RESUMO
Diabetes, a prevalent chronic condition, significantly increases the risk of mortality from COVID-19, yet the underlying mechanisms remain elusive. Emerging evidence implicates Cathepsin L (CTSL) in diabetic complications, including nephropathy and retinopathy. Our previous research identified CTSL as a pivotal protease promoting SARS-CoV-2 infection. Here, we demonstrate elevated blood CTSL levels in individuals with diabetes, facilitating SARS-CoV-2 infection. Chronic hyperglycemia correlates positively with CTSL concentration and activity in diabetic patients, while acute hyperglycemia augments CTSL activity in healthy individuals. In vitro studies reveal high glucose, but not insulin, promotes SARS-CoV-2 infection in wild-type cells, with CTSL knockout cells displaying reduced susceptibility. Utilizing lung tissue samples from diabetic and non-diabetic patients, alongside Leprdb/dbmice and Leprdb/+mice, we illustrate increased CTSL activity in both humans and mice under diabetic conditions. Mechanistically, high glucose levels promote CTSL maturation and translocation from the endoplasmic reticulum (ER) to the lysosome via the ER-Golgi-lysosome axis. Our findings underscore the pivotal role of hyperglycemia-induced CTSL maturation in diabetic comorbidities and complications.
People with diabetes are at greater risk of developing severe COVID-19 and dying from the illness, which is caused by a virus known as SARS-CoV-2. The high blood sugar levels associated with diabetes appear to be a contributing factor to this heightened risk. However, diabetes is a complex condition encompassing a range of metabolic disorders, and it is therefore likely that other factors may contribute. Previous research identified a link between an enzyme called cathepsin L and more severe COVID-19 in people with diabetes. Elevated cathepsin L levels are known to contribute to diabetes complications, such as kidney damage and vision loss. It has also been shown that cathepsin L helps SARS-CoV-2 to enter and infect cells. This raised the question of whether elevated cathepsin L is responsible for the increased COVID-19 vulnerability in patients with diabetes. To investigate, He, Zhao et al. monitored disease severity and cathepsin L levels in patients with COVID-19. This confirmed that people with diabetes had more severe COVID-19 and that higher levels of cathepsin L are linked to more severe disease. Analysis also revealed that cathepsin L activity increases as blood glucose levels increase. In laboratory experiments, cells exposed to glucose or fluid from the blood of people with diabetes were more easily infected with SARS-CoV-2, with cells genetically modified to lack cathepsin L being more resistant to infection. Further experiments revealed this was due to glucose promoting maturation and migration of cathepsin L in the cells. The findings of He, Zhao et al. help to explain why people with diabetes are more likely to develop severe or fatal COVID-19. Therefore, controlling blood glucose levels in people with diabetes may help to prevent or reduce the severity of the disease. Additionally, therapies targeting cathepsin L could also potentially help to treat COVID-19, especially in patients with diabetes, although more research is needed to develop and test these treatments.
Assuntos
COVID-19 , Catepsina L , Hiperglicemia , SARS-CoV-2 , COVID-19/mortalidade , COVID-19/metabolismo , Catepsina L/metabolismo , Catepsina L/genética , Humanos , Animais , Camundongos , SARS-CoV-2/genética , Masculino , Feminino , Complicações do Diabetes , Pessoa de Meia-Idade , Comorbidade , Diabetes Mellitus , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Adulto , Idoso , Complexo de Golgi/metabolismoRESUMO
MicroRNAs (miRNAs) are essential modulators of gene expression and are associated with various pathological processes, including spinal cord injury (SCI). This investigation aimed to elucidate miR-10a activity in SCI and its potential interaction with sirtuin 1 (SIRT1). The SCI rat model was established to assess hind limb movement, measure levels of miR-10a, SIRT1, neuronal survival, and inflammatory factors. An in-vitro SCI cell model was also developed to evaluate cell viability and inflammatory factor levels. The interaction between miR10a and SIRT1 was verified. Upregulated miR-10a and downregulated SIRT1 expression were found in the tissues of SCI rats. miR-10a knockdown in SCI rats enhanced the recovery of motor function, increased neuronal survival, and reduced the levels of inflammatory cytokines. Luciferase reporter assays confirmed that miR-10a targeted SIRT1 directly. In PC12 cells, downregulation of miR-10a increased SIRT1 expression, enhanced cell viability, and reduced inflammatory factor levels after LPS stimulation. Conversely, SIRT1 knockdown inhibited the protective effects of downregulated miR-10a on cell viability and inflammatory responses. The results suggest that miR-10a downregulation protects against SCI by upregulating SIRT1 expression, improving functional recovery, and reducing inflammation. Targeting the miR-10a/SIRT1 axis is a promising strategy for SCI treatment.
Assuntos
COVID-19 , MicroRNAs , Sirtuína 1 , Animais , Ratos , Sirtuína 1/metabolismo , Sirtuína 1/genética , Células PC12 , MicroRNAs/metabolismo , MicroRNAs/genética , COVID-19/metabolismo , Masculino , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/genética , Ratos Sprague-Dawley , Sobrevivência CelularRESUMO
Lung diseases affect pulmonary and respiratory function and are caused by bacterial viral and fungal infection as well as environmental factors. Unfortunately, symptom overlap between various pulmonary diseases often prevents clear differentiation and uncertain diagnosis. Accordingly, identification of specific markers of inflammatory activity in early disease stage could potential unveil the intrinsic molecular mechanisms of the underlying pathology. Proteomic studies aimed at understanding the genetic/environmental contributions to the development and progression of lung diseases represent a promising approach for diagnosis and treatment. The fluid phase of sputum represents a rich protein source and is frequently used in these studies. This chapter addresses causes of lung disorders, sputum composition, collection and processing as well as the clinical significance and challenges associated with the presence of interfering factors. Basics of proteomics and mass spectrometry are also described, together with the analytical approaches to investigate the sputum proteome. Finally, we explore the application of sputum proteomics in severe lung disorders including COVID-19 infection, chronic obstructive pulmonary disease, asthma, cystic fibrosis, lung cancer and tuberculosis.
Assuntos
COVID-19 , Pneumopatias , Proteômica , Escarro , Humanos , Escarro/química , Escarro/metabolismo , Proteômica/métodos , Pneumopatias/diagnóstico , Pneumopatias/metabolismo , COVID-19/diagnóstico , COVID-19/metabolismo , SARS-CoV-2/isolamento & purificação , Biomarcadores/análise , Biomarcadores/metabolismo , Proteoma/análiseRESUMO
To date, not many studies have presented evidence of SARS-CoV-2 infecting the female reproductive system. Furthermore, so far, no effect of the administration of anti-COVID 19 vaccines has been reported to affect the quality of oocytes retrieved from women who resorted to assisted reproduction technology (ART). The FF metabolic profiles of women who had been infected by SARS-CoV-2 before IVF treatments or after COVID-19 vaccination were examined by 1H NMR. Immunochemical characterization of proteins and cytokines involved in the redox and inflammatory pathways was performed. The increased expression of SOD2 and NQO1, the lack of alteration of IL-6 and CXCL10 levels, as well as the increased expression of CD39, suggested that, both sharing similar molecular mechanisms or proceeding along different routes, the redox balance is controlled in the FF of both vaccinated and recovered women compared to controls. The lower amount of metabolites known to have proinflammatory activity, i.e., TMAO and lipids, further supported the biochemical results, suggesting that the FF microenvironment is controlled so as to guarantee oocyte quality and does not compromise the outcome of ART. In terms of the number of blastocysts obtained after ICSI and the pregnancy rate, the results are also comforting.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Líquido Folicular , Metabolômica , Oxirredução , SARS-CoV-2 , Humanos , Feminino , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/metabolismo , Líquido Folicular/metabolismo , Adulto , Vacinas contra COVID-19/imunologia , SARS-CoV-2/imunologia , Gravidez , Metabolômica/métodos , Superóxido Dismutase/metabolismo , Inflamação/metabolismo , Citocinas/metabolismo , Vacinação , Antígenos CD/metabolismo , Metaboloma , ApiraseRESUMO
TMEM16 proteins, which function as Ca2+activated Cl channels are involved in regulating a wide variety of cellular pathways and functions. The modulators of Cl channels can be used for the moleculebased treatment of respiratory diseases, cystic fibrosis, tumors, cancer, osteoporosis and coronavirus disease 2019. The TMEM16 proteins link Ca2+ signaling, cellular electrical activity and lipid transport. Thus, deciphering these complex regulatory mechanisms may enable a more comprehensive understanding of the physiological functions of the TMEM16 proteins and assist in ascertaining the applicability of these proteins as potential pharmacological targets for the treatment of a range of diseases. The present review examined the structures, functions and characteristics of the different types of TMEM16 proteins, their association with the pathogenesis of various diseases and the applicability of TMEM16 modulatorbased treatment methods.
Assuntos
Anoctaminas , Proteínas de Transferência de Fosfolipídeos , Humanos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/metabolismo , Anoctaminas/genética , Animais , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Terapia de Alvo Molecular , Sinalização do Cálcio/efeitos dos fármacosRESUMO
SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NFκB, and unexpectedly, ENaC, the epithelial sodium channel. Post-infection inflammation may also contribute to "Long COVID"/PASC. Enhanced signaling by NFκB and ENaC also marks the airway of patients suffering from cystic fibrosis, a life-limiting proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 spike protein, and therefore activation of both NFκB and ENaC signaling. We used western blot and electrophysiological techniques, and an organoid model of normal airway epithelia, differentiated on an air-liquid-interface (ALI). We found that CFTR protein expression and CFTR cAMP-activated chloride channel activity were lost when the model epithelium was exposed to SARS-CoV-2 spike proteins. As hypothesized, the absence of CFTR led to activation of both TNFα/NFκB signaling and α and γ ENaC. We had previously shown that the cardiac glycoside drugs digoxin, digitoxin and ouabain blocked interaction of spike protein and ACE2. Consistently, addition of 30 nM concentrations of the cardiac glycoside drugs, prevented loss of both CFTR protein and CFTR channel activity. ACE2 and CFTR were found to co-immunoprecipitate in both basal cells and differentiated epithelia. Thus spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane. Thus, failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. Finally, we found that authentic SARS-CoV-2 virus infection induced loss of CFTR protein, which was rescued by the cardiac glycoside drugs digitoxin and ouabain. Based on experiments with this organoid model of small airway epithelia, and comparisons with 16HBE14o- and other cell types expressing normal CFTR, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by the SARS-CoV-2 spike protein, thus inducing a cystic fibrosis-like clinical phenotype. To our knowledge this is the first time COVID-19 airway inflammation has been experimentally traced in normal subjects to a contribution from SARS-CoV-2 spike-dependent inhibition of CFTR signaling.
Assuntos
COVID-19 , Regulador de Condutância Transmembrana em Fibrose Cística , Inflamação , SARS-CoV-2 , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , COVID-19/metabolismo , COVID-19/virologia , SARS-CoV-2/fisiologia , Inflamação/metabolismo , NF-kappa B/metabolismo , Canais Epiteliais de Sódio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ouabaína/farmacologiaRESUMO
BACKGROUND: Currently, pathophysiological mechanisms of post-acute sequelae of coronavirus disease-19-cardiovascular syndrome (PASC-CVS) remain unknown. METHODS AND RESULTS: Patients with PASC-CVS exhibited significantly higher circulating levels of severe acute respiratory syndrome-coronavirus-2 spike protein S1 than the non-PASC-CVS patients and healthy controls. Moreover, individuals with high plasma spike protein S1 concentrations exhibited elevated heart rates and normalized low frequency, suggesting cardiac ß-adrenergic receptor (ß-AR) hyperactivity. Microscale thermophoresis (MST) assay revealed that the spike protein bound to ß1- and ß2-AR, but not to D1-dopamine receptor. These interactions were blocked by ß1- and ß2-AR blockers. Molecular docking and MST assay of ß-AR mutants revealed that the spike protein interacted with the extracellular loop 2 of both ß-ARs. In cardiomyocytes, spike protein dose-dependently increased the cyclic adenosine monophosphate production with or without epinephrine, indicating its allosteric effects on ß-ARs. CONCLUSION: Severe acute respiratory syndrome-coronavirus-2 spike proteins act as an allosteric ß-AR agonist, leading to cardiac ß-AR hyperactivity, thus contributing to PASC-CVS.
Assuntos
COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , COVID-19/complicações , COVID-19/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Síndrome de COVID-19 Pós-Aguda , Idoso , Simulação de Acoplamento Molecular , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Agonistas Adrenérgicos beta/farmacologia , Agonistas Adrenérgicos beta/uso terapêuticoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired multiple mutations since its emergence. Analyses of the SARS-CoV-2 genomes from infected patients exhibit a bias toward C-to-U mutations, which are suggested to be caused by the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) cytosine deaminase proteins. However, the role of A3 enzymes in SARS-CoV-2 replication remains unclear. To address this question, we investigated the effect of A3 family proteins on SARS-CoV-2 replication in the myeloid leukemia cell line THP-1 lacking A3A to A3G genes. The Wuhan, BA.1, and BA.5 variants had comparable viral replication in parent and A3A-to-A3G-null THP-1 cells stably expressing angiotensin-converting enzyme 2 (ACE2) protein. On the other hand, the replication and infectivity of these variants were abolished in A3A-to-A3G-null THP-1-ACE2 cells in a series of passage experiments over 20 days. In contrast to previous reports, we observed no evidence of A3-induced SARS-CoV-2 mutagenesis in the passage experiments. Furthermore, our analysis of a large number of publicly available SARS-CoV-2 genomes did not reveal conclusive evidence for A3-induced mutagenesis. Our studies suggest that A3 family proteins can positively contribute to SARS-CoV-2 replication; however, this effect is deaminase-independent.
Assuntos
Desaminases APOBEC , COVID-19 , Citidina Desaminase , SARS-CoV-2 , Replicação Viral , Humanos , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , SARS-CoV-2/metabolismo , Desaminases APOBEC/metabolismo , Desaminases APOBEC/genética , COVID-19/virologia , COVID-19/metabolismo , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Células THP-1 , Mutação , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Genoma ViralRESUMO
Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.
Assuntos
Antígenos de Diferenciação de Linfócitos B , Linfócitos T CD4-Positivos , COVID-19 , Antígenos de Histocompatibilidade Classe II , Oxirredutases Intramoleculares , Ativação Linfocitária , Fatores Inibidores da Migração de Macrófagos , SARS-CoV-2 , Humanos , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Ativação Linfocitária/imunologia , SARS-CoV-2/metabolismo , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/metabolismo , COVID-19/patologia , Oxirredutases Intramoleculares/metabolismo , Oxirredutases Intramoleculares/genética , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Movimento Celular , Masculino , Feminino , Pessoa de Meia-Idade , Receptores ImunológicosRESUMO
Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.
Assuntos
Peptídeos , Proteômica , SARS-CoV-2 , Proteômica/métodos , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , Peptídeos/metabolismo , Peptídeos/química , COVID-19/metabolismo , COVID-19/virologia , Células HEK293 , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/química , Plasmídeos/genética , Plasmídeos/metabolismo , Espectrometria de Massas/métodos , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Mapeamento de Interação de Proteínas/métodosRESUMO
BACKGROUND: Patients with non-small cell lung cancer (NSCLC) are susceptible to coronavirus disease-2019 (COVID-19), but current treatments are limited. Icariside II (IS), a flavonoid compound derived from the plant epimedin, showed anti-cancer,anti-inflammation and immunoregulation effects. The present study aimed to evaluate the possible effect and underlying mechanisms of IS on NSCLC patients with COVID-19 (NSCLC/COVID-19). METHODS: NSCLC/COVID-19 targets were defined as the common targets of NSCLC (collected from The Cancer Genome Atlas database) and COVID-19 targets (collected from disease database of Genecards, OMIM, and NCBI). The correlations of NSCLC/COVID-19 targets and survival rates in patients with NSCLC were analyzed using the survival R package. Prognostic analyses were performed using univariate and multivariate Cox proportional hazards regression models. Furthermore, the targets in IS treatment of NSCLC/COVID-19 were defined as the overlapping targets of IS (predicted from drug database of TMSCP, HERBs, SwissTarget Prediction) and NSCLC/COVID-19 targets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of these treatment targets were performed aiming to understand the biological process, cellular component, molecular function and signaling pathway. The hub targets were analyzed by a protein-protein interaction network and the binding capacity with IS was characterized by molecular docking. RESULTS: The hub targets for IS in the treatment of NSCLC/COVID-19 includes F2, SELE, MMP1, MMP2, AGTR1 and AGTR2, and the molecular docking results showed that the above target proteins had a good binding degree to IS. Network pharmacology showed that IS might affect the leucocytes migration, inflammation response and active oxygen species metabolic process, as well as regulate the interleukin-17, tumor necrosus factor and hypoxia-inducible factor-1 signaling pathway in NSCLC/COVID-19. CONCLUSIONS: IS may enhance the therapeutic efficacy of current clinical anti-inflammatory and anti-cancer therapy to benefit patients with NSCLC combined with COVID-19.
Assuntos
COVID-19 , Carcinoma Pulmonar de Células não Pequenas , Flavonoides , Neoplasias Pulmonares , Simulação de Acoplamento Molecular , Farmacologia em Rede , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , COVID-19/virologia , COVID-19/metabolismo , Flavonoides/uso terapêutico , Flavonoides/química , Flavonoides/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Tratamento Farmacológico da COVID-19 , Mapas de Interação de Proteínas/efeitos dos fármacos , PrognósticoRESUMO
Virus receptors determine the tissue tropism of viruses and have a certain relationship with the clinical outcomes caused by viral infection, which is of great importance for the identification of virus receptors to understand the infection mechanism of viruses and to develop entry inhibitor. Proximity labeling (PL) is a new technique for studying protein-protein interactions, but it has not yet been applied to the identification of virus receptors or co-receptors. Here, we attempt to identify co-receptor of SARS-CoV-2 by employing TurboID-catalyzed PL. The membrane protein angiotensin-converting enzyme 2 (ACE2) was employed as a bait and conjugated to TurboID, and a A549 cell line with stable expression of ACE2-TurboID was constructed. SARS-CoV-2 pseudovirus were incubated with ACE2-TurboID stably expressed cell lines in the presence of biotin and ATP, which could initiate the catalytic activity of TurboID and tag adjacent endogenous proteins with biotin. Subsequently, the biotinylated proteins were harvested and identified by mass spectrometry. We identified a membrane protein, AXL, that has been functionally shown to mediate SARS-CoV-2 entry into host cells. Our data suggest that PL could be used to identify co-receptors for virus entry.
Assuntos
Enzima de Conversão de Angiotensina 2 , Receptores Virais , SARS-CoV-2 , Internalização do Vírus , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Células A549 , Receptores Virais/metabolismo , Receptor Tirosina Quinase Axl , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , COVID-19/virologia , COVID-19/metabolismo , Coloração e Rotulagem/métodos , Células HEK293 , Biotinilação , Mapeamento de Interação de Proteínas , Biotina/metabolismoRESUMO
Considering the high evolutionary rate and great harmfulness of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is imperative to develop new pharmacological antagonists. Human angiotensin-converting enzyme-2 (ACE2) functions as a primary receptor for the spike protein (S protein) of SARS-CoV-2. Thus, a novel functional peptide, KYPAY (K5), with a boomerang structure, was developed to inhibit the interaction between ACE2 and the S protein by attaching to the ACE2 ligand-binding domain (LBD). The inhibition property of K5 was evaluated via molecular simulations, cell experiments, and adsorption kinetics analysis. The molecular simulations showed that K5 had a high affinity for ACE2 but a low affinity for the cell membrane. The umbrella sampling (US) simulations revealed a significant enhancement in the binding potential of this functional peptide to ACE2. The fluorescence microscopy and cytotoxicity experiments showed that K5 effectively prevented the interaction between ACE2 and the S protein without causing any noticeable harm to cells. Further flow cytometry research indicated that K5 successfully hindered the interaction between ACE2 and the S protein, resulting in 78% inhibition at a concentration of 100 µM. This work offers an innovative perspective on the development of functional peptides for the prevention and therapy of SARS-CoV-2.
Assuntos
Enzima de Conversão de Angiotensina 2 , Peptídeos , Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Simulação de Dinâmica Molecular , COVID-19/virologia , COVID-19/metabolismo , Tratamento Farmacológico da COVID-19 , Antivirais/química , Antivirais/farmacologia , CinéticaRESUMO
Although SARS-CoV-2 induces mucin hypersecretion in the respiratory tract, hyposalivation/xerostomia has been reported by COVID-19 patients. We evaluate the submandibular gland (SMGs) pathogenesis in SARS-CoV-2-infected K18-hACE2 mice, focusing on the impact of infection on the mucin production and structural integrity of acini, ductal system, myoepithelial cells (MECs) and telocytes. The spike protein, the nucleocapsid protein, hACE2, actin, EGF, TNF-α and IL-1ß were detected by immunofluorescence, and the Egfr and Muc5b expression was evaluated. In the infected animals, significant acinar hypertrophy was observed in contrast to ductal atrophy. Nucleocapsid proteins and/or viral particles were detected in the SMG cells, mainly in the nuclear membrane-derived vesicles, confirming the nuclear role in the viral formation. The acinar cells showed intense TNF-α and IL-1ß immunoexpression, and the EGF-EGFR signaling increased, together with Muc5b upregulation. This finding explains mucin hypersecretion and acinar hypertrophy, which compress the ducts. Dying MECs and actin reduction were also observed, indicating failure of contraction and acinar support, favoring acinar hypertrophy. Viral assembly was found in the dying telocytes, pointing to these intercommunicating cells as viral transmitters in SMGs. Therefore, EGF-EGFR-induced mucin hypersecretion was triggered by SARS-CoV-2 in acinar cells, likely mediated by cytokines. The damage to telocytes and MECs may have favored the acinar hypertrophy, leading to ductal obstruction, explaining xerostomia in COVID-19 patients. Thus, acinar cells, telocytes and MECs may be viral targets, which favor replication and cell-to-cell viral transmission in the SMG, corroborating the high viral load in saliva of infected individuals.
Assuntos
COVID-19 , Receptores ErbB , SARS-CoV-2 , Glândula Submandibular , Xerostomia , COVID-19/patologia , COVID-19/virologia , COVID-19/metabolismo , Animais , Glândula Submandibular/virologia , Glândula Submandibular/patologia , Glândula Submandibular/metabolismo , SARS-CoV-2/fisiologia , Camundongos , Xerostomia/etiologia , Xerostomia/patologia , Xerostomia/virologia , Xerostomia/metabolismo , Receptores ErbB/metabolismo , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , Mucina-5B/metabolismo , Células Acinares/patologia , Células Acinares/metabolismo , Células Acinares/virologia , Interleucina-1beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Modelos Animais de DoençasRESUMO
BACKGROUND: In patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), momentary cortisol concentrations in blood, urine, and saliva are lower compared to healthy controls. Long-term cortisol concentration can be assessed through hair, but it is unclear whether these concentrations are also lower. Additionally, it is unknown if lower cortisol extends to other patients suffering from persistent fatigue and how hair cortisol concentration (HCC) relates to fatigue levels. Therefore, this study examines HCC in fatigued patients with ME/CFS, Q fever Fatigue Syndrome (QFS), Post-COVID-19 condition (PCC), and Juvenile Idiopathic Arthritis (JIA). METHODS: Adolescent and young adult patients with ME/CFS (n=12), QFS (n=20), PCC (n=8), JIA (n=19), and controls (n=57) were included. Patients participated in a randomized cross-over trial (RCT) targeting fatigue through lifestyle and dietary self-management strategies. HCC was measured pre-post RCT in patients and once in controls, quantified using a LC-MS/MS-based method. Fatigue severity was measured with the Checklist Individual Strength-8. HCC was compared between groups with ANOVAs. Relations between HCC, fatigue severity, and other variables were investigated using linear regression analyses. RESULTS: The ME/CFS (p=.009) and QFS (p=.047) groups had lower HCC compared to controls. Overall, HCC was negatively associated with the presence of symptoms related to chronic fatigue syndromes (e.g., sleeping issues, often feeling tired, trouble thinking clearly; ß=-0.018, p=.035), except in the QFS group (ß=.063, p<.001). Baseline HCC did not predict fatigue improvement during the RCT (p=.449), and HCC increased during the trial (Mdif=.076, p=.021) regardless of clinically relevant fatigue improvement (p=.658). CONCLUSION: Lower cortisol concentration can also be observed in the long-term. Lower HCC is not limited to ME/CFS, as it was also observed in QFS. The role of cortisol may differ between these diagnoses and appears to be unrelated to fatigue levels.
Assuntos
Síndrome de Fadiga Crônica , Cabelo , Hidrocortisona , Humanos , Síndrome de Fadiga Crônica/metabolismo , Hidrocortisona/metabolismo , Hidrocortisona/análise , Masculino , Feminino , Adolescente , Adulto Jovem , Cabelo/química , Cabelo/metabolismo , Adulto , COVID-19/metabolismo , COVID-19/complicações , Fadiga/metabolismo , Estudos Cross-Over , Artrite Juvenil/metabolismo , Artrite Juvenil/complicações , SARS-CoV-2RESUMO
The 14-3-3 family of proteins are highly conserved acidic eukaryotic proteins (25-32 kDa) abundantly present in the body. Through numerous binding partners, the 14-3-3 is responsible for many essential cellular pathways, such as cell cycle regulation and gene transcription control. Hence, its dysregulation has been linked to the onset of critical illnesses such as cancers, neurodegenerative diseases and viral infections. Interestingly, explorative studies have revealed an inverse correlation of 14-3-3 protein in cancer and neurodegenerative diseases, and the direct manipulation of 14-3-3 by virus to enhance infection capacity has dramatically extended its significance. Of these, COVID-19 has been linked to the 14-3-3 proteins by the interference of the SARS-CoV-2 nucleocapsid (N) protein during virion assembly. Given its predisposition towards multiple essential host signalling pathways, it is vital to understand the holistic interactions between the 14-3-3 protein to unravel its potential therapeutic unit in the future. As such, the general structure and properties of the 14-3-3 family of proteins, as well as their known biological functions and implications in cancer, neurodegeneration, and viruses, were covered in this review. Furthermore, the potential therapeutic target of 14-3-3 proteins in the associated diseases was discussed.
Assuntos
Proteínas 14-3-3 , COVID-19 , Neoplasias , Doenças Neurodegenerativas , Proteínas 14-3-3/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/virologia , Neoplasias/metabolismo , Neoplasias/virologia , Neoplasias/genética , COVID-19/metabolismo , COVID-19/virologia , SARS-CoV-2/metabolismo , Viroses/metabolismo , Viroses/virologia , Viroses/genéticaRESUMO
Thymocyte selection-associated high-mobility group box (TOX) is a transcription factor that is crucial for T cell exhaustion during chronic antigenic stimulation, but its role in inflammation is poorly understood. Here, we report that TOX extracellularly mediates drastic inflammation upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by binding to the cell surface receptor for advanced glycation end-products (RAGE). In various diseases, including COVID-19, TOX release was highly detectable in association with disease severity, contributing to lung fibroproliferative acute respiratory distress syndrome (ARDS). Recombinant TOX-induced blood vessel rupture, similar to a clinical signature in patients experiencing a cytokine storm, further exacerbating respiratory function impairment. In contrast, disruption of TOX function by a neutralizing antibody and genetic removal of RAGE diminished TOX-mediated deleterious effects. Altogether, our results suggest an insight into TOX function as an inflammatory mediator and propose the TOX-RAGE axis as a potential target for treating severe patients with pulmonary infection and mitigating lung fibroproliferative ARDS.
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COVID-19 , Receptor para Produtos Finais de Glicação Avançada , SARS-CoV-2 , Humanos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , COVID-19/imunologia , COVID-19/metabolismo , COVID-19/patologia , COVID-19/complicações , COVID-19/virologia , Animais , Camundongos , Inflamação/metabolismo , Inflamação/patologia , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/virologia , Lesão Pulmonar/imunologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Masculino , Pulmão/patologia , Pulmão/metabolismo , Pulmão/imunologia , FemininoRESUMO
SARS-CoV-2 infection has been recently shown to induce cellular senescence in vivo. A senescence-like phenotype has been reported in cystic fibrosis (CF) cellular models. Since the previously published data highlighted a low impact of SARS-CoV-2 on CFTR-defective cells, here we aimed to investigate the senescence hallmarks in SARS-CoV-2 infection in the context of a loss of CFTR expression/function. We infected WT and CFTR KO 16HBE14o-cells with SARS-CoV-2 and analyzed both the p21 and Ki67 expression using immunohistochemistry and viral and p21 gene expression using real-time PCR. Prior to SARS-CoV-2 infection, CFTR KO cells displayed a higher p21 and lower Ki67 expression than WT cells. We detected lipid accumulation in CFTR KO cells, identified as lipolysosomes and residual bodies at the subcellular/ultrastructure level. After SARS-CoV-2 infection, the situation reversed, with low p21 and high Ki67 expression, as well as reduced viral gene expression in CFTR KO cells. Thus, the activation of cellular senescence pathways in CFTR-defective cells was reversed by SARS-CoV-2 infection while they were activated in CFTR WT cells. These data uncover a different response of CF and non-CF bronchial epithelial cell models to SARS-CoV-2 infection and contribute to uncovering the molecular mechanisms behind the reduced clinical impact of COVID-19 in CF patients.
Assuntos
Brônquios , COVID-19 , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21 , Regulador de Condutância Transmembrana em Fibrose Cística , Células Epiteliais , Antígeno Ki-67 , SARS-CoV-2 , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Senescência Celular/genética , SARS-CoV-2/fisiologia , COVID-19/virologia , COVID-19/metabolismo , COVID-19/patologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Antígeno Ki-67/metabolismo , Brônquios/virologia , Brônquios/metabolismo , Brônquios/patologia , Brônquios/citologia , Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/virologia , Fibrose Cística/patologia , Linhagem CelularRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever recorded in human history, causing significant losses to both life and economies globally. However, the available evidence is currently insufficient to establish the effectiveness and safety of antiviral drugs or vaccines. The entry of the virus into host cells involves binding to angiotensin-converting enzyme 2 (ACE2), a cell surface receptor, via its spike protein. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a host surface protease, cleaves and activates the virus's S protein, thus promoting viral infection. Plant protease inhibitors play a crucial role in protecting plants against insects and/or microorganisms. The major storage proteins in sweet potato roots include sweet potato trypsin inhibitor (SWTI), which accounts for approximately 60% of the total water-soluble protein and has been found to possess a variety of health-promoting properties, including antioxidant, anti-inflammatory, ACE-inhibitory, and anticancer functions. Our study found that SWTI caused a significant reduction in the expression of the ACE2 and TMPRSS2 proteins, without any adverse effects on cells. Therefore, our findings suggest that the ACE2 and TMPRSS2 axis can be targeted via SWTI to potentially inhibit SARS-CoV-2 infection.
Assuntos
Enzima de Conversão de Angiotensina 2 , Antivirais , Ipomoea batatas , SARS-CoV-2 , Serina Endopeptidases , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Animais , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Ipomoea batatas/virologia , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , COVID-19/virologia , COVID-19/metabolismo , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/metabolismo , Internalização do Vírus/efeitos dos fármacos , Chlorocebus aethiops , Células Vero , Regulação para Baixo/efeitos dos fármacos , CamundongosRESUMO
Carnivorous pitcher plants from the genus Nepenthes are renowned for their ethnobotanical uses. This research explores the therapeutic potential of Nepenthes miranda leaf extract against nonstructural protein 9 (Nsp9) of SARS-CoV-2 and in treating human non-small cell lung carcinoma (NSCLC) cell lines. Nsp9, essential for SARS-CoV-2 RNA replication, was expressed and purified, and its interaction with ssDNA was assessed. Initial tests with myricetin and oridonin, known for targeting ssDNA-binding proteins and Nsp9, respectively, did not inhibit the ssDNA-binding activity of Nsp9. Subsequent screenings of various N. miranda extracts identified those using acetone, methanol, and ethanol as particularly effective in disrupting Nsp9's ssDNA-binding activity, as evidenced by electrophoretic mobility shift assays. Molecular docking studies highlighted stigmast-5-en-3-ol and lupenone, major components in the leaf extract of N. miranda, as potential inhibitors. The cytotoxic properties of N. miranda leaf extract were examined across NSCLC lines H1975, A549, and H838, focusing on cell survival, apoptosis, and migration. Results showed a dose-dependent cytotoxic effect in the following order: H1975 > A549 > H838 cells, indicating specificity. Enhanced anticancer effects were observed when the extract was combined with afatinib, suggesting synergistic interactions. Flow cytometry indicated that N. miranda leaf extract could induce G2 cell cycle arrest in H1975 cells, potentially inhibiting cancer cell proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled the tentative identification of the 19 most abundant compounds in the leaf extract of N. miranda. These outcomes underscore the dual utility of N. miranda leaf extract in potentially managing SARS-CoV-2 infection through Nsp9 inhibition and offering anticancer benefits against lung carcinoma. These results significantly broaden the potential medical applications of N. miranda leaf extract, suggesting its use not only in traditional remedies but also as a prospective treatment for pulmonary diseases. Overall, our findings position the leaf extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and antiviral therapies, warranting further investigation into its molecular mechanisms and potential clinical applications.