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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.

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.

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.

Understanding the structural mechanisms of protein–ligand binding and their dependence on protein sequence and conformation is of fundamental importance for biomedical research. Here we investigate the interplay of conformational change and ligand-binding kinetics for the serine protease Trypsin and its competitive inhibitor Benzamidine with an extensive set of 150 μs molecular dynamics simulation data, analysed using a Markov state model. Seven metastable conformations with different binding pocket structures are found that interconvert at timescales of tens of microseconds. These conformations differ in their substrate-binding affinities and binding/dissociation rates. For each metastable state, corresponding solved structures of Trypsin mutants or similar serine proteases are contained in the protein data bank. Thus, our wild-type simulations explore a space of conformations that can be individually stabilized by adding ligands or making suitable changes in protein sequence. These findings provide direct evidence of conformational plasticity in receptors. Conformational plasticity influences several aspects of protein function. Here the authors combine extensive MD simulations with Markov state models—using trypsin as model—to reveal new mechanistic details of how conformational plasticity influence ligand-receptors interactions.

Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks. In vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of autonomous synthetic systems. Now, a methodology has been developed to construct an enzymatic reaction network producing oscillations of active trypsin. The modular approach allows amplification or analog-to-digital conversion of the oscillations, and control over a self-assembly process.

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.

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.

Aeruginosins are common metabolites of cyanobacteria. In the course of re-isolation of the known aeruginosins KT608A and KT608B for bioassay studies, we isolated three new sulfated aeruginosins, named aeruginosins KT688 (1), KT718 (2), and KT575 (3), from the extract of a Microcystis cell mass collected during the 2016 spring bloom event in Lake Kinneret, Israel. The structures of the new compounds were established on the basis of analyses of the 1D and 2D NMR, as well as HRESIMS data. Marfey’s method, coupled with HR ESI LCMS and chiral HPLC, was used to establish the absolute configuration of the amino acid and hydroxyphenyl lactic acid residues, respectively. Compounds 1–3 were tested for inhibition of the serine protease trypsin, and compounds 1 and 2 were found to exhibit IC50 values of 2.38 and 1.43 µM, respectively.

Key messageHere, we reported that a pathogen- and herbivore-induced Kunitz trypsin inhibitor gene, NaKTI2, is required for herbivore resistance, and transcriptionally regulated mainly by NaWRKY3 and NaWRKY6 but not Jasmonate signaling.Plant protease inhibitor (PI) occurs widely in plant species, and is considered as an important part of plant defense arsenal against herbivores. Transcriptome analysis of Nicotiana attenuata leaves revealed that a Kunitz trypsin inhibitor gene, NaKTI2, was highly elicited after inoculation of Alternaria alternata (tobacco pathotype). However, the roles of NaKTI2 in pathogen- and herbivore resistance and its regulation were unclear. NaKTI2 had typical domains of Kunitz trypsin inhibitors and exhibited a high level of trypsin protease inhibitor activities when transiently over-expressed. The transcripts of NaKTI2 could be induced by A. alternata and Spodoptera litura oral secretions (OS). Silencing NaKTI2 via virus-induced gene silencing technique has no influence on lesion diameters developed on N. attenuata leaves after A. alternata inoculation, but S. litura larvae gained more mass and had higher survivorship on NaKTI2-silenced plants. Meanwhile, the expression of NaPI, a PI gene essential for herbivore resistance previously identified in N. attenuata, was not affected in NaKTI2-silenced plants. Unlike NaPI, which was predominantly regulated by jasmonate (JA) signaling, OS-elicited NaKTI2 transcripts were only slightly reduced in JA-deficient plants, but were dramatically decreased in NaWRKY3- and NaWRKY6- silenced plants, respectively. Further electromobility shift assays indicated that NaWRKY3 and NaWRKY6 could directly bind to the promoter regions of NaKTI2 in vitro. Taken together, our results demonstrate that in addition to NaPI, NaKTI2, a pathogen- and herbivore-induced Kunitz trypsin inhibitor gene, is also required for herbivore resistance, and mainly regulated by NaWRKY3 and NaWRKY6.

Purpose Ulinastatin, a serine protease inhibitor, inhibits several pro-inflammatory proteases and decreases inflammatory cytokine levels and mortality in experimental sepsis. We studied the effect of ulinastatin on 28-day all-cause mortality in a double-blind trial in patients with severe sepsis in seven Indian hospitals. Methods Patients with sepsis were randomized within 48 h of onset of one or more organ failures to receive intravenous administration of ulinastatin (200,000 IU) or placebo 12 hourly for 5 days. Results Of 122 randomized subjects, 114 completed the study (55 receiving ulinastatin, 59 receiving placebo). At baseline, the mean APACHE II score was 13.4 (SD = 4.4), 48 (42 %) patients were receiving mechanical ventilation, 58 (51 %) were on vasopressors, and 35 % had multiple organ failure. In the modified intention-to-treat analysis (patients receiving six or more doses of study drugs), 28-day all-cause mortality was 7.3 % with ulinastatin (4 deaths) versus 20.3 % (12 deaths) with placebo ( p  = 0.045). On multivariate analysis too, treatment with ulinastatin (odds ratio 0.26, 95 % CI 0.07–0.95; p  = 0.042) independently decreased 28-day all-cause mortality. However, the mortality difference did not reach statistical significance in the intention-to-treat analysis [10.2 % (6/59 deaths) with ulinastatin versus 20.6 % (13/63 deaths) in the placebo group; p  = 0.11]. The ulinastatin group had lower incidence of new-onset organ failure (10 vs. 26 patients, p  = 0.003), more ventilator-free days (mean ± SD 19.4 ± 10.6 days vs. 10.2 ± 12.5 days, p  = 0.019), and shorter hospital stay (11.8 ± 7.1 days vs. 24.2 ± 7.2 days, p  < 0.001). Conclusions In this pilot study, intravenous administration of ulinastatin reduced mortality in patients with severe sepsis in the modified intention-to-treat analysis, but not in the intention-to-treat analysis.