Search Results (54)

β-Lytic protease (Blp) and protease L5 are enzymes from Lysobacter bacteria with distinct proteolytic and bacteriolytic activities. To characterize their substrate specificity, we employed liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis following hydrolysis of fractionated protein mixtures. Heatmaps and sequence logos revealed a pronounced specificity of Blp towards glycine and lysine residues, while L5 preferentially cleaved non-polar residues such as methionine, phenylalanine, and leucine. Notably, proline was frequently observed at the P2 position in L5 substrates. Comparative analysis with trypsin revealed that L5 generated significantly shorter peptides, whereas Blp produced fragments similar in length to tryptic peptides. These findings indicate different cleavage preferences and suggest potential applications for these enzymes in proteomic analysis. Full article

To systematically elucidate the chronological patterns of embryonic development and morphological changes in the larval and juvenile stages of Silurus glanis, and provide fundamental biological insights into this species, in this study, fertilized eggs were obtained through artificial spawning induction technology. After removing adhesiveness from fertilized eggs using trypsin, a detailed developmental study was conducted. The study systematically analyzed the chronological sequence of embryonic development and the morphological change patterns of larval and juvenile fish. The results showed the following: The fertilized eggs of S. glanis are yellow, spherical, and sticky, and the stickiness allows eggs to attach to spawning substrates, enhancing hatching success. The egg diameter after water absorption was (2.88 ± 0.13) mm. The embryonic development took 47 h and 55 min, with a total accumulated temperature of 1245.56 h degrees Celsius, the developmental process includes seven stages and twenty-six periods, namely the zygophase stage, cleavage stage, blastula stage, gastrula stage, neurula stage, organogenesis stage, and hatching stage. At a temperature of (26.0 ± 0.9) °C, the hatched individuals went through the pre-yolk sac larval stage, late larval stage, juvenile fry stage, and juvenile stage. In the pre-yolk sac larval stage, otoliths appeared in the bilateral otic vesicles, a pair of barbel primordia emerged under the mandible, a short and thin straight intestine formed in the abdominal cavity, and the oral fissure first appeared. In the late larval stage, the fin rays were initially formed, the intestine became thicker and longer, the oral fissure, anus, and cloaca were formed, and the larvae could float and start feeding on exogenous food. In the juvenile fry stage, the differentiation of various organs was basically complete, the nostrils became larger, and both the anal fin and caudal fin had dark black markings. In the juvenile stage, the maxillary barbels elongated, the mucus layer thickened on the body and back, the abdomen is light white, and it had the external morphological characteristics of an adult fish. By measuring and calculating the total length, body length, body height, and head length of S. glanis larvae and juveniles (0–40 days), the results showed that the growth characteristics conformed to the following fish growth formula: TL = 0.0141x² + 0.8096x + 8.2421 (R² = 0.9916), where x denotes days after hatching. This study has preliminarily mastered the chronological patterns of the embryonic development, growth, and formation of the morphological characteristics in larval and juvenile S. glanis, providing scientific data and laying a theoretical foundation for the division of early developmental stages, reproduction, hatching, and fry cultivation. Full article

Angiotensin-converting enzyme 2 (ACE2) is a cell-surface receptor that helps the body regulate blood pressure and endocrine secretions. Transmembrane serine protease 2 (TMPRSS2) is a cell surface protein expressed mainly by endothelial cells of the respiratory and digestive tract, which participates in the cleavage of protein peptide bonds with serine as the active site. These two proteins have been studied to be highly associated with infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Soybean trypsin inhibitor (SBTI) has special bioactivities such as anticarcinogenic and anti-inflammatory functions, which can be widely used in functional foods or drugs. Our study involved in vitro and in vivo experiments to elucidate the effect of SBTI on SARS-CoV-2 host invasion. First, it was confirmed that being under 250 μg/mL of SBTI was not toxic to HepG2, HEK293T, and Calu-3 cells. The animal study administered SBTI to mice once daily for 14 days. In the lungs, liver, and kidneys, the histopathologic findings of the SBTI group were not different from those of the control group, but the expression of ACE2, TMPRSS2, and CD147 was reduced. Thus, our findings suggest that the inhibition of ACE2, TMPRSS,2 and CD147 proteins by SBTI shows promise in potentially inhibiting SARS-CoV-2 infection. Full article

C. perfringens type F isolates are a leading cause of food poisoning and antibiotic-associated diarrhea. Type F isolate virulence requires production of C. perfringens enterotoxin [CPE], which acts by forming large pore complexes in host cell plasma membranes. During GI disease, CPE is produced in the intestines when type F strains undergo sporulation. The toxin is then released into the intestinal lumen when the mother cell lyses at the completion of sporulation. Once present in the lumen, CPE encounters proteases. This study examined the in vitro, ex vivo, and in vivo processing of CPE by intestinal proteases and the effects of this processing on CPE activity. Results using purified trypsin or mouse intestinal contents detected the rapid cleavage of CPE to a major band of ~32 kDa and studies with Caco-2 cells showed that this processed CPE still forms large complexes and retains cytotoxic activity. When mouse small intestinal loops were challenged with CPE, the toxin caused intestinal histologic damage, despite rapid proteolytic processing of most CPE to 32 kDa within 15 min. Intestinal large CPE complexes became more stable with longer treatment times. These results indicate that CPE processing involving trypsin occurs in the intestines and the processed toxin retains enterotoxicity. Full article

In this study, we report the synthesis and characterization of aminated poly(methyl silsesquioxane)-based hydrogels ((AP/MS)SO-hydrogels) as potential enzyme-sensitive vehicles for antianemic drugs. The hydrogels were synthesized via sol–gel polymerization and functionalized with amine groups. Characterization techniques included Congo red assay, Brunauer–Emmett–Teller (BET) surface area analysis, scanning electron microscopy, elemental analysis, ¹³C NMR, ²⁹Si NMR, and ATR-FTIR spectroscopy and microscopy of hydrogels. The sorption of ferric chloride and ferrous D-gluconate, as well as complexes of ferrous D-gluconate with HPCD, was evaluated. Crosslinking of the gel with bifunctional agents was performed to create a new amide enzyme-sensitive bond, followed by infrared characterization of the crosslinked product. Trypsin-mediated degradation studies demonstrated the sensitivity of the hydrogel to enzymatic cleavage under model conditions. Iron release experiments in gastric and intestine-simulating media confirmed prolonged release. Overall, our findings suggest that aminated PMSSO-hydrogels hold promise as versatile and biocompatible carriers for targeted delivery of antianemic agents, warranting further exploration in preclinical and clinical applications. Full article

The insecticidal Cry proteins from Bacillus thuringiensis are used in biopesticides or transgenic crops for pest control. The Cry1I protein family has unique characteristics of being produced during the vegetative rather than sporulation phase, its protoxins forming dimers in solution, and exhibiting dual toxicity against lepidopteran and coleopteran pests. The Cry1Ia protoxin undergoes sequential proteolysis from the N- and C-terminal ends, producing intermediate forms with insecticidal activity, while in some cases, the fully processed toxin is inactive. We investigated the oligomerization and toxicity of Cry1Ia intermediate forms generated through trypsinization (T-Int) and larval gut fluid (GF-Int) treatments, as well as the fully trypsinized protein (toxin). Heterologously expressed intermediate forms assembled into oligomers and showed similar toxicity to Cry1Ia protoxin against Ostrinia nubilalis (European corn borer) larvae, while the toxin form was ~30 times less toxic. In contrast, bioassays with Leptinotarsa decemlineata (Colorado potato beetle) larvae did not show significant differences in toxicity among Cry1Ia protoxin, T-Int, GF-Int, and fully processed toxin. These results suggest that the Cry1I mode of action differs by insect order, with N-terminal cleavage affecting toxicity against lepidopteran but not coleopteran larvae. This knowledge is essential for designing pest control strategies using Cry1I insecticidal proteins. Full article

Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, the subcellular structure that links the intermediate filament network of one cell to its neighbor. A mutation “hot-spot” within the NH₂-terminal of the DSP protein (residues 299–515) is associated with arrhythmogenic cardiomyopathy. In a subset of DSP variants, disease is linked to calpain hypersensitivity. Previous studies show that calpain hypersensitivity can be corrected in vitro through the addition of a bulky residue neighboring the cleavage site, suggesting that physically blocking calpain accessibility is a viable strategy to restore DSP levels. Here, we aim to find drug-like molecules that also block calpain-dependent degradation of DSP. To do this, we screened ~2500 small molecules to identify compounds that specifically rescue DSP protein levels in the presence of proteases. We find that several molecules, including sodium dodecyl sulfate, palmitoylethanolamide, GW0742, salirasib, eprosarten mesylate, and GSK1838705A prevent wildtype and disease-variant-carrying DSP protein degradation in the presence of both trypsin and calpain without altering protease function. Computational screenings did not predict which molecules would protect DSP, likely due to a lack of specific DSP–drug interactions. Molecular dynamic simulations of DSP–drug complexes suggest that some long hydrophobic molecules can bind in a shallow hydrophobic groove that runs alongside the protease cleavage site. Identification of these compounds lays the groundwork for pharmacological treatment for individuals harboring these hypersensitive DSP variants. Full article

Pigs are important animals for meat production but can carry several zoonotic diseases, including the Japanese encephalitis virus, Nipah virus, and influenza viruses. Several Orthomyxoviridae and Coronavirinae respiratory viruses require cleavage of envelope proteins to acquire viral infectivity and consequently, need a host protease or the addition of exogenous trypsin for efficient propagation. Host TMPRSS2 is a key protease responsible for viral cleavage. Stable expression of human TMPRSS2 in African green monkey-derived Vero cells can enhance the porcine epidemic diarrhea virus. However, considering the narrow host tropism of viruses, a porcine cell line expressing pig TMPRSS2 could be optimal for replicating pig-derived viruses. Herein, we generated and evaluated a pig-derived PK-15 cell line stably expressing pig TMPRSS2. This cell line markedly (>1000-fold) and specifically enhanced the growth of influenza viruses. Furthermore, we demonstrated the usefulness of a PK-15 cell line lacking the Stat2 gene with a stable expression of pig TMPRSS2 for efficient virus isolation from clinical samples in the presence of type I interferons. Therefore, PK-15 cells expressing pig TMPRSS2 could be a valuable and promising tool for virus isolation, vaccine production, and virological studies of TMPRSS2-dependent viruses. Full article

Shiga toxins (Stxs), especially the Stx2a subtype, are the major virulence factors involved in enterohemorrhagic Escherichia coli (EHEC)-associated hemolytic uremic syndrome (eHUS), a life-threatening disease causing acute kidney injury, especially in children. After oral transmission and colonization in the gut, EHEC release Stx. Intracellular cleavage of the Stx A subunit, when followed by reduction, boosts the enzymatic activity that causes damage to targeted cells. This cleavage was assumed to be mostly mediated by furin during Stx intracellular trafficking. To investigate whether this cleavage could occur in the intestine, even prior to entering target cells, Stx2a A subunit structure (intact or cleaved) was characterized after its exposure to specific host factors present in human stool. The molecular weight of Stx2a A subunit/fragments was determined by immunoblotting after electrophoretic separation under reducing conditions. In this study, it was demonstrated that Stx2a is cleaved by certain human stool components. Trypsin and chymotrypsin-like elastase 3B (CELA3B), two serine proteases, were identified as potential candidates that can trigger the extracellular cleavage of Stx2a A subunit directly after its secretion by EHEC in the gut. Whether the observed cleavage indeed translates to natural infections and plays a role in eHUS pathogenesis has yet to be determined. If so, it seems likely that a host’s protease profile could affect disease development by changing the toxin’s biological features. Full article

SARS-CoV-2, responsible for the COVID-19 pandemic, invades host cells via its spike protein, which includes critical binding regions, such as the receptor-binding domain (RBD), the S1/S2 cleavage site, the S2 cleavage site, and heptad-repeat (HR) sections. Peptides targeting the RBD and HR1 inhibit binding to host ACE2 receptors and the formation of the fusion core. Other peptides target proteases, such as TMPRSS2 and cathepsin L, to prevent the cleavage of the S protein. However, research has largely ignored peptides targeting the S1/S2 cleavage site. In this study, bioinformatics was used to investigate the binding of the S1/S2 cleavage site to host proteases, including furin, trypsin, TMPRSS2, matriptase, cathepsin B, and cathepsin L. Peptides targeting the S1/S2 site were designed by identifying binding residues. Peptides were docked to the S1/S2 site using HADDOCK (High-Ambiguity-Driven protein–protein DOCKing). Nine peptides with the lowest HADDOCK scores and strong binding affinities were selected, which was followed by molecular dynamics simulations (MDSs) for further investigation. Among these peptides, BR582 and BR599 stand out. They exhibited relatively high interaction energies with the S protein at −1004.769 ± 21.2 kJ/mol and −1040.334 ± 24.1 kJ/mol, respectively. It is noteworthy that the binding of these peptides to the S protein remained stable during the MDSs. In conclusion, this research highlights the potential of peptides targeting the S1/S2 cleavage site as a means to prevent SARS-CoV-2 from entering cells, and contributes to the development of therapeutic interventions against COVID-19. Full article

Glycosylation is an important post-translational modification of proteins, contributing to protein function, stability and subcellular localization. Fungal immunomodulatory proteins (FIPs) are a group of small proteins with notable immunomodulatory activity, some of which are glycoproteins. In this study, the impact of glycosylation on the bioactivity and biochemical characteristics of FIP-nha (from Nectria haematococca) is described. Three rFIP-nha glycan mutants (N5A, N39A, N5+39A) were constructed and expressed in Pichia pastoris to study the functionality of the specific N-glycosylation on amino acid N5 and N39. Their protein characteristics, structure, stability and activity were tested. WT and mutants all formed tetramers, with no obvious difference in crystal structures. Their melting temperatures were 82.2 °C (WT), 81.4 °C (N5A), 80.7 °C (N39A) and 80.1 °C (N5+39A), indicating that glycosylation improves thermostability of rFIP-nha. Digestion assays showed that glycosylation on either site improved pepsin resistance, while 39N-glycosylation was important for trypsin resistance. Based on the 3D structure and analysis of enzyme cleavage sites, we conclude that glycosylation might interfere with hydrolysis via increasing steric hindrance. WT and mutants exerted similar bioactivity on tumor cell metabolism and red blood cells hemagglutination. Taken together, these findings indicate that glycosylation of FIP-nha impacts its thermostability and digestion resistance. Full article

Bioinformatics software, allowing the identification of peptides by the comparison of peptide fragmentation spectra obtained by mass spectrometry versus targeted databases or directly by de novo sequencing, is now mandatory in peptidomics/proteomics approaches. Programming the identification software requires specifying, among other things, the mass measurement accuracy of the instrument and the digestion enzyme used with the number of missed cleavages allowed. Moreover, these software algorithms are able to identify a large number of post-translational modifications (PTMs). However, peptide and PTM identifications are challenging in the agrofood field due to non-specific cleavage sites of physiological- or food-grade enzymes and the number and location of PTMs. In this study, we show the importance of customized software programming to obtain a better peptide and PTM identification rate in the agrofood field. A gelatine product and one industrial gelatine hydrolysate from three different sources (beef, pork, and fish), each digested by simulated gastrointestinal digestion, MS-grade trypsin, or both, were used to perform the comparisons. Two main points are illustrated: (i) the impact of the set-up of specific enzyme versus no specific enzyme use and (ii) the impact of a maximum of six PTMs allowed per peptide versus the standard of three. Prior knowledge of the composition of the raw proteins is an important asset for better identification of peptide sequences. Full article

Celiac peptide-generating α- and γ-gliadins consist of a disordered N-terminal domain extended by an α-helical-folded C-terminal domain. Celiac peptides, primarily located along the disordered part of α- and γ-gliadin molecules, are nicely exposed and directly accessible to proteolytic enzymes occurring in the gastric (pepsin) and intestinal (trypsin, chymotrypsin) fluids. More than half of the potential celiac peptides identified so far in gliadins exhibit cleavage sites for pepsin. However, celiac peptides proteolytically truncated by one or two amino acid residues could apparently retain some activity toward HLA-DQ2 and HLA-DQ8 receptors in docking experiments. Together with the uncleaved peptides, these still active partially degraded CD peptides account for the incapacity of the digestion process to inactivate CD peptides from gluten proteins. In contrast, sourdough fermentation processes involve other proteolytic enzymes susceptible to the deep degradation of celiac peptides. In particular, sourdough supplemented by fungal prolyl endoproteases enhances the degrading capacities of the sourdough fermentation process toward celiac peptides. Nevertheless, since tiny amounts of celiac peptides sufficient to trigger deleterious effects on CD people can persist in sourdough-treated bread and food products, it is advisable to avoid consumption of sourdough-treated food products for people suffering from celiac disease. As an alternative, applying the supplemented sourdough process to genetically modified low gluten or celiac-safe wheat lines should result in food products that are safer for susceptible and CD people. Full article

Background, Aims, Methods, Results, Conclusions: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global challenge due to its ability to mutate into variants that spread more rapidly than the wild-type virus. The molecular biology of this virus has been extensively studied and computational methods applied are an example paradigm for novel antiviral drug therapies. The rapid evolution of SARS-CoV-2 in the human population is driven, in part, by mutations in the receptor-binding domain (RBD) of the spike (S-) protein, some of which enable tighter binding to angiotensin-converting enzyme (ACE2). More stable RBD-ACE2 association is coupled with accelerated hydrolysis by proteases, such as furin, trypsin, and the Transmembrane Serine Protease 2 (TMPRSS2) that augment infection rates, while inhibition of the 3-chymotrypsin-like protease (3CL^pro) can prevent the viral replication. Additionally, non-RBD and non-interfacial mutations may assist the S-protein in adopting thermodynamically favorable conformations for stronger binding. This study aimed to report variant distribution of SARS-CoV-2 across European Union (EU)/European Economic Area (EEA) countries and relate mutations with the driving forces that trigger infections. Variants’ distribution data for SARS-CoV-2 across EU/EEA countries were mined from the European Centre for Disease Prevention and Control (ECDC) based on the sequence or genotyping data that are deposited in the Global Science Initiative for providing genomic data (GISAID) and The European Surveillance System (TESSy) databases. Docking studies performed with AutoDock VINA revealed stabilizing interactions of putative antiviral drugs, e.g., selected anionic imidazole biphenyl tetrazoles, with the ACE2 receptor in the RBD-ACE2 complex. The driving forces of key mutations for Alpha, Beta, Gamma, Delta, Epsilon, Kappa, Lambda, and Omicron variants, which stabilize the RBD-ACE2 complex, were investigated by computational approaches. Arginine is the critical amino acid in the polybasic furin cleavage sites S1/S2 (681-PRRARS-686) S2′ (814-KRS-816). Critical mutations into arginine residues that were found in the delta variant (L452R, P681R) and may be responsible for the increased transmissibility and morbidity are also present in two widely spreading omicron variants, named BA.4.6 and BQ.1, where mutation R346T in the S-protein potentially contributes to neutralization escape. Arginine binders, such as Angiotensin Receptor Blockers (ARBs), could be a class of novel drugs for treating COVID-19. Full article

Gram-negative bacteria have a large variety of channel-forming proteins in their outer membrane, generally referred to as porins. Some display weak voltage dependence. A similar trimeric channel former, named Triplin, displays very steep voltage dependence, rivaling that responsible for the electrical excitability of mammals, and high inter-subunit cooperativity. We report detailed insights into the molecular basis for these very unusual properties explored at the single-molecule level. By using chemical modification to reduce the charge on the voltage sensors, they were shown to be positively charged structures. Trypsin cleavage of the sensor eliminates voltage gating by cleaving the sensor. From asymmetrical addition of these reagents, the positively charged voltage sensors translocate across the membrane and are, thus, responsible energetically for the steep voltage dependence. A mechanism underlying the cooperativity was also identified. Theoretical calculations indicate that the charge on the voltage sensor can explain the rectification of the current flowing through the open pores if it is located near the pore mouth in the open state. All results support the hypothesis that one of the three subunits is oriented in a direction opposite to that of the other two. These properties make Triplin perhaps the most complex pore-forming molecular machine described to date. Full article