Search Results (158)

Background/Objectives: Global warming and concomitant extreme weather events have markedly increased the incidence of heat stroke. Heat stroke (HS) poses a substantial threat to cerebral health by triggering neuroinflammation and accelerating neurodegenerative processes. The activation of the Nod-like receptor protein 3 (NLRP3) inflammasome for interleukin-1β (IL-1β) secretion has been implicated as a critical mechanism underlying HS-related fatalities. However, the potential role of specific dietary factors to counteract heat stroke-induced neurotoxicity remains largely underexplored. We previously reported that eicosapentaenoic acid (EPA) and urolithin A (UroA), a gut metabolite of ellagic acid, effectively suppress NLRP3 inflammasome activation against metabolic or pathogenic insults. This study aimed to assess the impact of eicosapentaenoic acid (EPA), urolithin A (UroA), and their combination on mitigating heatstroke-mediated NLRP3 inflammasome activation in microglial cells. Methods: In vitro heatstroke conditions were replicated by subjecting murine BV2 microglial cells to a high temperature (41 °C) under hypoxic conditions. To achieve nutrient loading, BV2 cells were preincubated with either EPA (50 µM) or UroA (10 µM). NLRP3 inflammasome activation was evaluated by proinflammatory gene expression, caspase-1 cleavage in cells, and IL-1β secretion to the medium. The caspase-1 activation was determined using a luciferase-based inflammasome and protease activity reporter (iGLuc) assay. Results: Exposure to high temperatures under hypoxia successfully mimicked HS conditions and promoted NLRP3 inflammasome activation in BV2 cells. Both EPA and UroA substantially attenuated the heat stroke-induced priming of proinflammatory genes. More importantly, EPA and UroA demonstrated a synergistic effect in mitigating HS-induced active caspase-1 production, leading to a dramatic decrease in IL-1β secretion. This synergistic effect between EPA and UroA was further confirmed by the iGLuc reporter assay. Conclusions: Dietary enrichment with EPA and UroA precursors may constitute an efficacious strategy for mitigating heat stroke-mediated neuroinflammation and neurodegenerative diseases. Full article

CD9 protein belongs to a family of proteins called tetraspanins, so named for their four-transmembrane-spanning architectures. These proteins are located in domains in the plasmatic membrane, called tetraspanin-enriched microdomains (TEMs). Several proteases and cellular receptors for virus entry cluster into TEMs, suggesting that TEMs are preferred virus entry portals. Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates virus attachment and entry into cells by binding to human angiotensin-converting enzyme 2 (ACE-2). In addition, the secretory, type-I membrane-bound SARS-CoV-2 S protein is synthesized as a precursor (proS) that undergoes posttranslational cleavages by host cell proteases, such as furin and TMPRSS2. Moreover, it has been shown that neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates, potentiates SARS-CoV-2 infectivity. Our results indicate that CD9 facilitates SARS-CoV-2 infection. In addition, we show how knocking out CD9 leads to a decrease in the expression of NRP1, a protein that improves SARS-CoV-2 infection. Furthermore, we show that CD9 colocalizes with ACE-2, NRP1, furin, and TMPRSS2 at the plasma membrane; that the absence of CD9 decreases the expression of these proteins on the plasma membrane CD9-enriched microdomains, and that CD9 interacts with ACE2. In conclusion, our data suggest that CD9 facilitates SARS-CoV-2 infection and that CD9 brings together different host proteins involved in SARS-CoV-2 attachment and entry into host cells, such as ACE2, NRP1, furin, and TMPRSS2. Importantly, the fact that a blocking antibody targeting CD9 can effectively reduce SARS-CoV-2 titers highlights not only the mechanistic role of CD9 in viral entry but also offers translational potential, suggesting that tetraspanin-targeting antibodies could be developed as therapeutic agents against SARS-CoV-2 and possibly other coronaviruses, with meaningful implications for clinical intervention. Full article

Current marine mackerel (Scomberomorus niphonius) products predominantly involve low-value-added processing, while high-value-added products like fish floss remain underdeveloped. This study utilized mackerel dorsal muscle treated with flavor protease (FP), papain (PP), and neutral protease (NP) (10 U/g, 30 min), followed by steaming and stir-frying. Combined with sensory evaluation, HS-GC-IMS, and automatic amino acid analysis, the characteristic flavor was evaluated by multi-omics. The results showed that FP and NP significantly enhanced odor by reducing fishy compounds (e.g., hexanal) and increasing pyrazines/furans. PP enhanced taste by elevating umami and sweet amino acids (26.68% and 25.98%, respectively). Correlation analysis revealed the following potential pathways: Val and Leu served as precursors for furan, suppressing 2-methyl-3-(methylthio)furan formation, while Asp, Tyr, Phe, Gly, Cys, and Ile promoted 2,5-dimethylpyrazine and 2-methyl-3-(methylthio)furan generation while inhibiting furan. This study demonstrates that minimal protease addition effectively optimizes dried mackerel floss flavor, providing a novel approach for high-quality marine product development. Full article

Chilli veinal mottle virus (ChiVMV) severely compromises the quality and yield of solanaceous crops. The helper component protease (HCPro) of ChiVMV functions as a multifunctional RNA silencing suppressor that subverts host antiviral defenses through diverse strategies, However, the underlying mechanisms remain mechanistically unresolved. In this study, HCPro-overexpressing (HCPro-OX) and wild-type (WT) plants were inoculated with ChiVMV to monitor the physiological and molecular changes. Transcriptome analysis identified 11,815 differentially expressed genes (DEGs) under viral infection, among which 1115 genes were specifically regulated by HCPro. KEGG enrichment analysis revealed that the DEGs were significantly associated with plant hormone signal transduction pathways, indicating their crucial role in host–virus interactions. Furthermore, functional clustering of HCPro-regulated DEGs specifically identified key components in ethylene biosynthesis pathways. GO analysis of DEGs between virus-inoculated WT and HCPro-OX plants annotated ethylene biosynthesis-related genes NtACO and NtACS. qPCR validation confirmed that the expression of ethylene biosynthesis-related genes was suppressed by HCPro. Exogenous treatments with the ethylene precursor ACC demonstrated that ethylene suppressed viral accumulation, enhanced POD activity, and reduced the ROS accumulation induced by viral infection. In conclusion, our results demonstrate that HCPro promotes viral infection by suppressing ethylene biosynthesis, which in turn attenuates peroxidase activity, leading to ROS accumulation. Full article

This study investigates the impact of fermentation temperature on the physicochemical properties, bioactive compound retention, and in vitro digestion profile of cacao seeds (Theobroma cacao L.). Three fermentation conditions were evaluated: low (F40, 40 °C), medium (Control, 50 °C), and high (F60, 60 °C). The study assessed macronutrient composition, phenolic compound retention, antioxidant activity, enzymatic activity, structural changes, and glucose release during in vitro digestion. Fermentation temperature significantly influenced cacao seed quality and functionality. F40 preserved the highest levels of phenolic compounds (61% reduction compared to raw seeds) and antioxidant activity (73% reduction), offering a pronounced hypoglycemic effect through enzyme inhibition. In contrast, F60 facilitated extensive enzymatic activity, particularly protease and lipase, promoting flavor precursor formation and structural changes like cracking. However, this high-temperature treatment resulted in significant losses of phenolic compounds (76%) and antioxidant capacity (88%). Structural analysis revealed that higher fermentation temperatures enhanced cellular breakdown, increasing enzymatic access and glucose bioavailability. Digestion studies confirmed that roasted cacao fermented at higher temperatures released more glucose, driven by enzymatic hydrolysis and structural modifications. Conversely, the cacao from F40 exhibited slower glucose release due to the retention of bioactive compounds that inhibit carbohydrate-hydrolyzing enzymes. This research underscores the trade-offs in cacao processing: fermentation temperature significantly modulates cacao seed properties. At higher temperatures (60 °C), enhanced enzymatic activity (protease, lipase) facilitates the release of flavor precursors and structural modifications, increasing digestibility and glucose bioavailability, making it ideal for chocolate production. Conversely, fermentation at lower temperatures (40 °C) preserves bioactive compounds, including phenolics and antioxidants (with 61% retention compared to raw seeds), which may offer functional food applications for glycemic control. Roasting reversed some fermentation effects, reducing phenolic retention while increasing glucose bioavailability. This work tailors cacao fermentation for diverse end uses, from premium chocolate to nutraceutical products aimed at glycemic control. Full article

Circular bacteriocins are potent antimicrobials against pathogenic Gram-positives. In searching for marine bacteriocins, an antibacterial peptide (flexusin A) was purified from the fermentation broth of marine bacterium Bacillus flexus R29-2. Genome sequencing and gene annotation revealed the chromosome contained an unknown circular bacteriocin gene cluster. Approaches including shot-gun proteomics analysis, AntiSMASH and BAGEL4 predication as well as the comprehensive sequence alignment, were then conducted, respectively, to verify the correlation of flexusin A with the gene-encoded precursor peptide. The results confirmed that flexusin A was the mature circular bacteriocin of the predicated precursor peptide with six amino acids as leader peptide. Flexusin A was 6098.4 Da in size, with a net charge of +3 and PI of 9.60. It shared the typical saposin-like fold spatial conformation features as commonly found in other circular bacteriocins. Flexusin A was pH, thermal, and protease tolerant. It exhibited a narrow antimicrobial spectrum against Gram-positives, and it can strongly inhibit Staphylococcus aureus by causing cell destruction via membrane destabilization. Taken together, a novel circular bacteriocin flexusin A was identified in this work. The characterization of flexusin A has extended circular bacteriocins family to 26 members. This is also the first report on bacteriocin production by B. flexus. Full article

Background: Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products biosynthesized from a genetically encoded precursor peptide. RiPPs have attracted attention for the ability to generate and screen libraries of these compounds for useful biological activities. To facilitate this screening, it is useful to be able to do so with the leader peptide still present. We assessed the suitability of the microviridin family for these screening experiments by determining their activity with the leader peptide still present. Methods: Modified precursor peptides with the leader present were heterologously expressed in Escherichia coli. Their ability to inhibit elastase was tested with a fluorogenic substrate. HPLC was used to monitor degradation of the modified precursor peptides by elastase. SDS-PAGE was used to determine the ability of immobilized modified precursor peptide to pull down elastase. Results: We found that the fully modified precursor peptide of microviridin B can inhibit the serine protease elastase with a low nanomolar IC₅₀, and that the fully modified precursor with an N-terminal His-tag can mediate interactions between elastase and Ni-NTA resin, all indicating leader peptide removal is not necessary for microviridins to bind their target proteases. Additionally, we found that a bicyclic variant was able to inhibit elastase with the leader peptide still present, although with a roughly 100-fold higher IC₅₀ and being subject to hydrolysis by elastase. Conclusions: These results open a pathway to screening libraries of microviridin variants for improved protease inhibition or other characteristics that can serve as, or as inspirations for, new pharmaceuticals. Full article

To investigate the substrate recognition mechanism of trypsin-like protease cocoonase (CCN), mutational analyses were conducted at key substrate recognition sites, Asp187 and Ser188, and their effects on substrate specificity and enzymatic activity were evaluated. Mutants with the Asp187 substitution exhibited a significant reduction in catalytic activity compared with the wild-type enzyme, whereas the Ser188 mutants displayed a comparatively minor effect on activity. This indicates that Asp187 plays a crucial role in catalytic function, whereas Ser188 serves a complementary role in substrate recognition. Interestingly, the substitution of the Asp187 to Glu or Ser caused novel substrate specificities, resulting in the recognition of Orn and His residues. In addition, when Asp187 and Ser188 were substituted with acidic residues (Glu or Asp), both the precursor proCCN and mature CCN proteins retained highly similar secondary and tertiary structures. This reveals that the structural characteristics of precursor proteins are maintained in the mature proteins, potentially influencing substrate recognition and catalytic function. These findings suggest that the pro-regions of these mutants interact much more tightly with the mature enzyme than in the wild-type CCN. These results provide fruitful insights into the structural determinants governing substrate recognition in enzyme variants. Full article

The functional properties of pumpkin seed proteins remain unutilized in numerous food and industrial applications. Several current approaches aim to improve the functional properties of pumpkin seed proteins, allowing their innovative potential to develop and modify significantly. Several strategies can be implemented to alter the functional properties of proteins isolated from pumpkin seeds. The first is enzymatic hydrolysis, regardless of whether, proteases may free peptide binding and profoundly impact the protein structure and functionality. Thermal treatment can include heating and cooling to replace protein conformation and increase solubility, emulsification, and gelation properties. Chemical modification techniques, including acylation and glycation, can also be used to improve stability, viscosity, and foaming ability. Functional properties and, where possible, ingredients with many applications may include exceptional possibilities for proteins modified in food preparations, such as dairy replacements, plant-based meat analogues, and free gluten that have an outstanding aspect, satisfactory quality, and nutritional profiles. As multiple different proteins act as precursors of active peptides, they can also be used to generate bio-specific foods. This review briefly provides information about various types of protein extraction techniques and functional properties that are modified by different types of processing technologies. Full article

The conservation of the main protease in viral genomes, combined with the absence of a homologous protease in humans, makes this enzyme family an ideal target for developing broad-spectrum antiviral drugs with minimized host toxicity. GC-376, a peptidomimetic 3CL protease inhibitor, has shown significant efficacy against coronaviruses. Recently, a GC-376-based PROTAC was developed to target and induce the proteasome-mediated degradation of the dimeric SARS-CoV-2 3CL^Pro protein. Extending this approach, the current study investigates the application of the GC-376 PROTAC to the 3C^Pro protease of enteroviruses, specifically characterizing its interaction with CVB3 3C^Pro through X-ray crystallography, NMR (Nuclear Magnetic Resonance) and biochemical techniques. The crystal structure of CVB3 3C^Pro bound to the GC-376 PROTAC precursor was obtained at 1.9 Å resolution. The crystallographic data show that there are some changes between the binding of CVB3 3C^Pro and SARS-CoV-2 3CL^Pro, but the overall similarity is strong (RMSD on C-alpha 0.3 Å). The most notable variation is the orientation of the benzyloxycarbonyl group of GC-376 with the S4 subsite of the proteases. NMR backbone assignment of CVB3 3C^Pro bound and unbound to the GC-376 PROTAC precursor (80% and 97%, respectively) was obtained. This information complemented the investigation, by NMR, of the interaction of CVB3 3C^Pro with the GC-376 PROTAC, and its precursor allows us to define that the GC-376 PROTAC binds to CVB3 3C^Pro in a mode very similar to that of the precursor. The NMR relaxation data indicate that a quench of dynamics of a large part of the protein backbone involving the substrate-binding site and surrounding regions occurs upon GC-376 PROTAC precursor binding. This suggests that the substrate cavity, by sampling different backbone conformations in the absence of the substrate, is able to select the suitable one necessary to covalently bind the substrate, this being the latter reaction, which is the fundamental step required to functionally activate the enzymatic reaction. The inhibition activity assay showed inhibition potency in the micromolar range for GC-376 PROTAC and its precursor. Overall, we can conclude that the GC-376 PROTAC fits well within the binding sites of both proteases, demonstrating its potential as a broad-spectrum antiviral agent. Full article

Immobilizing different enzymes on membranes can result in biocatalytic active membranes with a self-cleaning capacity toward a complex mixture of foulants. The membrane modification can reduce fouling and enhance filtration performance. Protease, lipase, and amylase were immobilized on poly(vinylidene fluoride) (PVDF) microfiltration membranes using a polydopamine coating in a one-step method. The concentrations of polydopamine precursor and enzymes were optimized during the immobilization. The higher hydrolytic activities were obtained using 0.2 mg/mL of dopamine hydrochloride and 4 mg/mL of enzymes: 0.90 mg_starch/min·cm² for amylase, 10.16 nmol_tyrosine/min·cm² for protease, and 20.48 µmol_{p-nitrophenol}/min·cm² for lipase. Filtration tests using a protein, lipid, and carbohydrate mixture showed that the modified membrane retained 41%, 29%, and 28% of its initial water permeance (1808 ± 39 L/m²·h·bar) after three consecutive filtration cycles, respectively. In contrast, the pristine membrane (initial water permeance of 2016 ± 40 L/m²·h·bar) retained only 23%, 12%, and 8%. Filtrations of milk powder solution were also performed to simulate dairy industry wastewater: the modified membrane maintained 28%, 26%, and 26% of its initial water permeance after three consecutive filtration cycles, respectively, and the pristine membrane retained 34%, 21%, and 7%. The modified membrane showed increased fouling resistance against a mixture of foulants and presented a similar water permeance after three cycles of simulated dairy wastewater filtration. Membrane fouling is reduced by the immobilized enzymes through two mechanisms: increased membrane hydrophilicity (evidenced by the reduced water contact angle after modification) and the enzymatic hydrolysis of foulants as they accumulate on the membrane surface. Full article

Cancer cells can release EGF-like peptides, acquiring the capacity of autocrine stimulation via EGFR-mediated signaling. One of these peptides (HBEGF) was found to be released from a membrane-bound precursor protein and is critically implicated in the proliferative potential of cancer cells. We observed that the increased lactate levels characterizing neoplastic tissues can induce the release of uPA, a protease promoting HBEGF shedding. This effect led to EGFR activation and increased ERK1/2 phosphorylation. Since EGFR-mediated signaling potentiates glycolytic metabolism, this phenomenon can induce a self-sustaining deleterious loop, favoring tumor growth. A well characterized HBEGF inhibitor is CRM197, a single-site variant of diphtheria toxin. We observed that, when administered individually, CRM197 did not trigger evident antineoplastic effects. However, its association with a uPA inhibitor caused dampening of EGFR-mediated signaling and apoptosis induction. Overall, our study highlights that the increased glycolytic metabolism and lactate production can foster the activated state of EGFR receptor and suggests that the inhibition of EGFR-mediated signaling can be attempted by means of CRM197 administered with an appropriate protease inhibitor. This attempt could help in overcoming the problem of the acquired resistance to the conventionally used EGFR inhibitors. Full article

Furin, a serine protease enzyme located in the Golgi apparatus of animal cells, plays a crucial role in cleaving precursor proteins into their mature, active forms. It is ubiquitously expressed across various tissues, including the brain, lungs, gastrointestinal tract, liver, pancreas, and reproductive organs. Since its discovery in 1990, furin has been recognized as a significant therapeutic target, leading to the active development of furin inhibitors for potential use in antiviral, antibacterial, anticancer, and other therapeutic applications. This review provides a comprehensive overview of the progress in the development and characterization of furin inhibitors, encompassing peptides, linear and macrocyclic peptidomimetics, and non-peptide compounds, highlighting their potential in the treatment of both infectious and non-infectious diseases. Full article

The SARS-CoV-2 main protease (M^pro) is initially synthesized as part of polyprotein precursors that undergo autoproteolysis to release the free mature M^pro. To investigate the autoprocessing mechanism in transfected mammalian cells, we examined several fusion precursors, with the mature SARS-CoV-2 M^pro along with the flanking amino acids (to keep the native substrate sequences) sandwiched between different tags. Our analyses revealed differential proteolysis kinetics at the N- and C-terminal cleavage sites. Particularly, N-terminal processing is differentially influenced by various upstream fusion tags (GST, sGST, CD63, and Nsp4) and amino acid variations at the N-terminal P1 position, suggesting that precursor catalysis is flexible and subject to complex regulation. Mutating Q to E at the N-terminal P1 position altered both precursor catalysis and the properties of the released M^pro. Interestingly, the wild-type precursors exhibited different enzymatic activities compared to those of the released M^pro, displaying much lower susceptibility to known inhibitors targeting the mature form. These findings suggest the precursors as alternative targets for antiviral development. Accordingly, we developed and validated a high-throughput screening (HTS)-compatible platform for functional screening of compounds targeting either the N-terminal processing of the SARS-CoV-2 M^pro precursor autoprocessing or the released mature M^pro through different mechanisms of action. Full article

Monascus pigments (MPs) and monacolin K (MK) are important secondary metabolites produced by Monascus spp. This study aimed to investigate the effect of soybean protein isolate (SPI) on the biosynthesis of MPs and MK based on the analysis of physiological indicators, transcriptomes, and metabolomes. The results indicated that the growth, yellow MPs, and MK production of Monascus pilosus MS-1 were significantly enhanced by SPI, which were 8.20, 8.01, and 1.91 times higher than that of the control, respectively. The utilization of a nitrogen source, protease activity, the production and utilization of soluble protein, polypeptides, and free amino acids were also promoted by SPI. The transcriptomic analysis revealed that the genes mokA, mokB, mokC, mokD, mokE, mokI, and mokH which are involved in MK biosynthesis were significantly up-regulated by SPI. Moreover, the glycolysis/gluconeogenesis, pyruvate metabolism, fatty acid degradation, tricarboxylic acid (TCA) cycle, and amino acid metabolism were effectively up-regulated by SPI. The metabolomic analysis indicated that metabolisms of amino acid, lipid, pyruvate, TCA cycle, glycolysis/gluconeogenesis, starch and sucrose, and pentose phosphate pathway were significantly disturbed by SPI. Thus, MPs and MK production promoted by SPI were mainly attributed to the increased biomass, up-regulated gene expression level, and more precursors and energies. Full article