RESUMEN

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.

Asunto(s)

Gangliósidos/metabolismo , Glicoproteína Asociada a Mielina/metabolismo , Neuritas , Animales , Arginina/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , GTP Fosfohidrolasas/metabolismo , Inmunohistoquímica , Glicoproteína Asociada a Mielina/química , Unión Proteica , Ratas

RESUMEN

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.

Asunto(s)

Proteínas de Choque Térmico , Proteínas Periplasmáticas , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Alanina/química , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Secuencia de Bases , Sitios de Unión , Northern Blotting , Western Blotting , Células COS , Proteínas Portadoras/química , Proteínas Portadoras/genética , Caseínas/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Cromatografía Líquida de Alta Presión , Clonación Molecular , Retículo Endoplásmico/metabolismo , Escherichia coli/genética , Fibroblastos/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas , Serina Peptidasa A2 que Requiere Temperaturas Altas , Calor , Humanos , Proteínas de la Membrana/genética , Ratones , Microscopía Fluorescente , Proteínas Mitocondriales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Presenilina-1 , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Serina/química , Serina Endopeptidasas/biosíntesis , Fracciones Subcelulares/metabolismo , Temperatura , Factores de Tiempo , Distribución Tisular , Tunicamicina/farmacología , Técnicas del Sistema de Dos Híbridos , Regulación hacia Arriba

RESUMEN

The initiation of DNA replication in eukaryotes is regulated in a minimum of at least two ways. First, several proteins, including origin recognition complex (ORC), Cdc6 protein, and the minichromosome maintenance (MCM) protein complex, need to be assembled on chromatin before initiation. Second, cyclin-dependent kinases regulate DNA replication in both a positive and a negative way by inducing the initiation of DNA replication at G(1)/S transition and preventing further rounds of origin firing within the same cell cycle. Here we characterize a link between the two levels. Immunoprecipitation of Xenopus origin recognition complex with anti-XOrc1 or anti-XOrc2 antibodies specifically co-immunoprecipitates a histone H1 kinase activity. The kinase activity is sensitive to several inhibitors of cyclin-dependent kinases including 6-dimethylaminopurine (6-DMAP), olomoucine, and p21(Cip1). This kinase activity also copurifies with ORC over several fractionation steps and was identified as a complex of the Cdc2 catalytic subunit and cyclin A1. Neither Cdk2 nor cyclin E could be detected in ORC immunoprecipitations. Reciprocal immunoprecipitations with anti-Xenopus Cdc2 or anti-Xenopus cyclin A1 antibodies specifically co-precipitate XOrc1 and XOrc2. Our results indicate that Xenopus ORC and Cdc2 x cyclin A1 physically interact and demonstrate a physical link between an active cyclin-dependent kinase and proteins involved in the initiation of DNA replication.

Asunto(s)

Proteína Quinasa CDC2/metabolismo , Ciclina A/metabolismo , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Xenopus , Adenina/análogos & derivados , Adenina/farmacología , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/farmacología , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Cinetina , Complejo de Reconocimiento del Origen , Fosforilación , Pruebas de Precipitina , Proteínas Quinasas/metabolismo , Purinas/farmacología , Xenopus

RESUMEN

During late mitosis and early G1, a series of proteins are assembled onto replication origins that results in them becoming 'licensed' for replication in the subsequent S phase. In Xenopus this first involves the assembly onto chromatin of the Xenopus origin recognition complex XORC, and then XCdc6, and finally the RLF-M component of the replication licensing system. In this paper we examine changes in the way that XORC associates with chromatin in the Xenopus cell-free system as origins become licensed. Restricting the quantity of XORC on chromatin reduced the extent of replication as expected if a single molecule of XORC is sufficient to specify a single replication origin. During metaphase, XOrc1 associated only weakly with chromatin. In early interphase, XOrc1 formed a strong complex with chromatin, as evidenced by its resistance to elution by 200 mM salt, and this state persisted when XCdc6 was assembled onto the chromatin. As a consequence of origins becoming licensed the association of XOrc1 and XCdc6 with chromatin was destabilised, and XOrc1 became susceptible to removal from chromatin by exposure to either high salt or high Cdk levels. At this stage the essential function for XORC and XCdc6 in DNA replication had already been fulfilled. Since high Cdk levels are required for the initiation of DNA replication, this 'licensing-dependent origin inactivation' may contribute to mechanisms that prevent re-licensing of replication origins once S phase has started.

Asunto(s)

Cromatina/química , Replicación del ADN , Proteínas de Xenopus , Animales , Ciclo Celular/fisiología , Sistema Libre de Células/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Xenopus

RESUMEN

Eukaryotic DNA replication is regulated at least in part by the assembly of initiation proteins onto origins of replication. The origin recognition complex (ORC) is bound to origins throughout most of the cell cycle. Other initiation proteins, such as Cdc6 and the MCM/P1 proteins, are assembled onto ORC-containing chromatin during G1 to define a prereplicative complex. During S phase, these proteins are displaced from chromatin and their reassembly is inhibited by protein-dependent kinases.

Asunto(s)

Cromatina , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Origen de Réplica , Proteínas de Saccharomyces cerevisiae , Animales , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteína 1 de Mantenimiento de Minicromosoma , Proteínas de Schizosaccharomyces pombe , Factores de Transcripción/metabolismo

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Replicación del ADN , ADN/biosíntesis , Oocitos/fisiología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Proteínas de Ciclo Celular/aislamiento & purificación , Núcleo Celular/efectos de los fármacos , Núcleo Celular/fisiología , Sistema Libre de Células , Centrifugación , Cromatina/fisiología , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cromatografía Liquida , ADN/análisis , Inhibidores Enzimáticos/farmacología , Femenino , Fertilización , Indicadores y Reactivos , Meiosis , Metafase , Modelos Biológicos , Oocitos/citología , Polietilenglicoles , Origen de Réplica , Transducción de Señal , Moldes Genéticos , Xenopus laevis

RESUMEN

BACKGROUND: The origin recognition complex (ORC) and the minichromosome maintenance (MCM) protein complex were initially discovered in yeast and shown to be essential for DNA replication. Homologues of ORC and MCM proteins exist in higher eukaryotes, including Xenopus. The Xenopus MCM proteins and the Xenopus homologues of Saccharomyces cerevisiae Orc 1p and Orc2p (XOrc1 and XOrc2) have recently been shown to be essential for DNA replication. Here, we describe the different but interdependent functions of the ORC and MCM complexes in DNA replication in Xenopus egg extracts. RESULTS: The XOrc1 and XOrc2 proteins are present in the same multiprotein complex in Xenopus egg extracts. Immunodepletion of ORC inhibits DNA replication of Xenopus sperm nuclei. Mixing MCM-depleted and ORC-depleted extracts restores replication capacity. ORC does not co-localize with sites of DNA replication during elongation. However, at initiation the two staining patterns overlap. In contrast to MCMs, which are displaced from chromatin during S phase, XOrc1 and XOrc2 are nuclear chromatin-bound proteins throughout interphase and move to the cytoplasm in mitosis. Permeable HeLa G1- and G2-phase nuclei can replicate in ORC-depleted extract, consistent with the presence of chromatin-bound ORC in both pre-replicative and post-replicative nuclei. Interestingly, the binding of ORC to chromatin does not require the presence of MCMs; however, the binding of MCM proteins to chromatin is dependent on the presence of ORC. CONCLUSIONS: The Xenopus ORC and the MCM protein complex perform essential, non-redundant functions in DNA replication. Xenopus ORC is bound to chromatin throughout interphase but, in contrast to S. cerevisiae ORC, it appears to be, at least partly, displaced from chromatin during mitosis. The binding of MCM proteins requires the presence of ORC. Thus, the assembly of replication-competent chromatin involves the sequential binding of ORC and MCMs to DNA.

Asunto(s)

Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Xenopus , Animales , Núcleo Celular , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Fase G2 , Células HeLa , Humanos , Interfase , Proteínas Nucleares/genética , Complejo de Reconocimiento del Origen , Conejos , Xenopus/metabolismo

RESUMEN

The origin recognition complex (ORC) binds to origins of replication in budding yeast. We have cloned a Xenopus homolog of the largest ORC polypeptide (XORC1). Immunodepletion of XOrc1 from Xenopus egg extracts blocks the initiation of DNA replication. We have purified Xenopus ORC, consisting of a protein complex similar to yeast ORC. In Xenopus egg extracts, ORC associates with chromatin throughout G1 and S phases. RLF-M, a component of the replication licensing system, also associates with chromatin early in the cell cycle but dissociates during S phase. We show that the assembly of RLF-M onto chromatin is dependent on the presence of chromatin-bound ORC, leading to sequential assembly of initiation proteins onto replication origins during the cell cycle.

Asunto(s)

Replicación del ADN , Proteínas de Unión al ADN/fisiología , Proteínas de Xenopus , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ciclo Celular , Sistema Libre de Células , Cromatina/metabolismo , Cromatina/ultraestructura , Clonación Molecular , Humanos , Técnicas Inmunológicas , Masculino , Datos de Secuencia Molecular , Oocitos , Complejo de Reconocimiento del Origen , Alineación de Secuencia , Espermatozoides