Asunto(s)
Ataque Isquémico Transitorio , Janus Quinasa 2 , Policitemia Vera , Policitemia , Humanos , Ataque Isquémico Transitorio/etiología , Ataque Isquémico Transitorio/genética , Janus Quinasa 2/genética , Mutación , Policitemia/diagnóstico , Policitemia/etiología , Policitemia/genética , Policitemia Vera/complicaciones , Policitemia Vera/diagnóstico , Policitemia Vera/genética , Reacción en Cadena de la PolimerasaRESUMEN
Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca(2+)-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca(2+) overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC ß-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavß subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain-binding pocket in Cavß and interferes with the association between Cavß and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavß via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca(2+) channel activity at the plasma membrane, resulting in the inhibition of Ca(2+)-evoked exocytosis. Thus, BARP can modulate the localization of Cavß and its association with the Cavα1 subunit to negatively regulate VGCC activity.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Neuroendocrinas/metabolismo , Neuronas/metabolismo , Animales , Sitios de Unión , Células COS , Canales de Calcio Tipo L/genética , Chlorocebus aethiops , Cricetinae , Humanos , Glicoproteínas de Membrana/genética , Ratones , Proteínas del Tejido Nervioso/genética , Células Neuroendocrinas/citología , Neuronas/citología , Células PC12 , Unión Proteica , Estructura Terciaria de Proteína , RatasRESUMEN
RGK proteins (Kir/Gem, Rad, Rem, and Rem2) form a small subfamily of the Ras superfamily. Despite a conserved GTP binding core domain, several differences suggest that structure, mechanism of action, and functional regulation differ from Ras. RGK proteins down-regulate voltage-gated calcium channel activity by binding in a GTP-dependent fashion to the Cavbeta subunits. Mutational analysis combined with homology modeling reveal a novel effector binding mechanism distinct from that of other Ras GTPases. In this model the Switch 1 region acts as an allosteric activator that facilitates electrostatic interactions between Arg-196 in Kir/Gem and Asp-194, -270, and -272 in the nucleotide-kinase (NK) domain of Cavbeta3 and wedging Val-223 and His-225 of Kir/Gem into a hydrophobic pocket in the NK domain. Kir/Gem interacts with a surface on the NK domain that is distinct from the groove where the voltage-gated calcium channel Cavalpha1 subunit binds. A complex composed of the RGK protein and the Cavbeta3 and Cav1.2 subunits could be revealed in vivo using coimmunoprecipitation experiments. Intriguingly, docking of the RGK protein to the NK domain of the Cavbeta subunit is reminiscent of the binding of GMP to guanylate kinase.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleótidos/metabolismo , Fosfotransferasas/química , Fosfotransferasas/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Canales de Calcio/genética , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Línea Celular , Chlorocebus aethiops , Humanos , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Ratas , Homología Estructural de ProteínaRESUMEN
The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as cis-11-methyl-2-dodecenoic acid, as a cell-cell communication signal to synchronize virulence gene expression and biofilm dispersal. In this study, we showed that despite the existance of phenotype variations in different Xcc isolates, the DSF-mediated functions were in general conserved. To investigate the genomic profiles of DSF regulation, we designed and conducted oligomicroarray analysis by comparison of the gene expression patterns of wild-type strain XC1 and its DSF-deficient mutant XC1dF, as well as those of XC1dF in the presence or absence of DSF signals. The analyses led to identification of 165 genes, whose expression was significantly influenced by DSF signals. These genes encode proteins and enzymes belonging to at least 12 functional groups. In addition to those previously known DSF-dependent activities such as production of extracellular enzymes and extracellular polysaccharides, microarray analyses also revealed new functions mediated by DSF, such as flagellum synthesis, resistance to toxins and oxidative stress, and aerobic respiration. Phenotype analyses confirmed that DSF signalling contributed to resistance to toxin acriflavin and hydrogen peroxide, and to the survival of bacterial cells at different temperatures. We conclude that DSF cell-cell signalling is not only essential for co-ordinating the expression of virulence genes but also plays a vital role in keeping up the general competence of the pathogen in ecosystems.
