RESUMEN
We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.
Asunto(s)
Antivirales , Gripe Humana , Fenoles , Animales , Perros , Humanos , Antivirales/farmacología , Antivirales/química , Proteínas Mitocondriales/metabolismo , Prohibitinas , Espectrometría de Masas en Tándem , Canal Aniónico 1 Dependiente del Voltaje , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Canales Aniónicos Dependientes del Voltaje , Línea CelularRESUMEN
In eukaryotes, CREB-binding protein (CBP), a coactivator of CREB, functions both as a platform for recruiting other components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. We previously showed that the transcriptional activity of cAMP-responsive element binding protein (CREB) plays a crucial role in neuronal plasticity in the pond snail Lymnaea stagnalis. However, there is no information on the molecular structure and HAT activity of CBP in the Lymnaea central nervous system (CNS), hindering an investigation of its postulated role in long-term memory (LTM). Here, we characterize the Lymnaea CBP (LymCBP) gene and identify a conserved domain of LymCBP as a functional HAT. Like CBPs of other species, LymCBP possesses functional domains, such as the KIX domain, which is essential for interaction with CREB and was shown to regulate LTM. In-situ hybridization showed that the staining patterns of LymCBP mRNA in CNS are very similar to those of Lymnaea CREB1. A particularly strong LymCBP mRNA signal was observed in the cerebral giant cell (CGC), an identified extrinsic modulatory interneuron of the feeding circuit, the key to both appetitive and aversive LTM for taste. Biochemical experiments using the recombinant protein of the LymCBP HAT domain showed that its enzymatic activity was blocked by classical HAT inhibitors. Preincubation of the CNS with such inhibitors blocked cAMP-induced synaptic facilitation between the CGC and an identified follower motoneuron of the feeding system. Taken together, our findings suggest a role for the HAT activity of LymCBP in synaptic plasticity in the feeding circuitry.
Asunto(s)
Proteína de Unión a CREB , Lymnaea , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Sistema Nervioso Central/metabolismo , Cromatina/metabolismo , Lymnaea/genética , Lymnaea/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Personal protective equipment (PPE), including medical masks, should be worn for preventing the transmission of respiratory pathogens via infective droplets and aerosols. In medical masks, the key layer is the filter layer, and the melt-blown nonwoven fabric (NWF) is the most used fabric. However, the NWF filter layer cannot kill or inactivate the pathogens spread via droplets and aerosols. Povidone-iodine (PVP-I) has been used as an antiseptic solution given its potent broad-spectrum activity against pathogens. To develop PPE (e.g., medical masks) with anti-pathogenic activity, we integrated PVP-I into nylon-66 NWF. We then evaluated its antiviral activity against influenza A viruses by examining the viability of Madin-Darby canine kidney (MDCK) cells after inoculation with the virus strains exposed to the PVP-I-integrated nylon-66 NWF. The PVP-I nylon-66 NWF protected the MDCK cells from viral infection in a PVP-I concentration-dependent manner. Subsequently, we found to integrate PVP-I into nylon-66 and polyurethane materials among various materials. These PVP-I materials were also effective against influenza virus infection, and treatment with PVP-I nylon-66 NWF showed the highest cell survival among all the tested materials. PVP-I showed anti-influenza A virus activity when used in conjunction with PPE materials. Moreover, nylon-66 NWF integrated with PVP-I was found to be the best material to ensure anti-influenza activity. Therefore, PVP-I-integrated masks could have the potential to inhibit respiratory virus infection. Our results provide new information for developing multi-functional PPEs with anti-viral activity by integrating them with PVP-I to prevent the potential transmission of respiratory viruses.
Asunto(s)
Gripe Humana , Orthomyxoviridae , Animales , Perros , Humanos , Povidona Yodada/farmacología , Povidona Yodada/uso terapéutico , Nylons , Aerosoles y Gotitas Respiratorias , Gripe Humana/prevención & controlRESUMEN
trans-Banglene and cis(c)-banglene possess neurotrophin-like activity in rat neurons. However, the molecular mechanisms underlying t-banglene-induced neurotrophic activity in rat and human neurons remain unclear. Here, we performed transcriptome analysis in PC12 cells, a rat adrenal gland pheochromocytoma cell line treated with t-banglene, using comprehensive RNA sequencing. The differentially expressed gene analysis of the sequencing data revealed that the expression of RT1 class I, locus CE1 (RT1-CE1) was upregulated, and that of ATP binding cassette subfamily A member 1 (abca1), myosin light chain 6, and hippocampus abundant transcript 1 was downregulated in t-banglene-treated PC12 cells, with statistically significant differences. We also confirmed the RT1-CE1 upregulation and abca1 downregulation in t-banglene-treated PC12 cells by real-time reverse transcription quantitative PCR. RT1-CEl is a major histocompatibility complex class I (MHCI) protein. ABCAl is a major cholesterol transporter that regulates efflux of intracellular cholesterol and phospholipids. Thus, our results suggest an exciting link between MHCI, cholesterol regulation, and neural development.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Colesterol , Animales , Humanos , Ratas , Células PC12 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Colesterol/metabolismo , Regulación hacia Arriba , Perfilación de la Expresión Génica , Transportador 1 de Casete de Unión a ATP/genéticaRESUMEN
Recently, the novel coronavirus (SARS-CoV-2), which has spread from China to the world, was declared a global public health emergency, which causes lethal respiratory infections. Acetylation of several proteins plays essential roles in various biological processes, such as viral infections. We reported that the nucleoproteins of influenza virus and Zaire Ebolavirus were acetylated, suggesting that these modifications contributed to the molecular events involved in viral replication. Similar to influenza virus and Ebolavirus, the coronavirus also contains single-stranded RNA, as its viral genome interacts with the nucleocapsid (N) proteins. In this study, we report that SARS-CoV and SARS-CoV-2 N proteins are strongly acetylated by human histone acetyltransferases, P300/CBP-associated factor (PCAF), and general control nonderepressible 5 (GCN5), but not by CREB-binding protein (CBP) in vitro. Liquid chromatography-mass spectrometry analyses identified 2 and 12 acetyl-lysine residues from SARS-CoV and SARS-CoV-2 N proteins, respectively. Particularly in the SARS-CoV-2 N proteins, the acetyl-lysine residues were localized in or close to several functional sites, such as the RNA interaction domains and the M-protein interacting site. These results suggest that acetylation of SARS-CoV-2 N proteins plays crucial roles in their functions.
Asunto(s)
COVID-19/metabolismo , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Histona Acetiltransferasas/metabolismo , SARS-CoV-2/metabolismo , Síndrome Respiratorio Agudo Grave/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Proteína de Unión a CREB/metabolismo , Proteínas de la Nucleocápside de Coronavirus/química , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , SARS-CoV-2/químicaRESUMEN
Histone acetylation plays crucial roles in transcriptional regulation and chromatin organization. Viral RNA of the influenza virus interacts with its nucleoprotein (NP), whose function corresponds to that of eukaryotic histones. NP regulates viral replication and has been shown to undergo acetylation by the cAMP-response element (CRE)-binding protein (CBP) from the host. However, whether NP is the target of other host acetyltransferases is unknown. Here, we show that influenza virus NP undergoes acetylation by the two host acetyltransferases GCN5 and P300/CBP-associated factor (PCAF) and that this modification affects viral polymerase activities. Western blot analysis with anti-acetyl-lysine antibody on cultured A549 human lung adenocarcinoma epithelial cells infected with different influenza virus strains indicated acetylation of the viral NP. A series of biochemical analyses disclosed that the host lysine acetyltransferases GCN5 and PCAF acetylate NP in vitro MS experiments identified three lysine residues as acetylation targets in the host cells and suggested that Lys-31 and Lys-90 are acetylated by PCAF and GCN5, respectively. RNAi-mediated silencing of GCN5 and PCAF did not change acetylation levels of NP. However, interestingly, viral polymerase activities were increased by the PCAF silencing and were decreased by the GCN5 silencing, suggesting that acetylation of the Lys-31 and Lys-90 residues has opposing effects on viral replication. Our findings suggest that epigenetic control of NP via acetylation by host acetyltransferases contributes to regulation of polymerase activity in the influenza A virus.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Virus de la Influenza A/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas del Núcleo Viral/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Células A549 , Acetilación , Secuencia de Aminoácidos , Western Blotting , Cromatografía Liquida , Epigénesis Genética , Células Epiteliales/virología , Histona Acetiltransferasas/genética , Humanos , Virus de la Influenza A/enzimología , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Lisina/metabolismo , Proteínas de la Nucleocápside , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Viral/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Espectrometría de Masas en Tándem , Transcripción Genética , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Replicación Viral , Factores de Transcripción p300-CBP/genéticaRESUMEN
Jiadifenolide has been reported to have neurotrophin-like activity in primary rat cortical neurons, and also possesses neurotrophic effects in neuronal precursor cells derived from human induced pluripotent stem cells (hiPSCs), as we have previously reported. However, the molecular mechanisms by which jiadifenolide exerts its neurotrophic effects in rat and human neurons are unknown. Thus, we aimed to investigate the molecular mechanisms and pathways by which jiadifenolide promotes neurotrophic effects. Here, we found that jiadifenolide activated cellular communication network factor (CCN) signaling pathways by up-regulating mRNA level expression of CCN genes in human neuronal cells. We also found that CCN2 (also known as connective tissue growth factor, CTGF) protein promotes neurotrophic effects through activation of the p44/42 mitogen-activated protein kinase signaling pathway. This is the first discovery which links neurotrophic activity with CCN signaling.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Sesquiterpenos/farmacología , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Sesquiterpenos/síntesis química , Sesquiterpenos/químicaRESUMEN
Acetylation of histones and other proteins plays crucial roles in transcriptional regulation, chromatin organization, and other biological processes. It has been recently reported that the nucleoprotein (NP) of influenza virus is acetylated in infected cells, and this modification contributes to the RNA polymerization activity of the virus. As the influenza virus, the Ebolavirus contains single-stranded negative-sense RNA as its viral genome, which interacts with NP and other viral proteins. In this study, we performed a series of biochemical experiments and revealed that the recombinant Ebolavirus NP and the viral matrix protein VP40, which binds with NP, were acetylated by eukaryotic histone acetyltransferases, such as P300/CREB-binding protein (P300/CBP) and P300/CBP-associated factor (PCAF), in vitro. Mass spectrometry was used to identify the lysine residues that were potential acetylation targets in NP and VP40. The identified lysine residues in NP were located in the RNA-binding cleft and the VP35-binding domain. Potentially acetylated lysine targets in VP40 were identified in the basic patch, which is necessary for constructing oligomers. These results suggest that the acetylation of these lysine residues is involved in the interactions between viral proteins.
Asunto(s)
Ebolavirus/metabolismo , Lisina/metabolismo , Nucleoproteínas/metabolismo , Proteínas de la Matriz Viral/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Ebolavirus/genética , Humanos , Espectrometría de Masas , Nucleoproteínas/genética , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo , Proteínas de la Matriz Viral/genéticaRESUMEN
BACKGROUND: In the search for novel antidepressive drug candidates, bioguided fractionation of nonpolar constituents present in the oleoresin from ginger rhizomes (Zingiber officinale Roscoe, Zingiberaceae) was performed. This particular direction of the research was chosen due to the existing reports on the antidepressive properties of ginger total extract. The search for individual metabolites acting as MAO-A inhibitors, which correspond to the apparent effect of the total extract, is the subject of this work. METHODS: Hexane extracts from ginger rhizomes were fractionated by using column chromatography (including silica gel impregnated with silver nitrate) and semi-preparative high-performance chromatography. For the activity assessment, an in vitro monoamine oxidase A (MAO-A) inhibition luminescence assay was performed on 10 purified terpenes: 1,8-cineole, α-citronellal, geraniol, ß-sesquiphellandrene, γ-terpinen, geranyl acetate, isobornyl acetate, terpinen-4-ol, (E,E)-α-farnesene, and α-zingiberene. RESULTS: Geraniol and (-)-terpinen-4-ol were found to be the strongest enzyme inhibitors with inhibition of 44.1% and 42.5%, respectively, at a concentration of 125 µg/mL. No differences in the inhibition potential were observed for the different groups of terpenes: sesquiterpenes, monoterpenes, or sesquiterpene alcohols; however, the two most active compounds contained a hydroxyl moiety. CONCLUSIONS: Terpene constituents from ginger's extract were found to exhibit moderate inhibitory properties against the MAO-A enzyme in in vitro tests.
Asunto(s)
Antidepresivos/química , Inhibidores de la Monoaminooxidasa/química , Monoaminooxidasa/química , Terpenos/química , Zingiber officinale/química , Monoterpenos Acíclicos , Antidepresivos/aislamiento & purificación , Pruebas de Enzimas , Hexanos/química , Humanos , Extracción Líquido-Líquido/métodos , Inhibidores de la Monoaminooxidasa/aislamiento & purificación , Rizoma/química , Solventes/química , Terpenos/clasificación , Terpenos/aislamiento & purificaciónRESUMEN
BACKGROUND: To contribute to the development of novel anti-influenza drugs, we investigated the anti-influenza activity of crude extracts from 118 medicinal plants collected in Myanmar. We discovered that extract from the stems of Jatropha multifida Linn. showed anti-influenza activity. J. multifida has been used in traditional medicine for the treatment of various diseases, and the stem has been reported to possess antimicrobial, antimalarial, and antitumor activities. However, the anti-influenza activity of this extract has not yet been investigated. METHODS: We prepared water (H2O), ethyl acetate (EtOAc), n-hexane (Hex), and chloroform (CHCl3) extracts from the stems of J. multifida collected in Myanmar, and examined the survival of Madin-Darby canine kidney (MDCK) cells infected with the influenza A (H1N1) virus, and the inhibitory effects of these crude extracts on influenza A viral infection and growth in MDCK cells. RESULTS: The H2O extracts from the stems of J. multifida promoted the survival of MDCK cells infected with the influenza A H1N1 virus. The EtOAc and CHCl3 extracts resulted in similar, but weaker, effects. The H2O, EtOAc, and CHCl3 extracts from the stems of J. multifida inhibited influenza A virus H1N1 infection; the H2O extract possessed the strongest inhibitory effect on influenza infection in MDCK cells. The EtOAc, Hex, and CHCl3 extracts all inhibited the growth of influenza A H1N1 virus, and the CHCl3 extract demonstrated the strongest activity in MDCK cells. CONCLUSION: The H2O or CHCl3 extracts from the stems of J. multifida collected in Myanmar demonstrated the strongest inhibition of influenza A H1N1 viral infection or growth in MDCK cells, respectively. These results indicated that the stems of J. multifida could be regarded as an anti-influenza herbal medicine as well as a potential crude drug source for the development of anti-influenza compounds.
Asunto(s)
Antivirales/uso terapéutico , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/uso terapéutico , Animales , Antivirales/farmacología , Línea Celular , Perros , Humanos , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Medicina Tradicional , Mianmar , Extractos Vegetales/farmacología , Tallos de la PlantaRESUMEN
Influenza represents a substantial threat to human health and requires novel therapeutic approaches. Bakuchiol is a phenolic isoprenoid compound present in Babchi (Psoralea corylifolia L.) seeds. We examined the anti-influenza viral activity of synthetic bakuchiol using Madin-Darby canine kidney cells. We found that the naturally occurring form, (+)-(S)-bakuchiol, and its enantiomer, (-)-(R)-bakuchiol, inhibited influenza A viral infection and growth and reduced the expression of viral mRNAs and proteins in these cells. Furthermore, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-ß and myxovirus-resistant protein 1. Interestingly, (+)-(S)-bakuchiol had greater efficacy than (-)-(R)-bakuchiol, indicating that chirality influenced anti-influenza virus activity. In vitro studies indicated that bakuchiol did not strongly inhibit the activities of influenza surface proteins or the M2 ion channel, expressed in Chinese hamster ovary cells. Analysis of luciferase reporter assay data unexpectedly indicated that bakuchiol may induce some host cell factor(s) that inhibited firefly and Renilla luciferases. Next generation sequencing and KeyMolnet analysis of influenza A virus-infected and non-infected cells exposed to bakuchiol revealed activation of transcriptional regulation by nuclear factor erythroid 2-related factor (Nrf), and an Nrf2 reporter assay showed that (+)-(S)-bakuchiol activated Nrf2. Additionally, (+)-(S)-bakuchiol up-regulated the mRNA levels of two Nrf2-induced genes, NAD(P)H quinone oxidoreductase 1 and glutathione S-transferase A3. These findings demonstrated that bakuchiol had enantiomer-selective anti-influenza viral activity involving a novel effect on the host cell oxidative stress response.
Asunto(s)
Antivirales/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/virología , Estrés Oxidativo/efectos de los fármacos , Fenoles/farmacología , Terpenos/farmacología , Animales , Antivirales/química , Células CHO , Cricetinae , Cricetulus , Perros , Glutatión Transferasa/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Interferón beta/metabolismo , Células de Riñón Canino Madin Darby , NAD(P)H Deshidrogenasa (Quinona) , Factor 2 Relacionado con NF-E2/metabolismo , Infecciones por Orthomyxoviridae/virología , Fenoles/química , ARN Mensajero/efectos de los fármacos , ARN Viral/efectos de los fármacos , Terpenos/química , Transcripción GenéticaRESUMEN
Although jiadifenolide has been reported to neurotrophin-like activity in primary cultured rat cortical neurons, it is unknown on that of activity in human neurons. Thus, we aimed to assess neurotrophin-like activity by jiadifenolide in human neuronal cells. We analyzed neuronal precursor cells derived from human induced pluripotent stem cells for microtuble-associated-protein-2 expression by immunofluorescence and western blot, following jiadifenolide treatment. Jiadifenolide promoted dendrite outgrowth, facilitated growth, and prevented death in neuronal cells derived from human induced pluripotent stem cells. Interestingly, jiadifenolide also increased postsynaptic density-95 protein expression suggesting that jiadifenolide promotes neuronal maturation and post-synaptic formation. We demonstrate for the first time that jiadifenolide exhibits neurotrophic effects on human neuronal precursor cells.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Factores de Crecimiento Nervioso/administración & dosificación , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Sesquiterpenos/administración & dosificación , Diferenciación Celular , Aumento de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacosRESUMEN
The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m(7)GTP)) and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m(7)GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m(7)GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.
Asunto(s)
Acetilcoenzima A/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Acetiltransferasas/química , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular Tumoral , Perros , Células Eucariotas/enzimología , Humanos , Subtipo H3N2 del Virus de la Influenza A/enzimología , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/crecimiento & desarrollo , Células de Riñón Canino Madin Darby , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutación , Células Procariotas/enzimología , Unión Proteica , Estructura Terciaria de Proteína , Caperuzas de ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Ehlers-Danlos syndrome spondylodysplastic type 3 (EDSSPD3, OMIM 612350) is an inherited recessive connective tissue disorder that is caused by loss of function of SLC39A13/ZIP13, a zinc transporter belonging to the Slc39a/ZIP family. We previously reported that patients with EDSSPD3 harboring a homozygous loss of function mutation (c.221G > A, p.G64D) in ZIP13 exon 2 (ZIP13G64D) suffer from impaired development of bone and connective tissues, and muscular hypotonia. However, whether ZIP13 participates in the early differentiation of these cell types remains unclear. In the present study, we investigated the role of ZIP13 in myogenic differentiation using a murine myoblast cell line (C2C12) as well as patient-derived induced pluripotent stem cells (iPSCs). We found that ZIP13 gene expression was upregulated by myogenic stimulation in C2C12 cells, and its knockdown disrupted myotubular differentiation. Myocytes differentiated from iPSCs derived from patients with EDSSPD3 (EDSSPD3-iPSCs) also exhibited incomplete myogenic differentiation. Such phenotypic abnormalities of EDSSPD3-iPSC-derived myocytes were corrected by genomic editing of the pathogenic ZIP13G64D mutation. Collectively, our findings suggest the possible involvement of ZIP13 in myogenic differentiation, and that EDSSPD3-iPSCs established herein may be a promising tool to study the molecular basis underlying the clinical features caused by loss of ZIP13 function.
Asunto(s)
Proteínas Portadoras , Síndrome de Ehlers-Danlos , Osteocondrodisplasias , Animales , Humanos , Ratones , Diferenciación Celular/genéticaRESUMEN
Human-induced pluripotent stem cells (hiPSCs) are useful tools to examine human neuronal maturation processes. In this chapter, we describe the maturation of human neuronal precursor cells derived from hiPSCs by cellular communication network family member 2, also known as connective tissue growth factor. We describe the (1) preparation of feeder cells for undifferentiated culture of hiPSCs, (2) undifferentiated culture of hiPSCs, (3) induction of neuronal precursor cells from hiPSCs, (4) maturation of neuronal precursor cells from hiPSCs, (5) immunofluorescent staining of neuronal cells from hiPSCs, and (6) immunofluorescence analysis.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/farmacología , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Células Nutrientes , Neuronas , Comunicación Celular , Diferenciación CelularRESUMEN
The endonuclease activity of influenza A virus RNA polymerase allows the digestion of host mRNA. The PA endonuclease domain could be a target of anti-influenza A virus drugs such as glycoconjugates. To test this activity, purified viral PA endonuclease domain protein is necessary. Here, we describe a method for the expression and purification of recombinant influenza A virus PA endonuclease domain protein, and a PA endonuclease assay to test glycoconjugates for potential inhibitory activity. Using influenza A virus PA cDNA as a template, DNA from the open reading frame of the PA endonuclease domain protein was amplified with PCR and cloned into an expression vector. Six His-tagged PA endonuclease domain proteins were expressed in Escherichia coli and purified with Ni2+-agarose resin and imidazole using an ion-exchange column. Using the recombinant PA endonuclease domain protein, an endonuclease assay was performed.
Asunto(s)
Glicómica , Virus de la Influenza A , ADN Complementario , ARN Polimerasas Dirigidas por ADN , Endonucleasas/genética , Escherichia coli/genética , Imidazoles , Virus de la Influenza A/genética , SefarosaRESUMEN
The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity. Specifically, the full-length PA subunit expressed in cultured 293T cells was found to be strongly acetylated. Moreover, the partial recombinant protein of the PA N-terminal region containing the endonuclease domain was also acetylated by PCAF and GCN5 in vitro, which facilitated its endonuclease activity. Mass spectrometry analyses identified K19 as a candidate acetylation target in the PA N-terminal region. Notably, the substitution of the lysine residue at position 19 with glutamine, a mimic of the acetyl-lysine residue, enhanced its endonuclease activity in vitro; this point mutation also accelerated influenza A virus RNA-dependent RNA polymerase activity in the cell. Our findings suggest that PA acetylation is important for the regulation of the endonuclease and RNA polymerase activities of the influenza A virus.
Asunto(s)
Histona Acetiltransferasas/genética , Virus de la Influenza A/genética , Gripe Humana/genética , ARN Polimerasa Dependiente del ARN/genética , Factores de Transcripción p300-CBP/genética , Acetilación , Secuencia de Aminoácidos/genética , Humanos , Gripe Humana/virología , Nucleoproteínas/genética , Unión Proteica/genética , Procesamiento Proteico-Postraduccional/genética , ARN Viral/genética , Proteínas Virales/genética , Transcripción Viral/genéticaRESUMEN
Novel antiviral agents for influenza, which poses a substantial threat to humans, are required. Cyclobakuchiols A and B have been isolated from Psoralea glandulosa, and cyclobakuchiol C has been isolated from P. corylifolia. The structural differences between cyclobakuchiol A and C arise due to the oxidation state of isopropyl group, and these compounds can be derived from (+)-(S)-bakuchiol, a phenolic isoprenoid compound present in P. corylifolia seeds. We previously reported that bakuchiol induces enantiospecific anti-influenza A virus activity involving nuclear factor erythroid 2-related factor 2 (Nrf2) activation. However, it remains unclear whether cyclobakuchiols A-C induce anti-influenza A virus activity. In this study, cyclobakuchiols A, B, and C along with cyclobakuchiol D, a new artificial compound derived from cyclobakuchiol B, were synthesized and examined for their anti-influenza A virus activities using Madin-Darby canine kidney cells. As a result, cyclobakuchiols A-D were found to inhibit influenza A viral infection, growth, and the reduction of expression of viral mRNAs and proteins in influenza A virus-infected cells. Additionally, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-ß and myxovirus-resistant protein 1. In addition, cyclobakuchiols A-D upregulated the mRNA levels of NAD(P)H quinone oxidoreductase 1, an Nrf2-induced gene, in influenza A virus-infected cells. Notably, cyclobakuchiols A, B, and C, but not D, induced the Nrf2 activation pathway. These findings demonstrate that cyclobakuchiols have anti-influenza viral activity involving host cell oxidative stress response. In addition, our results suggest that the suitably spatial configuration between oxidized isopropyl group and phenol moiety in the structure of cyclobakuchiols is required for their effect.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Técnicas de Química Sintética , Ciclohexanos/síntesis química , Ciclohexanos/farmacología , Virus de la Influenza A/efectos de los fármacos , Animales , Antivirales/química , Supervivencia Celular/efectos de los fármacos , Ciclohexanos/química , Ciclohexanos/toxicidad , Perros , Regulación Viral de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador , Virus de la Influenza A/crecimiento & desarrollo , Interferón beta/genética , Interferón beta/metabolismo , Células de Riñón Canino Madin Darby , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Virales/metabolismoRESUMEN
Human influenza virus infections occur annually worldwide and are associated with high morbidity and mortality. Hence, development of novel anti-influenza drugs is urgently required. Rice Power® extract developed by the Yushin Brewer Co. Ltd. is a novel aqueous extract of rice obtained via saccharization and fermentation with various microorganisms, such as Aspergillus oryzae, yeast [such as Saccharomyces cerevisiae], and lactic acid bacteria, possessing various biological and pharmacological properties. In our previous experimental screening with thirty types of Rice Power® extracts, we observed that the 30th Rice Power® (Y30) extract promoted the survival of influenza A virus-infected Madin-Darby canine kidney (MDCK) cells. Therefore, to identify compounds for the development of novel anti-influenza drugs, we aimed to investigate whether the Y30 extract exhibits anti-influenza A virus activity. In the present study, we demonstrated that the Y30 extract strongly promoted the survival of influenza A H1N1 Puerto Rico 8/34 (A/PR/8/34), California 7/09, or H3N2 Aichi 2/68 (A/Aichi/2/68) viruses-infected MDCK cells and inhibited A/PR/8/34 or A/Aichi/2/68 viruses infection and growth in the co-treatment and pre-infection experiments. The pre-treatment of Y30 extract on MDCK cells did not induce anti-influenza activity in the cell. The Y30 extract did not significantly affect influenza A virus hemagglutination, and neuraminidase and RNA-dependent RNA polymerase activities. Interestingly, the electron microscopy experiment revealed that the Y30 extract disrupts the integrity of influenza A virus particles by permeabilizing the viral membrane envelope, suggesting that Y30 extract has a direct virucidal effect against influenza A virus. Furthermore, we observed that compared to the ethyl acetate (EtOAc) extract, the water extract of Y30 extract considerably promoted the survival of cells infected with A/PR/8/34 virus. These results indicated that more anti-influenza components were present in the water extract of Y30 extract than in the EtOAc extract. Our results highlight the potential of a rice extract fermented with A. oryzae and S. cerevisiae as an anti-influenza medicine and a drug source for the development of anti-influenza compounds.
Asunto(s)
Aspergillus oryzae/metabolismo , Virus de la Influenza A/efectos de los fármacos , Oryza/química , Oryza/microbiología , Extractos Vegetales/farmacología , Saccharomyces cerevisiae/metabolismo , Agua/química , Acetatos/química , Animales , Antivirales/farmacología , Perros , Fermentación , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , Viabilidad Microbiana/efectos de los fármacosRESUMEN
Swine-origin influenza A virus has caused pandemics throughout the world and influenza A is regarded as a serious global health issue. Hence, novel drugs that will target these viruses are very desirable. Influenza A expresses an RNA polymerase essential for its transcription and replication which comprises PA, PB1, and PB2 subunits. We identified potential novel anti-influenza agents from a screen of 34 synthesized phenethylphenylphthalimide analogs derived from thalidomide (PPT analogs). For this screen we used a PA endonuclease inhibition assay, a PB2 pathogenicity-determinant domain-binding assay, and an anti-influenza A virus assay. Three PPT analogs, PPT-65, PPT-66, and PPT-67, were found to both inhibit PA endonuclease activity and retard the growth of influenza A, suggesting a correlation between their activities. PPT-28 was also found to inhibit the growth of influenza A. These four analogs have a 3,4-dihydroxyphenethyl group in common. We also discuss the possibility that 3,4-dihydroxyphenethyl group flexibility may play an important functional role in PA endonuclease inhibition. Another analog harboring a dimethoxyphenethyl group, PPT-62, showed PB2 pathogenicity-determinant domain-binding activity, but did not inhibit the growth of the virus. Our present results indicate the utility of the PA endonuclease assay in the screening of anti-influenza drugs and are therefore useful for future strategies to develop novel anti-influenza A drugs and for mapping the function of the influenza A RNA polymerase subunits.