RESUMEN
Programmed cell death protein 1 (PD-1) inhibitors have modest efficacy as a monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. We present results from a single-arm, open-label, phase 1/2 study of a DNA plasmid PTCV (GNOS-PV02) encoding up to 40 neoantigens coadministered with plasmid-encoded interleukin-12 plus pembrolizumab in patients with advanced HCC previously treated with a multityrosine kinase inhibitor. Safety and immunogenicity were assessed as primary endpoints, and treatment efficacy and feasibility were evaluated as secondary endpoints. The most common treatment-related adverse events were injection-site reactions, observed in 15 of 36 (41.6%) patients. No dose-limiting toxicities or treatment-related grade ≥3 events were observed. The objective response rate (modified intention-to-treat) per Response Evaluation Criteria in Solid Tumors 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response. Clinical responses were associated with the number of neoantigens encoded in the vaccine. Neoantigen-specific T cell responses were confirmed in 19 of 22 (86.4%) evaluable patients by enzyme-linked immunosorbent spot assays. Multiparametric cellular profiling revealed active, proliferative and cytolytic vaccine-specific CD4+ and CD8+ effector T cells. T cell receptor ß-chain (TCRß) bulk sequencing results demonstrated vaccination-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed posttreatment T cell clonal expansion of cytotoxic T cell phenotypes. TCR complementarity-determining region cloning of expanded T cell clones in the tumors following vaccination confirmed reactivity against vaccine-encoded neoantigens. Our results support the PTCV's mechanism of action based on the induction of antitumor T cells and show that a PTCV plus pembrolizumab has clinical activity in advanced HCC. ClinicalTrials.gov identifier: NCT04251117 .
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Vacunas , Humanos , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/efectos adversos , Vacunas/uso terapéuticoRESUMEN
We have previously described a multiprotein complex termed the BHC or BRAF-HDAC complex, which is required for the repression of neuronal-specific genes. We have shown that the BHC complex is recruited by a neuronal silencer, REST (RE1-silencing transcription factor), and mediates the repression of REST-responsive genes. BHC is a multiprotein complex consisting of two enzymatic activities: a histone deacetylase (HDAC1 or 2) and a recently described histone demethylase (BHC110, also known as LSD1 or AOF2). Here we show that BHC110-containing complexes show a nearly fivefold increase in demethylation of histone H3 lysine 4 (H3K4) compared to recombinant BHC110. Furthermore, recombinant BHC110 is unable to demethylate H3K4 on nucleosomes, but BHC110-containing complexes readily demethylate nucleosomes. In vitro reconstitution of the BHC complex using recombinant subunits reveals an essential role for the REST corepressor CoREST, not only in stimulating demethylation on core histones but also promoting demethylation of nucleosomal substrates. We find that nucleosomal demethylation is the result of CoREST enhancing the association between BHC110 and nucleosomes. Depletion of CoREST in in vivo cell culture results in de-repression of REST-responsive gene expression and increased methylation of H3K4. Together, these results highlight an essential role for CoREST in demethylation of H3K4 both in vitro and in vivo.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Acetilación , Secuencia de Aminoácidos , Línea Celular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Histona Desacetilasas/metabolismo , Histona Demetilasas , Humanos , Lisina/genética , Metilación , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Oxidorreductasas N-Desmetilantes/genéticaRESUMEN
MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex. These pre-miRNAs are cleaved by the RNase III Dicer to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2), the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.
Asunto(s)
Silenciador del Gen , Péptidos y Proteínas de Señalización Intracelular/metabolismo , MicroARNs/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Ribonucleasa III/metabolismo , Proteínas Argonautas , Línea Celular , Factor 2 Eucariótico de Iniciación , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , MicroARNs/biosíntesis , MicroARNs/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Coactivadores de Receptor Nuclear , Factores de Iniciación de Péptidos/genética , Unión Proteica , Ribonucleasa III/genética , Transcripción GenéticaRESUMEN
Neoantigens are tumor-specific antigens that arise due to somatic mutations in the DNA of tumor cells. They represent ideal targets for cancer immunotherapy since there is minimal risk for on-target, off-tumor toxicities. Additionally, these are foreign antigens that should be immunogenic due to lack of central immune tolerance. Tumor neoantigens are predominantly passenger mutations, which do not contribute to tumorigenesis. In cases of multi-focal or metastatic tumors, different foci can have significantly different mutation profiles. This suggests that it is important to target as many neoantigens as possible to better control tumors and target multi-focal tumors within the same patient. Herein, we report a study targeting up to 40 neoantigens using a single DNA plasmid. We observed significant plasticity in the epitope strings arranged in the vaccine with regard to immune induction and tumor control. Different vaccines elicited T cell responses against multiple epitopes on the vaccine string and controlled growth of multi-focal, heterogeneous tumors in a therapeutic tumor challenge. Additionally, the multi-epitope antigens induced long-term immunity and rejected a tumor re-challenge several weeks after the final vaccination. These data provide evidence that DNA-encoded long antigen strings can be an important tool for immunotherapeutic vaccination against neoantigens with implications for other in vivo-delivered antigen strings.
RESUMEN
Estrogen actions are mediated by a complex interface of direct control of gene expression (the so-called "genomic action") and by regulation of cell signaling/phosphorylation cascades, referred to as the "nongenomic," or extranuclear, action. We have previously described the identification of MNAR (modulator of nongenomic action of estrogen receptor) as a novel scaffold protein that regulates estrogen receptor alpha (ERalpha) activation of cSrc. In this study, we have investigated the role of MNAR in 17beta-estradiol (E2)-induced activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Consistent with our previous results, a direct correlation was established between MNAR expression levels and E2-induced activation of PI3 and Akt kinases. Endogenous MNAR, ERalpha, cSrc, and p85, the regulatory subunit of PI3 kinase, interacted in MCF7 cells treated with E2. The interaction between p85 and MNAR required activation of cSrc and MNAR phosphorylation on Tyr 920. Consequently, the mutation of this tyrosine to alanine (Y920A) abrogated the interaction between MNAR and p85 and the E2-induced activation of the PI3K/Akt pathway, which was required for the E2-induced protection of MCF7 cells from apoptosis. Nonetheless, the Y920A mutant potentiated the E2-induced activation of the Src/MAPK pathway and MCF7 cell proliferation, as observed with the wild-type MNAR. These results provide new and important insights into the molecular mechanisms of E2-induced regulation of cell proliferation and apoptosis.
Asunto(s)
Estrógenos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Estrógenos/metabolismo , Alanina/metabolismo , Sustitución de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Activación Enzimática/efectos de los fármacos , Estradiol/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/metabolismo , Transfección , Tirosina/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
MicroRNAs (miRNAs) are a growing family of small non-protein-coding regulatory genes that regulate the expression of homologous target-gene transcripts. They have been implicated in the control of cell death and proliferation in flies, haematopoietic lineage differentiation in mammals, neuronal patterning in nematodes and leaf and flower development in plants. miRNAs are processed by the RNA-mediated interference machinery. Drosha is an RNase III enzyme that was recently implicated in miRNA processing. Here we show that human Drosha is a component of two multi-protein complexes. The larger complex contains multiple classes of RNA-associated proteins including RNA helicases, proteins that bind double-stranded RNA, novel heterogeneous nuclear ribonucleoproteins and the Ewing's sarcoma family of proteins. The smaller complex is composed of Drosha and the double-stranded-RNA-binding protein, DGCR8, the product of a gene deleted in DiGeorge syndrome. In vivo knock-down and in vitro reconstitution studies revealed that both components of this smaller complex, termed Microprocessor, are necessary and sufficient in mediating the genesis of miRNAs from the primary miRNA transcript.
Asunto(s)
MicroARNs/biosíntesis , Procesamiento Postranscripcional del ARN , Ribonucleasa III/metabolismo , Línea Celular , Cromatografía de Afinidad , Cromatografía en Gel , Células HeLa , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Peso Molecular , Complejos Multiproteicos , Unión Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas de Unión al ARN , Ribonucleasa III/química , Ribonucleasa III/genética , Ribonucleasa III/aislamiento & purificaciónRESUMEN
Overcoming tolerance to tumor-associated antigens remains a hurdle for cancer vaccine-based immunotherapy. A strategy to enhance the anti-tumor immune response is the inclusion of adjuvants to cancer vaccine protocols. In this report, we generated and systematically screened over twenty gene-based molecular adjuvants composed of cytokines, chemokines, and T cell co-stimulators for the ability to increase anti-tumor antigen T cell immunity. We identified several robust adjuvants whose addition to vaccine formulations resulted in enhanced T cell responses targeting the cancer antigens STEAP1 and TERT. We further characterized direct T cell stimulation through CD80-Fc and indirect T cell targeting via the dendritic cell activator Flt3L-Fc. Mechanistically, intramuscular delivery of Flt3L-Fc into mice was associated with a significant increase in infiltration of dendritic cells at the site of administration and trafficking of activated dendritic cells to the draining lymph node. Gene expression analysis of the muscle tissue confirmed a significant up-regulation in genes associated with dendritic cell signaling. Addition of CD80-Fc to STEAP1 vaccine formulation mimicked the engagement provided by DCs and increased T cell responses to STEAP1 by 8-fold, significantly increasing the frequency of antigen-specific cells expressing IFNγ, TNFα, and CD107a for both CD8+ and CD4+ T cells. CD80-Fc enhanced T cell responses to multiple tumor-associated antigens including Survivin and HPV, indicating its potential as a universal adjuvant for cancer vaccines. Together, the results of our study highlight the adjuvanting effect of T cell engagement either directly, CD80-Fc, or indirectly, Flt3L-Fc, for cancer vaccines.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígeno B7-1/inmunología , Vacunas contra el Cáncer/inmunología , Proteínas de la Membrana/inmunología , Neoplasias/terapia , Linfocitos T/inmunología , Tetraspanina 28/inmunología , Animales , Antígenos de Neoplasias , Antígeno B7-1/genética , Movimiento Celular/inmunología , Citocinas/metabolismo , ADN/genética , Células Dendríticas/inmunología , Femenino , Humanos , Inmunoterapia/métodos , Activación de Linfocitos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Neoplasias/inmunología , Plásmidos/genética , Vacunas de ADN/genética , Vacunas de ADN/inmunologíaRESUMEN
Respiratory Syncytial virus (RSV) is a major threat to many vulnerable populations. There are currently no approved vaccines, and RSV remains a high unmet global medical need. Here we describe the employment of a novel synthetic DNA-encoded antibody technology platform to develop and deliver an engineered human DNA-encoded monoclonal antibody (dMAbTM) targeting the fusion protein (F) of RSV as a new approach to prevention or therapy of at risk populations. In in vivo models, a single administration of synthetic DNA-encoding the single-chain fragment variable-constant fragment (scFv-Fc) RSV-F dMAb resulted in robust and durable circulating levels of a functional antibody systemically and in mucosal tissue. In cotton rats, which are the gold-standard animals to model RSV infection, we observed sustained scFv-Fc RSV-F dMAb in the sera and lung-lavage samples, demonstrating the potential for both long-lasting immunity to RSV and effective biodistribution. The scFv-Fc RSV-F dMAb harbored in the sera exhibited RSV antigen-specific binding and potent viral neutralizing activity. Importantly, in vivo delivery of synthetic DNA-encoding, the scFv-Fc RSV-F dMAb protected animals against viral challenge. Our findings support the significance of dMAbs as a potential platform technology for durable protection against RSV disease.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio , Vacunas contra Virus Sincitial Respiratorio , Animales , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/genética , Sigmodontinae , Distribución Tisular , Proteínas Virales de Fusión/genéticaRESUMEN
Lyme disease is the most common vector-borne disease in North America. The etiological agent is the spirochete Borreliella burgdorferi, transmitted to mammalian hosts by the Ixodes tick. In recent years there has been an increase in the number of cases of Lyme disease. Currently, there is no vaccine on the market for human use. We describe the development of a novel synthetically engineered DNA vaccine, pLD1 targeting the outer-surface protein A (OspA) of Borreliella burgdorferi. Immunization of C3 H/HeN mice with pLD1 elicits robust humoral and cellular immune responses that confer complete protection against a live Borreliella burgdorferi bacterial challenge. We also assessed intradermal (ID) delivery of pLD1 in Hartley guinea pigs, demonstrating the induction of robust and durable humoral immunity that lasts at least 1 year. We provide evidence of the potency of pLD1 by showing that antibodies targeting the OspA epitopes which have been associated with protection are prominently raised in the immunized guinea pigs. The described study provides the basis for the advancement of pDL1 as a potential vaccine for Lyme disease control.
Asunto(s)
Grupo Borrelia Burgdorferi , Borrelia burgdorferi , Enfermedad de Lyme , Vacunas de ADN , Animales , Anticuerpos Antibacterianos , Antígenos de Superficie , Proteínas de la Membrana Bacteriana Externa , Vacunas Bacterianas , Borrelia burgdorferi/genética , Cobayas , Enfermedad de Lyme/prevención & control , Ratones , América del NorteRESUMEN
Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors.
Asunto(s)
Histona Desacetilasas/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Acetilación/efectos de los fármacos , Proteínas de Unión al ADN/química , Inhibidores Enzimáticos/farmacología , Células HeLa , Histona Desacetilasa 1 , Histona Demetilasas , Humanos , Metilación/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , Nucleosomas/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismoRESUMEN
Estrogens play a critical role in the regulation of cellular proliferation, differentiation, and apoptosis. Evidence indicates that this regulation is mediated by a complex interface of direct control of gene expression (so-called "genomic action") and by regulation of cell-signaling/phosphorylation cascades (referred to as the "non-genomic", or "extranuclear" action). However, the mechanisms of the non-genomic action of estrogens are not well defined. We have recently described the identification of a novel scaffold protein termed MNAR (modulator of non-genomic action of estrogen receptor), that couples conventional steroid receptors with extranuclear signal transduction pathways, thus potentially providing additional and tissue- or cell-specific level of steroid hormone regulation of cell functions. We have demonstrated that the MNAR is required for ER alpha (ERa) interaction with p60(src) (Src), which leads to activation of Src/MAPK pathway. Our new data also suggest that activation of cSrc in response to E2 leads to MNAR phosphorylation, interaction with p85, and activation of the PI3 and Akt kinases. These data therefore suggest that MNAR acts as an important scaffold that integrates ERa action in regulation of important signaling pathways. ERa non-genomic action has been suggested to play a key role in estrogen-induced cardio-, neuro-, and osteo-protection. Therefore, evaluation of the molecular crosstalk between MNAR and ERa may lead to development of functionally selective ER modulators that can separate between beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS and the "detrimental", proliferative effects in reproductive tissues and organs.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Regulación Neoplásica de la Expresión Génica , Fosfatidilinositol 3-Quinasas/metabolismo , Transactivadores/fisiología , Familia-src Quinasas/metabolismo , Dominio Catalítico , Línea Celular Tumoral , Proliferación Celular , Proteínas Co-Represoras , Activación Enzimática , Humanos , Fosforilación , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Transactivadores/metabolismo , Factores de TranscripciónRESUMEN
Notch signaling blocks differentiation of 3T3-L1 preadipocytes, and this can be mimicked by constitutive expression of the Notch target gene Hes-1. Although considered initially to function only as a repressor, recent evidence indicates that Hes-1 can also activate transcription. We show here that the domains of Hes-1 needed to block adipogenesis coincide with those necessary for transcriptional repression. HRT1, another basic-helix-loop-helix protein and potential Hes-1 partner, was also induced by Notch in 3T3-L1 cells but did not block adipogenesis, suggesting that Hes-1 functions primarily as a homodimer or possibly as a heterodimer with an unknown partner. Purification of Hes-1 identified the Groucho/transducin-like enhancer of split family of corepressors as the only significant Hes-1 interacting proteins in vivo. An evaluation of global gene expression in preadipocytes identified approximately 200 Hes-1-responsive genes comprising roughly equal numbers of up-regulated and down-regulated genes. However, promoter analyses indicated that the down-regulated genes were significantly more likely to contain Hes-1 binding sites, indicating that Hes-1 is more likely to repress transcription of its direct targets. We conclude that Notch most likely blocks adipogenesis through the induction of Hes-1 homodimers, which repress transcription of key target genes.
Asunto(s)
Adipocitos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Homeodominio/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Co-Represoras , Dimerización , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Ratones , Células 3T3 NIH/metabolismo , Regiones Promotoras Genéticas , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Células Madre/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factor de Transcripción HES-1RESUMEN
The biochemical pathways that are disrupted in the genesis of sporadic breast cancers remain unclear. Moreover, the present prognosticating markers used to determine the prognosis of node-negative patient leads to probabilistic results, and the eventual clinical course is far from certain. Here we identified the human TREX complex, a multiprotein complex that links transcription elongation to mRNA transport, as culprit of aggressive human breast cancers. We show that whereas p84N5 (called hTREX84) is expressed at very low levels in normal breast epithelial cells, it is highly expressed in breast tumors. Importantly, hTREX84 expression correlates with tumor size and the metastatic state of the tumor progression. Reduction of hTREX84 levels in breast cancer cell lines by small interfering RNA result in inhibition of cellular proliferation and abrogation of mRNA export. These results not only identify hTREX84 as a prognosticator of breast cancer but also delineate human TREX complex as a target for therapeutic drugs against breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , ARN Mensajero/genética , Factores de Elongación Transcripcional/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN , Progresión de la Enfermedad , Femenino , Células HeLa , Humanos , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN , TransfecciónRESUMEN
WAY-255348 is a potent nonsteroidal progesterone receptor (PR) antagonist previously characterized in rodents and nonhuman primates. This report describes the novel mechanism by which WAY-255348 inhibits the activity of progesterone. Most PR antagonists bind to and block PR action by inducing a unique "antagonist" conformation of the PR. However, WAY-255348 lacks the bulky side chains or chemical groups that have been associated with the conformation changes of helix 12 that lead to functional antagonism. We show that WAY-255348 achieves antagonist activity by binding to and subsequently preventing progesterone-induced nuclear accumulation, phosphorylation and promoter interactions of the PR. This effect was concentration dependent, as high concentrations of WAY-255348 alone are able to induce nuclear translocation, phosphorylation and subsequent promoter interactions resulting in partial agonist activity at these concentrations. However, at lower concentrations where nuclear accumulation and phosphorylation are prevented, the progesterone-induced DNA binding is blocked along with PR-dependent gene expression. Analysis of the PR conformation induced by WAY-255348 using a limited protease digestion assay, suggested that the WAY-255348 bound PR conformation was similar to that of a progesterone agonist-bound PR and distinct from steroidal antagonist-bound PR conformations. Furthermore, the recruitment and binding of peptides derived from nuclear receptor co-activators is consistent with WAY-255348 inducing an agonist-like conformation. Taken together, these data suggest that WAY-255348 inhibits PR action through a novel molecular mechanism that is distinct from previously studied PR modulators and may be a useful tool to further understanding of PR signaling pathways. Development of therapeutic molecules with this 'passive' antagonism mechanism may provide distinct advantages for patients with reproductive disorders or PR positive breast cancers.
Asunto(s)
Indoles/farmacología , Pirroles/farmacología , Receptores de Progesterona/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Unión Competitiva , Línea Celular Tumoral , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Proteínas Co-Represoras/metabolismo , Agonismo Parcial de Drogas , Humanos , Modelos Moleculares , Coactivadores de Receptor Nuclear/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Conformación Proteica , Ensayo de Unión Radioligante , Receptores de Progesterona/agonistas , Receptores de Progesterona/genéticaRESUMEN
The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is an essential component of transcriptional regulation and RNA processing of protein-coding genes. A large body of data also implicates the CTD in the transcription and processing of RNAPII-mediated small nuclear RNAs (snRNAs). However, the identity of the complex (or complexes) that associates with the CTD and mediates the processing of snRNAs has remained elusive. Here, we describe an RNA polymerase II complex that contains at least 12 novel subunits, termed the Integrator, in addition to core RNAPII subunits. Two of the Integrator subunits display similarities to the subunits of the cleavage and polyadenylation specificity factor (CPSF) complex. We show that Integrator is recruited to the U1 and U2 snRNA genes and mediates the snRNAs' 3' end processing. The Integrator complex is evolutionarily conserved in metazoans and directly interacts with the C-terminal domain of the RNA polymerase II largest subunit.
Asunto(s)
ARN Polimerasa II/metabolismo , Procesamiento Postranscripcional del ARN , ARN Nuclear Pequeño/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Western Blotting , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Línea Celular , Inmunoprecipitación de Cromatina , Secuencia Conservada , Endorribonucleasas , Escherichia coli/genética , Evolución Molecular , Gliceraldehído-3-Fosfato Deshidrogenasas/análisis , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Subunidades de Proteína/química , ARN/biosíntesis , ARN Polimerasa II/química , ARN Mensajero/análisis , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , ARN Nuclear Pequeño/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Although Argonaute2 has been identified as the catalytic center of RISC, the RISC polypeptide composition and assembly using short interfering RNA (siRNA) duplexes has remained elusive. Here we show that RISC is composed of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2. We demonstrate that this complex can cleave target RNA using precursor microRNA (pre-miRNA) hairpin as the source of siRNA. Although RISC can also utilize duplex siRNA, it displays a nearly 10-fold greater activity using the pre-miRNA Dicer substrate. RISC distinguishes the guide strand of the siRNA from the passenger strand and specifically incorporates the guide strand. Importantly, ATP is not required for miRNA processing, RISC assembly, or multiple rounds of target-RNA cleavage. These results define the composition of RISC and demonstrate that miRNA processing and target-RNA cleavage are coupled.
Asunto(s)
MicroARNs/metabolismo , Interferencia de ARN , Complejo Silenciador Inducido por ARN/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Proteínas Argonautas , Secuencia de Bases , Catálisis , Línea Celular , Difosfatos/metabolismo , Factor 2 Eucariótico de Iniciación , Humanos , MicroARNs/genética , Modelos Genéticos , Factores de Iniciación de Péptidos/metabolismo , Fosfatos/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismoRESUMEN
Chromatin remodeling complexes play critical roles in development. Here we describe a transcription factor, CECR2, which is involved in neurulation and chromatin remodeling. CECR2 shows complex alternative splicing, but all variants contain DDT and bromodomain motifs. A mutant mouse line was generated from an embryonic stem cell line containing a genetrap within Cecr2. Reporter gene expression demonstrated Cecr2 expression to be predominantly neural in the embryo. Mice homozygous for the Cecr2 genetrap-induced mutation show a high penetrance of the neural tube defect exencephaly, the human equivalent of anencephaly, in a strain-dependent fashion. Biochemical isolation of CECR2 revealed the presence of this protein as a component of a novel heterodimeric complex termed CECR2-containing remodeling factor (CERF). CERF comprises CECR2 and the ATP-dependent chromatin remodeler SNF2L, a mammalian ISWI ortholog expressed predominantly in the central nervous system. CERF is capable of remodeling chromatin in vitro and displays an ATP hydrolyzing activity that is stimulated by nucleosomes. Together, these data identify a novel chromatin remodeling complex with a critical role in neurulation.
Asunto(s)
Sistema Nervioso Central/embriología , Ensamble y Desensamble de Cromatina , Cromatina , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Empalme Alternativo , Animales , Células Cultivadas , Sistema Nervioso Central/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , Defectos del Tubo Neural , Nucleosomas/metabolismo , Factores de Transcripción/genéticaRESUMEN
Mammalian genomes encode two imitation switch family chromatin remodeling proteins, SNF2H and SNF2L. In the mouse, SNF2H is expressed ubiquitously, whereas SNF2L expression is limited to the brain and gonadal tissue. This pattern of SNF2L expression suggests a critical role for SNF2L in neuronal physiology. Indeed, SNF2L was shown to promote neurite outgrowth as well as regulate the human engrailed homeotic genes, important regulators of brain development. Here we identify a novel splice variant of human SNF2L we call SNF2L+13, which contains a nonconserved in-frame exon within the conserved catalytic core domain of SNF2L. SNF2L+13 retains the ability to incorporate into multiprotein complexes; however, it is devoid of enzymatic activity. Most interestingly, unlike mouse SNF2L, human SNF2L is expressed ubiquitously, and regulation is mediated by isoform variation. The human SNF2L+13 null variant is predominant in non-neuronal tissue, whereas the human wild type active SNF2L isoform is expressed in neurons. Thus, like the mouse, active human SNF2L is limited to neurons and a few other tissues.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Adenosina Trifosfatasas/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Dominio Catalítico , Línea Celular , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Cromatografía , Enzimas de Restricción del ADN/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Exones , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Modelos Biológicos , Modelos Genéticos , Datos de Secuencia Molecular , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
We have isolated a holoenzyme complex termed BRCC containing BRCA1, BRCA2, and RAD51. BRCC not only displays increased association with p53 following DNA damage but also ubiquitinates p53 in vitro. BRCC36 and BRCC45 are novel components of the complex with sequence homology to a subunit of the signalosome and proteasome complexes. Reconstitution of a recombinant four-subunit complex containing BRCA1/BARD1/BRCC45/BRCC36 revealed an enhanced E3 ligase activity compared to that of BRCA1/BARD1 heterodimer. In vivo, depletion of BRCC36 and BRCC45 by the small interfering RNAs (siRNAs) resulted in increased sensitivity to ionizing radiation and defects in G2/M checkpoint. BRCC36 shows aberrant expression in sporadic breast tumors. These findings identify BRCC as a ubiquitin E3 ligase complex that enhances cellular survival following DNA damage.