Search Results (63)

Chickpea (Cicer arietinum L.) generally contains lower levels of these compounds than many other legumes, yet information on Hungarian chickpea cultivars is scarce. This study aimed to characterize protein-based antinutritional factors in twenty chickpeas grown under different agroclimatic conditions over three consecutive years (15 samples from seven Hungarian cultivars from three cultivation areas, and five commercially available foreign genotypes). Protein profiles were examined by SDS-PAGE and native PAGE, while trypsin inhibitor activity (TIA) was quantified spectrophotometrically according to ISO 14902, and lectin activity was determined using a hemagglutination assay. SDS-PAGE revealed highly similar protein patterns among samples, indicating comparable overall protein composition. Native PAGE combined with activity staining confirmed the presence of Kunitz-type trypsin inhibitors, with multiple isoforms detected, but no Bowman–Birk-type inhibitor activity was observed. TIA values were low (0.49–4.07 mg inhibited trypsin/g), and lectin activities were generally low (1–2.5 HU/mg flour; only one sample reached 5 HU/mg) or undetectable. Neither cultivation area nor growing year had a significant effect on TIA or lectin activity, confirmed by statistical analyses. Overall, Hungarian chickpea varieties exhibited low and stable levels of antinutritional proteins, supporting their favorable nutritional quality and suitability for human consumption and expanded cultivation under Hungarian agroclimatic conditions. Full article

Pericellular proteolysis is essential for maintaining tissue homeostasis. Central to this process are hepatocyte growth factor activator inhibitor-1 (HAI-1) and HAI-2, membrane-bound inhibitors that regulate type II transmembrane serine proteases, including matriptase and prostasin, through high-affinity Kunitz domains. This review summarizes current understanding of their molecular structures, physiological roles, and cancer-related clinical relevance. Genetic models reveal HAI-1 is critical for placental and skin development, while HAI-2 is crucial for neural tube closure and intestinal integrity. In cancer, HAIs generally act as tumor suppressors. Their downregulation, often via promoter hypermethylation, leads to excessive activation of hepatocyte growth factor/c-MET or protease-activated receptor-2/NF-κB signaling, promoting epithelial–mesenchymal transition and cancer progression. Clinically, reduced HAI levels in tumors correlate with metastasis and poor prognosis in several carcinomas. Paradoxically, elevated HAI expression in certain cancers suggests context-dependent pro-tumor functions. Emerging evidence links HAI loss to immune suppression, notably via M2 macrophage polarization in lung cancer. Finally, we highlight future directions for identifying tissue-specific serine proteases, downstream signaling, and therapeutic strategies, including recombinant mimetics and epigenetic reactivation, in precision oncology. In conclusion, HAI-1 and HAI-2 are key regulators of tissue homeostasis and cancer, with overlapping yet distinct functions, which present promising opportunities for therapeutic targeting. Full article

Acanthophis antarcticus, commonly known as the death adder, is a venomous Australian snake and a member of the Elapidae family. Due to its robust body and triangular head, it was historically misclassified as a viper. Its venom is known for neurotoxic, hemorrhagic, and hemolytic effects but displays low anticoagulant activity. Although key toxins such as three-finger toxins (3FTxs) and phospholipase A₂ (PLA₂) have been previously described, no study has integrated proteomic and functional analyses to date. In this study, we conducted a comprehensive characterization of A. antarcticus venom. Reverse-phase high-performance liquid chromatography (RP-HPLC) followed by LC-MS/MS enabled the identification of nine toxin families, with 3FTxs and PLA₂ as the most abundant. Less abundant but functionally relevant toxins included Kunitz-type inhibitors, CRISP, SVMP, LAAO, NGF, natriuretic peptides, and nucleotidases, the latter being reported here for the first time based on proteomic evidence. Hydrophilic interaction chromatography (HILIC) coupled with MALDI-TOF was used to analyze polar, non-retained venom components, revealing the presence of low-molecular-weight peptides (2–4 kDa). Functional assays confirmed the enzymatic activity of HYAL, PLA₂, and LAAO and, for the first time, demonstrated inhibitory activity on serine peptidases and fibrinogenolytic activity in the venom of this species. These findings expand our understanding of the biochemical and functional diversity of this venom. Full article

Soybean meal (SBM) is extensively used as a predominant protein source in animal feed. However, raw soybean cannot be directly utilized in animal feed, due to the presence of the Kunitz trypsin inhibitor (KTi) and the Bowman–Birk protease inhibitor (BBi). These antinutritional factors inhibit the digestive enzymes in animals, trypsin and chymotrypsin, resulting in poor animal performance. To inactivate the activity of protease inhibitors, SBM is subjected to heat processing, a procedure that can negatively impact the soybean protein quality. Thus, it would be beneficial to develop soybean varieties with little or no trypsin inhibitors. In this study, we report on the creation of experimental soybean lines with significantly reduced levels of Bowman–Birk protease inhibitors. RNA interference (RNAi) technology was employed to generate several transgenic soybean lines. Some of these BBi knockdown soybean lines showed significantly lower amounts of both trypsin and chymotrypsin inhibitor activities. Western blot analysis revealed the complete absence of BBi in selected RNAi-derived lines. RNA sequencing (RNAseq) analysis demonstrated a drastic reduction in the seed-specific expression of BBi genes in the transgenic soybean lines during seed development. Confocal fluorescence immunolabeling studies showed that the accumulation of BBi was drastically diminished in BBi knockdown lines compared to wild-type soybeans. The absence of BBi in the transgenic soybean did not alter the overall protein, oil, and sulfur amino acid content of the seeds compared to wild-type soybeans. The seed protein from the BBi knockdown lines were more rapidly hydrolyzed by trypsin and chymotrypsin compared to the wild type, indicating that the absence of BBi enhances protein digestibility. Our study suggests that these BBi knockdown lines could be a valuable resource in order for plant breeders to incorporate this trait into commercial soybean cultivars, potentially enabling the use of raw soybeans in animal feed. Full article

The functional significance of the choroid plexus (CP), such as in the control of circadian rhythm as well as production of cerebrospinal fluid, is attracting attention. Transepithelial and junctional transport between epithelial cells of CP plays an important role in its function. Recently, an epithelial cadherin, E-cadherin, as well as non-epithelial cadherins were confirmed to be expressed in CP epithelial cells. The serine protease inhibitor Kunitz type 1 (SPINT1) is expressed in many kinds of epithelial cells and affects epithelial developmental function by controlling E-cadherin expression. However, it has not been confirmed whether SPINT1 is expressed in epithelial cells of CP. Thus, in this study, we examined whether SPINT1 is expressed in CP epithelial cells by immunohistochemistry and RT-PCR. Immunohistochemical expression of SPINT1 was noted in the cytoplasm of epithelial cells of humans and mice, and mRNA of SPINT1 was expressed in samples derived from the CP of mice. SPINT1 was typically expressed in CP epithelial cells with E-cadherin and Smad-interacting protein (SIP1), an E-cadherin transcriptional repressor. Some enlarged epithelial cells showed strong SPINT1 signals. These findings indicate that SPINT1 is expressed in epithelial cells of CP in relation to E-cadherin expression. Full article

Aedes aegypti (Linnaeus, 1762) is Brazil’s primary vector of epidemiologically significant arboviruses such as yellow fever, dengue, Zika, and chikungunya. Despite using conventional chemical control measures, this species has developed resistance to standard chemical insecticides, prompting the search for natural larvicidal compounds. Plant protease inhibitors offer an insecticidal alternative as the primary digestive enzymes in the midgut of Ae. aegypti are proteases (trypsin and chymotrypsin). Ae. aegypti larvae fed with ILTI, a Kunitz-type trypsin inhibitor derived from Inga laurina seeds, at concentrations between 0.03 mg of protein per mL (mgP/mL) and 0.12 mgP/mL, exhibited delayed larval development, with a lethal concentration (LC₅₀) of 0.095 mgP mL⁻¹ of ILTI for 50% of fourth-instar larvae (L₄). The ex vivo assay indicated that ILTI effectively inhibited the activity of larval trypsin, which remained susceptible to the inhibitor. Additionally, molecular modelling and docking studies were conducted to predict the three-dimensional ILTI/enzyme molecular complexes at the atomic level. Therefore, the results demonstrate that ILTI functions as a protease inhibitor in this species, presenting itself as a promising larvicidal tool in the control of Ae. aegypti. Full article

Understanding plant-insect interactions can help control the harm of herbivorous pests. According to transcriptome data, transcripts of Solanum lycopersicum responding to feeding by Tuta absoluta were screened for important endopeptidase inhibitors. These genes were annotated as serine-type endopeptidase inhibitors from the potato inhibitor I family, potato type II proteinase inhibitor family, and soybean trypsin inhibitor (Kunitz) family. Based on the analysis of expression patterns, Solyc09g084480.2, Solyc03g020080.2, Solyc03g098760.1, and Solyc01g009020.1 were identified as key genes in the defense system of S. lycopersicum. The major endopeptidase genes such as Tabs008250, Tabs007396, and Tabs005701 in the larval stages of T. absoluta were also detected as potential targets of the plant endopeptidase inhibitors. The interaction mode between these endopeptidase and endopeptidase inhibitors was predicted based on the protein structure construction. This study aims to reveal the molecular response of S. lycopersicum to feeding by T. absoluta with high throughput sequencing and bioinformatics analysis. Full article

Inflammation is a physiological response of the immune system to infectious agents or tissue injury, which involves a cascade of vascular and cellular events and the activation of biochemical pathways depending on the type of harmful agent and the stimulus generated. The Kunitz peptide HCIQ2c1 of sea anemone Heteractis magnifica is a strong protease inhibitor and exhibits neuroprotective and analgesic activities. In this study, we investigated the anti-inflammatory potential of HCIQ2c1 in histamine- and lipopolysaccharide (LPS)-activated RAW 264.7 macrophages as well as in LPS-induced systemic inflammation and carrageenan-induced paw edema models in CD-1 mice. We found that 10 μM HCIQ2c1 dramatically decreases histamine-induced intracellular Ca²⁺ release and LPS-induced reactive oxygen species (ROS) production in RAW 264.7 macrophages. Moreover, HCIQ2c1 significantly inhibited the production of LPS-induced tumor necrosis factor α (TNF-α), inducible NO-synthase (iNOS), and 5-lipoxygenase (5-LO) but slightly influenced the IL-1β and cyclooxygenase-2 (COX-2) expression level in macrophages. Furthermore, intravenous administration by HCIQ2c1 at 0.1 mg/kg dose reduced LPS-induced TNF-α, IL-1β, COX-2, and iNOS gene expression in CD-1 mice. The subplantar administration of HCIQ2c1 at 0.1 mg/kg dose to mice significantly reduced carrageenan-induced paw edema by a factor of two, which is comparable to the effect of diclofenac at 1 mg/kg dose. Thus, peptide HCIQ2c1 has a strong anti-inflammatory potential by the attenuation of systemic and local inflammatory effects through the inhibition of intracellular Ca²⁺ release, the production of ROS and pro-inflammatory cytokines, and enzymes involved in arachidonic acid metabolism. Full article

Coagulation factor XIa is a new serine-protease family drug target for next-generation anticoagulants. With the snake venom Kunitz-type peptide BF9 as the scaffold, we obtained a highly active XIa inhibitor BF9-N17K in our previous work, but it also inhibited the hemostatic target plasmin. Here, in order to enhance the selectivity of BF9-N17K toward XIa, four mutants, BF9-N17K-L19A, BF9-N17K-L19S, BF9-N17K-L19D, and BF9-N17K-L19K, were further designed using the P2′ amino acid classification scanning strategy. The anticoagulation assay showed that the four P2′ single-point mutants still had apparent inhibitory anticoagulation activity that selectively inhibited the human intrinsic coagulation pathway and had no influence on the extrinsic coagulation pathway or common coagulation pathway, which indicated that the single-point mutants had minimal effects on the anticoagulation activity of BF9-N17K. Interestingly, the enzyme inhibitor assay experiments showed that the XIa and plasmin inhibitory activities were significantly changed by the P2′ amino acid replacements. The XIa inhibitory activity of BF9-N17K-L19D was apparently enhanced, with an IC₅₀ of 19.28 ± 2.53 nM, and its plasmin inhibitory was significantly weakened, with an IC₅₀ of 459.33 ± 337.40 nM. BF9-N17K-L19K was the opposite to BF9-N17K-L19D, which had enhanced plasmin inhibitory activity and reduced XIa inhibitory activity. For BF9-N17K-L19A and BF9-N17K-L19S, no apparent changes were found in the serine protease inhibitory activity, and they had similar XIa and plasmin inhibitory activities to the template peptide BF9-N17K. These results suggested that the characteristics of the charge of the P2′ site might be associated with the drug selectivity between the anticoagulant target XIa and hemostatic target plasmin. In addition, according to the molecular diversity and sequence conservation, a common motif GR/PCR/KA/SXIP-XYGGC is proposed in the XIa-inhibitory Kunitz-type peptides, which might provide a new clue for further peptide engineering. In conclusion, through P2′ amino acid classification scanning with the snake venom Kunitz-type peptide scaffold, a new potent and selective XIa inhibitor, BF9-N17K-L19D, was discovered, which provides a new XIa-targeting lead drug template for the treatment of thrombotic-related diseases. Full article

Plant protease inhibitors are a ubiquitous feature of plant species and exert a substantial influence on plant stress responses. However, the KTI (Kunitz trypsin inhibitor) family responding to abiotic stress has not been fully characterized in Populus yunnanensis. In this study, we conducted a genome-wide study of the KTI family and analyzed their gene structure, gene duplication, conserved motifs, cis-acting elements, and response to stress treatment. A total of 29 KTIs were identified in the P. yunnanensis genome. Based on phylogenetic analysis, the PyKTIs were divided into four groups (1,2, 3, and 4). Promoter sequence analysis showed that the PyKTIs contain many cis-acting elements related to light, plant growth, hormone, and stress responses, indicating that PyKTIs are widely involved in various biological regulatory processes. RNA sequencing and real-time quantitative polymerase chain reaction analysis showed that KTI genes were differentially expressed under the inverted cutting stress of P. yunnanensis. Transcriptome analysis of P. yunnanensis leaves revealed that PyKTI16, PyKTI18, and PyKTI19 were highly upregulated after inverted cutting. Through the GEO query of Populus transcriptome data, KTI genes played a positive defense role in MeJa, drought, time series, and pathogen stress. This study provided comprehensive information for the KTI family in P. yunnanensis, which should be helpful for the functional characterization of P. yunnanensis KTI genes in the future. Full article

TRPA1 is a homotetrameric non-selective calcium-permeable channel. It contributes to chemical and temperature sensitivity, acute pain sensation, and development of inflammation. HCIQ2c1 is a peptide from the sea anemone Heteractis magnifica that inhibits serine proteases. Here, we showed that HCIQ2c1 significantly reduces AITC- and capsaicin-induced pain and inflammation in mice. Electrophysiology recordings in Xenopus oocytes expressing rat TRPA1 channel revealed that HCIQ2c1 binds to open TRPA1 and prevents its transition to closed and inhibitor-insensitive ‘hyperactivated’ states. NMR study of the ¹⁵N-labeled recombinant HCIQ2c1 analog described a classical Kunitz-type structure and revealed two dynamic hot-spots (loops responsible for protease binding and regions near the N- and C-termini) that exhibit simultaneous mobility on two timescales (ps–ns and μs–ms). In modelled HCIQ2c1/TRPA1 complex, the peptide interacts simultaneously with one voltage-sensing-like domain and two pore domain fragments from different channel’s subunits, and with lipid molecules. The model explains stabilization of the channel in the open conformation and the restriction of ‘hyperactivation’, which are probably responsible for the observed analgetic activity. HCIQ2c1 is the third peptide ligand of TRPA1 from sea anemones and the first Kunitz-type ligand of this channel. HCIQ2c1 is a prototype of efficient analgesic and anti-inflammatory drugs. Full article

Kraits are venomous snakes of the genus Bungarus from the family Elapidae. Their venom typically demonstrates neurotoxicity; however, the toxicity is significantly influenced by the snake’s species and geographical origin. Among the Bungarus species, Bungarus suzhenae and B. bungaroides have been poorly studied, with little to no information available regarding their venom composition. In this study, a proteomic approach was employed using LC-MS/MS to identify proteins from trypsin-digested peptides. The analysis revealed 102 venom-related proteins from 18 distinct functional protein families in the venom of B. suzhenae, with the primary components being three-finger toxins (3-FTx, 25.84%), phospholipase A₂ (PLA₂, 40.29%), L-amino acid oxidase (LAAO, 10.33%), Kunitz-type serine protease inhibitors (KUN, 9.48%), and snake venom metalloproteinases (SVMPs, 6.13%). In the venom of B. bungaroides, 99 proteins from 17 families were identified, with primary components being 3-FTx (33.87%), PLA₂ (37.91%), LAAO (4.21%), and KUN (16.60%). Enzymatic activity assays confirmed the presence of key venom enzymes. Additionally, the LD50 values for B. suzhenae and B. bungaroides were 0.0133 μg/g and 0.752 μg/g, respectively, providing a reference for toxicity studies of these two species. This research elucidates the proteomic differences in the venoms of these two species, offering a foundation for developing antivenoms and clinical treatments for envenomation. Full article

Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of Acinetobacter baumannii and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system. Full article

Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a similar 3D fold and disulphide bond topology, they have remarkably different amino acid sequence (only ~24% sequence identity), thermal stability, folding pathways, protease specificity, and even mechanism of protease inhibition. Here, fully active and correctly folded TAP was produced in reasonably high yields (~20%) by solid-phase peptide chemical synthesis and thoroughly characterised with respect to its chemical identity, disulphide pairing, folding kinetics, conformational dynamics, and fXa inhibition. The versatility of the chemical synthesis was exploited to perform structure–activity relationship studies on TAP by incorporating non-coded amino acids at positions 1 and 3 of the inhibitor. Using Hydrogen–Deuterium Exchange Mass Spectrometry, we found that TAP has a remarkably higher conformational flexibility compared to BPTI, and propose that these different dynamics could impact the different folding pathway and inhibition mechanisms of TAP and BPTI. Hence, the TAP/BPTI pair represents a nice example of divergent evolution, while the relative facility of TAP synthesis could represent a good starting point to design novel synthetic analogues with improved pharmacological profiles. Full article

Kunitz trypsin inhibitor genes play important roles in stress resistance. In this study, we investigated RpKTI2 cloned from Robinia pseudoacacia and its effect on tobacco. RpKTI2 was introduced into the tobacco cultivar NC89 using Agrobacterium-mediated transformation. Six RpKTI2-overexpressing lines were obtained. Transgenic and wild-type tobacco plants were then compared for photosynthetic characteristics and endogenous hormone levels. Transgenic tobacco showed minor changes in chlorophyll content, fluorescence, and photosynthetic functions. However, the maximum photochemical efficiency (F_v/F_m) increased significantly while intercellular CO₂ concentration (C_i) decreased significantly. Stomatal size and hormone content (indole-3-acetic acid, zeatin riboside, gibberellin, and indole-3-propionic acid) were reduced, while brassinosteroid content increased. Random forest regression revealed that RpKTI2 overexpression had the biggest impact on carotenoid content, initial fluorescence, C_i, stomatal area, and indole-3-acetic acid. Overall, RpKTI2 overexpression minimally affected chlorophyll synthesis and photosynthetic system characteristics but influenced stomatal development and likely enhanced the antioxidant capacity of tobacco. These findings provide a basis for future in-depth research on RpKTI2. Full article