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The stratum corneum (SC), the outermost epidermal layer, plays a pivotal role in skin barrier function. This review delves into the intricate process of protein degradation within the stratum corneum, elucidating the roles of specific enzymes, regulatory mechanisms and the consequent impact on various skin conditions. Protein degradation is a finely tuned process, orchestrated by a suite of proteolytic enzymes like kallikreins. These enzymes are responsible for the breakdown of corneodesmosomes and the orderly desquamation of corneocytes, a process essential for skin homeostasis. Another critical enzymatic process is the breakdown of proteins like filaggrin and the generation of amino acids and their derivatives, required in the physiological water-handling properties of the SC. Regulation of these proteolytic activities is complex, involving a balance between endogenous inhibitors and other factors like pH, hydration and environmental stressors. Dysregulation of protease activity is linked to a spectrum of skin conditions, ranging from xerosis to inflammatory diseases like atopic dermatitis and psoriasis. Aberrant protein degradation can lead to compromised skin barrier function, increased tissue water loss and heightened susceptibility to infections and allergens. Understanding the factors affecting protein degradation can inform the development of targeted skincare products. Advances in biochemistry and dermatology have paved the way for the search for active ingredients designed to modulate protease activity. Such innovations may offer promising therapeutic avenues for enhancing skin barrier function and treating skin disorders. This review underscores the significance of enzymatic protein degradation in the SC and its regulatory mechanisms. It provides insights into the pathophysiology of skin diseases and outlines the potential for novel skincare interventions. By bridging the gap between fundamental research and practical applications, this article aims to inspire further investigation for better understanding of skin physiology and innovation in the realm of skincare product development.
La couche cornée (stratum corneum, SC), la couche épidermique la plus externe, joue un rôle essentiel dans la fonction de barrière cutanée. Cette revue se penche sur le processus complexe de dégradation des protéines au sein de la couche cornée, ce qui explique les rôles des enzymes spécifiques, les mécanismes de régulation et l'impact qui en résulte sur diverses affections cutanées. La dégradation des protéines est un processus subtil, orchestré par une série d'enzymes protéolytiques telles que les kallikréines. Ces enzymes sont responsables de la décomposition des cornéodesmosomes et de la desquamation ordonnée des cornéocytes, un processus essentiel à l'homéostasie de la peau. Un autre processus enzymatique essentiel est la dégradation des protéines telles que la filaggrine et la génération d'acides aminés et de leurs dérivés, nécessaires aux propriétés physiologiques de traitement de l'eau de la SC. La régulation de ces activités protéolytiques est complexe, impliquant un équilibre entre les inhibiteurs endogènes et d'autres facteurs tels que le pH, l'hydratation et les facteurs de stress environnementaux. Le dérèglement de l'activité de la protéase est lié à un spectre d'affections cutanées, allant de la xérose à des maladies inflammatoires telles que la dermatite atopique et le psoriasis. Une dégradation aberrante des protéines peut compromettre la fonction de barrière cutanée, augmenter la perte d'eau tissulaire et augmenter la sensibilité aux infections et aux allergènes. Comprendre les facteurs affectant la dégradation des protéines peut contribuer au développement de produits de soins de la peau ciblés. Les progrès en biochimie et en dermatologie ont ouvert la voie à la recherche de principes actifs conçus pour moduler l'activité de la protéase. Ces innovations peuvent offrir des pistes thérapeutiques prometteuses pour améliorer la fonction de la barrière cutanée et traiter les troubles cutanés. Cette revue souligne l'importance de la dégradation enzymatique des protéines dans la SC et ses mécanismes de régulation. Elle fournit des informations sur la physiopathologie des maladies cutanées et souligne le potentiel de nouvelles interventions pour soins de la peau. En comblant le fossé entre la recherche fondamentale et les applications pratiques, cet article vise à inspirer des recherches supplémentaires pour mieux comprendre la physiologie de la peau et l'innovation dans le domaine du développement de produits de soins de la peau.
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Epiderme , Proteínas Filagrinas , Humanos , Epiderme/metabolismo , Proteólise , Pele/metabolismo , Dermatopatias/metabolismoRESUMO
Linseed, also known as flax is an important oilseed crop with many potential uses in paint, textile, food and pharmaceutical industries. Susceptibility to bud fly (Dasyneura lini Barnes) infestation is a serious biotic concern leading to severe yield penalty in linseed. Protease inhibitors (PIs) are potential candidates that activate during the insect-pest attack and modulate the resistance. In the present study, we explored the PI candidates in the linseed genome and a total of 100 LuPI genes were identified and grouped into five distinct subgroups. The analysis of cis-acting elements revealed that almost all LuPI promoters contain several regulatory elementary related to growth and development, hormonal regulation and stress responses. Across the subfamilies of PIs, the specific domains are consistently found conserved in all protein sequences. The tissue-specific in-silico expression pattern via RNA-seq revealed that all the genes were regulated during different stress. The expression through qRT-PCR of 15 genes revealed the significant up-regulation of LuPI-24, LuPI-40, LuPI-49, LuPI-53, and LuPI-63 upon bud fly infestation in resistant genotype EC0099001 and resistant check variety Neela. This study establishes a foundation resource for comprehending the structural, functional, and evolutionary dimensions of protease inhibitors in linseed.
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Dípteros , Linho , Regulação da Expressão Gênica de Plantas , Inibidores de Proteases , Linho/genética , Linho/metabolismo , Animais , Dípteros/genética , Inibidores de Proteases/farmacologia , Inibidores de Proteases/metabolismo , Mapas de Interação de Proteínas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Doenças das Plantas/parasitologia , Doenças das Plantas/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Família Multigênica , FilogeniaRESUMO
Introduction: Galactose-deficient IgA1 (GdIgA1) is critical in the formation of immunodeposits in IgA nephropathy (IgAN), whereas the origin of GdIgA1 is unknown. We focused on the immune response to fecal microbiota in patients with IgAN. Methods: By running 16S ribosomal RNA gene sequencing, we compared IgAN samples to the control samples from household-matched or non-related individuals. Levels of plasma GdIgA1 and poly-IgA complexes were measured, and candidate microbes that can either incite IgA-directed antibody response or degrade IgA through specific IgA protease activities were identified. Results: The IgAN group showed a distinct composition of fecal microbiota as compared to healthy controls. Particularly, high abundance of Escherichia-Shigella was associated with the disease group based on analyses using receiver operating characteristic (area under curve, 0.837; 95% CI, 0.738-0.914), principle coordinates, and the linear discriminant analysis effect size algorithm (linear discriminant analysis score, 4.56; p < 0.001). Accordingly, the bacterial levels directly correlated with high titers of plasma GdIgA1(r = 0.36, p < 0.001), and patients had higher IgA1 against stx2(2.88 ± 0.46 IU/mL vs. 1.34 ± 0.35 IU/mL, p = 0.03), the main antigen of Escherichia-Shigella. Conversely, the healthy controls showed relatively higher abundance of the commensal bacteria that produce IgA-degrading proteases. Particularly, the abundance of some intestinal bacteria expressing IgA proteases showed an inverse correlation with the levels of plasma GdIgA1 in IgAN. Conclusion: Our data suggest that mucosal IgA production, including those of GdIgA1, is potentially linked to the humoral response to gut Escherichia-Shigella as one of the sources of plasma GdIgA1. Conversely, the IgA protease-producing microbiota in the gut are suppressed in patients with IgAN.
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Galactose , Microbioma Gastrointestinal , Glomerulonefrite por IGA , Imunidade Humoral , Imunoglobulina A , Glomerulonefrite por IGA/imunologia , Glomerulonefrite por IGA/microbiologia , Humanos , Microbioma Gastrointestinal/imunologia , Imunoglobulina A/imunologia , Imunoglobulina A/sangue , Masculino , Feminino , Adulto , Fezes/microbiologia , Pessoa de Meia-Idade , RNA Ribossômico 16S/genéticaRESUMO
Recurrent epidemics of coronaviruses have posed significant threats to human life and health. The mortality rate of patients infected with the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is 35 %. The main protease (Mpro) plays a crucial role in the MERS-CoV life cycle, and Mpro exhibited a high degree of conservation among different coronaviruses. Therefore inhibition of Mpro has become an effective strategy for the development of broad-spectrum anti-coronaviral drugs. The inhibition of SARS-CoV-2 Mpro by the anti-tumor drug carmofur has been revealed, but structural studies of carmofur in complex with Mpro from other types of coronavirus have not been reported. Hence, we revealed the structure of the MERS-CoV Mpro-carmofur complex, analysed the structural basis for the binding of carmofur to MERS-CoV Mpro in detail, and compared the binding patterns of carmofur to Mpros of two different coronaviruses, MERS-CoV and SARS-CoV-2. Considering the importance of Mpros for coronavirus therapy, structural understanding of Mpro inhibition by carmofur could contribute to the design and development of novel antiviral drugs with safe and broad-spectrum efficacy.
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Pseudomonas aeruginosa has become a top-priority pathogen in the health sector because it is ubiquitous, has high metabolic/genetic versatility, and is identified as an opportunistic pathogen. The production of numerous virulence factors by P. aeruginosa was reported to act individually or cooperatively to make them robots invasion, adherences, persistence, proliferation, and protection against host immune systems. P. aeruginosa produces various kinds of extracellular proteases such as alkaline protease, protease IV, elastase A, elastase B, large protease A, Pseudomonas small protease, P. aeruginosa aminopeptidase, and MucD. These proteases effectively allow the cells to invade and destroy host cells. Thus, inhibiting these protease activities has been recognized as a promising approach to controlling the infection caused by P. aeruginosa. The present review discussed in detail the characteristics of these proteases and their role in infection to the host system. The second part of the review discussed the recent updates on the multiple strategies for attenuating or inhibiting protease activity. These strategies include the application of natural and synthetic molecules, as well as metallic/polymeric nanomaterials. It has also been reported that a propeptide present in the middle domain of protease IV also attenuates the virulence properties and infection ability of P. aeruginosa.
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Since coronavirus disease infection-19 (COVID-19) entry to the cells is angiotensin enzyme receptor (ACEII) dependent, extrapulmonary manifestations have been suspected. Ocular manifestations reported in several studies to involve the anterior as well as posterior eye segments. However, the predominance of the anterior eye segment reduced the attention of the scientific community on the posterior eye segment. Our results showed that the incidence of changes in the posterior eye segment is 1/5 of the anterior eye segment. Posterior eye segment manifestations include acute macular neuroretinopathy and paracentral middle maculopathy, central retinal vein/artery occlusion, reactivation of previous uveitis, varicella zoster virus-related acute retinal necrosis in an immunocompromised patient, chorioretinitis, macular hemorrhage, paracentral acute middle maculopathy, retinal detachment, and vitritis with outer retinal abnormalities. The pathogenesis of posterior eye segment manifestations under COVID-19 includes viremia, autoimmune vasculitis, hyperimmune response, coagulopathy, and cytokine storm. A full ophthalmological examination is crucial for patients recovering from COVID-19. The paper provided up-to-date manifestations with potential underlying pathophysiological mechanisms of development, as well as pathogenetic therapy.
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Cancer cells depend on specific oncogenic pathways or present a genetic alteration that leads to a particular disturbance. Still, personalized and targeted biological therapy remains challenging, with current efforts generally yielding disappointing results. Carefully assessing onco-target molecular pathways can, however, potently assist with such efforts for the selection of patient populations that would best respond to a given drug treatment. RNF43, an E3 ubiquitin ligase that negatively regulates Wnt/frizzled (FZD) receptors by their ubiquitination, internalization, and degradation, controls a key pathway in cancer. Recently, additional target proteins of RNF43 were described, including p85 of the PI3K/AKT/mTOR signaling pathway and protease-activated receptor 2 (PAR2), a G-protein-coupled receptor that potently induces ß-catenin stabilization, independent of Wnts. RNF43 mutations with impaired E3 ligase activity were found in several types of cancers (e.g., gastrointestinal system tumors and endometrial and ovarian cancer), pointing to a high dependency on FZD receptors and possibly PAR2 and the PI3K/AKT/mTOR signaling pathway. The development of drugs toward these targets is essential for improved treatment of cancer patients.
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Neoplasias , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Transdução de Sinais/efeitos dos fármacos , Terapia de Alvo Molecular , Animais , Serina-Treonina Quinases TOR/metabolismo , Ubiquitinação , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Frizzled/metabolismo , Receptores Frizzled/genética , Receptor PAR-2/metabolismo , Receptor PAR-2/genética , Mutação , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Thrombotic thrombocytopenic purpura (TTP) is a life-threatening, often immune-mediated disease that affects 2-13 persons per million per year. Hemolytic anemia, thrombocytopenia, and end-organ damage due to the formation of microthrombi are characteristic of TTP. ADAMTS13 is a disintegrin, metalloproteinase, cleaving protein of von Willebrand factor (VWF) that processes the VWF multimers to prevent them from interacting with platelets and, in turn, to microvascular thrombosis. Prompt diagnosis of TTP is critical yet challenging. Thrombotic microangiopathies have similar clinical presentation. Measurement of ADAMTS13 activity helps in the differential diagnosis. Less than 10% ADAMTS13 activity is indicative of TTP. Laboratory ADAMTS13 activity assays include incubating the test plasma with the substrate (full-length VWM multimers) and detection with direct or indirect measurement of the cleavage product. The purpose of this study is to examine the diagnostic potential, advantages, and weaknesses of the ADAMTS13 potency in TTP.
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Proteína ADAMTS13 , Púrpura Trombocitopênica Trombótica , Proteína ADAMTS13/metabolismo , Humanos , Púrpura Trombocitopênica Trombótica/diagnóstico , Púrpura Trombocitopênica Trombótica/sangue , Fator de von Willebrand/metabolismoRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in compromised hosts. P. aeruginosa infections are difficult to treat because of the inherent ability of the bacteria to develop antibiotic resistance, secrete a variety of virulence factors, and form biofilms. The secreted aminopeptidase (PaAP) is an emerging virulence factor, key in providing essential low molecular weight nutrients and a cardinal modulator of biofilm development. PaAP is therefore a new potential target for therapy of P. aeruginosa infections. The present review summarizes the current knowledge of PaAP, with special emphasis on its biochemical and enzymatic properties, activation mechanism, biological roles, regulation, and structure. Recently developed specific inhibitors and their potential as adjuncts in the treatment of P. aeruginosa infections are also described.
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Aminopeptidases , Pseudomonas aeruginosa , Fatores de Virulência , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/metabolismo , Aminopeptidases/metabolismo , Humanos , Fatores de Virulência/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/tratamento farmacológico , AnimaisRESUMO
BACKGROUND AND AIMS: Inflammatory cells within atherosclerotic lesions secrete proteolytic enzymes that contribute to lesion progression and destabilization, increasing the risk for an acute cardiovascular event. Elastase is a serine protease, secreted by macrophages and neutrophils, that may contribute to the development of unstable plaque. We previously reported interaction of endogenous protease-inhibitor proteins with high-density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as a modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression. METHODS: We designed an HDL-targeting protease inhibitor (HTPI) that binds to HDL and confers elastase inhibitor activity. Lipoprotein binding and the impact of HTPI on atherosclerosis were examined using mouse models. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features. RESULTS: HTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with human plasma ex vivo, HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in its binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after administration to Apoe-/- mice. To examine the effect of HTPI treatment on atherosclerosis, prevention and progression studies were performed using Ldlr-/- mice fed Western diet. In both study designs, HTPI-treated mice had reduced lipid deposition in plaque. CONCLUSIONS: These data support the hypothesis that HDL-associated anti-elastase activity can improve the atheroprotective potential of HDL and highlight the potential utility of HDL enrichment with anti-protease activity as an approach for stabilization of atherosclerotic lesions.
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Cathepsin L (CTSL), a cysteine cathepsin protease of the papain superfamily, plays a crucial role in cancer progression and metastasis. Dysregulation of CTSL is frequently observed in tumor malignancies, leading to the degradation of extracellular matrix and facilitating epithelial-mesenchymal transition (EMT), a key process in malignant cancer metastasis. This review mainly provides a comprehensive information about recent findings on natural inhibitors targeting CTSL and their anticancer effects, which have emerged as potent anticancer therapeutic agents or metastasis-suppressive adjuvants. Specifically, inhibitors are categorized into small-molecule and macromolecule inhibitors, with a particular emphasis on cathepsin propeptide-type macromolecules. Additionally, the article explores the molecular mechanisms of CTSL involvement in cancer metastasis, highlighting its regulation at transcriptional, translational, post-translational, and epigenetic levels. This work underscores the importance of understanding natural CTSL inhibitors and provides researchers with practical insights to advance the relevant fields and discover novel CTSL-targeting inhibitors from natural sources.
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Overexpression of aspartic proteases, as cathepsin D, is an independent marker of poor prognosis in breast cancer, correlated with the incidence of clinical metastasis. We aimed to find if HIV-1 aspartic protease (PR) can play a similar role. Murine adenocarcinoma 4T1luc2 cells were transduced with lentivirus encoding inactivated drug-resistant PR, generating subclones PR20.1 and PR20.2. Subclones were assessed for production of reactive oxygen species (ROS), expression of epithelial-mesenchymal transition (EMT) factors, and in vitro migratory activity in the presence or absence of antioxidant N-acetyl cysteine and protease inhibitors. Tumorigenic activity was evaluated by implanting cells into BALB/c mice and following tumor growth by calipering and bioluminescence imaging in vivo, and metastases, by organ imaging ex vivo. Both subclones expressed PR mRNA, and PR20.2, also the protein detected by Western blotting. PR did not induce production of ROS, and had no direct effect on cell migration rate, however, treatment with inhibitors of drug-resistant PR suppressed the migratory activity of both subclones. Furthermore, expression of N-cadherin and Vimentin in PR20.2 cells and their migration were enhanced by antioxidant treatment. Sensitivity of in vitro migration to protease inhibitors and to antioxidant, known to restore PR activity, related the effects to the enzymatic activity of PR. In vivo, PR20.2 cells demonstrated higher tumorigenic and metastatic activity than PR20.1 or parental cells. Thus, HIV-1 protease expressed in breast cancer cells determines their migration in vitro and metastatic activity in vivo. This effect may aggravate clinical course of cancers in people living with HIV-1.
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Azurin is a small periplasmic blue copper protein found in bacterial strains such as Pseudomonas and Alcaligenes where it facilitates denitrification. Azurin is extensively studied for its ability to mediate electron-transfer processes, but it has also sparked interest of the pharmaceutical community as a potential antimicrobial or anticancer agent. Here we offer a novel approach for expression and single-step purification of azurin in Escherichia coli with high yields and optimal metalation. A fusion tag strategy using an N-terminal GST tag was employed to obtain pure protein without requiring any additional purification steps. After the on-column cleavage by HRV 3C Protease, azurin is collected and additionally incubated with copper sulphate to ensure sufficient metalation. UV-VIS absorption, mass spectroscopy, and circular dichroism analysis all validated the effective production of azurin, appropriate protein folding and the development of an active site with an associated cofactor. MD simulations verified that incorporation of the N-terminal GPLGS segment does not affect azurin structure. In addition, the biological activity of azurin was tested in HeLa cells.
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The assembly of two monomeric constructs spanning segments 1-199 (MPro1-199) and 10-306 (MPro10-306) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by â¼7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro1-199 and MPro10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201-214 onto a second helical domain of MPro10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at â¼6 µM MPro1-199. Catalytic activity arises primarily from MPro1-199 and is dependent on the interface interactions involving the N-finger residues 1-9 of MPro1-199 and E290 of MPro10-306. Importantly, our results confirm that a single N-finger region with its associated inter-subunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.
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Macroautophagy/autophagy plays a crucial role in inhibiting viral replication and regulating the host's immune response. The autophagy receptor SQSTM1/p62 (sequestosome 1) restricts viral replication by directing specific viral proteins to phagophores for degradation. In this study, we investigate the reciprocal relationship between Zika virus (ZIKV) and selective autophagy mediated by SQSTM1/p62. We show that NS2B3 protease encoded by ZIKV cleaves human SQSTM1/p62 at arginine 265 (R265). This cleavage also occurs with endogenous SQSTM1 in ZIKV-infected cells. Furthermore, overexpression of SQSTM1 inhibits ZIKV replication in A549 cells, while its absence increases viral titer. We have also shown that SQSTM1 impedes ZIKV replication by interacting with NS3 and NS5 and directing them to autophagic degradation, and that NS2B3-mediated cleavage could potentially alter this antiviral function of SQSTM1. Taken together, our study highlights the role of SQSTM1-mediated selective autophagy in the host's antiviral defense against ZIKV and uncovers potential viral evasion strategies that exploit the host's autophagic machinery to ensure successful infection.
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A new thermophilic strain, designated as Bacillus sp. LMB3902, was isolated from Hammam Debagh, the hottest spring in Algeria (up to 98 °C). This isolate showed high protease production in skim milk media at 55 °C and exhibited significant specific protease activity by using azocasein as a substrate (157.50 U/mg). Through conventional methods, chemotaxonomic characteristics, 16S rRNA gene sequencing, and comparative genomic analysis with the closely related strain Bacillus licheniformis DSM 13 (ATCC 14580 T), the isolate Bacillus sp. LMB3902 was identified as a potentially new strain of Bacillus licheniformis. In addition, the gene functions of Bacillus sp. LMB3902 strain were predicted using the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups, Non-Redundant Protein Sequence Database, Swiss-Prot, and Pfam databases. The results showed that the genome size of Bacillus sp. LMB3902 was 4.279.557 bp, with an average GC content of 46%. The genome contained 4.760 predicted genes, including 8 rRNAs, 78 tRNAs, and 24 sRNAs. A total of 235 protease genes were annotated including 50 proteases with transmembrane helix structures and eight secreted proteases with signal peptides. Additionally, the majority of secondary metabolites found by antiSMASH platform showed low similarity to identified natural products, such as fengicin (53%), lichenysin (57%), and surfactin (34%), suggesting that this strain may encode for novel uncharacterized natural products which can be useful for biotechnological applications. This study is the first report that describes the complete genome sequence, taxono-genomics, and gene annotation as well as protease production of the Bacillus genus in this hydrothermal vent.
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Reports have described SARS-CoV-2 rebound in COVID-19 patients treated with nirmatrelvir, a 3CL protease inhibitor. The cause remains a mystery, although drug resistance, re-infection, and lack of adequate immune responses have been excluded. We now present virologic findings that provide a clue to the cause of viral rebound, which occurs in â¼20% of the treated cases. Persistence of infectious SARS-CoV-2 was experimentally documented in vitro after treatment with nirmatrelvir or another 3CL protease inhibitor, but not with a polymerase inhibitor, remdesivir. This infectious form decayed slowly with a half-life of â¼1 day, suggesting that its persistence could outlive the treatment course to re-ignite SARS-CoV-2 infection as the drug is eliminated. Notably, extending nirmatrelvir treatment beyond 8 days abolished viral rebound in vitro. Our findings point in a particular direction for future investigation of virus persistence and offer a specific treatment recommendation that should be tested clinically.
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Two series of macrocyclic inhibitors addressing the S1 pocket and the prime site of the fibrinolytic serine protease plasmin have been developed. In the first series the P1 tranexamoyl residue was coupled to 4-aminophenylalanine in P1' position, which provided moderately potent inhibitors with inhibition constants around 1 µM. In the second series, a substituted biphenylalanine was incorporated as P1' residue leading to approximately 1000-fold stronger plasmin inhibitors, the best compounds possess subnanomolar inhibition constants. The most effective compounds already exhibit a certain selectivity as plasmin inhibitors compared to other trypsin-like serine proteases such as trypsin, plasma kallikrein, thrombin, activated protein Ca, as well as factors XIa and Xa. For inhibitor 28 of the second series, the co-crystal structure in complex with a Ser195Ala microplasmin mutant revealed the P2' residue adopts multiple conformations. Most polar contacts to plasmin and surrounding water molecules are mediated through the P1 tranexamoyl residue, whereas the bound conformation of the macrocycle is mainly stabilized by two intramolecular hydrogen bonds.
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A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.
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The presence of infective larvae (L3) of gastrointestinal nematode (GIN) parasites in pastures directly contributes to the constant recurrence of infections in ruminant herds. This study aimed to evaluate the nematophagous fungus Duddingtonia flagrans (AC001) (proteolytic crude extract and/or conidia) in the in vitro control of GIN L3 in coprocultures. To produce the proteolytic crude extract, a suspension (107 conidia/mL) of D. flagrans was inoculated into a liquid medium. After 6 days, the medium was filtered, centrifuged, and its proteolytic activity was measured. For the experimental assay, fecal samples were collected directly from the rectal ampulla of naturally infected sheep, and egg counts per gram of feces (EPG) were performed. Coprocultures were prepared using 10 g of fecal material with the groups defined as follows: control group G1 (1.0 mL of denatured proteolytic crude extract); treated group G2 (1.0 mL of active proteolytic crude extract); treated group G3 (1.0 mL of active proteolytic crude extract + 1.0 mL of AC001 conidia). The coprocultures were maintained at room temperature (25ºC), for 7 days, and then the L3 larvae were recovered. The results demonstrated that AC001 successfully produced protease (56.34 U/mL). The treatments with active proteolytic crude extract (G2) and active proteolytic crude extract + AC001 conidia (G3) were significantly different (p < 0.01) from the control group with denatured proteolytic crude extract (G1). AC001 and its proteolytic crude extract acted concomitantly on helminths directly in the fecal environment, suggesting potential future applications in the field.