RESUMEN
Although respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants, immunosuppressed adults and the elderly worldwide, there is no licensed RSV vaccine or widely applicable antiviral therapeutics We previously reported a staged redistribution of mitochondria with compromised respiratory activities and increased reactive oxygen species (ROS) generation during RSV infection. Here, we show for the first time that the RSV matrix protein (M) is sufficient and necessary to induce these effects. Ectopically expressed M, but not other RSV proteins, was able to induce mitochondrial perinuclear clustering, inhibition of mitochondrial respiration, loss of mitochondrial membrane potential (Δψm), and enhanced generation of mitochondrial ROS (mtROS) in infection. Truncation and mutagenic analysis revealed that the central nucleic acid-binding domain of M is essential for the effects on host mitochondria, with arginine/lysine residues 170/172 being critically important. Recombinant RSV carrying the arginine/lysine mutations in M was unable to elicit effects on host mitochondria. Further, wild-type but not mutant RSV was found to inhibit the mRNA expression of genes encoding mitochondrial proteins, including Complex I subunits. Importantly, the RSV mutant was impaired in virus production, underlining the importance of M-dependent effects on mitochondria to RSV infection. Together, our results highlight M's unique ability to remodel host cell mitochondria and its critical role in RSV infection, representing a novel, potential target for future anti-RSV strategies.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Humanos , Anciano , Especies Reactivas de Oxígeno/metabolismo , Lisina , Mitocondrias/metabolismo , ArgininaRESUMEN
Bimolecular fluorescence complementation utilizes the ability of two complementary nonfluorescent fragments to reconstitute and emit fluorescence when brought together through specific interaction of attached protein fragments of interest. It has been used in several different contexts to study protein-protein interaction. Here we apply the method for the first time to study interaction of the nuclear transporter importin α and its cargoes in a cellular context. By using image analysis to quantify the extent of nuclear complexation, it is possible to gain insight into the strength of interaction in cells.
Asunto(s)
Comunicación Celular , Proteínas , Espectrometría de Fluorescencia , alfa Carioferinas , Comunicación Celular/fisiología , Núcleo Celular/metabolismo , Unión Proteica , Proteínas/metabolismo , Espectrometría de Fluorescencia/métodos , alfa Carioferinas/metabolismoRESUMEN
The expanded polyglutamine (polyQ) tract form of ataxin-1 drives disease progression in spinocerebellar ataxia type 1 (SCA1). Although known to form distinctive intranuclear bodies, the cellular pathways and processes that polyQ-ataxin-1 influences remain poorly understood. Here we identify the direct and proximal partners constituting the interactome of ataxin-1[85Q] in Neuro-2a cells, pathways analyses indicating a significant enrichment of essential nuclear transporters, pointing to disruptions in nuclear transport processes in the presence of elevated levels of ataxin-1. Our direct assessments of nuclear transporters and their cargoes confirm these observations, revealing disrupted trafficking often with relocalisation of transporters and/or cargoes to ataxin-1[85Q] nuclear bodies. Analogous changes in importin-ß1, nucleoporin 98 and nucleoporin 62 nuclear rim staining are observed in Purkinje cells of ATXN1[82Q] mice. The results highlight a disruption of multiple essential nuclear protein trafficking pathways by polyQ-ataxin-1, a key contribution to furthering understanding of pathogenic mechanisms initiated by polyQ tract proteins.
Asunto(s)
Ataxina-1/metabolismo , Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Células de Purkinje/metabolismo , Transporte Activo de Núcleo Celular/genética , Animales , Ataxina-1/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células HeLa , Humanos , Ratones , Mutación , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Péptidos/genética , Unión Proteica , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismo , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Respiratory syncytial virus (RSV) is one of the leading causes of viral respiratory tract infection in infants, the elderly, and the immunocompromised worldwide, causing more deaths each year than influenza. Years of research into RSV since its discovery over 60 yr ago have elucidated detailed mechanisms of the host-pathogen interface. RSV infection elicits widespread transcriptomic and proteomic changes, which both mediate the host innate and adaptive immune responses to infection, and reflect RSV's ability to circumvent the host stress responses, including stress granule formation, endoplasmic reticulum stress, oxidative stress, and programmed cell death. The combination of these events can severely impact on human lungs, resulting in airway remodeling and pathophysiology. The RSV membrane envelope glycoproteins (fusion F and attachment G), matrix (M) and nonstructural (NS) 1 and 2 proteins play key roles in modulating host cell functions to promote the infectious cycle. This review presents a comprehensive overview of how RSV impacts the host response to infection and how detailed knowledge of the mechanisms thereof can inform the development of new approaches to develop RSV vaccines and therapeutics.
Asunto(s)
Antivirales/farmacología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano , Vacunas Virales/inmunología , Humanos , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/inmunologíaRESUMEN
Infection by RNA viruses such as human immunodeficiency virus (HIV)-1, influenza, and dengue virus (DENV) represent a major burden for human health worldwide. Although RNA viruses replicate in the infected host cell cytoplasm, the nucleus is central to key stages of the infectious cycle of HIV-1 and influenza, and an important target of DENV nonstructural protein 5 (NS5) in limiting the host antiviral response. We previously identified the small molecule ivermectin as an inhibitor of HIV-1 integrase nuclear entry, subsequently showing ivermectin could inhibit DENV NS5 nuclear import, as well as limit infection by viruses such as HIV-1 and DENV. We show here that ivermectin's broad spectrum antiviral activity relates to its ability to target the host importin (IMP) α/ß1 nuclear transport proteins responsible for nuclear entry of cargoes such as integrase and NS5. We establish for the first time that ivermectin can dissociate the preformed IMPα/ß1 heterodimer, as well as prevent its formation, through binding to the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity. We show that ivermectin inhibits NS5-IMPα interaction in a cell context using quantitative bimolecular fluorescence complementation. Finally, we show for the first time that ivermectin can limit infection by the DENV-related West Nile virus at low (µM) concentrations. Since it is FDA approved for parasitic indications, ivermectin merits closer consideration as a broad spectrum antiviral of interest.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Ivermectina/farmacología , alfa Carioferinas/antagonistas & inhibidores , beta Carioferinas/antagonistas & inhibidores , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Infecciones por Flavivirus/tratamiento farmacológico , Riñón/citología , Unión Proteica , Células Vero , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismoRESUMEN
A mutant form of the ataxin-1 protein with an expanded polyglutamine (polyQ) tract is the underlying cause of the inherited neurodegenerative disease spinocerebellar ataxia 1 (SCA1). In probing the biophysical features of the nuclear bodies (NBs) formed by polyQ-ataxin-1, we defined ataxin-1 NBs as spherical liquid protein/RNA droplets capable of rapid fusion. We observed dynamic exchange of the ataxin-1 protein into these NBs; notably, cell exposure to a pro-oxidant stress could trigger a transition to slower ataxin-1 exchange, typical of a hydrogel state, which no longer showed the same dependence on RNA or sensitivity to 1,6-hexanediol. Furthermore, we could alter ataxin-1 exchange dynamics either through modulating intracellular ATP levels, RNA helicase inhibition, or siRNA-mediated depletion of select RNA helicases. Collectively, these findings reveal the tunable dynamics of the liquid RNA/protein droplets formed by polyQ-ataxin-1.
Asunto(s)
Ataxina-1/metabolismo , Gotas Lipídicas/metabolismo , ARN/metabolismo , Ataxias Espinocerebelosas/metabolismo , Animales , Ataxina-1/genética , Línea Celular Tumoral , Humanos , Fusión de Membrana , Ratones , Modelos Moleculares , Mutación/genética , Péptidos/química , Unión Proteica , Ataxias Espinocerebelosas/genéticaRESUMEN
Respiratory syncytial virus (RSV) is a key cause of severe respiratory infection in infants, immunosuppressed adults, and the elderly worldwide, but there is no licensed vaccine or effective, widely-available antiviral therapeutic. We recently reported staged redistribution of host cell mitochondria in RSV infected cells, which results in compromised respiratory activities and increased reactive oxygen species (ROS) generation. Here, bioenergetic measurements, mitochondrial redox-sensitive dye, and high-resolution quantitative imaging were performed, revealing for the first time that mitochondrial complex I is key to this effect on the host cell, whereby mitochondrial complex I subunit knock-out (KO) cells, with markedly decreased mitochondrial respiration, show elevated levels of RSV infectious virus production compared to wild-type cells or KO cells with re-expressed complex I subunits. This effect correlates strongly with elevated ROS generation in the KO cells compared to wild-type cells or retrovirus-rescued KO cells re-expressing complex I subunits. Strikingly, blocking mitochondrial ROS levels using the mitochondrial ROS scavenger, mitoquinone mesylate (MitoQ), inhibits RSV virus production, even in the KO cells. The results highlight RSV's unique ability to usurp host cell mitochondrial ROS to facilitate viral infection and reinforce the idea of MitoQ as a potential therapeutic for RSV.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Interacciones Huésped-Patógeno , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/fisiología , Células Epiteliales Alveolares/metabolismo , Biomarcadores , Supervivencia Celular , Humanos , Consumo de Oxígeno , Replicación ViralRESUMEN
Although respiratory syncytial virus (RSV) is responsible for more human deaths each year than influenza, its pathogenic mechanisms are poorly understood. Here high-resolution quantitative imaging, bioenergetics measurements and mitochondrial membrane potential- and redox-sensitive dyes are used to define RSV's impact on host mitochondria for the first time, delineating RSV-induced microtubule/dynein-dependent mitochondrial perinuclear clustering, and translocation towards the microtubule-organizing centre. These changes are concomitant with impaired mitochondrial respiration, loss of mitochondrial membrane potential and increased production of mitochondrial reactive oxygen species (ROS). Strikingly, agents that target microtubule integrity the dynein motor protein, or inhibit mitochondrial ROS production strongly suppresses RSV virus production, including in a mouse model with concomitantly reduced virus-induced lung inflammation. The results establish RSV's unique ability to co-opt host cell mitochondria to facilitate viral infection, revealing the RSV-mitochondrial interface for the first time as a viable target for therapeutic intervention.
Asunto(s)
Interacciones Huésped-Patógeno , Mitocondrias/patología , Virus Sincitiales Respiratorios/crecimiento & desarrollo , Replicación Viral , Células A549 , Animales , Modelos Animales de Enfermedad , Dineínas/metabolismo , Humanos , Pulmón/patología , Pulmón/virología , Ratones , Microtúbulos/metabolismo , Infecciones por Virus Sincitial Respiratorio/patologíaRESUMEN
Mutations of the microtubule (MT)-associated protein Doublecortin X (DCX) gene disrupt cortical layering in brain development. Whilst many of these pathogenic DCX mutations are within the doublecortin domains (DC1 and DC2) that mediate direct DCX-MT association, a pathogenic mutation DCX E2K that causes cognitive impairment and pachygyria in human patients lies within the regulatory DCX N-terminus (DCX-N) preceding the DC1 domain. Here, we characterise the impact of DCX E2K on cytoskeletal association and regulation in neuronal cells. We show that the DCX E2K mutant protein retains the ability to interact with and bundle MTs, but these MTs show a reduced sensitivity to nocodazole-induced depolymerisation as well as slower α-tubulin exchange rates. Furthermore, we showed increased association of DCX E2K mutant with the actin filament (F-ACT) network. These results highlight the importance of the N-terminus of DCX in regulating association and co-ordination of MT and F-ACT networks.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Mutación , Neuropéptidos/genética , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptidos/metabolismo , Nocodazol/farmacología , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
Signal transducer and activator of transcription 3 (STAT3), a transcription factor responsive to the activation of cytokine receptors, is known for its oncogenic actions. Whilst STAT3α is the predominant spliceform in most tissues, alternative splicing of the STAT3 gene can generate a shorter STAT3ß spliceform. Redirecting splicing to enhance STAT3ß levels can result in tumor suppression in vivo, and so we evaluated the cellular basis underlying the anti-tumorigenic properties of STAT3ß. To investigate the impact of increased STAT3ß levels in cancer cells, we implemented a Morpholino-based antisense oligonucleotide strategy to modulate STAT3 spliceform expression in the MCF10CA1h cancer cells of the MCF10 series of human breast cancer cells. We employed nonsense-mediated decay (NMD) oligonucleotides and STAT3α-to-ß expression switching (SWI) oligonucleotides to successfully induce STAT3 knockdown and redirect alternative splicing to increase STAT3ß levels in MCF10CA1h cells, respectively. Importantly, assessment of the impacts of STAT3 splicing modulation on tumor cell biology showed that the SWI treatment significantly reduced MCF10CA1h cell growth, viability, and migration, whereas NMD treatment was without significant impact, although neither NMD nor SWI oligonucleotides significantly inhibited MCF10CA1h cell invasion through a semi-solid matrix. In conclusion, our data demonstrate that reduced breast cancer cell growth, viability and migration, but not invasion, follow the redirection of STAT3α-to-ß expression switching to favour STAT3ß expression.
Asunto(s)
Empalme Alternativo , Neoplasias de la Mama/patología , Oligonucleótidos/farmacología , Factor de Transcripción STAT3/metabolismo , Carcinogénesis , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Invasividad Neoplásica , Isoformas de Proteínas/farmacologíaRESUMEN
Doublecortin X (DCX) plays essential roles in neuronal development via its regulation of cytoskeleton dynamics. This is mediated through direct interactions between its doublecortin (DC) domains (DC1 and DC2) with microtubules (MTs) and indirect association with actin filaments (F-ACT). While the regulatory role of the DCX C-terminus following DC2 (i.e. DCX residues 275-366) has been established, less is known of the possible contributions made by the DCX N-terminus preceding DC1 (i.e. DCX residues 1-44). Here, we assessed the influence of DCX Ser28 within the DCX N-terminus, on the association of DCX with MTs and F-ACT. We compared the cytoskeletal interactions of the DCX S28E phosphomimetic and DCX S28A phospho-resistant mutants and wild-type DCX. Immunoprecipitation and colocalisation analyses indicated increased association of DCX S28E with F-ACT but decreased interaction with MTs, and conversely enhanced DCX S28A association with MTs but decreased association with F-ACT. To evaluate the impact of DCX mutants on cytoskeletal filaments we performed fluorescence recovery after photobleaching (FRAP) studies on SiR-tubulin and ß-actin-mCherry and observed comparable tubulin and actin exchange rates in the presence of DCX WT and DCX S28A. However, we observed faster tubulin exchange rates but slower actin exchange rates in the presence of DCX S28E. Moreover, DCX S28E enhanced the association with the actin-binding protein spinophilin (Spn) suggesting the shift to favour association with both F-ACT and Spn in the presence of DCX S28E. Taken together, our results highlight a new role for DCX S28 as a regulatory switch for cytoskeletal organisation.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuropéptidos/metabolismo , Actinas/metabolismo , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Quinasa 5 Dependiente de la Ciclina/metabolismo , Citoesqueleto/ultraestructura , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/química , Fosforilación , Serina/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Ataxin-1 mutation, arising from a polyglutamine (polyQ) tract expansion, is the underlying genetic cause of the late-onset neurodegenerative disease Spinocerebellar ataxia type 1 (SCA1). To identify protein partners of polyQ-ataxin-1 in neuronal cells under control or stress conditions, here we report our complementary proteomics strategies of proximity-dependent biotin identification (BioID) and affinity purification (via GFP-Trap pulldown) in Neuro-2a cells expressing epitope-tagged forms of ataxin-1[85Q]. These approaches allowed our enrichment of proximal proteins and interacting partners, respectively, with the subsequent protein identification performed by liquid chromatography-MS/MS. Background proteins, not dependent on the presence of the polyQ-ataxin-1 protein, were additionally defined by their endogenous biotinylation (for the BioID protocol) or by their non-specific interaction with GFP only (in the GFP-Trap protocol). All datasets were generated from biological replicates. Following the removal of the identified background proteins from the acquired protein lists, our experimental design has captured a comprehensive polyQ-ataxin-1 proximal and direct protein partners under normal and stress conditions. Data are available via ProteomeXchange, with identifier PXD010352.
Asunto(s)
Ataxina-1 , Péptidos , Mapas de Interacción de Proteínas , Proteómica/métodos , Animales , Ataxina-1/metabolismo , Ataxina-1/fisiología , Línea Celular , Ratones , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Neuronas/metabolismo , Espectrometría de Masas en Tándem , Repeticiones de TrinucleótidosRESUMEN
BACKGROUND: A strong focus of the post-genomic era is mining of the non-coding regulatory genome in order to unravel the function of regulatory elements that coordinate gene expression (Nat 489:57-74, 2012; Nat 507:462-70, 2014; Nat 507:455-61, 2014; Nat 518:317-30, 2015). Whole-genome approaches based on next-generation sequencing (NGS) have provided insight into the genomic location of regulatory elements throughout different cell types, organs and organisms. These technologies are now widespread and commonly used in laboratories from various fields of research. This highlights the need for fast and user-friendly software tools dedicated to extracting cis-regulatory information contained in these regulatory regions; for instance transcription factor binding site (TFBS) composition. Ideally, such tools should not require prior programming knowledge to ensure they are accessible for all users. RESULTS: We present TrawlerWeb, a web-based version of the Trawler_standalone tool (Nat Methods 4:563-5, 2007; Nat Protoc 5:323-34, 2010), to allow for the identification of enriched motifs in DNA sequences obtained from next-generation sequencing experiments in order to predict their TFBS composition. TrawlerWeb is designed for online queries with standard options common to web-based motif discovery tools. In addition, TrawlerWeb provides three unique new features: 1) TrawlerWeb allows the input of BED files directly generated from NGS experiments, 2) it automatically generates an input-matched biologically relevant background, and 3) it displays resulting conservation scores for each instance of the motif found in the input sequences, which assists the researcher in prioritising the motifs to validate experimentally. Finally, to date, this web-based version of Trawler_standalone remains the fastest online de novo motif discovery tool compared to other popular web-based software, while generating predictions with high accuracy. CONCLUSIONS: TrawlerWeb provides users with a fast, simple and easy-to-use web interface for de novo motif discovery. This will assist in rapidly analysing NGS datasets that are now being routinely generated. TrawlerWeb is freely available and accessible at: http://trawler.erc.monash.edu.au .
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Animales , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , ADN/química , ADN/metabolismo , Humanos , Internet , Mesotelina , Ratones , Motivos de Nucleótidos , Ratas , Factores de Transcripción/metabolismoRESUMEN
Doublecortin X (DCX), known to be essential for neuronal migration and cortical layering in the developing brain, is a 40 kDa microtubule (MT)-associated protein. DCX directly interacts with MTs via its two structured doublecortin (DC) domains, but the dynamics of this association and the possible regulatory roles played by the flanking unstructured regions remain poorly defined. Here, we employ quantitative fluorescence recovery after photobleaching (FRAP) protocols in living cells to reveal that DCX shows remarkably rapid and complete exchange within the MT network but that the removal of the C-terminal region significantly slows this exchange. We further probed how MT organization or external stimuli could additionally modulate DCX exchange dynamics. MT depolymerisation (nocodazole treatment) or stabilization (taxol treatment) further enhanced DCX exchange rates, however the exchange rates for the C-terminal truncated DCX protein were resistant to the impact of taxol-induced stabilization. Furthermore, in response to a hyperosmotic stress stimulus, DCX exchange dynamics were slowed, and again the C-terminal truncated DCX protein was resistant to the stimulus. Thus, the DCX dynamically associates with MTs in living cells and its C-terminal region plays important roles in the MT-DCX association.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuroblastoma/metabolismo , Neuropéptidos/metabolismo , Animales , Células COS , Movimiento Celular , Células Cultivadas , Chlorocebus aethiops , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Humanos , Proteínas Asociadas a Microtúbulos/genética , Neuroblastoma/patología , Neuropéptidos/genética , Dominios ProteicosRESUMEN
Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly, leading to more deaths than influenza each year, but there is no antiviral or efficacious vaccine currently available. Here we examine the role in infection of the host mitochondrial protein p32 (HABP/gC1qR/C1qbp) for the first time. RSV replication as well as infectious virus production was significantly reduced by p32 siRNA knockdown, consistent with an important role for p32 in RSV infection. p32 showed distinct mitochondrial localization throughout RSV infection, but immunostaining and high resolution confocal imaging for p32 as well as MitoTracker Red and cytochrome c, revealed clear changes in mitochondrial organization in RSV infection, with perinuclear mitochondrial compaction and asymmetric distribution at 8 and 18 h post-infection, respectively. The results implicate p32 as a key host factor for RSV virus production, and bring to light the potential importance of mitochondria in RSV infection.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Mitocondriales/metabolismo , Virus Sincitiales Respiratorios/crecimiento & desarrollo , Virus Sincitiales Respiratorios/metabolismo , Células A549 , Proteínas Portadoras/genética , Humanos , Proteínas Mitocondriales/genética , ARN Interferente Pequeño/genética , Células Tumorales CultivadasRESUMEN
Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly, leading to more deaths than influenza each year worldwide. With no RSV antiviral or efficacious vaccine currently available, improved understanding of the host-RSV interaction is urgently required. Here we examine the contribution to RSV infection of the host stress-regulated c-Jun N-terminal kinase (JNK), for the first time. Peak JNK1/2 phosphoactivation is observed at â¼24 h post-infection, correlating with the time of virus assembly. The release of infectious RSV virions from infected cells was significantly reduced by either JNK1/2 siRNA knockdown or treatment with the JNK-specific inhibitor, JNK-IN-VIII. High resolution microscopy confirmed RSV accumulation in the host cell cytoplasm. The results implicate JNK1/2 as a key host factor for RSV virus production, raising the possibility of agents targeting JNK activity as potential anti-RSV therapeutics.
Asunto(s)
Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Virus Sincitial Respiratorio Humano/fisiología , Replicación Viral/fisiología , Células A549 , Activación Enzimática , Técnicas de Silenciamiento del Gen , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 9 Activada por Mitógenos/genética , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/genética , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Virus Sincitial Respiratorio Humano/patogenicidad , Virión/fisiología , Ensamble de Virus/fisiología , Liberación del Virus/fisiologíaRESUMEN
The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states.
Asunto(s)
Células Eucariotas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Animales , Células Eucariotas/enzimología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Fosforilación/fisiología , Isoformas de Proteínas/genéticaRESUMEN
Oligomerization of transcription factors controls their translocation into the nucleus and DNA-binding activity. Here we present a fluorescence microscopy analysis termed pCOMB (pair correlation of molecular brightness) that tracks the mobility of different oligomeric species within live cell nuclear architecture. pCOMB amplifies the signal from the brightest species present and filters the dynamics of the extracted oligomeric population based on arrival time between two locations. We use this method to demonstrate a dependence of signal transducer and activator of transcription 3 (STAT3) mobility on oligomeric state. We find that on entering the nucleus STAT3 dimers must first bind DNA to form STAT3 tetramers, which are also DNA-bound but exhibit a different mobility signature. Examining the dimer-to-tetramer transition by a cross-pair correlation analysis (cpCOMB) reveals that chromatin accessibility modulates STAT3 tetramer formation. Thus, the pCOMB approach is suitable for mapping the impact oligomerization on transcription factor dynamics.
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Microscopía Fluorescente/métodos , Multimerización de Proteína , Factor de Transcripción STAT3/metabolismo , Calibración , Supervivencia Celular/efectos de los fármacos , Cromatina/metabolismo , Citocinas/farmacología , ADN/metabolismo , Células HeLa , Humanos , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacosRESUMEN
Mitotic spindle organization is regulated by centrosomal kinases that potentiate recruitment of spindle-associated proteins required for normal mitotic progress including the microcephaly protein WD40-repeat protein 62 (WDR62). WDR62 functions underlie normal brain development as autosomal recessive mutations and wdr62 loss cause microcephaly. Here we investigate the signaling interactions between WDR62 and the mitotic kinase Aurora A (AURKA) that has been recently shown to cooperate to control brain size in mice. The spindle recruitment of WDR62 is closely correlated with increased levels of AURKA following mitotic entry. We showed that depletion of TPX2 attenuated WDR62 localization at spindle poles indicating that TPX2 co-activation of AURKA is required to recruit WDR62 to the spindle. We demonstrated that AURKA activity contributed to the mitotic phosphorylation of WDR62 residues Ser49 and Thr50 and phosphorylation of WDR62 N-terminal residues was required for spindle organization and metaphase chromosome alignment. Our analysis of several MCPH-associated WDR62 mutants (V65M, R438H and V1314RfsX18) that are mislocalized in mitosis revealed that their interactions and phosphorylation by AURKA was substantially reduced consistent with the notion that AURKA is a key determinant of WDR62 spindle recruitment. Thus, our study highlights the role of AURKA signaling in the spatiotemporal control of WDR62 at spindle poles where it maintains spindle organization.
Asunto(s)
Aurora Quinasa A/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Huso Acromático/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Segregación Cromosómica , Células HeLa , Humanos , Metafase , Ratones , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Src-family kinases (SFKs) are involved in neuronal survival and their aberrant regulation contributes to neuronal death. However, how they control neuronal survival and death remains unclear. OBJECTIVE: To define the effect of inhibition of Src activity and expression on neuronal survival. RESULTS: In agreement with our previous findings, we demonstrated that Src was cleaved by calpain to form a 52-kDa truncated fragment in neurons undergoing excitotoxic cell death, and expression of the recombinant truncated Src fragment induced neuronal death. The data confirm that the neurotoxic signaling pathways are intact in the neurons we used for our study. To define the functional role of neuronal SFKs, we treated these neurons with SFK inhibitors and discovered that the treatment induced cell death, suggesting that the catalytic activity of one or more of the neuronal SFKs is critical to neuronal survival. Using small hairpin RNAs that suppress Src expression, we demonstrated that Src is indispensable to neuronal survival. Additionally, we found that neuronal death induced by expression of the neurotoxic truncated Src mutant, treatment of SFK inhibitors or knock-down of Src expression caused inhibition of the neuroprotective protein kinases Erk1/2, or Akt. CONCLUSIONS: Src is critical to both neuronal survival and death. Intact Src sustains neuronal survival. However, in the excitotoxic condition, calpain cleavage of Src generates a neurotoxic truncated Src fragment. Both intact Src and the neurotoxic truncated Src fragment exert their biological actions by controlling the activities of neuroprotective protein kinases.