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Trypsin inhibitor from the root-tuber of underutilized legume Winged bean (Psophocarpus tetragonolobus (L.) DC.) (WbT-TI) was purified using ion exchange chromatography followed by size-exclusion chromatography. The purified WbT-TI showed a molecular mass of 20,609 Da and an isoelectric point of 5.10. Ultraviolet circular dichroism (UV-CD) and intrinsic fluorescence reported, that WbT-TI interacts with trypsin. Domain-wise analysis of WbT-TI revealed it to belong to the Kunitz-type soybean trypsin inhibitor (STI) family with a specific β-trefoil fold. The sequence of WbT-TI showed 44% sequence coverage to acidic trypsin inhibitor from the seed of the same plant. Protein interaction similarity analysis (PIPSA) evaluated the electrostatic properties of WbT-TI and provided information about the interacting partners of trypsin inhibitors. The purified protein was quantified and tested for in vitro anticancer activity using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay against the human osteosarcoma (MG-63) cell line. At 5 µg/ml of WbT-TI, the highest inhibition was seen. These studies may lead to the development of winged bean protease inhibitor-based preventive and therapeutic strategies for different kinds of cancers.

We have previously demonstrated multidirectional changes in trypsin activity and some serum biochemical parameters in rats under conditions of water and food deprivation with following satiation. In the present study, administration of soybean trypsin inhibitor (STI) to animals subjected to water and food deprivation caused a moderate decrease in trypsin activity and an increase in the serum concentration of total protein, which attested to deceleration of protein metabolism. Significant changes in the content of cholesterol and triglycerides against the background of STI administration were also found indicating intensification of lipid and energy metabolism. These findings suggest that STI prevents disturbances of metabolic processes in the body under conditions of water and food deprivation. However, the peculiarities of STI influence on metabolic processes require further study.

We have previously reported in a series of papers that a Kunitz-type protease inhibitor, bikunin, suppresses up-regulation of urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) expression, phosphorylation of ERK1/2 and cancer cell invasion in vitro and peritoneal disseminated metastasis in vivo. In the present study, we investigated the effects of soy bean trypsin inhibitor (SBTI) on the net enzymatic activity of secreted, extracellular uPA, signal transduction involved in the expression of uPA and invasion in HRA human ovarian cancer cells. SBTI contains a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk inhibitor (BBI). Here, we show 1) that KTI and BBI were purified separately from soybeans; 2) that neither KTI nor BBI effectively inhibits enzymatic activity of uPA; 3) that uPA upregulation observed in HRA cells was inhibited by preincubation of the cells with KTI with an IC50 of approximately 2 microM, whereas BBI failed to repress uPA upregulation, as measured by enzyme-linked immunosorbent assay; 4) that cell invasiveness was inhibited by treatment of the cells with KTI with an IC50 of approximately 3 microM, whereas BBI failed to suppress cell invasion, as measured by an in vitro invasion assay; 5) KTI suppresses HRA cell invasion by blocking uPA up-regulation which may be mediated by a binding protein(s) other than a bikunin binding protein and/or its receptor; and 6) that transforming growth factor-beta 1 (TGF-beta1)-mediated activation of ERK1/2 was significantly reduced by preincubation of the cells with KTI. In conclusion, KTI, but not BBI, could inhibit cell invasiveness at least through suppression of uPA signaling cascade, although the mechanisms of KTI may be different from those of bikunin.

Dietary antinutritional factors have been reported to adversely affect the digestibility of protein, bioavailability of amino acids and protein quality of foods. Published data on these negative effects of major dietary antinutritional factors are summarized in this manuscript. Digestibility and the quality of mixed diets in developing countries are considerably lower than of those in developed regions. For example, the digestibility of protein in traditional diets from developing countries such as India, Guatemala and Brazil is considerably lower compared to that of protein in typical North American diets (54–78 versus 88–94 %). Poor digestibility of protein in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, is due to the presence of less digestible protein fractions, high levels of insoluble fibre, and/or high concentrations of antinutritional factors present endogenously or formed during processing. Examples of naturally occurring antinutritional factors include glucosinolates in mustard and canola protein products, trypsin inhibitors and haemagglutinins in legumes, tannins in legumes and cereals, gossypol in cottonseed protein products, and uricogenic nucleobases in yeast protein products. Heat/alkaline treatments of protein products may yield Maillard reaction compounds, oxidized forms of sulphur amino acids, D-amino acids and lysinoalanine (LAL, an unnatural nephrotoxic amino acid derivative). Among common food and feed protein products, soyabeans are the most concentrated source of trypsin inhibitors. The presence of high levels of dietary trypsin inhibitors from soyabeans, kidney beans or other grain legumes have been reported to cause substantial reductions in protein and amino acid digestibility (up to 50 %) and protein quality (up to 100 %) in rats and/or pigs. Similarly, the presence of high levels of tannins in sorghum and other cereals, fababean and other grain legumes can cause significant reductions (up to 23 %) in protein and amino acid digestibility in rats, poultry, and pigs. Normally encountered levels of phytates in cereals and legumes can reduce protein and amino acid digestibility by up to 10 %. D-amino acids and LAL formed during alkaline/heat treatment of lactalbumin, casein, soya protein or wheat protein are poorly digestible (less than 40 %), and their presence can reduce protein digestibility by up to 28 % in rats and pigs, and can cause a drastic reduction (100 %) in protein quality, as measured by rat growth methods. The adverse effects of antinutritional factors on protein digestibility and protein quality have been reported to be more pronounced in elderly rats (20-months old) compared to young (5-weeks old) rats, suggesting the use of old rats as a model for assessing the protein digestibility of products intended for the elderly.

Inhibitors of enzymes involved in carbohydrate digestion may be a potential option for glycemic control in Diabetes Mellitus. This study aimed to evaluate the effect of the trypsin inhibitor isolated from tamarind seed ( Tamarindus indica L.) (TTI) on α-amylase. After confirmation of the obtaining and characterization of the TTI, the in vitro inhibitory activity of the TTI against α-amylase was analyzed. The interaction of the modeled structures’ theoretical TTI (TTIp 56/287) and five of its derived peptides with α-amylase was also evaluated in silico using Docking and Molecular Dynamics, and their functional properties were examined. The Interaction Potential Energy (IPE) and the main interactions of the peptide-α-amylase complex were described using three-dimensional representations. TTI presented 100% antitryptic activity and a molecular mass of approximately 21 kDa. In vitro, inhibition of α-amylase was higher than 37%. These results were corroborated by computational analyses, which demonstrated strong interaction between the TTIp 56/287 complex and its peptides with the enzyme. The Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) analyses showed good stability. IPE was −705.08 kJ/mol for DTVHDTDGQVPL and −584.11 kJ/mol for TIAPACAPKPAR. Electrostatic interactions stand out, especially the salt bridge, between the main residues that interacted in the complexes (DTVHDTDGQVPL, TIAPACAPKPAR, and TVSQTPIDIPIGLPVR). Additionally, the bioactive potential predicted two candidates with good stability, a long half-life, and bioactivity in an intestinal simulation environment. This is the first report of tamarind trypsin inhibitor or its peptides inhibiting α-amylase. Thus, the amino acid sequences DTVHDTDGQVPL and TIAPACAPKPAR were revealed as candidates that could be tested for action against α-amylase and possibly for glycemic control.

Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [ DE3 ] . The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10 –7  mol L −1 . The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70–80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and β-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL −1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao , so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.

BackgroundWheat amylase trypsin inhibitors (ATI) are dietary non-gluten proteins that activate the toll-like receptor 4 on myeloid cells, promoting intestinal inflammation.Aim of the studyWe investigated the effects of dietary ATI on experimental allergic airway inflammation.MethodsMice on a gluten and ATI-free diet (GAFD), sensitized with PBS or ovalbumin (OVA) and challenged with OVA, were compared to mice on a commercial standard chow, a gluten diet naturally containing ~ 0.75% of protein as ATI (G+AD), a gluten diet containing ~ 0.19% of protein as ATI (G−AD) and a GAFD with 1% of protein as ATI (AD). Airway hyperreactivity (AHR), inflammation in bronchoalveolar lavage (BAL) and pulmonary tissue sections were analyzed. Allergic sensitization was assessed ex vivo via proliferation of OVA-stimulated splenocytes.ResultsMice on a GAFD sensitized with PBS did not develop AHR after local provocation with methacholine. Mice on a GAFD or on a G−AD and sensitized with OVA developed milder AHR compared to mice fed a G+AD or an AD. The increased AHR was paralleled by increased BAL eosinophils, IL-5 and IL-13 production, and an enhanced ex vivo splenocyte activation in the ATI-fed groups.ConclusionsDietary ATI enhance allergic airway inflammation in OVA-challenged mice, while an ATI-free or ATI-reduced diet has a protective effect on AHR. Nutritional wheat ATI, activators of intestinal myeloid cells, may be clinically relevant adjuvants to allergic airway inflammation.

Modification of catalytic expression of enzymes and regulating their in vivo activity are the goals of novel treatment strategies. A green synthetic nanostructured silver with potent trypsin inhibitory properties has not yet been developed, despite the fact that silver nanoparticles possess unique properties that allow them to efficiently block enzymes. The present study demonstrates for the first time a facile, safe, economic, and eco-friendly synthetic route for silver nanoparticles using an aqueous extract of Holigarna arnottiana bark engineered to interact with trypsin and hinder its activity effectively. The studies carried out to examine the interaction between these biofabricated AgNPs (HaAgNPs) and trypsin by UV-visible spectrophotometry and FTIR spectroscopy suggest that the formation of trypsin-HaAgNP complex is responsible for diminishing the catalytic efficiency of trypsin. In vivo studies on Aedes aegypti larval serum support these instrumental results of HaAgNP-induced trypsin inhibition and proves its application as a biopesticide. It is noteworthy that the bioengineered HaAgNPs were also found to have good inhibition potential against pepsin and urease as well. A variety of methods have been employed to characterize the synthesized biocompatible HaAgNPs and it possesses a characteristic absorption maximum of 420 nm. Their shelf life of above 7 years is noticeable, since none of the reported green synthesized AgNPs possess a shelf life of more than 1 year. Altogether, this work demonstrates that biofabricated HaAgNPs are multifunctional and cost-resilient biological tools that can be used as enzyme regulators possessing antioxidant, antimicrobial, and insecticidal features.

This study was aimed at investigating the purification and identification of serine protease inhibitors, F. tataricum trypsin inhibitor (FtTI) from tartary buckwheat ( Fagopyrum tataricum ) seeds. The FtTI was isolated by anion exchange chromatography, affinity chromatography, and centrifugal ultrafiltration. Under reducing and nonreducing conditions, an SDS-PAGE analysis showed that the isolated protein consists of a single polypeptide chain with a molecular mass of approximately 14 kDa. The two isoforms of FtTI were confirmed by the mass spectrometric profile where the two peaks corresponded to 11.487 and 13.838 kDa. The complete amino acid sequence of FtTI has been established by automatic Edman degradation and mass spectrometry. The molecule of FtTI consists of 86 amino acid residues containing two disulfide bonds which connect Cys8 to Cys65 and Cys49 to Cys58. The active site of FtTI contains an Asp66–Arg67 bond. The Ki value was calculated using the equation for slow tight binding inhibition which was 1.6 nM for trypsin. FtTI retained its inhibitory activity over a wide range of pH (3–10) and temperature (20–80 °C). FtTI can be rapidly inactivated by the combination of high temperature and high pressure. An analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor Ι family. Furthermore, FtTI exhibited a strong inhibitory activity against phytopathogenic fungi.

Protease inhibitors are biomolecules with growing biotechnological and biomedical relevance, including those derived from plants. This study investigated strong trypsin inhibitors in quinoa, amaranth, and lupine seeds, plant grains traditionally used in Andean South America. Amaranth seeds displayed the highest trypsin inhibitory activity, despite having the lowest content of aqueous soluble and thermostable protein material. This activity, directly identified by enzymatic assay, HPLC, intensity-fading mass spectrometry (IF-MS), and MS/MS, was attributed to a single protein of 7889.1 Da, identified as identical in Amaranthus caudatus and A. hybridus, with a Ki of 1.2 nM for the canonical bovine trypsin. This form of the inhibitor, which is highly homogeneous and scalable, was selected, purified, and structurally–functionally characterized due to the high nutritional quality of amaranth seeds as well as its promising agriculture–biotech–biomed applicability. The protein was crystallized in complex with bovine trypsin, and its 3D crystal structure resolved at 2.85 Å, revealing a substrate-like transition state interaction. This verified its classification within the potato I inhibitor family. It also evidenced that the single disulfide bond of the inhibitor constrains its binding loop, which is a key feature. Cell culture assays showed that the inhibitor did not affect the growth of distinct plant microbial pathogen models, including diverse bacteria, fungi, and parasite models, such as Mycoplasma genitalium and Plasmodium falciparum. These findings disfavour the notion that the inhibitor plays an antimicrobial role, favouring its potential as an agricultural insect deterrent and prompting a redirection of its functional research.