RESUMO
Although the brain functions of specific acetyltransferases such as the CREB-binding protein (CBP) and p300 have been well documented using mutant transgenic mice models, studies based on their direct pharmacological activation are still missing due to the lack of cell-permeable activators. Here we present a small-molecule (TTK21) activator of the histone acetyltransferases CBP/p300, which, when conjugated to glucose-based carbon nanosphere (CSP), passed the blood-brain barrier, induced no toxicity, and reached different parts of the brain. After intraperitoneal administration in mice, CSP-TTK21 significantly acetylated histones in the hippocampus and frontal cortex. Remarkably, CSP-TTK21 treatment promoted the formation of long and highly branched doublecortin-positive neurons in the subgranular zone of the dentate gyrus and reduced BrdU incorporation, suggesting that CBP/p300 activation favors maturation and differentiation of adult neuronal progenitors. In addition, mRNA levels of the neuroD1 differentiation marker and BDNF, a neurotrophin required for the terminal differentiation of newly generated neurons, were both increased in the hippocampus concomitantly with an enrichment of acetylated-histone on their proximal promoter. Finally, we found that CBP/p300 activation during a spatial training, while not improving retention of a recent memory, resulted in a significant extension of memory duration. This report is the first evidence for CBP/p300-mediated histone acetylation in the brain by an activator molecule, which has beneficial implications for the brain functions of adult neurogenesis and long-term memory. We propose that direct stimulation of acetyltransferase function could be useful in terms of therapeutic options for brain diseases.
Assuntos
Proteína de Ligação a CREB/metabolismo , Ativadores de Enzimas/farmacologia , Memória/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismo , Acetiltransferases/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/crescimento & desenvolvimento , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Contagem de Células , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Dendritos/metabolismo , Dendritos/ultraestrutura , Imunofluorescência , Hipocampo/citologia , Hipocampo/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/isolamento & purificação , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanosferas , Neurônios/metabolismo , Neurônios/ultraestrutura , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The recent developments in the field of epigenetics have changed the way the covalent modifications were perceived from mere chemical tags to important biological recruiting platforms as well as decisive factors in the process of transcriptional regulation and gene expression. Over the years, the parallel investigations in the area of epigenetics and disease have also shown the significance of the epigenetic modifications as important regulatory nodes that exhibit dysfunction in disease states. In the present scenario where epigenetic therapy is also being considered at par with the conventional therapeutic strategies, this article reviews the role of histone acetylation as an epigenetic mark involved in different biological processes associated with normal as well as abnormal gene expression states, modulation of this acetylation by small molecules and warrants the possibility of acetylation as a therapeutic target.
Assuntos
Epigênese Genética/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetilação , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Humanos , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, "KAPRK," where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.
Assuntos
Arginina/metabolismo , Ácido Elágico/metabolismo , Histonas/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Ácido Elágico/química , Regulação da Expressão Gênica , Histonas/química , Histonas/genética , Humanos , Lythraceae/química , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Prolina/metabolismo , Estrutura Terciária de Proteína , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Termodinâmica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Xenopus laevisRESUMO
Neurodegenerative diseases, such as polyglutamine-related diseases, amyotrophic lateral sclerosis, and Alzheimer's disease are accompanied by transcriptional dysfunctions, leading to neuronal death. It is becoming more evident that the chromatin acetylation status is impaired during the lifetime of neurons, by a common mechanism related to the loss of function of histone acetyltransferase (HAT) activity. Notably, the HAT termed cAMP response element binding protein (CREB)-binding protein (CBP) was shown to display neuroprotective functions. Several other HATs have now been shown to participate in basic but vital neuronal functions. In addition, there is increasing evidence of several HATs (including CBP), as essential regulators of neuronal plasticity and memory formation processes. In order to counteract neuronal loss and/or memory deficits in neurodegenerative diseases, the current therapeutic strategies involve the use of small molecules antagonizing histone deacetylase (HDAC) activity (i.e. HDAC inhibitors). Although this strategy lacks specificity, some of these molecules display promising therapeutic properties. With the rapidly evolving literature on HATs and their respective functions in neuronal survival and memory formation, it seems essential to envisage direct stimulation of the acetyltransferase function as a new therapeutic tool in neurodegenerative diseases. In this review, we will highlight the present understanding and the future prospects of such therapeutic approach.
Assuntos
Cromatina/metabolismo , Ativadores de Enzimas/uso terapêutico , Histona Acetiltransferases/metabolismo , Memória/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Plasticidade Neuronal/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativadores de Enzimas/química , Histona Acetiltransferases/química , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/patologia , Neurônios/enzimologia , Neurônios/patologiaRESUMO
Chromatin modifications have gained immense significance in the past few decades as key regulators of gene expression. The enzymes responsible for these modifications along with the other non-histone proteins, remodeling factors and small RNAs modulate the chromatin dynamicity, which in turn directs the chromatin function. A concerted action of different modifying enzymes catalyzes these modifications, which are read by effector modules and converted to functional outcomes by various protein complexes. Several small molecules in the physiological system such as acetyl CoA, NAD(+), and ATP are actively involved in regulating these functional outcomes. Recent understanding in the field of epigenetics indicate the possibility of the existence of a network, 'the epigenetic language' involving cross talk among different modifications that could regulate cellular processes like transcription, replication and repair. Hence, these modifications are essential for the cellular homeostasis, and any alteration in this balance leads to a pathophysiological condition or disease manifestation. Therefore, it is becoming more evident that modulators of these modifying enzymes could be an attractive therapeutic strategy, popularly referred to as 'Epigenetic therapy.' Although this field is currently monopolized by DNA methylation and histone deacetylase inhibitors, this review highlights the modulators of the other modifications namely histone acetylation, lysine methylation and arginine methylation and argues in favor of their therapeutic potential.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Cromatina/química , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Ativadores de Enzimas/química , Ativadores de Enzimas/uso terapêutico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Histonas/química , Humanos , Metilação/efeitos dos fármacos , NAD/química , NAD/metabolismo , Transcrição Gênica/efeitos dos fármacosAssuntos
Epigênese Genética , Lisina/metabolismo , Metabolismo , Acetilação , Humanos , Terapêutica/métodosRESUMO
Coactivator-associated arginine methyltransferase (CARM1/PRMT4)-mediated transcriptional coactivation and arginine methylation is known to regulate various tissue-specific differentiation events. Although CARM1 is expressed in the neural crest region in early development, coinciding with early neuronal progenitor specification, the role of CARM1 in any neuronal developmental pathways has been unexplored. Using a specific small-molecule inhibitor of CARM1-mediated H3R17 methylation in human embryonic stem cell line, we find that H3R17 methylation contributes to the maintenance of the astroglial cell population. A network of regulation was observed on the miR92a promoter by which H3R17-responsive Nanog bound to the miR92a promoter decreased upon inhibition, resulting in an abnormal gene expression program influencing the glial lineage. This was also true in zebrafish, in which, with the help of CARM1 inhibitor and CARM1 morpholinos, we show that inhibition of H3R17 methylation results in defective glial cell morphology and a sensory defect in a subpopulation. A gain-of-function strategy in which mCARM1 was introduced in the morpholino-treated embryos exhibited recovery of the sensory defect phenotype. This study thus establishes the functional cooperation between arginine methylation and microRNA expression in the neuronal developmental process, with potential implications in sensory development pathways.
Assuntos
Astrócitos/metabolismo , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Proteína-Arginina N-Metiltransferases/genética , Ativação Transcricional , Animais , Arginina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Metilação , MicroRNAs/metabolismo , Microscopia de Fluorescência , Proteína Homeobox Nanog , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
The acetylation of histone and non-histone proteins controls a great deal of cellular functions, thereby affecting the entire organism, including the brain. Acetylation modifications are mediated through histone acetyltransferases (HAT) and deacetylases (HDAC), and the balance of these enzymes regulates neuronal homeostasis, maintaining the pre-existing acetyl marks responsible for the global chromatin structure, as well as regulating specific dynamic acetyl marks that respond to changes and facilitate neurons to encode and strengthen long-term events in the brain circuitry (e.g., memory formation). Unfortunately, the dysfunction of these finely-tuned regulations might lead to pathological conditions, and the deregulation of the HAT/HDAC balance has been implicated in neurological disorders. During the last decade, research has focused on HDAC inhibitors that induce a histone hyperacetylated state to compensate acetylation deficits. The use of these inhibitors as a therapeutic option was efficient in several animal models of neurological disorders. The elaboration of new cell-permeant HAT activators opens a new era of research on acetylation regulation. Although pathological animal models have not been tested yet, HAT activator molecules have already proven to be beneficial in ameliorating brain functions associated with learning and memory, and adult neurogenesis in wild-type animals. Thus, HAT activator molecules contribute to an exciting area of research.
Assuntos
Acetiltransferases/genética , Histonas/genética , Doenças do Sistema Nervoso/terapia , Neurônios/metabolismo , Acetilação , Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismoRESUMO
Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.
Assuntos
Inibidores Enzimáticos/química , Naftoquinonas/química , Raízes de Plantas/química , Plumbaginaceae/química , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Acetilação , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Naftoquinonas/farmacologia , Estrutura Terciária de Proteína/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. We report the dynamic interaction of the DNA intercalator and potential anticancer plant alkaloid, sanguinarine (SGR), with chromatin. Association of SGR with different levels of chromatin structure was enthalpy driven with micromolar dissociation constant. Apart from DNA, it binds with comparable affinity with core histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR-mediated repression of epigenetic marks and the alteration of chromatin geography (nucleography) also result in the modulation of global gene expression. These data, conclusively, show an anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context.
Assuntos
Benzofenantridinas/metabolismo , Cromatina/genética , Cromatina/metabolismo , Isoquinolinas/metabolismo , Acetilação/efeitos dos fármacos , Animais , Cromatina/química , DNA/química , DNA/genética , DNA/metabolismo , Epigênese Genética , Células HeLa , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Metilação/efeitos dos fármacos , Conformação Molecular , Ratos , Transcrição GênicaRESUMO
In this report, we demonstrate glucose-derived carbon nanospheres to be an emerging class of intracellular carriers. The surfaces of these spheres are highly functionalized and do not need any further modification. Besides, the intrinsic fluorescence property of carbon nanospheres helps in tracking their cellular localization without any additional fluorescent tags. The spheres are found to target the nucleus of the mammalian cells, causing no toxicity. Interestingly, the in vivo experiments show that these nanospheres have an important ability to cross the blood-brain barrier and localize in the brain besides getting localized in the liver and the spleen. There is also evidence to show that they are continuously being removed from these tissues over time. Furthermore, these nanospheres were used as a carrier for the membrane-impermeable molecule CTPB (N-(4-chloro-3-trifluoromethylphenyl)-2-ethoxybenzamide), the only known small-molecule activator of histone acetyltransferase (HAT) p300. Biochemical analyses such as Western blotting, immunohistochemistry, and gene expression analysis show the induction of the hyperacetylation of histone acetyltransferase (HAT) p300 (autoacetylation) as well as histones both in vitro and in vivo and the activation of HAT-dependent transcription upon CTPB delivery. These results establish an alternative path for the activation of gene expression mediated by the induction of HAT activity instead of histone deacetylase (HDAC) inhibition.