Unsaponifiable Compounds and Phenols Content, Antioxidant and Antitrypsin Activities of Prunus persica Kernel Oil.

Although peach kernels are rich in oil, there is a lack of information about its chemical and biological properties. Therefore, the purpose of this study was to determine the lipid profile, antioxidant capacity, and trypsin inhibitory propriety of peach oil extracted from two varieties (sweet cap and O'Henry) cultivated in Tunisia. The investigated peach kernel oil contains significant amount of unsaponifiable (2.1±0.5-2.8±0.2% of oil) and phenolic compounds (45.8±0.92-74.6±1.3 mg GAE/g of oil). Its n-alkane profile was characterized by the predominance of tetracosane n-C24 (47.24%) followed by tricosane n-C23 (34.43%). An important total tocopherol content (1192.83±3.1 mg/kg oil) has been found in sweet cap cultivar. Although rich in polyphenols and tocopherols, the tested oil did not display an inhibitory effect on trypsin. However, all peach oil samples showed effective antioxidant capacity and the highest values (86.34±1.3% and 603.50±2.6 µmol TE/g oil for DPPH test and ORAC assay, respectively) were observed for sweet cap oil. Peach oil has an excellent potential for application in the food and pharmaceutical industries as source of naturally-occurring bioactive substances.

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.

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.

Expression and Purification of Recombinant Bowman-Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo.

Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-ß-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.

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.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Serum interα-trypsin inhibitor heavy chain H4 may be an anti-inflammatory marker reflecting disease risk, activity and treatment outcome of ankylosing spondylitis.

Interα-trypsin inhibitor heavy chain H4 (ITIH4) modulates inflammation and immunity, which take part in the pathogenesis of ankylosing spondylitis (AS). The current research intended to discover the clinical value of serum ITIH4 quantification for AS management. Serum ITIH4 among 80 AS patients before current treatment initiation (baseline) at weeks (W) 4, 8 and 12 after treatment was detected by ELISA. Serum ITIH4 from 20 disease controls (DCs) and 20 healthy controls (HCs) was detected. ITIH4 expression was lower in AS patients than in DCs (p = 0.002) and HCs (p < 0.001). Among AS patients, ITIH4 was negatively associated with C-reactive protein (CRP) (r = -0.311, p = 0.005), bath AS disease activity index (BASDAI) (r = -0.223, p = 0.047), total pack pain (r = -0.273, p = 0.014) and AS disease activity score (ASDAS) (CRP) (r = -0.265, p = 0.018). Meanwhile, ITIH4 was negatively related to tumor necrosis factor (TNF)-α (r = -0.364, p = 0.001), interleukin (IL)-1ß (r = -0.251, p = 0.025), IL-6 (r = -0.292, p = 0.009) and IL-17A (r = -0.254, p = 0.023). After treatment, the assessment of the spondylitis arthritis international society 40 response rate was 28.7% at W4, 46.3% at W8 and 55.0% at W12; ITIH4 showed an increasing trend from baseline to W12 (p < 0.001). Furthermore, ITIH4 at W8 (p = 0.020) and W12 (p = 0.035), but not at baseline or W4 (both p > 0.05), was enhanced in response patients vs. nonresponse patients. Additionally, ITIH4 at W12 was increased in AS patients receiving TNF inhibitors vs. those receiving nonsteroidal anti-inflammatory drugs (NSAIDs) (p = 0.024). Serum ITIH4 increases after treatment, and its augmentation is correlated with lower disease activity, decreased inflammation and enhanced treatment response in AS patients.

A fluorescent quantum dot conjugate to probe the interaction of Enterolobium contortisiliquum trypsin inhibitor with cancer cells.

Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.

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.

Clinical efficacy of ulinastatin in the treatment of unliquefied pyogenic liver abscess complicated by septic shock: A randomized controlled trial.

INTRODUCTION: This study determined the therapeutic effect of ulinastatin (UTI) on unliquefied pyogenic liver abscesses complicated by septic shock (UPLA-SS). METHODS: This was a randomized controlled trial involving patients with UPLA-SS who underwent treatment at our hospital between March 2018 and March 2022. The patients were randomly divided into control (n = 51) and study groups (n = 48). Both groups received routine treatment, but the study group received UTI (200,000 units q8h for >3 days). Differences in liver function, inflammatory indices, and effectiveness between the two groups were recorded. RESULTS: Following treatment, the white blood cell count, and lactate, C-reactive protein, procalcitonin, tumor necrosis factor-α, and interleukin-6 levels were significantly decreased in all patients compared to the admission values (p < .05). The study group had a faster decline with respect to the above indices compared to the control group (p < .05). The study group length of intensive care unit stay, fever duration, and vasoactive drug maintenance time were all significantly shorter than the control group (p < .05). The total bilirubin, alanine aminotransferase, and aspartate aminotransferase levels were significantly lower in the study and control groups after treatment compared to before treatment (p < .05); however, the study group had a faster recovery of liver function than the control group (p < .05). The overall mortality rate was 14.14% (14/99); 10.41% of the study group patients died and 17.65% of the control group patients died, but there was no statistically significant difference between the two groups (p > .05). CONCLUSION: UTI combined with conventional treatment significantly controlled the infection symptoms, improved organ function, and shortened the treatment time in patients with UPLA-SS.

Assessment of Protein Nutritional Quality of Novel Hairless Canary Seed in Comparison to Wheat and Oat Using In Vitro Static Digestion Models.

Hairless canary seed (Phalaris canariensis L.) is a novel true cereal that is now approved for human consumption in Canada and the United States. This true cereal grain has higher protein content (22%) than oat (13%) and wheat (16%) and represents a valuable source of plant proteins. Assessment of canary seed protein quality is therefore essential to evaluate its digestibility and ability to provide sufficient amounts of essential amino acids for human requirements. In this study, the protein nutritional quality of four hairless canary seed varieties (two brown and two yellow) were evaluated in comparison to oat and wheat. The assessment of anti-nutrients contents (phytate, trypsin inhibitor activity, and polyphenols) showed that brown canary seed varieties had the highest content in phytate and oat the highest in polyphenols. Trypsin inhibitor level was comparable among studied cereals, but slightly higher in the brown canary seed Calvi variety. In regard to protein quality, canary seed had a well-balanced amino acid profile and was particularly high in tryptophan, an essential amino acid normally lacking in cereals. The in vitro protein digestibility of canary seeds as determined by both the pH-drop and INFOGEST (international network of excellence on the fate of food in the gastrointestinal tract) protocols appears slightly lower than wheat and higher than oat. The yellow canary seed varieties showed better overall digestibility than the brown ones. For all studied cereal flours, the limiting amino acid was lysine. The calculated in vitro PDCAAS (protein digestibility corrected amino acid score) and DIAAS (digestible indispensable amino acid score) were higher for the yellow C05041 cultivar than the brown Bastia, similar to those of wheat, but lower than those of oat proteins. This study demonstrates the feasibility and utility of in vitro human digestion models for the assessment of protein quality for comparison purpose.

NaMLP, a new identified Kunitz trypsin inhibitor regulated synergistically by JA and ethylene, confers Spodoptera litura resistance in Nicotiana attenuata.

KEY MESSAGE: We identified a miraculin-like protein (NaMLP) who is a new Kunitz trypsin inhibitor regulated synergistically by JA and ethylene signals and confers Spodoptera litura resistance in wild tobacco Nicotiana attenuata. The findings revealed a new source of trypsin inhibitor activities after herbivory, and provide new insights into the complexity of the regulation of trypsin inhibitor-based defense after insect herbivore attack. Upon insect herbivore attack, wild tobacco Nicotiana attenuata accumulates trypsin protease inhibitor (TPI) activities as a defense response from different protease inhibitor (PI) coding genes, including WRKY3-regulated NaKTI2, and JA-dependent NaPI. However, whether any other TPI gene exists in N. attenuata is still unclear. A miraculin-like protein gene (NaMLP) was highly up-regulated in N. attenuata after Alternaria alternata infection. However, silencing or overexpression of NaMLP had no effect on the lesion diameter developed on N. attenuata leaves after A. alternata inoculation. Meanwhile, the transcripts of NaMLP could be induced by wounding and amplified by Spodoptera litura oral secretions (OS). S. litura larvae gained significantly more biomass on NaMLP-silenced plants but less on NaMLP overexpressed plants. Although NaMLP showed low sequence similarity to NaKTI2, it had conserved reaction sites of Kunitz trypsin inhibitors, and exhibited TPI activities when its coding gene was overexpressed transiently or stably in N. attenuata. This was consistent with the worst performance of S. litura larvae on NaMLP overexpressed lines. Furthermore, NaMLP-silenced plants had reduced TPI activities and better S. litura performance. Finally, OS-elicited NaMLP was dramatically reduced in JA-deficient AOC silencing and ethylene-reduced ACO-silencing plants, and the expression of NaMLP could be significantly induced by methyl jasmonate or ethephon alone, but dramatically amplified by co-treatment of both methyl jasmonate and ethephon. Thus, our results demonstrate that in addition to JA-regulated NaPI, and WRKY3/6-dependent NaKTI2, N. attenuata plants also up-regulates TPI activities via NaMLP, which confers S. litura resistance through JA and ethylene signaling pathways in a synergistic way.

Prospecting in silico antibacterial activity of a peptide from trypsin inhibitor isolated from tamarind seed.

Bacterial infections have become a global concern, stimulating the growing demand for natural and biologically safe therapeutic agents with antibacterial action. This study was evaluated the genotoxicity of the trypsin inhibitor isolated from tamarind seeds (TTI) and the antibacterial effect of TTI theoric model, number 56, and conformation number 287 (TTIp 56/287) and derived peptides in silico. TTI (0.3 and 0.6 mg.mL-1) did not cause genotoxicity in cells (p > 0.05). In silico, a greater interaction of TTIp 56/287 with the Gram-positive membrane (GP) was observed, with an interaction potential energy (IPE) of -1094.97 kcal.mol-1. In the TTIp 56/287-GP interaction, the Arginine, Threonine (Thr), and Lysine residues presented lower IPE. In molecular dynamics (MD), Peptidotrychyme59 (TVSQTPIDIPIGLPVR) showed an IPE of -518.08 kcal.mol-1 with the membrane of GP bacteria, and the Thr and Arginine residues showed the greater IPE. The results highlight new perspectives on TTI and its derived peptides antibacterial activity.

Morphology and Enzyme-Mimicking Activity of Copper Nanoassemblies Regulated by Peptide: Mechanism, Ultrasensitive Assaying of Trypsin, and Screening of Trypsin Inhibitors.

To regulate nanostructure synthesis is of crucial importance for developing various applications, including catalysis, bioanalysis, and optical devices. Herein, the morphology and peroxidase (POD)-mimicking activity of peptide-templated copper nanoassemblies (Cu NAs) are regulable with peptide types. The Cu NAs templated with peptide containing single cysteine are uniform nanoclusters with strong POD-like activity. However, the Cu NAs templated with peptide containing two cysteines are fusiform-like with very weak POD-like activity. Unexpectedly, the POD-like activity of Cu NAs templated with peptide containing two cysteines with lysine between the cysteines is significantly enhanced when trypsin is incubated, which is unchanged for the Cu NAs templated with peptide containing two cysteines without lysine between the cysteines. The remarkably enhanced POD-mimicking activity originates from trypsin specifically shearing the peptide bond on the lysine, thereby allowing the aggregated Cu NAs to unravel into individual nanoclusters. Therefore, a robust colorimetric sensing platform was constructed for sensitive and selective detection of trypsin, which showed a linear concentration range of 3-1000 nM and a detection limit of 0.82 nM (S/N = 3). More interestingly, featured by trypsin inhibitor restraining trypsin activity, it enabled us to screen trypsin inhibitors as well. Subsequently, the developed assay was applied to detect trypsin in serum samples with good accuracy and reproducibility. Thus, this strategy shows great potential application in the clinic for diagnosis of trypsin-indicating diseases as well as the screening of trypsin inhibitor-based anti-cancer drugs.

Identification of Pinelliae Rhizoma and its counterfeit species based on enzymatic signature peptides from toxic proteins.

BACKGROUND: Pinelliae Rhizoma (PR), a toxic medication, with long history, is commonly used for eliminating phlegm. Due to the shortage of wild resources and the relative lacking of cultivation technology, it is often confused with its counterfeit species in the market, such as Typhonii Rhizoma (TR), Arisaematis Rhizoma (AR) and tubers of Typhonium flagelliforme (TF) and Pinellia pedatisecta (PP). PURPOSE: It was aimed to screen signature enzymatic peptides from toxic proteins to identify PR and its four counterfeit species. STUDY DESIGN: A comparative proteogenomics strategy based on open-source transcriptome data was applied for screening signature peptides from toxic proteins, which were applied for species authentication of PR and its counterfeit species. METHODS: Firstly, the open-source transcriptome data was used for constructing the annotated protein database, which was used for peptides identification. Secondly, the toxicity of different fractions of PR were evaluated by the rat peritoneal inflammation model. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to profile the main proteins bands of five species, whose sequences were identified based on the in-gel digestion experiment by using ultra-high-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry. Finally, the label-free proteomic analysis was performed to character the proteins and screen the signature peptides of five species, which were validated in commercially available products by dynamic multi reaction monitor (DMRM). RESULTS: The results in this study confirmed that protein was the main toxic components of PR. Both Pinellia ternata agglutinin (PTA) and trypsin inhibitor (TI) like proteins are the main proteins, which were characterized by proteomic analysis based on four annotated protein database. Meanwhile, seven signature peptides from toxic proteins were screened and validated with good repeatability and specificity in commercial products. CONCLUSION: Seven signature enzymatic peptides from toxic protein screened by the comparative proteogenomics strategy based on open-source transcriptome data achieved good identification ability of PR and its four counterfeit species.

Trypsin-like Inhibitor Domain (TIL)-Harboring Protein Is Essential for Aedes aegypti Reproduction.

Cysteine-rich trypsin inhibitor-like domain (TIL)-harboring proteins are broadly distributed in nature but remain understudied in vector mosquitoes. Here we have explored the biology of a TIL domain-containing protein of the arbovirus vector Aedes aegypti, cysteine-rich venom protein 379 (CRVP379). CRVP379 was previously shown to be essential for dengue virus infection in Ae. aegypti mosquitoes. Gene expression analysis showed CRVP379 to be highly expressed in pupal stages, male testes, and female ovaries. CRVP379 expression is also increased in the ovaries at 48 h post-blood feeding. We used CRISPR-Cas9 genome editing to generate two mutant lines of CRVP379 with mutations inside or outside the TIL domain. Female mosquitoes from both mutant lines showed severe defects in their reproductive capability; mutant females also showed differences in their follicular cell morphology. However, the CRVP379 line with a mutation outside the TIL domain did not affect male reproductive performance, suggesting that some CRVP379 residues may have sexually dimorphic functions. In contrast to previous reports, we did not observe a noticeable difference in dengue virus infection between the wild-type and any of the mutant lines. The importance of CRVP379 in Ae. aegypti reproductive biology makes it an interesting candidate for the development of Ae. aegypti population control methods.

Green tea polyphenols bind to soy proteins and decrease the activity of soybean trypsin inhibitors (STIs) in heated soymilk.

The interaction between epigallocatechin gallate (EGCG) and soy proteins at room temperature (25 °C) and after heating at 100 and 121 °C, and their effects on the inactivation of soybean trypsin inhibitors (STIs) in soymilk were investigated. The results of the nitroblue tetrazolium (NBT) staining assay showed that soy proteins can covalently bind to EGCG. The α/α' and A subunits in heated soymilk preferred to bind to EGCG because of their soluble state. More thiols were trapped when EGCG was added before thermal processing, and the free amino groups were depleted more with EGCG addition after heating. Circular dichroism and fluorescence spectroscopy showed that EGCG addition before or after heating induced different secondary and tertiary structural changes for soy proteins. The exposed aromatic amino acids preferred to react with EGCG before protein aggregation in the heating process. The random coil of soymilk proteins increased more when EGCG was added in soymilk after heating, resulting in more disordered structures in protein conformation. The binding between EGCG and soy proteins promoted protein aggregation, which was confirmed by the particle size distribution and gel electrophoresis. The trypsin and chymotrypsin inhibitory activity (TIA and CIA) in soymilk significantly reduced to 693 U mL-1 and 613 U mL-1, respectively, under the conditions of 2 mM EGCG addition after 100 °C heating for 10 min (p < 0.05). Consequently, the influence of EGCG on STI inactivation in soymilk only worked when EGCG was added after heating.

Proliferation and Invasion of Melanoma Are Suppressed by a Plant Protease Inhibitor, Leading to Downregulation of Survival/Death-Related Proteins.

Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment.

Targeting protein-protein interaction for immunomodulation: A sunflower trypsin inhibitor analog peptidomimetic suppresses RA progression in CIA model.

Protein-protein interactions (PPI) of co-stimulatory molecules CD2-CD58 are important in the early stage of an immune response, and increased expression of these co-stimulatory molecules is observed in the synovial region of joints in rheumatoid arthritis (RA) patients. A CD2 epitope region that binds to CD58 was grafted on to sunflower trypsin inhibitor (SFTI) template structure to inhibit CD2-CD58 PPI. The peptide was incorporated with an organic moiety dibenzofuran (DBF) in its structure. The designed peptidomimetic was studied for its ability to inhibit CD2-CD58 interactions in vitro, and its thermal and enzymatic stability was evaluated. Stability studies indicated that the grafted peptidomimetic was stable against trypsin cleavage. In vivo studies using the collagen-induced arthritis (CIA) model in mice indicated that the peptidomimetic was able to slow down the progress of arthritis, an autoimmune disease in the mice model. These studies suggest that with the grafting of organic functional groups in the stable peptide template SFTI stabilizes the peptide structure, and these peptides can be used as a template to design stable peptides for therapeutic purposes.

JcTI-PepI, a synthetic peptide bioinspired in the trypsin inhibitor from Jatropha curcas, presents potent inhibitory activity against C. krusei, a neglected pathogen.

Antimicrobial resistance has been increasing globally, posing a global public health risk. It has prompted the scientific community to look for alternatives to traditional drugs. Antimicrobial Peptides (AMPs) have stood out in this context because they have the potential to control infectious diseases while causing no or little harm to mammalian cells. In the present study, three peptides, JcTI-PepI, JcTI-PepII, and JcTI-PepIII, were designed and tested for antimicrobial activity based on the primary sequence of JcTI-I, a 2S albumin with trypsin inhibitory activity from Jatropha curcas. JcTI-PepI strongly inhibited C. krusei growth, and it caused severe disruptions in cellular processes and cell morphology. C. krusei cells treated with JcTI-PepI showed indicative of membrane permeabilization and overproduction of Reactive Oxygen Species. Moreover, the yeast's ability to acidify the medium was severely compromised. JcTI-PepI was also effective against pre-formed biofilm and did not harm human erythrocytes and Vero cells. Overall, these characteristics indicate that JcTI-PepI is both safe and effective against C. krusei, an intrinsically resistant strain that causes serious health problems and is frequently overlooked. It implies that this peptide has a high potential for use as a new antimicrobial agent in the future.