RESUMEN
Amyloidogenesis, self-propagation of protein or peptide monomers to amyloid fibrils, has been linked to incurable pathogenesis of neurodegenerative diseases such as Alzheimer's disease and prion diseases. Investigations of amyloid structures and how monomers are transformed through seeding are therefore crucial for developing therapeutics toward these diseases. Here we describe a cross-seeding method to explore the amyloid core in prion fibrils that uses preformed amyloid fibrils as a seed to induce the transformation of other protein or peptide monomers to amyloid fibrils.
Asunto(s)
Amiloidosis , Enfermedades por Prión , Priones , Humanos , Amiloide/química , Priones/química , Proteínas AmiloidogénicasRESUMEN
Disaggregation of amyloid fibrils is a fundamental biological process required for amyloid propagation. However, due to the lack of experimental systems, the molecular mechanism of how amyloid is disaggregated by cellular factors remains poorly understood. Here, we established a robust in vitro reconstituted system of yeast prion propagation and found that heat-shock protein 104 (Hsp104), Ssa1 and Sis1 chaperones are essential for efficient disaggregation of Sup35 amyloid. Real-time imaging of single-molecule fluorescence coupled with the reconstitution system revealed that amyloid disaggregation is achieved by ordered, timely binding of the chaperones to amyloid. Remarkably, we uncovered two distinct prion strain conformation-dependent modes of disaggregation, fragmentation and dissolution. We characterized distinct chaperone dynamics in each mode and found that transient, repeated binding of Hsp104 to the same site of amyloid results in fragmentation. These findings provide a physical foundation for otherwise puzzling in vivo observations and for therapeutic development for amyloid-associated neurodegenerative diseases.
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Priones , Proteínas de Saccharomyces cerevisiae , Amiloide/química , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Prion diseases are transmissible fatal neurodegenerative disorders affecting humans and other mammals. The disease transmission can occur between different species but is limited by the sequence homology between host and inoculum. The crucial molecular event in the progression of this disease is prion formation, starting from the conformational conversion of the normal, membrane-anchored prion protein (PrPC) into the misfolded, ß-sheet-rich and aggregation-prone isoform (PrPSc), which then self-associates into the infectious amyloid form called prion. Amyloid is the aggregate formed from one-dimensional protein association. As amyloid formation is a key hallmark in prion pathogenesis, studying which segments in prion protein are involved in the amyloid formation can provide molecular details in the cross-species transmission barrier of prion diseases. However, due to the difficulties of studying protein aggregates, very limited knowledge about prion structure or prion formation was disclosed by now. In this study, cross-seeding assay was used to identify the segments involved in the amyloid fibril formation of full-length hamster prion protein, SHaPrP(23-231). Our results showed that the residues in the segments 108-127, 172-194 (helix 2 in PrPC) and 200-227 (helix 3 in PrPC) are in the amyloid core of hamster prion fibrils. The segment 127-143, but not 107-126 (which corresponds to hamster sequence 108-127), was previously reported to be involved in the amyloid core of full-length mouse prion fibrils. Our results indicate that hamster prion protein and mouse prion protein use different segments to form the amyloid core in amyloidogenesis. The sequence-dependent core formation can be used to explain the seeding barrier between mouse and hamster.
Asunto(s)
Amiloide/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Priónicas/metabolismo , Animales , Cricetinae , Ratones , Multimerización de ProteínaRESUMEN
Genome-wide association studies have identified 40 ovarian cancer risk loci. However, the mechanisms underlying these associations remain elusive. In this study, we conducted a two-pronged approach to identify candidate causal SNPs and assess underlying biological mechanisms at chromosome 9p22.2, the first and most statistically significant associated locus for ovarian cancer susceptibility. Three transcriptional regulatory elements with allele-specific effects and a scaffold/matrix attachment region were characterized and, through physical DNA interactions, BNC2 was established as the most likely target gene. We determined the consensus binding sequence for BNC2 in vitro, verified its enrichment in BNC2 ChIP-seq regions, and validated a set of its downstream target genes. Fine-mapping by dense regional genotyping in over 15,000 ovarian cancer cases and 30,000 controls identified SNPs in the scaffold/matrix attachment region as among the most likely causal variants. This study reveals a comprehensive regulatory landscape at 9p22.2 and proposes a likely mechanism of susceptibility to ovarian cancer. SIGNIFICANCE: Mapping the 9p22.2 ovarian cancer risk locus identifies BNC2 as an ovarian cancer risk gene.See related commentary by Choi and Brown, p. 439.
Asunto(s)
Carcinoma Epitelial de Ovario/genética , Cromosomas Humanos Par 9 , Neoplasias Ováricas/genética , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Mapeo Cromosómico , Cistadenocarcinoma Seroso/genética , ADN de Neoplasias/genética , Proteínas de Unión al ADN/genética , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido SimpleRESUMEN
Alzheimer's disease (AD) is the most common neurodegenerative disease. Imbalance between the production and clearance of amyloid ß (Aß) peptides is considered to be the primary mechanism of AD pathogenesis. This amyloid hypothesis is supported by the recent success of the human anti-amyloid antibody aducanumab, in clearing plaque and slowing clinical impairment in prodromal or mild patients in a phase Ib trial. Here, a peptide combining polyarginines (polyR) (for charge repulsion) and a segment derived from the core region of Aß amyloid (for sequence recognition) was designed. The efficacy of the designed peptide, R8-Aß(25-35), on amyloid reduction and the improvement of cognitive functions were evaluated using APP/PS1 double transgenic mice. Daily intranasal administration of PEI-conjugated R8-Aß(25-35) peptide significantly reduced Aß amyloid accumulation and ameliorated the memory deficits of the transgenic mice. Intranasal administration is a feasible route for peptide delivery. The modular design combining polyR and aggregate-forming segments produced a desirable therapeutic effect and could be easily adopted to design therapeutic peptides for other proteinaceous aggregate-associated diseases.
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Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/uso terapéutico , Encéfalo/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Péptidos/uso terapéutico , Administración Intranasal , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/patología , Amiloide/antagonistas & inhibidores , Amiloide/ultraestructura , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/ultraestructura , Animales , Encéfalo/patología , Línea Celular , Cognición/efectos de los fármacos , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/patología , Modelos Animales de Enfermedad , Femenino , Trastornos de la Memoria/complicaciones , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/patología , Ratones Endogámicos C57BL , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/ultraestructura , Péptidos/administración & dosificación , Péptidos/químicaRESUMEN
Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10(-30)), OSECs (P = 2.4 × 10(-23)) and HMECs (P = 6.7 × 10(-15)) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer.
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Predisposición Genética a la Enfermedad , Neoplasias Ováricas/genética , Cromatina/genética , Cromatina/metabolismo , Femenino , Estudio de Asociación del Genoma Completo , Histonas/genética , Histonas/metabolismo , Humanos , Especificidad de Órganos , Neoplasias Ováricas/metabolismo , Polimorfismo de Nucleótido Simple , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Genome-wide association studies (GWAS) have identified 12 epithelial ovarian cancer (EOC) susceptibility alleles. The pattern of association at these loci is consistent in BRCA1 and BRCA2 mutation carriers who are at high risk of EOC. After imputation to 1000 Genomes Project data, we assessed associations of 11 million genetic variants with EOC risk from 15,437 cases unselected for family history and 30,845 controls and from 15,252 BRCA1 mutation carriers and 8,211 BRCA2 mutation carriers (3,096 with ovarian cancer), and we combined the results in a meta-analysis. This new study design yielded increased statistical power, leading to the discovery of six new EOC susceptibility loci. Variants at 1p36 (nearest gene, WNT4), 4q26 (SYNPO2), 9q34.2 (ABO) and 17q11.2 (ATAD5) were associated with EOC risk, and at 1p34.3 (RSPO1) and 6p22.1 (GPX6) variants were specifically associated with the serous EOC subtype, all with P < 5 × 10(-8). Incorporating these variants into risk assessment tools will improve clinical risk predictions for BRCA1 and BRCA2 mutation carriers.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo/métodos , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Alelos , Carcinoma Epitelial de Ovario , Femenino , Genes Reporteros , Genotipo , Heterocigoto , Humanos , Mutación , Sitios de Carácter Cuantitativo , Riesgo , Adulto JovenRESUMEN
PURPOSE: Galeterone inhibits the enzyme CYP17A1 and is currently in phase II clinical trials for castration-resistant prostate cancer (CRPC). Galeterone is also a direct androgen receptor (AR) antagonist and may enhance AR degradation. This study was undertaken to determine the molecular basis for AR effects and their therapeutic potential. EXPERIMENTAL DESIGN: Effects of galeterone on AR expression and activities were examined in prostate cancer cell lines. RESULTS: Similar to the AR antagonist enzalutamide, but in contrast to bicalutamide, galeterone did not induce binding of a constitutively active VP16-AR fusion protein to reporter genes and did not induce AR recruitment to endogenous androgen-regulated genes based on chromatin immunoprecipitation. Galeterone at low micromolar concentrations that did not induce cellular stress responses enhanced AR protein degradation in LNCaP and C4-2 cells, which express a T878A mutant AR, but not in prostate cancer cells expressing wild-type AR. Further transfection studies using stable LNCaP and PC3 cell lines ectopically expressing wild-type or T878A-mutant ARs confirmed that galeterone selectively enhances degradation of the T878A-mutant AR. CONCLUSIONS: Similar to enzalutamide, galeterone may be effective as a direct AR antagonist in CRPC. It may be particularly effective against prostate cancer cells with the T878A AR mutation but may also enhance degradation of wild-type AR in vivo through a combination of direct and indirect mechanisms. Finally, these findings show that conformational changes in AR can markedly enhance its degradation and thereby support efforts to develop further antagonists that enhance AR degradation.
Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Androstadienos/farmacología , Bencimidazoles/farmacología , Cromatina/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Neoplasias de la Próstata/tratamiento farmacológico , Proteolisis/efectos de los fármacos , Receptores Androgénicos/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromatina/genética , Inmunoprecipitación de Cromatina , Humanos , Masculino , Proteínas Mutantes/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Unión Proteica/efectos de los fármacos , Receptores Androgénicos/genética , Células Tumorales CultivadasRESUMEN
Epithelial ovarian cancer (EOC) has a heritable component that remains to be fully characterized. Most identified common susceptibility variants lie in non-protein-coding sequences. We hypothesized that variants in the 3' untranslated region at putative microRNA (miRNA)-binding sites represent functional targets that influence EOC susceptibility. Here, we evaluate the association between 767 miRNA-related single-nucleotide polymorphisms (miRSNPs) and EOC risk in 18,174 EOC cases and 26,134 controls from 43 studies genotyped through the Collaborative Oncological Gene-environment Study. We identify several miRSNPs associated with invasive serous EOC risk (odds ratio=1.12, P=10(-8)) mapping to an inversion polymorphism at 17q21.31. Additional genotyping of non-miRSNPs at 17q21.31 reveals stronger signals outside the inversion (P=10(-10)). Variation at 17q21.31 is associated with neurological diseases, and our collaboration is the first to report an association with EOC susceptibility. An integrated molecular analysis in this region provides evidence for ARHGAP27 and PLEKHM1 as candidate EOC susceptibility genes.
Asunto(s)
Cromosomas Humanos Par 17 , Predisposición Genética a la Enfermedad , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Carcinoma Epitelial de Ovario , Femenino , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
TERT-locus SNPs and leukocyte telomere measures are reportedly associated with risks of multiple cancers. Using the Illumina custom genotyping array iCOGs, we analyzed â¼480 SNPs at the TERT locus in breast (n = 103,991), ovarian (n = 39,774) and BRCA1 mutation carrier (n = 11,705) cancer cases and controls. Leukocyte telomere measurements were also available for 53,724 participants. Most associations cluster into three independent peaks. The minor allele at the peak 1 SNP rs2736108 associates with longer telomeres (P = 5.8 × 10(-7)), lower risks for estrogen receptor (ER)-negative (P = 1.0 × 10(-8)) and BRCA1 mutation carrier (P = 1.1 × 10(-5)) breast cancers and altered promoter assay signal. The minor allele at the peak 2 SNP rs7705526 associates with longer telomeres (P = 2.3 × 10(-14)), higher risk of low-malignant-potential ovarian cancer (P = 1.3 × 10(-15)) and greater promoter activity. The minor alleles at the peak 3 SNPs rs10069690 and rs2242652 increase ER-negative (P = 1.2 × 10(-12)) and BRCA1 mutation carrier (P = 1.6 × 10(-14)) breast and invasive ovarian (P = 1.3 × 10(-11)) cancer risks but not via altered telomere length. The cancer risk alleles of rs2242652 and rs10069690, respectively, increase silencing and generate a truncated TERT splice variant.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/etiología , Sitios Genéticos/genética , Neoplasias Ováricas/etiología , Polimorfismo de Nucleótido Simple/genética , Telomerasa/genética , Telómero/genética , Empalme Alternativo , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Cromatina/genética , Metilación de ADN , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Luciferasas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de RiesgoRESUMEN
Previously available androgen receptor (AR) antagonists (bicalutamide, flutamide, and nilutamide) have limited activity against AR in prostate cancers that relapse after castration [castration resistant prostate cancer (CRPC)]. However, recent AR competitive antagonists such as MDV3100, generated through chemical modifications to the current AR ligands, appear to have increased activity in CRPC and have novel mechanisms of action. Using pharmacophore models and a refined homology model of the antagonist-liganded AR ligand binding domain, we carried out in silico screens of small molecule libraries and report here on the identification of a series of structurally distinct nonsteroidal small molecule competitive AR antagonists. Despite their unique chemical architectures, compounds representing each of six chemotypes functioned in vitro as pure AR antagonists. Moreover, similarly to MDV3100 and in contrast to previous AR antagonists, these compounds all prevented AR binding to chromatin, consistent with each of the six chemotypes stabilizing a similar AR antagonist conformation. Additional studies with the lead chemotype (chemotype A) showed enhanced AR protein degradation, which was dependent on helix 12 in the AR ligand binding domain. Significantly, chemotype A compounds functioned as AR antagonists in vivo in normal male mice and suppressed AR activity and tumor cell proliferation in human CRPC xenografts. These data indicate that certain ligand-induced structural alterations in the AR ligand binding domain may both impair AR chromatin binding and enhance AR degradation and support continued efforts to develop AR antagonists with unique mechanisms of action and efficacy in CRPC.
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Antagonistas de Receptores Androgénicos/análisis , Antagonistas de Receptores Androgénicos/uso terapéutico , Biología Computacional/métodos , Descubrimiento de Drogas , Orquiectomía , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/cirugía , Antagonistas de Receptores Androgénicos/química , Antagonistas de Receptores Androgénicos/farmacología , Animales , Células COS , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , ADN de Neoplasias/metabolismo , Humanos , Masculino , Ratones , Modelos Biológicos , Modelos Moleculares , Neoplasias de la Próstata/patología , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Receptores Androgénicos/química , Reproducibilidad de los Resultados , Homología Estructural de Proteína , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Androgen receptor (AR) continues to play a central role in prostate cancers that relapse after androgen deprivation therapy, but these tumors are refractory to available AR antagonists. In a recent issue of Science, Tran et al. describe an antagonist that prevents AR recruitment to chromatin and shows efficacy in relapsed prostate cancer.
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Antineoplásicos/uso terapéutico , Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/fisiología , Antagonistas de Receptores Androgénicos , Andrógenos , Antineoplásicos/farmacología , Benzamidas , Cromatina/metabolismo , Humanos , Masculino , Recurrencia Local de Neoplasia , Nitrilos , Orquiectomía , Feniltiohidantoína/farmacología , Feniltiohidantoína/uso terapéutico , Neoplasias de la Próstata/patología , Transducción de Señal , Activación Transcripcional/efectos de los fármacosRESUMEN
Androgen receptor (AR) recruitment of transcriptional corepressors NCoR and SMRT can be enhanced by antagonists such as mifepristone. This study shows that enhanced NCoR binding to the mifepristone-liganded AR is mediated by the NCoR COOH-terminal N1 CoRNR box and that this selectivity is due to charged residues unique to the COOH-terminal CoRNR boxes of NCoR and SMRT. Significantly, these residues are on a helical face adjacent to oppositely charged residues in helix 4 of the AR ligand-binding domain. Mutagenesis of these AR residues in helix 4, as well as mutation of lysine 720 in helix 3 (predicted to interact with the CoRNR box), markedly impaired AR recruitment of NCoR, indicating that N1 CoRNR box binding is being stabilized by these ionic interactions in the AR ligand-binding domain coactivator/corepressor binding site. Finally, results using a helix 12-deleted AR indicate that mifepristone induces allosteric changes in addition to helix 12 displacement that are critical for NCoR binding. These findings show that AR antagonists can enhance corepressor recruitment by stabilizing a distinct antagonist conformation of the AR coactivator/corepressor binding site and support the development of additional antagonists that may be able to further enhance AR recruitment of corepressors.
Asunto(s)
Antagonistas de Receptores Androgénicos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Mifepristona/farmacología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Secuencia Conservada , Humanos , Isoleucina/metabolismo , Ligandos , Datos de Secuencia Molecular , Co-Represor 1 de Receptor Nuclear , Co-Represor 2 de Receptor Nuclear , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Androgénicos/químicaRESUMEN
BACKGROUND: The androgen receptor (AR) plays a pivotal role in prostate cancer (PCa) initiation and progression. To date, studies have focused disproportionately on androgen-stimulated genes such as prostate-specific antigen (PSA), while repressed genes have gained little attention, even though they too may be involved in regulating cell growth, differentiation, and apoptosis. METHODS: ChIP Display was used to identify putative AR target genes in the ablation-resistant human PCa cell line, C4-2B. Quantitative real-time reverse transcription-PCR analysis was used to measure gene expression in cells subjected to dihydrotestosterone (DHT) timecourse and dose-response, as well as AR knock-down and bicalutamide-treatments. RESULTS: We report on three genes, KIAA1217, CHRM1, and WBSCR28, which were newly identified in a screen for AR-occupied regions in C4-2B PCa cells, and which were repressed by treatment with DHT. AR knock-down resulted in increased KIAA1217, CHRM1, and WBSCR28 mRNA, indicating that, like PSA stimulation, AR represses these three genes even in the absence of added ligand. DHT decreased KIAA1217 and CHRM1 pre-mRNA levels, suggesting AR-mediated transcriptional inhibition. Cycloheximide attenuated DHT-mediated repression of CHRM1, suggesting the requirement of new protein synthesis. Furthermore, bicalutamide treatment did not mimic, but rather antagonized DHT-mediated KIAA1217 repression. Unlike the handful of androgen-repressed genes studied thus far, AR occupancy at KIAA1217, CHRM1, and WBSCR28 was mapped outside their respective 5'-promoter regions. CONCLUSIONS: Many more genes likely share AR-mediated gene repression through distal regulatory elements. Further study of such targets and their transcriptional regulation may help explain the receptor's tumorigenicity in PCa.
Asunto(s)
Neoplasias de la Próstata/genética , Receptores Androgénicos/fisiología , Antagonistas de Andrógenos/farmacología , Anilidas/farmacología , Línea Celular Tumoral , Cicloheximida/farmacología , Dihidrotestosterona/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Nitrilos/farmacología , Neoplasias de la Próstata/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Receptor Muscarínico M1 , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Receptores Muscarínicos/biosíntesis , Receptores Muscarínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Compuestos de Tosilo/farmacología , TransfecciónRESUMEN
Prostate cancers (PCas) become resistant to hormone withdrawal through increased androgen receptor (AR) signaling. Here we show increased AR-mediated transcription efficiency in PCa cells that have acquired the ability to grow in low concentrations of androgen. Compared to androgen-dependent PCa cells, these cells showed increased activity of transiently transfected reporters and increased mRNA synthesis relative to levels of AR occupancy of the prostate-specific antigen (PSA) gene. The locus also displayed up to 10-fold-higher levels of histone H3-K9/K14 acetylation and H3-K4 methylation across the entire body of the gene. Although similar increased mRNA expression and locus-wide histone acetylation were also observed at another kallikrein locus (KLK2), at a third AR target locus (TMPRSS2) increased gene expression and locus-wide histone acetylation were not seen in the absence of ligand. Androgen-independent PCa cells have thus evolved three distinctive alterations in AR-mediated transcription. First, increased RNA polymerase initiation and processivity contributed to increased gene expression. Second, AR signaling was more sensitive to ligand. Third, locus-wide chromatin remodeling conducive to the increased gene expression in the absence of ligand was apparent and depended on sustained AR activity. Therefore, increased AR ligand sensitivity as well as locus-specific chromatin alterations contribute to basal gene expression of a subpopulation of specific AR target genes in androgen-independent PCa cells. These features contribute to the androgen-independent phenotype of these cells.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromatina/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismo , Transcripción Genética , Acetilación/efectos de los fármacos , Andrógenos/metabolismo , Animales , Cromatina/efectos de los fármacos , Dihidrotestosterona/farmacología , Genes Reporteros/genética , Histonas/metabolismo , Humanos , Ligandos , Luciferasas/genética , Masculino , Metilación/efectos de los fármacos , Ratones , Ratones Desnudos , Antígeno Prostático Específico/genética , Elementos de Respuesta/efectos de los fármacos , Elementos de Respuesta/genética , Células Tumorales CultivadasRESUMEN
Whereas the androgen receptor (AR) protein shares similarities in the structure of its DNA- and hormone-binding domains with other members of the steroid nuclear receptor family, the molecule in its unliganded form has a seemingly unordered amino-terminal transactivation domain unique to the AR. A comprehensive understanding of the specific sub-structures and protein-protein interactions inherent to this domain in both its inactive and activated states remains un-achieved. Therefore, the malleability of this peptide region in accommodating the diverse repertoire of transcription-modulating AR cofactors creates a great challenge for those intent on generating relevant three-dimensional molecular models. The AR transactivation domain achieves this flexibility through a series of conformational steps dependent on the presence of cofactors that induce allosteric changes, and thus has evolved several conserved peptide motifs representing key protein-protein interaction surfaces. Elucidation of these signaling regions, including their involvement in inducing AR transactivation domain structural changes, is of foremost interest in understanding how the AR achieves its pivotal role in regulating the androgen signaling axis particularly during the progression of prostate cancer.
Asunto(s)
Receptores Androgénicos/química , Receptores Androgénicos/fisiología , Activación Transcripcional , Secuencia de Aminoácidos , Sitios de Unión , Núcleo Celular/química , ADN/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Fosforilación , Neoplasias de la Próstata , Secuencias Repetitivas de Ácidos Nucleicos , Transducción de SeñalRESUMEN
The androgen receptor (AR) mediates transactivation of target genes by acting as a dimer in which its amino-terminal domain (AR-NTD) interacts with its carboxyl-terminal, ligand-binding domain (AR-LBD) (N/C interaction). Here we assessed if and how AR N/C interaction relates to AR transactivation activity and how the p160 coactivator GRIP1 participates in both processes. The concentration of dihydrotestosterone needed for half-maximal N/C interaction was approximately 10-fold higher than for half-maximal transactivation, indicating a disparity between the two processes. Although a mutation of an LXXLL-like motif, 23 FQNLF 27 --> 23 FQNAA 27 , in the AR-NTD abolished AR N/C interaction, it could be restored by the co-expression of the coactivator GRIP1. Co-expression of mutated forms of GRIP1, possessing alterations known to abolish either of the two AR interaction domains, could not restore AR N/C interaction, suggesting that wild-type GRIP1 normally bridges the two AR domains. Although AR transactivation activity can proceed without AR N/C interaction, we propose that part of the GRIP1 coactivation activity resides in its ability to bind both AR-NTD and -LBD, to stabilize the N/C complex and allow for secondary cofactors to be recruited more efficiently. Our results also indicate that AR N/C interaction enhances but is not necessary for AR transactivation activity.