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Haemonchus contortus, a blood-feeding nematode, inhibits blood coagulation at the site of infection to facilitate blood-sucking and digesting for successful parasitism. However, the mechanism underlying anti-coagulation at the host-parasite interface is largely unknown. In the current study, Hc-spi-i8, which has two greatly different transcripts named Hc-spi-i8a and Hc-spi-i8b, respectively, was described. Hc-SPI-I8A was a serine protease inhibitor containing a trypsin inhibitor-like cysteine rich (TIL) domain, while Hc-SPI-I8B was not. Hc-SPI-I8A/B were primarily expressed in the hypodermis, intestines and gonads in the parasitic stages of H. contortus. Hc-SPI-I8A interacted with Ovis aries TSP1-containing protein (OaTSP1CP), which was determined by yeast two-hybrid, co-immunoprecipitation (Co-IP), pull down and co-localization experiments. The blood clotting time contributed by the TIL domain was prolonged by Hc-SPI-I8A. Hc-SPI-I8A is most likely interfering in the extrinsic coagulation cascade by interacting with OaTSP1CP through its TIL domain and intrinsic coagulation cascade by an unknown mechanism. These findings depict a crucial point in the host-parasite interaction during H. contortus colonization, which should contribute to drug discovery and vaccine development in fighting against this important parasite worldwide.

The mechanisms enabling fish tapeworms to avoid proteolytic attacks by digestive enzymes of their fish host have been studied in less detail compared with mammalian cestodes. This study aimed to assess the inhibitory ability towards trypsin and chymotrypsin in Eubothrium rugosum, an intestinal parasite of burbot Lota lota, and establish its localization in the tapeworm. To this end, the worms were treated with Triton X-100 followed by differential centrifugation to isolate the tegumental brush border membrane. The protease inhibitory abilities of the worms were mostly determined by their excretory/secretory products released into the incubation medium. These inhibitory abilities proved to be linked mainly with the brush border fractions. Notably, the per cent inhibition of both studied digestive enzymes (trypsin and chymotrypsin) hardly depended on the duration of the parasite exposure in the incubation medium, probably due to intermittent glycocalyx renewal. Improved knowledge on functions of the excretory/secretory proteins produced by fish tapeworms may contribute to a better understanding of host-parasite relations and development of new approaches to the treatment and prevention of diseases caused by pathogenic helminths.

Trypsin is a protein-digesting enzyme that is essential for the growth and regeneration of bone, muscle, cartilage, skin, and blood. The trypsin inhibitors have various role in diseases such as inflammation, Alzheimer's disease, pancreatitis, rheumatoid arthritis, cancer prognosis, metastasis and so forth. From 10 endophytic fungi isolated, we were able to screen only one strain with the required activity. The fungus with activity was obtained as an endophyte from Dendrophthoe falcata and was later identified as Nigrospora sphaerica. The activity was checked by enzyme assays using trypsin. The fungus was fermented and the metabolites were extracted and further purified by bioassay-guided chromatographic methods and the compound isolated was identified using gas chromatography-mass spectrometry. The compound was identified as quercetin. Docking studies were employed to study the interaction. The absorption, distribution, metabolism, and excretion analysis showed satisfactory results and the compound has no AMES and hepatotoxicity. This study reveals the ability of N. sphaerica to produce bioactive compound quercetin has been identified as a potential candidate for trypsin inhibition. The present communication describes the first report claiming that N. sphaerica strain AVA-1 can produce quercetin and it can be considered as a sustainable source of trypsin active-site inhibitors.

Background Recent studies indicate that amylase-trypsin inhibitors (ATIs) and certain carbohydrates referred to as FODMAPs (fermentable oligo-, di-, monosaccharides and polyols) play an important role in promoting wheat sensitivity. Hitherto, no study has investigated the accumulation of ATIs during the development of the wheat caryopsis. We collected caryopses of common wheat cv. ‘Arnold’ at eight different grain developmental stages to study compositional changes in ATI and FODMAP content. Results The harvested caryopses were analysed for their size, protein and carbohydrate concentrations. ATIs were further characterized by MALDI-TOF MS, and their trypsin inhibition was evaluated by an enzymatic assay. The results showed that ATI accumulation started about 1 week after anthesis and subsequently increased steadily until physiological maturity. However, the biological activity of ATIs in terms of enzyme inhibition was not detectable before about 4 weeks after anthesis. Carbohydrate analysis revealed the abundance of short-chain fructans in early stages of grain development, whereas non-water-soluble carbohydrates increased during later developmental stages. Conclusions The results provide new insights into the complex metabolisms during grain filling and maturation, with particular emphasis on the ATI content as well as the inhibitory potential towards trypsin. The time lag between ATI accumulation and development of their biological activity is possibly attributed to the assembling of ATIs to dimers and tetramers, which seems to be crucial for their inhibitory potential.

Wheat amylase/trypsin inhibitors (ATIs) have gained significant relevance as inducers of intestinal and extra-intestinal inflammation. In this study, we present a novel hybrid data-independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS) approach, combining QconCAT technology with short microflow LC gradients and DIA and apply the method toward the quantitative proteome analysis of ATI extracts. The presented method is fast, robust, and reproducible and provides precise QconCAT-based absolute quantification of major ATI proteins while simultaneously quantifying the proteome by label-free quantification (LFQ). We analyzed extracts of 60 varieties of common wheat grown in replication and evaluated the reproducibility and precision of the workflow for the quantification of ATIs. Applying the method to analyze different wheat species (i.e., common wheat, spelt, durum wheat, emmer, and einkorn) and comparing the results to published data, we validated inter-laboratory and cross-methodology reproducibility of ATI quantification, which is essential in the context of large-scale breeding projects. Additionally, we applied our workflow to assess environmental effects on ATI expression, analyzing ATI content and proteome of same varieties grown at different locations. Finally, we explored the potential of combining QconCAT-based absolute quantification with DIA-based LFQ proteome analysis for the generation of new hypotheses or assay development.

This study explored the effects of lotus seedpod oligomeric procyanidins (LSOPC) and their main monomer catechin (CC) on the formation of advanced glycation end products (AGEs) and Caco-2 cytotoxicity during gastrointestinal digestion. Studies have found that LSOPC and CC inhibited the AGEs formation effectively in simulated gastrointestinal digestion and protected Caco-2 cells from AGEs attack. The effect of CC on the inhibition of AGEs formation was significantly better than that of LSOPC. Further, they could effectively inhibit the digestive enzyme activity, reactive oxygen species, RAGE-p38MAPK-NF-κB signaling pathway, inflammatory factors (tumor necrosis factor alpha, interleukin 6), and adhesion factors (intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1) to protect Caco-2 cells.

α-Amylase/trypsin inhibitors (ATIs) may have a role in nonceliac wheat sensitivity (NCWS) and celiac disease (CD), but the ATI content and diversity across a range of wheat cultivars are not well characterized. Discovery proteomics was used to detect ATIs across two wheat cultivars: Chara and Magenta. Comprehensive mapping of detected ATIs with the ATIs from the recently published wheat genome RefSeq v1.0 shows the presence of three major subclasses: monomeric (9%), dimeric (61%), and chloroform-methanol (CM) type (30%). Subsequently, the level of 18 ATI isoforms (63 peptides) grouped into four subtypes was monitored across 15 commercial wheat cultivars and the eight parental lines from a multiparent advanced-generation intercross (MAGIC) population using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS). The ATI content of wheat cultivars Janz, Sunvale, Diamond Bird, and Longreach Scout was significantly lower than that of other wheat cultivars. The MAGIC parental cultivars Baxter and Xiaoyan 54 contain higher levels (∼115% relative to the average wheat ATI content), whereas cultivar Pastor contained the lowest levels (∼87%). Comprehensive sequence analysis, annotation, chromosomal locations, and epitope mapping enabled us to build an LC-MRM-MS method to monitor and quantify the immunostimulatory ATI proteins potentially related to NCWS, autoimmune diseases, and metabolic disorders. This provides an opportunity to select wheat cultivars with significantly lower levels of ATIs.

Although digestive resistance of Kunitz protease inhibitors has been reported extensively, the molecular mechanism is not well established. In the present study, the first X-ray structure of Cassia obtusifolia trypsin inhibitor (COTI), a member of Kunitz protease inhibitors, was solved at a resolution of 1.9 Å. The structure adopted a classic β-trefoil fold with the inhibitory loop protruding from the hydrophobic core. The role of Phe139, a unique residue in Kunitz protease inhibitors, and Arg63 in the COTI structure was verified by F139A and R63E mutants. COTI was a specific inhibitor of bovine trypsin and the result was also verified by COTI-trypsin complex formation. Meanwhile, COTI showed equivalent inhibitory activity with that of soybean trypsin inhibitor against bovine trypsin and midgut trypsin from Pieris rapae. The F139 and R63E mutants further indicated that inhibitory specificity and efficiency of COTI were closely related to the global framework, the conformation and the amino acid composition of reactive loop. Finally, a midgut trypsin from P. rapae (PrSP40), which might be involve in the food digestion, was proposed to be a potential target of COTI and might be a promising target for future crop-protection strategy. The results supported the digestive resistance of COTI.

A novel thermostable trypsin inhibitor was obtained from Geoffroea decorticans seeds. G. decorticans trypsin inhibitor (GdTI) is a protein with molecular mass of 6743.7 Da, with a potent inhibitory activity (Ki of 2.1 nM) even at high temperatures and extreme pHs (100% after 5 h at 100 °C and 80% after 60 min at pH 2–12) constituting one of the most powerful serine protease inhibitors isolated from a plant source. GdTI displays anticoagulant activity against both extrinsic and intrinsic coagulation pathways, representing the first report of a plant serine protease inhibitor with anticoagulant activity against the extrinsic pathway. Finally, GdTI showed inhibitory activity against α-glucosidase (IC50 of 0.18 μM) evidencing the hypoglycemic effect of this inhibitor. Our results evidence the discovery of a natural molecule with unique features: i) GdTI is one of the most potent serine protease inhibitors founded to date, ii) with the most powerful thermostability reported in literature, iii) with anticoagulant effect against both coagulation pathways and hypoglycemic activity. This report suggest that GdTI could be exploited as a natural and hyperstable antidiabetic drug, in behalf of its antithrombotic and hypoglycemic activities, encouraging future studies with high impact on biomedical research and potential pharmaceutical applications.

Amylase/trypsin inhibitors (ATIs) are widely consumed in cereal-based foods and have been implicated in adverse reactions to wheat exposure, such as respiratory and food allergy, and intestinal responses associated with coeliac disease and non-coeliac wheat sensitivity. ATIs occur in multiple isoforms which differ in the amounts present in different types of wheat (including ancient and modern ones). Measuring ATIs and their isoforms is an analytical challenge as is their isolation for use in studies addressing their potential effects on the human body. ATI isoforms differ in their spectrum of bioactive effects in the human gastrointestinal (GI), which may include enzyme inhibition, inflammation and immune responses and of which much is not known. Similarly, although modifications during food processing (exposure to heat, moisture, salt, acid, fermentation) may affect their structure and activity as shown in vitro, it is important to relate these changes to effects that may present in the GI tract. Finally, much of our knowledge of their potential biological effects is based on studies in vitro and in animal models. Validation by human studies using processed foods as commonly consumed is warranted. We conclude that more detailed understanding of these factors may allow the effects of ATIs on human health to be better understood and when possible, to be ameliorated, for example by innovative food processing. We therefore review in short our current knowledge of these proteins, focusing on features which relate to their biological activity and identifying gaps in our knowledge and research priorities.

Erythrina poeppigiana belongs to Fabaceae family (subfamily Papillionoideae) and is commonly found in tropical and subtropical regions in Brazil. Herein, we described the purification and characterization of a new Kunitz-type inhibitor, obtained from E. poeppigiana seeds (EpTI). EpTI is composed by three isoforms of identical amino-terminal sequences with a molecular weight ranging from 17 to 20 kDa. The physicochemical features showed by EpTI are common to Kunitz inhibitors, including the dissociation constant (13.1 nM), stability against thermal (37–100 °C) and pH (2–10) ranging, and the presence of disulfide bonds stabilizing its reactive site. Furthermore, we investigated the antimicrobial, anti-adhesion, and anti-biofilm properties of EpTI against Gram-positive and negative bacteria. The inhibitor showed antimicrobial activity with a minimum inhibitory concentration (MIC, 5–10 µM) and minimum bactericidal concentration (MBC) of 10 µM for Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus haemolyticus. The combination of EpTI with ciprofloxacin showed a marked synergistic effect, reducing the antibiotic concentration by 150%. The increase in crystal violet uptake for S. aureus and K. pneumoniae strains was approximately 30% and 50%, respectively, suggesting that the bacteria plasma membrane is targeted by EpTI. Treatment with EpTI at 1x and 10 x MIC significantly reduced the biofilm formation and prompted the disruption of a mature biofilm. At MIC/2, EpTI decreased the bacterial adhesion to polystyrene surface within 2 h. Finally, EpTI showed low toxicity in animal model Galleria mellonella. Given its antimicrobial and anti-biofilm properties, the EpTI sequence might be used to design novel drug prototypes.

The primary objective of the current study was to evaluate the impact of trypsin inhibitor (TI) and exogenous protease supplementation on endogenous loss of amino acids (AA) in broiler chickens. A total of 384 Cobb-500 broiler chicks were allocated to 4 nitrogen-free diets, each with 8 replicate cages and 12 birds per replicate. The diets were arranged as a 2 × 2 factorial with factors being dietary TI (0 or 8,000 TIU/g) and exogenous protease (0 or 15,000 PROT/kg). Desired dietary TI concentration was achieved by addition of commercially available, purified soybean TI. There was no effect of TI or exogenous protease or their interaction on growth performance of birds. However, the endogenous loss of nitrogen (N) and all AA increased (P < 0.05) due to dietary TI concentration except for Cys. The increase in endogenous AA due to TI ranged from 17% for Thr to 52.2% for Trp. Exogenous protease had no effect on endogenous loss of N and all AA. There was no effect of TI or exogenous protease or their interaction on the AID of P, however AID of Ca, Fe, Mg, Mn, and Cu was reduced (P < 0.05) due to dietary TI. The AID of Cu (P < 0.01) and K (P < 0.05) improved with exogenous protease supplementation. Significant interactions (P < 0.05) between exogenous protease and TI existed for Zn, Mg, Cu, and Na. The concentration (g/kg DM intake) of crude mucin and sialic acid increased (P < 0.05) with increased dietary TI. Decreased trypsin (P < 0.001) and increased chymotrypsin (P < 0.001) activity in the pancreas were observed as a result of exogenous protease supplementation. In conclusion, the current study showed that TI increases the endogenous loss of AA and reduced the digestibility of minerals in broiler chickens. Furthermore, exogenous protease did not affect endogenous AA flow, irrespective of added purified dietary TI.

Kazal-type protease inhibitor specificity is believed to be determined by sequence of the reactive-site loop that make most, if not all, contacts with the serine protease. Here, we determined the complex crystal structure of Aedes aegypti trypsin inhibitor (AaTI) with μ-plasmin, and compared its reactivities with other Kazal-type inhibitors, infestin-1 and infestin-4. We show that the shortened 99-loop of plasmin creates an S2 pocket, which is filled by phenylalanine at the P2 position of the reactive-site loop of infestin-4. In contrast, AaTI and infestin-1 retain a proline at P2, rendering the S2 pocket unfilled, which leads to lower plasmin inhibitions. Furthermore, the protein scaffold of AaTI is unstable, due to an elongated Cys-V to Cys-VI region leading to a less compact hydrophobic core. Chimeric study shows that the stability of the scaffold can be modified by swapping of this Cys-V to Cys-VI region between AaTI and infestin-4. The scaffold instability causes steric clashing of the bulky P2 residue, leading to significantly reduced inhibition of plasmin by AaTI or infestin-4 chimera. Our findings suggest that surface loops of protease and scaffold stability of Kazal-type inhibitor are both necessary for specific protease inhibition, in addition to reactive site loop sequence. PDB ID code: 7E50.

Aims This study aimed to quantify α-amylase/trypsin inhibitor (ATI) CM3 and glutathione (GSH) during wheat sourdough breadmaking. Methods and results Breads were made with two wheat cultivars and fermented with Fructilactobacillus sanfranciscensis, F. sanfranciscensis ΔgshR or Latilactobacillus sakei; chemically acidified and straight doughs served as controls. Samples were analysed after mixing, after proofing and after baking. GSH and CM3 were quantified by multi-reaction-monitoring-based methods on an LC-QTRAP mass spectrometer. Undigested ATI extracts were further examined by SDS-PAGE. Conclusions GSH abundance was similar after mixing and after proofing but increased after baking (p < 0.001), regardless of fermentation. In breads baked with cv. Brennan, the samples fermented with lactobacilli had higher GSH abundance (p < 0.001) than in the controls. CM3 relative abundance remained similar after mixing and after proofing but decreased after baking (p < 0.001) across all treatments. This trend was supported by the SDS-PAGE analysis in which ATI band intensities decreased after baking (p < 0.001) in all experimental conditions. The overall effect of baking exerted a greater effect on the abundances of GSH and CM3 than fermentation conditions. Significance and Impact of the Study This is the first report to quantify ATI over the course of breadmaking by LC-MS/MS in sourdough and straight dough processes.

The present study investigated the effect of varying trypsin inhibitor activity (TIA) in differently processed soybean expellers on apparent prececal amino acid (AA) digestibility in male broiler chickens. Two different raw soybean batches were treated using varying processing techniques and intensities. In this way, 45 expeller extracted soybean meal (ESBM) variants were created. The processed soybean variants were then merged into a basal diet (160 g/kg crude protein [CP]) at 2 inclusion levels (15%, 30%) resulting in 90 different diets plus one basal diet (0.4 mg/g-8.5 mg/g TIA). All diets contained 0.5% titanium dioxide. A total of 5,460-day-old male broilers (Ross 308) were allocated on d 14 to 546 pens (10 birds/pen) after a starter phase (CP 215 g/kg, 14 g/kg Lysine, 12.5 MJ ME/kg). The 91 experimental diets were fed ad libitum until d 22. Subsequently, birds were euthanized and digesta of the terminal ileum was collected for determination of AA digestibility. TIA depressed the prececal digestibility of every single AA significantly in a straight linear fashion (P0.001). Sulfur-containing AA expressed the strongest suppression by TIA with cystine showing the lowest apparent prececal digestibility measured (10.6% at 23.6 mg/g TIA in raw ESBM). The present data demonstrate that TIA severely depresses digestibility of essential and nonessential AA in a straight linear fashion. On the one hand, this questions the usefulness of defined upper limits of TIA in soy products whereas on the other hand, TIA must be considered when testing raw components for their feed protein value in vivo.

Several studies have sought new therapies for obesity and liver diseases. This study investigated the effect of the trypsin inhibitor isolated from tamarind seeds (TTI), nanoencapsulated in chitosan and whey protein isolate (ECW), on the liver health status of the Wistar rats fed with a high glycemic index (HGLI) diet. The nanoformulations without TTI (CW) and ECW were obtained by nanoprecipitation technique, physically and chemically characterized, and then administered to the animals. The adult male Wistar rats (n = 20) were allocated to four groups: HGLI diet + water, standard diet + water, HGLI diet + ECW (12.5 mg/kg), and HGLI diet + CW (10.0 mg/kg), 1 mL per gagave, for ten days. They were evaluated using biochemical and hematological parameters, Fibrosis-4 Index for Liver Fibrosis (FIB-4), AST to Platelet Ratio Index (APRI) scores, and liver morphology. Both nanoparticles presented spherical shape, smooth surface, and nanometric size [120.7 nm (ECW) and 136.4 nm (CW)]. In animals, ECW reduced (p &lt, 0.05) blood glucose (17%), glutamic oxalacetic transaminase (39%), and alkaline phosphatase (24%). Besides, ECW reduced (p &lt, 0.05) APRI and FIB-4 scores and presented a better aspect of hepatic morphology. ECW promoted benefits over a liver injury caused by the HGLI diet.

Background/aim We developed an experimental method to reproduce insulin secretion from isolated rat pancreas preparations using an organ bath system. However, secretion of trypsin, another pancreatic enzyme, interferes with insulin production in such systems. We aimed to ascertain the minimum trypsin inhibitor (TI), dose for obtaining a sustained, stable rate of insulin secretion. Materials and methods The action of TI (1-10 μg/ml) on pancreatic preparations of male Wistar-Imamichi rats in organ bath experiments was assessed by measuring insulin, amylase, and trypsin activity. Results The level of insulin outflow remained steady in the TI-treated samples, in contrast to that in the untreated control, where insulin secretion decreased over time. The level of amylase outflow did not change significantly. Trypsin activity was significantly lower in the TI-treated samples than in the control. Conclusion Even low concentrations of TI can maintain insulin secretion by inhibiting trypsin activity in organ bath experiments.

Peptidase inhibitors (PIs) are defense proteins of plants which are active against gut peptidases of different insects. Sapindus mukorossi was identified as a source of bioactive PIs which could confer resistance against Bactrocera cucurbitae, a most devastating pest of several economically important crops. In the present study, a trypsin inhibitor was purified from mature dry seeds of S. mukorossi and characterized for its biochemical properties as well as its potential for bio control of B. cucurbitae. The purified fractions from RP- HPLC through SDS-PAGE gave an apparent molecular weight of ~29 kDa. S. mukorossi trypsin inhibitor (SMTI) was found to be a non-competitive inhibitor which was active over a broad range of temperature (10–100 °C) and pH (6–11). SMTI when incorporated in artificial diet inhibited the growth and development of B. cucurbitae larvae. Gene expression analysis of trypsin and chymotrypsin genes via qRT-PCR indicated that their mRNA expression was down-regulated while that of other genes namely, Catalase, Elastase, Superoxide Dismutase, Glutathione –S-transferase and Alkaline Phosphatase was up regulated. SMTI also showed deleterious effects against different bacterial strains. The results of this study indicated that S. mukorossi trypsin inhibitor has potential to be used as a bio control agent that can reduce the harm caused by melon fruit fly and other devastating pests.

A novel chymotrypsin inhibitor, named ClCI, was purified from coix seed (Coix lacryma-jobi L.) by aqueous two-phase extraction, chymotrypsin–Sepharose 4B affinity chromatography and centrifugal ultrafiltration. ClCI was a 7.9 kDa competitive inhibitor with pI 6.54. The inhibition constants (Ki) for bovine pancreatic chymotrypsin and bacterial subtilisin were 1.27 × 10−10 M and 1.57 × 10−9 M respectively. ClCI had no inhibitory activity against bovine trypsin and porcine elastase. ClCI had wide pH stability and good heat resistance. It can maintain >90% inhibition activity against chymotrypsin at 20–80 °C for 1 h. The primary structure of ClCI was highly similar (57%–92%) to those of several inhibitors belonging to the Gramineae crop potato protease inhibitor- I superfamily and showed the typical sequence motif of the protease inhibitor of the seed storage protein group. ClCI (12.5 mg) inhibited mycelial growth of the phytopathogenic fungi Mycosphaerella melonis, Helminthosporium turcicum, Alternaria solani, Phytophthora capsici, Isariopsis griseola, and Colletotrichum gloeosporioides, and caused 89% inhibition of the proteases from spore germination of plant-pathogenic fungi. The results of the present study indicate that ClCI had biotechnological potential as an alternative agent to combat the important phytopathogenic fungi.

Acute glucose fluctuation during the postprandial period causes a risk for type 2 diabetes mellitus (T2DM). α-Glucosidase inhibitors have been approved as therapeutic agents for diabetes. In the present study, a protein with α-glucosidase inhibitory activity from Flax (Linum usitatissimum) seeds was isolated using a one-step purification with Q-Sepharose4B column, followed by Sephacryl S-200 size-exclusion chromatography. It was identified as a trypsin inhibitor, named L. usitatissimum trypsin inhibitor (LUTI). The half maximal inhibitory concentration (IC50) of LUTI was 113.92 μM for α-glucosidase and 6.17 μM for trypsin. Lineweaver-Burk kinetic experiment showed that the protein exhibited two distinct inhibitory modes, a competitive inhibitor type for α-glucosidase and a non-competitive type for trypsin. The interaction between LUTI and α-glucosidase was detected through gel filtration chromatography and dynamic light scattering. Increased glucose consumption and lactic acid production were also observed following LUTI treatment in Caco-2 and HepG2 cells. LUTI inhibits not only the activity of trypsin but also the activity of α-glucosidase. It is expected that LUTI will become an oral hypoglycemic polypeptide drug for T2DM.

Subtilisin-like proteases play crucial roles in host-pathogen interactions. Thus, protease inhibitors constitute important tools in the regulation of this interaction. CmPI-II is a Kazal proteinase inhibitor isolated from Cenchritis muricatus that inhibits subtilisin A, trypsin and elastases. Based on sequence analysis it defines a new group of non-classical Kazal inhibitors. Lacking solved 3D structures from this group prevents the straightforward structural comparison with other Kazal inhibitors. The 3D structure of CmPI-II, solved in this work using NMR techniques, shows the typical fold of Kazal inhibitors, but has significant differences in its N-terminal moiety, the disposition of the CysI-CysV disulfide bond and the reactive site loop (RSL) conformation. The high flexibility of its N-terminal region, the RSL, and the α-helix observed in NMR experiments and molecular dynamics simulations, suggest a coupled motion of these regions that could explain CmPI-II broad specificity. The 3D structure of the CmPI-II/subtilisin A complex, obtained by modeling, allows understanding of the energetic basis of the subtilisin A inhibition. The residues at the P2 and P2′ positions of the inhibitor RSL were predicted to be major contributors to the binding free energy of the complex, rather than those at the P1 position. Site directed mutagenesis experiments confirmed the Trp14 (P2′) contribution to CmPI-II/subtilisin A complex formation. Overall, this work provides the structural determinants for the subtilisin A inhibition by CmPI-II and allows the designing of more specific and potent molecules. In addition, the 3D structure obtained supports the existence of a new group in non-classical Kazal inhibitors.

Momordica trypsin inhibitors (TIs) such as those isolated from the seeds of the gấc fruit, Momordica cochinchinensis (MCoTI-I and MCoTI-II), are widely used as scaffolds for drug design studies. To more effectively exploit these molecules in the development of therapeutics, there is a need for wider discovery of the natural sequence diversity among TIs from other species in the Momordica subfamily. Here we report the discovery of the encoding gene and six TIs from the seeds of the spiny gourd, Momordica dioica, four of which possess novel sequences (Modi 1, 3, 5, and 6) and two (Modi 2 and 4) of which are known peptides (TI-14, TI-17) previously identified in Momordica subangulata. Modi 6 is an acyclic peptide featuring a pyrrolidone carboxylic acid modification, whereas the remaining five TIs are cyclic. All Modi peptides display similar overall structures and trypsin inhibitory activities. No toxicity was observed for these peptides when tested against cancer and insect cells. All Modi peptides were exceptionally stable over 24 h in human serum, indicating a dual strategy to stabilize the peptides in nature, either head-to-tail cyclization or N-pyrolation, which suggests these peptides might be excellent candidates as scaffolds for epitope stabilization in drug design studies.

A Bowman–Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purified and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that differentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman–Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1–10) and temperatures (10–100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains β sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) values (16.6 × 10−8 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high affinity towards trypsin with dissociation constant (Kd) 2.28 µM.

Key message Wheat cultivars largely differ in the content and composition of ATI proteins, but heritability was quite low for six out of eight ATIs. The genetic architecture of ATI proteins is built up of few major and numerous small effect QTL. Abstract Amylase trypsin inhibitors (ATIs) are important allergens in baker’s asthma and suspected triggers of non-celiac wheat sensitivity (NCWS) inducing intestinal and extra-intestinal inflammation. As studies on the expression and genetic architecture of ATI proteins in wheat are lacking, we evaluated 149 European old and modern bread wheat cultivars grown at three different field locations for their content of eight ATI proteins. Large differences in the content and composition of ATIs in the different cultivars were identified ranging from 3.76 pmol for ATI CM2 to 80.4 pmol for ATI 0.19, with up to 2.5-fold variation in CM-type and up to sixfold variation in mono/dimeric ATIs. Generally, heritability estimates were low except for ATI 0.28 and ATI CM2. ATI protein content showed a low correlation with quality traits commonly analyzed in wheat breeding. Similarly, no trends were found regarding ATI content in wheat cultivars originating from numerous countries and decades of breeding history. Genome-wide association mapping revealed a complex genetic architecture built of many small, few medium and two major quantitative trait loci (QTL). The major QTL were located on chromosomes 3B for ATI 0.19-like and 6B for ATI 0.28, explaining 70.6 and 68.7% of the genotypic variance, respectively. Within close physical proximity to the medium and major QTL, we identified eight potential candidate genes on the wheat reference genome encoding structurally related lipid transfer proteins. Consequently, selection and breeding of wheat cultivars with low ATI protein amounts appear difficult requiring other strategies to reduce ATI content in wheat products.

© 2021 by the authors., The common bean (Phaseolus vulgaris L.) represents a sustainable and affordable source of protein, namely, to populations with vegetarian dietary habits. Despite the national germplasm genetic diversity, little is known about the Portuguese accessions’ nutritional and protein quality, leading to their underuse in breeding programs. To fill this gap, a representative collection (106 accessions) was cropped under two contrasting environments (traditional versus heat stress) and evaluated in terms of nutritional quality by near-infrared spectroscopy. Protein quality was assessed, under the stressful environment, considering the individual amino acid contents and the activity of trypsin inhibitors through mass spectrometry (LC-MS/MS) and spectrophotometry, respectively. On top of strong genotypic control, the nutritional composition (protein, fat, fiber, moisture and ash) was also highly influenced by the environment and by genotype × environment interaction, with a clear nutritional quality ranking change for the accessions in heat stress conditions. Classified into three clusters, the accessions from the cluster with the highest individual amino acid and protein contents also showed higher trypsin inhibitor activity (TIA). Since different levels of TIA had no translation into contrasting protein digestibility, breeders focusing on common beans’ protein quality improvement, especially under challenging warming climate conditions, may take advantage of this group of accessions., This research was funded by FCT, Portugal, through BEGEQA project (PTDC/AGRTEC/3555/2012), E.M. PhD fellowship (SFRH/BD/89287/2012 and R&D unit, UIDB/04551/2020 (GREEN-IT–Bioresources for sustainability), PORTUGAL 2020, grant number LISBOA-01-0145- FEDER-402-022125 and PDR2020 Operação 7.8.4, Recursos genéticos (PDR2020-784-042734).

The main anti-nutritive substances present in rye grains include alkylresorcinols, water-soluble pentosans, and trypsin inhibitors. The content of these compounds in the grains can be influenced by genetic factors, habitat conditions and the crop management technology used in cultivation. The aim of the study was to determine the variability of the concentration of anti-nutritive compounds in rye grains depending on the variety, weather conditions and production technology. The field research was conducted at the IUNG-PIB Experimental Station in Osiny (Poland) in three growing seasons 2010/2011, 2011/2012 and 2012/2013. The experiment was located on lessive soil, on plots of 45 m2 in three replications. The first factor was the type of production technology (integrated and intensive), while the second was a variety of winter rye. Two hybrid varieties (Brasetto, Visello) and two population varieties (Dańkowskie Diament, Kier) were included. The research showed that the content of anti-nutritive compounds in rye grains depended significantly on the genotype and weather conditions. Stress conditions during the rye growing season in 2011 and 2013 were the cause of higher synthesis of alkylresorcinols, soluble pentosans and trypsin inhibitors in rye grains. The content of alkylresorcinols and water-soluble pentosans also depended significantly on the intensity of the production technology. The higher content of these compounds was found in rye grains from intensive technology. The production technology had no significant impact on the activity of trypsin inhibitors.

Funding Information: We would like to thank A. Löfhjelm and L. Saari for excellent technical assistance. This work was supported by a Sigrid Jusélius Foundation grant to H.K. and the Academy of Finland funding (321809) to T.S. We would also like to thank the Erkko Foundation and Nordforsk Nordic center of Excellency NordAqua (project number #82845) and University of Helsinki’s Doctoral Programme in Microbiology and Biotechnology funding to M.N.A. D.O.A. was supported by a postdoctoral research fellowship from the São Paulo Research Foundation (FAPESP #2018/01563-2). We thank Biocenter Kuopio for the use of their facilities for molecular modeling and MD simulations. We thank the DNA Sequencing and Genomics laboratory, Institute of Biotechnology, University of Helsinki for assistance in sequencing, assembly, and annotation of DNA. We thank the Language Center of University of Helsinki for their help in language revisions. Publisher Copyright: © Serine proteases regulate many physiological processes and play a key role in a variety of cancers. Aeruginosins are a family of natural products produced by cyanobacteria that exhibit pronounced structural diversity and potent serine protease inhibition. Here, we sequenced the complete genome of Nodularia sphaerocarpa UHCC 0038 and identified the 43.7 kb suomilide biosynthetic gene cluster. Bioinformatic analysis demonstrated that suomilide belongs to the aeruginosin family of natural products. We identified 103 complete aeruginosin biosynthetic gene clusters from 12 cyanobacterial genera and showed that they encode an unexpected chemical diversity. Surprisingly, purified suomilide inhibited human trypsin-2 and -3, with IC50 values of 4.7 and 11.5 nM, respectively, while trypsin-1 was inhibited with an IC50 of 104 nM. Molecular dynamics simulations suggested that suomilide has a long residence time when bound to trypsins. This was confirmed experimentally for trypsin-1 and -3 (residence times of 1.5 and 57 min, respectively). Suomilide also inhibited the invasion of aggressive and metastatic PC-3M prostate cancer cells without affecting cell proliferation. The potent inhibition of trypsin-3, together with a long residence time and the ability to inhibit prostate cancer cell invasion, makes suomilide an attractive drug lead for targeting cancers that overexpress trypsin-3. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and suggest that aeruginosins may be a source of selective inhibitors of human serine proteases.

Nutritional properties of inulin-enriched tofu obtained after hydrothermal cooking of soymilk, using chymosin-pepsin rennet and inulin as a functional ingredient, were assessed. This procedure significantly differs from the traditional one. The residual activity (rTIA) of trypsin inhibitors (TIs) and lectins, content of proteins, carbohydrates, fats and their energy values (EV) were suitable for human nutrition. Inulin-enriched tofu was characterised with low rTIA (3.08–5.71%) and TIs content of 3.62–18.99%. Content of Kunitz and Bowman-Birk TIs as well as total TIs content (r = 0.98) in tofu were strongly correlated with tofu protein content. Content of Bowman-Birk polymeric forms (3.11–5.36%) was higher than Bowman-Birk monomeric forms (0.51–2.31%) in inulin-enriched tofu. Low urease activity (0.60–1.78%) indicated that soybean was heated adequately to inactivate TIs. Increasing content of inulin did not increase tofu EV (~18 kJ per g tofu). The proximate composition of inulin-enriched tofu, advantageous rTIA and a very low EV qualifies this product for human nutrition.

BACKGROUND Lignin extracted from Canna edulis Ker residues shows a strong inhibitory effect on α-glucosidase and a promoting effect on α-amylase. Protease activity inhibition may play a key role in disease processes, such as metastasis, tumor invasion and bacterial colonization. Hence, in the present study, the inhibitory mechanism of lignin on trypsin was examined, including the interaction type, thermodynamic parameters, structure, reaction site and molecular docking. RESULTS The isolated lignin presented an inhibitory effect on trypsin activity with an IC50 value of 1.35 μmol L-1 . This inhibition was a mixed linear type with a constant Ki of 3.92 μmol L-1 . The lignin could bind with the key amino acid residue Ser195 on the active site of the trypsin molecule to inhibit its activity, and the phenolic hydroxyl group and -OH on the β-O-4 structure of the lignin molecule were the major groups bound with trypsin. CONCLUSION These results illustrate the inhibitory effects of Canna edulis residue lignin on protease, which helps with respect to understanding the possible application of lignin in the food industry in functional foods. © 2020 Society of Chemical Industry.

Natural product extracts present inherently complex matrices in which the identification of novel bioactive peptide species is challenged by low abundance masses and significant structural and sequence diversity. Additionally, discovery efforts often result in the re-identification of known compounds, where modifications derived in vivo or during sample handling may obscure true sequence identity. Herein, we identify mass spectral (MS(2)) “fingerprint” ions characteristic of cyclotides, a diverse and biologically active family of botanical cysteine-rich peptides, based on regions of high sequence homology. We couple mass shift analysis with MS(2) spectral fingerprint ions cross referenced with CyBase - a cyclotide database - to discern unique mass species in Viola communis extracts from mass species that are likely already characterized and those with common modifications. The approach is extended to a related class of cysteine-rich peptide, the trypsin inhibitors, using the characterized botanical species Lagenaria siceraria. Coupling the observation of highly abundant MS(2) ions with mass shift analysis, we identify a new set of small, highly disulfide bound cysteine-rich L. siceraria peptides.

The widespread commercialization of genetically modified (GM) cotton makes it important to assess the potential impact of this recombinant crop on non-target organisms. As important natural enemies of cotton field predators, green lacewing Chrysoperla sinica larvae are exposed to Bt insecticidal proteins expressed by GM cotton by feeding on herbivorous pests, and adults are directly exposed to Bt proteins by cotton pollen consumption. However, potential impacts of transgenic Bt cotton on C. sinica remain unclear. In this study, we evaluated the effects of two transgenic cotton varieties, CCRI41 and CCRI45, which express Cry1Ac (Bt toxin) and CpTI (Cowpea Trypsin Inhibitor), on C. sinica larvae and adults. After being fed with cotton aphids Aphis gossypii reared on transgenic cotton, the survival rate, developmental duration, pupation rate, and emergence rate of larvae were not adversely affected. After being fed two types of transgenic cotton pollen, the 7-day weight of adults and the preoviposition period and the cumulative oviposition of females were not significantly different from control specimen. Taken together, these results indicate that the potential risks of the two tested GM cotton varieties for the predator C. sinica are negligible. Capsule: Our study indicated that GM cotton varieties CCRI41 and CCRI45 have no adverse effects on insect predator C. sinica.

A range of guanidine-based pyridines, and related compounds, have been prepared (19 examples). These compounds were evaluated in relation to their competitive inhibition of bovine pancreatic trypsin. Results demonstrate that compounds in which the guanidinyl substituent can form an intramolecular hydrogen bond (IMHB) with the pyridinyl nitrogen atom (6a–p) are better trypsin inhibitors than their counterparts (10–13) that are unable to form an IMHB. Among the compounds 6a–p, examples containing a 5-halo substituent were, generally, found to be better trypsin inhibitors. This trend was inversely related to electronegativity, thus, 1-(5-iodopyridin-2-yl)guanidinium ion 6e ( K i = 0.0151 mM) was the optimum inhibitor in the 5-halo series. Amongst the isomeric methyl substituted compounds, 1-(3-methylpyridin-2-yl)guanidinium ion 6h demonstrated optimum levels of trypsin inhibition ( K i = 0.0140 mM). In order to rationalise the measured enzyme inhibition, selected compounds were docked with bovine and human trypsin with a view to understanding active site occupancy and taken together with the K i values the order of inhibitory ability suggests that the 5-halo 2-guanidinyl pyridine inhibitors form a halogen bond with the catalytically active serine hydroxy group.

The economic loss in soybean crops caused by the Lepidoptera insects has encouraged the search for new strategies to control this pest, which are currently based on synthetic insecticides. This paper evaluated the ability of ApTI (Adenanthera pavonina trypsin inhibitor) to inhibit trypsin-like proteins from Anticarsia gemmatalis by docking, molecular dynamics, and enzymatic and survival assay. The docking and molecular dynamic simulation between trypsin and ApTI were performed using the program CLUSPRO and NAMD, respectively. The inhibitory constant Ki and the inhibition type were determined through chromogenic assays. The survival assay of neonatal larvae under treatment with artificial diet supplemented with ApTI was also performed. The ApTI binding site was predicted to block substrate access to trypsin due to four interactions with the enzyme, producing a complex with a surface area of 1,183.7 A2 . The kinetic analysis revealed a noncompetitive tight-binding mechanism. The survival curves obtained using Kaplan-Meier estimators indicated that the highest larvae mortality was 60%, using 1.2 mg of ApTI per 100 ml of artificial diet. The in vitro, in vivo, and in silico studies demonstrated that ApTI is a strong noncompetitive inhibitor of trypsin with biotechnological potential for the control of A. gemmatalis insect.

Peptidase inhibitors (PIs) are plant proteins that are found to be effective against various digestive peptidases of insects. The present study isolated and characterized a trypsin inhibitor from mature dry seeds of Mucuna pruriens and investigated its effect against Bactrocera cucurbitae larvae, a major pest of cucurbitaceae crops, for its inhibitory activity. The purified trypsin inhibitor from M. pruriens seeds gave a molecular weight of ~11 kDa on SDS-PAGE. M. pruriens trypsin inhibitor (MPTI) exhibited inhibitory effect on growth of melon fruit fly larvae (64-72 h old) as it resulted in prolongation of larval, pupal and total development period. There was a significant increase in larval mortality with increase in concentration of MPTI. Nutritional indices decreased significantly at all the concentrations of MPTI. Quantitative RT- PCR revealed that the mRNA expression level of trypsin and chymotrypsin genes was reduced while that of GST, esterases, AP, SOD and catalase were enhanced. It can therefore be inferred that MPTI can serve as a promising agent for biocontrol that can reduce the problems caused by fruit fly and other similar catastrophic pests. This study provides the fundamental information for future successful strategies for pest management.

The common bean is an important caloric-protein food source. However, its nutritional value may be affected by the presence of non-nutritional compounds, which decrease the assimilation of some nutrients, however, at low concentrations, they show a beneficial effect. Germination and treatment by controlled pressure-drop (DIC, French acronym of D&eacute, tente Instantan&eacute, e Contr&ocirc, l&eacute, e) are methods that modify the concentration of these components. The objective of this work was to evaluate the change in the non-nutritional composition of bean seeds and sprouts by DIC treatment. The results show that with the germination, the concentration of phenolic and tannin compounds increased 99% and 73%, respectively, as well as the quantity of saponins (65.7%), while phytates and trypsin inhibitors decreased 26% and 42%, respectively. When applying the DIC treatment, the content of phytates (23&ndash, 29%), saponins (44%) and oligosaccharides increased in bean sprouts and decreased phenolic compounds (4&ndash, 14%), tannins (23% to 72%), and trypsin inhibitors (95.5%), according to the pressure and time conditions applied. This technology opens the way to new perspectives, especially to more effective use of legumes as a source of vegetable protein or bioactive compounds.

Background Damage-associated molecular patterns (DAMPs) generated by major surgery can induce global inflammation response and may degrade the vascular endothelial glycocalyx layer (EGL); in turn, the resulting EGL fragments can act as DAMPs, in a destructive positive feedback loop, to promote exacerbation of inflammation. Ulinastatin (UTI) may attenuate EGL shedding by inhibiting serine proteases and hyaluronidase. Objective This trail evaluates whether EGL shedding elicited by Traditional Whipple Procedure (TWP) could be decreased by using UTI. Methods We divided 60 patients undergoing TWP into a control group and a UTI group (n = 30 for both). Blood samples were collected before (T0), near the end (T1), and 1 hour after (T2) surgery. Levels of syndecan-1, ICAM-1, VCAM-1, IL-6, C-reactive protein, thrombomodulin, Hbg and serum albumin were measured and plasma albumin leakage was estimated. Results IL-6 levels significantly elevated at T1 and T2 in the control group compared with T0, but not the UTI group. Syndecan-1 levels significantly elevated at T1 and T2 in the control group but only T2 in the UTI group compared with T0. Conclusions We found global inflammation reaction and EGL degradation during TWP. Perioperative UTI treatment can attenuate this EGL shedding and might alleviate plasma albumin leakage.

Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesotrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymotrypsin C (CTRC) mitigates the harmful effects of mesotrypsin by cleaving the autolysis loop. As human trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesotrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in K(m), it digested β-casein poorly and bound soybean trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesotrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesotrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesotrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic trypsin activity.

BackgroundWitches’ broom disease (WBD) of cacao (Theobroma cacaoL.), caused byMoniliophthora perniciosa, is the most important limiting factor for the cacao production in Brazil. Hence, the development of cacao genotypes with durable resistance is the key challenge for control the disease. Proteomic methods are often used to study the interactions between hosts and pathogens, therefore helping classical plant breeding projects on the development of resistant genotypes. The present study compared the proteomic alterations between two cacao genotypes standard for WBD resistance and susceptibility, in response toM. perniciosainfection at 72 h and 45 days post-inoculation; respectively the very early stages of the biotrophic and necrotrophic stages of the cacao xM. perniciosainteraction.ResultsA total of 554 proteins were identified, being 246 in the susceptible Catongo and 308 in the resistant TSH1188 genotypes. The identified proteins were involved mainly in metabolism, energy, defense and oxidative stress. The resistant genotype showed more expressed proteins with more variability associated with stress and defense, while the susceptible genotype exhibited more repressed proteins. Among these proteins, stand out pathogenesis related proteins (PRs), oxidative stress regulation related proteins, and trypsin inhibitors. Interaction networks were predicted, and a complex protein-protein interaction was observed. Some proteins showed a high number of interactions, suggesting that those proteins may function as cross-talkers between these biological functions.ConclusionsWe present the first study reporting the proteomic alterations of resistant and susceptible genotypes in theT. cacaoxM. perniciosapathosystem. The important altered proteins identified in the present study are related to key biologic functions in resistance, such as oxidative stress, especially in the resistant genotype TSH1188, that showed a strong mechanism of detoxification. Also, the positive regulation of defense and stress proteins were more evident in this genotype. Proteins with significant roles against fungal plant pathogens, such as chitinases, trypsin inhibitors and PR 5 were also identified, and they may be good resistance markers. Finally, important biological functions, such as stress and defense, photosynthesis, oxidative stress and carbohydrate metabolism were differentially impacted withM. perniciosainfection in each genotype.

Ecotin, first described inEscherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in variousCampylobacterspecies, includingCampylobacter rectusandCampylobacter showaeresiding in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotinsin vitro, the orthologs fromC.rectusandC.showaewere recombinantly expressed and purified fromE.coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins fromC.rectusandC.showaeinhibit NE, factor Xa and trypsin, but not theCampylobacter jejuniserine protease HtrA or its ortholog inE.coli, DegP. To further evaluate ecotin functionin vivo, anE.coliecotin-deficient mutant was complemented with theC.rectusandC.showaehomologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of theE.coliecotin-deficient mutant can be rescued upon expression of ecotins fromC.rectusandC.showae. In addition, theC.rectusandC.showaeecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen,Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.

Purpose: Early application of protease inhibitors through the intestinal lumen could increase survival following experimental shock by blocking the pancreatic digestive enzymes. Hence, it was hypothesized that two-route injection (intraintestinal + intravenous) of ulinastatin (UTI), a broad-spectrum protease inhibitor, could better alleviate intestinal injury than single-route injection (either intravenous or intraintestinal). Methods: A sepsis model induced by lipopolysaccharide on rats was established. The rats were randomly divided into five groups: sham, sepsis, UTI intravenous injection (Uiv), UTI intraintestinal injection (Uii), and UTI intraintestinal + intravenous injection (Uii + Uiv) groups. The mucosal barrier function, enzyme-blocking effect, levels of systemic inflammatory cytokines, and 5-day survival rate were compared among groups. The small intestinal villus height (VH), crypt depth (CD), and two components of mucosal barrier (E-cadherin and mucin-2) were measured to evaluate the mucosal barrier function. The levels of trypsin and neutrophil elastase (NE) in the intestine, serum, and vital organs were measured to determine the enzyme-blocking effect. Results: Compared with the single-route injection group (Uiv or Uii), the two-route injection (Uii + Uiv) group displayed: (1) significantly higher levels of VH, VH/CD, E-cadherin, and mucin-2; (2) decreased trypsin and NE levels in intestine, plasma, and vital organs; (3) reduced systemic inflammatory cytokine levels; and (4) improved survival of septic rats. Conclusion: Two-route UTI injection was superior to single-route injection in terms of alleviating intestinal injury, which might be explained by extensive blockade of proteases through different ways. Keywords: Sepsis, Ulinastatin, Intestines, Trypsin, Elastase

The recognition of protease inhibitors with insecticidal activity is important as a basis for the development of mimetic peptides with potential use as biorational insecticides. We sprayed benzamidine on soybean plants and assessed whether this potent synthetic trypsin-inhibitor has protease inhibitory, insecticidal and deterrent effects on the velvetbean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae). Activity of trypsin inhibition in soybean leaves was increased and total proteolytic activity in the midgut extract from larvae fed on these leaves was reduced by benzamidine. Different concentrations of benzamidine sprayed on the plant caused approximately 50 % of larval mortality, and larval choice and moth preference and oviposition were all negatively affected. Low concentrations of benzamidine increased mortality and hindered insect choice and oviposition as well as higher doses. Since many synthetic protease inhibitors are usually expensive, small doses of benzamidine may be effective to protect soybean against A. gemmatalis attack. Our results highlight the potential of synthetic protease inhibitors for insecticidal and deterrent purposes in insect pest management.

The interaction of trypsin with Gensenoside-Rg1 (G-Rg1) was studied using fluorescence, ultraviolet-visible (UV-vis), and circular dichroism (CD) spectroscopies along with enzyme activity assay and molecular docking. The enzyme activity assays showed that G-Rg1 inhibited the activity of trypsin effectively. The fluorescence experiments indicated that a complex of G-Rg1-trypsin was formed and that the fluorescence of trypsin was quenched by G-Rg1 via a mixed-quenching mechanism (both static and dynamic quenching). The thermodynamic analysis suggested that hydrophobic interaction and hydrogen bond were the major forces between G-Rg1 and trypsin. According to the theory of Förster's non-radiation energy transfer, the binding distance between trypsin and G-Rg1 was calculated to be 2.01 nm, which implies that energy transfer occurred within the complex. The experimental results obtained from UV-vis absorption spectra, synchronous fluorescence spectra, and CD spectra indicated that G-Rg1 was mainly located on tryptophan moiety and that the interaction between G-Rg1 and trypsin led to conformational changes of trypsin with some α-helix and unordered coil structures being transformed into β-sheet structures. In addition, docking results supported the above experimental findings and suggested the possible binding location of G-Rg1 on trypsin along with the possible hydrogen bonds and hydrophobic interactions between G-Rg1 and trypsin. The experimental results from this study should be useful to minimize the antinutritional effects and make full use of Genseng extracts in the food industry and also be helpful to the design of the drugs for the diseases related to overexpression of trypsin. Communicated by Ramaswamy H. Sarma.

Amylase/trypsin-inhibitors (ATIs) are putative triggers of nonceliac gluten sensitivity, but contents of ATIs in different wheat species were not available. Therefore, the predominant ATIs 0.19 + 0.53, 0.28, CM2, CM3, and CM16 in eight cultivars each of common wheat, durum wheat, spelt, emmer, and einkorn grown under the same environmental conditions were quantitated by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) and stable isotope dilution assays using specific marker peptides as internal standards. The results were compared to a label-free untargeted LC-MS/MS analysis, in which protein concentrations were determined by intensity based absolute quantitation. Both approaches yielded similar results. Spelt and emmer had higher ATI contents than common wheat, with durum wheat in between. Only three of eight einkorn cultivars contained ATIs in very low concentrations. The distribution of ATI types was characteristic for hexaploid, tetraploid, and diploid wheat species and suitable as species-specific fingerprint. The results point to a better tolerability of einkorn for NCGS patients, because of very low total ATI contents.

Cassia grandis trypsin inhibitor (CgTI) is a novel plant serine proteinase inhibitor. This study sets out to purify a thermostable inhibitor from the seeds of Cassia grandis and to provide biochemical information about a novel peptide belonging to the Kunitz family. Moreover, toxicity assays against Artemia, Aedes aegypti larvae-L4 and Nasutitermes corniger are evaluated. The purification process was performed using acetone precipitation, Trypsin-Sepharose-CL4B and Superdex-G75. The inhibitor showed an apparent molecular mass of around 19.8 kDa on Superdex-G75 gel filtration, and a mass of around 19.0 kDa visualized by SDS-PAGE under reducing conditions, and it also showed the protein consists of two polypeptide chains. N-terminal sequencing by Edman's degradation of 16 residues revealed a sequence of amino acids SVVLDTSGEPIRNGGG. 2D-electrophoresis identified a pI value of 6.3 and a 1:1 stoichiometric ratio was noted during CgTI-trypsin complex formation. The inhibitor retained the inhibitory activity over a broad range of pH (5–10) and showed thermostable activity at temperatures 30–80 °C. Furthermore, in vivo assays showed no lethality effect against Artemia and Aedes aegypti larvae, but mortality against Nasutitermes corniger with termiticidal activity LC50 of 0.685 mg/mL on workers and 0.765 mg/mL on soldiers. Preliminary investigations of CgTI revealed it to be a promising biotechnological and biomedical candidate.

Three out of 17 Streptomycetes strains - Streptomyces sp. 35 LBG09, Streptomyces sp. 36 LBG09, and Streptomyces sp. 39 LBG09, were selected based on the high production of proteinase inhibitors with trypsin serine proteinase activity. The strains were isolated from soil samples taken from the area around the Bulgarian station on Livingston Island, Antarctica. Biosynthesis of proteinase inhibitors by the promising strains started at different stages of their development but was generally not associated with the growth of the producers. Peak levels were reached in the stationary phase (96-120 h) of their cultivation. Inducing effects on strain development, biomass accumulation, and proteinase inhibitor biosynthesis were based on the composition of the nutrient medium: the polypeptones contained in Taguchi medium and glucose as a carbon source. The most productive out of the three strains was Streptomyces sp. 36 LBG09. Its maximum inhibitory activity was reached at 96 h in culturing media modified by three different carbon sources. The active proteinase inhibitor biosynthesis proceeded at pH values between 6.8 and 8.6 and the dynamics of production depended on the type of carbon source. Peak levels of extracellular protein and dry biomass were reached at 120 h in the stationary growth phase. The residual sugars were minimal at the end of the process when using soluble starch as a carbon source, and maximal when glucose was used.

Background Death due to cerebral stroke afflicts a large number of neuronal populations, including glial cells depending on the brain region affected. Drugs with a wide cellular range of protection are needed to develop effective therapies for stroke. Human alpha 1-antitrypsin (hAAT) is a serine proteinase inhibitor with potent anti-inflammatory, anti-apoptotic and immunoregulatory activities. This study aimed to test whether hAAT can protect different kind of neurons and glial cells after the oxygen and glucose deprivation (OGD). Methods Addition of hAAT to mouse neuronal cortical, hippocampal and striatal cultures, as well as glial cultures, was performed 30 min after OGD induction and cell viability was assessed 24 h later. The expression of different apoptotic markers and several inflammatory parameters were assessed by immunoblotting and RT-PCR. Results hAAT had a concentration-dependent survival effect in all neuronal cultures exposed to OGD, with a maximal effect at 1–2 mg/mL. The addition of hAAT at 1 mg/mL reduced the OGD-mediated necrotic and apoptotic death in all neuronal cultures. This neuroprotective activity of hAAT was associated with a decrease of cleaved caspase-3 and an increase of MAP2 levels. It was also associated with a reduction of pro-inflammatory cytokines protein levels and expression, increase of IL-10 protein levels and decrease of nuclear localization of nuclear factor-kappaB. Similar to neurons, addition of hAAT protected astrocytes and oligodendrocytes against OGD-induced cell death. Conclusions Human AAT protects neuronal and glial cells against OGD through interaction with cytokines. General significance Human AAT could be a good therapeutic neuroprotective candidate to treat ischemic stroke.

In this work, a facile, label-free, and sensitive fluorometric strategy for detection of trypsin and its inhibitor was established on the basis of the fluorescence resonance energy transfer (FRET) between mercaptoundecanoic acid functionalized gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs) via protamine as a bridge. Protamine can trigger the aggregation of AuNPs and link AuNCs with aggregated AuNPs through electrostatic interaction. Compared with monodisperse AuNPs, the UV–vis absorption band of aggregated AuNPs overlapped considerably with the emission spectrum of AuNCs. Thus, the fluorescence of AuNCs was obviously quenched by the aggregated AuNPs through FRET. In the presence of trypsin, protamine was hydrolyzed into small fragments, leading to the deaggregation of AuNPs and breaking of the short distance between AuNPs and AuNCs, so the FRET process was inhibited, and the fluorescence of AuNCs was recovered. The increase in the fluorescence intensity of AuNCs was directly related to the amount of trypsin. Hence trypsin can be determined on the basis of the variation of fluorescence intensity, with a linear range of 5–5000 ng mL-1 and a detection limit of 1.9 ng mL-1. In addition, this system was used for the detection of trypsin inhibitor by application of the inhibitor isolated from soybean as a model. The sensing method was applied for trypsin detection in human urine and commercial multienzyme tablet samples with satisfactory results.

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 (IC50 = 70 ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed

Techniques for rapidly measuring both the strength and mode of enzyme inhibitors are crucial to lead generation and optimization in drug development. Isothermal titration calorimetry (ITC) is emerging as a powerful tool for measuring enzyme kinetics with distinct advantages over traditional techniques. ITC measures heat flow, a feature of nearly all chemical reactions, and gives an instantaneous readout of enzyme velocity, eliminating the need for artificial substrates or postreaction processing. In principle, ITC is an ideal method for characterizing enzyme inhibition. However, existing ITC experiments are not well-suited to rapid throughput and few studies to date have employed this approach. We have developed a new ITC experiment, in which substrate and inhibitor are premixed in the injection syringe, that yields complete kinetic characterization of an enzyme inhibitor in an hour or less. This corresponds to savings in time and material of 5-fold or greater compared to previous ITC methods. We validated the approach using the trypsin inhibitor benzamidine as a model system, recapitulating both its competitive inhibition mode and binding constant. Our approach combines the rapid throughput of optimized spectroscopic assays with the universality and precision of ITC-based methods, providing substantially improved inhibitor characterization for biochemistry and drug development applications.

Staphylococcus aureus is a multidrug-resistant bacterium responsible for several cases of hospital-acquired infections, which constitute a global public health problem. The introduction of new healthcare strategies and/or the discovery of molecules capable of inhibiting the growth or killing S. aureus would have a huge impact on the treatment of S. aureus-mediated diseases. Herein, a Bowman–Birk protease inhibitor (LzaBBI), with strong in vitro antibacterial activity against S. aureus, was purified to homogeneity from Luetzelburgia auriculata seeds. LzaBBI in its native form is a 14.3 kDa protein and has a pI of 4.54, and its NH2-terminal sequence has high identity with other Bowman–Birk inhibitors. LzaBBI showed a mixed-type inhibitory activity against both trypsin and chymotrypsin, respectively, and it remained stable after both boiling at 98 °C for 120 min and incubation at various pHs. Scanning electron microscopy revealed that LzaBBI disrupted the S. aureus membrane integrity, leading to bacterial de...