RESUMEN
The process of cellular morphogenesis is highly conserved in eukaryotes and is dependent upon the function of proteins that are centrally involved in specification of the cell cycle. The human enhancer of invasion clone 10 (HEI10) protein was identified from a HeLa cell library based on its ability to promote yeast agar invasion and filamentation. Through two-hybrid screening, the mitotic cyclin B1 and an E2 ubiquitin-conjugating enzyme were isolated as HEI10-interacting proteins. Mutation of the HEI10 divergent RING finger motif (characteristic of E3 ubiquitin ligases) and Cdc2/cyclin binding and phosphorylation sites alter HEI10-dependent yeast phenotypes, including delay in G(2)/M transition. In vertebrates, the addition of HEI10 inhibits nuclear envelope breakdown and mitotic entry in Xenopus egg extracts. Mechanistically, HEI10 expression reduces cyclin B levels in cycling Xenopus eggs and reduces levels of the cyclin B ortholog Clb2p in yeast. HEI10 is itself a specific in vitro substrate of purified cyclin B/cdc2, with a TPVR motif as primary phosphorylation site. Finally, HEI10 is itself ubiquitinated in egg extracts and is also autoubiquitinated in vitro. These and other points lead to a model in which HEI10 defines a divergent class of E3 ubiquitin ligase, functioning in progression through G(2)/M.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Ciclina B/metabolismo , Fase G2/fisiología , Ligasas/fisiología , Mitosis/fisiología , Enzimas Ubiquitina-Conjugadoras , Dedos de Zinc/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Ciclina B1 , Proteínas del Huevo/metabolismo , Femenino , Biblioteca de Genes , Prueba de Complementación Genética , Humanos , Ligasas/genética , Ligasas/metabolismo , Metafase/fisiología , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Oocitos/citología , Oocitos/metabolismo , Fosforilación , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Relación Estructura-Actividad , Células Tumorales Cultivadas , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas , Xenopus laevisRESUMEN
INTRODUCTION: Hemophilia B is an inherited X chromosome-linked disorder characterized by impaired blood clotting owing to the absence of functional coagulation factor IX. Due to the relatively short half-life of factor IX, patients with hemophilia B require frequent factor IX infusions to maintain prophylaxis. We have developed a recombinant factor IX (rFIX) fused to the Fc region of IgG (rFIXFc) with an extended half-life in animals and humans. MATERIALS AND METHODS: Procoagulant properties of rFIXFc and rFIX (BENEFIX®) were compared to determine the effect of the Fc region on rFIXFc hemostatic function. Specifically, we assessed rFIXFc activation, intermolecular interactions within the Xase complex, inactivation by antithrombin III (AT) and thrombin generation potential compared with rFIX. We also assessed the acute and prophylactic efficacy profiles of rFIXFc and rFIX in vivo in hemophilia B mouse bleeding models. RESULTS AND CONCLUSIONS: The activation by factor XIa or factor VIIa/tissue factor, inhibition by AT, interaction profiles with phospholipids, affinities for factor VIIIa within the context of the Xase complex, and thrombin generation profiles were similar for rFIXFc and rFIX. Xase complexes formed with either molecule exhibited similar kinetic profiles for factor Xa generation. In acute efficacy models, mice infused with rFIXFc or rFIX were equally protected from bleeding. However, in prophylactic efficacy models, protection from bleeding was maintained approximately three times longer in rFIXFc-dosed mice than in those given rFIX; this prolonged efficacy correlates with the previously observed half-life extension. We conclude that rFIXFc retains critical FIX procoagulant attributes and that the extension in rFIXFc half-life translates into prolonged efficacy in hemophilia B mice.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Coagulantes/farmacología , Factor IX/farmacología , Hemofilia B/tratamiento farmacológico , Hemorragia/tratamiento farmacológico , Fragmentos Fc de Inmunoglobulinas/farmacología , Proteínas Recombinantes de Fusión/farmacología , Animales , Antitrombina III/farmacología , Pruebas de Coagulación Sanguínea , Modelos Animales de Enfermedad , Activación Enzimática/fisiología , Factor IX/genética , Factor VIIa/farmacología , Factor XIa/farmacología , Semivida , Fragmentos Fc de Inmunoglobulinas/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/genética , Trombina/biosíntesisRESUMEN
Bax-induced lethality in yeast is accompanied by morphological changes in mitochondria, giving rise to a reduced number of swollen tubules. Although these changes are completely abolished upon coexpression of the Bax inhibitor, Bcl-2, coexpression of Bax with Bax inhibiting-glutathione S-transferase (BI-GST) leads to aggregation, but not fusion of the mitochondria. In addition, Bax affects the integrity of yeast vacuoles, resulting in the disintegration and eventual loss of the organelles, and the disruption of intracellular protein traffic. While Bcl-2 coexpression only partially corrects this phenotype, coexpression of BI-GST fully restores the organelles, indicating a different mode of protection exerted by Bcl-2 and BI-GST.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Vacuolas/metabolismo , Levaduras/metabolismo , Supervivencia Celular , Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Membranas Intracelulares/metabolismo , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Mitocondrias/ultraestructura , Transporte de Proteínas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacuolas/ultraestructura , Levaduras/citología , Levaduras/genética , Proteína X Asociada a bcl-2RESUMEN
Recombinant factor VIII Fc (rFVIIIFc) is a fusion protein consisting of a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC), we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF), with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc.
Asunto(s)
Factor VIII/genética , Factor VIII/farmacocinética , Hemofilia A/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacocinética , Animales , Clonación Molecular/métodos , Modelos Animales de Enfermedad , Factor VIII/química , Factor VIII/uso terapéutico , Semivida , Hemofilia A/sangre , Hemorragia , Humanos , Técnicas In Vitro , Masculino , Ratones , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/uso terapéutico , Trombina/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
Genome sequencing has been completed for multiple organisms, and pilot proteomic analyses reported for yeast and higher eukaryotes. This work has emphasized the facts that proteins are frequently engaged in multiple interactions, and that governance of protein interaction specificity is a primary means of regulating biological systems. In particular, the ability to deconvolute complex protein interaction networks to identify which interactions govern specific signaling pathways requires the generation of biological tools that allow the distinction of critical from noncritical interactions. We report the application of an enhanced Dual Bait two-hybrid system to allow detection and manipulation of highly specific protein-protein interactions. We summarize the use of this system to detect proteins and peptides that target well-defined specific motifs in larger protein structures, to facilitate rapid identification of specific interactors from a pool of putative interacting proteins obtained in a library screen, and to score specific drug-mediated disruption of protein-protein interaction.