RESUMEN
The three human SNM1 metallo-ß-lactamase fold nucleases (SNM1A-C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site. Structure and turnover assay-guided optimization enabled the identification of potent quinazoline-hydroxamic acid containing inhibitors, which bind in a manner where the hydroxamic acid displaces the hydrolytic water and the quinazoline ring occupies a substrate nucleobase binding site. Cellular assays reveal that SNM1A inhibitors cause sensitisation to, and defects in the resolution of, cisplatin-induced DNA damage, validating the tractability of MBL fold nucleases as cancer drug targets.
RESUMEN
Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary prevention of arterial thrombosis, coincide with an increased bleeding risk. Targeting ITAM-linked receptors, such as the collagen receptor glycoprotein VI (GPVI), is expected to provide a better antithrombotic-hemostatic profile. Here, we developed and characterized an ultra-high-throughput (UHT) method based on intracellular [Ca2+]i increases to differentiate GPVI and GPCR effects on platelets. In 96-, 384-, or 1,536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca2+]i increase when stimulated with the GPVI agonist collagen-related peptide or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five parameters describing the Ca2+ responses. Verification of the UHT assay was done with a robustness compound library and clinically relevant platelet inhibitors. Taken together, these results present proof of principle of distinct receptor-type-dependent Ca2+ signaling curves in platelets, which allow identification of new inhibitors in a UHT way.
RESUMEN
Previously, we found that in t(X;1)(p11;q21)-positive renal cell carcinomas the bHLH-LZ transcription factor TFE3 is fused to a novel protein designated PRCC. In addition, we found that the PRCCTFE3 fusion protein, which has retained all known functional domains of TFE3, acts as a more potent transcriptional activator than wild type TFE3. We also found that PRCCTFE3 expression confers in vitro and in vivo transformation onto various cell types, including those of the kidney. Here we show that de novo expression of the PRCCTFE3 fusion protein provokes cell cycle delay. This delay, which is mediated by induction of the cyclin-dependent kinase inhibitor p21((WAF1/CIP1)), affects both the G1/S and the G2/M phases of the cell cycle and prevents the cells from undergoing polyploidization. We also show that the PRCCTFE3 fusion protein binds directly to the p21((WAF1/CIP1)) promoter and that the PRCCTFE3-induced up-regulation of p21((WAF1/CIP1)) leads to activation of the pRB pathway. Finally, we show that in t(X;1)(p11;q21)-positive renal tumor cells several processes that link PRCCTFE3 expression to p21((WAF1/CIP1))-mediated cell cycle delay are abrogated. Our data suggest a scenario in which, during the course of renal cell carcinoma development, an initial PRCCTFE3-induced cell cycle delay must be numbed, thus permitting continued proliferation and progression towards full-blown malignancy.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Carcinoma de Células Renales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Neoplasias Renales/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Carcinoma de Células Renales/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células HeLa , Humanos , Neoplasias Renales/patología , Nocodazol/farmacología , Poliploidía , Regiones Promotoras Genéticas/fisiologíaRESUMEN
Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the regulation of T-cell immunity in the tumor microenvironment. To enable the discovery of small-molecule Arginase-1 inhibitors by high-throughput screening, we developed a novel homogeneous (mix-and-measure) fluorescence-based activity assay. The assay measures the conversion of L-arginine into L-ornithine by a decrease in fluorescent signal due to quenching of a fluorescent probe, Arginase Gold. This way, inhibition of Arginase-1 results in a gain of signal when compared with the uninhibited enzyme. Side-by-side profiling of reference inhibitors in the fluorescence-based assay and a colorimetric urea formation assay revealed similar potencies and the same potency rank order among the two assay formats. The fluorescence-based assay was successfully automated for high-throughput screening of a small-molecule library in 384-well format with a good Z'-factor and hit confirmation rate. Finally, we show that the assay can be used to study the binding kinetics of inhibitors.
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Arginasa/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Neoplasias/terapia , Arginasa/antagonistas & inhibidores , Arginasa/inmunología , Arginina/genética , Arginina/metabolismo , Fluorescencia , Humanos , Neoplasias/inmunología , Ornitina/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24 h following reperfusion, which returned to base-line at 3 and 7 days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24 h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo.
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Activación de Complemento/inmunología , Complemento C3b/inmunología , Complemento C3c/inmunología , Riñón/inmunología , Daño por Reperfusión/inmunología , Animales , Activación de Complemento/genética , Complemento C3b/genética , Complemento C3b/metabolismo , Complemento C3c/genética , Complemento C3c/metabolismo , Complemento C9/inmunología , Complemento C9/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Estudios de Factibilidad , Riñón/irrigación sanguínea , Riñón/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
BACKGROUND: Although the mitotic arrest deficient protein MAD2B (MAD2L2) is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1 (FZR1), its exact role in cell cycle control still remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the human clathrin light chain A (CLTA) as a novel MAD2B binding protein. A direct interaction was established in mammalian cells via GST pull-down and endogenous co-immunoprecipitation during the G2/M phase of the cell cycle. Through subsequent confocal laser scanning microscopy we found that MAD2B and CLTA co-localize at the mitotic spindle. Clathrin forms a trimeric structure, i.e., the clathrin triskelion, consisting of three heavy chains (CLTC), each with an associated light chain. This clathrin structure has previously been shown to be required for the function of the mitotic spindle through stabilization of kinetochore fibers. Upon siRNA-mediated MAD2B depletion, we found that CLTA was no longer concentrated at the mitotic spindle but, instead, diffusely distributed throughout the cell. In addition, we found a marked increase in the percentage of misaligned chromosomes. CONCLUSIONS/SIGNIFICANCE: Previously, we identified MAD2B as an interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and a concomitant failure to shuttle MAD2B to the nucleus. Our current data show that MAD2B interacts with CLTA during the G2/M phase of the cell cycle and that depletion of MAD2B leads to a marked increase in the percentage of misaligned chromosomes and a redistribution of CLTA during mitosis.
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Cadenas Ligeras de Clatrina/metabolismo , Mitosis , Proteínas/metabolismo , Huso Acromático/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cadenas Ligeras de Clatrina/genética , Fase G2 , Células HEK293 , Humanos , Inmunoprecipitación , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Mad2 , Microscopía Confocal , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión Proteica , Proteínas/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
BACKGROUND: Previously, we identified the mitotic arrest deficient protein MAD2B (MAD2L2) as a bona fide interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and, concomitantly, an abrogation of cell cycle progression. Although MAD2B is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1(FZR1), its exact role in cell cycle control still remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the small GTPase RAN, a well-known cell cycle regulator, as a novel MAD2B binding protein. Endogenous interaction was established in mammalian cells via co-localization and co-immunoprecipitation of the respective proteins. The interaction domain of RAN could be assigned to a C-terminal moiety of 60 amino acids, whereas MAD2B had to be present in its full-length conformation. The MAD2B-RAN interaction was found to persist throughout the cell cycle. During mitosis, co-localization at the spindle was observed. CONCLUSIONS/SIGNIFICANCE: The small GTPase RAN is a novel MAD2B binding protein. This novel protein-protein interaction may play a role in (i) the control over the spindle checkpoint during mitosis and (ii) the regulation of nucleocytoplasmic trafficking during interphase.
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Mitosis , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína de Unión al GTP ran/química , Animales , Células COS , Proteínas Cdc20 , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Humanos , Proteínas Mad2 , Modelos Biológicos , Mapeo de Interacción de Proteínas , Técnicas del Sistema de Dos HíbridosRESUMEN
Fusion of the SS18 and either one of the SSX genes is a hallmark of human synovial sarcoma. The SS18 and SSX genes encode nuclear proteins that exhibit opposite transcriptional activities. The SS18 protein functions as a transcriptional coactivator and is associated with the SWI/SNF complex, whereas the SSX proteins function as transcriptional corepressors and are associated with the polycomb complex. The domains involved in these opposite transcriptional activities are retained in the SS18-SSX fusion proteins. Here, we set out to determine the direct transcriptional consequences of conditional SS18-SSX2 fusion protein expression using complementary DNA microarray-based profiling. By doing so, we identified several clusters of SS18-SSX2-responsive genes, including a group of genes involved in cholesterol synthesis, which is a general characteristic of malignancy. In addition, we identified a group of SS18-SSX2-responsive genes known to be specifically deregulated in primary synovial sarcomas, including IGF2 and CD44. Furthermore, we observed an uncoupling of EGR1, JUNB, and WNT signaling in response to SS18-SSX2 expression, suggesting that the SWI/SNF-associated coactivation functions of the SS18 moiety are impaired. Finally, we found that SS18-SSX2 expression affects histone modifications in the CD44 and IGF2 promoters and DNA methylation levels in the IGF2 imprinting control region. Together, we conclude that the SS18-SSX2 fusion protein may act as a so-called transcriptional "activator-repressor," which induces downstream target gene deregulation through epigenetic mechanisms. Our results may have implications for both the development and clinical management of synovial sarcomas.
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Epigénesis Genética/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/fisiología , Sarcoma Sinovial/genética , Transcripción Genética/genética , Colesterol/metabolismo , Metilación de ADN , Perfilación de la Expresión Génica , Histonas/metabolismo , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Proteínas de Neoplasias/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Fusión Oncogénica/química , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Sarcoma Sinovial/metabolismo , Somatomedinas/fisiología , Activación Transcripcional/genéticaRESUMEN
Molecular genetic analysis of familial and non-familial cases of conventional renal cell carcinoma (RCC) revealed a critical role(s) for multiple genes on human chromosome 3. For some of these genes, e.g. VHL, such a role has been firmly established, whereas for others, definite confirmation is still pending. Additionally, a novel role for constitutional chromosome 3 translocations as risk factors for conventional RCC development is rapidly emerging. Also, several candidate loci have been mapped to other chromosomes in both familial and non-familial RCCs of distinct histologic subtypes. The MET gene on chromosome 7, for example, was found to be involved in both forms of papillary RCC. A PRCC-TFE3 fusion gene is typically encountered in t(X;1)-positive non-familial papillary RCCs and results in abrogation of the cell cycle mitotic spindle checkpoint in a dominant-negative fashion, thus leading to RCC. Together, these data turn human RCC into a model system in which different aspects of both familial and non-familial syndromes may act as novel paradigms for cancer development.