RESUMEN
How the cuticles of the roughly 4.5 million species of ecdysozoan animals are constructed is not well understood. Here, we systematically mine gene expression datasets to uncover the spatiotemporal blueprint for how the chitin-based pharyngeal cuticle of the nematode Caenorhabditis elegans is built. We demonstrate that the blueprint correctly predicts expression patterns and functional relevance to cuticle development. We find that as larvae prepare to molt, catabolic enzymes are upregulated and the genes that encode chitin synthase, chitin cross-linkers, and homologs of amyloid regulators subsequently peak in expression. Forty-eight percent of the gene products secreted during the molt are predicted to be intrinsically disordered proteins (IDPs), many of which belong to four distinct families whose transcripts are expressed in overlapping waves. These include the IDPAs, IDPBs, and IDPCs, which are introduced for the first time here. All four families have sequence properties that drive phase separation and we demonstrate phase separation for one exemplar in vitro. This systematic analysis represents the first blueprint for cuticle construction and highlights the massive contribution that phase-separating materials make to the structure.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Muda , Proteínas , Larva/metabolismo , Quitina , Proteínas de Caenorhabditis elegans/metabolismoRESUMEN
Nucleolin is an essential cellular receptor to human respiratory syncytial virus (RSV). Pharmacological targeting of the nucleolin RNA binding domain RBD1,2 can inhibit RSV infections in vitro and in vivo; however, the site(s) on RBD1,2 which interact with RSV are not known. We undertook a series of experiments designed to: document RSV-nucleolin co-localization on the surface of polarized MDCK cells using immunogold electron microscopy, to identify domains on nucleolin that physically interact with RSV using biochemical methods and determine their biological effects on RSV infection in vitro, and to carry out structural analysis toward informing future RSV drug development. Results of immunogold transmission and scanning electron microscopy showed RSV-nucleolin co-localization on the cell surface, as would be expected for a viral receptor. RSV, through its fusion protein (RSV-F), physically interacts with RBD1,2 and these interactions can be competitively inhibited by treatment with Palivizumab or recombinant RBD1,2. Treatment with synthetic peptides derived from two 12-mer domains of RBD1,2 inhibited RSV infection in vitro, with structural analysis suggesting these domains are potentially feasible for targeting in drug development. In conclusion, the identification and characterization of domains of nucleolin that interact with RSV provide the essential groundwork toward informing design of novel nucleolin-targeting compounds in RSV drug development.
Asunto(s)
Fosfoproteínas/metabolismo , Dominios y Motivos de Interacción de Proteínas/fisiología , Proteínas de Unión al ARN/metabolismo , Receptores Virales/metabolismo , Virus Sincitiales Respiratorios/metabolismo , Animales , Antivirales/farmacología , Línea Celular , Perros , Humanos , Inmunohistoquímica , Células de Riñón Canino Madin Darby , Microscopía Electrónica , Palivizumab/farmacología , NucleolinaRESUMEN
Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children. Currently, there is no RSV vaccine or universally accessible antiviral treatment available. Addressing the urgent need for new antiviral agents, we have investigated the capacity of a non-coding single-stranded oligonucleotide (ssON) to inhibit RSV infection. By utilizing a GFP-expressing RSV, we demonstrate that the ssON significantly reduced the proportion of RSV infected A549 cells (lung epithelial cells). Furthermore, we show that ssON's antiviral activity was length dependent and that both RNA and DNA of this class of oligonucleotides have antiviral activity. We reveal that ssON inhibited RSV infection by competing with the virus for binding to the cellular receptor nucleolin in vitro. Additionally, using a recombinant RSV that expresses luciferase we show that ssON effectively blocked RSV infection in mice. Treatment with ssON in vivo resulted in the upregulation of RSV-induced interferon stimulated genes (ISGs) such as Stat1, Stat2, Cxcl10, and Ccl2. This study highlights the possibility of using oligonucleotides as therapeutic agents against RSV infection. We demonstrate that the mechanism of action of ssON is the inhibition of viral entry in vitro, likely through the binding of the receptor, nucleolin and that ssON treatment against RSV infection in vivo additionally results in the upregulation of ISGs.
Asunto(s)
ADN de Cadena Simple/genética , Oligonucleótidos/genética , Mucosa Respiratoria/metabolismo , Infecciones por Virus Sincitial Respiratorio/terapia , Virus Sincitiales Respiratorios/fisiología , Células A549 , Animales , Quimiocina CCL2/genética , Quimiocina CXCL10/genética , Femenino , Humanos , Interferones/genética , Interferones/metabolismo , Ratones , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Mucosa Respiratoria/patología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT2/genética , Internalización del Virus , NucleolinaRESUMEN
Nucleolin (NCL) has been reported as a cellular receptor for the human respiratory syncytial virus (RSV). We studied the effects of re-purposing AS1411, an anti-cancer compound that binds cell surface NCL, as a possible novel strategy for RSV therapy in vitro and in vivo. AS1411 was administered to RSV-infected cultures of non-polarized (HEp-2) and polarized (MDCK) epithelial cells and to virus-infected mice and cotton rats. Results of in vitro experiments showed that AS1411, used in micromolar concentrations, was associated with decreases in the number of virus-positive cells. Intranasal administration of AS1411 (50 mg/kg) to RSV-infected mice and cotton rats was associated with partial reductions in lung viral titers, decreased virus-associated airway inflammation, and decreased IL-4/IFN-γ ratios when compared to untreated, infected animals. In conclusion, our findings indicate that therapeutic use of AS1411 has modest effects on RSV replication and host response. While the results underscore the challenges of targeting cell surface NCL as a potential novel strategy for RSV therapy, they also highlight the potential of cell surface NCL as a therapeutic target.
RESUMEN
Human respiratory syncytial virus (RSV) infects airway epithelium and can cause serious illnesses such as bronchiolitis and pneumonia. With the discovery of cell-surface nucleolin as a fusion receptor for RSV, the question arose as to whether nucleolin could explain RSV tropism in vivo. Here, we report the distribution of cell-surface nucleolin expression in tissues of normal mice and how this distribution of expression relates to what is known about RSV tropism and its clinical manifestations. Our results show evidence of cell-surface nucleolin expression in the respiratory tract. In addition, cell-surface nucleolin is expressed in tissues outside of the respiratory tract, many of which correspond to previous reports of tissue-specific RSV infection, and others that may allude to additional potential sites for RSV infection in vivo. Furthermore, our work provides a foundation for the investigation of nucleolin's physiological function in various healthy mammalian tissues.
Asunto(s)
Fosfoproteínas/biosíntesis , Proteínas de Unión al ARN/biosíntesis , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/fisiología , Tropismo Viral/fisiología , Animales , Ratones , Infecciones por Virus Sincitial Respiratorio/metabolismo , NucleolinaRESUMEN
Intrachromosomal deletions linking Dpl expression to the PrP promoter produce cerebellar degeneration that can be abrogated by the introduction of wild-type PrP transgenes. Since Dpl-like truncated forms of PrP are neuropathogenic in mice and likewise counterbalanced by expression of PrP(C) we asked whether naturally occurring mutant forms of human PrP have Dpl-like attributes. Five PRNP missense mutations causing familial Creutzfeldt-Jakob disease (F-CJD) map to a helical region found in both PrP(C) and Dpl and result in amino acids identical to conserved residues in Dpl. These F-CJD alleles may cause mutant PrP to become a weak mimetic of Dpl structure and/or function.
Asunto(s)
Amiloide/genética , Síndrome de Creutzfeldt-Jakob/genética , Imitación Molecular , Mutación Missense , Priones/genética , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Animales , Bovinos , Secuencia Conservada , Evolución Molecular , Proteínas Ligadas a GPI , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Priónicas , Priones/química , Estructura Secundaria de Proteína , Alineación de Secuencia , OvinosRESUMEN
Respiratory syncytial virus (RSV) is a major worldwide pathogen for which there is still no effective vaccine or antiviral treatment available, and immunoprophylaxis with RSV-specific antibodies (e.g., palivizumab) is used in limited clinical settings. In this review, we discuss virus-host interactions relevant to RSV pathobiology and how advances in cell and systems biology have accelerated knowledge in this area. We also highlight recent advances in understanding the relationship between RSV bronchiolitis and sequelae of recurrent wheezing and asthma, new findings into an intriguing interaction between RSV and air pollution, and exciting developments toward the goal of realizing a safe and effective RSV vaccine.
RESUMEN
In this review we propose a partially hypothetical model of respiratory syncytial virus (RSV) binding and entry to the cell that includes the recently discovered RSV receptor nucleolin, in an attempt to stimulate further inquiry in this research area. RSV binding and entry is likely to be a two-step process, the first involving the attachment of the virus to the cell membrane, which may be enhanced by electrostatic interactions with cellular glycoproteins/heparin and the viral G protein, and the second involving fusion to the cell membrane mediated by the viral F protein and a specific cellular fusion receptor. With our recent discovery of nucleolin as a functional fusion receptor for RSV, comes the possibility of a number of new approaches to the development of novel strategies for RSV prophylaxis and therapy, as well as raising some new questions concerning the pathobiology of RSV infection and tropism.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/fisiología , Internalización del Virus , Animales , Humanos , Modelos Biológicos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , Infecciones por Virus Sincitial Respiratorio/genética , Infecciones por Virus Sincitial Respiratorio/metabolismo , Virus Sincitiales Respiratorios/genética , NucleolinaRESUMEN
This article reviews current knowledge about respiratory syncytial virus (RSV) binding and entry into cells. The recent discovery of Nucleolin as a fusion receptor for RSV opens new avenues for developing interventions, while raising questions concerning RSV pathobiology and tropism. We also discuss characteristics of a good RSV drug target.
Asunto(s)
Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores Virales/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/fisiología , Animales , Línea Celular , Humanos , Virus Sincitial Respiratorio Humano/metabolismo , Proteínas Virales de Fusión/metabolismo , Tropismo Viral , Acoplamiento Viral , Internalización del Virus , NucleolinaRESUMEN
Human respiratory syncytial virus (RSV) causes a large burden of disease worldwide. There is no effective vaccine or therapy, and the use of passive immunoprophylaxis with RSV-specific antibodies is limited to high-risk patients. The cellular receptor (or receptors) required for viral entry and replication has yet to be described; its identification will improve understanding of the pathogenesis of infection and provide a target for the development of novel antiviral interventions. Here we show that RSV interacts with host-cell nucleolin via the viral fusion envelope glycoprotein and binds specifically to nucleolin at the apical cell surface in vitro. We observed decreased RSV infection in vitro in neutralization experiments using nucleolin-specific antibodies before viral inoculation, in competition experiments in which virus was incubated with soluble nucleolin before inoculation of cells, and upon RNA interference (RNAi) to silence cellular nucleolin expression. Transfection of nonpermissive Spodoptera frugiperda Sf9 insect cells with human nucleolin conferred susceptibility to RSV infection. RNAi-mediated knockdown of lung nucleolin was associated with a significant reduction in RSV infection in mice (P = 0.0004), confirming that nucleolin is a functional RSV receptor in vivo.
Asunto(s)
Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Virus Sincitiales Respiratorios/metabolismo , Internalización del Virus , Análisis de Varianza , Animales , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoprecipitación , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Interferencia de ARN , Spodoptera , Transfección , Proteínas del Envoltorio Viral/metabolismo , NucleolinaRESUMEN
The cellular prion protein, PrP(C), is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP ('DeltaPrP'). This paradigm has facilitated mapping of activity determinants in PrP(C) and implicated a cryptic PrP(C)-like protein, 'pi'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrP(C), but is low in cerebellar granular neurons (CGNs) containing PrP(C) and high in PrP(C)-deficient dendritic processes. In Prnp(0/0) CGNs, Sho transgenes were PrP(C)-like in their ability to counteract neurotoxic effects of either Doppel or DeltaPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for pi, and since it engenders a PrP(C)-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.
Asunto(s)
Encéfalo/metabolismo , Glicoproteínas/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas PrPC/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Cerebelo/metabolismo , Proteínas Ligadas a GPI , Glicoproteínas/genética , Hipocampo/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Unión ProteicaRESUMEN
Gamma-secretase depends on presence of presenilins (PS), Nct, Aph-1, and PEN-2 within a core complex. This endoproteolytic activity cleaves within transmembrane domains of amyloid-beta precursor protein (APP) and Notch, and familial Alzheimer's disease (FAD) mutations in PS1 or PS2 genes shift APP cleavage from production of amyloid-beta (Abeta) 40 peptide to greater production of Abeta42. Although studies in PS1/PS2-deficient embryonic cells define overlapping activities for these proteins, in vivo complementation of PS1-deficient animals described here reveals an unexpected spectrum of activities dictated by PS1 and PS2 alleles. Unlike PS1 transgenes, wild-type PS2 transgenes expressed in the mouse CNS support little Abeta40 or Abeta42 production, and FAD PS2 alleles support robust production of only Abeta42. Although wild-type PS2 transgenes failed to rescue Notch-associated skeletal defects in PS1 hypomorphs, a "gained" competence in this regard was apparent for FAD alleles of PS2. The range of discrete and divergent processing activities in mice reconstituted with different PS genes and alleles argues against gamma-secretase being a single enzyme with intrinsically relaxed substrate and cleavage site specificities. Instead, our studies define functionally distinct gamma-secretase variants. We speculate that extrinsic components, in combination with core complexes, may tailor functional variants of this enzyme to their preferred substrates.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Huesos/anomalías , Huesos/patología , Endopeptidasas , Homocigoto , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Fragmentos de Péptidos/metabolismo , Fenotipo , Presenilina-1 , Presenilina-2RESUMEN
Although Cu(II) ions bind to the prion protein (PrP), there have been conflicting findings concerning the number and location of binding sites. We have combined diethyl pyrocarbonate (DEPC)-mediated carbethoxylation, protease digestion, and mass spectrometric analysis of apo-PrP and copper-coordinated mouse PrP23-231 to "footprint" histidine-dependent Cu(II) coordination sites within this molecule. At pH 7.4 Cu(II) protected five histidine residues from DEPC modification. No protection was afforded by Ca(II), Mn(II), or Mg(II) ions, and only one or two residues were protected by Zn(II) or Ni(II) ions. Post-source decay mapping of DEPC-modified histidines pinpointed residues 60, 68, 76, and 84 within the four PHGGG/SWGQ octarepeat units and residue 95 within the related sequence GGGTHNQ. Besides defining a copper site within the protease-resistant core of PrP, our findings suggest application of DEPC footprinting methodologies to probe copper occupancy and pathogenesis-associated conformational changes in PrP purified from tissue samples.
Asunto(s)
Cobre/metabolismo , Dietil Pirocarbonato/metabolismo , Priones/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Ratones , Datos de Secuencia Molecular , Mapeo Peptídico , Priones/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/metabolismoRESUMEN
Doppel (Dpl) is a glycosylphosphatidylinositol-anchored protein expressed in the testis. It exhibits 26% sequence identity with the prion protein (PrP) but lacks the octarepeat region implicated as the major copper-binding domain. Contrary to expectations, Cu(II) induced a 26% reduction in the intrinsic fluorescence of Dpl(27-154) and a calculated K(d) for a single-site model of 0.16 +/- 0.08 microm. Other metals had minimal effects on fluorescence quenching. Matrix-assisted laser desorption ionization mass spectrometry of a Dpl peptide revealed binding of copper (but not other metals) to the helical alphaB/B'-loop-alphaC subregion of Dpl. Fluorescence quenching and equilibrium dialysis analyses of this Dpl(101-145) peptide were compatible with a binding site of K(d) = 0.4 microm. Diethylpyrocarbonate footprinting (Qin, K., Yang, Y., Mastrangelo, P., and Westaway, D. (2002) J. Biol. Chem. 277, 1981-1990) of Dpl(27-154) defined one residue/molecule was protected by copper from diethylpyrocarbonate adduct formation, and reiteration of this analysis with Dpl(101-145) suggested that His(131) may contribute to Cu(II) binding. Taken together, our data indicate that the alpha-helical region of mouse Dpl possesses a selective copper-binding site with a submicromolar K(d) and perhaps one or more lower affinity sites. Although metallated forms of Dpl might exist in vivo, analyses of Tg(Dpl)10329 mice were inconsistent with reports that Dpl expression is associated with increased carbonylation and nitrosylation of brain proteins. Thus, rather than comprising an important source of free radical damage, copper binding may serve to modulate the activity, stability, or localization of the Dpl protein.
Asunto(s)
Cobre/metabolismo , Priones/metabolismo , Tirosina/análogos & derivados , Animales , Sitios de Unión , Unión Competitiva , Western Blotting , Carbono/metabolismo , Quimotripsina/farmacología , Dicroismo Circular , Cobre/química , Dietil Pirocarbonato/farmacología , Relación Dosis-Respuesta a Droga , Radicales Libres , Proteínas Ligadas a GPI , Histidina/química , Cinética , Ratones , Nitrógeno/metabolismo , Péptidos/química , Proteínas PrPC/química , Unión Proteica , Proteínas/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tirosina/químicaRESUMEN
The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrP(C). Internally deleted forms of PrP(C) resembling Dpl such as PrPDelta32-121 produce a similar PrP(C)-sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrP(C), and PrPDelta132-121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrP(C) was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrPDelta23-28), by deletion of all five octarepeats (PrPDelta51-90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp"H/G"). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B' (DplDelta101-125). These data suggest contributions of copper binding and neuronal trafficking to wt PrP(C) function in vivo and place constraints upon current hypotheses to explain Dpl/PrP(C) antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.
Asunto(s)
Apoptosis , Proteínas PrPC/genética , Proteínas PrPC/fisiología , Priones/genética , Priones/fisiología , Alelos , Animales , Muerte Celular , Línea Celular Tumoral , Cerebelo/metabolismo , Mapeo Cromosómico , Cobre , Análisis Mutacional de ADN , Endocitosis , Proteínas Ligadas a GPI , Eliminación de Gen , Glicina/química , Proteínas Fluorescentes Verdes/metabolismo , Iones , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Modelos Genéticos , Mutación , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Plásmidos/metabolismo , Mutación Puntual , Proteínas PrPC/química , Priones/química , Unión Proteica , Estructura Terciaria de Proteína , Transfección , TransgenesRESUMEN
The presenilins and nicastrin form high molecular mass, multimeric protein complexes involved in the intramembranous proteolysis of several proteins. Post-translational glycosylation and trafficking of nicastrin is necessary for the activity of these complexes. We report here that although there are differences in the post-translational processing of nicastrin in neurons and glia, both of the presenilins are required for the physiological post-translational modification and for the correct subcellular distribution of nicastrin. Absence of presenilin 1 (PS1) is associated with dramatic reductions in the level of mature glycosylated nicastrin and with redistribution of nicastrin away from the cell surface. In contrast, absence of presenilin 2 (PS2) is associated with only modest reductions in the levels of immature nicastrin. It is notable that these differential effects parallel the differential effects of null mutations in PS1 and PS2 on APP and Notch processing. Our data therefore suggest that the differential interactions of PS1 and PS2 with nicastrin reflect different functions for the PS1 and PS2 complexes.