structure containing two separate inhibitory loops that can bind and inhibit trypsin and chymotrypsin proteases simultaneously. By contrast, monocot BBIs have a non-functional chymotrypsin inhibitory loop, although they have undergone internal duplication events giving rise to proteins with multiple BBI domains. Results We used a Hidden Markov Model (HMM) profile-based search to identify 57 BBI genes in the common wheat (Triticum aestivum L.) genome. The BBI genes are unevenly distributed, with large gene clusters in the telomeric regions of homoeologous group 1 and 3 chromosomes that likely arose through a series of tandem gene duplication events. The genomes of wheat progenitors also contain contiguous clusters of BBI genes, suggesting this family underwent expansion before the domestication of common wheat. However, the BBI gene family varied in size among different cultivars, showing this family remains dynamic. Because of these expansions, the BBI gene family is larger in wheat than other monocots such as maize, rice and Brachypodium. We found BBI proteins in common wheat with intragenic homologous duplications of cysteine-rich functional domains, including one protein with four functional BBI domains. This diversification may expand the spectrum of target substrates. Expression profiling suggests that some wheat BBI proteins may be involved in regulating endogenous proteases during grain development, while others were induced in response to biotic and abiotic stresses, suggesting a role in plant defense. Conclusions Genome-wide characterization reveals that the BBI gene family in wheat is subject to a high rate of homologous tandem duplication and deletion events, giving rise to a diverse set of encoded proteins. This information will facilitate the functional characterization of individual wheat BBI genes to determine their role in wheat development and stress responses, and their potential application in breeding.

A Bowman-Birk protease, i.e., Mucuna pruriens trypsin inhibitor (MPTI), was purified from the seeds by 55.702-fold and revealed a single trypsin inhibitor on a zymogram with a specific activity of 202.31 TIU/mg of protein. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under non-reducing conditions, the protease trypsin inhibitor fraction [i.e., trypsin inhibitor non-reducing (TINR)] exhibited molecular weights of 74 and 37 kDa, and under reducing conditions [i.e., trypsin inhibitor reducing (TIR)], 37 and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 and 18 kDa protein bands on SDS–PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 56.085, and 74.78 kDa on ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization/quadrupole time-of-flight-mass spectrometry (ESI/QTOF-MS). Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Furthermore, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by the UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of Bowman-Birk inhibitors (BBIs). The multimeric association helps to achieve more stability, thus enhancing their functional efficiency. MPTI was found to be a competitive inhibitor which again suggested that it belongs to the BBI family of inhibitors, displayed an inhibitor constant of 1.3 × 10–6 M, and further demonstrates potent anti-inflammatory activity. The study provided a comprehensive basis for the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in the native state from M. pruriens.

A Bowman-Birk protease, i.e., Mucuna pruriens trypsin inhibitor (MPTI), was purified from the seeds by 55.702-fold and revealed a single trypsin inhibitor on a zymogram with a specific activity of 202.31 TIU/mg of protein. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under non-reducing conditions, the protease trypsin inhibitor fraction [i.e., trypsin inhibitor non-reducing (TINR)] exhibited molecular weights of 74 and 37 kDa, and under reducing conditions [i.e., trypsin inhibitor reducing (TIR)], 37 and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 and 18 kDa protein bands on SDS–PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 56.085, and 74.78 kDa on ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization/quadrupole time-of-flight-mass spectrometry (ESI/QTOF-MS). Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Furthermore, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by the UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of Bowman-Birk inhibitors (BBIs). The multimeric association helps to achieve more stability, thus enhancing their functional efficiency. MPTI was found to be a competitive inhibitor which again suggested that it belongs to the BBI family of inhibitors, displayed an inhibitor constant of 1.3 × 10–6 M, and further demonstrates potent anti-inflammatory activity. The study provided a comprehensive basis for the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in the native state from M. pruriens. [ABSTRACT FROM AUTHOR]

Plant proteinase/protease inhibitors (PIs) have therapeutic potential besides their role in regulating endogenous protease activity in several physiological processes. They exert their function by inhibiting the catalytic activity of specific proteases. The use of PIs for inhibiting coagulation factors and slowing down the blood coagulation process has emerged as a critical intervention point for developing antithrombotic agents. The present study has demonstrated the characterization of bacterially expressed recombinant peanut Bowman-Birk inhibitor (rPnBBI) and evaluation of its anticoagulant potential compared to native PnBBI (isoinhibitors pool) purified from peanuts. The full-length BBI gene was cloned, and rPnBBI (7.53 kDa) expressed in the soluble fraction of Escherichia coli BL21 (DE3) strain was isolated by passing through the trypsin affinity column. The purified rPnBBI with β-sheets as major secondary structural elements exhibited inhibitory activity towards bovine trypsin and chymotrypsin at diverse pH and temperatures. Both, rPnBBI and PnBBI showed anticoagulant effects by prolonging the activated partial thromboplastin time (aPTT) and inhibiting the activity of proteases/factors (plasma kallikrein, FXIIa, FXIa, FIXa, and FXa) involved in the blood coagulation pathway. Besides, rPnBBI and PnBBI showed a similar inhibitory tendency towards FXIIa, FXIa, and FIXa, while rPnBBI exhibited a higher inhibition potential than PnBBI towards FXa. Contrarily, PnBBI showed higher inhibitory potential than rPnBBI on plasma kallikrein. Altogether, the bioactive BBI molecules from the natural food source peanuts showed significant anticoagulant potential, which could be exploited in the prophylaxis of thromboembolic disorders associated with various human diseases. [Display omitted] [ABSTRACT FROM AUTHOR]

Oral mucosal melanomas (OMM) are aggressive cancers in dogs, and are good models for human OMM. Gap junctions are composed of connexin units, which may have altered expression patterns and/or subcellular localization in cancer cells. Cell-to-cell communication by gap junctions is often impaired in cancer cells, including in melanomas. Meanwhile, the upregulated expression of the gap junction protein connexin 43 (Cx43) inhibits melanoma progression. The α-connexin carboxyl-terminal (aCT1) peptide reportedly maintains Cx43 expression and cell-cell communication in human mammary cells and increases the communication activity through gap junctions in functional assays, therefore causing decreased cell proliferation. The Bowman-Birk protease inhibitor (BBI), a component of soybeans, induces Cx43 expression in several tumor cells as a trypsin–chymotrypsin inhibition function, with antineoplastic effects. This study investigated the effect of aCT1 peptide and BBI treatment, alone or in combination, on TLM1 canine melanoma cell viability. Cell viability after treatment with aCT1, the reverse sequence peptide (R-pep), and/or BBI for 5 days was analyzed by PrestoBlue assay. Immunofluorescence was used to observe Cx43 localization and expression. aCT1 (200 μM) alone did not significantly decrease cell viability in TLM1 cells, whereas BBI (400 μg/ml) alone significantly decreased the TLM1 viability. Combined treatment with both aCT1 (200 μM) and BBI (400 μg/ml) significantly decreased cell viability in TLM1 cells. Cx43 expression, as identified by immunostainings in TLM1 cells, was increased in the cell membrane after the combination treatment with BBI and aCT1. This dual treatment can be combined to achieve the anticancer activity, possibly by increasing Cx 43 expression and affecting Cx43 migration to the cell membrane. In conclusion, a treatment strategy targeting Cx43 with BBI and aCT1 may possibly lead to new effective therapies for canine OMM.

Bowman-Birk inhibitor (BBI) is a soybean seed serine protease inhibitor whose nutraceutical properties far exceed its anti-nutritional property arising from its trypsin and chymotrypsin inhibitor activity. High BBI soybean genotypes are sought for the commercial preparation of this nutraceutical biomolecule while low BBI content in Kunitz trypsin inhibitor (KTI)-free genetic background is desirable in soymeal manufacturing. In the present investigation, monoclonal antibody assay of 95 soybean genotypes revealed 11-fold genetic variation for BBI concentration. The study led to the identification of 3 very high (>20 mg/g defatted soy flour) and 5 very low BBI (< 4 mg/g defatted soy flour) soybean genotypes. Contribution of BBI to total trypsin inhibition activity ranged from 2.2 to 53.5% with average value of 11.6%. Genotypes with varying level of BBI raised consecutively for two years exhibited non-significant (p>0.05) effect of growing year on the trait. BBI content in BC3F2 derived KTI-free lines was at par with the recurrent parent. Low and high BBI content genotypes with diverse genetic background identified in the study may be exploited to develop mapping population to identify genomic regions underlying BBI in soybean.

Background: Bowman-Birk inhibitors (BBI) are a family of serine-type protease inhibitors that modulate endogenous plant proteolytic activities during different phases of development. They also inhibit exogenous proteases as a component of plant defense mechanisms, and their overexpression can confer resistance to phytophagous herbivores and multiple fungal and bacterial pathogens. Dicot BBIs are multifunctional, with a "double-headed" structure containing two separate inhibitory loops that can bind and inhibit trypsin and chymotrypsin proteases simultaneously. By contrast, monocot BBIs have a non-functional chymotrypsin inhibitory loop, although they have undergone internal duplication events giving rise to proteins with multiple BBI domains. Results: We used a Hidden Markov Model (HMM) profile-based search to identify 57 BBI genes in the common wheat (Triticum aestivum L.) genome. The BBI genes are unevenly distributed, with large gene clusters in the telomeric regions of homoeologous group 1 and 3 chromosomes that likely arose through a series of tandem gene duplication events. The genomes of wheat progenitors also contain contiguous clusters of BBI genes, suggesting this family underwent expansion before the domestication of common wheat. However, the BBI gene family varied in size among different cultivars, showing this family remains dynamic. Because of these expansions, the BBI gene family is larger in wheat than other monocots such as maize, rice and Brachypodium. We found BBI proteins in common wheat with intragenic homologous duplications of cysteine-rich functional domains, including one protein with four functional BBI domains. This diversification may expand the spectrum of target substrates. Expression profiling suggests that some wheat BBI proteins may be involved in regulating endogenous proteases during grain development, while others were induced in response to biotic and abiotic stresses, suggesting a role in plant defense. Conclusions: Genome-wide characterization reveals that the BBI gene family in wheat is subject to a high rate of homologous tandem duplication and deletion events, giving rise to a diverse set of encoded proteins. This information will facilitate the functional characterization of individual wheat BBI genes to determine their role in wheat development and stress responses, and their potential application in breeding. [ABSTRACT FROM AUTHOR]

Bowman-Birk inhibitor (BBI) is the main type of soybean trypsin inhibitor, which is a kind of antinutritional factor. It can cause indigestion and hinder growth and development of human and animal. Herein, a rapid immunochromatographic strip was developed in a sandwich format using the anti-BBI monoclonal antibody (mAb) in order to specifically detect BBI. The mAbs showed high affinity towards BBI and no cross-reactivity with other soybean allergenic proteins and antinutritional factors. The assay could be performed within 10 min. The limit of detection of the test strip was 0.5 µg/mL for visual and 0.23 µg/mL for strip reader. The assay showed high specificity for BBI and recovery efficacy of BBI in spiked milk samples ranged between 83.24% and 89.86% with the coefficient of variation of less than 7.98%. Therefore, the test strip could be used as a rapid and reliable detection method for BBI qualitative or quantitative assessment.

Bowman-Birk inhibitor (BBI) is a protease inhibitor that affects protein digestibility; however, it is increasingly being recognised as anutraceutical and cosmeceutical molecule. In the present study, BBI concentration during soybean seed development, its loss during processing treatments, and the level in commercial soy products were determined. Significant differences for BBI concentration were observed across the genotypes and seed development stages. Genotype × seed development stage interaction was also found to be significant (P<0.05) for BBI concentration. Boiling, autoclaving, microwave irradiation, and sprouting resulted in significant (P<0.05) loss of BBI. Minimum loss was observed in sprouting, while autoclaving for 5 min completely deactivated BBI. Microwave irradiation of the soaked seeds resulted in higher BBI loss than of dry seeds. Among the commercial soy products, BBI concentration was high in soy flour brands, minuscule in ready-to-cook miso soup and undetectable in extruded soy products and roasted soy nuts. [ABSTRACT FROM AUTHOR]

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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. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors (SPIs) play an important role in cell survival, development and host defense. In plants, serine protease inhibitors such as the Kunitz-type inhibitor (KTI) and the Bowman-Birk inhibitor (BBI) have been shown to be induced in response to abiotic stress such as salinity and drought resulting in tolerance to these stresses. In this study, Arabidopsis thaliana (T3) plants overexpressing the BBI gene from maize were generated and subjected to drought stress in order to study the role of BBI protease inhibitor in drought tolerance. Drought treatment of four-week-old Arabidopsis plants was performed by withholding water from plants for nine days and harvested plant material was used for physiological and biochemical analysis. The transgenic lines exhibited normal growth after nine days of drought as compared to the wild-type. The results also showed a higher leaf relative water content (RWC) in transgenic lines when compared to the wild-type (WT), with line 2 having the highest RWC of 72% and the WT having the lowest RWC of 32%. Trypsin-inhibitor activity indicated that the total protein of the positive transgenic plants had stronger protease inhibitory activity than the wild-type. Transgenic lines overexpressing BBI also showed reduced lipid peroxidation (MDA content) as well as enhanced activity of antioxidants glutathione-s-transferase (GST) and ascorbate peroxidase (APX). These results suggest that BBI protease inhibitor leads to drought tolerance associated with reduction in drought-induced oxidative stress. • Generated transgenic lines 1 and 2 exhibited more drought tolerance. • Higher leaf relative water content (RWC) was maintained in transgenic lines. • Trypsin-inhibitor activity was higher in transgenic lines. • Transgenic lines showed reduced lipid peroxidation (MDA content). • Antioxidants (GST and APX) were enhanced in transgenic lines. [ABSTRACT FROM AUTHOR]

Leukoplakia in the oral cavity has been used as a putative surrogate marker of head and neck cancer development. A class of chemoprevention compounds, called protease inhibitors, has been shown in vitro and in animal models to effectively suppress premalignant lesions. Bowman-Birk inhibitor (BBI) is a protease inhibitor derived from soybeans that has demonstrated chemoprevention activity in many in vitro and animal systems, including the hamster cheek pouch model. Pilot, Phase I and Phase IIa studies of Bowman-Birk Inhibitor in patients with oral leukoplakia have demonstrated no detectable side effects. In the Phase IIa trial, changes in the protease activity in oral mucosal cells after BBI Concentratec (BBIC) treatment correlated with the changes in neu protein levels. Additionally, evidence for a dose-related treatment effect of BBIC on oral leukoplakia was demonstrated. These results indicate that BBIC should be investigated for chemopreventive activity in a randomized clinical trial.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables ( C = dB + eK and T = xB + yK ). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

Bowman-Birk inhibitor (BBI) is the main type of soybean trypsin inhibitor, which is a kind of antinutritional factor. It can cause indigestion and hinder growth and development of human and animal. Herein, a rapid immunochromatographic strip was developed in a sandwich format using the anti-BBI monoclonal antibody (mAb) in order to specifically detect BBI. The mAbs showed high affinity towards BBI and no cross-reactivity with other soybean allergenic proteins and antinutritional factors. The assay could be performed within 10 min. The limit of detection of the test strip was 0.5 µg/mL for visual and 0.23 µg/mL for strip reader. The assay showed high specificity for BBI and recovery efficacy of BBI in spiked milk samples ranged between 83.24% and 89.86% with the coefficient of variation of less than 7.98%. Therefore, the test strip could be used as a rapid and reliable detection method for BBI qualitative or quantitative assessment. [ABSTRACT FROM AUTHOR]

Abstract Rhynchosia sublobata , a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC 50 = 24 ng) and RsKI (IC 50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively. Graphical abstract Image 1 Highlights • Trypsin specific protease inhibitors (RsPI) were purified from seeds of R. sublobata. • A quick method (<2 h) was developed to separate Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI) from RsPI. • Both RsBBI and RsKI existed as small oligomers. • The structural stability of RsBBI and RsKI varied with heat and dithiothreitol (DTT) treatment. • In vitro studies revealed the inhibitory activity of RsBBI against AjGPs and RsKI against HaGPs. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors are found in plants, animals and microorganisms, where they play important roles in many physiological and pathological processes. Inhibitor scaffolds based on natural proteins and peptides have gradually become the focus of current research as they tend to bind to their targets with greater specificity than small molecules. In this report, a novel Bowman–Birk type inhibitor, named ranacyclin-NF (RNF), is described and was identified in the skin secretion of the East Asian frog, Pelophylax nigromaculatus. A synthetic replicate of the peptide was subjected to a series of functional assays. It displayed trypsin inhibitory activity with an inhibitory constant, Ki, of 447 nM and had negligible direct cytotoxicity. No observable direct antimicrobial activity was found but RNF improved the therapeutic potency of Gentamicin against Methicillin-resistant Staphylococcus aureus (MRSA). RNF shared significant sequence similarity to previously reported and related inhibitors from Odorrana grahami (ORB) and Rana esculenta (ranacyclin-T), both of which were found to be multi-functional. Two analogues of RNF, named ranacyclin-NF1 (RNF1) and ranacyclin-NF3L (RNF3L), were designed based on some features of ORB and ranacyclin-T to study structure–activity relationships. Structure–activity studies demonstrated that residues outside of the trypsin inhibitory loop (TIL) may be related to the efficacy of trypsin inhibitory activity.

Black‐eyed pea trypsin and chymotrypsin inhibitor (BTCI) is a small protein from Bowman–Birk protease inhibitor family, which simultaneously inhibits trypsin and chymotrypsin. Through the inhibition of trypsin‐ and chymotrypsin‐like sites on the 20S subunit of human proteasome, BTCI acts as a potent anticarcinogenic agent inducing apoptosis in breast cancer cells. Because of the lack of crystallographic information of the BTCI‐proteasome complex, we analyze here the BTCI‐chymotrypsin and BTCI‐trypsin interfaces using computations. We adopt the corrected semiempirical quantum‐mechanical methods in combination with implicit description of the water environment. Firstly, we carefully check the representativeness of smaller systems by fragmentation and analyzing the convergence of the overall interaction energies. Then, we use the "virtual glycine scan" technique to understand the binary complex formation, identifying the most contributing amino acid side chains in the interfaces. Besides detailed quantification of all important residue contributions, the importance of Lys26 and Phe53 in the BTCI and Asp186 (trypsin) and Ser195 (chymotrypsin) residues is confirmed. In summary, we have worked out an accurate and efficient in silico protocol for protein–protein interfaces, which can be later used for studying the inhibition of 20S proteasome. We describe the interfaces of the Bowman–Birk inhibitor (BTCI) with proteases trypsin and chymotrypsin by semiempirical quantum‐mechanical methods and give an implicit description of the water environment. The importance of amino acids at the interfaces, for example Lys26 and Phe53 in the BTCI and Asp186 (trypsin) and Ser195 (chymotrypsin), was identified and the side‐chain contributions were quantified. [ABSTRACT FROM AUTHOR]

Trypsin inhibitor activity (TIA) in soybean is attributed to two polypeptides, namely, Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Standard spectrophotometric protocol widely followed for estimation of TIA is cumbersome and does not distinguish KTI from BBI. In the present investigation, extraction conditions for KTI were optimized and different forms of this polypeptide were resolved in 180 soybean genotypes of Indian and exotic origin through native PAGE. This led to the identification of three KTI alleles, namely, Tia, Tib, and Tic, with Tia occurring in most of the Indian genotypes. Trypsin-KTI complex assay exhibited binding of Tia polypeptide with 2.51 fold concentration of trypsin. Subsequently, seeds of selected genotypes were subjected to estimation of KTI and BBI activity through densitometry and enzyme-linked immunosorbent assay (ELISA), respectively; and total TIA through standard spectrophotometric protocol. Summation of KTI and BBI was significantly (P < 0.05) lower than that of TIA determined through the spectrophotometric method. [ABSTRACT FROM AUTHOR]

Crude proteinase inhibitors (CPIs) extracted from the seeds of Rhynchosia sublobata , a wild relative of pigeon pea showed pronounced inhibitory activity on the larval gut trypsin-like proteases of lepidopteran insect pest – Achaea janata . Consequently, a full-length cDNA of Bowman-Birk inhibitor gene ( RsBBI1 ) was cloned from the immature seeds of R. sublobata . It contained an ORF of 360 bp encoding a 119-amino acid polypeptide (13.3 kDa) chain with an N-terminus signal sequence comprising of 22 amino acids. The amino acid sequence and phylogenetic analysis together revealed that RsBBI1 exhibited a close relation with BBIs from soybean and Phaseolus spp. A cDNA sequence corresponding to RsBBI1 mature protein (89 amino acid stretch) was expressed in E. coli . The recombinant rRsBBI1 protein with a molecular mass of 9.97 kDa was purified using trypsin affinity chromatography. The purified rRsBBI1 exhibited non-competitive mode of inhibition of both bovine trypsin ( Ki of 358 ± 11 nM) and chymotrypsin ( Ki of 446 ± 9 nM). Its inhibitory activity against these proteases was stable at high temperatures (>95 °C) and a wide pH range but sensitive to reduction with dithiothreitol (DTT), indicating the importance of disulphide bridges in exhibiting its activity. Also, rRsBBI1 showed significant inhibitory activity ( IC 50  = 70 ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed <1% inhibitory activity ( IC 50  = 8 μg) on H. armigera larval gut trypsin-like proteases (HaGPs) than it has against AjGPs. Besides, in vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on larval growth and development in A. janata which suggests it can be further exploited for such properties. [ABSTRACT FROM AUTHOR]

One of the major challenges in the administration of therapeutic proteins involves delivery limitations. Liposomes are well-known drug delivery systems (DDS) that have been used to overcome this drawback; nevertheless, low protein entrapment efficiency (EE) still limits their wide biomedical application on a commercial scale. In the present work, different methods for protein entrapment into liposomes were tested in order to obtain tailored DDS platforms for multiple biomedical applications. The protein used as model was the Black-eyed pea Trypsin and Chymotrypsin Inhibitor (BTCI), a member of the Bowman-Birk protease inhibitor family (BBIs), which has been largely explored for its potential application in many biomedical therapies. We optimized reverse-phase evaporation (REV) and freeze/thaw (F/T) entrapment methods, using a cationic lipid matrix to entrap expressive amounts of BTCI (∼100 μM) in stable liposomes without affecting its protease inhibition activity. The influence of various parameters ( e.g. entrapment method, liposome composition, buffer type) on particle size, charge, polydispersity, and EE of liposomes was investigated to provide an insight on how to control such parameters in view of obtaining a high entrapment yield. In addition, BTCI liposome platforms obtained herein showed to be versatile vesicles, allowing surface modification with moieties/polymers of interest ( e.g. PEG, transferrin). The aforementioned results are relevant to focusing on the entrapment of other promising BBIs or protein agents sharing similar structural features. These findings encourage future studies to investigate the advantages of using the liposome platforms presented herein to broaden the use of this type of DDS for BBI biomedical applications. [ABSTRACT FROM AUTHOR]

The hydrolysis of bradykinin (Bk) by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI) and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M−1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus) ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ.

Leguminous seeds (Fabaceae) have a high content of inhibitors of which serine protease inhibitors are the most widely studied. However, there are only a few studies related to the investigation of these proteins in tree species belonging to the Amazon flora. The protein content presented in seeds of four Amazonian Leguminosae species, Parkia pendula, P. discolor, P. multijuga and P. Nitida, was extracted by using NaCl 0.15 mol L-1 and then partially fractionated by using affinity chromatography performed on a trypsin-Sepharose 4B. These inhibitors presented different affinities between trypsin and chymotrypsin serine proteases, showing a higher inhibition to trypsin compared to chymotrypsin, except for P. nitida, which showed high inhibition against both enzymes. The SDS-PAGE analysis showed that the species from Parkia genus have a main band corresponding to partially purified trypsin inhibitors. The apparent molecular mass inhibitors (approximately 13-18 kDa) and the high specificity for trypsin suggest the occurrence of Bowman-Birk and Kunitz type inhibitors.

Bowman-Birk inhibitor (BBI) is a soybean-derived protease inhibitor that has anti-inflammation and anti-HIV effect. Here, we further investigated the anti-HIV action of BBI in macrophages, focusing on its effect on viral entry. We found that BBI could significantly block HIV entry into macrophages. Investigation of the mechanism(s) of the BBI action on HIV inhibition showed that BBI down-regulated the expression of CD4 receptor (as much as 80%) and induced the production of the CC chemokines (up to 60 folds at protein level) in macrophages. This inhibitory effect of BBI on HIV entry could be blocked by the neutralization antibodies to CC chemokines. These findings indicate that BBI may have therapeutic potential as a viral entry inhibitor for the prevention and treatment of HIV infection. [ABSTRACT FROM AUTHOR]

Due to the complications of the soymilk system, the heat-induced Bowman-Birk inhibitor (BBI) inactivation mechanism is not well known. In this study, two BBI samples with low and high purities were prepared from soymilk. It was confirmed that three groups (A, C, and D) of BBI, which are contained in soybean seeds, were transferred into soymilk during processing. On heating, it was found that 1) the two subdomains of BBI were not equally heat stable, 2) the conformation of BBI gradually changed, 3) some amino acid residues (namely, cystine, serine and lysine) in BBI were degraded, 4) BBI did not tend to form intermolecular cross-links with another BBI, but did slightly with non-BBI proteins. Based on some previous studies, the conformational change of BBI was attributed to β-elimination reactions on the amino acid residues of BBI and the subsequent intramolecular reactions induced by the products yielded by the β-elimination reactions. [ABSTRACT FROM AUTHOR]

A divergent protein synthesis strategy was executed to effectively synthesize Bowman-Birk protease inhibitor (BBI) analogues using native chemical ligation of peptide hydrazides. Grafting selected residues from a potent trypsin inhibitor, sunflower trypsin inhibitor-1, onto the α-chymotrypsin-binding loop of BBI, resulted in a fourfold improvement of α-chymotrypsin inhibition. The crystal structure of a synthetic BBI analogue co-crystallized with α-chymotrypsin confirmed the correct protein fold and showed a similar overall structure to unmodified BBI in complex with α-chymotrypsin. Dynamic light scattering showed that C-terminal truncation of BBI led to increased self-association. [ABSTRACT FROM AUTHOR]

The black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) forms concentration dependent homomultimers, as previously demonstrated by Light scattering and Atomic Force Microscopy. Considering that these self-aggregates might influence their binding to cognate enzymes, we investigated the interaction of BTCI at picomolar concentrations using surface immobilized Chymotrypsin (α-CT) and Trypsin (T) by Surface Plasmon Resonance. Our results indicate that BTCI has subnanomolar affinity to both immobilized enzymes, which is approximately two orders of magnitude higher than previously reported. Moreover, we probed the influence of temperature on protein binding equilibria in order to investigate their interaction energetics. While the BTCI/T interaction concurs with the canonical entropy-driven mechanism described for BBI interactions with serine proteinases, the BTCI/α-CT interaction does not. Our measurements indicate that bimolecular BTCI/α-CT complexes form with a negative enthalpy change and a moderate entropic increase. Direct calorimetric evaluation is in accord with the van't Hoff approximation obtained by SPR. We demonstrate that as protein concentrations increase to the micromolar range, secondary endothermic events become prevalent and affect both the kinetics and thermodynamics of protein associations. Our study reinforces that BBI interactions with serine proteinases should be studied in dilute solutions to abridge often neglected secondary interactions. [ABSTRACT FROM AUTHOR]

Main conclusion : A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3′RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M cm. MpBBI inhibits strongly trypsin with K in the 10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants. [ABSTRACT FROM AUTHOR]

Complex behavior of Bowman–Birk protease inhibitor (BBI) with ι -carrageenan (LC) as a function of pH, protein to polysaccharides ratio and salt concentration was studied by turbidimetric titration, dynamic light scattering (DLS) and isothermal titration calorimetry (ITC). At fixed BBI/LC weight ratio of 5:1, turbidity and DLS results showed that pH c and pH φ1 shift to the lower pH values with the increase in ionic strength ( I ), whereas the former occurred at the pH lower than isoelectric point (pI = 4.2) of BBI at I ≥ 100 mM NaCl. ITC results showed that BBI binding to LC involves a two-step process with an increasing exothermic enthalpy at the first binding step. The further insight of BBI-LC complexation was studied as a function of BBI concentration, ionic strength and temperatures using ITC. The critical molar ratio ( r critical ) between two binding steps was independent of protein concentration, ionic strength and temperature, although the heat flow obviously decreased with the increasing I (0–200 mM) and slightly increased with the elevated temperature (25–45 °C). The negative heat capacity (Δ C p ) and the gain in nonionic contribution (Δ G no ) indicated the involvement of nonelectrostatic interactions (e.g., hydrophobic effect) for the first binding step. [ABSTRACT FROM AUTHOR]

We report for the first time the recombinant expression of bioactive wild-type sunflower trypsin inhibitor 1 (SFTI-1) inside E. coli cells by making use of intracellular protein trans-splicing in combination with a high efficient split-intein. SFTI-1 is a small backbone-cyclized polypeptide with a single disulfide bridge and potent trypsin inhibitory activity. Recombinantly produced SFTI-1 was fully characterized by NMR and was observed to actively inhibit trypsin. The in-cell expression of SFTI-1 was very efficient reaching intracellular concentration ≈ 40 µ M. This study clearly demonstrates the possibility of generating genetically encoded SFTI-based peptide libraries in live E. coli cells, and is a critical first step for developing in-cell screening and directed evolution technologies using the cyclic peptide SFTI-1 as a molecular scaffold. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 818-824, 2016. [ABSTRACT FROM AUTHOR]

Sugarcane's (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The upand downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory. [ABSTRACT FROM AUTHOR]

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Background In the present study, we investigated the immuno-regulatory and therapeutic effects of Bowman–Birk inhibitor concentrate (BBIC) on experimental autoimmune neuritis (EAN), an animal model of Guillain–Barré syndrome (GBS) in human. Methods EAN in Lewis rats induced by inoculation with peripheral nerve myelin P0 protein peptide 180-199 (P0 peptide) was treated with BBIC at two different therapeutic regimens. Results Our data indicated that the administration of BBIC daily orally effectively inhibited and ameliorated the clinical and pathological signs of EAN. The suppression of EAN was associated with an insufficiency of autoreactive T cells, as reflected by inhibited P0 peptide-specific mononuclear cell proliferation and decreased in CD4 and CD8 T cells infiltrating into the peripheral nervous system (PNS). BBIC might mediate its therapeutic effects by shifting macrophages from M1 to M2 subtype as evidenced by increasing Arg-1, CD206 and IL-10 and inhibiting IFN-γ, TNF-α, IL-12, iNOS and CD40 expressions on macrophages as well as enhancing anti-inflammatory cytokines IL-4 and IL-10 and decreasing inflammatory cytokines, IFN-γ, TNF-α and IL-17 in the PNS. Conclusion Our results suggest that BBIC may have therapeutic potential in human GBS and other autoimmune diseases in the future. [ABSTRACT FROM AUTHOR]

[Display omitted] • High-fat diet and alcohol induced inflammation involved in chronic diseases. • Soybean exhibits anti-inflammatory properties due to bioactive compounds. • Anti-inflammatory mechanism works mostly through inhibition of NF-κB pathway. • Soybean bioactive compounds have the potential to mitigate inflammation. Chronic inflammation refers to long-lasting inflammation that occurs over a period of several months to years, and it is associated with the progression of other chronic diseases. It may be induced by alcohol consumption and a high-fat diet. Soybean bioactive compounds prevent chronic inflammation by primarily targeting the nuclear factor kappa B (NF-κB) pathway, which inhibits the phosphorylation of IkappaB kinase complex (IκB) and reduces inflammatory marker levels. We performed a systematic review of studies published between 2012 and 2022 on the impact of soybeans on diet-induced chronic inflammation. Soy bioactive compounds may mitigate chronic inflammation. However, more human intervention studies are needed to assess their efficacy as potential modulating agents for inflammation and inflammation-related diseases. The objective was to review the impact of soy-derived bioactive compounds on high-fat diet-induced and alcohol-induced inflammation. To our knowledge, it is the first review to look specifically at high-fat diet-induced and alcohol-induced inflammation and how it is modulated by specific bioactive compounds in soybean. [ABSTRACT FROM AUTHOR]

Trypsin inhibitor SFTI-1 is the smallest and the most potent among BBI inhibitors. It is also an interesting object for SAR studies since it is cyclic 14 amino acid molecule which additionally contains disulfide bridge. We showed that elimination of head-to-tail cycliztion did not influence its activity. Moreover peptoid monomers of Nlys and Nphe introduced in the substrate specificity P 1 position of monocyclic SFTI-1 preserved trypsin and chymotripsin inhibitory activity respectively and made P 1 –P 1 ′ bond proteolytically stable. These findings motivated us to perform conformational analysis of [Nphe 5 ]SFTI-1 by means of 2D NMR spectroscopy and molecular dynamics calculations. Obtained structure occurred to be in a good agreement with published structures for wild-type SFTI-1, its monocyclic analog with disulfide bridge only as well as one containing Nlys peptoid monomer in P 1 position. [ABSTRACT FROM AUTHOR]

Lunasin is a 5-kDa soybean bioactive peptide with demonstrated anti-cancer and anti-inflammatory properties. Recently, purification methods have been developed to obtain gram quantities of lunasin. However, these methods are cumbersome, time consuming and cost-prohibitive. To overcome these constrains we have developed a novel method which involves extraction of soybean flour with 30% ethanol followed by preferential precipitation of lunasin and protease inhibitors by calcium. The calcium precipitated protein fraction, which we termed as Lunasin Protease Inhibitor Concentrate (LPIC), contains three abundant proteins with molecular weights of 21, 14 and 5 kDa. This simple procedure yields 3.2 g of LPIC from 100 g of soybean flour and the entire isolation procedure can be completed in less than 2 h. Treatment of THP-1 human monocyte cell lines with LPIC resulted in suppression of lipopolysaccharide-stimulated cytokine expression, demonstrating that the LPIC isolated by our simple procedure is biologically active. [ABSTRACT FROM AUTHOR]

Common bean ( Phaseolus vulgaris L.) is a good source of protein, vitamins, minerals and complex carbohydrates. The objective was to compare protein profile, including anti-nutrient proteins, and potential bioactive peptides of improved common bean cultivars grown in Mexico and Brazil. Bean protein isolates (BPI) were prepared from 15 common bean cultivars and hydrolyzed using pepsin/pancreatin. Thirteen proteins were identified by SDS-PAGE and protein in-gel tryptic-digestion-LC/MS. Protein profile was similar among common bean cultivars with high concentrations of defense-related proteins. Major identified proteins were phaseolin, lectin, protease and α-amylase inhibitors. Lectin (159.2 to 357.9 mg lectin/g BPI), Kunitz trypsin inhibitor (inh) (4.3 to 75.5 mg trypsin inh/g BPI), Bowman-Birk inhibitor (5.4 to 14.3 μg trypsin-chymotrypsin inh/g BPI) and α-amylase inhibitor activity (2.5 to 14.9 % inhibition relative to acarbose/mg BPI) were higher in Mexican beans compared to Brazilian beans. Abundant peptides were identified by HPLC-MS/MS with molecular masses ranging from 300 to 1500 Da and significant sequences were SGAM, DSSG, LLAH, YVAT, EPTE and KPKL. Potential bioactivities of sequenced peptides were angiotensin converting enzyme inhibitor (ACE), dipeptidyl peptidase IV inhibitor (DPP-IV) and antioxidant capacity. Peptides from common bean proteins presented potential biological activities related to control of hypertension and type-2 diabetes. [ABSTRACT FROM AUTHOR]

ABSTRACT Protease inhibitors of the Bowman-Birk (BBI) family are commonly found in plants and animals where they play a protective role against invading pathogens. Here, we report an atomic resolution (1Å) crystal structure of a peptide inhibitor isolated from a skin secretion of a Chinese bamboo odorous frog Huia versabilis (HV-BBI) in complex with trypsin. HV-BBI shares significant similarities in sequence with a previously described inhibitor from a diskless-fingered odorous frog Odorrana graham (ORB). However, the latter is characterized by more than a 16,000 fold higher Ki against trypsin than HV-BBI. Comparative analysis of trypsin cocrystal structures of HV-BBI and ORB and additionally that of Sunflower Trypsin Inhibitor (SFTI-1) together with accessory information on the affinities of inhibitor variants allowed us to pinpoint the inhibitor moiety responsible for the observed large difference in activity and also to define the extent of modifications permissible within the common protease-binding loop scaffold of BBI inhibitors. We suggest that modifications outside of the inhibitory loop permit the evolution of specificity toward different enzymes characterized by trypsin-like specificity. Proteins 2015; 83:582-589. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The hydrolysis of bradykinin (Bk) by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI) and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M-1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus) ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ. [ABSTRACT FROM AUTHOR]

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman–Birk protease inhibitor ( RbTI ) from rice bean ( Vigna umbellata ). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus , Vigna trilobata and Vigna vexilata . Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichia coli and the highest expression was recorded after 5.5 h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes. [ABSTRACT FROM AUTHOR]

Highlights: [•] Isolation of major serine protease inhibitors, Bowman-Birk and Kunitz, from soybean. [•] Quantification of active Bowman-Birk isoinhibitors from soymilk. [•] Significant variation in amounts of Bowman-Birk inhibitors among soymilks. [•] Physiologically relevant amounts of active Bowman-Birk inhibitors in soymilks. [Copyright &y& Elsevier]