The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites.

Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.

StMLP1, as a Kunitz trypsin inhibitor, enhances potato resistance and specifically expresses in vascular bundles during Ralstonia solanacearum infection.

Miraculin-like proteins (MLPs), members of the Kunitz trypsin inhibitor (KTI) family that are present in various plants, have been discovered to have a role in defending plants against pathogens. In this study, we identified a gene StMLP1 in potato that belongs to the KTI family. We found that the expression of StMLP1 gradually increases during Ralstonia solanacearum (R. solanacearum) infection. We characterized the promoter of StMLP1 as an inducible promoter that can be triggered by R. solanacearum and as a tissue-specific promoter with specificity for vascular bundle expression. Our findings demonstrate that StMLP1 exhibits trypsin inhibitor activity, and that its signal peptide is essential for proper localization and function. Overexpression of StMLP1 in potato can enhance the resistance to R. solanacearum. Inhibiting the expression of StMLP1 during infection accelerated the infection by R. solanacearum to a certain extent. In addition, the RNA-seq results of the overexpression-StMLP1 lines indicated that StMLP1 was involved in potato immunity. All these findings in our study reveal that StMLP1 functions as a positive regulator that is induced and specifically expressed in vascular bundles in response to R. solanacearum infection.

Structural basis for the binding of famotidine, cimetidine, guanidine, and pimagedine with serine protease.

Serine proteases are among the important groups of enzymes having significant roles in cell biology. Trypsin is a representative member of the serine superfamily of enzymes, produced by acinar cells of pancreas. It is a validated drug target for various ailments including pancreatitis and colorectal cancer. Premature activation of trypsin is involved in the lysis of pancreatic tissues, which causes pancreatitis. It is also reported to be involved in colorectal carcinoma by activating other proteases, such as matrix metalloproteinase (MMPs). The development of novel trypsin inhibitors with good pharmacokinetic properties could play important roles in pharmaceutical sciences. This study reports the crystal structures of bovine pancreatic trypsin with four molecules; cimetidine, famotidine, pimagedine, and guanidine. These compounds possess binding affinity towards the active site (S1) of trypsin. The structures of all four complexes provided insight of the binding of four different ligands, as well as the dynamics of the active site towards the bind with different size ligands. This study might be helpful in designing of new potent inhibitors of trypsin and trypsin like serine proteases.

Residual protein analysis by SDS-PAGE in clinically manufactured BM-MSC products.

Residual substances that are considered hazardous to the recipient must be removed from final cellular therapeutic products manufactured for clinical purposes. In doing so, quality rules determined by competent authorities (CAs) for the clinical use of tissue- and cell-based products can be met. In our study, we carried out residual substance analyses, and purity determination studies of trypsin and trypsin inhibitor in clinically manufactured bone marrow-derived mesenchymal stromal/stem cell products, using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Despite being a semiquantitative method, SDS-PAGE has several benefits over other methods for protein analysis, such as simplicity, convenience of use, and affordability. Due to its convenience and adaptability, SDS-PAGE is still a commonly used method in many laboratories, despite its limits in dynamic range and quantitative precision. Our goal in this work was to show that SDS-PAGE may be used effectively for protein measurement, especially where practicality and affordability are the major factors. The results of our study suggest a validated method to guide tissue and cell manufacturing sites for making use of an agreeable, accessible, and cost-effective method for residual substance analyses in clinically manufactured cellular therapies.

A new trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative.

Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.

Prebound State Discovered in the Unbinding Pathway of Fluorinated Variants of the Trypsin-BPTI Complex Using Random Acceleration Molecular Dynamics Simulations.

The serine protease trypsin forms a tightly bound inhibitor complex with the bovine pancreatic trypsin inhibitor (BPTI). The complex is stabilized by the P1 residue Lys15, which interacts with negatively charged amino acids at the bottom of the S1 pocket. Truncating the P1 residue of wildtype BPTI to α-aminobutyric acid (Abu) leaves a complex with moderate inhibitor strength, which is held in place by additional hydrogen bonds at the protein-protein interface. Fluorination of the Abu residue partially restores the inhibitor strength. The mechanism with which fluorination can restore the inhibitor strength is unknown, and accurate computational investigation requires knowledge of the binding and unbinding pathways. The preferred unbinding pathway is likely to be complex, as encounter states have been described before, and unrestrained umbrella sampling simulations of these complexes suggest additional energetic minima. Here, we use random acceleration molecular dynamics to find a new metastable state in the unbinding pathway of Abu-BPTI variants and wildtype BPTI from trypsin, which we call the prebound state. The prebound state and the fully bound state differ by a substantial shift in the position, a slight shift in the orientation of the BPTI variants, and changes in the interaction pattern. Particularly important is the breaking of three hydrogen bonds around Arg17. Fluorination of the P1 residue lowers the energy barrier of the transition between the fully bound state and prebound state and also lowers the energy minimum of the prebound state. While the effect of fluorination is in general difficult to quantify, here, it is in part caused by favorable stabilization of a hydrogen bond between Gln194 and Cys14. The interaction pattern of the prebound state offers insights into the inhibitory mechanism of BPTI and might add valuable information for the design of serine protease inhibitors.

A comparative analysis of sivelestat sodium hydrate and ulinastatin combination therapy in the treatment of sepsis with acute respiratory distress syndrome.

OBJECTIVE: This comparative analysis aimed to investigate the efficacy of Sivelestat Sodium Hydrate (SSH) combined with Ulinastatin (UTI) in the treatment of sepsis with acute respiratory distress syndrome (ARDS). METHODS: A control group and an observation group were formed with eighty-four cases of patients with sepsis with ARDS, with 42 cases in each group. The control group was intravenously injected with UTI based on conventional treatment, and the observation group was injected with SSH based on the control group. Both groups were treated continuously for 7 days, and the treatment outcomes and efficacy of both groups were observed. The Murray Lung Injury Score (MLIS), Sequential Organ Failure Assessment (SOFA), and Acute Physiology and Chronic Health Evaluation II (APACHE II) were compared. Changes in respiratory function, inflammatory factors, and oxidative stress indicators were assessed. The occurrence of adverse drug reactions was recorded. RESULTS: The total effective rate in the observation group (95.24%) was higher than that in the control group (80.95%) (P < 0.05). The mechanical ventilation time, intensive care unit (ICU) hospitalization time, and duration of antimicrobial medication in the observation group were shorter and multiple organ dysfunction syndrome incidence was lower than those in the control group (P < 0.05). The mortality rate of patients in the observation group (35.71%) was lower than that in the control group (52.38%), but there was no statistically significant difference between the two groups (P > 0.05). MLIS, SOFA, and APACHE II scores in the observation group were lower than the control group (P < 0.05). After treatment, respiratory function, inflammation, and oxidative stress were improved in the observation group (P < 0.05). Adverse reactions were not significantly different between the two groups (P > 0.05). CONCLUSION: The combination of SSH plus UTI improves lung injury and pulmonary ventilation function, and reduces inflammation and oxidative stress in patients with sepsis and ARDS.

Sulfated Aeruginosins from Lake Kinneret: Microcystis Bloom, Isolation, Structure Elucidation, and Biological Activity.

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.

MDR49 coding for both P-glycoprotein and TMOF transporter functions in ivermectin resistance, trypsin activity inhibition, and fertility in the yellow fever mosquito, Aedes aegypti.

This study investigated the function of the MDR49 gene in Aedes aegypti. MDR49 mutants were constructed using CRISPR/Cas9 technology; the mutation led to increased sensitivity to ivermectin (LC50: from 1.3090 mg L-1 to 0.5904 mg L-1), and a reduction in midgut trypsin activity. These findings suggest that the P-gp encoded by MDR49 confers resistance to ivermectin and impacts the reproductive function in Ae. aegypti. RNA interference technology showed that knockdown of MDR49 gene resulted in a significant decrease in the expression of VGA1 after a blood meal, as well as a decrease in the number of eggs laid and their hatching rate. LC-MS revealed that following ivermectin treatment, the MDR493d+2s/3d+2s strain larvae exhibited significantly higher drug concentrations in the head and fat body compared to the wild type. Modeling of inward-facing P-gp and molecular docking found almost no difference in the affinity of P-gp for ivermectin before and after the mutation. However, modeling of the outward-facing conformation demonstrated that the flexible linker loop between TM5 and TM6 of P-gp undergoes changes after the mutation, resulting in a decrease in trypsin activity and an increase in sensitivity to ivermectin. These results provide useful insights into ivermectin resistance and the other roles played by the MDR49 gene.

The Preventive Effect of Urinary Trypsin Inhibitor on Postoperative Cognitive Dysfunction, on the Aspect of Behavior, Evaluated by Y-Maze Test, via Modulation of Microglial Activity.

This experimental study was designed to evaluate the effect of ulinastatin, a urinary trypsin inhibitor, on postoperative cognitive dysfunction (POCD) in rats under general anesthesia with isoflurane, on the aspect of behavior, as evaluated using a Y-maze test and focusing on microglial activity. Ulinastatin (50,000 U/mL) and normal saline (1 mL) were randomly (1:1) administered intraperitoneally to the ulinastatin and control groups, respectively, before general anesthesia. Anesthesia with isoflurane 1.5 volume% was maintained for 2 h. The Y-maze test was used to evaluate cognitive function. Neuronal damage using caspase-1 expression, the degree of inflammation through cytokine detection, and microglial activation with differentiation of the phenotypic expression were evaluated. Twelve rats were enrolled in the study and evenly allocated into the two groups, with no dropouts from the study. The Y-maze test showed similar results in the two groups before general anesthesia (63 ± 12% in the control group vs. 64 ± 12% in the ulinastatin group, p = 0.81). However, a significant difference was observed between the two groups after general anesthesia (17 ± 24% in the control group vs. 60 ± 12% in the ulinastatin group, p = 0.006). The ulinastatin group showed significantly lower expression of caspase-1. Pro-inflammatory cytokine levels were significantly lower in the ulinastatin group than in the control group. The ulinastatin group had a significantly lower microglial activation (41.74 ± 10.56% in the control group vs. 4.77 ± 0.56% in the ulinastatin, p < 0.001), with a significantly lower activation of M1 phenotypes (52.19 ± 7.83% in the control group vs. 5.58 ± 0.76% in the ulinastatin group, p < 0.001). Administering ulinastatin before general anesthesia prevented neuronal damage and cognitive decline after general anesthesia, in terms of the aspect of behavior, as evaluated by the Y-maze test. The protective effect of ulinastatin was associated with the inhibition of microglial activation, especially the M1 phenotype.

Correlation of Experimental and Calculated Inhibition Constants of Protease Inhibitor Complexes.

Predicting the potency of inhibitors is key to in silico screening of promising synthetic or natural compounds. Here we describe a predictive workflow that provides calculated inhibitory values, which concord well with empirical data. Calculations of the free interaction energy ΔG with the YASARA plugin FoldX were used to derive inhibition constants Ki from PDB coordinates of protease-inhibitor complexes. At the same time, corresponding KD values were obtained from the PRODIGY server. These results correlated well with the experimental values, particularly for serine proteases. In addition, analyses were performed for inhibitory complexes of cysteine and aspartic proteases, as well as of metalloproteases, whereby the PRODIGY data appeared to be more consistent. Based on our analyses, we calculated theoretical Ki values for trypsin with sunflower trypsin inhibitor (SFTI-1) variants, which yielded the more rigid Pro14 variant, with probably higher potency than the wild-type inhibitor. Moreover, a hirudin variant with an Arg1 and Trp3 is a promising basis for novel thrombin inhibitors with high potency. Further examples from antibody interaction and a cancer-related effector-receptor system demonstrate that our approach is applicable to protein interaction studies beyond the protease field.

Bowman-Birk Inhibitor Mutants of Soybean Generated by CRISPR-Cas9 Reveal Drastic Reductions in Trypsin and Chymotrypsin Inhibitor Activities.

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

Anti-SARS-CoV-2 Viral Activity of Sweet Potato Trypsin Inhibitor via Downregulation of TMPRSS2 Activity and ACE2 Expression In Vitro and In Vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever recorded in human history, causing significant losses to both life and economies globally. However, the available evidence is currently insufficient to establish the effectiveness and safety of antiviral drugs or vaccines. The entry of the virus into host cells involves binding to angiotensin-converting enzyme 2 (ACE2), a cell surface receptor, via its spike protein. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a host surface protease, cleaves and activates the virus's S protein, thus promoting viral infection. Plant protease inhibitors play a crucial role in protecting plants against insects and/or microorganisms. The major storage proteins in sweet potato roots include sweet potato trypsin inhibitor (SWTI), which accounts for approximately 60% of the total water-soluble protein and has been found to possess a variety of health-promoting properties, including antioxidant, anti-inflammatory, ACE-inhibitory, and anticancer functions. Our study found that SWTI caused a significant reduction in the expression of the ACE2 and TMPRSS2 proteins, without any adverse effects on cells. Therefore, our findings suggest that the ACE2 and TMPRSS2 axis can be targeted via SWTI to potentially inhibit SARS-CoV-2 infection.

Enrichment of Trypsin Inhibitor from Soybean Whey Wastewater Using Different Precipitating Agents and Analysis of Their Properties.

Recovering valuable active substances from the by-products of agricultural processing is a crucial concern for scientific researchers. This paper focuses on the enrichment of soybean trypsin inhibitor (STI) from soybean whey wastewater using either ammonium sulfate salting or ethanol precipitation, and discusses their physicochemical properties. The results show that at a 60% ethanol content, the yield of STI was 3.983 mg/mL, whereas the yield was 3.833 mg/mL at 60% ammonium sulfate saturation. The inhibitory activity of STI obtained by ammonium sulfate salting out (A-STI) was higher than that obtained by ethanol precipitation (E-STI). A-STI exhibited better solubility than E-STI at specific temperatures and pH levels, as confirmed by turbidity and surface hydrophobicity measurements. Thermal characterization revealed that both A-STI and E-STI showed thermal transition temperatures above 90 °C. Scanning electron microscopy demonstrated that A-STI had a smooth surface with fewer pores, while E-STI had a rough surface with more pores. In conclusion, there was no significant difference in the yield of A-STI and E-STI (p < 0.05); however, the physicochemical properties of A-STI were superior to those of E-STI, making it more suitable for further processing and utilization. This study provides a theoretical reference for the enrichment of STI from soybean whey wastewater.

A Novel Kunitz Trypsin Inhibitor from Enterolobium gummiferum Seeds Exhibits Antibiofilm Properties against Pathogenic Yeasts.

Plant peptidase inhibitors play crucial roles in plant defence mechanisms and physiological processes. In this study, we isolated and characterised a Kunitz trypsin inhibitor from Enterolobium gummiferum seeds named EgPI (E. gummiferum peptidase inhibitor). The purification process involved two chromatography steps using size exclusion and hydrophobic resins, resulting in high purity and yield. EgPI appeared as a single band of ~20 kDa in SDS-PAGE. Under reducing conditions, the inhibitor exhibited two polypeptide chains, with 15 and 5 kDa. Functional characterisation revealed that EgPI displayed an inhibition stoichiometry of 1:1 against trypsin, with a dissociation constant of 8.4 × 10-9 mol·L-1. The amino-terminal sequencing of EgPI revealed the homology with Kunitz inhibitors. Circular dichroism analysis provided insights into the secondary structure of EgPI, which displayed the signature typical of Kunitz inhibitors. Stability studies demonstrated that EgPI maintained the secondary structure necessary to exhibit its inhibitory activity up to 70 °C and over a pH range from 2 to 8. Microbiological screening revealed that EgPI has antibiofilm properties against pathogenic yeasts at 1.125 µmol·L-1, and EgPI reduced C. albicans biofilm formation by 82.7%. The high affinity of EgPI for trypsin suggests potential applications in various fields. Furthermore, its antibiofilm properties recommended its usefulness in agriculture and antimicrobial therapy research, highlighting the practical implications of our research.

Relationships between haptoglobin and inter-alpha trypsin inhibitor heavy chain 4 levels in local and peripheral blood and systemic inflammatory response syndrome in bitches with pyometra.

We aimed to assess the usefulness of monitoring inter-alpha trypsin inhibitor heavy chain 4 (ITIH4) and haptoglobin (Hp) in peripheral and local blood in canine pyometra, and evaluation the relationships among acute phase proteins (APPs), systemic inflammatory response syndrome (SIRS) and the presence of bacteria. The material was collected from bitches with pyometra and from healthy ones. Blood was taken from the cephalic and uterine veins. APPs levels were quantified by ELISA. In the peripheral circulation, the Hp was higher in animals with open-cervix pyometra (OCP) than in the closed-cervix pyometra (CCP) and the control group. The Hp concentration was not correlated with age, with the presence of SIRS or with the type of bacteria (Gram-negative, Gram-positive or mixed flora). The ITIH4 concentrations in the peripheral blood did not differ significantly in the cases of pyometra. The Hp concentration in the local circulation increased in the OCP but not in the CCP groups, although the histopathological changes in the endometrium were similar. Peripheral Hp concentrations may be a useful tool in differentiating between the types of pyometra.

The impact of ulinastatin on wound infection and healing in patients with burn wounds: A meta-analysis.

Burn injuries result in localised tissue damage and precipitate systemic responses; routine clinical treatments, which typically include metabolic nutritional support and anti-infection therapies, do not yield optimal outcomes. Therefore, we aimed to systematically evaluate the effects of ulinastatin on wound infection and healing in patients with burns to provide reliable evidence-based recommendations for burn treatment. An electronic search of the Web of Science, PubMed, Cochrane Library, Embase, Wanfang, Chinese Biomedical Literature Database, and China National Knowledge Infrastructure databases, supplemented by manual searches, was conducted from database inception to October 2023 to collect randomised controlled trials (RCTs) assessing the efficacy of ulinastatin for the treatment of burns. Two researchers screened all retrieved articles according to the inclusion and exclusion criteria; the included studies were evaluated for quality, and the relevant data were extracted. Stata 17.0 software was employed for data analysis. Overall, 8 RCTs with 803 patients were included, with 404 and 399 in the ulinastatin and conventional treatment groups, respectively. The analysis revealed that wound infections (odds ratio [OR] = 0.08, 95% CI: 0.02-0.35, p = 0.001) and complications (OR = 0.21, 95% CI: 0.10-0.42, p < 0.001) were significantly lower, and wound healing time (standardised mean differences [SMD] = -1.31, 95% CI: -2.05 to -0.57, p = 0.001) was significantly shorter, in the ulinastatin groups than in the control group. This meta-analysis revealed that ulinastatin can effectively reduce the incidence of wound infections and complications and significantly shorten the duration of wound healing in patients with burns, thereby promoting early recovery in these patients.

Dietary wheat amylase trypsin inhibitors exacerbate CNS inflammation in experimental multiple sclerosis.

OBJECTIVE: Wheat has become a main staple globally. We studied the effect of defined pro-inflammatory dietary proteins, wheat amylase trypsin inhibitors (ATI), activating intestinal myeloid cells via toll-like receptor 4, in experimental autoimmune encephalitis (EAE), a model of multiple sclerosis (MS). DESIGN: EAE was induced in C57BL/6J mice on standardised dietary regimes with defined content of gluten/ATI. Mice received a gluten and ATI-free diet with defined carbohydrate and protein (casein/zein) content, supplemented with: (a) 25% of gluten and 0.75% ATI; (b) 25% gluten and 0.19% ATI or (c) 1.5% purified ATI. The effect of dietary ATI on clinical EAE severity, on intestinal, mesenteric lymph node, splenic and central nervous system (CNS) subsets of myeloid cells and lymphocytes was analysed. Activation of peripheral blood mononuclear cells from patients with MS and healthy controls was compared. RESULTS: Dietary ATI dose-dependently caused significantly higher EAE clinical scores compared with mice on other dietary regimes, including on gluten alone. This was mediated by increased numbers and activation of pro-inflammatory intestinal, lymph node, splenic and CNS myeloid cells and of CNS-infiltrating encephalitogenic T-lymphocytes. Expectedly, ATI activated peripheral blood monocytes from both patients with MS and healthy controls. CONCLUSIONS: Dietary wheat ATI activate murine and human myeloid cells. The amount of ATI present in an average human wheat-based diet caused mild intestinal inflammation, which was propagated to extraintestinal sites, leading to exacerbation of CNS inflammation and worsening of clinical symptoms in EAE. These results support the importance of the gut-brain axis in inflammatory CNS disease.

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor.

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman-Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P41, with Rwork /Rfree values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme-inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.

Exploring Photoswitchable Binding Interactions with Small-Molecule- and Peptide-Based Inhibitors of Trypsin.

The ability to photochemically activate a drug, both when and where needed, requires optimisation of the difference in biological activity between each isomeric state. As a step to this goal, we report small-molecule- and peptide-based inhibitors of the same protease-trypsin-to better understand how photoswitchable drugs interact with their biological target. The best peptidic inhibitor displayed a more than fivefold difference in inhibitory activity between isomeric states, whereas the best small-molecule inhibitor only showed a 3.4-fold difference. Docking and molecular modelling suggest this result is due to a large change in 3D structure in the key binding residues of the peptidic inhibitor upon isomerisation; this is not observed for the small-molecule inhibitor. Hence, we demonstrate that significant structural changes in critical binding motifs upon irradiation are essential for maximising the difference in biological activity between isomeric states. This is an important consideration in the design of future photoswitchable drugs for clinical applications.