RESUMEN
UNLABELLED: As a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently, lysosomal cell death has been defined as "lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol." Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosome-associated membrane proteins (LAMPs; the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells. The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry, and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus, however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death. IMPORTANCE: The integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosome-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function, as well as for influenza inhibitors' mechanism of action, and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings and why NA inhibitors have more potent therapeutic effects for lethal avian influenza virus infections at early stage.
Asunto(s)
Membrana Celular/enzimología , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/enzimología , Neuraminidasa/metabolismo , Proteínas Virales/metabolismo , Ácidos Carbocíclicos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/química , Ciclopentanos/farmacología , Citosol/efectos de los fármacos , Citosol/enzimología , Citosol/virología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Guanidinas/farmacología , Humanos , Hidrólisis , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H5N1 del Virus de la Influenza A/química , Subtipo H5N1 del Virus de la Influenza A/enzimología , Proteínas de Membrana de los Lisosomas/química , Lisosomas/efectos de los fármacos , Lisosomas/virología , Unión Proteica , Proteolisis , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/virología , Especificidad de la Especie , Internalización del Virus/efectos de los fármacos , Zanamivir/farmacologíaRESUMEN
The clinical efficacy of cisplatin in esophageal squamous cell carcinoma (ESCC) treatment remains undesirable. Src, a non-receptor tyrosine kinase involved in multiple fields of tumorigenesis, recently has been indicated as a promising therapeutic target in the treatment of solid tumors including ESCC. However, whether inhibition of Src activity can increase cisplatin efficacy in ESCC cells remains unknown. The present study found that inhibition of Src by its inhibitor-dasatinib sensitized ESCC cells to cisplatin in vitro. Our data also suggest a likely mechanism for this synergy that dasatinib reduces expression of critical oncogenic members of the signaling pathways, such as AKT or Stat3, and cisplatin-resistant molecules, such as ERCC1 and BRCA1, under the control of Src. Furthermore, dasatinib could sensitize ESCC cells to another platin-based agent, carboplatin. Therefore, this study provides a potential target for improving cisplatin efficacy in ESCC therapy.
Asunto(s)
Carcinoma de Células Escamosas/patología , Cisplatino/farmacología , Neoplasias Esofágicas/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Pirimidinas/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Tiazoles/farmacología , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Dasatinib , Sinergismo Farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
BACKGROUND: Nanoparticles have become a key technology in multiple industries. However, there are growing reports of the toxicity of nanomaterials to humans. In particular, nanomaterials have been linked to lung diseases. The molecular mechanisms of nanoparticle toxicity are largely unexplored. METHODS: Acute lung injury was induced in wild-type mice and angiotensin-coverting enzyme 2 (ACE2) knockout mice by the intratracheal instillation of cationic polyamidoamine dendrimer (PAMAM) nanoparticles. For rescue experiments, losartan (15 mg/kg in PBS) was injected intraperitoneally 30 min before nanoparticle administration. RESULTS: Some PAMAM nanoparticles, but not anionic PAMAM nanoparticles or carbon nanotubes, triggered acute lung failure in mice. Mechanistically, cationic nanoparticles can directly bind ACE2, decrease its activity and down-regulate its expression level in lung tissue, resulting in deregulation of the renin-angiotensin system. Gene inactivation of Ace2 can exacerbate lung injury. Importantly, the administration of losartan, which is an angiotensin II type I receptor antagonist, can ameliorate PAMAM nanoparticle-induced lung injury. CONCLUSIONS: Our data provide molecular insight into PAMAM nanoparticle-induced lung injury and suggest potential therapeutic and screening strategies to address the safety of nanomaterials.
Asunto(s)
Lesión Pulmonar Aguda/inducido químicamente , Dendrímeros/toxicidad , Nanopartículas/toxicidad , Peptidil-Dipeptidasa A/metabolismo , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Enzima Convertidora de Angiotensina 2 , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Cationes , Dendrímeros/administración & dosificación , Dendrímeros/química , Regulación hacia Abajo , Instilación de Medicamentos , Losartán/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Nanopartículas/administración & dosificación , Nanopartículas/química , Peptidil-Dipeptidasa A/genética , Unión Proteica , Análisis de SupervivenciaRESUMEN
Innate immunity plays critical antiviral roles. The highly virulent avian influenza viruses (AIVs) H5N1, H7N9, and H5N6 can better escape host innate immune responses than the less virulent seasonal H1N1 virus. Here, we report a mechanism by which transcriptional readthrough (TRT)-mediated suppression of innate immunity occurs post AIV infection. By using cell lines, mouse lungs, and patient PBMCs, we showed that genes on the complementary strand ("trans" genes) influenced by TRT were involved in the disruption of host antiviral responses during AIV infection. The trans-TRT enhanced viral lethality, and TRT abolishment increased cell viability and STAT1/2 expression. The viral NS1 protein directly bound to SSU72, and degradation of SSU72 induced TRT. SSU72 overexpression reduced TRT and alleviated mouse lung injury. Our results suggest that AIVs infection induce TRT by reducing SSU72 expression, thereby impairing host immune responses, a molecular mechanism acting through the NS1-SSU72-trans-TRT-STAT1/2 axis. Thus, restoration of SSU72 expression might be a potential strategy for preventing AIV pandemics.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Subtipo H5N1 del Virus de la Influenza A , Subtipo H7N9 del Virus de la Influenza A , Gripe Humana , Animales , Antivirales , Humanos , Inmunidad Innata , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Subtipo H7N9 del Virus de la Influenza A/metabolismo , Ratones , Fosfoproteínas Fosfatasas , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Cell entry of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by the viral spike (S) protein. Amino acids 319-510 on the S protein have been mapped as the receptor-binding domain (RBD), which mediates binding to the SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) on SARS-CoV susceptible cells. In this study, we expressed a fusion protein containing the human codon-optimized RBD of the SARS-CoV spike protein linked to the Fc portion of human IgG1 (named RBD-Fc) in HEK293 cells. The RBD-Fc protein was purified by affinity chromatography. The flow cytometry assay showed that the purified RBD-Fc protein could bind to ACE2. We demonstrated that the RBD spike protein alone could be internalized into SARS-CoV susceptible cells together with ACE2. We also showed that the removal of N-glycans from the RBD spike protein did not abolish this phenomenon. Our discoveries may have some implications for the development of the SARS vaccine.
Asunto(s)
Endocitosis , Glicoproteínas de Membrana/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Receptores Virales/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Proteínas del Envoltorio Viral/metabolismo , Enzima Convertidora de Angiotensina 2 , Animales , Línea Celular , Chlorocebus aethiops , Citometría de Flujo , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Glicoproteínas de Membrana/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/genéticaRESUMEN
B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 µl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/química , Antígenos/química , Linfocitos B/química , Hibridomas/química , Inmunoglobulina G/química , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Antígenos/inmunología , Linfocitos B/inmunología , Hibridomas/inmunología , Inmunoglobulina G/inmunología , RatonesRESUMEN
Platelet-activating factor receptor (PAFR), a G-protein-coupled receptor, has been implicated in tumorigenesis, but its contributions to metastatic progression have not been investigated. Here, we show that PAFR is overexpressed in non-small cell lung cancer (NSCLC) as well as in breast, colorectal, and gastric carcinomas. Expression of PAFR correlates closely with clinical stages, survival time, and distant metastasis. In human NSCLC cells, activation of the PAF/PAFR signaling axis accentuated malignant character, including by stimulating epithelial-mesenchymal transition (EMT). In contrast, silencing PAFR in aggressive NSCLC cells inhibited these effects. Mechanistic investigations showed that PAFR stimulated EMT by activating STAT3 via upregulation of G-protein-dependent SRC or JAK2 kinase activity. Notably, STAT3 transcriptionally elevated PAFR expression. Thus, activation of PAFR in NSCLC cells initiated a forward feedback loop responsible for mediating the aggressive malignant character of NSCLC cells in vitro and in vivo. Reinforcing this reciprocal activation loop, PAF/PAFR signaling also upregulated IL6 expression and thereby STAT3 activation. Overall, our results elucidated an important role for PAFR dysregulation in the pathogenicity of NSCLC and unraveled a forward feedback loop between PAFR and STAT3 that acts to drive the malignant progression of NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Transición Epitelial-Mesenquimal/fisiología , Neoplasias Pulmonares/patología , Proteínas de Neoplasias/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Receptores Acoplados a Proteínas G/fisiología , Factor de Transcripción STAT3/fisiología , Adulto , Anciano , Animales , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Progresión de la Enfermedad , Activación Enzimática , Retroalimentación Fisiológica , Femenino , Proteínas de Unión al GTP/fisiología , Xenoinjertos , Humanos , Interleucina-6/biosíntesis , Interleucina-6/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Trasplante de Neoplasias , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/farmacología , Transducción de Señal , Neoplasias Gástricas/metabolismo , Regulación hacia ArribaRESUMEN
Ebola virus (EBOV) causes a highly lethal hemorrhagic fever syndrome in humans and has been associated with mortality rates of up to 91% in Zaire, the most lethal strain. Though the viral envelope glycoprotein (GP) mediates widespread inflammation and cellular damage, these changes have mainly focused on alterations at the protein level, the role of microRNAs (miRNAs) in the molecular pathogenesis underlying this lethal disease is not fully understood. Here, we report that the mi-RNAs hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p were induced in human umbilical vein endothelial cells (HUVECs) following expression of EBOV GP. Among the proteins encoded by predicted targets of these miRNAs, the adhesion-related molecules tissue factor pathway inhibitor (TFPI), dystroglycan1 (DAG1) and the caspase 8 and FADD-like apoptosis regulator (CFLAR) were significantly downregulated in EBOV GP-expressing HUVECs. Moreover, inhibition of hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p, or overexpression of TFPI, DAG1 and CFLAR rescued the cell viability that was induced by EBOV GP. Our results provide a novel molecular basis for EBOV pathogenesis and may contribute to the development of strategies to protect against future EBOV pandemics.
Asunto(s)
Fiebre Hemorrágica Ebola/genética , MicroARNs/genética , Proteínas del Envoltorio Viral/metabolismo , Western Blotting , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/metabolismo , Supervivencia Celular , Distroglicanos/metabolismo , Ebolavirus , Regulación de la Expresión Génica , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Inflamación , Lipoproteínas/metabolismo , MicroARNs/fisiologíaRESUMEN
The threat of a new influenza pandemic has existed since 1997, when the highly pathogenic H5N1 strain of avian influenza A virus infected humans in Hong Kong and spread across Asia, where it continued to infect poultry and people. The human mortality rate of H5N1 infection is about 60%, whereas that of seasonal H1N1 infection is less than 0.1%. The high mortality rate associated with H5N1 infection is predominantly a result of respiratory failure caused by acute lung injury; however, how viral infection contributes to this disease pathology is unclear. Here, we used electron microscopy to show the accumulation of autophagosomes in H5N1-infected lungs from a human cadaver and mice, as well as in infected A549 human epithelial lung cells. We also showed that H5N1, but not seasonal H1N1, induced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase Akt, the tumor suppressor protein TSC2, and the mammalian target of rapamycin. Additionally, we suggest that the hemagglutinin protein of H5N1 may be responsible for stimulating autophagy. When applied prophylactically, reagents that blocked virus-induced autophagic signaling substantially increased the survival rate of mice and substantially ameliorated the acute lung injury and mortality caused by H5N1 infection. We conclude that the autophagic cell death of alveolar epithelial cells likely plays a crucial role in the high mortality rate of H5N1 infection, and we suggest that autophagy-blocking agents might be useful as prophylactics and therapeutics against infection of humans by the H5N1 virus.
Asunto(s)
Autofagia/fisiología , Subtipo H5N1 del Virus de la Influenza A , Gripe Humana/patología , Pulmón/ultraestructura , Transducción de Señal/fisiología , Análisis de Varianza , Animales , Autofagia/efectos de los fármacos , Proteína 5 Relacionada con la Autofagia , Western Blotting , Línea Celular , Cartilla de ADN/genética , Células Epiteliales/fisiología , Técnicas de Silenciamiento del Gen , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/farmacología , Humanos , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/genética , Proteína Oncogénica v-akt/metabolismo , Fagosomas/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Serina-Treonina Quinasas TOR/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
It has been reported that lactoferrin (LF) participates in the host immune response against Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) invasion by enhancing NK cell activity and stimulating neutrophil aggregation and adhesion. We further investigated the role of LF in the entry of SARS pseudovirus into HEK293E/ACE2-Myc cells. Our results reveal that LF inhibits SARS pseudovirus infection in a dose-dependent manner. Further analysis suggested that LF was able to block the binding of spike protein to host cells at 4°C, indicating that LF exerted its inhibitory function at the viral attachment stage. However, LF did not disrupt the interaction of spike protein with angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV. Previous studies have shown that LF colocalizes with the widely distributed cell-surface heparan sulfate proteoglycans (HSPGs). Our experiments have also confirmed this conclusion. Treatment of the cells with heparinase or exogenous heparin prevented binding of spike protein to host cells and inhibited SARS pseudovirus infection, demonstrating that HSPGs provide the binding sites for SARS-CoV invasion at the early attachment phase. Taken together, our results suggest that, in addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry. LF may play a protective role in host defense against SARS-CoV infection through binding to HSPGs and blocking the preliminary interaction between SARS-CoV and host cells. Our findings may provide further understanding of SARS-CoV pathogenesis and aid in treatment of this deadly disease.
Asunto(s)
Proteoglicanos de Heparán Sulfato/metabolismo , Lactoferrina/farmacología , Síndrome Respiratorio Agudo Grave/prevención & control , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Internalización del Virus , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Lactoferrina/inmunología , Lactoferrina/metabolismo , Unión Proteica/inmunología , Síndrome Respiratorio Agudo Grave/etiología , Síndrome Respiratorio Agudo Grave/inmunología , Internalización del Virus/efectos de los fármacosRESUMEN
BACKGROUND: Numerous Asian cases of avian influenza virus infection, especially the highly pathogenic strain H5N1, in humans have raised the concern that another influenza pandemic is close. However, there are no effective therapeutic drugs or preventative vaccines available. Hemagglutinin is the membrane glycoprotein of avian influenza virus responsible for receptor binding to human cells and the main immunogenic protein that elicits a strong immune response. Although this protein is of great importance to the study of pathogenesis and vaccine development, its expression and purification are difficult due to high levels of glycosylation. METHODS: In this study, we expressed codon-optimized, full-length hemagglutinin 5 (H5) protein fused with a human IgG Fc tag (H5-Fc) in HEK293 cells. To enhance secretion of this protein, we also deleted the transmembrane domain and the intracellular domain of the H5 protein (H5DeltaTM-Fc). Purified proteins were obtained using a protein A column. RESULTS: ELISA revealed that the yield of soluble H5DeltaTM-Fc protein in the supernatant was about 20 mg/L. Western blotting and fluorescence activated cell sorter (FACS) indicated that the purified H5 protein was correctly folded and biologically active. CONCLUSION: Purification of H5 proteins from mammalian cells could be used for large-scale production of recombinant H5 protein for basic scientific research or the development of vaccines.
Asunto(s)
Codón/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Proteínas Recombinantes/metabolismo , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Pliegue de Proteína , Proteínas Recombinantes/genéticaRESUMEN
While severe acute respiratory syndrome coronavirus (SARS-CoV) was initially thought to enter cells through direct fusion with the plasma membrane, more recent evidence suggests that virus entry may also involve endocytosis. We have found that SARS-CoV enters cells via pH- and receptor-dependent endocytosis. Treatment of cells with either SARS-CoV spike protein or spike-bearing pseudoviruses resulted in the translocation of angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV, from the cell surface to endosomes. In addition, the spike-bearing pseudoviruses and early endosome antigen 1 were found to colocalize in endosomes. Further analyses using specific endocytic pathway inhibitors and dominant-negative Eps15 as well as caveolin-1 colocalization study suggested that virus entry was mediated by a clathrin- and caveolae-independent mechanism. Moreover, cholesterol- and sphingolipid-rich lipid raft microdomains in the plasma membrane, which have been shown to act as platforms for many physiological signaling pathways, were shown to be involved in virus entry. Endocytic entry of SARS-CoV may expand the cellular range of SARS-CoV infection, and our findings here contribute to the understanding of SARS-CoV pathogenesis, providing new information for anti-viral drug research.