RESUMEN

Coronavirus disease 2019 (COVID-19) has led to a significant number of infections and deaths worldwide, yet its pathogenesis and severity remain incompletely understood. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2), play crucial roles as receptors and molecules responsible for the virus's entry into host cells, initiating the infection process. Their polymorphisms have been extensively studied in relation to COVID-19 severity. The aim of this study was to examine the association of ACE2 (rs2074192) and TMPRSS2 (rs12329760) polymorphisms with COVID-19 outcome and severity. A prospective cohort study was conducted in 2022 at Haji Adam Malik Hospital, Medan, Indonesia. We randomly recruited hospitalized adult patients with COVID-19, confirmed by real-time polymerase chain reaction (RT-PCR). The baseline demographic data, disease severity, underlying disease, comorbidities, and COVID-19 vaccination status were collected. The single-nucleotide polymorphism (SNP) was assessed using TaqMan SNP genotyping assay, and the levels of TMPRSS2 and ACE2 proteins were measured using enzyme-linked immunosorbent assay (ELISA). A total of 151 COVID-19 patients were recruited and there were significant associations between age and severity with mortality outcomes. The age, kidney and lung diseases, and vaccination status were associated with severity levels. The results showed the CC genotype of ACE2 had the highest proportion, followed by TT and CT genotypes among patients, while CT was the most prevalent genotype, followed by CC and TT for TMPRSS2. This study did not find a significant association between ACE2 and TMPRSS2 genetic variants with disease severity and outcome but highlighted a specific association of TMPRSS2 SNP with mortality within the group. In addition, ACE2 concentration was significant different between mild-moderate and severe-critical COVID-19 groups (p=0.033).

Asunto(s)

Enzima Convertidora de Angiotensina 2 , COVID-19 , Polimorfismo de Nucleótido Simple , Serina Endopeptidasas , Índice de Severidad de la Enfermedad , Humanos , COVID-19/genética , COVID-19/mortalidad , COVID-19/epidemiología , Serina Endopeptidasas/genética , Enzima Convertidora de Angiotensina 2/genética , Masculino , Femenino , Estudios Prospectivos , Persona de Mediana Edad , Indonesia/epidemiología , Adulto , Centros de Atención Terciaria , Anciano , SARS-CoV-2

RESUMEN

Fibrous dysplasia (FD) is a mosaic skeletal disorder involving the development of benign, expansile fibro-osseous lesions during childhood that cause deformity, fractures, pain, and disability. There are no well-established treatments for FD. Fibroblast activation protein (FAPα) is a serine protease expressed in pathological fibrotic tissues that has promising clinical applications as a biomarker and local pro-drug activator in several pathological conditions. In this study, we explored the expression of FAP in FD tissue and cells through published genetic expression datasets and measured circulating FAPα in plasma samples from patients with FD and healthy donors. We found that FAP genetic expression was increased in FD tissue and cells, and present at higher concentrations in plasma from patients with FD compared to healthy donors. Moreover, FAPα levels were correlated with skeletal disease burden in patients with FD. These findings support further investigation of FAPα as a potential imaging and/or biomarker of FD, as well as a pro-drug activator specific to FD tissue.

Asunto(s)

Endopeptidasas , Displasia Fibrosa Ósea , Gelatinasas , Proteínas de la Membrana , Serina Endopeptidasas , Humanos , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Femenino , Masculino , Endopeptidasas/metabolismo , Endopeptidasas/genética , Gelatinasas/metabolismo , Gelatinasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Displasia Fibrosa Ósea/metabolismo , Displasia Fibrosa Ósea/genética , Displasia Fibrosa Ósea/patología , Adulto , Adolescente , Niño , Biomarcadores/metabolismo , Biomarcadores/sangre , Osteoblastos/metabolismo , Osteoblastos/patología , Persona de Mediana Edad

RESUMEN

This study aimed to investigate the impact of different types of nasal inflammation on the regulation of entry-associated genes of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), and influenza virus, in the nasal epithelium. Subjects were classified into three groups: control, eosinophilic chronic rhinosinusitis (ECRS), and noneosinophilic CRS (NECRS) groups. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2), alanyl aminopeptidase (ANPEP), dipeptidyl peptidase 4 (DPP4), and beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1), and beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) were selected as key entry-associated genes for SARS-CoV-2, HCoV-229E, MERS-CoV, and influenza, respectively, and were evaluated. Brushing samples obtained from each group and human nasal epithelial cells cultured using an air-liquid interface system were treated for 7 days with typical inflammatory cytokines and analyzed using real-time polymerase chain reaction. Western blot analysis and confocal microscopy were performed. The entry-associated genes showed distinct regulation patterns in response to each interleukin-4 (IL-4), interleukin-13 (IL-13), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Specifically, ACE2 significantly decreased in type 2 cytokines (IL-4 and IL-13), while TMPRSS2 significantly decreased in type 1 cytokines (TNF-α and IFN-γ). ANPEP significantly decreased in both types of cytokines. Remarkably, DPP4 significantly increased in type 2 cytokines and decreased in type 1 cytokines. Moreover, ST6GAL1 and ST3GAL4 significantly increased in type 2 cytokines and decreased in type 1 cytokines, particularly IFN-γ. These findings were supported by western blot analysis and confocal imaging results, especially for ACE2 and DPP4. The findings regarding differential regulation suggest that patients with ECRS, primarily mediated by type 2 inflammation, may have lower susceptibility to SARS-CoV-2 and HCoV-229E infections but higher susceptibility to MERS-CoV and influenza infections.

Asunto(s)

Citocinas , Mucosa Nasal , Internalización del Virus , Humanos , Citocinas/genética , Citocinas/metabolismo , Mucosa Nasal/virología , Adulto , Masculino , Femenino , Persona de Mediana Edad , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Sinusitis/virología , Sinusitis/genética , Sinusitis/inmunología , SARS-CoV-2/inmunología , Rinitis/virología , Rinitis/genética , Rinitis/inmunología , Regulación de la Expresión Génica , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , COVID-19/inmunología , COVID-19/virología , Coronavirus Humano 229E/genética , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología

RESUMEN

BACKGROUND: Serum corin has been associated with stroke in observational studies, but the underlying causality is uncertain. This study examined the causal association between corin and stroke through Mendelian randomization study. METHODS AND RESULTS: In the Gusu cohort, serum corin was assayed at baseline, and stroke incidents were prospectively obtained during 10 years of follow-up. Single-nucleotide polymorphisms (SNPs) in CORIN were genotyped by MassArray for 2310 participants (mean age, 53 years; 39% men). Seventeen SNPs passed the Hardy-Weinberg test and were considered the potential instruments. Only 1 SNP (rs2271037) determined variability of serum corin was significantly associated with stroke even after adjusting for conventional risk factors (hazard ratio [HR], 1.36 [95% CI, 1.00-1.85]). The weighted genetic risk score generated from the SNP-corin associations was significantly associated with stroke (HR, 2.01 [95% CI, 1.15-3.51]). Using this genetic risk score as the instrument, 1-sample Mendelian randomization analysis found a significant HR of stroke per-SD higher log2-transformed corin (HR, 1.37 [95% CI, 1.07-1.76]). The inverse variance-weighted analysis based on the SNP-corin and SNP-stroke associations found that the HR of stroke pre-SD higher log2-transformed corin was 5.92 (95% CI, 2.23-15.72). The effect estimates stayed consistent regardless of an individual SNP being removed from the instruments. An almost identical effect estimate was also confirmed by multiple other 2-sample Mendelian randomization methods. CONCLUSIONS: Genetically determined variations of serum corin concentration were significantly associated with the risk of stroke in Chinese adults. Elevated serum corin may be a risk factor for stroke.

Asunto(s)

Análisis de la Aleatorización Mendeliana , Polimorfismo de Nucleótido Simple , Serina Endopeptidasas , Accidente Cerebrovascular , Humanos , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/epidemiología , Femenino , Persona de Mediana Edad , Masculino , China/epidemiología , Serina Endopeptidasas/genética , Serina Endopeptidasas/sangre , Factores de Riesgo , Estudios Prospectivos , Predisposición Genética a la Enfermedad , Pueblo Asiatico/genética , Adulto , Medición de Riesgo , Biomarcadores/sangre , Anciano , Incidencia , Pueblos del Este de Asia

RESUMEN

Early in the SARS-CoV2 pandemic, in this journal, Hou et al. (BMC Med 18:216, 2020) interpreted public genotype data, run through functional prediction tools, as suggesting that members of particular human populations carry potentially COVID-risk-increasing variants in genes ACE2 and TMPRSS2 far more often than do members of other populations. Beyond resting on predictions rather than clinical outcomes, and focusing on variants too rare to typify population members even jointly, their claim mistook a well known artifact (that large samples reveal more of a population's variants than do small samples) as if showing real and congruent population differences for the two genes, rather than lopsided population sampling in their shared source data. We explain that artifact, and contrast it with empirical findings, now ample, that other loci shape personal COVID risks far more significantly than do ACE2 and TMPRSS2-and that variation in ACE2 and TMPRSS2 per se unlikely exacerbates any net population disparity in the effects of such more risk-informative loci.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Serina Endopeptidasas , Humanos , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/genética , COVID-19/epidemiología , Predisposición Genética a la Enfermedad , SARS-CoV-2/genética , Serina Endopeptidasas/genética

RESUMEN

Introduction: Fibroblast activation protein (FAP) overexpression on cancer-associated fibroblasts (CAFs) is associated with poor prognosis and worse clinical outcomes. Selective ablation of pro-tumorgenic FAP+ stromal cells with CAR-T cells may be a new therapeutic strategy. However, the clinical use of FAP-CAR T cells is suggested to proceed with caution for occasional poor efficacy and induction of on-target off-tumor toxicity (OTOT), including lethal osteotoxicity and cachexia. Hence, more investigations and preclinical trials are required to optimize the FAP-CAR T cells and to approve their safety and efficacy. Methods: In this study, we designed second-generation CAR T cells targeting FAP with 4-1BB as a co-stimulatory molecule, and tested their cytotoxicity against FAP-positive cells (hFAP-HT1080 cells and a variety of primary CAFs) in vitro and in Cell line-derived xenograft (CDX) and a patient-derived xenograft (PDX) model. Results: Results showed that our FAP-CAR T cells were powerfully potent in killing human and murine FAP-positive tumor cells and CAFs in multiple types of tumors in BALB/c and C57BL/6 mice and in patient-derived xenografts (PDX) model. And they were proved to be biologically safe and exhibit low-level OTOT. Discussion: Taken together, the human/murine cross-reactive FAP-CAR T cells were powerfully potent in killing human and murine FAP positive tumor cells and CAFs. They were biologically safe and exhibit low-level OTOT, warranting further clinical investigation into our FAP-CAR T cells.

Asunto(s)

Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Animales , Femenino , Humanos , Ratones , Fibroblastos Asociados al Cáncer/inmunología , Fibroblastos Asociados al Cáncer/metabolismo , Línea Celular Tumoral , Reacciones Cruzadas/inmunología , Endopeptidasas , Gelatinasas/inmunología , Gelatinasas/metabolismo , Inmunoterapia Adoptiva/métodos , Inmunoterapia Adoptiva/efectos adversos , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/terapia , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development.

Asunto(s)

Proteínas no Estructurales Virales , Virus del Nilo Occidental , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Virus del Nilo Occidental/enzimología , Virus del Nilo Occidental/fisiología , Humanos , Animales , Replicación Viral , ARN Helicasas/metabolismo , ARN Helicasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Chlorocebus aethiops , Citoplasma/metabolismo , Células Vero , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Fiebre del Nilo Occidental/virología , Línea Celular , Proteasas Virales , Nucleósido-Trifosfatasa , ARN Helicasas DEAD-box

RESUMEN

Dengue virus (DENV) infection is a worldwide public health concern infecting approximately 400 million individuals and about 40,000 mortalities yearly. Despite this, no licensed or readily available antiviral medication is currently available specifically for DENV infection, and therapy is typically symptomatic. Therefore, the objective of the study was to investigate the antiviral activity of Beta vulgaris L. phytoconstituents against DENV-2 targeting NS3 protein. The antiviral activity of phytochemicals was examined through virtual ligand-based screening, antiviral inhibition and dosage response assays, western blotting analysis and MD simulations. We conducted toxicological, and pharmacokinetic analysis to assess plant-based natural compound's efficacy, safety, and non-toxic doses. Molecular docking and MD simulation results revealed that the nonstructural protein-3 (NS3) might prove as a funamental target for Betanin and Glycine Betaine against Dengue virus. Betanin and Glycine betaine were initially studied for their non-toxic doses in HeLa, CHO, and Vero cells via MTT assay. HeLa cells were transiently transfected with cloned vector pcDNA3.1/Zeo(+)/DENV-2 NS3 along with non-toxic doses (80 µM-10 µM) of selected phytochemicals. The dose-response assay illustrated downregulated expression of DENV-2 NS3 gene after administration of Betanin (IC50 = 4.35 µM) and Glycine Betaine (IC50 = 4.49 µM). Dose response analysis of Betanin (80 µM-10 µM) depicted the significant inhibition of NS3 protein expression as well. These results suggested downregulated expression of DENV-2 NS3 at mRNA and protein level portraying the DENV replication inhibition. Based on our study findings, NS3 protease is depicted as distinctive DENV-2 inhibitor target. We will channel our study further into in vitro characterization employing the mechanistic study to understand the role of host factors in anti-flavi therapeutic.

Asunto(s)

Antivirales , Betaína , Virus del Dengue , Simulación del Acoplamiento Molecular , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Humanos , Antivirales/farmacología , Células HeLa , Animales , Chlorocebus aethiops , Células Vero , Betaína/farmacología , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Betacianinas/farmacología , Células CHO , Cricetulus , Fitoquímicos/farmacología , Simulación de Dinámica Molecular , Replicación Viral/efectos de los fármacos , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Dengue/tratamiento farmacológico , Dengue/virología , Proteasas Virales

RESUMEN

OBJECTIVE: To explore the role and molecular mechanism of cancer-associated fibroblasts (CAFs) in the tumor microenvironment of gastric cancer (GC). METHODS: The expression of CAFs in GC patients was first assessed for abundance, and survival analysis was performed. Subsequently, The Cancer Genome Atlas (TCGA) data were used for differential analysis, survival analysis, and EPIC analysis, while single-cell data (GSE183904) were downloaded for differential analysis of CAFs. Clinical data pooling, univariate and multivariate Cox analysis, and immunofluorescence were carried out on clinical GC tissue samples to explore RCN3 expression within patient CAFs. Western blot and quantitative polymerase chain reaction (qPCR) were used to detect the expression of RCN3. The relationship between RCN3, PCSK6, and STAT1 was explored by chromatin immunoprecipitation (CHIP) experiments, and the effects of the genes on macrophage polarization were detected by detecting biomarkers of biological M1/M2. RESULTS: CAFs in GC were found to be significantly higher compared to the normal group. Revealing the results of TCGA differential analysis, it was observed that GC exhibited a substantial upregulation in the expression levels of RCN3. The clinical statistics indicate a positive correlation between an elevated level of RCN3 expression and the T-stage classification of tumor size. In addition, RCN3 was found to have a significant impact on the overall survival of patients with gastric cancer, acting as an independent prognostic indicator. Analysis of single-cell data showed high expression of PCSK6 in macrophages, and immunofluorescence staining of samples from GC patients showed increased expression of PCSK6 on the cell membranes of macrophages in GC tissues. The subsequent cellular experiments confirmed RCN3 protein can regulate the expression of PCSK6, and PCSK6 regulates macrophage polarization through STAT1. CONCLUSIONS: CAFs regulate macrophage polarization through the RCN3/PCSK6/STAT1 pathway in GC.

Asunto(s)

Fibroblastos Asociados al Cáncer , Macrófagos , Neoplasias Gástricas , Microambiente Tumoral , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Microambiente Tumoral/genética , Macrófagos/metabolismo , Femenino , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Regulación Neoplásica de la Expresión Génica , Masculino , Transducción de Señal , Persona de Mediana Edad , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Activación de Macrófagos/genética

RESUMEN

Celiac disease (CD) is a common autoimmune disorder in which the patients are unable to digest gluten, which is present in foods made up of wheat, barley and rye. Whilst diagnosis happens late in 80% of the cases, avoidance of such foods appears to be the common solution. Alternative management strategies are required for the patients and their families since CD is also genetically carried over. Probiotic therapeutics and the consumption of appropriate enzymes, such as prolyloligopeptidases (POPs), from gut-friendly bacteria could reduce the disease burden and provide a better lifestyle for CD patients. We have examined around 5000 gut bacterial genomes and identified nearly 4000 non-redundant putative POPs. A select set of 10 gut bacterial POP sequences were subject to three-dimensional modelling, ligand docking and molecular dynamics simulations where stable interactions were observed between the POPs and gluten peptides. Our study provides sequence and structural analysis of potential POP enzymes in gut bacterial genomes, which form a strong basis to offer probiotic solutions to CD patients. In particular, these enzymes could be lead future therapeutics for this disease.

Asunto(s)

Enfermedad Celíaca , Microbioma Gastrointestinal , Glútenes , Prolil Oligopeptidasas , Enfermedad Celíaca/genética , Enfermedad Celíaca/microbiología , Enfermedad Celíaca/tratamiento farmacológico , Humanos , Prolil Oligopeptidasas/metabolismo , Glútenes/metabolismo , Simulación de Dinámica Molecular , Simulación del Acoplamiento Molecular , Biología Computacional/métodos , Bacterias/genética , Bacterias/enzimología , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/química , Probióticos/uso terapéutico

RESUMEN

Objective:This study aims to identify the genetic etiology underlying late-onset hearing loss in two unrelated Chinese families. Methods:Detailed clinical data of recruited participants of two families were collected and analyzed using next-generation sequencing, combined with Sanger sequencing and bioinformatics tools. Results:Patients in both families manifested as down-sloping audiograms, mainly with severe mid-to-high frequency hearing loss as well as decreased speech recognition rate, both of which occurred during the second decade. Next-generation sequencing panels succeeded in identifying mutations in gene TMPRSS3, and three heterozygous mutations were screened out, among which c. 383T>C was the first reported mutation. In silico functional analysis and molecular modeling defined the five mutations as "pathogenic" or "likely pathogenic" according to official guideline. Conclusion:The novel mutation combinations in TMPRSS3 gene segregated with an exclusive auditory phenotype in the two pedigrees. Our results provided new data regarding the characteristic deafness caused by TMPRSS3 mutations during adolescent period when hearing should be closely monitored.

Asunto(s)

Pérdida Auditiva , Heterocigoto , Proteínas de la Membrana , Serina Endopeptidasas , Humanos , Edad de Inicio , Sordera/genética , Pérdida Auditiva/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de la Membrana/genética , Mutación , Proteínas de Neoplasias , Linaje , Serina Endopeptidasas/genética , Pueblos del Este de Asia/genética

RESUMEN

Serine protease 50 (PRSS50/TSP50) is highly expressed in spermatocytes. Our study investigated its role in testicular development and spermatogenesis. Initially, PRSS50 knockdown was observed to impair DNA synthesis in spermatocytes. To further explore this, we generated PRSS50 knockout ( Prss50 -/- ) mice ( Mus musculus), which exhibited abnormal spermatid nuclear compression and reduced male fertility. Furthermore, dysplastic seminiferous tubules and decreased sex hormones were observed in 4-week-old Prss50 -/- mice, accompanied by meiotic progression defects and increased apoptosis of spermatogenic cells. Mechanistic analysis indicated that PRSS50 deletion resulted in increased phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and elevated levels of MAP kinase phosphatase 3 (MKP3), a specific ERK antagonist, potentially accounting for testicular dysplasia in adolescent Prss50 -/- mice. Taken together, these findings suggest that PRSS50 plays an important role in testicular development and spermatogenesis, with the MKP3/ERK signaling pathway playing a significant role in this process.

Asunto(s)

Sistema de Señalización de MAP Quinasas , Meiosis , Ratones Noqueados , Espermatozoides , Animales , Masculino , Ratones , Meiosis/fisiología , Espermatozoides/fisiología , Espermatogénesis/fisiología , Fosfatasa 6 de Especificidad Dual/genética , Fosfatasa 6 de Especificidad Dual/metabolismo , Testículo/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo

RESUMEN

A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRR→AGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP_AGTAA mutant entry. Furthermore, we show that the EBOV GP_AGTAA mutant is cleaved into fusion-competent GP2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell. Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed.

Asunto(s)

Catepsina L , Ebolavirus , Furina , Serina Endopeptidasas , Proteínas del Envoltorio Viral , Internalización del Virus , Catepsina L/metabolismo , Catepsina L/genética , Furina/metabolismo , Furina/genética , Ebolavirus/genética , Ebolavirus/fisiología , Ebolavirus/metabolismo , Animales , Humanos , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Chlorocebus aethiops , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Proteolisis , Células Vero , Línea Celular , Endosomas/metabolismo , Endosomas/virología

RESUMEN

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with neurological sequelae including haemorrhage, thrombosis and ischaemic necrosis and encephalitis. However, the mechanism by which this occurs is unclear. Neurological disease associated with COVID-19 has been proposed to occur following direct infection of the central nervous system and/or indirectly by local or systemic immune activation. We evaluated the expression of angiotensin-converting enzyme-2 and transmembrane protease, serine 2 (TMPRSS2) in brain tissue from five healthy human donors and observed low-level expression of these proteins in cells morphologically consistent with astrocytes, neurons and choroidal ependymal cells within the frontal cortex and medulla oblongata. Primary human astrocytes, neurons, choroid plexus epithelial cells and pericytes supported productive SARS-CoV-2 infection with ancestral, Alpha, Delta and Omicron variants. Infected cells supported the full viral life cycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 can infect human brain cells, and the mechanism of viral entry warrants further investigation.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Astrocitos , COVID-19 , Plexo Coroideo , Células Epiteliales , Neuronas , Pericitos , SARS-CoV-2 , Serina Endopeptidasas , Humanos , Pericitos/virología , SARS-CoV-2/fisiología , Astrocitos/virología , Plexo Coroideo/virología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Neuronas/virología , COVID-19/virología , COVID-19/patología , Células Epiteliales/virología , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Células Cultivadas , Encéfalo/virología , Encéfalo/patología , Sistema Nervioso Central/virología

RESUMEN

Reelin (RELN) is a secreted glycoprotein essential for cerebral cortex development. In humans, recessive RELN variants cause cortical and cerebellar malformations, while heterozygous variants were associated with epilepsy, autism, and mild cortical abnormalities. However, the functional effects of RELN variants remain unknown. We identified inherited and de novo RELN missense variants in heterozygous patients with neuronal migration disorders (NMDs) as diverse as pachygyria and polymicrogyria. We investigated in culture and in the developing mouse cerebral cortex how different variants impacted RELN function. Polymicrogyria-associated variants behaved as gain-of-function, showing an enhanced ability to induce neuronal aggregation, while those linked to pachygyria behaved as loss-of-function, leading to defective neuronal aggregation/migration. The pachygyria-associated de novo heterozygous RELN variants acted as dominant-negative by preventing WT RELN secretion in culture, animal models, and patients, thereby causing dominant NMDs. We demonstrated how mutant RELN proteins in vitro and in vivo predict cortical malformation phenotypes, providing valuable insights into the pathogenesis of such disorders.

Asunto(s)

Moléculas de Adhesión Celular Neuronal , Movimiento Celular , Proteínas de la Matriz Extracelular , Mutación Missense , Proteínas del Tejido Nervioso , Proteína Reelina , Serina Endopeptidasas , Humanos , Animales , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Ratones , Femenino , Masculino , Movimiento Celular/genética , Neuronas/metabolismo , Neuronas/patología , Polimicrogiria/genética , Polimicrogiria/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Heterocigoto , Lisencefalia/genética , Lisencefalia/patología , Alelos

RESUMEN

Introduction: This study investigates the role of Fibroblast Activation Protein (FAP)-positive cancer-associated fibroblasts (FAP+CAF) in shaping the tumor immune microenvironment, focusing on its association with immune cell functionality and cytokine expression patterns. Methods: Utilizing immunohistochemistry, we observed elevated FAP+CAF density in metastatic versus primary renal cell carcinoma (RCC) tumors, with higher FAP+CAF correlating with increased T cell infiltration in RCC, a unique phenomenon illustrating the complex interplay between tumor progression, FAP+CAF density, and immune response. Results: Analysis of immune cell subsets in FAP+CAF-rich stromal areas further revealed significant correlations between FAP+ stroma and various T cell types, particularly in RCC and non-small cell lung cancer (NSCLC). This was complemented by transcriptomic analyses, expanding the range of stromal and immune cell subsets interrogated, as well as to additional tumor types. This enabled evaluating the association of these subsets with tumor infiltration, tumor vascularization and other components of the tumor microenvironment. Our comprehensive study also encompassed cytokine, angiogenesis, and inflammation gene signatures across different cancer types, revealing heterogeneous cellular composition, cytokine expressions and angiogenic profiles. Through cytokine pathway profiling, we explored the relationship between FAP+CAF density and immune cell states, uncovering potential immunosuppressive circuits that limit anti-tumor activity in tumor-resident immune cells. Conclusions: These findings underscore the complexity of tumor biology and the necessity for personalized therapeutic and patient enrichment approaches. The insights gathered from FAP+CAF prevalence, immune infiltration, and gene signatures provide valuable perspectives on tumor microenvironments, aiding in future research and clinical strategy development.

Asunto(s)

Fibroblastos Asociados al Cáncer , Inmunoterapia , Serina Endopeptidasas , Microambiente Tumoral , Microambiente Tumoral/inmunología , Humanos , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/inmunología , Inmunoterapia/métodos , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Citocinas/metabolismo , Endopeptidasas , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Gelatinasas/metabolismo , Gelatinasas/genética , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Neoplasias Renales/terapia , Neoplasias Renales/metabolismo , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/terapia , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/metabolismo , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/metabolismo

RESUMEN

Dengue virus (DENV2) is the cause of dengue disease and a worldwide health problem. DENV2 replicates in the host cell using polyproteins such as NS3 protease in conjugation with NS2B cofactor, making NS3 protease a promising antiviral drug-target. This study investigated the efficacy of 'Niloticin' against NS2B/NS3-protease. In silico and in vitro analyses were performed which included interaction of niloticin with NS2B/NS3-protease, protein stability and flexibility, mutation effect, betweenness centrality of residues and analysis of cytotoxicity, protein expression and WNV NS3-protease activity. Similar like acyclovir, niloticin forms strong H-bonds and hydrophobic interactions with residues LEU149, ASN152, LYS74, GLY148 and ALA164. The stability of the niloticin-NS2B/NS3-protease complex was found to be stable compared to the apo NS2B/NS3-protease in structural deviation, PCA, compactness and FEL analysis. The IC50 value of niloticin was 0.14 µM in BHK cells based on in vitro cytotoxicity analysis and showed significant activity at 2.5 µM in a concentration-dependent manner. Western blotting and qRT-PCR analyses showed that niloticin reduced DENV2 protein transcription in a dose-dependent manner. Besides, niloticin confirmed the inhibition of NS3-protease by the SensoLyte 440 WNV protease detection kit. These promising results suggest that niloticin could be an effective antiviral drug against DENV2 and other flaviviruses.

Asunto(s)

Antivirales , Virus del Dengue , Serina Endopeptidasas , Proteínas no Estructurales Virales , Virus del Dengue/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Animales , Dengue/tratamiento farmacológico , Dengue/virología , Humanos , Proteasas Virales

RESUMEN

Proteolytic activation of the haemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro. IAV infection in vivo resulted in a significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to wild-type pigs. Our findings support the commercial use of GE pigs to mitigate influenza A virus infection in pigs, as an alternative approach to prevent zoonotic influenza A transmissions from pigs to humans.

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Subtipo H3N2 del Virus de la Influenza A , Infecciones por Orthomyxoviridae , Serina Endopeptidasas , Enfermedades de los Porcinos , Replicación Viral , Animales , Porcinos , Infecciones por Orthomyxoviridae/virología , Infecciones por Orthomyxoviridae/prevención & control , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Subtipo H3N2 del Virus de la Influenza A/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/prevención & control , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/fisiología , Humanos , Esparcimiento de Virus , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Virus de la Influenza A/patogenicidad , Técnicas de Inactivación de Genes

RESUMEN

Iron-refractory iron deficiency anaemia (IRIDA) is a rare autosomal recessive disorder, distinguished by hypochromic microcytic anaemia, low transferrin levels and inappropriately elevated hepcidin (HEPC) levels. It is caused by mutations in TMPRSS6 gene. Systematic screening of 500 pregnant women with iron deficiency anaemia having moderate to severe microcytosis with no other causes of anaemia were enrolled to rule out oral iron refractoriness. It identified a final cohort of 10 (2.15% prevalence) individuals with IRIDA phenotype. Haematological and biochemical analysis revealed significant differences between iron responders and iron non-responders, with iron non-responders showing lower haemoglobin, red blood cell count, serum iron and serum ferritin levels, along with elevated HEPC (9.47 ± 2.75 ng/mL, p = 0.0009) and erythropoietin (4.58 ± 4.07 µ/mL, p = 0.0196) levels. Genetic sequencing of the TMPRSS6 gene in this final cohort identified 10 novel variants, including seven missense and three frame-shift mutations, with four missense variants showing high functional impact defining the IRIDA phenotype. Structural analysis revealed significant damage caused by two variants (p.L83R and p.S235R). This study provides valuable insights into IRIDA among pregnant women in the Indian subcontinent, unveiling its underlying causes of unresponsiveness, genetic mechanisms and prevalence. Furthermore, research collaboration is essential to validate these findings and develop effective treatments.

Asunto(s)

Anemia Ferropénica , Proteínas de la Membrana , Serina Endopeptidasas , Humanos , Femenino , Embarazo , Anemia Ferropénica/genética , Proteínas de la Membrana/genética , Adulto , Serina Endopeptidasas/genética , Complicaciones Hematológicas del Embarazo/genética , India/epidemiología , Fenotipo , Mutación Missense , Hierro/metabolismo , Genotipo , Mutación , Adulto Joven

RESUMEN

Staphylococcus aureus signal peptidase IB (SpsB) is an essential enzyme for protein secretion. While inhibition of its activity by small molecules is a well-precedented mechanism to kill bacteria, the mode of activation is however less understood. We here investigate the activation mechanism of a recently introduced activator, the antibiotic compound PK150, and demonstrate by combined experimental and Molecular Dynamics (MD) simulation studies a unique principle of enzyme stimulation. Mass spectrometric studies with an affinity-based probe of PK150 unravel the binding site of PK150 in SpsB which is used as a starting point for MD simulations. Our model shows the localization of the molecule in an allosteric pocket next to the active site which shields the catalytic dyad from excess water that destabilizes the catalytic geometry. This mechanism is validated by the placement of mutations aligning the binding pocket of PK150. While the mutants retain turnover of the SpsB substrate, no stimulation of activity is observed upon PK150 addition. Overall, our study elucidates a previously little investigated mechanism of enzyme activation and serves as a starting point for the development of future enzyme activators.

Asunto(s)

Proteínas Bacterianas , Simulación de Dinámica Molecular , Serina Endopeptidasas , Staphylococcus aureus , Staphylococcus aureus/enzimología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Activación Enzimática , Sitios de Unión , Antibacterianos/farmacología , Dominio Catalítico