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The complement system is the other major proteolytic cascade in the blood of vertebrates besides the coagulation–fibrinolytic system. Among the three main activation routes of complement, the lectin pathway (LP) has been discovered the latest, and it is still the subject of intense research. Mannose-binding lectin (MBL), other collectins, and ficolins are collectively termed as the pattern recognition molecules (PRMs) of the LP, and they are responsible for targeting LP activation to molecular patterns, e.g., on bacteria. MBL-associated serine proteases (MASPs) are the effectors, while MBL-associated proteins (MAps) have regulatory functions. Two serine protease components, MASP-1 and MASP-2, trigger the LP activation, while the third component, MASP-3, is involved in the function of the alternative pathway (AP) of complement. Besides their functions within the complement system, certain LP components have secondary ("moonlighting") functions, e.g., in embryonic development. They also contribute to blood coagulation, and some might have tumor suppressing roles. Uncontrolled complement activation can contribute to the progression of many diseases (e.g., stroke, kidney diseases, thrombotic complications, and COVID-19). In most cases, the lectin pathway has also been implicated. In this review, we summarize the history of the lectin pathway, introduce their components, describe its activation and regulation, its roles within the complement cascade, its connections to blood coagulation, and its direct cellular effects. Special emphasis is placed on disease connections and the non-canonical functions of LP components. [ABSTRACT FROM AUTHOR]

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases. [ABSTRACT FROM AUTHOR]

Background: Proprotein convertase subtilisin/kexin type-9 (PCSK-9) is a serine protease with profound effects on plasma LDL-C, the major risk factor for cardiovascular diseases (CVDs). However, plasma PCSK-9 level and its association with the biomarkers of CVDs, diabetes, and associated complications have not yet been reported in the northeastern population of India. Methods: Of the total cohort (n = 233), we analyzed healthy controls (HC; n = 50), freshly diagnosed type-2-diabetes mellitus (T2DM-FD; n = 46), T2DM treated (T2DM-T; n = 49), diabetic nephropathy (T2DM-N; n = 43), and diabetic dyslipidemia (T2DM-DL; n = 45) subjects. Plasma PCSK-9 and other biological determinants associated with T2DM, CVD, and nephrotic dysfunction were assessed. Results: The level of plasma PCSK-9 in HC, T2DM-FD, T2DM-T, T2DM-N, and T2DM-DL groups was found to be 184.1 ± 13.83, 183.1 ± 24.4.3, 241.8 ± 75.42, 403.7 ± 85.94, and 641.3 ± 135.5 ng/mL, respectively, indicating its role in the severity of the here-mentioned complications. Moreover, plasma PCSK-9 levels further showed a significant correlation with the biomarkers of hyperglycemia, particularly HbA1c, as well as LDL-C in T2DM-FD, T2DM-N, and T2DM-DL subjects of the Indian population, while moderate association in T2DM-T subjects. Conclusions: Our first-of-its-kind clinical study aiming to quantify the circulatory PCSK-9 level in the Indian population concluded that elevated PCSK-9 was significantly associated with the severity of diabetes and associated complications. Moreover, such elevation in PCSK-9 might be attributed to the lipid- and glucose-lowering medication-induced SREBP-2-dependent mechanisms. Since our conclusion is based on a pilot study, further cohort studies in larger populations of India are required to get a generalization regarding the role of PCSK-9 in DM and associated complications. [ABSTRACT FROM AUTHOR]

Background: Key actors in SARS-CoV-2 entrance into host cells include angiotensinconverting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Our objective was to study gene expression of those key actors in COVID-19 patients, with especial emphasis on patients having other comorbidities. Methods: The Expression of ACE2 and TMPRSS2 genes in nasopharyngeal swabs was assessed in 84 COVID-19 patients in comparison with 30 healthy controls using real-time PCR. We also compared expression levels of ACE2 and TMPRSS2 between different COVID-19 groups; moderate & severe, diabetic & non-diabetic, hypertensive & normotensive individuals, and between individuals belonging to different age groups. Results: Our results showed that upregulation of ACE2 and TMPRSS2 was observed in COVID-19 patients relative to healthy controls, and there was a positive correlation between ACE2 and TMPRSS2 expression levels. TMPRSS2 gene expression level was higher in older age groups and both genes showed higher expression levels in males. On the other hand, no difference was detected in the gene expression level of ACE2 and TMPRSS2 between moderate and severe COVID-19 patients. Also, alterations were detected in the two genes expression levels in patients having comorbidities. Conclusions: The ACE2 and TMPRSS2 gene expression levels varied between different patient groups, posing a greater risk of getting SARS-CoV2 infection. [ABSTRACT FROM AUTHOR]

The Manchineel, Hippomane mancinella ("Death Apple Tree") is one of the most toxic fruits worldwide and nevertheless is the host plant of the monophagous fruit fly species Anastrepha acris (Diptera: Tephritidae). Here we aimed at elucidating the detoxification mechanisms in larvae of A. acris reared on a diet enriched with the toxic fruit (6% lyophilizate) through comparative transcriptomics. We compared the performance of A. acris larvae with that of the sister species A. ludens, a highly polyphagous pest species that is unable to infest H. mancinella in nature. The transcriptional alterations in A. ludens were significantly greater than in A. acris. We mainly found two resistance mechanisms in both species: structural, activating cuticle protein biosynthesis (chitin-binding proteins likely reducing permeability to toxic compounds in the intestine), and metabolic, triggering biosynthesis of serine proteases and xenobiotic metabolism activation by glutathione-S-transferases and cytochrome P450 oxidoreductase. Some cuticle proteins and serine proteases were not orthologous between both species, suggesting that in A. acris, a structural resistance mechanism has been selected allowing specialization to the highly toxic host plant. Our results represent a nice example of how two phylogenetically close species diverged over recent evolutionary time related to resistance mechanisms to plant secondary metabolites. [ABSTRACT FROM AUTHOR]

Serine proteases regulate cell functions through G protein-coupled protease-activated receptors (PARs). Cleavage of one peptide bond of the receptor amino terminus results in the formation of a new N-terminus ("tethered ligand") that can specifically interact with the second extracellular loop of the PAR receptor and activate it. Activation of PAR1 by thrombin (canonical agonist) and activated protein C (APC, noncanonical agonist) was described as a biased agonism. Here, we have supposed that synthetic peptide analogs to the PAR1 tethered ligand liberated by APC could have neuroprotective effects like APC. To verify this hypothesis, a model of the ischemic brain impairment based on glutamate (Glu) excitotoxicity in primary neuronal cultures of neonatal rats has been used. It was shown that the nanopeptide NPNDKYEPF-NH2 (AP9) effectively reduced the neuronal death induced by Glu. The influence of AP9 on cell survival was comparable to that of APC. Both APC and AP9 reduced the dysregulation of intracellular calcium homeostasis in cultured neurons induced by excitotoxic Glu (100 µM) or NMDA (200 µM) concentrations. PAR1 agonist synthetic peptides might be noncanonical PAR1 agonists and a basis for novel neuroprotective drugs for disorders related to Glu excitotoxicity such as brain ischemia, trauma and some neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Background: Gut epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism by which larval penetration of gut epithelium is not completely elucidated. Previous studies showed that proteases secreted by T. spiralis intestinal infective larvae (IIL) degraded tight junctions (TJs) proteins of gut epithelium and mediated larval invasion. A new T. spiralis serine proteinase (TsSPc) was identified in the IIL surface proteins and ES proteins, rTsSPc bound to the intestinal epithelial cell (IECs) and promoted larval invasion of IECs. The aim of this study was to characterize the interacted proteins of TsSPc and IECs, and to investigate the molecular mechanisms of TsSPc mediating larval invasion of gut mucosa. Methodology/Principal finding: IIFT results showed natural TsSPc was detected in infected murine intestine at 6, 12 hours post infection (hpi) and 3 dpi. The results of GST pull-down, mass spectrometry (MS) and Co-IP indicated that rTsSPc bound and interacted specifically with receptor for activated protein C kinase 1 (RACK1) in Caco-2 cells. rTsSPc did not directly hydrolyze the TJs proteins. qPCR and Western blot showed that rTsSPc up-regulated RACK1 expression, activated MAPK/ERK1/2 pathway, reduced the expression levels of gut TJs (occludin and claudin-1) and adherent protein E-cad, increased the paracellular permeability and damaged the integrity of intestinal epithelial barrier. Moreover, the RACK1 inhibitor HO and ERK1/2 pathway inhibitor PD98059 abolished the rTsSPc activating ERK1/2 pathway, they also inhibited and abrogated the rTsSPc down-regulating expression of occludin, claudin-1 and E-cad in Caco-2 monolayer and infected murine intestine, impeded larval invasion and improved intestinal epithelial integrity and barrier function, reduced intestinal worm burdens and alleviated intestinal inflammation. Conclusions: rTsSPc bound to RACK1 receptor in gut epithelium, activated MAPK/ERK1/2 pathway, decreased the expression of gut epithelial TJs proteins and disrupted the epithelial integrity, consequently mediated T. spiralis larval invasion of gut epithelium. The results are valuable to understand T. spiralis invasion mechanism, and TsSPc might be regarded as a vaccine target against T. spiralis invasion and infection. Author summary: A novel T. spiralis serine proteinase (TsSPc) was identified in excretory/secretory proteins (ESP) of intestinal infective larvae (IIL). The purpose of this study was to determine the mechanism of TsSPc action in larval invasion of intestinal epithelium. IIFT showed that natural rTsSPc bound to intestinal epithelial cells (IECs). GST pull-down, co-immunoprecipitation (Co-IP), IFT and mass spectrometry verified that rTsSPc specifically bound to the RACK1 receptor in Caco-2 cells, activated MAPK/ERK1/2 signaling pathway, as demonstrated by increased expression of RACK1 and phosphorylated ERK1/2 (p-ERK1/2). Binding of rTsSPc to RACK1 and activation of MAPK/ERK1/2 pathway significantly decreased expression of gut epithelium TJs proteins (occludin, claudin-1 and E-cad), paracellular permeability was obviously increased. Besides, HO (RACK1 inhibitor) and PD98059 (ERK1/2 pathway inhibitor) suppressed and abolished the rTsSPc activating ERK1/2 pathway, and abrogated the rTsSPc down-regulating expression of occludin, claudin-1 and E-cad in Caco-2 monolayer and infected murine intestine, impeded larval invasion and improved intestinal epithelial integrity and barrier function, decreased intestinal worm burdens and meliorated intestinal inflammation. The results indicated that TsSPc binding to gut epithelial RACK1 receptor activated MAPK/ERK1/2 pathway, decreased expression of gut TJs proteins, and destroyed gut epithelial integrity, thereby mediated T. spiralis larval invasion of gut mucosa. rTsSPc might be a candidate vaccine target to block T. spiralis larval intrusion. [ABSTRACT FROM AUTHOR]

Background: Over the past decade, various research studies have proved the interconnection between the inflammatory pathways and diabetes complication in clinical condition. The present study evaluated the anti-inflammatory and antioxidant activity. Further, the sample was tested for its pharmacokinetics properties and the best compounds were docked with the diabetic markers (DPP IV (PDB-ID: IJ2E) and SGLT2 (PDB-ID: 7VSI)). Results: C.verticillata showed a good hydrogen peroxide (78.3 ± 0.34%, IC50 = 287.81 µg/ml) and superoxide scavenging activity (52.7 ± 1.26%, IC50 = 796.15 µg/ml). In addition, the sample was checked for its anti-inflammatory activity with protein denaturation (57.4 ± 0.19%, IC50 = 471.5 µg/ml) and proteinase inhibition assay (68.3 ± 0.48%, IC50 = 213.42 µg/ml). Further, the bioactive compounds detected from HPLC-ESI-MS/MS analyzed sample were checked for its drug likeliness by checking its ADME properties and toxicological parameters. It has been observed that except Loliolide, all the other compounds have followed the physicochemical parameters and proved to exhibit drug likeliness characteristics. The bioactive compounds that follow the Lipinski's rule were taken further for in silico molecular docking analysis with the diabetic protein markers (DPP IV and SGLT2). Docking results revealed that Pyro pheophorbide a with DPP IV and Dihydromonacolin L acid with SGLT2 have recorded a maximum docking score of (− 9.4 kcal/mol) and (− 9.2 kcal/mol), respectively. Conclusion: The observed results suggest that the identified and selected bioactive compounds from C.verticillata can be considered as a potential target molecule for the management of oxidative stress induced diabetic condition. Furthermore, the study also provides an insight on the effectiveness of the compounds on reducing the inflammation as well. [ABSTRACT FROM AUTHOR]

Activation of the lectin pathway of the complement system, as demonstrated by elevated levels of mannan-binding lectin proteins (MBL), contributes to vascular pathology in type 1 diabetes (T1D). Vascular complications are greatest in T1D individuals with concomitant insulin resistance (IR), however, whether IR amplifies activiation of the lectin pathway in T1D is unknown. We pooled pretreatment data from two RCTs and performed a cross-sectional analysis on 46 T1D individuals. We employed estimated glucose disposal rate (eGDR), a validated IR surrogate with cut-points of: <5.1, 5.1–8.7, and > 8.7 mg/kg/min to determine IR status, with lower eGDR values conferring higher degrees of IR. Plasma levels of MBL-associated proteases (MASP-1, MASP-2, and MASP-3) and their regulatory protein MAp44 were compared among eGDR classifications. In a subset of 14 individuals, we assessed change in MASPs and MAp44 following improvement in IR. We found that MASP-1, MASP-2, MASP-3, and MAp44 levels increased in a stepwise fashion across eGDR thresholds with elevated MASPs and MAp44 levels conferring greater degrees of IR. In a subset of 14 patients, improvement in IR was associated with significant reductions in MASPs, but not MAp44, levels. In conclusion, IR in T1D amplifies levels of MASP-1/2/3 and their regulator MAp44, and improvement of IR normalizes MASP-1/2/3 levels. Given that elevated levels of these proteins contribute to vascular pathology, amplification of the lectin pathway of the complement system may offer mechanistic insight into the relationship between IR and vascular complications in T1D. Plasma levels of mannan-binding lectin-associated serine proteases are increased in type 1 diabetes patients with insulin resistance. Graphical Abstract [ABSTRACT FROM AUTHOR]

Seed aging is a complex and irreversible process that occurs during seed development and storage. The quality of Allium mongolicum Regel seeds directly impacts its cultivation and production. However, the mechanism of aging seeds in A. mongolicum Regel is not well understood. Germination and physiological indicators were assessed, and RNA-Seq technique was performed on samples stored for 1, 4, and 8 years. In total, 5723, 7347 and 5293 differentially expressed genes (DEGs) were identified in 1 year old (S1) versus 4 year old (S2), S1 versus 8 year old (S4), and S2 versus S4, respectively. The number of DEGs increased as the storage time increased. The analysis revealed that the majority of the these DEGs were involved in a wide range of processes, such as protein synthesis, degradation and targeting; RNA processing and regulation; transport; DNA; signaling; stress; metabolism; amino acid metabolism; secondary metabolism; and hormone metabolism. The germination rate of A. mongolicum seeds remained consistently high, exceeding 80%, even after four years of storage. This observation indicates a potential correlation with the increased expression levels of aspartic proteases. However, as the storage time surpassed the seed's lifespan, there was a notable decline in the expression levels of genes that encode aspartic proteases, cysteine proteases, and serine proteases. Conducting further research on these candidate genes could enhance our comprehension of the regulatory mechanisms involved in seed aging during storage for varying durations. [ABSTRACT FROM AUTHOR]

This study was aimed to extract, purify and characterize the pancreatic elastase from the Catfish (silurus trioseegus) digestive duct. Different extraction solutions were used to optimize the extraction condition being (Distilled water, 200 mM NaCl, Phosphate buffer (100 mM, pH 8), the latter buffer Tis-HCl (100 mM, pH 8) both containing 0-100 mM NaCl or CaCl2 separately). Tris-HCl buffer containing 100 mM CaCl2 was the best among the rest extraction solutions. The crude extract was precipitated using a 30 % - 55 % of ammonium sulfate saturation ratio. The crude precipitate was dissolved in the same buffer and dialyzed. Then the dialyzed precipitate was concentrated and applied to CM-52 column (3.5 ×10 cm), elastase activity was appeared in two protein peaks. Each peak fraction were pooled individually and loaded on a Sephadex G-75 column (1.6×62 cm). Elastase activity was recovered only from one peak with 29.54 % yield, 530.70 U total activity and 17.89 U/mg specific activity. The optimum temperature for elastase stability and activity was 0-30 C° and 55 C° respectively. while the optimum pH for the enzyme activity and stability were 9 and 4-9 respectively. The molecular weight was 21898 Da as determined by Gel filtration. [ABSTRACT FROM AUTHOR]

Nummular eczema, a chronic dermatitis characterized by coin-shaped lesions, was first documented in 1857. However, its pathophysiological characteristics are still not well known. To investigate differences in the regulation of the desquamation process in the stratum corneum of lesional and nonlesional skin of patients with nummular eczema and healthy control subjects, tape-stripped stratum corneum samples from patients with nummular eczema and healthy volunteers were analysed using immunofluorescence staining and western blot analysis. In the nummular eczema lesional skin, expression of desmoglein-1, desmocollin-1, and corneodesmosin exhibited a disorganized, dense or partially diffuse non-peripheral pattern with increased intensity, compared with the peripheral patterns observed in healthy or nonlesional skin, suggesting the impaired desquamation process in nummular eczema. Furthermore, although the expression of the desquamation- related serine proteases, kallikrein-related peptidase 7 and 5, was increased in nummular eczema lesional skin, the immunofluorescence staining of lympho-epithelial Kazal-type-related inhibitor-1, an endogenous inhibitor of various kallikrein-related peptidases, and its fragments were significantly increased in the nummular eczema lesional skin, suggesting its contribution to the inhibition of corneodesmosomal degradation. Therefore, the increased detection of corneodesmosomal proteins in nummular eczema lesions may be due to the increased amount of the fragments of lympho-epithelial Kazal-type-related inhibitor-1, which could contribute to delayed desquamation. [ABSTRACT FROM AUTHOR]

Background Understanding the genotype of pest species provides an important baseline for designing integrated pest management (IPM) strategies. Recently developed long-read sequence technologies make it possible to compare genomic features of nonmodel pest species to disclose the evolutionary path underlying the pest species profiles. Here we sequenced and assembled genomes for 3 agricultural pest gelechiid moths: Phthorimaea absoluta (tomato leafminer), Keiferia lycopersicella (tomato pinworm), and Scrobipalpa atriplicella (goosefoot groundling moth). We also compared genomes of tomato leafminer and tomato pinworm with published genomes of Phthorimaea operculella and Pectinophora gossypiella to investigate the gene family evolution related to the pest species profiles. Results We found that the 3 solanaceous feeding species, P. absoluta, K. lycopersicella , and P. operculella , are clustered together. Gene family evolution analyses with the 4 species show clear gene family expansions on host plant–associated genes for the 3 solanaceous feeding species. These genes are involved in host compound sensing (e.g. gustatory receptors), detoxification (e.g. ABC transporter C family, cytochrome P450, glucose-methanol-choline oxidoreductase, insect cuticle proteins, and UDP-glucuronosyl), and digestion (e.g. serine proteases and peptidase family S1). A gene ontology enrichment analysis of rapid evolving genes also suggests enriched functions in host sensing and immunity. Conclusions Our results of family evolution analyses indicate that host plant adaptation and pathogen defense could be important drivers in species diversification among gelechiid moths. [ABSTRACT FROM AUTHOR]

Background: Chronic inflammation plays a vital role in the development of gestational diabetes mellitus (GDM). Studies in mouse models show that neutrophil serine proteases (NSPs), neutrophil elastase (NE) and proteinase-3 (PR3) are important drivers of chronic inflammation with consequent metabolic disturbances. This study evaluated the association of NE and PR3 with GDM development and adverse fetal outcomes. Method(s): This was a prospective cohort study. Serum PR3 and NE concentration was measured in all enrolled pregnant women in the first and the second trimester to determine the connection between NSPs and GDM and adverse fetal outcomes. Logistic regression, spline regression and linear regression analyses were applied to investigate the association of NE or PR3 with GDM development and adverse fetal outcomes. The concentration of NE and PR3 in placental biopsies was evaluated by semi-quantitative analysis of immunohistochemistry staining. Result(s): NE or PR3 concentration in the first trimester, rather than the second, increased more significantly in women with GDM than in those without, regardless of pre-pregnancy body mass index and age. There was a stepwise increase in GDM occurrence as well as comprehensive adverse fetal outcomes across tertiles of NE and PR3. NE and PR3 were positively associated with neutrophil count, pre-pregnancy BMI, plasma glucose level and newborn weight. Logistic regression revealed NE or PR3 to be independent risk factors for the development of GDM and comprehensive adverse fetal outcomes. Spline regression showed a significant increased risk of GDM occurrence and comprehensive adverse fetal outcomes when serum NE concentration exceeded 417.60 ng/mL and a similar result for PR3 and GDM occurrence when the latter exceeded 88.52 ng/mL. Immunohistochemistry data confirmed that enriched NE and PR3 content in placental tissue may have contributed to the development of GDM. Conclusion(s): This work demonstrates that excessive first-trimester NE and PR3 increase the risk of GDM development and comprehensive adverse fetal outcomes. [ABSTRACT FROM AUTHOR]

α1-Antitrypsin (AAT), an acute-phase reactant not unsimilar to C-reactive protein (CRP), is a serine protease inhibitor that harbors tissue-protective and immunomodulatory attributes. Its concentrations appropriately increase during conditions of extensive tissue injury, and it induces immune tolerance, in part, by inhibiting the enzymatic activity of the inflammatory serine protease, proteinase 3 (PR3). Typically administered to patients with genetic AAT deficiency, AAT treatment was recently shown to improve outcomes in patients with steroid-refractory graft-versus-host disease (GVHD). GVHD represents a grave outcome of allogeneic hematopoietic stem cell transplantation (HSCT), a potentially curative intervention for hematological diseases. The procedure requires radio/chemotherapy conditioning of the prospective marrow recipient, a cytotoxic process that causes vast tissue injury and, in some formats, interferes with liver production of AAT. To date, changes in the functional profile of AAT during allogeneic HSCT, and during the cytotoxic intervention that precedes HSCT, are unknown. The present study followed 53 patients scheduled for allogeneic HSCT (trial registration NCT03188601). Serum samples were tested before and after HSCT for AAT and CRP levels and for intrinsic anti-proteolytic activity. The ex vivo response to clinical-grade AAT was tested on circulating patient leukocytes and on a human epithelial cell line treated with patient sera in a gap closure assay. According to the ex vivo experiments, circulating leukocytes responded to AAT with a favorable immune-regulated profile, and epithelial gap closure was enhanced by AAT in sera from GVHD-free patients but not in sera from patients who developed GVHD. According to serum collected prior to HSCT, non-relapse mortality was reliably predicted by combining three components: AAT and CRP levels and serum anti-proteolytic activity. Taken together, HSCT outcomes are significantly affected by the anti-proteolytic function of circulating AAT, supporting early AAT augmentation therapy for allogeneic HSCT patients. [ABSTRACT FROM AUTHOR]

The extended cleavage specificity of catfish granzyme-like II has been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated by using a panel of recombinant substrates. This serine protease, which has previously been isolated as cDNA from a catfish natural killer-like cell line showed a preference for Ala in the P1 position of the substrate, and for multiple basic amino acids N-terminally of the cleavage site. A closely related zebrafish serine protease (zebrafish esterase-like) showed a very similar cleavage specificity, indicating an evolutionary conservation of this protease specificity among various fish species. Two catfish serine proteases, originating from NK-like cells, have now been isolated and characterized. One of them is highly specific met-ase with similar characteristics as the mammalian granzyme M. This enzyme may be involved in the induction of apoptosis in virus-infected cells, with a potential target in (catfish) caspase 6. In contrast to catfish granzyme-like I, the second enzyme analyzed here does not seem to have a direct counterpart in mammalian NK cells, and its role in the immune function of catfish NK cells is, therefore, still not known. However, this enzyme seems to be able to cleave a number of cytoskeletal proteins, indicating a separate strategy to induce apoptosis in target cells. Both of these enzymes are very interesting targets for further studies of their roles in catfish immunity, as enzymes with similar specificities have also been identified in zebrafish. [ABSTRACT FROM AUTHOR]

The prophenoloxidase (PPO) activation and Toll antimicrobial peptide synthesis pathways are two critical immune responses in the insect immune system. The activation of these pathways is mediated by the cascade of serine proteases, which is negatively regulated by serpins. In this study, we identified a typical serpin, BmSerpin-4, in silkworms, whose expression was dramatically up-regulated in the fat body and hemocytes after bacterial infections. The pre-injection of recombinant BmSerpin-4 remarkably decreased the antibacterial activity of the hemolymph and the expression of the antimicrobial peptides (AMPs) gloverin-3, cecropin-D, cecropin-E, and moricin in the fat body under Micrococcus luteus and Yersinia pseudotuberculosis serotype O: 3 (YP III) infection. Meanwhile, the inhibition of systemic melanization, PO activity, and PPO activation by BmSerpin-4 was also observed. Hemolymph proteinase 1 (HP1), serine protease 2 (SP2), HP6, and SP21 were predicted as the candidate target serine proteases for BmSerpin-4 through the analysis of residues adjacent to the scissile bond and comparisons of orthologous genes in Manduca sexta. This suggests that HP1, SP2, HP6, and SP21 might be essential in the activation of the serine protease cascade in both the Toll and PPO pathways in silkworms. Our study provided a comprehensive characterization of BmSerpin-4 and clues for the further dissection of silkworm PPO and Toll activation signaling. [ABSTRACT FROM AUTHOR]

Cathepsin C (CatC, syn. Dipeptidyl peptidase I) is a lysosomal cysteine proteinase expressed in several tissues including inflammatory cells. This enzyme is important for maintaining multiple cellular functions and for processing immune cell-derived proteases. While mutations in the CatC gene were reported in Papillon-Lefèvre syndrome, a rare autosomal recessive disorder featuring hyperkeratosis and periodontitis, evidence from clinical and preclinical studies points toward pro-inflammatory effects of CatC in various disease processes that are mainly mediated by the activation of neutrophil serine proteinases. Moreover, tumor-promoting effects were ascribed to CatC. The aim of this review is to highlight current knowledge of the CatC as a potential therapeutic target in inflammatory disorders. [Display omitted] • Cathepsin C activates granule serine proteinases involved in inflammation. • Cathepsin C contributes to the pathogenesis of different acute and chronic diseases. • Potential of cathepsin C inhibition to ameliorate inflammation in preclinical models. • Need for studies evaluating CatC inhibitors repurposing in chronic inflammatory diseases. [ABSTRACT FROM AUTHOR]

Hyperhomocysteinemia (HHcy) is now being actively studied as a potential risk factor and/or biomarker for numerous pathological conditions, including brain diseases. This study aimed to analyze the proteolytic processes in the brains of rats with HHcy. Total proteolytic activity, metal-dependent, and serine proteases activities, the content of matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases-1, cytokines, serine proteases, total protein and medium and low molecular-weight substances (MLMWS), were evaluated. HHcy was induced by DL-homocysteine thiolactone (HTL) daily intragastric administration (200 mg·kg-1 of body weight) to young and adult albino non-linear male rats for 8 weeks following rat sacrifice and brain harvesting. It was established that HHcy causes an increase in total proteolytic activity and a rise in MLMWS levels in rat brains. Serine protease activity increased to a greater extent compared to metal-dependent one, and bigger changes were observed in young rats. Rise in MMP-9 and -10 levels (in young animals), a decline in MMP-3 and -8 levels, and a decrease in the content of interleukin-1ß, interferon-γ, interleukin-4 and tumor necrosis factor-a (the last two in young animals) was also detected. No significant changes were found in serine protease content. Therefore, proteolysis intensification in the brain of rats with HHcy is more likely caused by protease up-regulation through mechanisms stimulated by homocysteine, HTL, and oxidative stress, without involving pro-inflammatory signaling pathways. [ABSTRACT FROM AUTHOR]

Venom components are invaluable in biomedical research owing to their specificity and potency. Many of these components exist in two genera of rattlesnakes, Crotalus and Sistrurus, with high toxicity and proteolytic activity variation. This review focuses on venom components within rattlesnakes, and offers a comparison and itemized list of factors dictating venom composition, as well as presenting their known characteristics, activities, and significant applications in biosciences. There are 64 families and subfamilies of proteins present in Crotalus and Sistrurus venom. Snake venom serine proteases (SVSP), snake venom metalloproteases (SVMP), and phospholipases A2 (PLA2) are the standard components in Crotalus and Sistrurus venom. Through this review, we highlight gaps in the knowledge of rattlesnake venom; there needs to be more information on the venom composition of three Crotalus species and one Sistrurus subspecies. We discuss the activity and importance of both major and minor components in biomedical research and drug development. [ABSTRACT FROM AUTHOR]

Shiga-toxin producing Escherichia coli (STEC) infections can cause from bloody diarrhea to Hemolytic Uremic Syndrome. The STEC intestinal infection triggers an inflammatory response that can facilitate the development of a systemic disease. We report here that neutrophils might contribute to this inflammatory response by secreting Interleukin 1 beta (IL-1β). STEC stimulated neutrophils to release elevated levels of IL-1β through a mechanism that involved the activation of caspase-1 driven by the NLRP3-inflammasome and neutrophil serine proteases (NSPs). Noteworthy, IL-1β secretion was higher at lower multiplicities of infection. This secretory profile modulated by the bacteria:neutrophil ratio, was the consequence of a regulatory mechanism that reduced IL-1β secretion the higher were the levels of activation of both caspase-1 and NSPs, and the production of NADPH oxidase-dependent reactive oxygen species. Finally, we also found that inhibition of NSPs significantly reduced STEC-triggered IL-1β secretion without modulating the ability of neutrophils to kill the bacteria, suggesting NSPs might represent pharmacological targets to be evaluated to limit the STEC-induced intestinal inflammation. Author summary: The enteric pathogen Shiga toxin (Stx)-producing Escherichia coli (STEC) is a food- and water-borne bacteria that upon ingestion colonizes the intestine where it releases Shiga-toxins (Stx) and other virulence factors. STEC not only can cause self-limited gastrointestinal infections and bloody diarrhea but also a severe and potentially deathly systemic condition, known as Hemolytic Uremic Syndrome (HUS). There are no vaccines available to prevent STEC infections or specific treatments. The most frequent clinical STEC isolate in HUS is E. coli O157:H7. Severe inflammation and damage to the mucosal membrane are common symptoms of gastroenteritis induced by STEC. Interleukin-1β (IL-1β) is a highly pro-inflammatory mediator. Neutrophils are professional phagocytes that are extensively recruited to the intestinal mucosa upon STEC infection. Here we determined that human neutrophils secrete IL-1β in response to STEC and identified a molecular mechanism that limits the secretion of this mediator as the dose of bacteria that interacts with the neutrophils increases. Our study provides new insights into the role of neutrophils in the inflammatory response against STEC infections and opens a new road to investigate therapeutic strategies to avoid the progression to HUS. [ABSTRACT FROM AUTHOR]

Epithelial sodium channel (ENaC) are integral to maintaining salt and water homeostasis in various biological tissues, including the kidney, lung, and colon. They enable the selective reabsorption of sodium ions, which is a process critical for controlling blood pressure, electrolyte balance, and overall fluid volume. ENaC activity is finely controlled through proteolytic activation, a process wherein specific enzymes, or proteases, cleave ENaC subunits, resulting in channel activation and increased sodium reabsorption. This regulatory mechanism plays a pivotal role in adapting sodium transport to different physiological conditions. In this review article, we provide an in-depth exploration of the role of proteolytic activation in regulating ENaC activity. We elucidate the involvement of various proteases, including furin-like convertases, cysteine, and serine proteases, and detail the precise cleavage sites and regulatory mechanisms underlying ENaC activation by these proteases. We also discuss the physiological implications of proteolytic ENaC activation, focusing on its involvement in blood pressure regulation, pulmonary function, and intestinal sodium absorption. Understanding the mechanisms and consequences of ENaC proteolytic activation provides valuable insights into the pathophysiology of various diseases, including hypertension, pulmonary disorders, and various gastrointestinal conditions. Moreover, we discuss the potential therapeutic avenues that emerge from understanding these mechanisms, offering new possibilities for managing diseases associated with ENaC dysfunction. In summary, this review provides a comprehensive discussion of the intricate interplay between proteases and ENaC, emphasizing the significance of proteolytic activation in maintaining sodium and fluid balance in both health and disease. [ABSTRACT FROM AUTHOR]

Simple Summary: Chronic ammonia-N exposure always exists in realistic cultivation, and it is important to know about the mechanisms of shrimp in response to it and to shorten the breeding cycle. The aim of this study was to investigate the influence of chronic ammonia-N exposure on the growth performance, morphological and physiological alterations, and transcriptome changes of shrimp and to evaluate their ammonia-N resistance after chronic ammonia-N stress. Results showed that chronic ammonia-N exposure affected growth, caused significant structural damage and vacuolation in hepatopancreas and gill tissues, and changed related antioxidant enzyme activities. The gills maintained cellular homeostasis mainly through the high expression of cytoskeleton and transcription genes, whereas the hepatopancreas down-regulated related genes in the ribosome, proteasome, and spliceosome pathways. These genes and pathways are important in the biosynthesis and transformation of living organisms. In addition, both tissues maintained organismal growth primarily through lipid metabolism, which may serve as an effective strategy for ammonia-N resistance in L. vannamei. Ammonia nitrogen is one of the main toxic substances in aquatic cultivation environments. Chronic exposure to excessive amounts of ammonia-N creates toxic consequences, retarding the growth of aquatic organisms. This study investigated the growth performance, morphological and physiological alterations, and transcriptome changes in the hepatopancreas and gills of white shrimp Litopenaeus vannamei. The results showed that there was no significant difference in the survival rate (p > 0.05), whereas growth performance was reduced significantly in the treated groups compared to the control groups (p < 0.05). Significant structural damage and vacuolation occurred in hepatopancreas and gill tissues in the treated groups. Superoxide dismutase (SOD) activity and Na+/K+-ATPase content were significantly increased by chronic ammonia-N exposure in the two tissue groups. In addition, catalase (CAT) activity and malondialdehyde (MDA) levels were significantly altered in the hepatopancreas groups (p < 0.05), whereas no differences were observed in the gill groups (p > 0.05). There were 890 and 1572 differentially expressed genes identified in the hepatopancreas (treated versus control groups) and gills (treated versus control groups), respectively, of L. vannamei under chronic ammonia-N exposure. Functional enrichment analysis revealed associations with oxidative stress, protein synthesis, lipid metabolism, and different serine proteases. The gills maintained cellular homeostasis mainly through high expression of cytoskeleton and transcription genes, whereas the hepatopancreas down-regulated related genes in the ribosome, proteasome, and spliceosome pathways. These genes and pathways are important in the biosynthesis and transformation of living organisms. In addition, both tissues maintained organismal growth primarily through lipid metabolism, which may serve as an effective strategy for ammonia-N resistance in L. vannamei. These results provided a new perspective in understanding the mechanisms of ammonia-N resistance in crustaceans. [ABSTRACT FROM AUTHOR]

Objective: The current study's objective is to characterize a new throm-bin-like enzyme called TLBro that was obtained from Bothrops roedingeris snake from a biochemical and hemostatic perspective. Methodology: One chromatographic step was used to purify it, producing the serine protease TLBro. Molecular mass was estimated by SDS-PAGE to be between reduced and unreduced by 35 kDa. Tryptic peptide sequencing using Swiss Prot provided the complete amino acid sequence. Expasy.org by conducting a search that is limited to Crotalinae snake serine proteases and displaying a high degree of amino acid sequence. Results: Ser (182) is inhibited by phenylmethylsulfonyl fluoride (PMSF), and TLBro demonstrated the presence of Asp (88) residues. It also deduced the positions of His (43) and Ser (182) in the set of three coordinated amino acids in serine proteases. It was discovered that this substrate had high specificity for BANA, Michaelis-Menten behavior with KM 0 point85 mM and Vmax 1 point89 nmoles -NA/L/min, and high stability between temperatures (15 to 70°C) and pHs (2 point0 to 10 point0). According to doses and incubation times, TLBro degraded fibrin preferentially on the B-chain; additionally, its activities were significantly diminished after preincubation with divalent ions (Zn2 and Cd2). When incubated with PMSF, a particular serine protease inhibitor, enzymatic activities and platelet aggregation were inhibited. Conclusion: The findings revealed distinct structural and functional differences between the serine proteases, adding to the information and assisting in the improvement of the structure-function relationship. [ABSTRACT FROM AUTHOR]

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly evolving pathogen that is frequently associated with outbreaks and sustained epidemics. This study investigated the population structure, resistome, virulome, and the correlation between antimicrobial resistance determinants with phenotypic resistance profiles of 36 representative hospital-acquired MRSA isolates recovered from hospital settings in Egypt. Results: The community-acquired MRSA lineage, clonal complex 1 (CC1) was the most frequently detected clone, followed by three other globally disseminated clones, CC121, CC8, and CC22. Most isolates carried SCCmec type V and more than half of isolates demonstrated multi-drug resistant phenotypes. Resistance to linezolid, a last resort antibiotic for treating multidrug resistant MRSA, was observed in 11.11% of the isolates belonging to different genetic backgrounds. Virulome analysis indicated that most isolates harboured a large pool of virulence factors and toxins. Genes encoding aureolysin, gamma hemolysins, and serine proteases were the most frequently detected virulence encoding genes. CC1 was observed to have a high pool of AMR resistance determinants including cfr, qacA, and qacB genes, which are involved in linezolid and quaternary ammonium compounds resistance, as well as high content of virulence-related genes, including both of the PVL toxin genes. Molecular clock analysis revealed that CC1 had the greatest frequency of recombination (compared to mutation) among the four major clones, supporting the role of horizontal gene transfer in modulating AMR and hypervirulence in this clone. Conclusions: This pilot study provided evidence on the dissemination success of CA-MRSA clone CC1 among Egyptian hospitals. Co-detection of multiple AMR and virulence genes in this lineage pose a broad public health risk, with implications for successful treatment. The results of this study, together with other surveillance studies in Egypt, should be used to develop strategies for controlling MRSA infections in Egyptian health-care settings. [ABSTRACT FROM AUTHOR]

Uncontrolled type 2 immunity by type 2 helper T (Th2) cells causes intractable allergic diseases; however, whether the interaction of CD4+ T cells shapes the pathophysiology of allergic diseases remains unclear. We identified a subset of Th2 cells that produced the serine proteases granzyme A and B early in differentiation. Granzymes cleave protease-activated receptor (Par)-1 and induce phosphorylation of p38 mitogen-activated protein kinase (MAPK), resulting in the enhanced production of IL-5 and IL-13 in both mouse and human Th2 cells. Ubiquitin-specific protease 7 (USP7) regulates IL-4-induced phosphorylation of STAT3, resulting in granzyme production during Th2 cell differentiation. Genetic deletion of Usp7 or Gzma and pharmacological blockade of granzyme B ameliorated allergic airway inflammation. Furthermore, PAR-1+ and granzyme+ Th2 cells were colocalized in nasal polyps from patients with eosinophilic chronic rhinosinusitis. Thus, the USP7-STAT3-granzymes-Par-1 pathway is a potential therapeutic target for intractable allergic diseases. [ABSTRACT FROM AUTHOR]

There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca2+]i dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of nonspecific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ion conductance microscopy, and patch clamp analysis. Goto-Kakizaki, Wistar, type 2 DN (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i compared with controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared with Wistar rats. T2DNPar1−/− rats were embryonically lethal. T2DNPar1+/− rats had a significant decrease in glomerular damage associated with DN lesions. Overall, these data provide evidence that, during the development of DN, elevated levels of serine proteases promote an excessive [Ca2+]i influx in podocytes through PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage. Article Highlights: Increased urinary serine proteases are associated with diabetic nephropathy. During the development of diabetic nephropathy in type 2 diabetes, the elevation of serine proteases could overstimulate protease-activated receptor 1 (PAR1). PAR1 signaling is involved in the development of DN via TRPC6-mediated intracellular calcium signaling. This study provides fundamental knowledge that can be used to develop efficient therapeutic approaches targeting serine proteases or corresponding PAR pathways to prevent or slow the progression of diabetes-associated kidney diseases. [ABSTRACT FROM AUTHOR]

Venoms are primarily believed to evolve under strong diversifying selection resulting from persistent coevolution between predator and prey. Recent research has challenged this hypothesis, proposing that venoms from younger venomous lineages (e.g., snakes and cone snails) are governed predominantly by diversifying selection, while venoms from older venomous lineages (e.g., centipedes, scorpions, and spiders) are under stronger purifying selection. However, most research in older lineages has tested selection at more diverse phylogenetic scales. Although these tests are important for evaluating broad macroevolutionary trends underlying venom evolution, they are less equipped to detect species-level evolutionary trends, which likely have large impacts on venom variation seen at more diverse phylogenetic scales. To test for selection among closely related species from an older venomous lineage, we generated high-throughput venom-gland transcriptomes and venom proteomes for four populations of Giant Desert Hairy Scorpions (Hadrurus), including three Hadrurus arizonensis populations and one Hadrurus spadix population. We detected significant episodic and pervasive diversifying selection across a highly abundant toxin family that likely has a major role in venom function (α KTxs), providing a contrast to the stronger purifying selection identified from other studies on scorpion venoms. Conversely, we detected weak episodic diversifying and/or stronger purifying selection in four toxin families (non-disulfide bridged peptides, phospholipase A2s, scorpine-like antimicrobial peptides, and serine proteases), most of which were less abundant and likely have ancillary functional roles. Finally, although we detected several major toxin families at disproportionate transcriptomic and/or proteomic abundances, we did not identify significant sex-based variation in Hadrurus venoms. [ABSTRACT FROM AUTHOR]

Summary: Rapid softening of texture is a major problem associated with quality loss and consumer rejection of harpiosquillid mantis shrimp (Harpiosquilla raphidea) (HMS). Effects of endogenous proteases in muscles and digestive tract on protein degradation and softening were investigated. Scanning electron microscopic and histological images indicated drastic destruction of muscle during iced storage. Based on autolysis, maximal activity was found at pH 8.5 and 55 °C, and serine proteases including trypsin were dominant. Cathepsin B and L + B in muscle were altered throughout storage, while trypsin remained constant in HMS meat for 1–3 days. Firmness and toughness decreased and coincided with augmented degradation of myofibrillar proteins and collagen. Trypsin from the digestive tract was contaminated into the abdomen during iced storage, therefore inducing drastic hydrolysis of muscle proteins. This phenomenon caused the undesirable pasty/mushy texture associated with the loss in firmness and toughness of meat within 3 days of storage. [ABSTRACT FROM AUTHOR]

Background: The purinergic receptor P2X7 plays a crucial role in infection, inflammation, and cell death. It is thought that P2X7 receptor stimulation triggers processing and release of the pro-inflammatory cytokine interleukin (IL)-1β by activation of the NLRP3 inflammasome; however, the underlying mechanisms remain poorly understood. Methods: Modulation of IL-1β secretion was studied in THP-1 macrophages. Adenosine 5'-triphosphate (ATP), BzATP, nigericin and pharmacological inhibitors of P2X receptors, inflammatory caspases and the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome were used to characterize signaling. Results: In primed macrophages, IL-1β release was increased after P2X7 receptor activation by ATP and 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP). Pharmacological inhibition or genetic knockout of NLRP3 does not completely inhibit IL-1β release in TLR2/1-primed macrophages. Increase in extracellular K+ as well as inhibition of caspase-1 or serine proteases maintained IL-1β release in macrophages stimulated with P2X7 receptor agonists at 50%. Conclusions: Our findings suggest a previously unrecognized mechanism of P2X7 receptor mediated IL-1β release and highlight the existence of an NLRP3-independent pathway in human macrophages. 4h-1gv1RJyWYx97JbdiPpX Video Abstract [ABSTRACT FROM AUTHOR]

Introduction: Overactivation of the lectin pathway of complement plays a pathogenic role in a broad range of immune-mediated and inflammatory disorders; mannan-binding lectin-associated serine protease-2 (MASP-2) is the key effector enzyme of the lectin pathway. We developed a fully human monoclonal antibody, narsoplimab, to bind to MASP-2 and specifically inhibit lectin pathway activation. Herein, we describe the preclinical characterization of narsoplimab that supports its evaluation in clinical trials. Methods and results: ELISA binding studies demonstrated that narsoplimab interacted with both zymogen and enzymatically active forms of human MASP-2 with high affinity (KD 0.062 and 0.089 nM, respectively) and a selectivity ratio of >5,000-fold relative to closely related serine proteases C1r, C1s, MASP-1, and MASP-3. Interaction studies using surface plasmon resonance and ELISA demonstrated approximately 100-fold greater binding affinity for intact narsoplimab compared to a monovalent antigen binding fragment, suggesting an important contribution of functional bivalency to high-affinity binding. In functional assays conducted in dilute serum under pathway-specific assay conditions, narsoplimab selectively inhibited lectin pathway-dependent activation of C5b-9 with high potency (IC50 ~ 1 nM) but had no observable effect on classical pathway or alternative pathway activity at concentrations up to 500 nM. In functional assays conducted in 90% serum, narsoplimab inhibited lectin pathway activation in human serum with high potency (IC50 ~ 3.4 nM) whereas its potency in cynomolgus monkey serum was approximately 10-fold lower (IC50 ~ 33 nM). Following single dose intravenous administration to cynomolgus monkeys, narsoplimab exposure increased in an approximately dose-proportional manner. Clear dose-dependent pharmacodynamic responses were observed at doses >1.5 mg/kg, as evidenced by a reduction in lectin pathway activity assessed ex vivo that increased in magnitude and duration with increasing dose. Analysis of pharmacokinetic and pharmacodynamic data revealed a well-defined concentration-effect relationship with an ex vivo EC50 value of approximately 6.1 µg/mL, which was comparable to the in vitro functional potency (IC50 33 nM; ~ 5 µg/mL). Discussion: Based on these results, narsoplimab has been evaluated in clinical trials for the treatment of conditions associated with inappropriate lectin pathway activation, such as hematopoietic stem cell transplantation-associated thrombotic microangiopathy. [ABSTRACT FROM AUTHOR]

We report the molecular basis of Aspergillus fumigatus oryzin, allergen Asp f 13, or alkaline proteinase ALP1, containing the sequence motif His–Asp–Ser of the subtilisin family, structure, and function at atomic detail. Given the resolution of the data (1.06 Å), we use fragment molecular replacement with ideal polyalanine α-helices to determine the first crystal structure of oryzin. We probe the catalytic serine through formation of an irreversible bond to a small molecule compound, specifically labeling it, describing the amino acid residues performing the catalytic function. Defined by a self-processed pro-peptide, the active site architecture shapes up pocket-like subsites that bind to and unveil the S1′–S4′ substrate binding preferences. We use molecular modeling to dock a model of the pro-peptide in the S1–S4 region and to dock collagen along the active site cleft. Opposite to the face harboring the catalytic serine, the enzyme binds to a calcium ion in a binding site created by backbone flipping. We use thermal unfolding to show that this metal ion provides structural stability. With no known host inhibitor identified thus far, this structure may hasten the progress of developing new therapeutic agents for diseases caused by pathogenic fungi. [ABSTRACT FROM AUTHOR]

Mycoparasitism is a key feature of Trichoderma (Hypocreales, Ascomycota) biocontrol agents. Recent studies of intracellular signal transduction pathways of the potent mycoparasite Trichoderma atroviride revealed the involvement of Tmk1, a mitogen-activated protein kinase (MAPK), in triggering the mycoparasitic response. We previously showed that mutants missing Tmk1 exhibit reduced mycoparasitic activity against several plant pathogenic fungi. In this study, we identified the most robustly regulated targets that were governed by Tmk1 during mycoparasitism using transcriptome and proteome profiling. Tmk1 mainly exerts a stimulating function for T. atroviride during its mycoparasitic interaction with the fungal plant pathogen Rhizoctonia solani, as reflected by 89% of strongly differently responding genes in the ∆tmk1 mutant compared to the wild type. Specifically, 54% of these genes showed strong downregulation in the response with a deletion of the tmk1 gene, whereas in the wild type the same genes were strongly upregulated during the interaction with the fungal host. These included the gene encoding the mycoparasitism-related proteinase Prb1; genes involved in signal transduction pathways such as a candidate coding for a conserved 14-3-3 protein, and a gene coding for Tmk2, the T. atroviride cell-wall integrity MAP kinase; genes encoding a specific siderophore synthetase, and multiple FAD-dependent oxidoreductases and aminotransferases. Due to the phosphorylating activity of Tmk1, different (phospho-)proteomics approaches were applied and identified proteins associated with cellular metabolism, energy production, protein synthesis and fate, and cell organization. Members of FAD- and NAD/NADP-binding-domain proteins, vesicular trafficking of molecules between cellular organelles, fungal translational, as well as protein folding apparatus were among others found to be phosphorylated by Tmk1 during mycoparasitism. Outstanding downregulation in the response of the ∆tmk1 mutant to the fungal host compared to the wild type at both the transcriptome and the proteome levels was observed for nitrilase, indicating that its defense and detoxification functions might be greatly dependent on Tmk1 during T. atroviride mycoparasitism. An intersection network analysis between the identified transcripts and proteins revealed a strong involvement of Tmk1 in molecular functions with GTPase and oxidoreductase activity. These data suggest that during T. atroviride mycoparasitism this MAPK mainly governs processes regulating cell responses to extracellular signals and those involved in reactive oxygen stress. [ABSTRACT FROM AUTHOR]

Background: Heart transplantation (HTX) is the standard treatment for end-stage heart failure. However, reperfusion following an ischemic period can contribute to myocardial injury. Neutrophil infiltration, along with the subsequent release of tissue-degrading neutrophil elastase (NE)-related serine proteases and oxygen-derived radicals, is associated with adverse graft outcomes. The inhibition of cathepsin C (CatC) has been shown to block NE-related protease activation. We hypothesized that the CatC inhibitor BI-9740 improves graft function after HTX. Methods: In a rat model of HTX, the recipient Lewis rats were orally administered with either a placebo (n = 12) or BI-9740 (n = 11, 20 mg/kg) once daily for 12 days. Donor hearts from untreated Lewis rats were explanted, preserved in a cardioplegic solution, and subsequently heterotopically implanted. In vivo left-ventricular (LV) graft function was assessed after 1 h of reperfusion. The proteolytic activity of neutrophil serine proteases was determined in bone marrow lysates from BI-9740-treated and control rats. Additionally, myocardial morphological changes were examined, and heart samples underwent immunohistochemistry and western blot analysis. Results: The NE-related proteolytic activity in bone marrow cell lysates was markedly decreased in the BI-9740-treated rats compared to those of the placebo group. Histopathological lesions, elevated CatC and myeloperoxidase-positive cell infiltration, and nitrotyrosine immunoreactivity with an increased number of poly(ADP-ribose) polymerase (PARP)-1-positive cells were lowered in the hearts of animals treated with BI-9740 compared to placebo groups. Regarding the functional parameters of the implanted graft, improvements were observed in both systolic function (LV systolic pressure 110 ± 6 vs 74 ± 6 mmHg; dP/dtmax 2782 ± 149 vs 2076 ± 167 mmHg/s, LV developed pressure, at an intraventricular volume of 200 µl, p < 0.05) and diastolic function in the hearts of BI-9740 treated animals compared with those receiving the only placebo. Furthermore, the administration of BI-9740 resulted in a shorter graft re-beating time compared to the placebo group. However, this study did not provide evidence of DNA fragmentation, the generation of both superoxide anions and hydrogen peroxide, correlating with the absence of protein alterations related to apoptosis, as evidenced by western blot in grafts after HTX. Conclusions: We provided experimental evidence that pharmacological inhibition of CatC improves graft function following HTX in rats. [ABSTRACT FROM AUTHOR]

Aims: This study aimed to explore associations of mannan-binding lectin-associated serine protease (MASP) levels in early pregnancy with gestational diabetes mellitus (GDM). We also examined interactions of MASPs and deoxycholic acid (DCA)/glycoursodeoxycholic acid (GUDCA) for the GDM risk and whether the interactive effects if any on the GDM risk were mediated via lysophosphatidylcholine (LPC) 18:0. Materials and methods: A 1:1 case-control study (n = 414) nested in a prospective cohort of pregnant women was conducted in Tianjin, China. Binary conditional logistic regressions were performed to examine associations of MASPs with the GDM risk. Additive interaction measures were used to examine interactions between MASPs and DCA/GUDCA for the GDM risk. Mediation analyses and Sobel tests were used to examine mediation effects of LPC18:0 between the copresence of MASPs and DCA/GUDCA on the GDM risk. Results: High MASP-2 was independently associated with GDM [odds ratio (OR): 2.62, 95% confidence interval (CI): 1.44-4.77], while the effect of high MASP-1 on GDM was attributable to high MASP-2 (P for Sobel test: 0.003). Low DCA markedly increased the OR of high MASP-2 alone from 2.53 (1.10-5.85) up to 10.6 (4.22-26.4), with a significant additive interaction. In addition, high LPC18:0 played a significant mediating role in the links from low DCA to GDM and from the copresence of high MASP-2 and low DCA to GDM (P for Sobel test <0.001) but not in the link from high MASP-2 to GDM. Conclusions: High MASP-1 and MASP-2 in early pregnancy were associated with GDM in Chinese pregnant women. MASP-2 amplifies the risk of low DCA for GDM, which is mediated via LPC18:0. [ABSTRACT FROM AUTHOR]

Plant-parasitic nematodes significantly threaten agriculture and forestry, causing various diseases. They cause annual losses of up to 178 billion dollars worldwide due to their parasitism. Nematophagous fungi (NF) are valuable in controlling or reducing parasitic nematode diseases by killing nematodes through predatory behavior. This article summarizes the strategic approaches adopted by NF to capture, poison, or consume nematodes for food. NF are classified based on their attacking strategies, including nematode trapping, endoparasitism, toxin production, and egg and female parasitism. Moreover, extracellular enzymes such as serine proteases and chitinases also play an important role in the fungal infection of nematodes by disrupting nematode cuticles, which act as essential virulence factors to target the chemical constituents comprising the nematode cuticle and eggshell. Based on the mentioned approaches, it is crucial to consider the mechanisms employed by NF to control nematodes focused on the use of NF as biocontrol agents. [ABSTRACT FROM AUTHOR]

The European catfish Silurus glanis is attracting growing interest as an object of fisheries and aquaculture, which is reinforced by the expansion of its natural range under climate change. Shaping the effective exploitation strategy for this valuable species requires detailed knowledge of its biology, including feeding and digestion processes, especially near the natural limits of the species range. Meanwhile, the digestion physiology of the European catfish remains poorly explored, including the activity of major digestive enzymes and the possible effect of intestinal parasites on this activity. In this regard, the activity of proteinases and α‐amylase in the intestinal mucosa of the catfish was studied. Adult catfish were collected in the Rybinsk reservoir (Upper Volga) located close to the northern limit of the species range. It was shown that all subclasses of intestinal digestive proteinases, including serine proteinases, metalloproteases and cysteine (thiol) proteinases, function in the gut mucosa of the catfish. The mucosal levels of total proteolytic activity depended on fish size, in contrast to those of trypsin, chymotrypsin and α‐amylase. The level of chymotrypsin activity was significantly higher than that of trypsin activity. It was also found that the incubation medium and extract of the cestodes Silurotaenia siluri parasitizing the catfish gut had a significant inhibitory effect on the activity of serine proteases (trypsin and chymotrypsin) operating in the intestines of the host fish. [ABSTRACT FROM AUTHOR]

Snakebite envenoming is one of the most significantly neglected tropical diseases in the world. The lack of diagnosis/prognosis methods for snakebite is one of our motivations to develop innovative technological solutions for Brazilian health. The objective of this work was to evaluate the protein and metallic ion composition of Crotalus durissus terrificus, Bothrops jararaca, B. alternatus, B. jararacussu, B. moojeni, B. pauloensis, and Lachesis muta muta snake venoms. Brazilian snake venoms were subjected to the shotgun proteomic approach using mass spectrometry, and metal ion analysis was performed by atomic spectrometry. Shotgun proteomics has shown three abundant toxin classes (PLA2, serine proteases, and metalloproteinases) in all snake venoms, and metallic ions analysis has evidenced that the Cu2+ ion is present exclusively in the L. m. muta venom; Ca2+ and Mg2+ ions have shown a statistical difference between the species of Bothrops and Crotalus genus, whereas the Zn2+ ion presented a statistical difference among all species studied in this work. In addition, Mg2+ ions have shown 42 times more in the C. d. terrificus venom when compared to the average concentration in the other genera. Though metal ions are a minor fraction of snake venoms, several venom toxins depend on them. We believe that these non-protein fractions are capable of assisting in the development of unprecedented diagnostic devices for Brazilian snakebites. [ABSTRACT FROM AUTHOR]

Serine proteases are involved in many biological processes and are associated with irritable bowel syndrome (IBS) pathology. An increase in serine protease activity has been widely reported in IBS patients. While most of the studies focused on host proteases, the contribution of microbial proteases are poorly studied. In the present study, we report the analysis of proteolytic activities in fecal samples from the first Tunisian cohort of IBS-M patients and healthy individuals. We demonstrated, for the first time, that metalloproteases activities were fourfold higher in fecal samples of IBS patients compared to controls. Of interest, the functional characterization of serine protease activities revealed a 50-fold increase in trypsin-like activities and a threefold in both elastase- and cathepsin G-like activities. Remarkably, we also showed a fourfold increase in proteinase 3-like activity in the case of IBS. This study also provides insight into the alteration of gut microbiota and its potential role in proteolytic modulation in IBS. Our results stressed the impact of the disequilibrium of serine proteases, metalloproteases and gut microbiota in IBS and the need of the further characterization of these targets to set out new therapeutic approaches. [ABSTRACT FROM AUTHOR]

Plant serpins are a superfamily of protein inhibitors that have been continuously studied in different species and have great biotechnological potential. However, despite ongoing studies with these inhibitors, the biological role of this family in the plant kingdom has not yet been fully clarified. In order to obtain new insights into the potential of plant serpins, this study presents the first systematic review of the topic, whose main objective was to scrutinize the published literature to increase knowledge about this superfamily. Using keywords and the eligibility criteria defined in the protocol, we selected studies from the Scopus, PubMed, and Web of Science databases. According to the eligible studies, serpins inhibit different serine and non-serine proteases from plants, animals, and pathogens, and their expression is affected by biotic and abiotic stresses. Moreover, serpins like AtSerpin1, OSP-LRS, MtSer6, AtSRP4, AtSRP5, and MtPiI4, act in resistance and are involved in stress-induced cell death in the plant. Also, the system biology analysis demonstrates that serpins are related to proteolysis control, cell regulation, pollen development, catabolism, and protein dephosphorylation. The information systematized here contributes to the design of new studies of plant serpins, especially those aimed at exploring their biotechnological potential. [ABSTRACT FROM AUTHOR]

Severe COVID-associated lung injury is a major confounding factor of hospitalizations and death with no effective treatments. Here, we describe a non-classical fibrin clotting mechanism mediated by SARS-CoV-2 infected primary lung but not other susceptible epithelial cells. This infection-induced fibrin formation is observed in all variants of SARS-CoV-2 infections, and requires thrombin but is independent of tissue factor and other classical plasma coagulation factors. While prothrombin and fibrinogen levels are elevated in acute COVID BALF samples, fibrin clotting occurs only with the presence of viral infected but not uninfected lung epithelial cells. We suggest a viral-induced coagulation mechanism, in which prothrombin is activated by infection-induced transmembrane serine proteases, such as ST14 and TMPRSS11D, on NHBE cells. Our finding reveals the inefficiency of current plasma targeted anticoagulation therapy and suggests the need to develop a viral-induced ARDS animal model for treating respiratory airways with thrombin inhibitors. Severe SARS-CoV-2 infection has been associated with extensive diffuse alveolar damage and fibrin formation. Here, Erickson et al describe an infection-induced coagulation mechanism which involves activation of prothrombin by members of TMPRSS genes. [ABSTRACT FROM AUTHOR]

Alkaline proteases from microbial sources have been found suitable for diverse industrial applications, with serine proteases being the most common enzymes used in the detergent industry. In the present study, we have purified and characterized an extracellular alkaline serine protease from Microbacterium paraoxydans sp. SKS10. The protease was purified using ammonium sulfate precipitation followed by different chromatography techniques (fold purification 6.919). Km and Vmax for the protease were determined to be 0.183 mg/mL and 4.904 U/mL, respectively. This enzyme is a thermostable high molecular weight (∼109.4 kDa) protease which has maximal activity at 60°C, and above pH 10. Inhibitor assays revealed the enzyme to be a serine protease whose activity increased by 2.5‐fold in the presence of EDTA. This enzyme remained active in the presence of various metal salts and organic solvents and was compatible with commercially available laundry detergents highlighting its potential for use in the detergent industry. [ABSTRACT FROM AUTHOR]

The serine proteases CAP1/Prss8 and CAP3/St14 are identified as ENaC channel-activating proteases in vitro, highly suggesting that they are required for proteolytic activation of ENaC in vivo. The present study tested whether CAP3/St14 is relevant for renal proteolytic ENaC activation and affects ENaC-mediated Na+ absorption following Na+ deprivation conditions. CAP3/St14 knockout mice exhibit a significant decrease in CAP1/Prss8 protein expression with altered ENaC subunit and decreased pNCC protein abundances but overall maintain sodium balance. RNAscope-based analyses reveal co-expression of CAP3/St14 and CAP1/Prss8 with alpha ENaC in distal tubules of the cortex from wild-type mice. Double CAP1/Prss8; CAP3/St14-deficiency maintained Na+ and K+ balance on a Na+-deprived diet, restored ENaC subunit protein abundances but showed reduced NCC activity under Na+ deprivation. Overall, our data clearly show that CAP3/St14 is not required for direct proteolytic activation of ENaC but for its protein abundance. Our study reveals a complex regulation of ENaC by these serine proteases on the expression level rather than on its proteolytic activation. [ABSTRACT FROM AUTHOR]

Collagenolytic proteases (collagenases) can be used in a variety of areas of human life: processing of leather, production of detergents, and food and beverage preparation, as well as pharmaceuticals and medicine. The aim of this study was to develop methods for the preparation and characterization of collagenolytic proteinase from the filamentous fungus Aspergillus fumigatus. A method for preparing A. fumigatus extracellular collagenolytic proteinase by two-stage chromatographic purification was developed. Analysis of the physicochemical and biochemical properties of the enzyme confirmed that a serine-type proteinase with a molecular weight of 40 kDa and maximum activity at 40°C, pH 7.5, was synthesized. The data obtained here suggest that A. fumigatus proteinase can be regarded as a basis for obtaining new drugs with collagenolytic effects. [ABSTRACT FROM AUTHOR]

Albuminuria in kidney transplant recipients (KTRs) is associated with hypertension and aberrant glomerular filtration of serine proteases that may proteolytically activate the epithelial Na+ channel (ENaC). The present nonrandomized, pharmacodynamic intervention study aimed to investigate if inhibition of ENaC increases Na+ excretion and reduces extracellular volume in KTRs dependent on the presence of albuminuria. KTRs with and without albuminuria (albumin-to-creatinine ratio > 300 mg/g, n = 7, and <30 mg/g, n = 7, respectively) were included and ingested a diet with fixed Na+ content (150 mmol/day) for 5 days. On the last day, amiloride at 10 mg was administered twice. Body weight, 24-h urine electrolyte excretion, body water content, and ambulatory blood pressure as well as plasma renin, angiotensin II, and aldosterone concentrations were determined before and after amiloride. Amiloride led to a significant decrease in body weight, increase in 24-h urinary Na+ excretion, and decrease in 24-h urinary K+ excretion in both groups. Urine output increased in the nonalbuminuric group only. There was no change in plasma renin, aldosterone, and angiotensin II concentrations after amiloride, whereas a significant decrease in nocturnal systolic blood pressure and increase in 24-h urine aldosterone excretion was observed in albuminuric KTRs only. There was a significant correlation between 24-h urinary albumin excretion and amiloride-induced 24-h urinary Na+ excretion. In conclusion, ENaC activity contributes to Na+ and water retention in KTRs with and without albuminuria. ENaC is a relevant pharmacological target in KTRs; however, larger and long-term studies are needed to evaluate whether the magnitude of this effect depends on the presence of albuminuria. NEW & NOTEWORTHY Amiloride has a significant natriuretic effect in kidney transplant recipients (KTRs) that relates to urinary albumin excretion. The epithelial Na+ channel may be a relevant direct pharmacological target to counter Na+ retention and hypertension in KTRs. Epithelial Na+ channel blockers should be further investigated as a mean to mitigate Na+ and water retention and to potentially obtain optimal blood pressure control in KTRs. [ABSTRACT FROM AUTHOR]

Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type plasminogen activator (uPA) and plasmin cleave and activate each other, constituting a positive feedback loop. How this mutual activation cycle begins has remained a mystery. We used hydrogen deuterium exchange mass spectrometry to characterize the dynamic differences between the inactive single-chain uPA (scuPA) and its active form two-chain uPA (tcuPA). The results show that the C-terminal ß-barrel and the area around the new N terminus have significantly reduced dynamics in tcuPA as compared with scuPA. We also show that the zymogen scuPA is inactive but can, upon storage, become active in the absence of external proteases. In addition to plasmin, the tcuPA can activate scuPA by cleavage at K158, a process called autoactivation. Unexpectedly, tcuPA can cleave at position 158 even when this site is mutated. TcuPA can also cleave scuPA after K135 or K136 in the disordered linker, which generates the soluble protease domain of uPA. Plasmin cleaves scuPA exclusively after K158 and at a faster rate than tcuPA. We propose a mechanism by which the uPA receptor dimerization could promote autoactivation of scuPA on cell surfaces. These results resolve long-standing controversies in the literature surrounding the mechanism of uPA activation. [ABSTRACT FROM AUTHOR]

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

Nanocarrier systems are widely used for drug delivery applications, but limitations such as the use of synthetic surfactants, leakage of toxic drugs, and a poor encapsulation capacity remain as challenges. We present a new hybrid nanocarrier system that utilizes natural materials to overcome these limitations and improve the safety and efficacy of drug delivery. The system comprises a biopolymeric shell and a lipid core, encapsulating the lipophilic anticancer drug paclitaxel. Bovine serum albumin and dextran, in various molecular weights, are covalently conjugated via Maillard reaction to form the shell which serves as a stabilizer to maintain nanoparticle integrity. The properties of the system, such as Maillard conjugate concentration, protein/polysaccharide molar ratio, and polysaccharide molecular weight, are optimized to enhance nanoparticle size and stability. The system shows high stability at different pH conditions, high drug loading capacity, and effective in vitro drug release through the trigger of enzymes and passive diffusion. Serine proteases are used to digest the protein portion of the nanoparticle shell to enhance the drug release. This nanocarrier system represents a significant advancement in the field of nanomedicine, offering a safe and effective alternative for the delivery of lipophilic drugs. [ABSTRACT FROM AUTHOR]

Human plasma kallikrein (PKa) is obtained by activating its precursor, prekallikrein (PK), historically named the Fletcher factor. Human PKa and tissue kallikreins are serine proteases from the same family, having high- and low-molecular weight kininogens (HKs and LKs) as substrates, releasing bradykinin (Bk) and Lys-bradykinin (Lys-Bk), respectively. This review presents a brief history of human PKa with details and recent observations of its evolution among the vertebrate coagulation proteins, including the relations with Factor XI. We explored the role of Factor XII in activating the plasma kallikrein-kinin system (KKS), the mechanism of activity and control in the KKS, and the function of HK on contact activation proteins on cell membranes. The role of human PKa in cell biology regarding the contact system and KSS, particularly the endothelial cells, and neutrophils, in inflammatory processes and infectious diseases, was also approached. We examined the natural plasma protein inhibitors, including a detailed survey of human PKa inhibitors' development and their potential market. [ABSTRACT FROM AUTHOR]