RESUMEN
The conjugation of proteins with ubiquitin plays numerous regulatory roles through both proteasomal-dependent and nonproteasomal-dependent functions. Alterations in ubiquitylation are observed in a wide range of pathologic conditions, including numerous malignancies. For this reason, there is great interest in targeting the ubiquitin-proteasome system in cancer. Several classes of proteasome inhibitors, which block degradation of ubiquitylated proteins, are widely used in research, and one, Bortezomib, is now in clinical use. Despite the well-defined and central role of the ubiquitin-activating enzyme (E1), no cell permeable inhibitors of E1 have been identified. Such inhibitors should, in principle, block all functions of ubiquitylation. We now report 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41) as the first such inhibitor. Unexpectedly, in addition to blocking ubiquitylation, PYR-41 increased total sumoylation in cells. The molecular basis for this is unknown; however, increased sumoylation was also observed in cells harboring temperature-sensitive E1. Functionally, PYR-41 attenuates cytokine-mediated nuclear factor-kappaB activation. This correlates with inhibition of nonproteasomal (Lys-63) ubiquitylation of TRAF6, which is essential to IkappaB kinase activation. PYR-41 also prevents the downstream ubiquitylation and proteasomal degradation of IkappaBalpha. Furthermore, PYR-41 inhibits degradation of p53 and activates the transcriptional activity of this tumor suppressor. Consistent with this, it differentially kills transformed p53-expressing cells. Thus, PYR-41 and related pyrazones provide proof of principle for the capacity to differentially kill transformed cells, suggesting the potential for E1 inhibitors as therapeutics in cancer. These inhibitors can also be valuable tools for studying ubiquitylation.
Asunto(s)
Benzoatos/farmacología , Furanos/farmacología , Pirazoles/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Citocinas/metabolismo , Células HeLa , Humanos , Quinasa I-kappa B/metabolismo , Células Jurkat , Ratones , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Conejos , Especificidad por Sustrato , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
The steroid receptor coactivator oncogene, amplified in breast cancer 1 (AIB1; also known as ACTR/RAC-3/TRAM-1/SRC-3/p/CIP), is amplified and overexpressed in a variety of epithelial tumors. AIB1 has been reported to have roles in both steroid-dependent and steroid-independent transcription during tumor progression. In this report, we describe that the cellular levels of AIB1 are controlled through regulated proteasomal degradation. We found that serum withdrawal or growth in high cell density caused rapid degradation of AIB1 protein, but not mRNA, in immortalized cell lines. Proteasome inhibitors prevented this process, and high molecular weight ubiquitylated species of AIB1 were detected. Nuclear export was required for proteasomal degradation of AIB1 and involved the ubiquitin ligase, E6AP. AIB1/E6AP complexes were detected in cellular extracts, and reduction of cellular E6AP levels with E6AP short interfering RNA prevented proteasomal degradation of AIB1. Conversely, overexpression of E6AP promoted AIB1 degradation. The COOH terminus of AIB1 interacted with E6AP in vitro and deletion of this region in AIB1 rendered it resistant to degradation in cells. From our results, we propose a model whereby signals promoted by changes in the cellular milieu initiate E6AP-mediated proteasomal degradation of AIB1 and thus contribute to the control of steady-state levels of this protein.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Histona Acetiltransferasas/genética , Transactivadores/genética , Ubiquitina-Proteína Ligasas/fisiología , Neoplasias de la Mama/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/fisiopatología , Línea Celular , Línea Celular Transformada , Línea Celular Tumoral , Medio de Cultivo Libre de Suero , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Riñón , Coactivador 3 de Receptor Nuclear , Plásmidos , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Interferente Pequeño/genética , Transfección , Ubiquitina/genéticaRESUMEN
A concept that has arisen over the last decade is that proteins can, in general, be covalently modified by polypeptides, resulting in alterations in their fate and function. The first-identified and most well studied of these modifying polypeptides is ubiquitin. Although targeting for proteasomal degradation is the best studied outcome of ubiquitylation, we now understand that modification of proteins with ubiquitin has numerous other cellular roles that alter protein function and that are unrelated to proteasomal degradation. Ubiquitylation is a complex process that is regulated at the level of both addition and removal of ubiquitin from target proteins. This unit includes a number of different basic protocols that will facilitate the study of components of the ubiquitin system and substrate ubiquitylation both in vitro and in cells. Because another protein modifier, NEDD8, itself regulates aspects of the ubiquitin system, basic protocols on neddylation are also included in this unit.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/análisis , Ubiquitina/análisis , Ubiquitinación , Animales , Conejos , Enzimas Activadoras de Ubiquitina/análisis , Enzimas Ubiquitina-Conjugadoras/análisis , Complejos de Ubiquitina-Proteína Ligasa/análisis , Ubiquitina-Proteína Ligasas/análisisRESUMEN
Ubiquitin-conjugating enzymes (E2s) play a central role in ubiquitylation. They function to bridge the first, nonspecific step of ubiquitin activation by E1 with the transfer of activated ubiquitin to substrates by substrate-specific E3s. While sharing a common core UBC domain, members of this family exhibit significant specificity in their physical and functional interactions with E3s. Among the families of E2s, members of the yeast Ubc4/5 family are particularly well conserved in higher metazoans. In humans, these are represented by the UbcH5 family. Members of this ubiquitously expressed family show a capacity to interact with a wide range of E3s from both HECT and RING finger families, making them particularly useful tools in the laboratory. Using the UbcH5 family as a prototype, this chapter describes methods for the expression, purification, and characterization of E2 enzymes in vitro and some of the basics for their use in experiments in cells.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/genética , Enzimas Ubiquitina-Conjugadoras/genética , Escherichia coli/enzimología , Humanos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/aislamiento & purificación , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
RING finger proteins represent the largest class of potential ubiquitin ligases. This chapter describes methods used to express and assess the activity of proteins containing RING fingers based on our experience with a number of different family members. In addition to general protocols for assessing activity, specific protocols are provided for evaluating the ubiquitylation of p53 by the RING finger E3 Hdm2/Mdm2. Use of these methods may help identify new E3s, dissect factors involved in ubiquitylation of substrates, and screen for molecules that affect ubiquitylation.
Asunto(s)
Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/genética , Animales , Línea Celular Tumoral , Glutatión , Humanos , Radioisótopos de Fósforo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina , Ubiquitina-Proteína Ligasas/metabolismo , Dedos de Zinc/genéticaRESUMEN
Covalent modification of proteins with ubiquitin regulates almost all aspects of eukaryotic cellular function. Ubiquitin protein ligases (E3s) play central regulatory roles in that they provide substrate specificity to this process and therefore, represent attractive molecular targets for disease therapy. We summarize recent advances in our understanding of RING finger and RING finger-related E3s with emphasis on BRCA1 and the tumor autocrine motility factor receptor (gp78), as well as discuss the potential for components of the ubiquitin pathway for proteasomal degradation as molecular targets.