RESUMEN
The generation of functional structures during development requires tight spatial regulation of signaling pathways. Thus, in Drosophila legs, in which Notch pathway activity is required to specify joints, only cells distal to ligand-producing cells are capable of responding. Here, we show that the asymmetric distribution of planar cell polarity (PCP) proteins correlates with this spatial restriction of Notch activation. Frizzled and Dishevelled are enriched at distal sides of each cell and hence localize at the interface with ligand-expressing cells in the non-responding cells. Elimination of PCP gene function in cells proximal to ligand-expressing cells is sufficient to alleviate the repression, resulting in ectopic Notch activity and ectopic joint formation. Mutations that compromise a direct interaction between Dishevelled and Notch reduce the efficacy of repression. Likewise, increased Rab5 levels or dominant-negative Deltex can suppress the ectopic joints. Together, these results suggest that PCP coordinates the spatial activity of the Notch pathway by regulating endocytic trafficking of the receptor.
Asunto(s)
Polaridad Celular/fisiología , Receptores Notch/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Polaridad Celular/genética , Proteínas Dishevelled , Drosophila , Proteínas de Drosophila , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Mutación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores Notch/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity.
Asunto(s)
Bioquímica/métodos , Cobre/química , Priones/química , Animales , Sitios de Unión , Calorimetría , Quelantes/farmacología , Escherichia coli/metabolismo , Glicina/química , Histidina/química , Concentración de Iones de Hidrógeno , Cinética , Ratones , Mutagénesis , Mutación , Priones/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Superóxido Dismutasa/química , TemperaturaRESUMEN
The Cip/Kip family of mammalian cyclin-dependent kinase (cdk) inhibitors plays important roles in development, particularly in cell fate determination and differentiation, in addition to their function of blocking cell cycle progression. We have identified two novel members of the Kip/Cip cdk inhibitor family, p16Xic2 and p17Xic3, from Xenopus laevis. Sequence analysis revealed that p16Xic2 and p17Xic3 are orthologues of mammalian p21Cip1 and p27Kip1, respectively. Overexpression of these inhibitors results in cell cycle arrest by inhibition of cdk2 activity. Interestingly, the expression of these inhibitors is highly developmentally regulated. p16Xic2 is highly expressed in differentiating somite, tail bud, lens, and cement gland, while p17Xic3 is expressed in the central nervous system. In a retinal cell fate determination assay, both p16Xic2 and p17Xic3 have an activity that influences cell fate determination. These observations suggest that p16Xic2 and p17Xic3 might be involved in cell fate determination in a tissue-specific manner by coordinating proliferation and differentiation as observed with p27Xic1.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Clonación Molecular , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Masculino , Microinyecciones , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Tiempo , Xenopus laevis/embriologíaRESUMEN
Mesoderm induction and patterning are primarily regulated by the concentration of locally expressed morphogens such as members of the TGFbetasuperfamily. Smad2 functions as a transcription factor to regulate expression of mesodermal genes downstream of such morphogens. We have identified Xenopus PIASy (XPIASy), a member of the PIAS family, by yeast two-hybrid screening using Xenopus Smad2 (XSmad2) as a bait. During mesoderm induction, XPIASy is expressed in the animal half of embryos with a ventral high-dorsal low gradient at the marginal zone. XPIASy expression is positively and negatively regulated by activities of the XSmad2 and Wnt pathways, respectively. Interestingly, inhibition of XPIASy by morpholinos induces elongation of animal caps with induction of mesoderm genes even in the absence of their morphogen-mediated activation. In addition, their introduction into the ventral marginal zone results in a secondary axis formation. Gain-of-function analysis revealed that XPIASy inhibits mesoderm induction by specific and direct downregulation of XSmad2 transcriptional activity. These observations indicate that XPIASy functions as an essential negative regulator of the XSmad2 pathway to ensure proper mesoderm induction at the appropriate time and in the appropriate region, and suggest that both the initial step of morphogen-mediated activation of the XSmad2 pathway and regulation of the final downstream transcription step have crucial roles in mesoderm induction and patterning.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Mesodermo/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mesodermo/citología , Datos de Secuencia Molecular , Fenotipo , Filogenia , Unión Proteica , Proteínas Inhibidoras de STAT Activados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Alineación de Secuencia , Transducción de Señal , Proteína Smad2 , Transactivadores/antagonistas & inhibidores , Transactivadores/química , Transactivadores/genética , Proteínas Wnt , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Cigoto/metabolismoRESUMEN
Prion diseases are neurodegenerative conditions. The main pathological alterations common to these diseases include the loss of neurones, gliosis and the deposition of an abnormal isoform of the prion protein in aggregates in the nervous tissue. Prevention of the devastating effects of prion disease requires prevention of neuronal death. Therefore, understanding the mechanism by which this occurs is essential. Cell culture studies using the synthetic peptide PrP106-126 have been central to developing a model of this mechanism. Using a coculture system, we have shown that PrP106-126 caused neuronal death mediated by glutamate. This neuronal death resulted from modification of the expression of NMDA receptor subtypes stimulated by the exposure of neurones to the combination of astrocytic factors, elevated Cu and PrP106-126. The results of these experiments suggest neuronal death in prion disease might be reduced by the use of NMDA receptor antagonists such as MK801 or inhibitors of the arachidonic acid metabolism pathway.
Asunto(s)
Astrocitos/metabolismo , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/toxicidad , Enfermedades por Prión/metabolismo , Priones/toxicidad , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Animales Recién Nacidos , Ácido Araquidónico/antagonistas & inhibidores , Ácido Araquidónico/metabolismo , Comunicación Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Cobre/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Ácido Glutámico/metabolismo , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/patología , Fragmentos de Péptidos/metabolismo , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/fisiopatología , Priones/metabolismo , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efectos de los fármacosRESUMEN
Prion diseases such as Creutzfeldt-Jakob disease are possibly caused by the conversion of a normal cellular glycoprotein, the prion protein (PrPc) into an abnormal isoform (PrPSc). The process that causes this conversion is unknown, but to understand it requires a detailed insight into the normal activity of PrPc. It has become accepted from results of numerous studies that PrPc is a Cu-binding protein and that its normal function requires Cu. Further work has suggested that PrPc is an antioxidant with an activity like that of a superoxide dismutase. We have shown in this investigation that this activity is optimal for the whole protein and that deletion of parts of the protein reduce or abolish this activity. The protein therefore contains an active domain requiring certain regions such as the Cu-binding octameric repeat region and the hydrophobic core. These regions show high evolutionary conservation fitting with the idea that they are important to the active domain of the protein.
Asunto(s)
Priones/genética , Animales , Anticuerpos/inmunología , Dicroismo Circular , Cobre/metabolismo , Ratones , Priones/química , Priones/metabolismo , Eliminación de Secuencia , Relación Estructura-Actividad , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/inmunología , Superóxido Dismutasa/metabolismoRESUMEN
The technology of gene knockout and transgenic mice has allowed the study of the role of genes and their proteins in animal physiology and metabolism. However, these techniques have often been found to be limited in that some genetic manipulations of mice led either to a fatal phenotype or to compensations that mask the loss of function of the target protein. The experimentation on neurons from transgenic mice is particularly critical in the study of key proteins that may be involved in neurodegeneration. The cell fusion technique has been implemented as a novel way to generate cell lines from prion protein knockout mice. Fusion between neonatal mouse neurons and a neuroblastoma cell line have led to a Prnp degrees / degrees cell line that facilitates the study of the knockout phenotype. These cells are readily transfectable and allowed us to study the expression of prion protein mutants on a PrP-knockout background. Using this cell line we have examined the effect of PrP mutations reported to alter PrPc to a transmembrane form. Our results suggest that these mutations do not create transmembrane forms of the protein, but block normal transport of PrP to the cell membrane.
Asunto(s)
Fusión Celular , Línea Celular , Neuronas/metabolismo , Priones/metabolismo , Animales , Transporte Biológico/genética , Membrana Celular/metabolismo , Resistencia a Medicamentos , Endopeptidasa K/farmacología , Expresión Génica , Ratones , Ratones Noqueados , Mutación/fisiología , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Priones/efectos de los fármacos , Priones/genética , TransfecciónRESUMEN
The outbreak of new variant Creutzfeldt-Jakob disease has raised the specter of a potentially large population being at risk to develop this prionosis. None of the prionoses currently have an effective treatment. Recently, vaccination has been shown to be effective in mouse models of another neurodegenerative condition, namely Alzheimer's disease. Here we report that vaccination with recombinant mouse prion protein delays the onset of prion disease in mice. Vaccination was performed both before peripheral prion exposure and after exposure. A delay in disease onset was seen in both groups, but was more prolonged in animals immunized before exposure. The increase in the incubation period closely correlated with the anti-prion protein antibody titer. This promising finding suggests that a similar approach may work in humans or other mammalian species at risk for prion disease.
Asunto(s)
Inmunización , Inmunoterapia , Enfermedades por Prión/prevención & control , Enfermedades por Prión/terapia , Animales , Encéfalo/patología , Femenino , Ratones , Ratones Endogámicos , Enfermedades por Prión/patología , Priones/inmunología , Proteínas Recombinantes/inmunología , Factores de TiempoRESUMEN
Prion diseases are associated with the conversion of the normal prion protein, PrP(C), to the infectious disease form PrP(Sc). Discrimination between these isoforms would significantly enhance diagnosis of these diseases, and it has recently been reported that PrP(Sc) is specifically recognized by the serine protease zymogen plasminogen (Fischer et al. (2000) Nature 408, 479). Here we have tested the hypothesis that PrP is a regulator of the plasminogen activation system. The effect of recombinant PrP, either containing copper (holo-PrP) or devoid of it (apo-PrP), on plasminogen activation by both uPA and tPA was determined. PrP had no effect on plasminogen activation by uPA. By contrast, the activity of tPA was stimulated by up to 280-fold. This was observed only with the apo-PrP isoforms. The copper-binding octapeptide repeat region of PrP was involved in the effects, as a mutant lacking this region failed to stimulate plasminogen activation, although a synthetic peptide corresponding to this region was unable to stimulate tPA activity. Competition experiments demonstrated that, in addition to plasminogen binding, the stimulation required a high-affinity interaction between tPA and PrP (K(d) < 2.5 nM). Kinetic analysis revealed a template mechanism for the stimulation, suggesting independent binding sites for tPA and plasminogen. Lack of copper-binding may be an early event in the conversion of PrP(C) to PrP(Sc), and our data therefore suggest that tPA-catalyzed plasminogen activation may provide the basis for a sensitive detection system for the early stages of prion diseases and also play a role in the pathogenesis of these diseases.
Asunto(s)
Plasminógeno/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Priones/farmacología , Activador de Tejido Plasminógeno/metabolismo , Animales , Apoproteínas/metabolismo , Cobre/metabolismo , Activación Enzimática/efectos de los fármacos , Ratones , Enfermedades por Prión/diagnóstico , Priones/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoAsunto(s)
Proteínas PrPC/aislamiento & purificación , Superóxido Dismutasa/aislamiento & purificación , Sinapsis/enzimología , Animales , Pollos , Dicroismo Circular , Cobre/química , Ratones , Proteínas PrPC/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Solubilidad , Espectrofotometría , Superóxido Dismutasa/genéticaRESUMEN
Depolarization of cerebellar granule cells with elevated potassium has been described as essential to maintain their survival in culture. There are several reports that this is only specific for rat cerebellar granule cells and not those of mouse. We reinvestigated this issue and found that although high potassium enhanced the survival of cerebellar granule cells from both rat and mouse it was not essential for the survival of those cultures. Further analysis of the culture system indicated that high potassium offered protection against the toxicity of glutamate and cytosine arabinose (Ara C), a standard antimitotic additive to cultures of granule cells. Ara C was found to be toxic to cerebellar cells after potassium withdrawal at concentrations standardly used in culturing these cells (10 microM). High potassium was found to diminish the expression of p53. Ara C toxicity is known to utilize the p53-dependent signaling pathway to initiate apoptosis. Another depolarizing agent, veratridine, offers no protection against Ara C but we provide evidence that the protective effect of high potassium against Ara C is mediated through calcium balance within the cells. We suggest that there is no requirement for high potassium in terms of cerebellar granule cell survival. The previously proposed role for high potassium in the survival cerebellar granule cells is rather a protective effect against toxic substances in serum such as glutamate or against agents such as Ara C.