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1.
Cancer Cell ; 7(6): 547-59, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15950904

RESUMEN

The p53 tumor suppressor protein is regulated by its interaction with HDM2, which serves as a ubiquitin ligase (E3) to target p53 for degradation. We have identified a family of small molecules (HLI98) that inhibits HDM2's E3 activity. These compounds show some specificity for HDM2 in vitro, although at higher concentrations effects on unrelated RING and HECT domain E3s are detectable, which could be due, at least in part, to effects on E2-ubiquitin thiol-ester levels. In cells, the compounds allow the stabilization of p53 and HDM2 and activation of p53-dependent transcription and apoptosis, although other p53-independent toxicity was also observed.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Inhibidores Enzimáticos/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Flavinas/química , Expresión Génica/efectos de los fármacos , Humanos , Ratones , Estructura Molecular , Ubiquitina-Proteína Ligasas Nedd4 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Transfección , Proteína p53 Supresora de Tumor/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo
2.
Cancer Res ; 67(19): 9472-81, 2007 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-17909057

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/metabolismo
3.
Methods Enzymol ; 398: 54-68, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16275319

RESUMEN

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/metabolismo
4.
Methods Enzymol ; 398: 103-12, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16275323

RESUMEN

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/genética
5.
Curr Protoc Cell Biol ; Chapter 15: Unit 15.9, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18228479

RESUMEN

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/análisis
6.
Semin Cancer Biol ; 13(1): 5-14, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12507552

RESUMEN

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.


Asunto(s)
Secuencias de Aminoácidos , Regulación Neoplásica de la Expresión Génica , Ligasas/genética , Neoplasias/genética , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Humanos , Ligasas/metabolismo , Metástasis de la Neoplasia , Neoplasias/terapia , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Ubiquitina-Proteína Ligasas
7.
Exp Cell Res ; 288(1): 84-93, 2003 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-12878161

RESUMEN

We previously identified BTBD1 and BTBD2 as novel topoisomerase I-interacting proteins that share 80% amino acid identity. Here we report the characterization of their subcellular localization. In a number of mouse and human cells, BTBD1 and BTBD2 (BTBD1/2) colocalized to punctate or elongated cytoplasmic bodies (< 5 microm long and several per cell) that were larger and more elongated in cancer cell lines than in fibroblasts and myoblasts. A search for potential colocalizing proteins identified TRIM family members that localize to morphologically similar cytoplasmic bodies, which were then tested for colocalization with BTBD1/2. TRIM5delta, expressed as a GFP fusion, colocalized with BTBD1/2 immunostaining and appeared to serve as a scaffold for the assembly of endogenous BTBD1/2 proteins. TRIM family members contain a RING domain, B-box(es), and coiled-coil regions, which have a characteristic order and spacing (RBCC domain). RING-dependent ubiquitin ligase activity and multimerization via the coiled-coil region may be defining properties of the RBCC/TRIM protein family. We found that TRIM5delta with a deleted coiled-coil region or a mutated RING domain failed to colocalize with BTBD1/2. Additionally, TRIM5delta ubiquitylated itself in a RING finger- and UbcH5B-dependent manner. BTBD1/2 each contain a PHR-similarity region, repeated twice on the putative ubiquitin ligases PAM, highwire and RPM-1, which also contain a RING and B-box. Thus, four protein modules found on each of these putative ubiquitin ligases, a RING, a B-box and two PHR repeats, are present on BTBD1/2 and TRIM5delta that are colocalized to cytoplasmic bodies.


Asunto(s)
Proteínas Portadoras/metabolismo , Citoplasma/ultraestructura , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Restricción Antivirales , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Humanos , Ratones , Mutación , Estructura Terciaria de Proteína , Transporte de Proteínas , Factores de Transcripción/genética , Transfección , Proteínas de Motivos Tripartitos , Células Tumorales Cultivadas , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas
8.
EMBO J ; 22(19): 5241-50, 2003 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-14517261

RESUMEN

Ubiquitin (Ub) regulates diverse functions in eukaryotes through its attachment to other proteins. The defining step in this protein modification pathway is the attack of a substrate lysine residue on Ub bound through its C-terminus to the active site cysteine residue of a Ub-conjugating enzyme (E2) or certain Ub ligases (E3s). So far, these E2 and E3 cysteine residues are the only enzyme groups known to participate in the catalysis of conjugation. Here we show that a strictly conserved E2 asparagine residue is critical for catalysis of E2- and E2/RING E3-dependent isopeptide bond formation, but dispensable for upstream and downstream reactions of Ub thiol ester formation. In contrast, the strictly conserved histidine and proline residues immediately upstream of the asparagine are dispensable for catalysis of isopeptide bond formation. We propose that the conserved asparagine side chain stabilizes the oxyanion intermediate formed during lysine attack. The E2 asparagine is the first non-covalent catalytic group to be proposed in any Ub conjugation factor.


Asunto(s)
Dominio Catalítico/genética , Secuencia Conservada , Ligasas/genética , Ubiquitina/metabolismo , Asparagina/metabolismo , Dominio Catalítico/fisiología , Histidina/metabolismo , Ligasas/metabolismo , Prolina/metabolismo , Estructura Terciaria de Proteína
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