RESUMO
hTERT, the catalytic component of the human telomerase enzyme, is regulated by post-translational modifications, like phosphorylation and ubiquitination by multiple proteins which remarkably affects the overall activity of the enzyme. Here we report that hTERT gets SUMOylated by SUMO1 and polycomb protein CBX4 acts as the SUMO E3 ligase of hTERT. hTERT SUMOylation positively regulates its telomerase activity which can be inhibited by SENP3-mediated deSUMOylation. Interestingly, we have established a new role of hTERT SUMOylation in the repression of E-cadherin gene expression and consequent triggering on the epithelial-mesenchymal-transition (EMT) program in breast cancer cells. We also observed that catalytically active CBX4, leads to retention of hTERT/ZEB1 complex onto E-cadherin promoter leading to its repression through hTERT-SUMOylation. Further through wound healing and invasion assays in breast cancer cells, we showed the tumor promoting ability of hTERT was significantly compromised upon overexpression of SUMO-defective mutant of hTERT. Thus our findings establish a new post-translational modification of hTERT which on one hand is involved in telomerase activity maintenance and on the other hand plays a crucial role in the regulation of gene expression thereby promoting migration and invasion of breast cancer cells.
Assuntos
Antígenos CD/metabolismo , Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Movimento Celular , Ligases/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Telomerase/metabolismo , Transcrição Gênica , Antígenos CD/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/genética , Feminino , Células HeLa , Humanos , Ligases/genética , Células MCF-7 , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas do Grupo Polycomb/genética , Telomerase/genéticaRESUMO
Transcription factor 19 (TCF19) has been reported as a type 1 diabetes-associated locus involved in maintenance of pancreatic ß cells through a fine-tuned regulation of cell proliferation and apoptosis. TCF19 also exhibits genomic association with type 2 diabetes, although the precise molecular mechanism remains unknown. It harbors both a plant homeodomain and a forkhead-associated domain implicated in epigenetic recognition and gene regulation, a phenomenon that has remained unexplored. Here, we show that TCF19 selectively interacts with histone 3 lysine 4 trimethylation through its plant homeodomain finger. Knocking down TCF19 under high-glucose conditions affected many metabolic processes, including gluconeogenesis. We found that TCF19 overexpression represses de novo glucose production in HepG2 cells. The transcriptional repression of key genes, induced by TCF19, coincided with NuRD (nucleosome-remodeling-deacetylase) complex recruitment to the promoters of these genes. TCF19 interacted with CHD4 (chromodomain helicase DNA-binding protein 4), which is a part of the NuRD complex, in a glucose concentration-independent manner. In summary, our results show that TCF19 interacts with an active transcription mark and recruits a co-repressor complex to regulate gluconeogenic gene expression in HepG2 cells. Our study offers critical insights into the molecular mechanisms of transcriptional regulation of gluconeogenesis and into the roles of chromatin readers in metabolic homeostasis.
Assuntos
Gluconeogênese , Hepatócitos/metabolismo , Histonas/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Linhagem Celular , Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/enzimologia , Histonas/química , Histonas/genética , Humanos , Lisina , Metilação , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Regiões Promotoras Genéticas , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
ZMYND8 (zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8), a newly identified component of the transcriptional coregulator network, was found to interact with the Nucleosome Remodeling and Deacetylase (NuRD) complex. Previous reports have shown that ZMYND8 is instrumental in recruiting the NuRD complex to damaged chromatin for repressing transcription and promoting double strand break repair by homologous recombination. However, the mode of transcription regulation by ZMYND8 has remained elusive. Here, we report that through its specific key residues present in its conserved chromatin-binding modules, ZMYND8 interacts with the selective epigenetic marks H3.1K36Me2/H4K16Ac. Furthermore, ZMYND8 shows a clear preference for canonical histone H3.1 over variant H3.3. Interestingly, ZMYND8 was found to be recruited to several developmental genes, including the all-trans-retinoic acid (ATRA)-responsive ones, through its modified histone-binding ability. Being itself inducible by ATRA, this zinc finger transcription factor is involved in modulating other ATRA-inducible genes. We found that ZMYND8 interacts with transcription initiation-competent RNA polymerase II phosphorylated at Ser-5 in a DNA template-dependent manner and can alter the global gene transcription. Overall, our study identifies that ZMYND8 has CHD4-independent functions in regulating gene expression through its modified histone-binding ability.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Receptores de Superfície Celular/metabolismo , Tretinoína/farmacologia , Acetilação/efeitos dos fármacos , Autoantígenos/genética , Autoantígenos/metabolismo , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Células HEK293 , Células HeLa , Histonas/genética , Humanos , Metilação/efeitos dos fármacos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Proteínas Supressoras de TumorRESUMO
BACKGROUND: NSAIDs are the most common class of painkillers and anti-inflammatory agents. They also show other functions like chemoprevention and chemosuppression for which they act at the protein but not at the genome level since they are mostly anions at physiological pH, which prohibit their approach to the poly-anionic DNA. Complexing the drugs with bioactive metal obliterate their negative charge and allow them to bind to the DNA, thereby, opening the possibility of genome level interaction. To test this hypothesis, we present the interaction of a traditional NSAID, Piroxicam and its copper complex with core histone and chromatin. METHODS: Spectroscopy, DLS, and SEM studies were applied to see the effect of the interaction on the structure of histone/chromatin. This was coupled with MTT assay, immunoblot analysis, confocal microscopy, micro array analysis and qRT-PCR. RESULTS: The interaction of Piroxicam and its copper complex with histone/chromatin results in structural alterations. Such structural alterations can have different biological manifestations, but to test our hypothesis, we have focused only on the accompanied modulations at the epigenomic/genomic level. The complex, showed alteration of key epigenetic signatures implicated in transcription in the global context, although Piroxicam caused no significant changes. We have correlated such alterations caused by the complex with the changes in global gene expression and validated the candidate gene expression alterations. CONCLUSION AND GENERAL SIGNIFICANCE: Our results provide the proof of concept that DNA binding ability of the copper complexes of a traditional NSAID, opens up the possibility of modulations at the epigenomic/genomic level.
Assuntos
Anti-Inflamatórios não Esteroides/química , Cromatina/química , Cobre/química , Epigenômica , Piroxicam/química , Cobre/metabolismo , DNA/metabolismo , Células HeLa , Histonas/química , Humanos , Piroxicam/metabolismo , Espectrometria de Fluorescência , TranscriptomaRESUMO
Phosphoinositide signaling has been implicated in the regulation of numerous cellular processes including cytoskeletal dynamics, cellular motility, vesicle trafficking, and gene transcription. Studies have also shown that nuclear phosphoinositide(s) regulates processes such as mRNA export, cell cycle progression, gene transcription, and DNA repair. We have shown previously that the nuclear form of phosphatidylinositol-4-phosphate 5-kinase 1α (PIP5K), the enzyme responsible for phosphatidylinositol 4,5-bisphosphate synthesis, is modified by small ubiquitin-like modifier (SUMO)-1. In this study, we have shown that due to the site-specific Lys to Ala mutations of PIP5K at Lys-244 and Lys-490, it is unable to localize in the nucleus and nucleolus, respectively. Furthermore, by using chromatin immunoprecipitation assays, we have observed that PIP5K associates with the chromatin silencing complex constituted of H3K9me3 and heterochromatin protein 1α at multiple ribosomal DNA (rDNA) loci. These interactions followed a definite cyclical pattern of occupancy (mostly G1) and release from the rDNA loci (G1/S) throughout the cell cycle. Moreover, the immunoprecipitation results clearly demonstrate that PIP5K SUMOylated at Lys-490 interacts with components of the chromatin silencing machinery, H3K9me3 and heterochromatin protein 1α. However, PIP5K does not interact with the gene activation signature protein H3K4me3. This study, for the first time, demonstrates that PIP5K, an enzyme actively associated with lipid modification pathway, has additional roles in rDNA silencing.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Ribossômico/metabolismo , Epigênese Genética , Histonas/metabolismo , Lisina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ciclo Celular , Linhagem Celular Tumoral , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , DNA Ribossômico/genética , Inativação Gênica , Células HEK293 , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/genética , Humanos , Células MCF-7 , Metilação , Fosfatidilinositol 4,5-Difosfato/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais , SumoilaçãoRESUMO
Recognition of core histone components of chromatin along with chromosomal DNA by a class of small molecule modulators is worth examining to evaluate their intracellular mode of action. A plant alkaloid ellipticine (ELP) which is a putative anticancer agent has so far been reported to function via DNA intercalation, association with topoisomerase II and binding to telomere region. However, its effect upon the potential intracellular target, chromatin is hitherto unreported. Here we have characterized the biomolecular recognition between ELP and different hierarchical levels of chromatin. The significant result is that in addition to DNA, it binds to core histone(s) and can be categorized as a 'dual binder'. As a sequel to binding with histone(s) and core octamer, it alters post-translational histone acetylation marks. We have further demonstrated that it has the potential to modulate gene expression thereby regulating several key biological processes such as nuclear organization, transcription, translation and histone modifications.
Assuntos
Cromatina/efeitos dos fármacos , Elipticinas/farmacologia , Acilação , Cromatina/metabolismo , Dicroísmo Circular , Histonas/metabolismo , Ligação Proteica , Espectrometria de FluorescênciaRESUMO
Bile acids (BAs) and BA receptors, including G protein-coupled bile acid receptor 1 (GPBAR1), represent novel targets for the treatment of metabolic and inflammatory disorders. However, BAs elicit myriad effects on cardiovascular function, although this has not been specifically ascribed to GPBAR1. This study was designed to test whether stimulation of GPBAR1 elicits effects on cardiovascular function that are mechanism based that can be identified in acute ex vivo and in vivo cardiovascular models, to delineate whether effects were due to pathways known to be modulated by BAs, and to establish whether a therapeutic window between in vivo cardiovascular liabilities and on-target efficacy could be defined. The results demonstrated that the infusion of three structurally diverse and selective GPBAR1 agonists produced marked reductions in vascular tone and blood pressure in dog, but not in rat, as well as reflex tachycardia and a positive inotropic response, effects that manifested in an enhanced cardiac output. Changes in cardiovascular function were unrelated to modulation of the levothyroxine/thyroxine axis and were nitric oxide independent. A direct effect on vascular tone was confirmed in dog isolated vascular rings, whereby concentration-dependent decreases in tension that were tightly correlated with reductions in vascular tone observed in vivo and were blocked by iberiotoxin. Compound concentrations in which cardiovascular effects occurred, both ex vivo and in vivo, could not be separated from those necessary for modulation of GPBAR1-mediated efficacy, resulting in project termination. These results are the first to clearly demonstrate direct and potent peripheral arterial vasodilation due to GPBAR1 stimulation in vivo through activation of large conductance Ca(2+) activated potassium channel K(Ca)1.1.
Assuntos
Artérias/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Vasodilatação/efeitos dos fármacos , Animais , Artérias/fisiologia , Fator Natriurético Atrial/sangue , Células CHO , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/patologia , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Citocinas/sangue , Dinitrofluorbenzeno/análogos & derivados , Cães , Endotelina-1/sangue , Humanos , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Óxido Nítrico/biossíntese , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Tiroxina/sangue , Triazóis/farmacologiaRESUMO
The discovery of a new series of selective S1P1 agonists is described. This series of piperazinyl-oxadiazole derivatives was rapidly optimized starting from high-throughput screening hit 1 to afford potent and selective lead compound 10d. Further SAR studies showed that 10d was converted to the active phosphate metabolite 29 in vivo. Oral administration of compound 10d to rats was shown to induce lymphopenia at 3 mg/kg.
Assuntos
Oxidiazóis/farmacologia , Piperazinas/farmacologia , Receptores de Lisoesfingolipídeo/agonistas , Administração Oral , Animais , Relação Dose-Resposta a Droga , Feminino , Linfopenia/induzido quimicamente , Linfopenia/patologia , Estrutura Molecular , Oxidiazóis/administração & dosagem , Oxidiazóis/química , Piperazinas/administração & dosagem , Piperazinas/química , Ratos , Ratos Endogâmicos Lew , Receptores de Esfingosina-1-Fosfato , Relação Estrutura-AtividadeRESUMO
During B cell development, cells progress through multiple developmental stages with the pro-B cell stage defining commitment to the B cell lineage. YY1 is a ubiquitous transcription factor that is capable of both activation and repression functions. We find here that knockout of YY1 at the pro-B cell stage eliminates B lineage commitment. YY1 knockout pro-B cells can generate T lineage cells in vitro using the OP9- DL4 feeder system, as well as in vivo after injection into sub-lethally irradiated Rag1 -/- mice. These T lineage-like cells lose their B lineage transcript profile and gain a T cell lineage profile. Single cell-RNA-seq experiments showed that as YY1 knockout pro-B cells transition into T lineage cells, various cell clusters adopt transcript profiles representing a multiplicity of hematopoietic lineages indicating unusual lineage plasticity. Given the ubiquitous nature of YY1 and its dual activation and repression functions, YY1 likely regulates commitment in multiple cell lineages.
RESUMO
Indoleamine-2,3-dioxygenase-1 (IDO1) has emerged as an attractive target for cancer immunotherapy. An automated ligand identification system screen afforded the tetrahydroquinoline class of novel IDO1 inhibitors. Potency and pharmacokinetic (PK) were key issues with this class of compounds. Structure-based drug design and strategic incorporation of polarity enabled the rapid improvement on potency, solubility, and oxidative metabolic stability. Metabolite identification studies revealed that amide hydrolysis in the D-pocket was the key clearance mechanism for this class. Strategic survey of amide isosteres revealed that carbamates and N-pyrimidines, which maintained exquisite potencies, mitigated the amide hydrolysis issue and led to an improved rat PK profile. The lead compound 28 is a potent IDO1 inhibitor, with clean off-target profiles and the potential for quaque die dosing in humans.
RESUMO
Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the post-translational symmetric dimethylation of protein substrates. PRMT5 plays a critical role in regulating biological processes including transcription, cell cycle progression, RNA splicing, and DNA repair. As such, dysregulation of PRMT5 activity is implicated in the development and progression of multiple cancers and is a target of growing clinical interest. Described herein are the structure-based drug designs, robust synthetic efforts, and lead optimization strategies toward the identification of two novel 5,5-fused bicyclic nucleoside-derived classes of potent and efficacious PRMT5 inhibitors. Utilization of compound docking and strain energy calculations inspired novel designs, and the development of flexible synthetic approaches enabled access to complex chemotypes with five contiguous stereocenters. Additional efforts in balancing bioavailability, solubility, potency, and CYP3A4 inhibition led to the identification of diverse lead compounds with favorable profiles, promising in vivo activity, and low human dose projections.
Assuntos
Aminoquinolinas/uso terapêutico , Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Nucleosídeos/uso terapêutico , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Aminoquinolinas/síntese química , Aminoquinolinas/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Feminino , Humanos , Camundongos SCID , Simulação de Acoplamento Molecular , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/metabolismo , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismo , Relação Estrutura-AtividadeRESUMO
Herein we have engineered a smart nuclear targeting thiol-modified riboflavin-gold nano assembly, RfS@AuNPs, which accumulates selectively in the nucleus without any nuclear-targeting peptides (NLS/RGD) and shows photophysically in vitro DNA intercalation. A theoretical model using Molecular Dynamics has been developed to probe the mechanism of formation and stability as well as dynamics of the RfS@AuNPs in aqueous solution and within the DNA microenvironment. The RfS@AuNPs facilitate the binucleated cell formation that is reflected in the significant increase of DNA damage marker, γ-H2AX as well as the arrest of most of the HeLa cells at the pre-G1 phase indicating cell death. Moreover, a significant upregulation of apoptotic markers confirms that the cell death occurs through the apoptotic pathway. Analyses of the microarray gene expression of RfS@AuNPs treated HeLa cells show significant alterations in vital biological processes necessary for cell survival. Taken together, our study reports a unique nuclear targeting mechanism through targeting the riboflavin receptors, which are upregulated in cancer cells and induce apoptosis in the targeted cells.
Assuntos
Dano ao DNA , Apoptose , Linhagem Celular Tumoral , Ouro , Células HeLa , Humanos , RiboflavinaRESUMO
Chelerythrine (CHL), a plant alkaloid, possesses antimicrobial, anti-inflammatory, and antitumor properties. Although CHL influences several key signal transduction pathways, its ability to interact directly with nucleoprotein complex chromatin, in eukaryotic cells has so far not been looked into. Here we have demonstrated its association with hierarchically assembled chromatin components, viz. long chromatin, chromatosome, nucleosome, chromosomal DNA, and histone H3 and the consequent effect on chromatin structure. CHL was found to repress acetylation at H3K9. It is more target-specific in terms of gene expression alteration and less cytotoxic compared to its structural analog sanguinarine.
Assuntos
Antineoplásicos/farmacologia , Benzofenantridinas/farmacologia , Eucromatina/efeitos dos fármacos , Histonas/metabolismo , Nucleossomos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Acetilação/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Epigênese Genética , Eucromatina/química , Eucromatina/metabolismo , Células HeLa , Histonas/genética , Humanos , Isoquinolinas/farmacologia , Nucleossomos/química , Nucleossomos/metabolismo , Regiões Promotoras GenéticasRESUMO
Mithramycin (MTR) is a clinically approved DNA-binding antitumor antibiotic currently in Phase 2 clinical trials at National Institutes of Health for treatment of osteosarcoma. In view of the resurgence in the studies of this generic antibiotic as a human medicine, we have examined the binding properties of MTR with the integral component of chromatin - histone proteins - as a part of our broad objective to classify DNA-binding molecules in terms of their ability to bind chromosomal DNA alone (single binding mode) or both histones and chromosomal DNA (dual binding mode). The present report shows that besides DNA, MTR also binds to core histones present in chromatin and thus possesses the property of dual binding in the chromatin context. In contrast to the MTR-DNA interaction, association of MTR with histones does not require obligatory presence of bivalent metal ion like Mg(2+). As a consequence of its ability to interact with core histones, MTR inhibits histone H3 acetylation at lysine 18, an important signature of active chromatin, in vitro and ex vivo. Reanalysis of microarray data of Ewing sarcoma cell lines shows that upon MTR treatment there is a significant down regulation of genes, possibly implicating a repression of H3K18Ac-enriched genes apart from DNA-binding transcription factors. Association of MTR with core histones and its ability to alter post-translational modification of histone H3 clearly indicates an additional mode of action of this anticancer drug that could be implicated in novel therapeutic strategies.
RESUMO
Eukaryotic DNA is compacted in the form of chromatin, in a complex with histones and other non-histone proteins. The intimate association of DNA and histones in chromatin raises the possibility that DNA-interactive small molecules may bind to chromatin-associated proteins such as histones. Employing biophysical and biochemical techniques we have characterized the interaction of a classical intercalator, ethidium bromide (EB) and its structural analogue propidium iodide (PI) with hierarchical genomic components: long chromatin, chromatosome, core octamer and chromosomal DNA. Our studies show that EB and PI affect both chromatin structure and function, inducing chromatin compaction and disruption of the integrity of the chromatosome. Calorimetric studies and fluorescence measurements of the ligands demonstrated and characterized the association of these ligands with core histones and the intact octamer in absence of DNA. The ligands affect acetylation of histone H3 at lysine 9 and acetylation of histone H4 at lysine 5 and lysine 8 ex vivo. PI alters the post-translational modifications to a greater extent than EB. This is the first report showing the dual binding (chromosomal DNA and core histones) property of a classical intercalator, EB, and its longer analogue, PI, in the context of chromatin.