RESUMEN
The repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) modulates the expression of genes with RE1/neuron-restrictive silencing element (RE1/NRSE) sites by recruiting the switch independent 3 (SIN3) factor and the REST corepressor (COREST) to its N and C-terminal repressor domain, respectively. Both, SIN3 and COREST assemble into protein complexes that are composed of multiple subunits including a druggable histone deacetylase (HDAC) enzyme. The SIN3 core complex comprises the eponymous proteins SIN3A or SIN3B, the catalytically active proteins HDAC1 or HDAC2, the histone chaperone retinoblastoma-associated protein 46/retinoblastoma-binding protein 7 (RBAP46/RBBP7) or RBAP48/RBBP4, the SIN3-associated protein 30 (SAP30), and the suppressor of defective silencing 3 (SDS3). Here, we overcome a bottleneck limiting the molecular characterization of the REST/NRSF-SIN3 transcriptional corepressor complex. To this end, SIN3 genes were amplified from the complementary DNA of neural stem/progenitor cells, and expressed in a baculovirus/insect cell expression system. We show that the isolates bind to DNA harboring RE1/NRSE sites and demonstrate that the histone deacetylase activity is blocked by small-molecule inhibitors. Thus, our isolates open up for future biomedical research on this critical transcriptional repressor complex and are envisioned as tool for drug testing.
Asunto(s)
Proteínas Co-Represoras/genética , Inhibidores de Histona Desacetilasas/farmacología , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/metabolismo , Proteínas Represoras/genética , Complejo Correpresor Histona Desacetilasa y Sin3/genética , Complejo Correpresor Histona Desacetilasa y Sin3/aislamiento & purificación , Animales , Baculoviridae/metabolismo , Benzamidas/farmacología , Proteínas Co-Represoras/aislamiento & purificación , Proteínas Co-Represoras/metabolismo , Depsipéptidos/farmacología , Biblioteca de Genes , Histona Desacetilasas/metabolismo , Humanos , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/enzimología , Pirimidinas/farmacología , Proteínas Recombinantes , Elementos Reguladores de la Transcripción/genética , Proteínas Represoras/aislamiento & purificación , Proteínas Represoras/metabolismo , Células Sf9 , Complejo Correpresor Histona Desacetilasa y Sin3/metabolismoRESUMEN
Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.
Asunto(s)
Cromatografía de Afinidad/métodos , Espectrometría de Masas/métodos , Modelos Moleculares , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas de Ciclo Celular/metabolismo , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/aislamiento & purificación , Proteínas Co-Represoras/metabolismo , Estudios de Factibilidad , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Microscopía Intravital , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Proteínas Asociadas a Microtúbulos/metabolismo , Imagen Molecular/métodos , Sondas Moleculares/química , Fosfoproteínas/genética , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The recruitment of co-repressors to the androgen receptor is an important mechanism for reducing androgen-mediated gene activation. Importantly, co-repressors play a major role in the treatment of hormone-dependent growing tissue, such as prostate cancer and breast cancer. In line with this, co-repressor dysfunction seems to be a major player for development of castration-resistant prostate cancer or therapy-resistant breast cancer. The molecular basis of hormone therapy by particular antihormones (antagonists) for the androgen receptor (AR) is mediated by enhanced recruitment and activity of co-repressors that cause repression of AR target genes that regulate proliferation and alteration of cancer cells. Therefore co-repressor recruitment is a crucial molecular mechanism of gene repression as well as inhibition of cancer growth. Here we describe different strategies to investigate co-repressor recruitment to the AR. First, we developed a modified mammalian two-hybrid system to investigate the recruitment of co-repressors to the AR within mammalian cells. This assay is very useful for the identification of the molecular mechanism of new AR antagonists and for molecular analysis of castration-resistant prostate cancer expressing the AR. Second, we describe a technique to analyze the interaction of AR isolated from human prostate cancer cells with a newly generated AR-specific co-repressor peptide, which is bacterially expressed and affinity purified by glutathione-S-transferase affinity precipitation assays in vitro. In summary, these methods can greatly facilitate the study of AR-co-repressor interactions.
Asunto(s)
Proteínas Co-Represoras/metabolismo , Receptores Androgénicos/metabolismo , Técnicas de Cultivo de Célula , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos , Humanos , Inmunoprecipitación , Ligandos , Receptores Androgénicos/genética , Receptores Androgénicos/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Mature osteoclasts are multinuclear, macrophage-like cells derived from hematopoietic stem cells in the bone marrow. Several transcription factors regulating osteoclast differentiation have been identified. However, the molecular basis of transcriptional regulation in osteoclasts at epigenetic levels is largely unknown. In fact, no osteoclast-specific transcriptional co-regulators have been characterized. Recently, selective ablation of estrogen receptor alpha (ERalpha) in mature osteoclasts derived from female mice (ERalpha(Deltaoc/Deltaoc)) exhibited trabecular bone loss due to induced apoptosis via upregulated expression of Fas ligand mRNA. In general, the component composition of the ERalpha-associated co-activator complex and its expression levels are distinct among tissues. However, ERalpha transcriptional co-regulators in mature osteoclasts remain unclear. In the present study, we achieved large-scale cultivation of mature, multinucleated osteoclasts and established a purification system for ERalpha-associated proteins. In addition to co-regulators previously found in other ERalpha target cells, several unexpected factors were found such as CAP-H. The mRNA expression level of CAP-H was high during osteoclast differentiation. These results demonstrate the existence of osteoclast-specific transcriptional co-regulators supporting ERalpha function.