Coxsackievirus B3 (CVB3) encodes proteinases that are essential for processing of the translated viral polyprotein. Viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. While some host protein substrates of the CVB3 3C and 2A cysteine proteinases have been identified, the full repertoire of targets is not known. Here, we utilize an unbiased quantitative proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) to conduct a global analysis of CVB3 protease-generated N-terminal peptides in both human HeLa and mouse cardiomyocyte (HL-1) cell lines infected with CVB3. We identified >800 proteins that are cleaved in CVB3-infected HeLa and HL-1 cells including the viral polyprotein, known substrates of viral 3C proteinase such as PABP, DDX58, and HNRNPs M, K, and D and novel cellular proteins. Network and GO-term analysis showed an enrichment in biological processes including immune response and activation, RNA processing, and lipid metabolism. We validated a subset of candidate substrates that are cleaved under CVB3 infection and some are direct targets of 3C proteinase in vitro. Moreover, depletion of a subset of TAILS-identified target proteins decreased viral yield. Characterization of two target proteins showed that expression of 3Cpro-targeted cleaved fragments of emerin and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 modulated autophagy and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, respectively. The comprehensive identification of host proteins targeted during virus infection provides insights into the cellular pathways manipulated to facilitate infection.
OBJECTIVE: RNA viruses encode proteases that are responsible for processing viral proteins into their mature form. Viral proteases also target and cleave host cellular proteins; however, the full catalog of these target proteins is incomplete. We use a technique called terminal amine isotopic labeling of substrates (TAILS), an N-terminomics to identify host proteins that are cleaved under virus infection. We identify hundreds of cellular proteins that are cleaved under infection, some of which are targeted directly by viral protease. Revealing these target proteins provides insights into the host cellular pathways and antiviral signaling factors that are modulated to promote virus infection and potentially leading to virus-induced pathogenesis.

In this study, it was aimed to investigate the direct release of BAPs from einkorn flour in one-step process. Thus, the protein extraction step was eliminated, thereby reducing processing cost. Commercial proteases (Alcalase, Flavourzyme, Neutrase, and Trypsin), and crude enzyme from Bacillus mojavensis sp. EBTA7 were used for hydrolyzing einkorn flour (30 %, w/v) solutions at 50-60 °C. The supernatants after centrifugation were used for bioactivity and techno-functionality tests. All hydrolysates demonstrated significant antioxidant capacities, with values ranging from 17.7 to 33.0 μmol TE/g for DPPH, 107 to 190 μmol TE/g for ABTS, and 0.09 to 3.08 mg EDTA/g for ion-chelating activities. Alcalase and Flavourzyme hydrolysis had the highest DPPH activities, while Bacillus mojavensis sp. EBTA7 enzyme yielded relatively high ABTS and ion-chelating activities. Notably, Bacillus mojavensis sp. EBTA7 crude enzyme hydrolysates demonstrated higher oil absorption capacity (2.94 g oil/g hydrolysate), robust emulsion (227 min), and foam stability (94 %) compared to commercial enzymes. FTIR spectroscopy confirmed variations in the secondary structure of peptides. All hydrolysates exhibited negative zeta potentials. The SDS-PAGE showcased MW ranged from 14 to 70 kDa, which was influenced by both the enzyme type and the degree of hydrolysis. Overall, Bacillus mojavensis sp. EBTA7 hydrolysates revealed considerable bio and techno-functional characteristics.

The objective of this research was to evaluate the efficiency of aqueous enzymatic extraction (AEE) to obtain oil from hemp seeds (Cannabis sativa L.) grown in northern Morocco. Optimisation of AEE extraction parameters, including pH, enzyme concentration (hemicellulase, protease and pectinase), temperature and incubation time, to maximize oil yield was achieved using response surface methodology with a central composite design. For comparison, the solvent extraction (Soxhlet) (SE) method was also used. Optimized hydrolysis conditions involved incubation for 4 hours at 60°C with a pH of 6.5, using a multi-enzyme preparation comprising protease, hemicellulase and pectinase at concentrations of 55, 202.5 and 234 U/mg, respectively. Referring to the conventional Soxhlet extraction (SE), Aqueous Enzymatic Extraction (AEE) achieved a 30.65% oil recovery rate under the optimized parameters mentioned above. The use of enzymes produced an oil that was more stable against oxidation than the solvent-extracted oil, with a peroxide value (PV) of 19.54 and 47.87 meq O 2 /kg, respectively. Furthermore, HPLC-DAD analysis of tocopherol content indicated a higher total tocopherol content (547.2 mg/kg) in Aqueous Enzymatic Extraction (AEE) compared to Soxhlet Extraction (SE) (513.51 mg/kg), with γ-tocopherol being the predominant form. No significant differences in fatty acid composition were observed between the two extraction methods with linoleic acid and alpha-linolenic acid being the predominant constituents.

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become the standard of care in cancer surgeries. One of the key parameters to optimize in contrast agents is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker, as well as the positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased overall signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

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UNASSIGNED: This study investigated the impact of supplementation of protease and organic acid on growth performance and other biological parameters in broilers fed poultry by-product meal (PBM) based diet.
UNASSIGNED: Five hundred-day-old broiler chicks (Ross 308) were distributed into five treatments with 5 replicates, each pen having 20 birds, and fed each group one of five isocaloric and isonitrogenous diets in two phases: stater phase (1-21 days) ME 3000 kcal/kg; CP 22%, and a finisher phase (22-35 days) ME 3200 kcal/kg; CP 19.5%. The dietary treatments were: 1) standard broiler ration (Cont); 2) The control diet with 25% of the soybean meal replaced by poultry by-product meal (PBM) on an equivalent protein basis (PBM); 3) PBM diet supplemented with 0.5 g/kg of protease (PBMP); 4) PBM diet supplemented with 1 g/kg organic acid (PBMO); and 5) PBM diet addition with 0.5 g/kg protease and 1 g/kg organic acid (PBMPO).
UNASSIGNED: The overall data showed that FCR was improved (P<0.05) in the PBMP group. Apparent crude protein digestibility was higher (P<0.05) in both Cont and PBMP groups. Jejunal villus height (VH) increased (P<0.05) in PBMP and PBMPO groups, while only the PBMO group exhibited a higher (P<0.05) crypt depth (CD). Lipase activity was increased (P<0.05) in the PBMP, PBMO and PBMPO dietary treatments. However, trypsin activity showed a significant increase (P<0.05) in the PBMP and PBMO groups. Serum biochemistry increased (P<0.05) globulin and total protein levels in the PBMP group.
UNASSIGNED: PBM could partially replace the soybean meal with supplementation of either protease or organic acid in broiler diets without impairing overall growth performance. Furthermore, careful optimization must be considered when combining protease and organic acids.

BACKGROUND: The rhizome of Dryopteris crassirhizoma Nakai (Dryopteridaceae, RDC), a traditional East Asian herbal medicine, possesses a broad spectrum of medicinal properties, including anti-inflammatory, anticancer, antibacterial, and antiviral activities.
OBJECTIVE: This study investigates the 30% ethanolic extract of RDC\'s antiviral potential against human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and its variants infections.
METHODS: A 30% ethanolic extract of RDC or its components, filixic acid ABA (PubChem CID: 15081408) and dryocrassin ABBA (PubChem CID: 3082025) were treated with Human Coronavirus infection (HCoV-OC43, SARS-CoV-2 and its variants). The base peak chromatogram of RDC was evaluated using UPLC-Q/TOF Mass to identify the RDC, and the quantitative analysis of RDC compounds was performed using LC-MS/MS. A cytopathic effect (CPE) reduction assay, Western blot, immunofluorescence staining of viral protein expression, and qRT-PCR were performed to quantify the viral RNA copy numbers and determine the antiviral activity. The time-of-addition assay, the virus attachment, penetration, and virucidal assays, and SARS-CoV-2 Mpro and PLpro activity assay were used to elucidate the mode of action.
RESULTS: RDC exhibited dose-dependent inhibition of HCoV-OC43-induced cytopathic effects, reducing viral RNA copy numbers and viral protein levels. Time-of-addition assays indicated that RDC targets the early stages of the HCoV-OC43 life cycle, inhibiting virion attachment and penetration with virucidal activity. Notably, filixic acid ABA and dryocrassin ABBA, constituents of RDC, reduced HCoV-OC43 viral RNA loads. Furthermore, RDC effectively blocked viral entry in pseudotyped lentivirus assays, involving spike proteins of SARS-CoV-2 Delta plus and South Africa variants, as well as control lentiviral particles expressing vesicular stomatitis virus glycoprotein G. Additionally, RDC demonstrated inhibition of SARS-CoV-2 infection and its variants by targeting viral proteases, namely main protease (Mpro) and papain-like protease (PLpro).
CONCLUSIONS: These findings underscore RDC\'s multistage approach to targeting viral infections by impeding virus entry and inhibiting viral protease activity. Therefore, RDC holds promise as a potent, broad-spectrum anticoronaviral therapeutic agent.

Brown seaweeds of the Fucus genus represent a rich source of natural antiviral products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme\'s proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 ± 6.4 to 76.8 ± 3.6% inhibition at 10 µM). This study is the first report identifying peptides from Fucus ceranoides with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease\'s active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices.

YabG is a sporulation-specific protease that is conserved among sporulating bacteria. C. difficile YabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in yabG were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabG C207A (catalytically inactive), C. difficile yabG A46D, C. difficile yabG G37E, and C. difficile yabG P153L strains germinated in response to TA alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the pre sequence from preproSleC. Interestingly, only YabGA46D showed any activity towards purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.

Proteases, enzymes that hydrolyze peptide bonds, have various applications in medicine, clinical applications, and pharmaceutical development. They are used in cancer treatment, wound debridement, contact lens cleaning, prion degradation, biofilm removal, and fibrinolytic agents. Proteases are also crucial in cardiovascular disease treatment, emphasizing the need for safe, affordable, and effective fibrinolytic drugs. Proteolytic enzymes and protease biosensors are increasingly used in diagnostic and therapeutic applications. Advanced technologies, such as nanomaterials-based sensors, are being developed to enhance the sensitivity, specificity, and versatility of protease biosensors. These biosensors are becoming effective tools for disease detection due to their precision and rapidity. They can detect extracellular and intracellular proteases, as well as fluorescence-based methods for real-time and label-free detection of virus-related proteases. The active utilization of proteolytic enzymatic biosensors is expected to expand significantly in biomedical research, in-vitro model systems, and drug development. We focused on journal articles and books published in English between 1982 and 2024 for this study.