RESUMEN
The ubiquitin-dependent proteolysis system (UPS) is the main driver of regulated protein degradation in all eukaryotic cells, and it is becoming increasingly clear that defects within this pathway drive a large number of human pathologies. Recent success in the use of proteasome inhibitors in the treatment of hematological malignancies validates the UPS as a viable therapeutic pathway, and substantial effort is now focused on the development of both second-generation proteasome inhibitors as well as novel strategies for the inhibition of upstream UPS enzymes. In this review we discuss the potential 'druggability' of key nodes within the UPS and summarize recent advances within the field.
Asunto(s)
Inhibidores de Proteasoma , Ubiquitina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Unión al Calcio , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Proteínas Cullin , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Replicación del ADN/fisiología , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor , Ubiquitinas/metabolismo , Proteínas de Xenopus , Animales , Proteínas Portadoras , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Femenino , Ligasas/genética , Ligasas/metabolismo , Oocitos/citología , Oocitos/metabolismo , Complejo de Reconocimiento del Origen , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas Asociadas a Fase-S , Proteínas Ligasas SKP Cullina F-box , Ubiquitina-Proteína Ligasas , Ubiquitinas/genética , Xenopus laevisRESUMEN
Recently, many new examples of E3 ubiquitin ligases or E3 enzymes have been found to regulate a host of cellular processes. These E3 enzymes direct the formation of multiubiquitin chains on specific protein substrates, and - typically - the subsequent destruction of those proteins. We discuss how the modular architecture of E3 enzymes connects one of two distinct classes of catalytic domains to a wide range of substrate-binding domains. In one catalytic class, a HECT domain transfers ubiquitin directly to substrate bound to a non-catalytic domain. Members of the other catalytic class, found in the SCF, VBC and APC complexes, use a RING finger domain to facilitate ubiquitylation. The separable substrate-recognition domains of E3 enzymes provides a flexible means of linking a conserved ubiquitylation function to potentially thousands of ubiquitylated substrates in eukaryotic cells.
Asunto(s)
Ligasas/metabolismo , Animales , Dominio Catalítico , Células Eucariotas/enzimología , Humanos , Ligasas/química , Especificidad por Sustrato/fisiología , Ubiquitina-Proteína LigasasRESUMEN
The SRm160/300 splicing coactivator, which consists of the serine/arginine (SR)-related nuclear matrix protein of 160 kDa and a 300-kDa nuclear matrix antigen, functions in splicing by promoting critical interactions between splicing factors bound to pre-mRNA, including snRNPs and SR family proteins. In this article we report the isolation of a cDNA encoding the 300-kDa antigen and investigate the activity of it and SRm160 in splicing. Like SRm160, the 300-kDa antigen contains domains rich in alternating S and R residues but lacks an RNA recognition motif; the protein is accordingly named "SRm300." SRm300 also contains a novel and highly conserved N-terminal domain, several unique repeated motifs rich in S, R, and proline residues, and two very long polyserine tracts. Surprisingly, specific depletion of SRm300 does not prevent the splicing of pre-mRNAs shown previously to require SRm160/300. Addition of recombinant SRm160 alone to SRm160/300-depleted reactions specifically activates splicing. The results indicate that SRm160 may be the more critical component of the SRm160/300 coactivator in the splicing of SRm160/300-dependent pre-mRNAs.
Asunto(s)
Antígenos Nucleares , Proteínas Asociadas a Matriz Nuclear , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Empalme del ARN/genética , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Núcleo Celular/metabolismo , Células Cultivadas , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN Complementario/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/inmunología , Pruebas de Precipitina , Empalme del ARN/fisiología , ARN Mensajero/metabolismo , Empalmosomas/metabolismoRESUMEN
Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. We have recently identified a splicing "coactivator," SRm160/300, which contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. In the present study, we show that SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independently of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound "activators" and the snRNP machinery of the spliceosome.