RESUMO
The anaphase-promoting complex/cyclosome (APC) ubiquitin ligase is activated by Cdc20 and Cdh1 and inhibited by Mad2 and the spindle assembly checkpoint complex, Mad2B, and the early mitotic inhibitor Emi1. Mad2 inhibits APC(Cdc20), whereas Mad2B preferentially inhibits APC(Cdh1). We have examined the mechanism of APC inhibition by Emi1 and find that unlike Mad2 proteins, Emi1 binds and inhibits both APC(Cdh1) and APC(Cdc20). Also unlike Mad2, Emi1 stabilizes cyclin A in the embryo and requires zinc for its APC inhibitory activity. We find that Emi1 binds the substrate-binding region of Cdc20 and prevents substrate binding to the APC, illustrating a novel mechanism of APC inhibition.
Assuntos
Desidrogenases de Carboidrato/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular/fisiologia , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Complexos Ubiquitina-Proteína Ligase , Ciclossomo-Complexo Promotor de Anáfase , Animais , Sítios de Ligação , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Proteínas F-Box , Proteínas Fúngicas/fisiologia , Humanos , Ligases/antagonistas & inibidores , Proteínas Mad2 , Mitose/fisiologia , Proteínas Nucleares , Fosfoproteínas/fisiologia , Proteínas/fisiologia , Proteínas Repressoras/fisiologia , Ubiquitina-Proteína Ligases , Proteínas de Xenopus , Xenopus laevis , Zinco/farmacologiaRESUMO
We have discovered an early mitotic inhibitor, Emi1, which regulates mitosis by inhibiting the anaphase promoting complex/cyclosome (APC). Emi1 is a conserved F box protein containing a zinc binding region essential for APC inhibition. Emi1 accumulates before mitosis and is ubiquitylated and destroyed in mitosis, independent of the APC. Emi1 immunodepletion from cycling Xenopus extracts strongly delays cyclin B accumulation and mitotic entry, whereas nondestructible Emi1 stabilizes APC substrates and causes a mitotic block. Emi1 binds the APC activator Cdc20, and Cdc20 can rescue an Emi1-induced block to cyclin B destruction. Our results suggest that Emi1 regulates progression through early mitosis by preventing premature APC activation, and may help explain the well-known delay between cyclin B/Cdc2 activation and cyclin B destruction.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila , Ligases/metabolismo , Mitose/fisiologia , Proteínas/metabolismo , Complexos Ubiquitina-Proteína Ligase , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas Cdc20 , Sequência Conservada , Ciclina A/metabolismo , Ciclina B/metabolismo , Drosophila , Técnicas In Vitro , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Oócitos/citologia , Oócitos/fisiologia , Coelhos , Ubiquitina-Proteína Ligases , Xenopus , Proteínas de XenopusRESUMO
Sibrafiban is a double prodrug that is converted to the inactive single prodrug and to the active GP IIb/IIIa antagonist after oral administration. This clinical investigation evaluated whether coadministration of oral aspirin or intravenous heparin would alter the pharmacokinetics or pharmacodynamics of oral sibrafiban. Twenty-four adult subjects received two of the following four combinations: sibrafiban alone, sibrafiban with ASA, sibrafiban with heparin, and sibrafiban with ASA and heparin, separated by a 2-week washout period. Concentration profiles of active drug in citrate and EDTA plasma were unchanged with coadministration of ASA or heparin. No pharmacodynamic interaction was seen with coadministration of heparin. Inhibition of platelet aggregation increased 4% to 55%, and Ivy bleeding time increased 58% to 87% with coadministration of sibrafiban and ASA. The combined pharmacodynamic effect of sibrafiban and ASA may indicate a potentially greater therapeutic effect but an increased risk of bleeding when these drugs are used in combination.