RESUMEN
USP7, also known as the hepes simplex virus associated ubiquitin-specific protease (HAUSP), deubiquitinates both mdm2 and p53, and plays an important role in regulating the level and activity of p53. Here, we report that deletion of the TRAF-like domain at the N-terminus of USP7, previously reported to contain the mdm2/p53 binding site, has no effect on USP7 mediated deubiquitination of Ub(n)-mdm2 and Ub(n)-p53. Amino acids 208-1102 were identified to be the minimal length of USP7 that retains proteolytic activity, similar to full length enzyme, towards not only a truncated model substrate Ub-AFC, but also Ub(n)-mdm2, Ub(n)-p53. In contrast, the catalytic domain of USP7 (amino acids 208-560) has 50-700 fold less proteolytic activity towards different substrates. Moreover, inhibition of the catalytic domain of USP7 by Ubal is also different from the full length or TRAF-like domain deleted proteins. Using glutathione pull-down methods, we demonstrate that the C-terminal domain of USP7 contains additional binding sites, a.a. 801-1050 and a.a. 880-1050 for mdm2 and p53, respectively. The additional USP7 binding site on mdm2 is mapped to be the C-terminal RING finger domain (a.a. 425-491). We propose that the C-terminal domain of USP7 is responsible for maintaining the active conformation for catalysis and inhibitor binding, and contains the prime side of the proteolytic active site.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/química , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina Tiolesterasa/química , Secuencias de Aminoácidos/genética , Sitios de Unión/genética , Dominio Catalítico/genética , Genes p53 , Humanos , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteína p53 Supresora de Tumor/genética , Peptidasa Específica de Ubiquitina 7 , UbiquitinaciónRESUMEN
BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.
Asunto(s)
Benzodiazepinas/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Apoptosis/genética , Benzodiazepinas/química , Benzodiazepinas/toxicidad , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Cinética , Ratones , Modelos Moleculares , Conformación Molecular , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/ß-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The "specificity domains" are structurally also completely different exhibiting a ß-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.
Asunto(s)
Dipeptidil Peptidasa 4/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Prolina/química , Aminoácidos/química , Animales , Secuencia de Bases , Células CHO , Catálisis , Dominio Catalítico , Cricetinae , Dimerización , Dipeptidil Peptidasa 4/química , Evolución Molecular , Humanos , Insectos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Baculovirus vectors engineered to contain mammalian cell-active promoter elements have been described as an efficient method for transduction of a broad spectrum of human cell lines at high frequency. In the first large-scale comparative study of secreted protein production using these viral vectors, we have evaluated production of 16 recombinant enzymes--specifically, we exploited these viral vectors, termed 'BacMam' viruses, to drive expression of a panel of proteases selected from all four major mechanistic classes, including secreted, lysosomal, endosomal, and type I transmembrane proteins. To allow a generic purification strategy, coding sequences were truncated to remove transmembrane and/or subcellular retention signals before introduction, in parallel, into a C-terminally Fc-tagged BacMam transfer vector. BacMam viruses were generated and subsequently evaluated for expression of Fc-tagged protein in virus-transduced HEK-F cells. The common Fc-tag enabled single-step affinity purification of secreted recombinant protein from the culture medium. Yields were excellent, with 14 of 16 genes expressed producing 10-30 mg or more purified protein per litre of culture using standardised transduction conditions. At this level, reagent demands for a typical protease high-throughput screen (HTS) could be met from expression cultures as small as 0.1-0.5 L. Our results indicate this expression system offers a highly efficient and scaleable method for production of enzymatically-active secreted proteases and may therefore represent a novel method of protein production for other secreted enzymes with significant advantages over the diverse approaches in current use.