A lava-inspired proteolytic enzyme therapy on cancer with a PEG-based hydrogel enhances tumor distribution and penetration of liposomes.

The enhanced permeability and retention (EPR) effect has become the guiding principle for nanomedicine against cancer for a long time. However, several biological barriers severely resist therapeutic agents' penetration and retention into the deep tumor tissues, resulting in poor EPR effect and high tumor mortality. Inspired by lava, we proposed a proteolytic enzyme therapy to improve the tumor distribution and penetration of nanomedicine. A trypsin-crosslinked hydrogel (Trypsin@PSA Gel) was developed to maintain trypsin's activity. The hydrogel postponed trypsin's self-degradation and sustained the release. Trypsin promoted the cellular uptake of nanoformulations in breast cancer cells, enhanced the penetration through endothelial cells, and degraded total and membrane proteins. Proteomic analysis reveals that trypsin affected ECM components and down-regulated multiple pathways associated with cancer progression. Intratumoral injection of Trypsin@PSA Gel significantly increased the distribution of liposomes in tumors and reduced tumor vasculature. Combination treatment with intravenous injection of gambogic acid-loaded liposomes and intratumoral injection of Trypsin@PSA Gel inhibited tumor growth. The current study provides one of the first investigations into the enhanced tumor distribution of liposomes induced by a novel proteolytic enzyme therapy.

Antioxidant and Antimicrobial Potential of Peptide from Rabbit Meat Hydrolysate Prepared by Trypsin and Zingibain.

<b>Background and Objective:</b> Rabbit meat is a livestock product potentially viable as a protein source to obtain peptides. Antioxidant and antimicrobial peptides are ingredients extracted from various foods through enzymatic hydrolysis, chemical hydrolysis and fermentation to produce health-promoting foods. This research aims to investigate the potential of rabbit meat as a source of antioxidant and antimicrobial peptides through hydrolysis using trypsin and zingibain enzymes. <b>Materials and Methods:</b> This research conducted an explorative-descriptive approach, focusing on antioxidant and antimicrobial activity. Rabbit meat was extracted using trypsin, zingibain and a combination of trypsin and crude extract zingibain. The hydrolyzed rabbit meat extract was tested at intervals of 0, 2, 6, 16, 24, 40 and 48 hrs to determine the degree of hydrolysis and the profile of hydrolyzed proteins with electrophoresis SDS PAGE. The antioxidant activity was tested using the DPPH method and the antimicrobial activity using agar well diffusion method. <b>Results:</b> The degree of hydrolysis increased with the hydrolysis time. The highest protein content of rabbit meat extract hydrolyzed with trypsin was 287.65 mg/mL, observed during 12 hrs hydrolysis. The optimum conditions for the hydrolysis of rabbit meat protein were obtained at 24 hrs, with an IC₅₀ value of 52.45% hydrolyzed by trypsin. As per antimicrobial activities, <i>Escherichia coli</i> and <i>Salmonella</i> sp. were more effective in inhibiting rabbit meat hydrolysates compared to <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i>. The inhibition of all pathogen increased until 12 hrs hydrolysis but decreased in 24 hrs hydrolysis. <b>Conclusion:</b> The combination zingibain enzyme and trypsin is feasible for hydrolyzing rabbit meat and the optimum hydrolysis time was 24 hrs with IC₅₀ 52.45 ppm, although accompanied by reduction in antibacterial activities.

Mechanism for improving the in vitro digestive properties of coconut milk by modifying the structure and properties of coconut proteins with monosodium glutamate.

In this paper, the effect of monosodium glutamate (MSG) on coconut protein (CP) solubility, surface hydrophobicity, emulsification activity, ultraviolet spectroscopy and fluorescence spectroscopy was investigated. Meanwhile, the changes in the in vitro digestive properties of coconut milk were also further analyzed. MSG treatment altered the solubility and surface hydrophobicity of CP, thereby improving protein digestibility. Molecular docking showed that CP bound to pepsin and trypsin mainly through hydrogen bonds and salt bridges. And MSG increased the cleavable sites of pepsin and trypsin on CP, thus further improving the protein digestibility. In addition, MSG increased the Na+ concentration in coconut milk, promoted flocculation and aggregation between coconut milk droplets, which prevented the binding of lipase and oil droplets and inhibited lipid digestion. These findings may provide new ideas and insights to improve the digestive properties of plant-based milk.

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.

Characteristics of Trypsin genes and their roles in insecticide resistance based on omics and functional analyses in the malaria vector Anopheles sinensis.

Trypsin is one of the most diverse and widely studied protease hydrolases. However, the diversity and characteristics of the Trypsin superfamily of genes have not been well understood, and their role in insecticide resistance is yet to be investigated. In this study, a total of 342 Trypsin genes were identified and classified into seven families based on homology, characteristic domains and phylogenetics in Anopheles sinensis, and the LY-Domain and CLECT-Domain families are specific to the species. Four Trypsin genes, (Astry2b, Astry43a, Astry90, Astry113c) were identified to be associated with pyrethroid resistance based on transcriptome analyses of three field resistant populations and qRT-PCR validation, and the knock-down of these genes significantly decrease the pyrethroid resistance of Anopheles sinensis based on RNAi. The activity of Astry43a can be reduced by five selected insecticides (indoxacarb, DDT, temephos, imidacloprid and deltamethrin); and however, the Astry43a could not directly metabolize these five insecticides, like the trypsin NYD-Tr did in earlier reports. This study provides the overall information frame of Trypsin genes, and proposes the role of Trypsin genes to insecticide resistance. Further researches are necessary to investigate the metabolism function of these trypsins to insecticides.

Improved Detection of Tryptic Peptides from Tissue Sections Using Desorption Electrospray Ionization Mass Spectrometry Imaging.

DESI-MSI is an ambient ionization technique used frequently for the detection of lipids, small molecules, and drug targets. Until recently, DESI had only limited use for the detection of proteins and peptides due to the setup and needs around deconvolution of data resulting in a small number of species being detected at lower spatial resolution. There are known differences in the ion species detected using DESI and MALDI for nonpeptide molecules, and here, we identify that this extends to proteomic species. DESI MS images were obtained for tissue sections of mouse and rat brain using a precommercial heated inlet (approximately 450 °C) to the mass spectrometer. Ion mobility separation resolved spectral overlap of peptide ions and significantly improved the detection of multiply charged species. The images acquired were of pixel size 100 µm (rat brain) and 50 µm (mouse brain), respectively. Observed tryptic peptides were filtered against proteomic target lists, generated by LC-MS, enabling tentative protein assignment for each peptide ion image. Precise localizations of peptide ions identified by DESI and MALDI were found to be comparable. Some spatially localized peptides ions were observed in DESI that were not found in the MALDI replicates, typically, multiply charged species with a low mass to charge ratio. This method demonstrates the potential of DESI-MSI to detect large numbers of tryptic peptides from tissue sections with enhanced spatial resolution when compared to previous DESI-MSI studies.

Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network.

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead-based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.

Characterization and Hydrolysis Mechanism Analysis of a Cold-Adapted Trypsin-Like Protease from Antarctic Krill.

Cold-adapted proteases are capable of efficient protein hydrolysis at reduced temperatures, which offer significant potential applications in the area of low temperature food processing. In this paper, we attempted to characterize cold-adapted proteases from Antarctic krill. Antarctic krill possesses an extremely active autolytic enzyme system in their bodies, and the production of peptides and free amino acids accompanies the rapid breakdown of muscle proteins following the death. The crucial role of trypsin in this process is recognized. A cold-adapted trypsin named OUC-Pp-20 from Antarctic krill genome was cloned and expressed in Pichia pastoris. Recombinant trypsin is a monomeric protein of 26.8 ± 1.0 kDa with optimum reaction temperature at 25 °C. In addition, the catalytic specificity of OUC-Pp-20 was assessed by identifying its hydrolysis sites through LC-MS/MS. OUC-Pp-20 appeared to prefer Gln and Asn at the P1 position, which is an amino acid with an amide group in its side chain. Hydrolysis reactions on milk and shrimp meat revealed that it can effectively degrade allergenic components in milk and arginine kinase in shrimp meat. These findings update the current knowledge of cold-adapted trypsin and demonstrate the potential application of OUC-Pp-20 in low temperature food processing.

Host-derived protease promotes aggregation of Staphylococcus aureus by cleaving the surface protein SasG.

Staphylococcus aureus is one of the leading causes of hospital-acquired infections, many of which begin following attachment and accumulation on indwelling medical devices or diseased tissue. These infections are often linked to the establishment of biofilms, but another often overlooked key characteristic allowing S. aureus to establish persistent infection is the formation of planktonic aggregates. Such aggregates are physiologically similar to biofilms and protect pathogens from innate immune clearance and increase antibiotic tolerance. The cell-wall-associated protein SasG has been implicated in biofilm formation via mechanisms of intercellular aggregation but the mechanism in the context of disease is largely unknown. We have previously shown that the expression of cell-wall-anchored proteins involved in biofilm formation is controlled by the ArlRS-MgrA regulatory cascade. In this work, we demonstrate that the ArlRS two-component system controls aggregation, by repressing the expression of sasG by activation of the global regulator MgrA. We also demonstrate that SasG must be proteolytically processed by a non-staphylococcal protease to induce aggregation and that strains expressing functional full-length sasG aggregate significantly upon proteolysis by a mucosal-derived host protease found in human saliva. We used fractionation and N-terminal sequencing to demonstrate that human trypsin within saliva cleaves within the A domain of SasG to expose the B domain and induce aggregation. Finally, we demonstrated that SasG is involved in virulence during mouse lung infection. Together, our data point to SasG, its processing by host proteases, and SasG-driven aggregation as important elements of S. aureus adaptation to the host environment.IMPORTANCEHere, we demonstrate that the Staphylococcus aureus surface protein SasG is important for cell-cell aggregation in the presence of host proteases. We show that the ArlRS two-component regulatory system controls SasG levels through the cytoplasmic regulator MgrA. We identified human trypsin as the dominant protease triggering SasG-dependent aggregation and demonstrated that SasG is important for S. aureus lung infection. The discovery that host proteases can induce S. aureus aggregation contributes to our understanding of how this pathogen establishes persistent infections. The observations in this study demonstrate the need to strengthen our knowledge of S. aureus surface adhesin function and processing, regulation of adhesin expression, and the mechanisms that promote biofilm formation to develop strategies for preventing chronic infections.

Accelerating Proteomics Using Broad Specificity Proteases.

Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000× less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow "on-the-fly" digestion of samples in the future.

High-throughput method for Peptide mapping and Amino acid sequencing for Calcitonin Salmon in Calcitonin Salmon injection using Ultra High Performance Liquid Chromatography - High Resolution Mass Spectrometry (UHPLC-HRMS) with the application of Bioinformatic tools.

BACKGROUND: Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic peptide drugs, and peptide drug products including side chain modifications in the Peptide drugs. This article explains a step-by-step guide to developing a high-throughput method using high resolution mass spectrometry for characterization of Calcitonin Salmon injection containing high proportion of UV-active excipients. METHODS: The major challenge in the method development of Amino acid sequencing and Peptide mapping was presence of phenol in drug product. Phenol is a UV-active excipient and reacts with both Dithiothreitol (DTT) and Trypsin. Hence Calcitonin Salmon was extracted from the Calcitonin Salmon injection using solid phase extraction after the extraction, Amino acid sequencing and peptide mapping study was performed. Upon incubation of Calcitonin Salmon with Trypsin and DTT, digested fragments were generated which were separated by mass compatible reverse phase chromatography and the molecular mass of each fragment was determined using HRMS. RESULTS: A reverse phase chromatographic method was developed using UHPLC-HRMS for the determination of direct mass, peptide mapping and to determine the amino acid sequencing in the Calcitonin Salmon injection. The method was found Specific and fragments after trypsin digest are well resolved from each other and the molecular mass of each fragment was determined using HRMS. Sequencing was performed using automated identification of b and y ions annotation and identifications based on MS/MS spectra using Biopharma finder and Proteome discoverer software. CONCLUSION: Using this approach 100% protein coverage was obtained and protein was identified as Calcitonin Salmon and the observed masses of tryptic digest of peptide was found similar with theoretical masses. The method can be used for both UV and MS based Peptide mapping and whereas the UV based peptide mapping method can be used as identification test for Calcitonin Salmon drug substance and drug product in quality control.

DbGTi: Thermostable trypsin inhibitor from Dioscorea bulbifera L. ground tubers: assessment of antioxidant and antibacterial properties and cytotoxicity evaluation using zebrafish model.

Oxidative stress disorders and diseases caused by drug-resistant bacteria have emerged as significant public health concerns. Plant-based medications like protease inhibitors are growing despite adverse effects therapies. Consecutively, in this study, trypsin inhibitors from Dioscorea bulbifera L. (DbGTi trypsin inhibitor) ground tubers were isolated, purified, characterized, and evaluated for their potential cytotoxicity, antibacterial, and antioxidant activities. DbGTi protein was purified by Q-Sepharose matrix, followed by trypsin inhibitory activity. The molecular weight of the DbGTi protein was found to be approximately 31 kDa by SDS-PAGE electrophoresis. The secondary structure analysis by circular dichroism (CD) spectroscopy revealed that the DbGTi protein predominantly comprises ß sheets followed by α helix. DbGTi protein showed competitive type of inhibition with Vmax = 2.1372 × 10-1 µM/min, Km = 1.1805 × 102 µM, & Ki = 8.4 × 10-9 M and was stable up to 70 °C. DbGTi protein exhibited 58 % similarity with Dioscorin protein isolated from Dioscorea alata L. as revealed by LC-MS/MS analysis. DbGTi protein showed a non-toxic effect, analyzed by MTT, Haemolytic assay and in vivo studies on zebrafish model. DbGTi protein significantly inhibited K. pneumoniae and has excellent antioxidant properties, confirmed by various antioxidant assays. The results of anti-microbial, cytotoxicity and antioxidant assays demonstrate its bioactive potential and non-toxic nature.

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.

Trypsin-induced aggregation of transthyretin Valine 30 variants associated with hereditary amyloidosis.

Amyloid fibrils of transthyretin (TTR) consist of full-length TTR and C-terminal fragments starting near residue 50. However, the molecular mechanism underlying the production of the C-terminal fragment remains unclear. Here, we investigated trypsin-induced aggregation and urea-induced unfolding of TTR variants associated with hereditary amyloidosis. Trypsin strongly induced aggregation of variants V30G and V30A, in each of which Val30 in the hydrophobic core of the monomer was mutated to less-bulky amino acids. Variants V30L and V30M, in each of which Val30 was mutated to bulky amino acids, also exhibited trypsin-induced aggregation. On the other hand, pathogenic variant I68L as well as the nonpathogenic V30I did not exhibit trypsin-induced aggregation. The V30G variant was extremely unstable compared with the other variants. The V30G mutation caused the formation of a cavity and the rearrangement of Leu55 in the hydrophobic core of the monomer. These results suggest that highly destabilized transthyretin variants are more susceptible to trypsin digestion.

Mirror-image trypsin digestion and sequencing of D-proteins.

The development of mirror-image biology systems and related applications is hindered by the lack of effective methods to sequence mirror-image (D-) proteins. Although natural-chirality (L-) proteins can be sequenced by bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS), the sequencing of long D-peptides and D-proteins with the same strategy requires digestion by a site-specific D-protease before mass analysis. Here we apply solid-phase peptide synthesis and native chemical ligation to chemically synthesize a mirror-image version of trypsin, a widely used protease for site-specific protein digestion. Using mirror-image trypsin digestion and LC-MS/MS, we sequence a mirror-image large subunit ribosomal protein (L25) and a mirror-image Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and distinguish between different mutants of D-Dpo4. We also perform writing and reading of digital information in a long D-peptide of 50 amino acids. Thus, mirror-image trypsin digestion in conjunction with LC-MS/MS may facilitate practical applications of D-peptides and D-proteins as potential therapeutic and informational tools.

A Multicomponent Mannich Reaction Catalyzed by Hydrolases Immobilized on Titanate Nanotubes.

This study presents an innovative method for synthesizing ß-amino carbonylated compounds, specifically 2-[phenyl(phenylamino)methyl] cyclohexanone, achieving high conversions and diastereomeric ratios. Using trypsin or α-chymotrypsin in both free and immobilized forms on titanate nanotubes (NtsTi), synthesized through alkaline hydrothermal methods, successful immobilization yields were attained. Notably, α-chymotrypsin, when free, displayed a diastereoselective synthesis of the anti-isomer with 97 % conversion and 16 : 84 (syn : anti) diastereomeric ratio, which slightly decreased upon immobilization on NtsTi. Trypsin, in its free form, exhibited diastereoselective recognition of the syn-isomer, while immobilization on NtsTi (trypsin/NtsTi) led to an inversion of diastereomeric ratio. Both trypsin/NtsTi and α-chymotrypsin/NtsTi demonstrated significant catalytic efficiency over five cycles. In conclusion, NtsTi serves as an effective support for trypsin and α-chymotrypsin immobilization, presenting promising prospects for diastereoselective synthesis and potential industrial applications. Furthermore, it offers promising prospects for the diastereoselective synthesis of 2-[phenyl(phenylamino)methyl] cyclohexanone through multicomponent Mannich reaction and future industrial application.

A novel trypsin of Trichinella spiralis mediates larval invasion of gut epithelium via binding to PAR2 and activating ERK1/2 pathway.

BACKGROUND: Proteases secreted by Trichinella spiralis intestinal infective larvae (IIL) play an important role in larval invasion and pathogenesis. However, the mechanism through which proteases mediate larval invasion of intestinal epithelial cells (IECs) remains unclear. A novel T. spiralis trypsin (TsTryp) was identified in IIL excretory/secretory (ES) proteins. It was an early and highly expressed protease at IIL stage, and had the potential as an early diagnostic antigen. The aim of this study was to investigate the biological characteristics of this novel TsTryp, its role in larval invasion of gut epithelium, and the mechanisms involved. METHODOLOGY/PRINCIPAL FINDING: TsTryp with C-terminal domain was cloned and expressed in Escherichia coli BL21 (DE3), and the rTsTryp had the enzymatic activity of natural trypsin, but it could not directly degrade gut tight junctions (TJs) proteins. qPCR and western blotting showed that TsTryp was highly expressed at the invasive IIL stage. Immunofluorescence assay (IFA), ELISA and Far Western blotting revealed that rTsTryp specifically bound to IECs, and confocal microscopy showed that the binding of rTsTryp with IECs was mainly localized in the cytomembrane. Co-immunoprecipitation (Co-IP) confirmed that rTsTryp bound to protease activated receptors 2 (PAR2) in Caco-2 cells. rTsTryp binding to PAR2 resulted in decreased expression levels of ZO-1 and occludin and increased paracellular permeability in Caco-2 monolayers by activating the extracellular regulated protein kinases 1/2 (ERK1/2) pathway. rTsTryp decreased TJs expression and increased epithelial permeability, which could be abrogated by the PAR2 antagonist AZ3451 and ERK1/2 inhibitor PD98059. rTsTryp facilitated larval invasion of IECs, and anti-rTsTryp antibodies inhibited invasion. Both inhibitors impeded larval invasion and alleviated intestinal inflammation in vitro and in vivo. CONCLUSIONS: TsTryp binding to PAR2 activated the ERK1/2 pathway, decreased the expression of gut TJs proteins, disrupted epithelial integrity and barrier function, and consequently mediated larval invasion of the gut mucosa. Therefore, rTsTryp could be regarded as a potential vaccine target for blocking T. spiralis invasion and infection.

Study of an inhibitory effect of plant polyphenolic compounds against digestive enzymes using bench-working experimental evidence predicted by molecular docking and dynamics.

The substantial nutritional content and diversified biological activity of plant-based nutraceuticals are due to polyphenolic chemicals. These chemicals are important and well-studied plant secondary metabolites. Their protein interactions are extensively studied. This relationship is crucial for the logical development of functional food and for enhancing the availability and usefulness of polyphenols. This study highlights the influence of protein types and polyphenols on the interaction, where the chemical bindings predominantly consist of hydrophobic interactions and hydrogen bonds. The interaction between polyphenolic compounds (PCs) and digestive enzymes concerning their inhibitory activity has not been fully studied. Therefore, we have examined the interaction of four digestive enzymes (α-amylase, pepsin, trypsin, and α-chymotrypsin) with four PCs (curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone) through in silico and in vitro approaches. In vitro plate assays, enzyme kinetics, spectroscopic assays, molecular docking, and simulations were performed. We observed all these PCs have significant docking scores and preferable interaction with the active site of the digestive enzymes, resulting in the reduction of enzyme activity. The enzyme-substrate binding mechanism was determined using the Lineweaver Burk plot, indicating that the inhibition occurred competitively. Among four PCs diosmin and morin has the highest interaction energy over digestive enzymes with IC50 value of 1.13 ± 0.0047 and 1.086 ± 0.0131 µM. Kinetic studies show that selected PCs inhibited pepsin, trypsin, and chymotrypsin competitively and inhibited amylase in a non-competitive manner, especially by 2',3',4'-trihydroxychalcone. This study offers insights into the mechanisms by which the selected PCs inhibit the enzymes and has the potential to enhance the application of curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone as natural inhibitors of digestive enzymes.

Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation.

To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.

MiR-2b-3p Downregulated PxTrypsin-9 Expression in the Larval Midgut to Decrease Cry1Ac Susceptibility of the Diamondback Moth, Plutella xylostella (L.).

Crystal (Cry) toxins, produced by Bacillus thuringiensis, are widely used as effective biological pesticides in agricultural production. However, insects always quickly evolve adaptations against Cry toxins within a few generations. In this study, we focused on the Cry1Ac protoxin activated by protease. Our results identified PxTrypsin-9 as a trypsin gene that plays a key role in Cry1Ac virulence in Plutella xylostella larvae. In addition, P. xylostella miR-2b-3p, a member of the micoRNA-2 (miR-2) family, was significantly upregulated by Cry1Ac protoxin and targeted to PxTrypsin-9 downregulated its expression. The mRNA level of PxTrypsin-9, regulated by miR-2b-3p, revealed an increased tolerance of P. xylostella larvae to Cry1Ac at the post-transcriptional level. Considering that miR-2b and trypsin genes are widely distributed in various pest species, our study provides the basis for further investigation of the roles of miRNAs in the regulation of the resistance to Cry1Ac and other insecticides.