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The process of viral entry into host cells is crucial for the establishment of infection and the determination of viral pathogenicity. A comprehensive understanding of entry pathways is fundamental for the development of novel therapeutic strategies. Standard techniques for investigating viral entry include confocal microscopy and flow cytometry, both of which provide complementary qualitative and quantitative data. Imaging flow cytometry, which integrates the advantages of both methodologies, offers significant potential in virological studies. In this investigation, we employed imaging flow cytometry coupled with immunostaining to monitor the entry of equine arteritis virus EAV into Vero cells via the endosomal trafficking route. Analysis provided an insight into the early infection dynamics across thousands of cells, revealing statistically significant alterations in internalization and uncoating process. Moreover, we evaluated the effectiveness of two inhibitors targeting cellular factors involved in facilitating viral entry: ammonium chloride, which disrupts endocytosis, and camostat mesylate, which inhibits the activity of serine proteases. The results demonstrated a clear distinction between effective and ineffective inhibitors. This study highlighted the potential of imaging flow cytometry to advance the study of viral entry and the evaluation of antiviral agents. [ABSTRACT FROM AUTHOR]

Whipworms (Trichuris spp) are ubiquitous parasites of humans and domestic and wild mammals that cause chronic disease, considerably impacting human and animal health. Egg hatching is a critical phase in the whipworm life cycle that marks the initiation of infection, with newly hatched larvae rapidly migrating to and invading host intestinal epithelial cells. Hatching is triggered by the host microbiota; however, the physical and chemical interactions between bacteria and whipworm eggs, as well as the bacterial and larval responses that result in the disintegration of the polar plug and larval eclosion, are not completely understood. Here, we examined hatching in the murine whipworm, Trichuris muris, and investigated the role of specific bacterial and larval structures and molecules in this process. Using scanning and transmission electron microscopy, we characterised the physical interactions of both fimbriated (Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa) and non-fimbriated (Staphylococcus aureus) bacteria with the egg polar plugs during the induction/initiation stage, and visualised the effects of structural changes in the polar plugs, leading to larval eclosion. Further, we found that protease inhibitors blocked whipworm hatching induced by both fimbriated and non-fimbriated bacteria in a dose-dependent manner, suggesting the partial involvement of bacterial enzymes in this process. In addition, we identified the minimal egg developmental timing required for whipworm hatching, and transcriptomic analysis of T. muris eggs through embryonation revealed the specific upregulation of serine proteases (S01A family) in fully embryonated eggs containing 'hatch-ready' L1 larvae. Finally, we demonstrated that inhibition of serine proteases with the serine-protease inhibitor Pefabloc ablated T. muris egg hatching induced by bacteria. Collectively, our findings unravel the temporal and physicochemical bacterial-egg interactions leading to whipworm hatching and indicate serine proteases of both bacterial and larval origin mediate these processes. Author summary: Human whipworms are parasites that cause the gastrointestinal disease trichuriasis in millions of people around the world. Infections occur when whipworm eggs, ingested in contaminated food and water, hatch in the intestine in response to gut bacteria (microbiota). The egg encloses a larva within an egg-shell and has a plug at each end. Hatching liberates the larva that burrows inside the cells that line the gut. Interactions between the microbiota of the gut and whipworm eggs are needed for hatching, but are poorly understood. In this study, using the natural mouse whipworm as an infection model, we show that bacteria bind the whipworm egg plugs during the initial stages of hatching, resulting in their degradation and leading to larval exit. We further show that disintegration of the egg plugs is caused by protein-degrading enzymes produced by the bacteria and the larvae. The production of those enzymes by the parasite is dependent on the full development of the larva inside the whipworm egg. These new mechanistic insights pave the way for future studies to understand human whipworm infection and develop new tools to tackle these globally important parasites. [ABSTRACT FROM AUTHOR]

Plant peptides, synthesized from larger precursor proteins, often undergo proteolytic cleavage and post-translational modifications to form active peptide hormones. This process involves several proteolytic enzymes (proteases). Among these, SBTs (serine proteases) are a major class of proteolytic enzymes in plants and play key roles in various regulatory mechanisms, including plant immune response, fruit development and ripening, modulating root growth, seed development and germination, and organ abscission. However, current knowledge about SBTs is largely limited to 'in vitro cleavage assays ,' with few studies exploring loss of function analyses for more in depth characterization. Research focused on economically significant horticultural crops, like tomato and pepper, remains scarce. Given this, leveraging SBTs for horticultural crop improvement through advanced gene-editing tools is critical for enhancing crop resilience to stress and pathogens. Over the past five years, research on proteolytic enzymes, especially SBTs, has increased markedly, yet reports involving loss- or gain-of function analyses aimed at improving crop yield and quality are still limited. This review summarizes recent findings on SBT enzymes, which act as 'protein scissors' in activating peptide hormones, and discusses the potential for using selected SBTs in CRISPR-Cas9 gene editing to enhance the growth and resilience of economically important Solanaceae crops, with a focus on pepper. [ABSTRACT FROM AUTHOR]

Background: We compared the enzymatic, coagulant, and neuromuscular activities of two variants (yellow—CDRy and white—CDRw) of Crotalus durissus ruruima venom with a sample of C. d. terrificus (CDT) venom and examined their neutralization by antivenom against CDT venom. Methods: The venoms were screened for enzymatic and coagulant activities using standard assays, and electrophoretic profiles were compared by SDS-PAGE. Neutralization was assessed by preincubating venoms with crotalic antivenom and assaying the residual activity. Results: SDS-PAGE showed that the venoms had similar electrophoretic profiles, with the main bands being phospholipase A2 (PLA2), serine proteinases, L-amino acid oxidase (LAAO), and phosphodiesterase. CDRy venom had the highest proteolytic and LAAO activities, CDRw venom had greater PLA2 and esterolytic activities at the highest quantity tested, and CDT had greater PLA2 activity than CDRy. CDRw and CDT venoms had similar proteolytic and LAAO activities, and CDRy and CDT venoms had comparable esterolytic activity. None of the venoms altered the prothrombin time (PT), but all of them decreased the activated partial thromboplastin time (aPPT); this activity was neutralized by antivenom. The minimum coagulant dose potency was CDRw >> CDRy > CDT. All venoms had thrombin-like activity that was attenuated by antivenom. CDRy and CDRw venoms showed α-fibrinogenolytic activity. All venoms partially cleaved the β-chain. CDRy and CDT venoms caused neuromuscular facilitation (enhanced muscle contractions) followed by complete blockade, whereas CDRw venom caused only blockade. Antivenom neutralized the neuromuscular activity to varying degrees. Conclusions: These findings indicate that while CDR and CDT venoms share similarities, they also differ in some enzymatic and biological activities and in neutralization by antivenom. Some of these differences could influence the clinical manifestations of envenomation by C. d. ruruima and their neutralization by the currently used therapeutic antivenom. [ABSTRACT FROM AUTHOR]

Aims: Bronchiectasis patients face an unmet need for treatment options that reduce inflammation. Cathepsin C inhibition is expected to achieve this by reducing the activation of neutrophil‐derived serine proteases. Here, we present safety and pharmacokinetic (PK) data from a Phase I trial evaluating the novel cathepsin C inhibitor BI 1291583 in healthy Japanese male subjects. Methods: This randomized, double‐blind, placebo‐controlled, parallel‐group study investigated BI 1291583 in healthy Japanese male subjects (jRCT2071210111) and consisted of a single‐rising‐dose (SRD) part and a multiple‐dose (MD) part. The primary endpoint was the percentage of subjects with drug‐related treatment‐emergent adverse events (AEs). Secondary PK endpoints (SRD: AUC0‐∞ and Cmax; MD: AUCτ,1 and Cmax,1 after first dose and AUCτ,ss and Cmax,ss after last dose), as well as further safety and PK endpoints, were also assessed. Results: Overall, 36 subjects (n = 24 for SRD part; n = 12 for MD part) entered this Phase I trial. BI 1291583 was safe and well tolerated across the doses tested. All AEs were of mild intensity, with no drug‐related treatment‐emergent AEs, deaths, serious AEs or AEs of special interest reported in either part of the trial. Following both SRD and MD administration, BI 1291583 was readily absorbed, and PK was supraproportional over the doses assessed. Conclusion: The results show that BI 1291583 has an appropriate benefit–risk ratio for Japanese patients, with no safety or exposure concerns at the doses studied. Japanese patients with bronchiectasis can be safely integrated into future global clinical trials of BI 1291583, with no dose adjustment required. [ABSTRACT FROM AUTHOR]

BACKGROUND: Sperm development and behavior present promising targets for environmentally safer, target‐specific biorational control strategies. Serine protease in seminal fluid proteins plays a crucial role in the post‐mating reproductive processes of lepidopteran pest insects. The serine protease 2 has been identified as the initiatorin of the seminal fluid protein in Lepidoptera, and its loss of function leads to male sterility. Nevertheless, the genetic pattern of this gene mutation and the impacts of various mutant genotypes on the hatchability of the eggs of pests remain unclear. RESULTS: This study focused on the cloning of Spodoptera exigua serine protease 2 (SeSer2), which is specifically expressed in male moths. The open reading frame of SeSer2 consists of 843 nucleotides, encoding 280 amino acids with structural characteristics typical of serine proteases in the S1 family. To validate the functional role of SeSer2 in the fertility of S. exigua, a targeted ~3574‐bp deletion of SeSer2 was introduced using the CRISPR/Cas9 genome editing system, leading to premature truncation of the SeSer2 protein. The SeSer2 mutation had no significant impact on the growth and development of individuals of either sex. However, disruption of SeSer2 resulted in heritable male sterility. Although females mated with SeSer2−/− (SeSer2 knockout homozygote) males laid eggs normally, these eggs failed to hatch. SeSer2+/− (SeSer2 knockout heterozygote) male moths crossed with female moths produced viable offspring, indicating the gene's recessive role in egg hatching. CONCLUSION: These findings strongly support the conclusion that the Ser2 gene is essential for male reproductive success in diverse lepidopterans. Targeting the Ser2 gene holds promise as a foundational element of a novel pest control strategy. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

During intestinal and liver invasion by the protozoan parasite Entamoeba histolytica , extensive tissue destruction linked to large neutrophil infiltrates is observed. It has been proposed that microbicidal components of neutrophils are responsible for the damage, however, the mechanism by which they are released and act in the extracellular space remains unknown. In previous studies, we have shown that E. histolytica trophozoites induce NET formation, leading to the release of neutrophil granule content into extruded DNA. In this work, we evaluate the possible participation of NETs in the development of amoeba-associated pathology and analyze the contribution of anti-microbial components of the associated granules. E. histolytica- induced NETs were isolated and their effect on the viability and integrity of HCT 116 colonic and Hep G2 liver cultures were evaluated. The results showed that simple incubation of cell monolayers with purified NETs for 24 h resulted in cell detachment and death in a dose-dependent manner. The effect was thermolabile and correlated with the amount of DNA and protein present in NETs. Pretreatment of NETs with specific inhibitors of some microbicidal components suggested that serine proteases, are mostly responsible for the damage caused by NETs on HCT 116 cells, while the MPO activity was the most related to Hep G2 cells damage. Our study also points to a very important role of DNA as a scaffold for the activity of these proteins. We show evidence of the development of NETs in amoebic liver abscesses in hamsters as a preamble to evaluate their participation in tissue damage. In conclusion, these studies demonstrate that amoebic-induced NETs have potent cytotoxic effects on target cells and, therefore, may be responsible for the intense damage associated with tissue invasion by this parasite. [ABSTRACT FROM AUTHOR]

The filamentous fungus Stachybotrys chartarum is rich in meroterpenoid secondary metabolites, some of which carry o -dialdehyde moieties, which are readily derivatized to isoindolinones by addition of primary amines. The structural diversity of phenylspirodrimanes, in particular, is linked to a wide range of biological activities, making them ideal candidates for semi-synthetic modification. In this study, acetoxystachybotrydial acetate was reacted with l-tryptophan and tryptamine, resulting in the detection of both regiospecific isomeric structures - a rare and significant finding that enabled the examination of four novel reaction products. Besides their successful purification, a detailed report on their isomer-specific behavior with regard to chromatographic retention, UV-spectral specificities, nuclear magnetic resonances, and mass spectrometric fragmentation is given. Furthermore, a comprehensive insight into each compounds' unique effect within the tested biological assays is provided, which include cytotoxicity, genotoxicity, their biological activity against serine proteases of the blood coagulation cascade, and in vitro hepatic metabolism, always in comparison to the non-derivatized substance. Ultimately, each isomer can be distinguished already during the purification process, which extends to the biological assays where we present one less cytotoxic, faster metabolized, and more active regio-isomeric phenylspirodrimane-derivative. [ABSTRACT FROM AUTHOR]

• ECM architecture is dynamically altered by serine proteases during carcinogenesis. • Protease-mediated proteolysis modulates activities of ECM components and integrins. • Degradation of ECM allows epithelial cancer cells to enter the blood circulation. • ECM remodeling causes the release of many ECM-associated growth factors. The extracellular matrix (ECM) serves as a physical scaffold for tissues that is composed of structural proteins such as laminins, collagens, proteoglycans and fibronectin, forming a three dimensional network, and a wide variety of other matrix proteins with ECM-remodeling and signaling functions. The activity of ECM-associated signaling proteins is tightly regulated. Thus, the ECM serves as a reservoir for water and growth regulatory signals. The ECM architecture is dynamically modulated by multiple serine proteases that process both structural and signaling proteins to regulate physiological processes such as organogenesis and tissue homeostasis but they also contribute to pathological events, especially cancer progression. Here, we review the current literature regarding the role of ECM remodeling by serine proteases (KLKs, uPA, furin, HtrAs, granzymes, matriptase, hepsin) in tumorigenesis. [ABSTRACT FROM AUTHOR]

Granzymes are serine proteases with intracellular cytotoxic and/or proinflammatory functions but also accumulate in the extracellular space in aging and disease, promoting proinflammatory phenotypes through the cleavage of receptors, cytokines, adhesion molecules, and autoantigens. Granzyme B transcripts and protein have recently been detected in aging immune cell populations and linked to inflammaging phenotypes and age-related pathologies. Granzyme B has been implicated in many age-related pathologies. Granzyme B is induced by environmental factors that exacerbate biological aging (UV exposure, poor diet, smoking, wildfire smoke, grain dust), while genetic deletion and/or pharmacological inhibition of granzyme B reduces premature aging and/or disease phenotypes in animal models. Aging is a major risk factor for pathologies that manifest later in life. Much attention is devoted towards elucidating how prolonged environmental exposures and inflammation promote biological (accelerated) tissue aging. Granzymes, a family of serine proteases, are increasingly recognized for their emerging roles in biological aging and disease. Widely recognized as intracellular mediators of cell death, granzymes, particularly granzyme B (GzmB), also accumulate in the extracellular milieu of tissues with age, contributing to chronic tissue injury, inflammation, and impaired healing. Consequently, this has prompted the field to reconsider how GzmB regulation, accumulation, and proteolysis impact health and disease with age. While GzmB is observed in numerous age-related conditions, the current review focuses on mechanistic studies where proof-of-concept has been forwarded. [ABSTRACT FROM AUTHOR]

The North African mountain viper (Vipera monticola) is a medically relevant venomous snake distributed in Morocco, Algeria, and Tunisia. Three subspecies of V. monticola , exhibiting differences in morphotypes and dietary regimes, are currently recognised: V. m. monticola , V. m. atlantica , and V. m. saintgironsi. Through the application of snake venomics, we analysed the venoms of specimens of Moroccan origin belonging to each of the three subspecies. Snake venom metalloproteinases (svMP), snake venom serine proteases (svSP), C-type lectin and C-type lectin-related proteins (CTL), and phospholipases A 2 (PLA 2) were predominant, with PLA 2 being the most abundant toxin family overall. Disintegrins (DI) and cysteine-rich secretory proteins (CRISP) were exclusive to V. m. monticola and V. m. atlantica , while l -amino-acid oxidases (LAAO) were only found in V. m. saintgironsi. The differences detected in the venom profiles, as well as in presence/absence and relative abundances of toxin families, indicate the occurrence of intraspecific venom variation within V. monticola. The identified patterns of venom similarity between subspecies seem to align more with their phylogenetic relationships than with the reported differences in their feeding habits. [Display omitted] • First proteomics analysis of the medically relevant Vipera monticola venom. • Evident venom variation across the three subspecies. • Characteristic viperine toxin families are predominant: svMP, svSP, CTL, and PLA 2. • DI and CRISP unique to "dwarf" subspecies V. m. monticola and V. m. atlantica. • LAAO exclusive to larger V. m. saintgironsi. [ABSTRACT FROM AUTHOR]

The intricate combination of organic and inorganic compounds found in snake venom includes proteins, peptides, lipids, carbohydrates, nucleotides, and metal ions. These components work together to immobilise and consume prey through processes such as paralysis and hypotension. Proteins, both enzymatic and non-enzymatic, form the primary components of the venom. Based on the effects they produce, venom can be classified as neurotoxic, hemotoxic, and cytotoxic. Studies have shown that, after envenomation, proteins in snake venom also contribute significantly to the induction of inflammatory responses which can either have systemic or localized consequences. This review delves into the mechanisms by which snake venom proteins trigger inflammatory responses, focusing on key families such as phospholipase A2, metalloproteinases, serine proteases, C-type lectins, cysteine-rich secretory proteins, and L-amino acid oxidase. In addition, the role of venom proteins in activating various inflammatory pathways, including the complement system, inflammasomes, and sterile inflammation are also summarized. The available therapeutic options are examined, with a focus on antivenom therapy and its side effects. In general, this review offers a comprehensive understanding of the inflammatory mechanisms that are triggered by snake venom proteins and the side effects of antivenom treatment. All these emphasize the need for effective strategies to mitigate these detrimental effects. [ABSTRACT FROM AUTHOR]

Previous proteomic studies of viperid venom revealed that it is mainly composed of metalloproteinases (SVMPs), serine proteinases (SVSPs), phospholipase A2 (PLA2), and C-type lectins (CTLs). However, other proteins appear in minor amounts that affect prey and need to be identified. This study aimed to identify novel toxic proteins in the venom gland transcriptome of Bothrops asper and Bothrops jararaca, using data from NCBI. Bioinformatics tools were used to assemble, identify, and compare potentially novel proteins in both species, and we performed functional annotation with BLASTX against the NR database. While previous assemblies have been performed for B. jararaca, this is the first assembly of the B. asper venom gland transcriptome. Proteins with potentially novel functions were identified, including arylsulfatase and dihydroorotate dehydrogenase, among others, that could have implications for venom toxicity. These results suggest that the identified proteins may contribute to venom toxic variation and provide new opportunities for antivenom research. The study improves the understanding of the protein composition of Bothrops venom and suggests new possibilities for the development of treatments and antivenoms. [ABSTRACT FROM AUTHOR]

Cytomegaloviruses are highly species-specific as they replicate only in cells of their own or a closely related species. For instance, human cytomegalovirus cannot replicate in rodent cells, and mouse cytomegalovirus (MCMV) cannot replicate in human and monkey cells. However, the mechanisms underlying the host species restriction remain poorly understood. We have previously shown that passaging MCMV in human retinal pigment epithelial cells allows the virus to replicate to high titers in these cells due to the accumulation of adaptive mutations, such as loss-of-function mutations in the viral M117 gene. The M117 protein interacts with E2F transcription factors and activates E2F-dependent transcription. Here, we show that activation of E2F3 is primarily responsible for MCMV's inability to replicate in human cells. By transcriptome analysis, we identified two E2F3-induced serine proteases, FAM111A and FAM111B, as potential host restriction factors. By using shRNA-mediated gene knockdown and CRISPR/Cas9-mediated gene knockout, we demonstrated that FAM111B, but not its paralog FAM111A, suppresses MCMV replication in human and rhesus macaque cells. By immunofluorescence, we detected FAM111B predominantly in the nucleus of infected cells with enrichment in viral replication compartments, suggesting that it might play a role during viral replication. The fact that the FAM111B gene is conserved in primates but absent in rodents suggests that MCMV has not evolved to evade or counteract this restriction factor, which is not present in its natural host. [ABSTRACT FROM AUTHOR]

Background: Plant serine proteases are emerging as viable alternatives to animal and microbial proteases, particularly for whey protein hydrolysate preparation to enhance their nutritional and bio-functional properties. Objectives: The present study thus aims to optimize the conditions for whey protein hydrolysis employing ash gourd protease (AGP) to achieve maximum 2, 2 diphenyl -1-picrylhydrazyl (DPPH) radical scavenging (antioxidant) activity and degree of hydrolysis of the generated hydrolysate. Methods: Optimization was accomplished using response surface methodology and treatment combinations applying generated through a central composite design. This was conducted in two steps: first, temperature (50-90°C) and pH (6- 9) was optimized adding same concentration of AGP (0.3% v/v) at reconstituted WPC (0.6% protein), and second, E/S ratio (2-6 %v/v) and hydrolysis time (1-6h) was optimized at reconstituted WPC (4% protein). Furthermore, antioxidant potency of hydrolysate and its ultra-centrifuged fractions ≤3kDa and >3kDa obtained from the optimized condition was also evaluated. The AGP (Adjusted to activity 2U/mL) was partially purification using the three phase partitioning method for hydrolysis experiments. Results: The optimum conditions for enzymatic hydrolysis of whey protein to achieve maximum degree of hydrolysis (13.99%) and DPPH radical scavenging activity (23.05%) were observed at pH 7.48 and 66.40°C. Similarly, the enzyme performed optimally at enzyme by substrate ratio of 5.85% (v/v) for 6 hours of hydrolysis, providing 19.31 % of degree of hydrolysis and 47.71 % of DPPH radical scavenging activity. The lower molecular weight peptide fraction ≤3kDa was found to be more effective (1.45 times) than the peptide fractions >3kDa. However, it was less effective (1.19 times) than the whole hydrolysate regarding antioxidant activity. Conclusion: Overall, the study showed that the AGP employed whey protein hydrolysate has the potential for use as a natural antioxidant. [ABSTRACT FROM AUTHOR]

Introduction: Increased fecal protease activity, which may induce visceral hypersensitivity, has been observed in patients with irritable bowel syndrome (IBS). Serine proteases modulate FK506 binding protein (FKBP)-type peptidylprolyl cis-trans isomerase (PPIase) activity associated with immune and glucocorticoid receptor functions. The aim was to investigate whether camostat mesilate (CM), a serine protease inhibitor, modifies fecal bacterial function related to FKBP-type PPIases in patients with IBS. Methods: Randomly assigned 16 patients with IBS received 200 mg po tid of CM and 16 patients received placebo for 14 days. Self-reported adequate relief (AR) as a primary endpoint, IBS Symptom Severity Scale (IBS-SSS), and colonic motor and pain thresholds to colorectal distention were assessed before and after treatment. The fecal bacterial content was inferred from 16S rRNA gene sequence data using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States and the Kyoto Encyclopedia of Genes and Genomes database. Results: CM significantly increased the relative abundance of Streptococcus and the functional abundances of serine protease and FKBP-type PPIase FkpA, FklB and SlyD more than placebo after treatment. CM treatment was not superior to placebo in proportion of AR although colonic motor response partially changed. Conclusion: CM modulated the fecal microbiome composition and functional potentials that are related to FKBP-type PPIase activity in IBS patients. These findings suggest that protease inhibitors may modify gut microbial function along with abnormal immunological and/or stress responses that underlie pathophysiology of IBS. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) plays a central role in the structural integrity and functionality of the cardiovascular system. Moreover, the ECM is involved in atherosclerotic plaque formation and stability. In fact, ECM remodeling affects plaque stability, cellular migration, and inflammatory responses. Collagens, fibronectin, laminin, elastin, and proteoglycans are crucial proteins during atherosclerosis development. This dynamic remodeling is driven by proteolytic enzymes such as matrix metalloproteinases (MMPs), cathepsins, and serine proteases. Exploring and investigating ECM dynamics is an important step to designing innovative therapeutic strategies targeting ECM remodeling mechanisms, thus offering significant advantages in the management of cardiovascular diseases. This review illustrates the structure and role of vascular ECM, presenting a new perspective on ECM remodeling and its potential as a therapeutic target in atherosclerosis treatments. [ABSTRACT FROM AUTHOR]

Subtilisin-like proteins are serine proteases that use two types of catalytic triads: Ser-His-Asp and Ser-Glu-Asp. Here, we investigate the two known families of subtilisin-like proteins, the subtilases (Ser-His-Asp triad) and the serine-carboxyl proteinases (Ser-Glu-Asp triad), and describe the local structural arrangements (cores) that govern the catalytic residues in these proteins. We show the separation of the cores into conserved structural zones, which can be repeatedly found in different structures, and compare the structural cores in subtilisin-like proteins with those in trypsin-like serine proteases and alpha/beta-hydrolases. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic, caused by SARS-CoV-2, demonstrated that zoonotic transmission of animal sarbecoviruses threatens human health but the determinants of transmission are incompletely understood. Here, we show that most spike (S) proteins of horseshoe bat and Malayan pangolin sarbecoviruses employ ACE2 for entry, with human and raccoon dog ACE2 exhibiting broad receptor activity. The insertion of a multibasic cleavage site into the S proteins increased entry into human lung cells driven by most S proteins tested, suggesting that acquisition of a multibasic cleavage site might increase infectivity of diverse animal sarbecoviruses for the human respiratory tract. In contrast, two bat sarbecovirus S proteins drove cell entry in an ACE2-independent, trypsin-dependent fashion and several ACE2-dependent S proteins could switch to the ACE2-independent entry pathway when exposed to trypsin. Several TMPRSS2-related cellular proteases but not the insertion of a multibasic cleavage site into the S protein allowed for ACE2-independent entry in the absence of trypsin and may support viral spread in the respiratory tract. Finally, the pan-sarbecovirus antibody S2H97 enhanced cell entry driven by two S proteins and this effect was reversed by trypsin while trypsin protected entry driven by a third S protein from neutralization by S2H97. Similarly, plasma from quadruple vaccinated individuals neutralized entry driven by all S proteins studied, and availability of the ACE2-independent, trypsin-dependent pathway reduced neutralization sensitivity. In sum, our study reports a pathway for entry into human cells that is ACE2-independent, can be supported by TMPRSS2-related proteases and may be associated with antibody evasion. Author summary: Bats host a wide range of coronaviruses, including those related to SARS-CoV-1 and SARS-CoV-2 (subgenus Sarbecovirus), but their ability to infect human cells remains poorly understood. Identifying sarbecoviruses with zoonotic potential and understanding the factors controlling their entry into human cells are crucial for risk assessment and antiviral development. In this study, we examined how bat sarbecovirus spike proteins facilitate entry into human cells and identified key host factors involved. Using pseudovirus particles, we show that several bat sarbecovirus spike proteins use human and animal ACE2 receptors for entry. Additionally, we confirm that some spike proteins utilize an ACE2-independent entry pathway requiring trypsin treatment and demonstrate that this process is controlled by the spike protein receptor binding domain. Furthermore, we reveal that a subset of ACE2-using bat sarbecovirus spike proteins can switch to ACE2-independent entry following trypsin exposure and show that certain human type II transmembrane serine proteases can substitute for trypsin, enabling ACE2-independent entry. Finally, we demonstrate that repeated COVID-19 vaccination generates cross-neutralizing activity against bat sarbecoviruses, though ACE2-independent entry reduces neutralization sensitivity. [ABSTRACT FROM AUTHOR]

Objective: B-type natriuretic peptide (BNP) is a neurohormone primarily secreted from cardiac ventricles in reaction to increased volume and pressure. The plasma level of BNP is used to measure the mechanical function of the heart and the risk of developing chronic obstructive pulmonary disease (COPD). Alpha-1 antitrypsin (AAT) is a glycoprotein that acts as an inhibitor of serine proteases and plays a crucial role in protecting the lungs against potential harm. AAT deficiency also causes COPD. The data on the prevalence of BNP and AAT in African Americans (AA) are limited and inconsistent. No previous studies have investigated whether any association exists between BNP and AAT. We collected fasting blood samples from 114 AA subjects who provided informed consent. The plasma levels of the biomarkers were measured using ELISA. Results: Our findings revealed a significant negative correlation between AAT and BNP (r=-0.266, p = 0.01) as well as between AAT and TNF-α (r=-0.242, p = 0.01). Sex hormones (estrogen and testosterone) have a significant relationship with the levels of AAT and BNP. Increasing the AAT and reducing the levels of inflammation can lower the levels of BNP, which in turn could help lower the risk of heart disease in the AA population. [ABSTRACT FROM AUTHOR]

Proteases catalyze hydrolysis of amide bonds within peptides and proteins, therefore they play crucial functions for organism functioning, but also in industry to facilitate numerous processes. Feather-degrading fungus Onygena corvina (O. corvina) is loaded with numerous proteases that can be utilized for variety of applications. The most active species of these enzymes is heat-sensitive serine protease (NHSSP), from O. corvina fungi and due to its potential applications in industry is an alternative to proteinase K. The uniqueness of NHSSP relies on the ability of this enzyme to hydrolyze peptides at neutral to acidic pH values between 5.0 and 8.5, with an optimum of 6.8 and a temperature activity ranging from 15 to 50 °C making NHSSP exceptionally universal enzyme. Thus, we have performed the in-depth characterization of NHSSP substrate specificity by using a positional scanning substrate combinatorial library (PS-SCL). Afterward, we obtained a set of fluorescent substrates hydrolyzed by NHSSP that served as a leading sequence for the first tailored covalent inhibitor of this enzyme, containing a diphenylphosphonate as a warhead and MeOSuc amine protecting group. Our first inhibitor for NHSSP binds potently with target protease and is a tool for future study of this enzyme functions. [Display omitted] • Catalytic preferences of NHSSP at the S1–S4 pockets were determined. • NHSSP recognizes many amino acids at the P1 position, including hydrophobic, bulky, and aliphatic residues. • NHSSP possesses broad substrate specificity at the P2 and P3 positions, while the S4 pocket is the most restricted. • Potent substrate for NHSSP was designed, synthesized, and validated. • First inhibitor for NHSSP protease was designed and synthesized and inhibits NHSSP with k obs /I = 5.3 × 106 M−1s−1. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) and related dementias constitute an important global health challenge. Detailed understanding of the multiple molecular mechanisms underlying their pathogenesis constitutes a clue for the management of the disease. Kallikrein-related peptidases (KLKs), a lead family of serine proteases, have emerged as potential biomarkers and therapeutic targets in the context of AD and associated cognitive decline. Hence, KLKs were proposed to display multifaceted impacts influencing various aspects of neurodegeneration, including amyloid-beta aggregation, tau pathology, neuroinflammation, and synaptic dysfunction. We propose here a comprehensive survey to summarize recent findings, providing an overview of the main kallikreins implicated in AD pathophysiology namely KLK8, KLK6 and KLK7. We explore the interplay between KLKs and key AD molecular pathways, shedding light on their significance as potential biomarkers for early disease detection. We also discuss their pertinence as therapeutic targets for disease-modifying interventions to develop innovative therapeutic strategies aimed at halting or ameliorating the progression of AD and associated dementias. [Display omitted] • Serine proteases among which kallikrein-related peptidases (KLK) are key players in AD patho-physiological pathways. • Some KLKs may serve as emerging biomarkers in combination with established ones such as A β and tau. • Some KLKs such as KLK6 and KLK8 were more extensively investigated and are discussed as potential therapeutical targets. [ABSTRACT FROM AUTHOR]

Members of the widely conserved high temperature requirement A (HtrA) family of serine proteases are involved in multiple aspects of protein quality control. In this context, they have been shown to efficiently degrade misfolded proteins or protein fragments. However, recent reports suggest that folded proteins can also be native substrates. To gain a deeper understanding of how folded proteins are initially processed and subsequently degraded into short peptides by human HTRA1, we established an integrated and quantitative approach using time-resolved mass spectrometry, CD spectroscopy, and bioinformatics. The resulting data provide high-resolution information on up to 178 individual proteolytic sites within folded ANXA1 (consisting of 346 amino acids), the relative frequency of cuts at each proteolytic site, the preferences of the protease for the amino acid sequence surrounding the scissile bond, as well as the degrees of sequential structural relaxation and unfolding of the substrate that occur during progressive degradation. Our workflow provides precise molecular insights into protease-substrate interactions, which could be readily adapted to address other posttranslational modifications such as phosphorylation in dynamic protein complexes. [ABSTRACT FROM AUTHOR]

Cantharidin, a natural toxin produced by the blister beetle, is a topical agent that induces acantholysis of the epidermis, breaking down desmosome plaques through the release of serine proteases. Cantharidin is available in three liquid forms: Ycanth (0.7%), Canthacur (0.7%), and Canthacur PS (1% cantharidin, 30% salicylic acid, 2% podophyllotoxin). Ycanth is used to treat molluscum contagiosum (MC). Canthacur is routinely used to treat a variety of dermatologic conditions including MC, plantar warts, and common warts, whereas Canthacur PS is a more potent formulation indicated for treatment of plantar warts only. The objective of this review is to highlight the efficacy, safety, and diverse use of topical cantharidin in the treatment of various skin conditions. Conditions in which treatment with topical cantharidin yielded a good-to-excellent response include MC, plantar warts, and common warts. Topical cantharidin treatment of anogenital warts yielded mixed results. None of the indications reviewed herein yielded a poor response to topical cantharidin. Overall, topical cantharidin resulted in a good-to-excellent clinical response in several conditions with mild and transient adverse events. The results of this review suggest the safe and efficacious use of topical cantharidin in the field of dermatology and highlight the potential for future use. [ABSTRACT FROM AUTHOR]

Background/Aim: The serine proteinase inhibitor 1 (SERPINE1) gene codes for the plasminogen activator inhibitor 1 (PAI1) protein and is thought to play a tumor supportive role in various cancers. In this work we aimed to uncover the role PAI1 plays in the proliferation, migration, and invasion of vulvar cancer (VC), and define the protein’s function as an oncogene or tumor suppressor. Materials and Methods: Through treatment with an agonist (G1) and antagonist (G36) of G-coupled estrogen receptor 1 (GPER1), an upstream regulator of SERPINE1 expression, and a forward transfection knockdown protocol, the expression of SERPINE1/PAI1 in VC cells was altered. The effects these altered SERPINE1/PAI1 levels had on tumor cell functions were then examined. Proliferation was analyzed using the resazurin assay, while migration was studied via the gap closure assay. Through colony- and tumor sphere- formation assays clonogenicity was tested, and western blots showed protein expression. Results: In A431 VC cells, when the levels of PAI1 were reduced via knockdown or treatment with G1, migration, proliferation, and colony growth was reduced. Treatment with G36 increased expression of PAI1 and increased migration and colony size in CAL39 cells. Conclusion: Based on the findings in this study, suppressing PAI1 expression in VC cells appears to reduce their progression and tumorigenic potential. Therefore, PAI1 could possibly function as an oncogene in VC. GPER1 appears to be a suitable target for suppressing PAI1 in VC. [ABSTRACT FROM AUTHOR]

Dioryctria abietella Denis Schiffermuller (Lepidoptera: Pyralidae) is an oligophagous pest that mainly damages Pinaceae plants. Here, we investigated the effects of the Bacillus thuringiensis 2913 strain (Bt 2913), which carries the Cry1Ac, Cry2Ab, and Vip3Aa genes, on the D. abietella midgut transcriptome at 6, 12, and 24 h after infection. In total, 7497 differentially expressed genes (DEGs) were identified from the midgut transcriptome of D. abietella larvae infected with Bt 2913. Among these DEGs, we identified genes possibly involved in Bt 2913-induced perforation of the larval midgut. For example, the DEGs included 67 genes encoding midgut proteases involved in Cry/Vip toxin activation, 74 genes encoding potential receptor proteins that bind to insecticidal proteins, and 19 genes encoding receptor NADH dehydrogenases that may bind to Cry1Ac. Among the three transcriptomes, 88 genes related to metabolic detoxification and 98 genes related to immune defense against Bt 2913 infection were identified. Interestingly, 145 genes related to the 60S ribosomal protein were among the DEGs identified in the three transcriptomes. Furthermore, we performed bioinformatic analysis of zonadhesin, GST, CYP450, and CarE in the D. abietella midgut to determine their possible associations with Bt 2913. On the basis of the results of this analysis, we speculated that trypsin and other serine proteases in the D. abietella larval midgut began to activate Cry/Vip prototoxin at 6 h to 12 h after Bt 2913 ingestion. At 12 h after Bt 2913 ingestion, chymotrypsin was potentially involved in degrading the active core fragment of Vip3Aa toxin, and the detoxification enzymes in the larvae contributed to the metabolic detoxification of the Bt toxin. The ABC transporter and several other receptor-protein-related genes were also downregulated to increase resistance to Bt 2913. However, the upregulation of 60S ribosomal protein and heat shock protein expression weakened the resistance of larvae to Bt 2913, thereby enhancing the expression of NADH dehydrogenase and other receptor proteins that are highly expressed in the larval midgut and bind to activating toxins, including Cry1Ac. At 24 h after Bt 2913 ingestion, many activated toxins were bound to receptor proteins such as APN in the larval midgut, resulting in membrane perforation. Here, we clarified the mechanism of Bt 2913 infection in D. abietella larvae, as well as the larval immune defense response to Bt 2913, which provides a theoretical basis for the subsequent control of D. abietella using B. thuringiensis. [ABSTRACT FROM AUTHOR]

Angiostrongylus vasorum is a cardiopulmonary nematode of canids and is, among others, associated with bleeding disorders in dogs. The pathogenesis of such coagulopathies remains unclear. A deep proteomic characterization of sex specific A. vasorum excretory/ secretory proteins (ESP) and of cuticular surface proteins was performed, and the effect of ESP on host coagulation and fibrinolysis was evaluated in vitro. Proteins were quantified by liquid chromatography coupled to mass spectrometry and functionally characterized through gene ontology and pathway enrichment analysis. In total, 1069 ESP (944 from female and 959 from male specimens) and 1195 surface proteins (705 and 1135, respectively) were identified. Among these were putative modulators of host coagulation, e.g., von Willebrand factor type D domain protein orthologues as well as several proteases, including serine type proteases, protease inhibitors and proteasome subunits. The effect of ESP on dog coagulation and fibrinolysis was evaluated on canine endothelial cells and by rotational thromboelastometry (ROTEM). After stimulation with ESP, tissue factor and serpin E1 transcript expression increased. ROTEM revealed minimal interaction of ESP with dog blood and ESP did not influence the onset of fibrinolysis, leading to the conclusion that Angiostrongylus vasorum ESP and surface proteins are not solely responsible for bleeding in dogs and that the interaction with the host's vascular hemostasis is limited. It is likely that coagulopathies in A. vasorum infected dogs are the result of a multifactorial response of the host to this parasitic infection. [ABSTRACT FROM AUTHOR]

The genus Mixcoatlus is composed of three species: Mixcoatlus barbouri , M. browni , and M. melanurus , of which the venom composition of M. melanurus , the most common species of the three, has only recently been described. However, very little is known about the natural history of M. barbouri and M. browni , and the venom composition of these two species has remained thus far unexplored. In this study we characterize the proteomic profiles and the main biochemical and toxic activities of these two venoms. Proteomic data obtained by shotgun analysis of whole venom identified 12 protein families for M. barbouri , and 13 for M. browni. The latter venom was further characterized by using a quantitative 'venomics' protocol, which revealed that it is mainly composed of 51.1 % phospholipases A 2 (PLA 2), 25.5 % snake venom serine proteases (SVSP), 4.6 % l -amino oxidases (LAO), and 3.6 % snake venom metalloproteases (SVMP), with lower percentages other six protein families. Both venoms contained homologs of the basic and acidic subunits of crotoxin. However, due to limitations in M. barbouri venom availability, we could only characterize the crotoxin-like protein of M. browni venom, which we have named Mixcoatlutoxin. It exhibited a lethal potency in mice like that described for classical rattlesnake crotoxins. These findings expand knowledge on the distribution of crotoxin-like heterodimeric proteins in viper snake species. Further investigation of the bioactivities of the venom of M. barbouri , on the other hand, remains necessary. [Display omitted] • The first proteomic characterization of Mixcoatlus browni and M. barbouri venoms is reported. • A new Crotoxin-like protein in the venom of Mixcoatlus browni is characterized. • Mixcoatlus barbouri venom contains homologs of acidic and basic subunits of Crotoxin. • The Antivipmyn antivenom can neutralize the lethality of M. browni. [ABSTRACT FROM AUTHOR]

Toxicofera reptile venoms are composed of several toxins, including serine proteases. These proteases are glycosylated enzymes that affect the prey's hemostatic system. Their actions extend across the coagulation cascade, the kallikrein–kinin system, and platelet activation. Despite their specificity for different substrates, these enzymes are homologous across all toxicoferans and display high sequence similarity. The aim of this review is to compile decades of knowledge about venom serine proteases, showing the diversity of biochemically and biophysically characterized enzymes, their structural characteristics, advances in understanding their origin and evolution, as well as methods of obtaining enzymes and their biotechnological applications. [ABSTRACT FROM AUTHOR]

The process of aging is a natural phenomenon characterized by gradual deterioration in biological functions and systemic homeostasis, which can be modulated by both genetic and environmental factors. Numerous investigations conducted on model organisms, including nematodes, flies, and mice, have elucidated several pivotal aging pathways, such as insulin signaling and AMPK signaling. However, it remains uncertain whether the regulation of the aging process is uniform or diverse across different tissues and whether manipulating the same aging factor can result in consistent outcomes in various tissues. In this study, we utilize the Drosophila organism to investigate tissue‐specific proteome signatures during the aging process. Although distinct proteins undergo changes in aged tissues, certain common altered functional networks are constituently identified across different tissues, including the decline of the mitochondrial ribosomal network, autophagic network, and anti‐ROS defense networks. Furthermore, downregulation of insulin receptor (InR) in the midguts, muscle, and central nervous system (CNS) of flies leads to a significant extension in fly lifespans. Notably, despite manipulating the same aging gene InR, diverse alterations in proteins are observed across different tissues. Importantly, knockdown of InR in the midguts leads to a distinct proteome compared with other tissues, resulting in enhanced actin nucleation and glutathione metabolism, while attenuating age‐related elevation of serine proteases. Consequently, knockdown of InR results in rejuvenation of the integrity of the midgut barrier and augmentation of anti‐ROS defense capabilities. Our findings suggest that the barrier function of the midgut plays a pivotal role in defending against aging, underscoring the paramount importance of maintaining optimal gut physiology to effectively delay the aging process. Moreover, when considering age‐related changes across various tissues, it is more reasonable to identify functional networks rather than focusing solely on individual proteins. [ABSTRACT FROM AUTHOR]

Background: Serine proteases represent over 1% of all proteins in humans. This family of proteins is found on cell surfaces, subcellular organelles like lysosomes or mitochondria, within the nucleus and the protoplasm. Among them, trypsin, chymotrypsin and elastase have aroused great interest because of their numerous functions in pathophysiological processes. Altered expression of these enzymes in experimental animal models and humans has been related to various pathologies, like developmental defects, metabolic dysfunctions, cancer, peripheral vascular diseases and infectious diseases. Trypsin and chymotrypsin-like proteases activate, or less oftentimes inactivate, numerous substrates, together with growth factors, receptors, adhesion molecules, angiogenic factors and metalloproteases. Among these substrates, a number of them are key factors in cancer progression, metastasis and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease. Elastin-degrading enzyme- elastase, slowly damages elastin over the lifetime of an organism. The physiological processes triggered by elastase leads to the progression of different conditions such as cancer, metabolic syndrome, pulmonary emphysema, atherosclerosis, and chronic obstructive pulmonary disease. These serine proteases are currently considered to be targets for the development of new potent therapeutics. Short conclusion: The cumulative knowledge that outlined the physiological functions and pathological implications of these proteases and the proposed strategies to regulate a number of their activities and their targeting for therapeutic application and validation in selected disease states are highlighted. These should enhance our appreciation of their roles in aetiology of some diseases as well as the chemotherapeutic benefits of their inhibition or modulation. [ABSTRACT FROM AUTHOR]

Protease-activated receptor-2 (PAR2) is a class-A G protein-coupled receptor (GPCR) activated by serine proteases and is expressed by multiple tissues, including the skin. PAR2 is involved in the skin inflammatory response, promoting Th2 inflammation, delaying skin barrier repair, and affecting the differentiation of keratinocytes. It also participates in the transmission of itch and pain sensations in the skin. Increasing evidence indicates that PAR2 plays an important role in the pathogenesis of inflammatory skin diseases such as acne vulgaris, rosacea, psoriasis, and atopic dermatitis. Additional focus will be placed on potential targeted therapies based on PAR2. The Goal of this review is to outline the emerging effects of PAR2 activation in inflammatory skin disease and highlight the promise of PAR2 modulators. [ABSTRACT FROM AUTHOR]

The collaboration between cellular proteases and host cells is pivotal in mounting an effective innate immune defense. Of particular interest is the synergistic interaction between cathepsin G (CatG) and neutrophil elastase (NE), which are proteases secreted by activated neutrophils, and the human alveolar basal epithelial cell line (A549) and the human lung epithelial-like cell line (H1299), because of the potential implications for viral infection. Our study aimed to investigate the binding capacity of CatG and NE on the surface of A549 and H1299 cells through preincubation with purified CatG and NE; thereby, the proteolytic activity could be detected using activity-based probes. Both CatG and NE were capable of binding to the cell surface and exhibited proteolytic activity, leading to increased cell surface levels of MHC I molecules, which is crucial for displaying the endogenous antigenic repertoire. In addition, CatG cleaved the S2′ site of the SARS-CoV-2 spike protein at two specific sites (815RS816 and 817FI818) as well as NE (813SK814 and 818IE819), which potentially leads to the destruction of the fusion peptide. Additionally, furin required the presence of Ca2+ ions for the distinct cleavage site necessary to generate the fusion peptide. Overall, the findings suggest that CatG and NE can fortify target cells against viral entry, underscoring the potential significance of cell surface proteases in protecting against viral invasion. [ABSTRACT FROM AUTHOR]

SERPINA11 is a hitherto poorly characterised gene belonging to Clade A of the SERPIN superfamily, with unknown expression pattern and functional significance. We report a perinatal lethal phenotype in two foetuses from the same family associated with a biallelic loss of function variant in SERPINA11, and provide functional evidence to support its candidature as a Mendelian disorder. The SERPINA11 variant‐associated foetal phenotype is characterised by gross and histopathological features of extracellular matrix disruption. Western blot and immunofluorescence analyses revealed SERPINA11 expression in multiple mouse tissues, with pronounced expression in the bronchiolar epithelium. We observed a significant decrease in SERPINA11 immunofluorescence in the affected foetal lung compared with a healthy gestation‐matched foetus. Protein expression data from HEK293T cell lines following site‐directed mutagenesis support the loss of function nature of the variant. Transcriptome analysis from the affected foetal liver indicated the possibility of reduced SERPINA11 transcript abundance. This novel serpinopathy appears to be a consequence of the loss of inhibition of serine proteases involved in extracellular matrix remodelling, revealing SERPINA11 as a protease inhibitor critical for embryonic development. [ABSTRACT FROM AUTHOR]

Simple Summary: The digestive enzymes present in the insect gut play a crucial role in digesting food and extracting essential nutrients. The diverse range of digestive enzymes found in the insect gut serves as a valuable source of functional proteins. With a significant population of herbivorous insects, there has been extensive research on cellulase in the insect gut. This study utilized both culture-independent and culture-dependent methods to investigate the protease resources responsible for digesting animal and plant proteins, laying a solid foundation for future research and utilization of proteases derived from insect sources. An insect's gut microbiome is an essential "organ" in their life cycle, playing a crucial role by aiding food digestion and nutrient absorption. This study employed both culture-independent and culture-dependent methods to explore the protease resources present in the gut of the omnivorous insect Gryllotalpa orientalis. The findings revealed that the gut extract of G. orientalis contained a diverse array of proteases, including cysteine proteases, pepsin, serine proteases, and trypsin, as well as some unidentified proteases. Furthermore, the protease gene htpX, derived from gut bacterium Priestia megaterium DX-3, has been cloned and recombinantly expressed. The recombinant DX-3-htpX protease exhibited a 61.9-fold increase in fermentation level compared to the DX-3 protease. This protease was characterized as a neutral, heat-resistant metalloprotease with an M48 peptidase domain, and it was observed that the binding of Ca2+ to the recombinant protease resulted in the formation of the largest active pocket. This study provides technical support for further development and utilization of functional protein resources in insect gut. [ABSTRACT FROM AUTHOR]

Background: Tissue factor pathway inhibitors (TFPI1 and TFPI2) are ubiquitously distributed in humans and exhibit inhibitory activity against serine proteinases. TFPI1 inhibits the tissue factor (TF)-dependent extrinsic coagulation pathway, while TFPI2 modulates extracellular matrix remodeling. TFPI2 has been reported to be an epigenetically silenced tumor suppressor and independent prognostic factor in various human cancers. However, elevated serum levels of TFPI2 have been observed in ovarian and endometrial cancers compared to healthy controls, with increased levels correlating with poor prognosis in endometrial cancer. This raises the question of why the tumor suppressor TFPI2 is elevated in the blood of patients with gynecological cancers and is associated with adverse outcomes. Methods: A comprehensive literature search was performed in PubMed and Google Scholar without time restriction. Results: TFPI2 gene expression may be influenced by both cancer cell-specific gene expression profiles (e.g., oncogenic signaling pathways) and epigenetic modifications (e.g., DNA methylation, histone modifications, and non-coding RNAs). Although TFPI2 generally exhibits an anti-invasion effect in most human cancers, it has been reported to have a paradoxical pro-invasive effect in certain cancers. TFPI2 facilitates cancer invasion through aberrant alternative splicing or through a pathophysiological process known as angiotropism or vasculogenic mimicry. The overproduction of TFPI2 in the tumor microenvironment may reinforce the extracellular matrix, thereby enhancing tumor cell adhesion and invasion. Conclusion: This review summarizes the current understanding of the seemingly contradictory functions of TFPI2 in human malignancies, primarily focusing on the mechanisms regulating its expression and function, and discusses future prospects for translational research. [ABSTRACT FROM AUTHOR]

BACKGROUND: The aim of this study was to address the diagnostic challenges associated with pancreatic disorders, such as chronic pancreatitis (CP) and pancreatic cancer (PC) and highlight the importance of protease activity as a crucial prognostic marker for these conditions. METHODS: Total proteolytic activity was determined using casein as a substrate. To measure the activity of metal-dependent and serine proteases, ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride were used. The content of low- and middle-molecular-weight (LMMW) substances was determined spectrophotometrically. The protein profile was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while zymography was performed to indicate the molecular weight of active enzymes. RESULTS: The pathogenesis of both pancreatic disorders was found to be accompanied by a proteolytic imbalance in pancreatic tissues, manifested in either the increase in the total proteolytic activity under pancreatitis or the decrease in the total proteolytic activity under pancreatic cancer. The increase in the levels of both metal-dependent and serine proteases was indicated in the blood plasma of patients in both studied groups. This proteolytic imbalance was accompanied by the accumulation of protein degradation fragments and LMMW substances. Results of zymography were also informative: pancreatic tissue and plasma samples from patients with either CP or PC were characterized by the presence of additional bands, indicating altered proteolytic activity in both pathological conditions. CONCLUSIONS: This study underscores the importance of proteolytic features in distinguishing CP and PC, offering potential diagnostic markers. Proteolytic shifts may aid in differentiating pathologies, detecting transitions, and developing specific protease inhibitors. Further research is crucial for advancing diagnostic and therapeutic strategies for CP and PC. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus expresses three high-affinity neutrophil serine protease (NSP) inhibitors known as the extracellular adherence protein domain (EAPs) proteins. Whereas EapH1 and EapH2 are comprised of a single EAP domain, the modular extracellular adherence protein (Eap) from S. aureus strain Mu50 consists of four EAP domains. We recently reported that EapH2 can simultaneously bind and inhibit cathepsin-G (CG) and neutrophil elastase (NE), which are the two most abundant NSPs. This unusual property of EapH2 arises from independent CG and NE-binding sites that lie on opposing faces of its EAP domain. Here we used X-ray crystallography and enzyme assays to show that all four individual domains of Eap (i.e. Eap1, Eap2, Eap3, and Eap4) exhibit an EapH2-like ability to form ternary complexes with CG and NE that inhibit both enzymes simultaneously. We found that Eap1, Eap2, and Eap3 have similar functional profiles insofar as NSP inhibition is concerned but that Eap4 displays an unexpected ability to inhibit two NE enzymes simultaneously. Using X-ray crystallography, we determined that this second NE-binding site in Eap4 arises through the same region of its EAP domain that also comprises its CG-binding site. Interestingly, small angle X-ray scattering data showed that stable tail-to-tail dimers of the NE/Eap4/NE ternary complex exist in solution. This arrangement is compatible with NSP-binding at all available sites in a twodomain fragment of Eap. Together, our work implies that Eap is a polyvalent inhibitor of NSPs. It also raises the possibility that higher-order structures of NSP-bound Eap may have unique functional properties. [ABSTRACT FROM AUTHOR]

Previously, elastase activities have been found in some microorganisms and presented by metalloproteases, which may play an important role in helping bacterium invasiveness and establishment of infection. The elastases from Bacillus species are serine proteases, however, to date there are few reports on systematic study of these enzymes producers among representatives of Bacillus, as well as studies of their properties. The aim of this work was to study some physicochemical characteristics of partially purified elastase enzyme from a number of Bacillus sp. strains. Methods. The objects of the study were strains of Bacillus sp. (L1, L2, L9) isolated from the dry grass of the coastal zone of the Kinburn split (Mykolaiv region). Cultures were grown under submerged cultivation at 28°C, with a shaking speed of the nutrient medium of 230 rpm for 2 days. Methods of determining elastase activity in the culture liquid supernatant were used. Results. It has been shown that Bacillus sp. L9, L1, and L2 are characterized by high levels of elastase activity (35.80, 28.0, and 33.80 U/mL, respectively). The maximum synthesis of elastase occurs on the second day of cultivation of the producer at 28 °C and shaking at 230 rpm. Maximum hydrolysis of elastin by Bacillus sp. L1 and L2 are carried out at 40 °C but at different pH optima. Thus, for the preparation of Bacillus sp. L1, optimal pH is 8, and for L2 - 10. For the complex enzyme preparation of Bacillus sp. L9, maximum hydrolysis of elastin occurs at pH 9 and temperature 50 °C. [ABSTRACT FROM AUTHOR]

Cathepsin C (CTSC) is a lysosomal cysteine protease constitutively expressed at high levels in the lung, kidney, liver, and spleen. It plays a key role in the activation of serine proteases in cytotoxic T cells, natural killer cells (granzymes A and B), mast cells (chymase and tryptase) and neutrophils (cathepsin G, neutrophil elastase, proteinase 3) underscoring its pivotal significance in immune and inflammatory defenses. Here, we comprehensively review the structural attributes, synthesis, and function of CTSC, with a focus on its variants implicated in the etiopathology of several syndromes associated with neutrophil serine proteases, including Papillon–Lefevre syndrome (PLS), Haim–Munk Syndrome (HMS), and aggressive periodontitis (AP). These syndromes are characterized by palmoplantar hyperkeratosis, and early-onset periodontitis (severe gum disease) resulting in premature tooth loss. Due to the critical role played by CTSC in these and several other conditions it is being explored as a potential therapeutic target for autoimmune and inflammatory disorders. The review also discusses in depth the gene variants of CTSC, and in particular their postulated association with chronic obstructive pulmonary disease (COPD), COVID-19, various cancers, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, sudden cardiac death (SCD), atherosclerotic vascular disease, and neuroinflammatory disease. Finally, the therapeutic potential of CTSC across a range of human diseases is discussed. [ABSTRACT FROM AUTHOR]

Serine proteases are important environmental contributors of enterovirus biocontrol. However, the structural features of molecular interaction accounting for the susceptibility of enteroviruses to proteases remains unexplained. Here, we describe the molecular mechanisms involved in the recruitment of serine proteases to viral capsids. Among the virus types used, coxsackievirus A9 (CVA9), but not CVB5 and echovirus 11 (E11), was inactivated by Subtilisin A in a host-independent manner, while Bovine Pancreatic Trypsin (BPT) only reduced CVA9 infectivity in a host-dependent manner. Predictive interaction models of each protease with capsid protomers indicate the main targets as internal disordered protein (IDP) segments exposed either on the 5-fold vertex (DE loop VP1) or at the 5/2-fold intersection (C-terminal end VP1) of viral capsids. We further show that a functional binding protease/capsid depends on both the strength and the evolution over time of protease-VP1 complexes, and lastly on the local adaptation of proteases on surrounding viral regions. Finally, we predicted three residues on CVA9 capsid that trigger cleavage by Subtilisin A, one of which may act as a sensor residue contributing to enzyme recognition on the DE loop. Overall, this study describes an important biological mechanism involved in enteroviruses biocontrol. A predictive molecular model sheds light on serine proteases and Enterovirus capsids interaction leading to viral inactivation. [ABSTRACT FROM AUTHOR]

This article is a personal reflection on the author's 20-year professional relationship and friendship with a consultant named Tony, who is highly regarded in the field of cosmetic science. The author highlights Tony's contributions to their company, including the development of the "Corneocare" concept and the creation of the SquameScan device for measuring the amount of stratum corneum protein. They also discuss their collaborative research on serine proteases in the stratum corneum and the impact of protease activity on different skin types. Additionally, the article mentions Tony's achievements as an editor and his collaborations with various universities. The author expresses their admiration for Tony's scientific expertise, pragmatism, and dedication to teaching. The article concludes with a personal anecdote about their friendship and Tony's appreciation for the author's culinary knowledge. [Extracted from the article]

Characteristics of the digestive enzymes of the fifth instar larvae of the beet moth were determined to achieve a superior understanding of the digestive physiology of the pest. After dissecting the larval digestive tube, optimum acidity for the activity of α-amylase and α-glucosidase was obtained 8–9, and 8 for β-glucosidase, respectively. The optimum temperatures were 35 °C and 35–40 °C for α-amylase activity and for α- and β-glucosidase, respectively. The zymogram displayed the existence of three α-amylase isoforms in the digestive tract of this insect. The maximum activity of general protease was determined in the digestive tract of this pest using azocasein substrate in the pH range of 10–11. Furthermore, the optimum temperature was 35 °C for general protease. The metal ions including iron, magnesium, manganese, zinc, and copper at 5 and 10mM concentrations had a significant influence on the activity of α-amylase, α- and β-glucosidase, trypsin, chymotrypsin, and elastase enzymes. Zymogram confirmed the significant inhibitory effect of PMSF and TLCK on the activity of serine proteases, especially trypsin. The results of this research have determined to some extent the digestive system physiology and the digestive enzymes activity range in S. ocellatella. Certainly, Study on the characteristics of digestive enzymes of beet moth can be the starting point for further studies on the use of enzyme inhibitors and the possibility of preparing new methods to control this key sugar beet pest. [ABSTRACT FROM AUTHOR]

Numerous Aspergillus fumigatus (Af) airborne spores are inhaled daily by humans and animals due to their ubiquitous presence. The interaction between the spores and the respiratory epithelium, as well as its impact on the epithelial barrier function, remains largely unknown. The epithelial barrier protects the respiratory epithelium against viral infections. However, it can be compromised by environmental contaminants such as pollen, thereby increasing susceptibility to respiratory viral infections, including alphaherpesvirus equine herpesvirus type 1 (EHV-1). To determine whether Af spores disrupt the epithelial integrity and enhance susceptibility to viral infections, equine respiratory mucosal ex vivo explants were pretreated with Af spore diffusate, followed by EHV-1 inoculation. Spore proteases were characterized by zymography and identified using mass spectrometry-based proteomics. Proteases of the serine protease, metalloprotease, and aspartic protease groups were identified. Morphological analysis of hematoxylin-eosin (HE)-stained sections of the explants revealed that Af spores induced the desquamation of epithelial cells and a significant increase in intercellular space at high and low concentrations, respectively. The increase in intercellular space in the epithelium caused by Af spore proteases correlated with an increase in EHV-1 infection. Together, our findings demonstrate that Af spore proteases disrupt epithelial integrity, potentially leading to increased viral infection of the respiratory epithelium. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors (serpins) participate in the regulation of inflammation, blood coagulation, and complement activation in humans. This research aimed to identify and characterize such inhibitors of the human liver fluke Opisthorchis viverrini. Parasite proteins that might contribute to the modulation of host physiology are of particular interest, especially as chronic opisthorchiasis increases the risk of developing biliary cancer. BLAST was used to find hypothetical serpins predicted from the parasite genome data. RNA extraction and reverse transcriptase PCR were used to isolate a serpin cDNA and to determine developmental transcript abundance. The evolutionary relation to other trematode serpins was revealed by phylogenetic analysis. Recombinant serpin was expressed in Escherichia coli and used to test the immunoreactivity of human opisthorchiasis sera and the inhibition of human serine proteases. A substantial serpin family with high sequence divergence among the members was found in the genus Opisthorchis. A serpin, different from previously analyzed trematode serpins, was cloned. The transcript was only detected in metacercariae and newly excysted juveniles. Human opisthorchiasis sera showed statistically significant reactivity to recombinant serpin. The serpin caused moderate inhibition of thrombin and low inhibition of kallikrein and chymotrypsin. This parasite serpin could be further evaluated as a diagnostic tool for early infection. Kallikrein and thrombin are involved in fibrinolysis; therefore, further research should explore the effects of the parasite serpin on this process. [ABSTRACT FROM AUTHOR]

The evolution of complex chemical inventory from Darwin's nutrient-rich warm pond necessitated rudimentary yet efficient catalytic folds. Short peptides and their self-organized microstructures, ranging from spherical colloids to amyloidogenic aggregates might have played a crucial role in the emergence of contemporary catalytic entities. However, the question of how short peptide fragments had functions akin to contemporary complex enzymes to catalyze cleavage and formation of highly stable peptide bonds that constitute the backbone of all proteins remains an unresolved yet fundamentally important question in terms of the origins of enzymes. We report short-peptide-based spherical assemblies that demonstrated residue-specific cleavage and formation of peptide bonds of diverse peptide-based substrates under aqueous environment. Despite the short sequence length, the assemblies utilized the synergistic collaboration of four residues which included the catalytic triad of extant serine proteases with a nonproteinogenic amino acid (quinone moiety), to facilitate proteolysis, ligation, and a three-step (hydrolysis-ligation-hydrolysis) cascade. Such short-peptide-based catalytic assemblies argue for their candidacy as the earliest protein folds and open up avenues for biotechnological applications. [ABSTRACT FROM AUTHOR]

Serine proteases (SPs) are distributed among all living cells accounting for almost one-third of all proteases. Dysregulation of SPs during inflammation and/or infection can result in devastating consequences, such as skin and lung inflammation, neuroinflammation, arthritis, as well as metastasis of cancerous cells. Such activities are tightly regulated by various inhibitors known as serine protease inhibitors (SERPIN). The thermodynamic investigations previously revealed that L-ascorbic acid binds to trypsin more firmly than pepsin and the binding force of L-ascorbic acid is driven by hydrogen bonds and van der Waals forces. However, the physiochemical effects of such interaction on trypsin and/or pepsin have not yet been reported. Ascorbic acid, also known as vitamin C, is one of the essential nutrients and most common food supplements, fortificants, and preservatives. The aim of this study was to explore the inhibitory effects of ascorbic acid on serine proteases at various concentrations on the in-vitro digestion and/or hydrolysis of intercellular matrix of cell monolayer and human serum albumin (HSA). The inhibitory effects of ascorbic on trypsin are investigated by qualitative and quantitative analysis using SDS-PAGE imaging and NIH densitometric software. Upon the addition of ascorbic acid in both indicator systems, the detachment and/or dissociation of cell monolayer and the digestion of HSA were inhibited in the presence of EDTA-Trypsin. The inhibitory effect of ascorbic acid on the digestion of intercellular matrix and/or hydrolysis of HSA showed a dose-dependent trend until it reached the maximum extent of inhibition. At an equal concentration (2.5mg/mL) ascorbic acid and EDTA-Trypsin exhibited the most potent inhibitory effect on the in vitro digestion of protein either in the form of intercellular matrix in cell monolayer and/or HSA respectively. Overall, our results based on two indicator systems strongly indicate that ascorbic acid may function as a serine protease inhibitor (SERPIN) beyond other important functions. [ABSTRACT FROM AUTHOR]

We report a chemoselective and site-selective precision engineering of lysine in proteases. The mild and physiological reaction conditions keep their auto-degradation under control. Furthermore, it enables single-site ordered immobilization, enhancing protein digestion and peptide mapping efficiency. [ABSTRACT FROM AUTHOR]

Certain corona- and influenza viruses utilize type II transmembrane serine proteases for cell entry, making these enzymes potential drug targets for the treatment of viral respiratory infections. In this study, the cytotoxicity and inhibitory effects of seven matriptase/TMPRSS2 inhibitors (MI-21, MI-463, MI-472, MI-485, MI-1900, MI-1903, and MI-1904) on cytochrome P450 enzymes were evaluated using fluorometric assays. Additionally, their antiviral activity against influenza A virus subtypes H1N1 and H9N2 was assessed. The metabolic depletion rates of these inhibitors in human primary hepatocytes were determined over a 120-min period by LC–MS/MS, and PK parameters were calculated. The tested compounds, with the exception of MI-21, displayed potent inhibition of CYP3A4, while all compounds lacked inhibitory effects on CYP1A2, CYP2C9, CYP2C19, and CYP2D6. The differences between the CYP3A4 activity within the series were rationalized by ligand docking. Elucidation of PK parameters showed that inhibitors MI-463, MI-472, MI-485, MI-1900 and MI-1904 were more stable compounds than MI-21 and MI-1903. Anti-H1N1 properties of inhibitors MI-463 and MI-1900 and anti-H9N2 effects of MI-463 were shown at 20 and 50 µM after 24 h incubation with the inhibitors, suggesting that these inhibitors can be applied to block entry of these viruses by suppressing host matriptase/TMPRSS2-mediated cleavage. [ABSTRACT FROM AUTHOR]

Serine proteases are important regulators of airway epithelial homeostasis. Altered serum or cellular levels of two serpins, Scca1 and Spink5, have been described for airway diseases but their function beyond antiproteolytic activity is insufficiently understood. To close this gap, we generated fly lines with overexpression or knockdown for each gene in the airways. Overexpression of both fly homologues of Scca1 and Spink5 induced the growth of additional airway branches, with more variable results for the respective knockdowns. Dysregulation of Scca1 resulted in a general delay in fruit fly development, with increases in larval and pupal mortality following overexpression of this gene. In addition, the morphological changes in the airways were concomitant with lower tolerance to hypoxia. In conclusion, the observed structural changes of the airways evidently had a strong impact on the airway function in our model as they manifested in a lower physical fitness of the animals. We assume that this is due to insufficient tissue oxygenation. Future work will be directed at the identification of key molecular regulators following the airway-specific dysregulation of Scca1 and Spink5 expression. [ABSTRACT FROM AUTHOR]