RESUMO
Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis PYCR gene and proline degradation PRODH gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of PYCR expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.
Assuntos
Transformação Celular Neoplásica/patologia , Herpesvirus Humano 8/metabolismo , Prolina/metabolismo , Pirrolina Carboxilato Redutases/metabolismo , Sarcoma de Kaposi/patologia , Proteínas Virais/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Metabolômica , Camundongos , Prolina Oxidase/metabolismo , Sarcoma de Kaposi/virologia , Esferoides Celulares , Ensaios Antitumorais Modelo de Xenoenxerto , delta-1-Pirrolina-5-Carboxilato RedutaseRESUMO
The presentation of antigenic peptides by major histocompatibility complex (MHC) class I and class II molecules is crucial for activation of the adaptive immune system. The nucleotide-binding domain and leucine-rich repeat receptor family members CIITA and NLRC5 function as the major transcriptional activators of MHC class II and class I gene expression, respectively. Since the identification of NLRC5 as the master regulator of MHC class I and class-I-related genes, there have been major advances in understanding the function of NLRC5 in infectious diseases and cancer. Here, we discuss the biological significance and mechanism of NLRC5-dependent MHC class I expression.
Assuntos
Imunidade Adaptativa , Antígenos de Histocompatibilidade Classe I/metabolismo , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/metabolismo , Transativadores/metabolismo , Animais , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Inflamassomos/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/genética , Neoplasias/imunologia , Transdução de Sinais , Evasão Tumoral , Microambiente TumoralRESUMO
RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).
Assuntos
RNA Helicases DEAD-box/imunologia , Infecções por Vírus de RNA/imunologia , Transdução de Sinais/imunologia , eIF-2 Quinase/imunologia , Grânulos Citoplasmáticos/imunologia , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Vírus de RNA/imunologia , RNA de Cadeia Dupla/imunologia , RNA Interferente Pequeno/genética , RNA Viral/imunologia , Receptores Imunológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico , TransfecçãoRESUMO
In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses.
Assuntos
Vírus da Encefalomiocardite/imunologia , Vírus da Encefalomiocardite/patogenicidade , Ribonucleoproteínas/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citocinas/genética , DNA Helicases , Vírus de DNA/patogenicidade , Vírus da Encefalomiocardite/fisiologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Interferons/genética , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Vírus de RNA/patogenicidade , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/imunologia , Estresse Fisiológico , Replicação ViralRESUMO
Cellular stress responses are crucial for maintaining cellular homeostasis. Stress granules (SGs), activated by eIF2α kinases in response to various stimuli, play a pivotal role in dealing with diverse stress conditions. Viral infection, as one kind of cellular stress, triggers specific cellular programs aimed at overcoming virus-induced stresses. Recent studies have revealed that virus-derived stress responses are tightly linked to the host's antiviral innate immunity. Virus infection-induced SGs act as platforms for antiviral sensors, facilitating the initiation of protective antiviral responses called "antiviral stress granules" (avSGs). However, many viruses, including coronaviruses, have evolved strategies to suppress avSG formation, thereby counteracting the host's immune responses. This review discusses the intricate relationship between cellular stress responses and antiviral innate immunity, with a specific focus on coronaviruses. Furthermore, the diverse mechanisms employed by viruses to counteract avSGs are described.
Assuntos
Infecções por Coronavirus , Coronavirus , Viroses , Humanos , Imunidade Inata , AntiviraisRESUMO
In the context of aging, the susceptibility to infectious diseases increases, leading to heightened morbidity and mortality. This phenomenon, termed immunosenescence, is characterized by dysregulation in the aging immune system, including abnormal alterations in lymphocyte composition, elevated basal inflammation, and the accumulation of senescent T cells. Such changes contribute to increased autoimmune diseases, enhanced infection severity, and reduced responsiveness to vaccines. Utilizing aging animal models becomes imperative for a comprehensive understanding of immunosenescence, given the complexity of aging as a physiological process in living organisms. Our investigation focuses on Cisd2, a causative gene for Wolfram syndrome, to elucidate on immunosenescence. Cisd2 knockout (KO) mice, serving as a model for premature aging, exhibit a shortened lifespan with early onset of aging-related features, such as decreased bone density, hair loss, depigmentation, and optic nerve degeneration. Intriguingly, we found that the Cisd2 KO mice present a higher number of neutrophils in the blood; however, isolated neutrophils from these mice display functional defects. Through mass spectrometry analysis, we identified an interaction between Cisd2 and Calnexin, a protein known for its role in protein quality control. Beyond this function, Calnexin also regulates calcium homeostasis through interaction with sarcoendoplasmic reticulum calcium transport ATPase (SERCA). Our study proposes that Cisd2 modulates calcium homeostasis via its interaction with Calnexin and SERCA, consequently influencing neutrophil functions. [BMB Reports 2024; 57(5): 256-261].
Assuntos
Proteínas Relacionadas à Autofagia , Cálcio , Homeostase , Proteínas do Tecido Nervoso , Neutrófilos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , Camundongos , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Knockout , Neutrófilos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
Sandfly-borne Toscana virus (TOSV) is an enveloped tri-segmented negative single-strand RNA Phlebovirus. It is an emerging virus predominantly endemic in southwestern Europe and Northern Africa. Although TOSV infection is typically asymptomatic or results in mild febrile disease, it is neurovirulent and ranks among the three most common causes of summer meningitis in certain regions. Despite this clinical significance, our understanding of the molecular aspects and host factors regulating phlebovirus infection is limited. This study characterized the early steps of TOSV infection. Our findings reveal that two members of the Numb-associated kinases family of Ser/Thr kinases, namely adaptor-associated kinase 1 (AAK1) and cyclin G-associated kinase (GAK), play a role in regulating the early stages of TOSV entry. FDA-approved inhibitors targeting these kinases demonstrated significant inhibition of TOSV infection. This study suggests that AAK1 and GAK represent druggable targets for inhibiting TOSV infection and, potentially, related Phleboviruses.
Assuntos
Vírus da Febre do Flebótomo Napolitano , Internalização do Vírus , Vírus da Febre do Flebótomo Napolitano/genética , Humanos , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Linhagem CelularRESUMO
Coronaviruses rely on host proteases to activate the viral spike protein, which facilitates fusion with the host cell membrane and the release of viral genomic RNAs into the host cell cytoplasm. The distribution of specific host proteases in the host determines the host, tissue, and cellular tropism of these viruses. Here, we identified the kallikrein (KLK) family member KLK5 as a major host protease secreted by human airway cells and exploited by multiple human betacoronaviruses. KLK5 cleaved both the priming (S1/S2) and activation (S2') sites of spike proteins from various human betacoronaviruses in vitro. In contrast, KLK12 and KLK13 displayed preferences for either the S2' or S1/S2 site, respectively. Whereas KLK12 and KLK13 worked in concert to activate SARS-CoV-2 and MERS-CoV spike proteins, KLK5 by itself efficiently activated spike proteins from several human betacoronaviruses, including SARS-CoV-2. Infection of differentiated human bronchial epithelial cells (HBECs) with human betacoronaviruses induced an increase in KLK5 that promoted virus replication. Furthermore, ursolic acid and other related plant-derived triterpenoids that inhibit KLK5 effectively suppressed the replication of SARS-CoV, MERS-CoV, and SARS-CoV-2 in HBECs and mitigated lung inflammation in mice infected with MERS-CoV or SARS-CoV-2. We propose that KLK5 is a pancoronavirus host factor and a promising therapeutic target for current and future coronavirus-induced diseases.
Assuntos
Calicreínas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Replicação Viral , Humanos , Calicreínas/metabolismo , Calicreínas/genética , Animais , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Camundongos , SARS-CoV-2/metabolismo , Betacoronavirus/metabolismo , Betacoronavirus/fisiologia , COVID-19/metabolismo , COVID-19/virologia , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Células HEK293 , Pulmão/virologia , Pulmão/metabolismoRESUMO
RIPK3-ZBP1-MLKL-mediated necroptosis is a proinflammatory cell death process that is crucial for antiviral host defence. RIPK3 self-oligomerization and autophosphorylation are prerequisites for executing necroptosis, yet the underlying mechanism of virus-induced RIPK3 activation remains elusive. Interferon-inducible 2'-5' oligoadenylate synthetase-like (OASL) protein is devoid of enzymatic function but displays potent antiviral activity. Here we describe a role of OASL as a virus-induced necroptosis promoter that scaffolds the RIPK3-ZBP1 non-canonical necrosome via liquid-like phase condensation. This liquid-like platform of OASL recruits RIPK3 and ZBP1 via protein-protein interactions to provide spatial segregation for RIPK3 nucleation. This process facilitates the amyloid-like fibril formation and activation of RIPK3 and thereby MLKL phosphorylation for necroptosis. Mice deficient in Oasl1 exhibit severely impaired necroptosis and attenuated inflammation after viral infection, resulting in uncontrolled viral dissemination and lethality. Our study demonstrates an interferon-induced innate response whereby OASL scaffolds RIPK3-ZBP1 assembly via its phase-separated liquid droplets to facilitate necroptosis-mediated antiviral immunity.
Assuntos
Necroptose , Proteínas Quinases , Animais , Camundongos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Morte Celular , Antivirais , Interferons/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Apoptose , Proteínas de Ligação a RNA/metabolismoRESUMO
With the emergence of multiple predominant SARS-CoV-2 variants, it becomes important to have a comprehensive assessment of their viral fitness and transmissibility. Here, we demonstrate that natural temperature differences between the upper (33°C) and lower (37°C) respiratory tract have profound effects on SARS-CoV-2 replication and transmissibility. Specifically, SARS-CoV-2 variants containing the NSP12 mutations P323L or P323L/G671S exhibit enhanced RNA-dependent RNA polymerase (RdRp) activity at 33°C compared with 37°C and high transmissibility. Molecular dynamics simulations and microscale thermophoresis demonstrate that the NSP12 P323L and P323L/G671S mutations stabilize the NSP12-NSP7-NSP8 complex through hydrophobic effects, leading to increased viral RdRp activity. Furthermore, competitive transmissibility assay reveals that reverse genetic (RG)-P323L or RG-P323L/G671S NSP12 outcompetes RG-WT (wild-type) NSP12 for replication in the upper respiratory tract, allowing markedly rapid transmissibility. This suggests that NSP12 P323L or P323L/G671S mutation of SARS-CoV-2 is associated with increased RdRp complex stability and enzymatic activity, promoting efficient transmissibility.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , SARS-CoV-2/genética , Furões , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/química , Mutação/genética , Replicação Viral/genéticaRESUMO
Lung mast cells are important in host defense, and excessive proliferation or activation of these cells can cause chronic inflammatory disorders like asthma. Two parallel pathways induced by KIT-stem cell factor (SCF) and FcεRI-immunoglobulin E interactions are critical for the proliferation and activation of mast cells, respectively. Here, we report that mast cell-expressed membrane protein1 (MCEMP1), a lung-specific surface protein, functions as an adaptor for KIT, which promotes SCF-mediated mast cell proliferation. MCEMP1 elicits intracellular signaling through its cytoplasmic immunoreceptor tyrosine-based activation motif and forms a complex with KIT to enhance its autophosphorylation and activation. Consequently, MCEMP1 deficiency impairs SCF-induced peritoneal mast cell proliferation in vitro and lung mast cell expansion in vivo. Mcemp1-deficient mice exhibit reduced airway inflammation and lung impairment in chronic asthma mouse models. This study shows lung-specific MCEMP1 as an adaptor for KIT to facilitate SCF-mediated mast cell proliferation.
Assuntos
Asma , Fator de Células-Tronco , Animais , Camundongos , Proliferação de Células , Pulmão/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/metabolismoRESUMO
Although several vaccines and antiviral drugs against SARS-CoV-2 are currently available, control and prevention of COVID-19 through these interventions is limited due to inaccessibility and economic issues in some regions and countries. Moreover, incomplete viral clearance by ineffective therapeutics may lead to rapid genetic evolution, resulting in the emergence of new SARS-CoV-2 variants that may escape the host immune system as well as currently available COVID-19 vaccines. Here, we report that phytochemicals extracted from Chlorella spp. and Psidium guajava possess broad-spectrum antiviral activity against a range of SARS-CoV-2 variants. Through chromatography-based screening, we identified four bioactive compounds and subsequently demonstrated their potential antiviral activities in vivo. Interestingly, in hACE2 mice, treatment with these compounds significantly attenuates SARS-CoV-2-induced proinflammatory responses, demonstrating their potential anti-inflammatory activity. Collectively, our study suggests that phytochemicals from edible plants may be readily available therapeutics and prophylactics against multiple SARS-CoV-2 strains and variants.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19 , Chlorella , Animais , Antivirais/uso terapêutico , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Camundongos , Compostos Fitoquímicos/farmacologia , SARS-CoV-2RESUMO
With the convergent global emergence of SARS-CoV-2 variants of concern (VOC), a precise comparison study of viral fitness and transmission characteristics is necessary for the prediction of dominant VOCs and the development of suitable countermeasures. While airway temperature plays important roles in the fitness and transmissibility of respiratory tract viruses, it has not been well studied with SARS-CoV-2. Here we demonstrate that natural temperature differences between the upper (33°C) and lower (37°C) respiratory tract have profound effects on SARS-CoV-2 replication and transmission. Specifically, SARS-COV-2 variants containing the P323L or P323L/G671S mutation in the NSP12 RNA-dependent RNA polymerase (RdRp) exhibited enhanced RdRp enzymatic activity at 33°C compared to 37°C and high transmissibility in ferrets. MicroScale Thermophoresis demonstrated that the NSP12 P323L or P323L/G671S mutation stabilized the NSP12-NSP7-NSP8 complex interaction. Furthermore, reverse genetics-derived SARS-CoV-2 variants containing the NSP12 P323L or P323L/G671S mutation displayed enhanced replication at 33°C, and high transmission in ferrets. This suggests that the evolutionarily forced NSP12 P323L and P323L/G671S mutations of recent SARS-CoV-2 VOC strains are associated with increases of the RdRp complex stability and enzymatic activity, promoting the high transmissibility.
RESUMO
Omicron has become the globally dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, creating additional challenges due to its ability to evade neutralization. Here, we report that neutralizing antibodies against Omicron variants are undetected following COVID-19 infection with ancestral or past SARS-CoV-2 variant viruses or after two-dose mRNA vaccination. Compared with two-dose vaccination, a three-dose vaccination course induces broad neutralizing antibody responses with improved durability against different SARS-CoV-2 variants, although neutralizing antibody titers against Omicron remain low. Intriguingly, among individuals with three-dose vaccination, Omicron breakthrough infection substantially augments serum neutralizing activity against a broad spectrum of SARS-CoV-2 variants, including Omicron variants BA.1, BA.2, and BA.5. Additionally, after Omicron breakthrough infection, memory T cells respond to the spike proteins of both ancestral and Omicron SARS-CoV-2 by producing cytokines with polyfunctionality. These results suggest that Omicron breakthrough infection following three-dose mRNA vaccination induces pan-SARS-CoV-2 immunity that may protect against emerging SARS-CoV-2 variants of concern.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Formação de Anticorpos , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral/genética , Anticorpos Antivirais , Anticorpos Amplamente Neutralizantes , COVID-19/prevenção & controle , Citocinas , RNA MensageiroRESUMO
The innate immune system is the first line of the host defense program against pathogens and harmful substances. Antiviral innate immune responses can be triggered by multiple cellular receptors sensing viral components. The activated innate immune system produces interferons (IFNs) and cytokines that perform antiviral functions to eliminate invading viruses. Coronaviruses are single-stranded, positive-sense RNA viruses that have a broad range of animal hosts. Coronaviruses have evolved multiple means to evade host antiviral immune responses. Successful immune evasion by coronaviruses may enable the viruses to adapt to multiple species of host organisms. Coronavirus transmission from zoonotic hosts to humans has caused serious illnesses, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease-2019 (COVID-19), resulting in global health and economic crises. In this review, we summarize the current knowledge of the mechanisms underlying host sensing of and innate immune responses against coronavirus invasion, as well as host immune evasion strategies of coronaviruses.
Assuntos
Coronaviridae/imunologia , Infecções por Coronavirus/imunologia , Evasão da Resposta Imune , Imunidade Inata , Animais , COVID-19/imunologia , Humanos , Interferons/imunologia , SARS-CoV-2/imunologiaRESUMO
The MHC class I-mediated antigen presentation pathway plays a critical role in antiviral immunity. Here we show that the MHC class I pathway is targeted by SARS-CoV-2. Analysis of the gene expression profile from COVID-19 patients as well as SARS-CoV-2 infected epithelial cell lines reveals that the induction of the MHC class I pathway is inhibited by SARS-CoV-2 infection. We show that NLRC5, an MHC class I transactivator, is suppressed both transcriptionally and functionally by the SARS-CoV-2 ORF6 protein, providing a mechanistic link. SARS-CoV-2 ORF6 hampers type II interferon-mediated STAT1 signaling, resulting in diminished upregulation of NLRC5 and IRF1 gene expression. Moreover, SARS-CoV-2 ORF6 inhibits NLRC5 function via blocking karyopherin complex-dependent nuclear import of NLRC5. Collectively, our study uncovers an immune evasion mechanism of SARS-CoV-2 that targets the function of key MHC class I transcriptional regulators, STAT1-IRF1-NLRC5.
Assuntos
COVID-19/imunologia , Genes MHC Classe I/imunologia , Fator Regulador 1 de Interferon/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , SARS-CoV-2/genética , Fator de Transcrição STAT1/antagonistas & inibidores , Proteínas Virais/metabolismo , COVID-19/genética , COVID-19/patologia , COVID-19/virologia , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/isolamento & purificação , Transdução de Sinais , Proteínas Virais/imunologiaRESUMO
Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with 12%-30% case mortality rates and is related to the Heartland virus (HRTV) identified in the United States. Together, SFTSV and HRTV are emerging segmented, negative-sense RNA viral (sNSV) pathogens with potential global health impact. Here, we characterize the amino-terminal cap-snatching endonuclease domain of SFTSV polymerase (L) and solve a 2.4-Å X-ray crystal structure. While the overall structure is similar to those of other cap-snatching sNSV endonucleases, differences near the C terminus of the SFTSV endonuclease suggest divergence in regulation. Influenza virus endonuclease inhibitors, including the US Food and Drug Administration (FDA) approved Baloxavir (BXA), inhibit the endonuclease activity in in vitro enzymatic assays and in cell-based studies. BXA displays potent activity with a half maximal inhibitory concentration (IC50) of â¼100 nM in enzyme inhibition and an EC50 value of â¼250 nM against SFTSV and HRTV in plaque assays. Together, our data support sNSV endonucleases as an antiviral target.
Assuntos
Antivirais/farmacologia , Endonucleases/química , Phlebovirus/efeitos dos fármacos , Phlebovirus/enzimologia , Animais , Antivirais/química , Cátions Bivalentes/farmacologia , Linhagem Celular , Sequência Conservada , Cristalografia por Raios X , Dibenzotiepinas/química , Dibenzotiepinas/farmacologia , Endonucleases/antagonistas & inibidores , Endonucleases/metabolismo , Humanos , Modelos Moleculares , Morfolinas/química , Morfolinas/farmacologia , Domínios Proteicos , Estrutura Secundária de Proteína , Piridonas/química , Piridonas/farmacologia , Triazinas/química , Triazinas/farmacologiaRESUMO
Non-small-cell lung cancer (NSCLC) is the most common lung cancer subtype and accounts for more than 80% of all lung cancer cases. Epidermal growth factor receptor (EGFR) phosphorylation by binding growth factors such as EGF activates downstream prooncogenic signaling pathways including KRAS-ERK, JAK-STAT, and PI3K-AKT. These pathways promote the tumor progression of NSCLC by inducing uncontrolled cell cycle, proliferation, migration, and programmed death-ligand 1 (PD-L1) expression. New cytotoxic drugs have facilitated considerable progress in NSCLC treatment, but side effects are still a significant cause of mortality. Gallic acid (3,4,5-trihydroxybenzoic acid; GA) is a phenolic natural compound, isolated from plant derivatives, that has been reported to show anticancer effects. We demonstrated the tumor-suppressive effect of GA, which induced the decrease of PD-L1 expression through binding to EGFR in NSCLC. This binding inhibited the phosphorylation of EGFR, subsequently inducing the inhibition of PI3K and AKT phosphorylation, which triggered the activation of p53. The p53-dependent upregulation of miR-34a induced PD-L1 downregulation. Further, we revealed the combination effect of GA and anti-PD-1 monoclonal antibody in an NSCLC-cell and peripheral blood mononuclear-cell coculture system. We propose a novel therapeutic application of GA for immunotherapy and chemotherapy in NSCLC.
RESUMO
BACKGROUND/AIM: Non-small cell lung cancer (NSCLC) is one among the most common cancers worldwide. Recently, dietary phytochemicals have been reported as an attractive approach to improve the symptoms of NSCLC patients. Tannic acid is a natural polyphenol, which is known to have anticancer effects on in vitro models of breast, gingival and colon cancer. However, the molecular mechanisms associated with the actions of tannic acid on A549 human lung cancer cells have not been elucidated. MATERIALS AND METHODS: In this study, we analyzed the effect of tannic acid on A549 cells and their underlying mechanisms using western blotting, flow cytometry, invasion assay and tumorsphere formation assay. RESULTS: Tannic acid treatment suppressed the viability of A549 cells through cell cycle arrest and induction of the intrinsic pathways of apoptosis. In addition, the various malignant phenotypes of A549 cells including invasion, migration, and stemness were inhibited by tannic acid treatment. CONCLUSION: Tannic acid could be used as an effective inhibitor of lung cancer progression.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Taninos/uso terapêutico , Células A549 , Apoptose , Linhagem Celular Tumoral , Humanos , Transdução de Sinais , Taninos/farmacologiaRESUMO
: Human embryonic carcinoma (EC; NCCIT) cells have self-renewal ability and pluripotency. Cancer stem cell markers are highly expressed in NCCIT cells, imparting them with the pluripotent nature to differentiate into other cancer types, including breast cancer. As one of the main cancer stem cell pathways, Wnt/ß-catenin is also overexpressed in NCCIT cells. Thus, inhibition of these pathways defines the ability of a drug to target cancer stem cells. Tannic acid (TA) is a natural polyphenol present in foods, fruits, and vegetables that has anti-cancer activity. Through Western blotting and PCR, we demonstrate that TA inhibits cancer stem cell markers and the Wnt/ß-catenin signaling pathway in NCCIT cells and through a fluorescence-activated cell sorting analysis we demonstrated that TA induces sub-G1 cell cycle arrest and apoptosis. The mechanism underlying this is the induction of mitochondrial reactive oxygen species (ROS) (mROS), which then induce the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated extrinsic apoptosis pathway instead of intrinsic mitochondrial apoptosis pathway. Moreover, ribonucleic acid sequencing data with TA in NCCIT cells show an elevation in TRAIL-induced extrinsic apoptosis, which we confirm by Western blotting and real-time PCR. The induction of human TRAIL also proves that TA can induce extrinsic apoptosis in NCCIT cells by regulating mROS.