Evidence that the Bowman-Birk inhibitor from Pisum sativum affects intestinal proteolytic activities in chickens.

Chicken diet essentially relies on soybean as the major source of proteins but there are increasing efforts to identify other protein-rich feedstuffs. Of these, some pea cultivars constitute interesting sources of proteins, although some of them contain antinutritional factors that may compromise the digestibility of their protein content. Consequently, chickens exhibit low performance, while undigested compounds rejected in feces have a negative environmental impact. In this article, we analyzed the intestinal content of chickens fed a pea diet (Pisum sativum) to decipher the mechanisms that could explain such a low digestibility. Using gelatin zymography, we observed that the contents of chicken fed the pea diet exhibit altered proteolytic activities compared with intestinal contents from chickens fed a rapeseed, corn, or soybean diet. This pea-specific profile parallels the presence of a 34 kDa protein band that resists proteolysis during the digestion process. Using mass spectrometry analysis, we demonstrated that this band contains the pea-derived Bowman-Birk protease inhibitor (BBI) and 3 chicken proteases, the well-known chymotrypsinogen 2-like (CTRB2) and trypsin II-P39 (PRSS2), and the yet uncharacterized trypsin I-P38 (PRSS3). All 3 proteases are assumed to be protease targets of BBI. Molecular modeling of the interaction of pea BBI with PRSS2 and PRSS3 trypsins reveals that electrostatic features of PRSS3 may favor the formation of a BBI-PRSS3 complex at physiological pH. We hypothesize that PRSS3 is specifically expressed and secreted in the intestinal lumen to form a complex with BBI, thereby limiting its inhibitory effects on PRSS2 and chymotrypsinogen 2-like proteases. These data clearly demonstrate that in chickens, feedstuff containing active pea BBI affects intestinal proteolytic activities. Further studies on the effects of BBI on the expression of PRSS3 by digestive segments will be useful to better appreciate the impact of pea on intestine physiology and function. From these results, we suggest that PRSS3 protease may represent an interesting biomarker of digestive disorders in chickens, similar to human PRSS3 that has been associated with gut pathologies.

Ultrasound-enhanced interfacial adsorption and inactivation of soy trypsin inhibitors.

In this study, liquid-liquid interfacial protein adsorption was proposed as a means of inactivating soy trypsin inhibitors (TIs, including Kunitz (KTI) and Bowman-Birk inhibitor (BBI)). Hexane-water was first selected as a model system to compare three emulsification methods (hand shaking, rotor-stator and ultrasound mixing). Ultrasound could generate the smallest and least polydisperse emulsion droplets, resulting in highest interfacial adsorption amount of KTI and BBI as well as the highest inactivation percentage of TIs (p < 0.05). Therefore, ultrasound was selected to further explore the effect of the non-aqueous phase on interfacial adsorption and inactivation kinetics of TIs in a food emulsion system containing vegetable oil (VTO). The adsorption amounts of KTI and BBI in the VTO-aqueous emulsion increased by âˆ¼ 25 % compared to the hexane-aqueous emulsion. In addition, the adsorption amounts of KTI and BBI were rapidly increased as a function of sonication time, especially for the hexane-aqueous emulsion system. This result suggests that such inactivation of TIs could be implemented in continuous systems for large-scale processing. Finally, the pathways of interface-induced inactivation of BBI and KTI were investigated based on separate experiments on individual BBI and KTI systems. The results showed that the interface adsorption caused the changes in the secondary and tertiary structure of KTI that led to its activitation. However, BBI was quite stable at the liquid-liquid interface without significant conformational change. Overall, ultrasound-assisted interfacial adsorption can be considered a rapid and highly efficient method to inactivate KTI.

Proteases, protease inhibitors and radiation carcinogenesis.

PURPOSE: The purpose of the studies described in this mini review article was to identify nontoxic compounds that could prevent or suppress the radiation induced malignant transformation of cells and be useful as human cancer preventive agents. CONCLUSIONS: (1) Many different types of potential anticarcinogenic substances were evaluated initially for their abilities to prevent or suppress radiation induced malignant transformation in vitro, and certain anticarcinogenic protease inhibitors (APIs) were observed to be the most powerful anticarcinogenic agents at suppressing this surrogate endpoint biomarker of radiation carcinogenesis. (2) Within the category of APIs, those that inhibited the activity of chymotrypsin were effective at far lower molar concentrations than other APIs. The soybean-derived protease inhibitor known as the Bowman-Birk inhibitor (BBI) is a particularly powerful chymotrypsin inhibitor that is able to prevent radiation induced transformation in vitro (at concentrations down to nanomolar levels) as well as radiation induced carcinogenesis in vivo without toxicity. (3) There were many other unusual characteristics of APIs that led to the selection of one of these APIs, BBI, as the most appropriate compound for us to develop as a human cancer preventive agent. As one example, the APIs have an irreversible effect on carcinogenesis, while the effects are reversible for most anticarcinogenic agents when they are removed from carcinogenesis assay systems. (4) Numerous studies were performed in attempts to determine the potential mechanisms by which the APIs could prevent or suppress radiation induced carcinogenesis in in vitro and in vivo systems, and the results of these studies are described in this review article. The APIs and the proteases which interact with them appear to play important roles in radiation carcinogenesis. (5) Preparations for human trials using BBI began decades ago. The cost of preparing purified BBI was far too high to consider performing human trials with this agent, so BBI Concentrate (BBIC), a soybean extract enriched in BBI, was developed for the specific purpose of performing human trials with BBI. BBIC achieved Investigational New Drug (IND) Status with the Food and Drug Administration in April,1992, and human BBIC trials began at that time. (6) Several human trials were performed using BBIC and they indicated many potentially beneficial health effects produced by BBIC administration to people in various forms (e.g. tablets). 7) It is hypothesized that BBI takes the place of α-1-antichymotrypsin, an important regulatory compound in the human body, and helps to maintain homeostasis.

Determination of protease inhibitors, glycinin, and beta-conglycinin in soybeans and their relationships.

In order to search for suitable soybean varieties for different applications, the protein contents of Kunitz trypsin inhibitor (KTI), Bowman-Birk trypsin inhibitor (BBI), glycinin (11S), and ß-conglycinin (7S) of 93 soybean samples from different sources and harvest years were quantified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Meanwhile, the protease inhibitory activities against trypsin and chymotrypsin were determined. Results showed that the individual protein contents and trypsin inhibitor activities differed significantly (p < 0.05) among soybean samples. KTI contents ranged from 5.25 to 14.60 mg·g-1 ; BBI contents ranged from 1.81 to 5.74 mg·g-1 ; 11S varied from 13.65% to 48.55% and 7S varied from 15.68% to 42.15% of total soluble protein; trypsin and chymotrypsin inhibitory activities were 8.93-20.95 mg TI·g-1 and 4.18 -12.79 mg CI·g-1 , respectively. Excellent linear relationships existed between trypsin inhibitor contents and their activities. The regression equations offer a rapid method for estimating the activity of KTI or BBI in raw soybeans. PRACTICAL APPLICATION: The regression equations established based on a large number of soybean varieties offered a rapid method to estimate the activity of trypsin inhibitors. The data presented here provided useful information for the food industry or breeders to select soybean varieties with different inhibitory activities or protein contents for different food processing applications.

Protease Inhibitors from Plants as Therapeutic Agents- A Review.

Plant-based diets are a great source of protease inhibitors (PIs). Two of the most well-known families of PIs are Bowman-Birk inhibitors (BBI) and Kunitz-type inhibitors (KTI). The first group acts mainly on trypsin, chymotrypsin, and elastase; the second is on serine, cysteine, and aspartic proteases. PIs can retard or inhibit the catalytic action of enzymes; therefore, they are considered non-nutritional compounds; nevertheless, animal studies and cell line experiments showed promising results of PIs in treating human illnesses such as obesity, cardiovascular diseases, autoimmune diseases, inflammatory processes, and different types of cancer (gastric, colorectal, breast, and lung cancer). Anticarcinogenic activity's proposed mechanisms of action comprise several inhibitory effects at different molecular levels, i.e., transcription, post-transcription, translation, post-translation, and secretion of cancer cells. This work reviews the potential therapeutic applications of PIs as anticarcinogenic and anti-inflammatory agents in human diseases and the mechanisms by which they exert these effects.

Inhibitory effect of Bowman-Birk protease inhibitor on autophagy in MDAMB231 breast cancer cell line.

BACKGROUND: Autophagy has an essential role in cellular energetic balance, cell cycle, and cell death, so the change in autophagy level is crucial in many human diseases such as cancer. Herbal medicine has been widely used to treat cancer. Bowman-Birk protease inhibitor (BBI), a protease inhibitor extracted from soybean, has antitumorigenic, anti-inflammatory, and anti-angiogenic activities. In this study, we evaluated the effect of BBI on the growth of breast cancer cell line and transcript level of autophagy and apoptosis-related genes. MATERIALS AND METHODS: BBI was purified from soybean by ion-exchange chromatography method. The viability of MDA-MB-231 cells that were treated with BBI was measured by MTT assay, and the transcript level of genes involved in autophagy and apoptosis was measured by real-time-polymerase chain reaction (PCR) technique. RESULTS: The results of BBI purification showed that 100 g of the ethanolic fraction yielded 300-mg BBI with more than 95% purity. MTT results revealed that BBI inhibited the cell growth of MDA-MB-231 cell line in a dose-dependent manner, with an IC50 of 200 µg/mL. The results of real-time reverse transcription-PCR exhibited that BBI altered the expression of Atg5, Beclin1, light chain 3-II, and sequestosome1 and increased the Bax/Bcl2 ratio in MDA-MB-231 cell line. CONCLUSION: According to our results, BBI could inhibit autophagy and induce apoptosis in MDA-MB-231 cell line. Thus, BBI may be used as a therapeutic drug in the treatment of breast cancer whether alone or with chemotherapeutic drugs.

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.

Glycation affects differently the main soybean Bowman-Birk isoinhibitors, IBB1 and IBBD2, altering their antiproliferative properties against HT29 colon cancer cells.

Naturally-occurring serine protease inhibitors of the Bowman-Birk family, particularly abundant in legume seeds, exert their potential chemopreventive and/or therapeutic properties via protease inhibition. Processing of legume seeds, including soybeans, has been proposed as a major cause for their loss of bioactivity due to glycation. In order to assess how glycation affected the protease inhibitory activities of major soybean Bowman-Birk isoinhibitors (BBI) and their antiproliferative properties, IBB1 and IBBD2 were purified and subjected to glycation under controlled conditions using glucose at high temperature. Both soybean isoinhibitors showed remarkable heat stability. In the presence of glucose, IBBD2 lost most of its trypsin inhibitory activity while IBB1 maintains similar trypsin and chymotrypsin inhibitory activities as in the absence of sugar. Glycation patterns of both BBI proteins were assessed by MALDI-TOF spectrometry. Our results show that the glycation process affects IBBD2, losing partially its antiproliferative activity against HT29 colon cancer cells, while glycated-IBB1 was unaffected.

Combination Effect of Bowman-Birk Inhibitor and α-Tocopheryl Succinate on Prostate Cancer Stem-Like Cells.

The reoccurrence of androgen-dependent prostate cancer after anti-androgen therapy mainly depends on prostate cancer stem-like cells. To reduce the risk, it is important to delete the cancer stem-like cells. Furthermore, to induce differentiation of cancer stem-like cells is critical to abrogate stemness of the cells. Therefore, we tried to investigate a possibility on the establishment of a new effective therapy to eradicate the cancer stem-like cells via the induction of differentiation in this study. Prostate cancer stem-like cells from an androgen-dependent prostate cancer cell line (LNCaP cell) had severe resistance against an anti-androgen therapeutic agent. We selected Bowman-Birk inhibitor (BBI) from soybeans reported as a chemopreventive agent in prostate cancer to differentiate the caner stem-like cells and α-tocopheryl succinate (TOS) known as a mitocan to induce effectively cytotoxic effect against the cancer stem-like cells. In fact, only TOS treatment had cytotoxic effect against the cancer stem-like cells, but the addition of BBI treatment to the cells treated with TOS reinforced TOS-mediated cytotoxicity in the cancer stem-like cells. This reinforcement coincided with the combination-enhanced apoptosis in the stem-like cells. Also, we confirmed caspase9-caspase3 cascade mainly contributed to the enhancement of the cytotoxicity in the stem-like cells caused by the combination, indicating that the reinforcement of BBI on TOS-mediated apoptosis via mitochondria related to the enhancing cytotoxic effect of the combination on the prostate cancer stem-like cells. Overall, it seems that the combination is an effective new approach to reduce the reoccurrence of prostate cancer targeting prostate cancer stem cells.

Amino Acid Scanning at P5' within the Bowman-Birk Inhibitory Loop Reveals Specificity Trends for Diverse Serine Proteases.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14-amino acid cyclic peptide that shares an inhibitory loop with a sequence and structure similar to a larger family of serine protease inhibitors, the Bowman-Birk inhibitors. Here, we focus on the P5' residue in the Bowman-Birk inhibitory loop and produce a library of SFTI variants to characterize the P5' specificity of 11 different proteases. We identify seven amino acids that are generally preferred by these enzymes and also correlate with P5' sequence diversity in naturally occurring Bowman-Birk inhibitors. Additionally, we show that several enzymes have divergent specificities that can be harnessed in engineering studies. By optimizing the P5' residue, we improve the potency or selectivity of existing inhibitors for kallikrein-related peptidase 5 and show that a variant with substitutions at 7 of the scaffold's 14 residues retains a similar structure to SFTI-1. These findings provide new insights into P5' specificity requirements for the Bowman-Birk inhibitory loop.

Soybean-derived Bowman-Birk Inhibitor (BBI) Inhibits HIV Replication in Macrophages.

The Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, is known to have anti-inflammatory effect in both in vitro and in vivo systems. Macrophages play a key role in inflammation and immune activation, which is implicated in HIV disease progression. Here, we investigated the effect of BBI on HIV infection of peripheral blood monocyte-derived macrophages. We demonstrated that BBI could potently inhibit HIV replication in macrophages without cytotoxicity. Investigation of the mechanism(s) of BBI action on HIV showed that BBI induced the expression of IFN-ß and multiple IFN stimulated genes (ISGs), including Myxovirus resistance protein 2 (Mx2), 2',5'-oligoadenylate synthetase (OAS-1), Virus inhibitory protein (viperin), ISG15 and ISG56. BBI treatment of macrophages also increased the expression of several known HIV restriction factors, including APOBEC3F, APOBEC3G and tetherin. Furthermore, BBI enhanced the phosphorylation of IRF3, a key regulator of IFN-ß. The inhibition of IFN-ß pathway by the neutralization antibody to type I IFN receptor (Anti-IFNAR) abolished BBI-mediated induction of the anti-HIV factors and inhibition of HIV in macrophages. These findings that BBI could activate IFN-ß-mediated signaling pathway, initialize the intracellular innate immunity in macrophages and potently inhibit HIV at multiple steps of viral replication cycle indicate the necessity to further investigate BBI as an alternative and cost-effective anti-HIV natural product.

Soybean Bowman-Birk protease inhibitor (BBI): identification of the mechanisms of BBI suppressive effect on growth of two adenocarcinoma cell lines: AGS and HT29.

BACKGROUND AND AIMS: Bowman-Birk protease inhibitor (BBI) has been well known to suppress the emergence and progression of different cancers. In the present study, the mechanisms by which BBI alters cancers have been addressed. To reach this goal, the effects of BBI on proliferation of and VEGF secretion by two cell lines (AGS: gastric adenocarcinoma and HT-29: colorectal adenocarcinoma) and also BBI effect on MMP-2 and 9 synthesis/secretion by AGS cells was evaluated. METHODS: ELISA method was used to assess VEGF concentration and gelatin zymography was used to address MMP-2 and 9 production/excretion. RESULTS: BBI had powerful inhibitory effect on proliferation and VEGF secretion by both cell lines. In addition, inhibition of MMP-2 and MMP-9 secreted by AGS cells suggests BBI as a potent inhibitor of gastric cancer progression. On the other hand, the results indicated that inhibition of MMP-2, MMP-9 and VEGF secretion is one of the mechanisms of anti-angiogenic effect of BBI. CONCLUSION: BBI expresses powerful suppressive effect on tumor progression of two prevalent cancers: gastric adenocarcinoma and colorectal adenocarcinoma.

Therapeutic potential of the peptide leucine arginine as a new nonplant bowman-birk-like serine protease inhibitor.

The peptide leucine arginine (pLR) belongs to a new class of cyclic peptides isolated from frog skin. Its primary sequence is similar to the reactive loop of plant Bowman-Birk inhibitors (BBI), and the recently discovered circular sunflower trypsin inhibitor-1 (SFTI-1). The conformational properties of pLR in solution were determined by NMR spectroscopy and revealed excellent structural similarity to BBI and SFTI-1. Moreover, pLR is a highly potent trypsin inhibitor, with Ki values in the nanomolar range, and, due to its small size, a potential inhibitor of the serine protease tryptase. Since tryptase plays a crucial role in the development of allergic airway inflammation, the therapeutic potential of pLR in a murine asthma model was investigated. Treatment of ovalbumin-sensitized mice with pLR during allergen challenge reduced the acute asthma phenotype. Most importantly, application even at the end of a long-lasting chronic asthma model decreased the development of chronic airway inflammation and tissue remodeling.

Prevention of cancer and inflammation by soybean protease inhibitors.

Several plant-based nutrients and non-nutrients that can inhibit mutagenesis and cell proliferation have been identified. Some of the most promising compounds identified as chemopreventive and anti-metastatic agents include soybean-derived protease inhibitors (PIs), Bowman-Birk Inhibitor (BBI) and Kunitz-Trypsin Inhibitor (KTI). A growing body of evidence suggests that BBI could act as anti-carcinogenic agent and KTI is considered to prevent cancer invasion and metastasis. These inhibitors are non-toxic, are of low cost and can be taken orally or as a part of the daily diet. PIs are undergoing investigation in the clinical setting as potential agents for chemoprevention and anti-metastasis. A complex scenario about the interaction between PIs and cell signaling has been emerging. Soybean PIs are not just anti-proteolytic proteins, but can also be modulators of cell signal transduction. Cancer and inflammatory treatment strategies modulating signal transduction need further investigation.

Stability and bioactivity of a Bowman-Birk inhibitor in orange juice during processing and storage.

The present research aimed to evaluate the feasibility of a soybean Bowman-Birk inhibitor (BBI) as a natural functional food ingredient. The influence of food processing, storage and digestion on the structure and bioactivity of BBI added to a food matrix was evaluated. The cytotoxic effect of the digested samples was also tested with the aim to provide information regarding the safe use of BBI as a functional food ingredient. Samples of freshly squeezed orange juice (OJ) and two orange juice model systems (OJ-ms) supplemented and non-supplemented with BBI were prepared and processed mimicking pasteurization and sterilization industrial conditions. Moreover, pasteurized samples were stored at 4 °C for two months. The samples were digested in vitro under oral-gastrointestinal physiological conditions. Results seem to indicate that the activity of BBI supplemented to the food matrix sufficiently survives thermal processing, storage and digestion processes. Changes in BBI bioactivity may be ascribed among others to the Maillard reaction. Formation of early Maillard reaction products also called Amadori products has been detected in the food sample. No cytotoxic effect was observed for the samples under study. In conclusion, our findings support that BBI has significant potential as a natural functional food ingredient in pasteurized orange juice stored at 4 °C.

De novo sequencing and characterization of a novel Bowman-Birk inhibitor from Lathyrus sativus L. seeds by electrospray mass spectrometry.

Bowman-Birk serine protease inhibitors (BBIs) from legume seeds are small proteins showing a two-head structure with distinct reactive site loops, which inhibit two molecules of the same enzyme or two different proteases. Purification and characterization of new BBIs is of broad interest for understanding the basic molecular mechanisms underlying natural defence against the action of proteolytic enzymes. In this study, two novel acidic BBIs (LSI-1a and LSI-2a) were isolated from L. sativus seeds using classical biochemical techniques and characterized for their inhibitory activity. In addition, the N-terminal sequencing of LSI-1a was performed by Edman degradation up to residue 10 and the complete primary structure of the most abundant form (LSI-2a) was determined by using a combination of mass spectrometry approaches, including MALDI-TOF MS, tandem MS and Electron Transfer Dissociation coupled with Proton Transfer Reaction (ETD/PTR) top-down sequencing of N- and C-termini. Furthermore, the LSI-2a dimerization surface has also been investigated by a combination of gel filtration, electrophoretic techniques and homology modelling. Knowing the structure of small proteins inhibiting proteolytic enzymes is of general importance for understanding the defence mechanisms against degradation for their use in biological applications as well as for designing artificial inhibitors.

Role of serine proteases in inflammation: Bowman-Birk protease inhibitor (BBI) as a potential therapy for autoimmune diseases.

Serine proteases, a sub-category of the protease family, participate in various physiologic and pathologic conditions. Serine proteases are involved in different arms of the immune system and play an important role in inflammation. They have been evaluated as therapeutic targets in several inflammatory diseases. The Bowman-Birk protease inhibitor (BBI), a soybean-derived serine protease inhibitor, is resistant to temperature and acidic conditions. These characteristics make it a good candidate for oral administration, with no major side effects. In addition, the therapeutic effect of BBI has been shown in inflammatory diseases and cancer. We have demonstrated the immunoregulatory and anti-inflammatory effects of BBI in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Here we review the role of serine proteases in inflammatory diseases, with emphasis on the potential of BBI as a novel oral therapy for multiple sclerosis.