Thymic Stromal Lymphopoietin (TSLP) Is Cleaved by Human Mast Cell Tryptase and Chymase.

Thymic stromal lymphopoietin (TSLP), mainly expressed by epithelial cells, plays a central role in asthma. In humans, TSLP exists in two variants: the long form TSLP (lfTSLP) and a shorter TSLP isoform (sfTSLP). Macrophages (HLMs) and mast cells (HLMCs) are in close proximity in the human lung and play key roles in asthma. We evaluated the early proteolytic effects of tryptase and chymase released by HLMCs on TSLP by mass spectrometry. We also investigated whether TSLP and its fragments generated by these enzymes induce angiogenic factor release from HLMs. Mass spectrometry (MS) allowed the identification of TSLP cleavage sites caused by tryptase and chymase. Recombinant human TSLP treated with recombinant tryptase showed the production of 1-97 and 98-132 fragments. Recombinant chymase treatment of TSLP generated two peptides, 1-36 and 37-132. lfTSLP induced the release of VEGF-A, the most potent angiogenic factor, from HLMs. By contrast, the four TSLP fragments generated by tryptase and chymase failed to activate HLMs. Long-term TSLP incubation with furin generated two peptides devoid of activating property on HLMs. These results unveil an intricate interplay between mast cell-derived proteases and TSLP. These findings have potential relevance in understanding novel aspects of asthma pathobiology.

Oral administration of egg ovalbumin allergen induces dysregulation of tryptophan metabolism in sensitized BALB/c mice.

Food allergy (FA), triggered by specific dietary allergens, has emerged as a substantial global concern for food safety and public health. While studies have elucidated changes in immune cells and cytokines associated with allergen exposure, a comprehensive analysis of the host's metabolic features and the interaction between metabolites and the gut microbiota has not been conducted. In this study, egg allergen ovalbumin (OVA) was administered by the oral route to sensitized BALB/c mice to faithfully replicate key aspects of human FA, including severe allergic diarrhea, mast cell infiltration, and elevated levels of serum IgE, mMCPT-1, and Th2 cell hallmark cytokines (such as IL-4, IL-5, and IL-13). Furthermore, the untargeted and targeted metabolomic analyses indicated that FA in mice precipitated a substantial decrease in the tryptophan metabolites indole-3-acrylic acid (IA) and indole-3-lactic acid (ILA). The integration of shotgun metagenome and metabolome data further unveiled that the dysregulation of indole metabolism is related to a decline in the abundance of beneficial bacteria such as Lactobacillus and Bifidobacterium. Additionally, disruption of the tryptophan indole derivative pathway compromises the maintenance of intestinal mucosal function through the AHR signaling pathway, manifested by decreased expression of Reg3g and IL22. Taken together, this study demonstrated that the anaphylaxis triggered by oral ingestion of food allergens can lead to disruptions in tryptophan metabolism, consequently impairing intestinal immune homeostasis.

Development of de-novo coronavirus 3-chymotrypsin-like protease (3CLpro) inhibitors since COVID-19 outbreak: A strategy to tackle challenges of persistent virus infection.

Although no longer a public health emergency of international concern, COVID-19 remains a persistent and critical health concern. The development of effective antiviral drugs could serve as the ultimate piece of the puzzle to curbing this global crisis. 3-chymotrypsin-like protease (3CLpro), with its substrate specificity mirroring that of the main picornavirus 3C protease and conserved across various coronaviruses, emerges as an ideal candidate for broad-spectrum antiviral drug development. Moreover, it holds the potential as a reliable contingency option to combat emerging SARS-CoV-2 variants. In this light, the approved drugs, promising candidates, and de-novo small molecule therapeutics targeting 3CLpro since the COVID-19 outbreak in 2020 are discussed. Emphasizing the significance of diverse structural characteristics in inhibitors, be they peptidomimetic or nonpeptidic, with a shared mission to minimize the risk of cross-resistance. Moreover, the authors propose an innovative optimization strategy for 3CLpro reversible covalent PROTACs, optimizing pharmacodynamics and pharmacokinetics to better prepare for potential future viral outbreaks.

GW0742 reduces mast cells degranulation and attenuates neurological impairments via PPARß/δ/CD300a/SHP1 pathway after GMH in neonatal rats.

BACKGROUND: Activation of mast cells plays an important role in brain inflammation. CD300a, an inhibitory receptor located on mast cell surfaces, has been reported to reduce the production of pro-inflammatory cytokines and exert protective effects in inflammation-related diseases. Peroxisome proliferator-activated receptor ß/δ (PPARß/δ), a ligand-activated nuclear receptor, activation upregulates the transcription of CD300a. In this study, we aim to investigate the role of PPARß/δ in the attenuation of germinal matrix hemorrhage (GMH)-induced mast cell activation via CD300a/SHP1 pathway. METHODS: GMH model was induced by intraparenchymal injection of bacterial collagenase into the right hemispheric ganglionic eminence in P7 Sprague Dawley rats. GW0742, a PPARß/δ agonist, was administered intranasally at 1 h post-ictus. CD300a small interfering RNA (siRNA) and PPARß/δ siRNA were injected intracerebroventricularly 5 days and 2 days before GMH induction. Behavioral tests, Western blot, immunofluorescence, Toluidine Blue staining, and Nissl staining were applied to assess post-GMH evaluation. RESULTS: Results demonstrated that endogenous protein levels of PPARß/δ and CD300a were decreased, whereas chymase, tryptase, IL-17A and transforming growth factor ß1 (TGF-ß1) were elevated after GMH. GMH induced significant short- and long-term neurobehavioral deficits in rat pups. GW0742 decreased mast cell degranulation, improved neurological outcomes, and attenuated ventriculomegaly after GMH. Additionally, GW0742 increased expression of PPARß/δ, CD300a and phosphorylation of SHP1, decreased phosphorylation of Syk, chymase, tryptase, IL-17A and TGF-ß1 levels. PPARß/δ siRNA and CD300a siRNA abolished the beneficial effects of GW0742. CONCLUSIONS: GW0742 inhibited mast cell-induced inflammation and improved neurobehavior after GMH, which is mediated by PPARß/δ/CD300a/SHP1 pathway. GW0742 may serve as a potential treatment to reduce brain injury for GMH patients.

Mast cells are upregulated in hidradenitis suppurativa tissue, associated with epithelialized tunnels and normalized by spleen tyrosine kinase antagonism.

Mast cells have traditionally been associated with allergic inflammatory responses; however, they play important roles in cutaneous innate immunity and wound healing. The Hidradenitis Suppurativa tissue transcriptome is associated with alterations in innate immunity and wound healing-associated pathways; however, the role of mast cells in the disease is unexplored. We demonstrate that mast cell-associated gene expression (using whole tissue RNAseq) is upregulated, and in-silico cellular deconvolution identifies activated mast cells upregulated and resting mast cells downregulated in lesional tissue. Tryptase/Chymase positive mast cells (identified using IHC) localize adjacent to epithelialized tunnels, fibrotic regions of the dermis and at perivascular sites associated with Neutrophil Extracellular Trap formation and TNF-alpha production. Treatment with Spleen Tyrosine Kinase antagonist (Fostamatinib) reduces the expression of mast cell-associated gene transcripts, associated biochemical pathways and the number of tryptase/chymase positive mast cells in lesional hidradenitis suppurativa tissue. This data indicates that although mast cells are not the most abundant cell type in Hidradenitis Suppurativa tissue, the dysregulation of mast cells is paralleled with B cell/plasma cell inflammation, inflammatory epithelialized tunnels and epithelial budding. This provides an explanation as to the mixed inflammatory activation signature seen in HS, the correlation with dysregulated wound healing and potential pathways involved in the development of epithelialized tunnels.

Characterization of novel truncated apolipoprotein A-I in human high-density lipoprotein generated by sequential treatment with myeloperoxidase and chymase.

High-density lipoprotein (HDL) cholesterol is a well-known biomarker, which has been associated with reduction in the risk of cardiovascular diseases (CVD). However, some HDL anti-atherosclerotic functions may be impaired without altered HDL-cholesterol (HDL-C) level via its dysfunctional proteins or other physiological reactions in vivo. We previously showed that activated mast cell-derived chymase could modestly cleave apolipoprotein A-I (apoA-I) in HDL3, and further easily cleave lipid-free apoA-I. In contrast, myeloperoxidase (MPO) secreted by macrophages, the main cell type in atherosclerotic plaques, could oxidize HDL proteins, which might modify their tertiary structures, increasing their susceptibility to other enzymes. Here we focused on the co-modification and impact of chymase and MPO, usually secreted during inflammation from cells with possible co-existence in atheromas, on HDL. Only after sequential treatment with MPO and then chymase, two novel truncated apoA-I fragments were generated from HDL. One fragment was 16.5 kDa, and the cleavage site by chymase after MPO modification was the C-terminal of Tyr100 in apoA-I, cross-validated by three different mass spectrometry methods. This novel apoA-I fragment can be trapped in HDL particles to avoid kidney glomerular filtration and has a specific site for antibody generation for ELISA tests. As such, its quantification can be useful in predicting patients with CVD having normal HDL-C levels.

Improved expression and purification of highly-active 3 chymotrypsin-like protease from SARS-CoV-2.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is the causative pathogen of coronavirus disease-19 (COVID-19). The COVID-19 pandemic has resulted in millions of deaths and widespread socio-economic damage worldwide. Therefore, numerous studies have been conducted to identify effective measures to control the spreading of the virus. Among various potential targets, the 3 chymotrypsin-like protease (3CLpro), also known as Mpro, stands out as the key protease of SARS-CoV-2, playing an essential role in virus replication and assembly, is the most prospective. In this study, we modified the commercial vector, pETM33-Nsp5-Mpro (plasmid # 156475, Addgene, USA), by inserting an autocleavage site (AVLQ) of 3CLpro and 6 × His-tag encoding sequences before and after the Nsp5-Mpro sequence, respectively. This modification enabled the expression of 3CLpro as an authentic N terminal protease (au3CLpro), which was purified to electrophoretic homogeneity by a single-step chromatography using two tandem Glutathione- and Ni-Sepharose columns. The enzyme au3CLpro demonstrated significantly higher activity (3169 RFU/min/µg protein) and catalytic efficiency (Kcat/Km of 0.007 µM-1.s-1) than that of the 3CLpro (com3CLpro) expressed from the commercial vector (pETM33-Nsp5-Mpro) with specific activity 889 RFU/min/µg and Kcat/Km of 0.0015 µM-1.s-1, respectively. Optimal conditions for au3CLpro activity included a 50 mM Tris-HCl buffer at pH 7, containing 150 mM NaCl and 0.1 mg/ml BSA at 37 °C.

Chymase-independent vascular Ang-(1-12)/Ang II pathway and TXA2 generation are involved in endothelial dysfunction in the murine model of heart failure.

The angiotensin (Ang)-(1-12)/Ang II pathway contributes to cardiac pathology. However, its involvement in the development of peripheral endothelial dysfunction associated with heart failure (HF) remains unknown. Therefore, this study aimed to characterise the effect of exogenous Ang-(1-12) and its conversion to Ang II on endothelial function using the murine model of HF (Tgαq*44 mice), focusing on the role of chymase and vascular-derived thromboxane A2 (TXA2). Ex vivo myographic assessments of isolated aorta showed impaired endothelium-dependent vasodilation in late-stage HF in 12-month-old Tgαq*44 mice. However, endothelium-dependent vasodilation was fully preserved in the early stage of HF in 4-month-old Tgαq*44 mice and 4- and 12-month-old FVB control mice. Ang-(1-12) impaired endothelium-dependent vasodilation in 4- and 12-month-old Tgαq*44 mice, that was associated with increased Ang II production. The chymase inhibitor chymostatin did not inhibit this response. Interestingly, TXA2 production reflected by TXB2 measurement was upregulated in response to Ang-(1-12) and Ang II in aortic rings isolated from 12-month-old Tgαq*44 mice but not from 4-month-old Tgαq*44 mice or age-matched FVB mice. Furthermore, in vivo magnetic resonance imaging showed that Ang-(1-12) impaired endothelium-dependent vasodilation in the aorta of Tgαq*44 mice and FVB mice. However, this response was inhibited by angiotensin I converting enzyme (ACE) inhibitor; perindopril, angiotensin II receptor type 1 (AT1) antagonist; losartan and TXA2 receptor (TP) antagonist-picotamide in 12-month-old-Tgαq*44 mice only. In conclusion, the chymase-independent vascular Ang-(1-12)/Ang II pathway and subsequent TXA2 overactivity contribute to systemic endothelial dysfunction in the late stage of HF in Tgαq*44 mice. Therefore, the vascular TXA2 receptor represents a pharmacotherapeutic target to improve peripheral endothelial dysfunction in chronic HF.

In Silico Screening of Natural Flavonoids against 3-Chymotrypsin-like Protease of SARS-CoV-2 Using Machine Learning and Molecular Modeling.

The "Long-COVID syndrome" has posed significant challenges due to a lack of validated therapeutic options. We developed a novel multi-step virtual screening strategy to reliably identify inhibitors against 3-chymotrypsin-like protease of SARS-CoV-2 from abundant flavonoids, which represents a promising source of antiviral and immune-boosting nutrients. We identified 57 interacting residues as contributors to the protein-ligand binding pocket. Their energy interaction profiles constituted the input features for Machine Learning (ML) models. The consensus of 25 classifiers trained using various ML algorithms attained 93.9% accuracy and a 6.4% false-positive-rate. The consensus of 10 regression models for binding energy prediction also achieved a low root-mean-square error of 1.18 kcal/mol. We screened out 120 flavonoid hits first and retained 50 drug-like hits after predefined ADMET filtering to ensure bioavailability and safety profiles. Furthermore, molecular dynamics simulations prioritized nine bioactive flavonoids as promising anti-SARS-CoV-2 agents exhibiting both high structural stability (root-mean-square deviation < 5 Å for 218 ns) and low MM/PBSA binding free energy (<-6 kcal/mol). Among them, KB-2 (PubChem-CID, 14630497) and 9-O-Methylglyceofuran (PubChem-CID, 44257401) displayed excellent binding affinity and desirable pharmacokinetic capabilities. These compounds have great potential to serve as oral nutraceuticals with therapeutic and prophylactic properties as care strategies for patients with long-COVID syndrome.

Ixodes scapularis nymph saliva protein blocks host inflammation and complement-mediated killing of Lyme disease agent, Borrelia burgdorferi.

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent.

Genetic and functional analysis of chymotrypsin-like protease (CTRL) in chronic pancreatitis.

BACKGROUND: Genetic predisposition is crucial in the pathogenesis of early-onset chronic pancreatitis (CP). So far, several genetic alterations have been identified as risk factors, predominantly in genes encoding digestive enzymes. However, many early-onset CP cases have no identified underlying cause. Chymotrypsins are a family of serine proteases that can cleave trypsinogen and lead to its degradation. Because genetic alterations in the chymotrypsins CTRC, CTRB1, and CTRB2 are associated with CP, we genetically and functionally investigated chymotrypsin-like protease (CTRL) as a potential risk factor. METHODS: We screened 1005 non-alcoholic CP patients and 1594 controls for CTRL variants by exome sequencing. We performed Western blots and activity assays to analyse secretion and proteolytic activity. We measured BiP mRNA expression to investigate the potential impact of identified alterations on endoplasmic reticulum (ER) stress. RESULTS: We identified 13 heterozygous non-synonymous CTRL variants: five exclusively in patients and three only in controls. Functionality was unchanged in 6/13 variants. Four alterations showed normal secretion but reduced (p.G20S, p.G56S, p.G61S) or abolished (p.S208F) activity. Another three variants (p.C201Y, p.G215R and p.C220G) were not secreted and already showed reduced or no activity intracellularly. However, intracellular retention did not lead to ER stress. CONCLUSION: We identified several CTRL variants, some showing potent effects on protease function and secretion. We observed these effects in variants found in patients and controls, and CTRL loss-of-function variants were not significantly more common in patients than controls. Therefore, CTRL is unlikely to play a relevant role in the development of CP.

Antcin-B, a phytosterol-like compound from Taiwanofungus camphoratus inhibits SARS-CoV-2 3-chymotrypsin-like protease (3CLPro) activity in silico and in vitro.

Despite the remarkable development of highly effective vaccines, including mRNA-based vaccines, within a limited timeframe, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not been entirely eradicated. Thus, it is crucial to identify new effective anti-3CLPro compounds, pivotal for the replication of SARS-CoV-2. Here, we identified an antcin-B phytosterol-like compound from Taiwanofungus camphoratus that targets 3CLPro activity. MTT assay and ADMET prediction are employed for assessing potential cytotoxicity. Computational molecular modeling was used to screen various antcins and non-antcins for binding affinity and interaction type with 3CLPro. Further, these compounds were subjected to study their inhibitory effects on 3CLPro activity in vitro. Our results indicate that antcin-B has the best binding affinity by contacting residues like Leu141, Asn142, Glu166, and His163 via hydrogen bond and salt bridge and significantly inhibits 3CLPro activity, surpassing the positive control compound (GC376). The 100 ns molecular dynamics simulation studies showed that antcin-B formed consistent, long-lasting water bridges with Glu166 for their inhibitory activity. In summary, antcin-B could be useful to develop therapeutically viable drugs to inhibit SARS-CoV-2 replication alone or in combination with medications specific to other SARS-CoV-2 viral targets.

Space-Flight- and Microgravity-Dependent Alteration of Mast Cell Population and Protease Expression in Digestive Organs of Mongolian Gerbils.

Mast cell (MC)-specific proteases are of particular interest for space biology and medicine due to their biological activity in regulating targets of a specific tissue microenvironment. MC tryptase and chymase obtain the ability to remodel connective tissue through direct and indirect mechanisms. Yet, MC-specific protease expression under space flight conditions has not been adequately investigated. Using immunohistochemical stainings, we analyzed in this study the protease profile of the jejunal, gastric, and hepatic MC populations in three groups of Mongolian gerbils-vivarium control, synchronous experiment, and 12-day orbital flight on the Foton-M3 spacecraft-and in two groups-vivarium control and anti-orthostatic suspension-included in the experiment simulating effects of weightlessness in the ground-based conditions. After a space flight, there was a decreased number of MCs in the studied organs combined with an increased proportion of chymase-positive MCs and MCs with a simultaneous content of tryptase and chymase; the secretion of specific proteases into the extracellular matrix increased. These changes in the expression of proteases were observed both in the mucosal and connective tissue MC subpopulations of the stomach and jejunum. Notably, the relative content of tryptase-positive MCs in the studied organs of the digestive system decreased. Space flight conditions simulated in the synchronous experiment caused no similar significant changes in the protease profile of MC populations. The space flight conditions resulted in an increased chymase expression combined with a decreased total number of protease-positive MCs, apparently due to participating in the processes of extracellular matrix remodeling and regulating the state of the cardiovascular system.

Substrate specificity of human chymotrypsin-like protease (CTRL) characterized by phage display-selected small-protein inhibitors.

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.

Two granzyme A/K homologs in Zebra mbuna have different specificities, one classical tryptase and one with chymase activity.

Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTLs) and natural killer (NK) cells. The locus encoding these two proteases is the first of the hematopoietic serine protease loci to appear during vertebrate evolution. This locus is found in all jawed vertebrates including the cartilaginous fishes. Granzyme A is the most abundant of the different granzymes expressed by CTLs and NK cells and its potential function has been studied extensively for many years. However, no clear conclusions concerning its primary role in the immune defense has been obtained. In all mammals, there are only one copy each of granzyme A and K, whereas additional copies are found in both cartilaginous and ray finned fishes. In cichlids two of these copies seem to encode new members of the granzyme A/K family. These two new members appear to have changed primary specificity and to be pure chymases based on the amino acids in their active site substrate binding pockets. Interestingly, one of these gene copies is located in the middle of the granzyme A/K locus, while the other copy is present in another locus, the met-ase locus. We here present a detailed characterization of the extended cleavage specificity of one of these non-classical granzymes, a Zebra mbuna granzyme positioned in the granzyme A/K locus. This enzyme, named granzyme A2, showed a high preference for tyrosine in the P1 position of substrates, thereby being a strict chymase. We have also characterized one of the classical granzyme A/Ks of the Zebra mbuna, granzyme A1, which is a tryptase with preference for arginine in the P1 position of substrates. Based on their extended specificities, the two granzymes showed major similarities, but also some differences in preferred amino acids in positions surrounding the cleavable amino acid. Fish lack one of the hematopoietic serine protease loci of mammals, the chymase locus, where one of the major mast cell enzymes is located. An interesting question is now if cichlids have by compensatory mechanisms generated a mast cell chymase from another locus, and if similar chymotryptic enzymes have appeared also in other fish species.

Is chymase 1 a therapeutic target in cardiovascular disease?

INTRODUCTION: Non-angiotensin converting enzyme mechanisms of angiotensin II production remain underappreciated in part due to the success of current therapies to ameliorate the impact of primary hypertension and atherosclerotic diseases of the heart and the blood vessels. This review scrutinize the current literature to highlight chymase role as a critical participant in the pathogenesis of cardiovascular disease and heart failure. AREAS COVERED: We review the contemporaneous understanding of circulating and tissue biotransformation mechanisms of the angiotensins focusing on the role of chymase as an alternate tissue generating pathway for angiotensin II pathological mechanisms of action. EXPERT OPINION: While robust literature documents the singularity of chymase as an angiotensin II-forming enzyme, particularly when angiotensin converting enzyme is inhibited, this knowledge has not been fully recognized to clinical medicine. This review discusses the limitations of clinical trials' that explored the benefits of chymase inhibition in accounting for the failure to duplicate in humans what has been demonstrated in experimental animals.

Angiotensinogen, Angiotensin-Converting Enzyme, and Chymase Gene Polymorphisms as Biomarkers for Basal Cell Carcinoma Susceptibility.

INTRODUCTION: The intake of angiotensin-converting enzyme (ACE) inhibitors and specific antagonists of angiotensin II receptors, widely used as antihypertensive drugs, significantly reduces the risk of developing basal cell carcinoma (BCC), highlighting the possible tumorigenic role of angiotensin II (AngII). We present here the investigated genetic association between the development of BCC and functional DNA polymorphisms M235T, I/D, and A1903G in the genes of angiotensinogen (AGT), angiotensin-converting enzyme (ACE), and chymase (CMA1), which mediate AngII production levels. METHODS: DNA samples of 203 unrelated Greeks were studied, including 100 patients with BCC and 103 matched healthy controls. RESULTS: The MT genotype of the AGT-M235T polymorphism was significantly more prevalent in the patient group (78.0%) versus the healthy control group (28.3%; p < 0.001). The DD genotype of the ACE-I/D polymorphism was also increased in BCC patients (72.8%) compared to controls (46.2%; p = 0.001). The heterozygous AG genotype of CMA1-A1903G was significantly more frequent in the BCC group (86%) than in the healthy controls (50.5%; p < 0.001). CONCLUSIONS: The MT, DD, and AG genotypes of the AGT- M235T, ACE-I/D, and CMA1-A1903G polymorphisms, respectively, were significantly increased in frequency within the group of cancer patients compared to the healthy controls. All three genotypes correspond to increased enzyme levels or activity and result in increased levels of AngII; therefore, they may be potentially utilized as reliable biomarkers associated with an individual's increased risk for BCC development.

Deciphering the role of fucoidan from brown macroalgae in inhibiting SARS-CoV-2 by targeting its main protease and receptor binding domain: Invitro and insilico approach.

The current study investigated the role of fucoidan from Padina tetrastromatica and Turbinaria conoides against 3-chymotrypsin like protease (3CLpro) and receptor binding domain (RBD) spike protein of SARS-CoV-2 using an invitro and computational approach. The 3CLpro and RBD genes were successfully cloned in pET28a vector, expressed in BL-21DE3 E. coli rosetta cells and purified by ion exchange affinity and size exclusion chromatography. Fucoidan extracted from both biomass using green approach, subcritical water, was found to inhibit 3CLpro of SARS-CoV-2 with an IC50 value of up to 0.35 mg mL-1. However, fucoidan was found to be inactive against the RBD protein. Molecular docking studies demonstrated that fucoidan binds to the active sites of 3CLpro with an affinity of -5.0 kcal mol-1. In addition, molecular dynamic simulations recorded stabilized interactions of protein-ligand complexes in terms of root mean square deviation, root mean square fluctuation, the radius of gyration, solvent accessible surface area and hydrogen bond interaction. The binding energy of fucoidan with 3CLpro was determined to be -101.821 ± 12.966 kJ mol-1 using Molecular Mechanic/Poisson-Bolt-Boltzmann Surface Area analysis. Fucoidan satisfies the Absorption, Distribution, Metabolism, and Excretion (ADME) properties, including Lipinski's rule of five, which play an essential role in drug design. According to the toxicity parameters, fucoidan does not exhibit skin sensitivity, hepatotoxicity, or AMES toxicity. Therefore, this work reveals that fucoidan from brown macroalgae could act as possible inhibitors in regulating the function of the 3CLpro protein, hence inhibiting viral replication and being effective against COVID-19.

Recombinant SARS-CoV-2 Spike Protein Stimulates Secretion of Chymase, Tryptase, and IL-1ß from Human Mast Cells, Augmented by IL-33.

SARS-CoV-2 infects cells via its spike (S) protein binding to its surface receptor angiotensin-converting enzyme 2 (ACE2) and results in the production of multiple proinflammatory cytokines, especially in the lungs, leading to what is known as COVID-19. However, the cell source and the mechanism of secretion of such cytokines have not been adequately characterized. In this study, we used human cultured mast cells that are plentiful in the lungs and showed that recombinant SARS-CoV-2 full-length S protein (1-10 ng/mL), but not its receptor-binding domain (RBD), stimulates the secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) as well as the proteolytic enzymes chymase and tryptase. The secretion of IL-1ß, chymase, and tryptase is augmented by the co-administration of interleukin-33 (IL-33) (30 ng/mL). This effect is mediated via toll-like receptor 4 (TLR4) for IL-1ß and via ACE2 for chymase and tryptase. These results provide evidence that the SARS-CoV-2 S protein contributes to inflammation by stimulating mast cells through different receptors and could lead to new targeted treatment approaches.