Search Results (945)

The serine protease prostasin on the surface of the exosomes released from epithelial cells can interact with ectopically over-expressed cell-surface serine protease matriptase in cancerous B cells to initiate the prostasin–matriptase proteolytic activation cascade. Matriptase activation and the ensuing self-activation result in its removal from cancer cells, reducing cell proliferation and migration. In this study, we tested the hypothesis that the matriptase in the lymphoid cells could be removed by the prostasin-initiated activation and self-activation using genetically engineered autologous cells carrying prostasin. In co-cultures with the prostasin-positive cells, the matriptase on the prostasin-negative vector-control cells was removed in a dose-dependent manner, as determined by flow cytometry. This paracrine phenotype requires the active sites of both proteases. In silico analysis of the RNA-seq profiles indicated an imbalanced expression of high matriptase and low prostasin, and their cognate protease inhibitors in B-cell lymphoma patient specimens. The impact of exosomal prostasin on the cluster of differentiation molecules in activated human peripheral blood mononuclear cells was investigated by flow cytometry, revealing candidate mechanisms for prostasin’s role in regulating cellular adaptive immunity. This autologous paracrine prostasin–matriptase interaction could be exploited as a method for targeting over-expressed matriptase in diseases such as B-cell lymphoma. Full article

Protease-activated receptor 2 (PAR2) is a seven-transmembrane, G-protein-coupled receptor that is activated by coagulation proteases such as factor VIIa and factor Xa and other serine proteases. It is a potential therapeutic target for kidney injury, as it enhances inflammatory and fibrotic responses via the nuclear factor-kappa B and mitogen-activated protein kinase cascades. The body of knowledge regarding the role of PAR2 in kidney disease is currently growing, and its role in various kidney disease models, such as acute kidney injury, renal fibrosis, diabetic kidney disease, aging, and thrombotic microangiopathy, has been reported. Here, we review the literature to better understand the various aspects of PAR2 in kidney disease. Full article

Complement factor B (CFB) is a crucial component for the activation of the alternative pathway due to the formation of the C3 convertase with C3b, which further produces C3b to enhance the overall complement activity. Although Cfb is expressed not only in the immune tissues, but also in the reproductive tract, the physiological role of the alternative complement pathway in reproduction remains unclear. In this study, we addressed this issue by producing Cfb-knockout (KO) mice and analyzing their phenotypes. Sperm function, number of ovulated oocytes, and litter size were normal in KO mice. In contrast, the diversity of microbiomes in the gut and vaginal tract significantly increased in KO mice. Some serine protease activity in the serum from KO mice was lower than that of wild-type mice. Since the serum from KO mice showed significantly lower activity of the alternative complement pathway, CFB was found to be essential for this pathway. Our results indicate that although the alternative pathway is dispensable for normal fertility and development, it maintains the gut and vaginal microbiomes by suppressing their diversity and activating the alternative complement pathway. Full article

Samples of marine surimi made from six different fish types have been comprehensively investigated and compared in terms of chemical composition, protease activity, gelling chemistry, gel properties and 3D printability. It has been found that surimi with high protein content, low fat concentration, low activity of endogenous protease (cathepsin B, cathepsin L, cathepsin D, calpain and serine protease) and low TVB-N showed better gelling capacity and gel properties. However, the 3D printability of surimi was more relevant to apparent viscosity. The surimi with viscosity between 5000 Pa·s and 12,000 Pa·s yielded better printing performance as indicated by higher printing accuracy than 97% and less cooking loss. This study clarified the effects of marine fish types on the corresponding surimi gelation and gel properties, and successfully established relationships between surimi properties and 3D printing performance, thus providing new insights on exploring new surimi sources and developing 3D surimi printing techniques. Full article

A novel caseinolytic protease (ClpP) of the S14 family from Cobetia amphilecti KMM 296 (CamClpP), comprising 206 amino acids, with a calculated molecular weight of 22.66 kDa and a pI of 4.88, was expressed in Escherichia coli cells to verify the functional annotation of the encoding gene that has low identity with known structures. The proteolytic activity of the purified recombinant enzyme was found to be 2824 U/mg, using 1% casein as a substrate. Enzyme activity was maximal at pH 5.6 and 7.4 in phosphate buffer and was maintained over a wide pH range of 4-10. The optimum temperature for protease activity was 45 °C. The enzyme in its optimal state required the presence of either NaCl or KCl at concentrations of 0.3 and 0.2 M, respectively. The addition of the metal ions Mg²⁺, Ca²⁺, Ni²⁺, Mn²⁺, Li⁺, and Zn²⁺ at 2 mM resulted in a significant inhibition of the protease activity. However, the presence of Co²⁺ led to a marked activation of the enzyme in the absence of ATP. The enzyme activity was inhibited by ethanol, isopropanol, glycerol, SDS, EGTA, and EDTA. The presence of Triton X-100, acetone, DTT, and PMSF resulted in a significant increase in the CamClpP protease activity. The protease CamClpP effectively and preferentially degrades high-polymer wheat and rye flour proteins. This new proteolytic enzyme with unique properties is of great ecological and biotechnological importance. Full article

Snakebite is a critical global public health issue, causing substantial mortality and morbidity, particularly in tropical and subtropical regions. The development of innovative antivenoms targeting snake venom toxins is therefore of paramount importance. In this study, we adopted an epitope-directed approach to design three degenerate 15-mer peptides based on amino acid sequence alignments of snake venom phospholipase A₂s (PLA₂s) and snake venom serine proteases (SVSPs) from snake (Crotalus atrox). By leveraging their immunogenic and inhibitory profiles, these peptides were specifically designed to target the Asp49 and Lys49 variants of PLA₂ and SVSP toxins. Groups of five mice were immunized with each peptide, and IgG mRNA was subsequently extracted from peripheral blood mononuclear cells (PBMCs) and spleen lymphocytes of the top three responders. The extracted mRNA was reverse-transcribed into complementary DNA (cDNA), and the variable regions of the IgG heavy and kappa chains were amplified using polymerase chain reaction (PCR). These amplified regions were then linked with a 66-nucleotide spacer to construct single-chain variable fragments (scFvs). Sequence analysis of 48 randomly selected plasmids from each PLA₂ and SVSP scFv library revealed that over 80% contained scFv sequences with notable diversity observed in the complementarity-determining regions (CDRs), particularly CDR3. Enzyme-linked immunosorbent assay (ELISA) results demonstrated that the SP peptide elicited a broader immune response in mice compared to the Asp49 peptide, implying the strong immunogenicity of the SP peptide. These scFvs represent a promising foundation for the development of recombinant human monoclonal antibodies targeting snake PLA₂ and SVSP toxins, providing a potential therapeutic strategy for the treatment of snakebites. Full article

Information on skin barrier in horses is limited. A study on the epidermal ultrastructure of normal and allergic horses documented disorganized amorphous intercellular lipids in the stratum corneum of allergic samples. These findings are similar to atopic canine and human skin. Currently, there is no published study comparing skin barrier function parameters between normal and allergic horses; thus, the functional implications of the ultrastructural changes are unknown. In normal horses, body location, gender, breed, and ambient conditions affect skin barrier parameters, such as Transepidermal Water Loss. Skin microbiome studies on normal horses have highlighted the importance of season and environmental conditions, since horses housed together share similar microbiomes. Skin dysbiosis and predominance of staphylococcus have been described in horses with pastern dermatitis. Transcriptomic studies of the epidermis of normal and allergic horses have found that lesional allergic skin has substantial transcriptomic differences when compared with healthy skin, namely downregulation of genes of tight junctions, keratins, and upregulation of serine proteases and IL-13. Keratinocytes harvested from horses with insect bite hypersensitivity show upregulation of IL-31 gene expression under stimulation. While more research is clearly needed, preliminary results seem to support skin barrier differences between normal and allergic horses. Full article

The outermost surface of wool is covered by a scale layer, posing challenges to some steps of fabric processing. This layer, primarily composed of keratin, resists degradation by conventional proteases due to its high disulfide bond content. Protease K, an extracellular serine endo-proteinase derived from Tritirachium album Limber (tPRK), is known for its ability to digest native keratin. However, its limited activity against keratin has restricted its application in wool scale layer treatment. In this study, the substrate-binding pocket of tPRK was engineered, yielding the mutant N162A, which demonstrated an 84% increase in catalytic activity toward keratin. Additionally, the catalytic efficiency (k_cat/K_m) of N162A on keratin improved by 44.52%. Structural analysis indicated that modifications in the substrate-binding pocket reduced steric hindrance during substrate entry while enhancing substrate binding. Additionally, 3.3 mg/mL of amino acids were released within 6 h, which were catalyzed by N162A, with a 61% increase compared to the native tPRK. Moreover, the N162A variant effectively reduced the scale layer thickness without compromising the tensile strength of the wool, maintaining its mechanical properties. The findings provide a sustainable strategy for the wool industry while broadening the scope of biotechnological applications in the textile sector. Full article

The accumulation of D-amino acid-containing peptides is associated with age-related diseases such as Alzheimer’s disease and cataracts, while glycosylation is an important modification of proteins and plays a key role in improving the physicochemical properties of peptides and facilitating their regulation in biological systems. This study investigates the effects of glycosylation position, glycan number, and monosaccharide structure on the conformation and enzymatic degradation of D-amino acid-containing peptides, using KYNEtWRSED (5-t) as a model peptide and six monosaccharides as model glycans. The results demonstrated that glycosylation inhibited the enzymatic degradation of 5-t in the presence of most serine-like proteases. However, in the presence of chymotrypsin, glycosylation with modified monosaccharides (except for β-D-GalNAc) promoted the degradation of 5-t. Furthermore, glycosylation had no effect on the cleavage site of 5-t. Molecular docking analysis revealed that the hydrogen bonding and electrostatic interactions between the glycopeptide and chymotrypsin were markedly strengthened, likely serving as a key determinant of the enzymatic effects. Collectively, these findings highlight the potential of glycosylation to enhance the therapeutic and biomedical applications of D-amino acid-containing peptides in disease treatment and drug design. Full article

Serine proteases are multifunctional and versatile venom components found in viper snakes, including the Bothrops species, a widely distributed genus notorious for causing the highest number of snakebites across Latin America. These enzymes, representing a significant fraction of Bothrops venom proteomes, exhibit a wide range of biological activities that influence blood coagulation, fibrinolysis, and inflammation. This review provides a comprehensive overview of serine proteases, with a particular focus on those found in the venom of Brazilian Bothrops snakes. The discussion begins with a summary of snake species found in Brazil and their medical relevance. Specifically addressing the Bothrops genus, this review explores the distribution of these species across Brazilian territory and their associated medical importance. Subsequently, the article investigates the biochemistry of Bothrops venoms and the clinical manifestations induced by envenomation. Finally, it offers an in-depth discussion on the serine proteases, highlighting their biochemical properties, mechanisms of action, and potential therapeutic applications. Furthermore, this review provides an in-depth exploration of the diverse serine proteases found in Bothrops venoms and their functional significance, from thrombin-like effects to potent fibrinogenolytic actions, which determine the clinical manifestations of envenomation. This review delves into the evolutionary adaptations and biochemical diversity of serine proteases in Bothrops venoms, emphasizing their critical roles in venom functionality and the resulting pathophysiological effects. Additionally, it opens new avenues for utilizing these enzymes in biomedical applications, underscoring their potential beyond toxinology. Full article

This study explores the potential of Tenebrio molitor protein hydrolysates as functional food ingredients, evaluating their bioactivity and consumer acceptance of the incorporation of edible insects into food across Poland and Spain. By aligning technical advancements with consumer preferences, this research bridges the gap between laboratory innovation and market feasibility, contributing to the development of sustainable functional foods. The study optimized the process of enzyme hydrolysis using serine protease from Cucurbita ficifolia, thereby enhancing DPPH scavenging capacity increased from 3.15 ± 0.53 to 8.17 ± 0.62 µM Trolox/mL and ABTS decolorization capacity increased from 4.29 ± 0.01 to 10.29 ± 0.01 µM Trolox/mL after 5 h of hydrolysis. Consumer surveys incorporating the Food Neophobia, Insect Phobia, and Entomophagy Scales revealed demographic and cultural influences on entomophagy acceptance. Among respondents, 27.1% in Poland and 25.7% in Spain had previously consumed insect-based products, while Polish participants showed a higher willingness to adopt insect-enriched foods. The study confirmed that hydrolysis enhances the antioxidant activity of T. molitor protein hydrolysates and that demographic and cultural factors significantly influence consumer acceptance of insect-based foods. Full article

The human transmembrane protease, serine 2 (TMPRSS2), essential for SARS-CoV-2 entry, is a key antiviral target. Here, we computationally profiled the TMPRSS2-binding affinities of 15 antiviral compounds. Molecular dynamics (MD) simulations for the docked complexes revealed that three compounds exited the substrate-binding cavity (SBC), suggesting noncompetitive inhibition. Of the remaining compounds, five charged ones exhibited reduced binding stability due to competing electrostatic interactions and increased solvent exposure, while seven neutral compounds showed stronger binding affinity driven by van der Waals (vdW) interactions compensating for unfavorable electrostatic effects (including electrostatic interactions and desolvation penalties). Positive and negative hotspot residues were identified as uncharged and charged, respectively, both lining the SBC. Despite forming diverse interactions with compounds, the burial of positive hotspots led to strong vdW interactions that overcompensated for unfavorable electrostatic effects, whereas negative hotspots incurred high desolvation penalties, negating any favorable contributions. Charged residues at the SBC’s outer rim can reduce binding affinity significantly when forming hydrogen bonds or salt bridges. These findings underscore the importance of enhancing vdW interactions with uncharged residues and minimizing the unfavorable electrostatic effects of charged residues, providing valuable insights for designing effective TMPRSS2 inhibitors. Full article

Alpha-1 antitrypsin (AAT) is a key serine protease inhibitor for regulating proteases such as neutrophil elastase. AAT restrains the pulmonary matrix from enzymatic degradation, and a deficiency in AAT leads to inflammatory tissue damage in the lungs, resulting in chronic obstructive pulmonary disease. Due to the crucial biological function of AAT, the emerging research interest in this protein has shifted to its role in cancer-associated inflammation and the dynamics of the tumor microenvironment. However, the lack of comprehensive reviews in this field hinders our understanding of AAT as an essential immune modulator with great potential in cancer immunotherapy. Therefore, in this review, we have elucidated the pivotal roles of AAT in inflammation and the tumor microenvironment, including the structure and molecular properties of AAT, its molecular functions in the regulation of the inflammatory response and tumor microenvironment, and its clinical implications in cancer including its diagnosis, prognosis, and therapeutic intervention. This review seeks to bridge the gap in the understanding of AAT between inflammatory diseases and cancer, and to foster deeper investigations into its translational potential in cancer immunotherapy in the future. Full article

Coagulation factor XIa is a new serine-protease family drug target for next-generation anticoagulants. With the snake venom Kunitz-type peptide BF9 as the scaffold, we obtained a highly active XIa inhibitor BF9-N17K in our previous work, but it also inhibited the hemostatic target plasmin. Here, in order to enhance the selectivity of BF9-N17K toward XIa, four mutants, BF9-N17K-L19A, BF9-N17K-L19S, BF9-N17K-L19D, and BF9-N17K-L19K, were further designed using the P2′ amino acid classification scanning strategy. The anticoagulation assay showed that the four P2′ single-point mutants still had apparent inhibitory anticoagulation activity that selectively inhibited the human intrinsic coagulation pathway and had no influence on the extrinsic coagulation pathway or common coagulation pathway, which indicated that the single-point mutants had minimal effects on the anticoagulation activity of BF9-N17K. Interestingly, the enzyme inhibitor assay experiments showed that the XIa and plasmin inhibitory activities were significantly changed by the P2′ amino acid replacements. The XIa inhibitory activity of BF9-N17K-L19D was apparently enhanced, with an IC₅₀ of 19.28 ± 2.53 nM, and its plasmin inhibitory was significantly weakened, with an IC₅₀ of 459.33 ± 337.40 nM. BF9-N17K-L19K was the opposite to BF9-N17K-L19D, which had enhanced plasmin inhibitory activity and reduced XIa inhibitory activity. For BF9-N17K-L19A and BF9-N17K-L19S, no apparent changes were found in the serine protease inhibitory activity, and they had similar XIa and plasmin inhibitory activities to the template peptide BF9-N17K. These results suggested that the characteristics of the charge of the P2′ site might be associated with the drug selectivity between the anticoagulant target XIa and hemostatic target plasmin. In addition, according to the molecular diversity and sequence conservation, a common motif GR/PCR/KA/SXIP-XYGGC is proposed in the XIa-inhibitory Kunitz-type peptides, which might provide a new clue for further peptide engineering. In conclusion, through P2′ amino acid classification scanning with the snake venom Kunitz-type peptide scaffold, a new potent and selective XIa inhibitor, BF9-N17K-L19D, was discovered, which provides a new XIa-targeting lead drug template for the treatment of thrombotic-related diseases. Full article

Background: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for causing the Coronavirus disease 2019 (COVID-19) outbreak. While mutations cause the emergence of new variants, the ancestral SARS-CoV-2 strain is unique among other strains. Methods: Various clinical parameters, the activity of cathepsin proteases, and the concentration of various proteins were measured in urine samples from COVID-19-negative participants and COVID-19-positive participants. Urinary extracellular vesicles (uEVs) were isolated from urine samples from the two groups and used for proteomic analysis and subsequent pathway analyses. Results: Activity levels of cathepsin S and L were greater in the urine of COVID-19-positive participants. The concentration of C-reactive protein, transmembrane serine protease 2, and klotho protein were significantly greater in the urine of COVID-19-positive participants. There was a greater amount of uEVs in the COVID-19 group and klotho protein was found to be enriched in uEVs from the COVID-19 group. Pathway analyses of the proteomics data showed most of the identified proteins were involved in signal transduction, stress response, protein metabolism, and transport. The identified proteins were predominantly associated with cellular membranes and with function of the cytoskeleton, enzyme regulation, and signal transduction. Conclusions: Taken together, our data identify novel urinary biomarkers that could be used to further investigate the long-term effects of SARS-CoV-2 infection. Full article