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1.
Mol Pharmacol ; 98(2): 88-95, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487734

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.


Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Isoenzimas/química , Isoenzimas/metabolismo , Sirtuína 1/genética , Sirtuína 2/genética , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Benzoatos/farmacologia , Proteína de Ligação a CREB/metabolismo , Cristalografia por Raios X , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Niacinamida/farmacologia , Conformação Proteica , Pirazóis/farmacologia , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Transfecção
2.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 45(2): 198-203, 2020 Feb 28.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-32386048

RESUMO

Rubinstein-Taybi syndrome (RSTS), also known as broad thumb-great toe syndrome or broad digits syndrome, is a rare autosomal dominant genetic disease. The main features of the patients are craniofacial dysmorphisms, skeletal malformations, and delay of growth and psychomotor development. In this case, the child has a typical RSTS specific face and growth retardation, with atypical indirect inguinalhemia. A heterozygous mutation, C. 4492 C>T (p. Arg1498Ter), was found in the exon of CREBBP gene by gene sequencing. It was a nonsense mutation, which leads to the premature termination of peptide synthesis. The mutation was not observed in the child's parents, which may be a de Novo mutation. The disease is lack of effective therapy so far.


Assuntos
Proteína de Ligação a CREB/genética , Síndrome de Rubinstein-Taybi , Códon sem Sentido , Éxons , Humanos , Lactente , Mutação
3.
Biochim Biophys Acta Gene Regul Mech ; 1863(8): 194576, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32389826

RESUMO

Juvenile hormones (JH) and ecdysone coordinately regulate metamorphosis in Aedes aegypti. We studied the function of an epigenetic regulator and multifunctional transactivator, CREB binding protein (CBP) in A. aegypti. RNAi-mediated knockdown of CBP in Ae. aegypti larvae resulted in suppression of JH primary response gene, Krüppel-homolog 1 (Kr-h1), and induction of primary ecdysone response gene, E93, resulting in multiple effects including early metamorphosis, larval-pupal intermediate formation, mortality and inhibition of compound eye development. RNA sequencing identified hundreds of genes, including JH and ecdysone response genes regulated by CBP. In the presence of JH, CBP upregulates Kr-h1 by acetylating core histones at the Kr-h1 promoter and facilitating the recruitment of JH receptor and other proteins. CBP suppresses metamorphosis regulators, EcR-A, USP-A, BR-C, and E93 through the upregulation of Kr-h1 and E75A. CBP regulates the expression of core eye specification genes including those involved in TGF-ß and EGFR signaling. These studies demonstrate that CBP is an essential player in JH and 20E action and regulates metamorphosis and compound eye development in Ae. aegypti.


Assuntos
Aedes/metabolismo , Proteína de Ligação a CREB/metabolismo , Olho/crescimento & desenvolvimento , Metamorfose Biológica/fisiologia , Organogênese/fisiologia , Aedes/genética , Animais , Proteína de Ligação a CREB/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Ecdisona/genética , Ecdisona/metabolismo , Ecdisona/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Hormônios Juvenis/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Larva , Organogênese/efeitos dos fármacos , Organogênese/genética , Regiões Promotoras Genéticas , Pupa/crescimento & desenvolvimento , Transdução de Sinais , Fatores de Transcrição/metabolismo , Febre Amarela/genética
4.
Cancer Sci ; 111(5): 1829-1839, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32162442

RESUMO

Lysine acetyltransferases (KATs) are a highly diverse group of epigenetic enzymes that play important roles in various cellular processes including transcription, signal transduction, and cellular metabolism. However, our knowledge of the genomic and transcriptomic alterations of KAT genes and their clinical significance in human cancer remains incomplete. We undertook a metagenomic analysis of 37 KATs in more than 10 000 cancer samples across 33 tumor types, focusing on breast cancer. We identified associations among recurrent genetic alteration, gene expression, clinicopathologic features, and patient survival. Loss-of-function analysis was carried out to examine which KAT has important roles in growth and viability of breast cancer cells. We identified that a subset of KAT genes, including NAA10, KAT6A, and CREBBP, have high frequencies of genomic amplification or mutation in a spectrum of human cancers. Importantly, we found that 3 KATs, NAA10, ACAT2, and BRD4, were highly expressed in the aggressive basal-like subtype, and their expression was significantly associated with disease-free survival. Furthermore, we showed that depletion of NAA10 inhibits basal-like breast cancer growth in vitro. Our findings provide a strong foundation for further mechanistic research and for developing therapies that target NAA10 or other KATs in human cancer.


Assuntos
Genoma Humano/genética , Lisina Acetiltransferases/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Proteína de Ligação a CREB/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Intervalo Livre de Doença , Proteína p300 Associada a E1A/genética , Dosagem de Genes , Expressão Gênica , Histona Acetiltransferases/genética , Humanos , Lisina Acetiltransferases/metabolismo , Mutação , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal E/genética , Acetiltransferase N-Terminal E/metabolismo , Neoplasias/mortalidade , Prognóstico , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Fatores de Transcrição/genética
5.
Ann Hematol ; 99(3): 487-500, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32006151

RESUMO

Fusion partners of KMT2A affect disease phenotype and influence the current World Health Organization classification of hematologic neoplasms. The t(11;16)(q23;p13)/KMT2A-CREBBP is considered presumptive evidence of a myelodysplastic syndrome (MDS) and a MDS-related cytogenetic abnormality in the classification of acute myeloid leukemia (AML). Here, we report 18 cases of hematologic neoplasms with t(11;16). There were 8 males and 10 females with a median age of 51.9 years at time of detection of t(11;16). Of 17 patients with enough clinical information and pathological materials for review, 16 had a history of cytotoxic therapies for various malignancies including 12/15 patients who received topoisomerase II inhibitors, and 15 were classified as having therapy-related neoplasms. The median interval from the diagnosis of primary malignancy to the detection of t(11;16) was 23.2 months. Dysplasia, usually mild, was observed in 7/17 patients. Blasts demonstrated monocytic differentiation in 8/8 patients who developed AML at the time or following detection of t(11;16). t(11;16) was observed as the sole chromosomal abnormality in 10/18 patients. KMT2A rearrangement was confirmed in 11/11 patients. The median survival from the detection of t(11;16) was 15.4 months. In summary, t(11;16)(q23;p13) is rare and overwhelmingly associated with prior exposure of cytotoxic therapy. Instead of being considered presumptive evidence of myelodysplasia, we suggest that the detection of t(11;16) should automatically prompt a search for a history of malignancy and cytotoxic therapy so that proper risk stratification and clinical management are made accordingly. The dismal outcome of patients with t(11;16) is in keeping with that of therapy-related neoplasms.


Assuntos
Proteína de Ligação a CREB/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 16/genética , Bases de Dados Factuais , Neoplasias Hematológicas , Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Proteína de Leucina Linfoide-Mieloide/genética , Segunda Neoplasia Primária , Proteínas de Fusão Oncogênica/genética , Inibidores da Topoisomerase II/administração & dosagem , Translocação Genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/mortalidade , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Segunda Neoplasia Primária/tratamento farmacológico , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/mortalidade , Medição de Risco
6.
J Agric Food Chem ; 68(1): 193-205, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31826610

RESUMO

Gynostemma pentaphyllum possesses neuroprotective bioactivity. However, the effect of gypenosides on hypoxia-induced neural damage remains obscure. In this study, Gyp, the active fraction extracted from G. pentaphyllum and its bioactive compounds as well as the underlying molecular mechanisms were investigated. Eighteen dammarane-type saponins were isolated from Gyp. The absolute configurations of six unreported compounds (13-18) were assessed via electron capture detection (ECD) analyses. The results of cell viability assay showed that Gyp and its bioactive compounds (13-16 and 18) effectively protected PC12 cells from hypoxia injury. Gyp pretreatment also improved mice spatial memory impairment caused by hypoxia exposure. At the molecular level, Gyp and its bioactive compounds could activate the signaling pathways of ERK, Akt, and CREB in vitro and in vivo. In summary, Gyp and its bioactive compounds could prevent hypoxia-induced injury via ERK, Akt, and CREB signaling pathways.


Assuntos
Proteína de Ligação a CREB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gynostemma/química , Hipóxia/tratamento farmacológico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteína de Ligação a CREB/genética , Sobrevivência Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Células PC12 , Extratos Vegetais/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Saponinas/administração & dosagem
7.
Int J Mol Sci ; 20(21)2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31717815

RESUMO

Previously, we found that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) model mice (PD mice) showed facilitation of hippocampal memory extinction via reduced cyclic adenosine monophosphate (cAMP)/cAMP-dependent response element-binding protein (CREB) signaling, which may cause cognitive impairment in PD. Serotonergic neurons in the median raphe nucleus (MnRN) project to the hippocampus, and functional abnormalities have been reported. In the present study, we investigated the effects of the serotonin 5-HT4 receptor (5-HT4R) agonists prucalopride and velusetrag on the facilitation of memory extinction observed in PD mice. Both 5-HT4R agonists restored facilitation of contextual fear extinction in PD mice by stimulating the cAMP/CREB pathway in the dentate gyrus of the hippocampus. A retrograde fluorogold-tracer study showed that γ-aminobutyric acid-ergic (GABAergic) neurons in the reticular part of the substantia nigra (SNr), but not dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc), projected to serotonergic neurons in the MnRN, which are known to project their nerve terminals to the hippocampus. It is possible that the degeneration of the SNpc DAergic neurons in PD mice affects the SNr GABAergic neurons, and thereafter, the serotonergic neurons in the MnRN, resulting in hippocampal dysfunction. These findings suggest that 5HT4R agonists could be potentially useful as therapeutic drugs for treating cognitive deficits in PD.


Assuntos
Hipocampo/metabolismo , Doença de Parkinson/metabolismo , Neurônios Serotoninérgicos/efeitos dos fármacos , Agonistas do Receptor 5-HT4 de Serotonina/uso terapêutico , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Medo/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/psicologia , Núcleos da Rafe/efeitos dos fármacos , Receptores 5-HT4 de Serotonina/metabolismo , Neurônios Serotoninérgicos/citologia , Neurônios Serotoninérgicos/metabolismo , Substância Negra/metabolismo
8.
Photochem Photobiol Sci ; 18(11): 2740-2747, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573014

RESUMO

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID-KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID(CREB)-KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.


Assuntos
Proteína de Ligação a CREB/análise , Medições Luminescentes/métodos , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Colforsina/química , Células HEK293 , Humanos , Luciferases/química , Luciferases/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos/genética , Análise de Célula Única , Imagem com Lapso de Tempo
9.
J Phys Chem Lett ; 10(19): 5963-5968, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31535860

RESUMO

Allosteric regulation by intrinsically disordered proteins (IDPs) is an important class of cellular processes, including transcription. Molecular dynamics (MD) simulation is a promising approach to unravel the complex molecular interactions involved in the allosteric regulation by IDPs. While allosteric regulation is often characterized by the effect of a ligand on the binding affinity of a distal ligand, the binding affinity is often challenging to calculate by MD simulations because of insufficient sampling of the rare events in this binding-unbinding process. In the current work, we present a new sampling approach based on Hamiltonian replica exchange that allows accurate and efficient calculation of binding affinities using a native-centric coarse-grained model. We also demonstrate the utility of the new method by studying the positive allostery in the kinase-inducible domain interacting domain of the CREB binding protein, in which a prebound ligand enhances the binding of the second ligand.


Assuntos
Proteína de Ligação a CREB/química , Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Transcrição Genética , Regulação Alostérica , Sítio Alostérico , Proteína de Ligação a CREB/genética , Proteínas Intrinsicamente Desordenadas/genética , Ligantes , Ligação Proteica , Conformação Proteica , Termodinâmica
10.
Stem Cell Res ; 40: 101553, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31491690

RESUMO

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder characterized by growth retardation, skeletal anomalies and intellectual disability, caused by heterozygous mutations in either CREBBP (RSTS1) or EP300 (RSTS2) genes. We characterized 3 iPSC lines generated by Sendai from blood of RSTS1 patients with unique non sense c.4435G > T, p.(Gly1479*), c.3474G > A, p.(Trp1158*) and missense c.4627G > T, p.(Asp1543Tyr) CREBBP mutations. All lines displayed iPSC morphology, pluripotency markers, trilineage differentiation potential, stable karyotype and specific mutations. Western-blot using a CREB-Binding Protein N-terminus antibody demonstrated the same amount of full length protein as control in the missense mutation line and reduced amount in lines with stop mutations.


Assuntos
Proteína de Ligação a CREB/genética , Linhagem Celular/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação de Sentido Incorreto , Síndrome de Rubinstein-Taybi/genética , Adolescente , Sequência de Bases , Proteína de Ligação a CREB/metabolismo , Diferenciação Celular , Linhagem Celular/citologia , Feminino , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Mutação Puntual , Síndrome de Rubinstein-Taybi/metabolismo , Síndrome de Rubinstein-Taybi/fisiopatologia
11.
Int J Biochem Cell Biol ; 116: 105620, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31561018

RESUMO

Diazepam is a medicament of the benzodiazepine family and it typically produces a sedative effect. Researchers have revealed that diazepam can induce melanogenesis and produce dendrite-like structures in B16 melanoma cells. However, the associated mechanisms of melanogenesis and phenotypic alterations have mostly remained unknown. In this study, we determined the effects of diazepam on melanogenesis, cellular phenotypic alterations, the location of melanosomes and the expression of relevant proteins in melanocytes using Masson-Fontana ammoniacal silver staining, scanning electron microscopy, immunocytochemistry and western blot analysis. Our results collectively indicated that diazepam had a pivotal role in melanocytes by enhancing melanin synthesis, melanocyte dendricity, melanosome trafficking, and capture at the dendrite tips. These functions might be attributed to the fact that diazepam activated the peripheral benzodiazepine receptor (PBR). This increased intracellular levels of cAMP, which stimulated the phosphorylation of cAMP response element-binding (CREB). As a result, this increased the tyrosinase, microphthalmia-associated transcription factor (MITF), Rab27a, Myosin Va, Rab17 and Cdc42 expression. This caused melanogenesis and melanosome transport. Therefore, our findings may provide a potential strategy for treating anti-hypopigmentation disorders.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Diazepam/farmacologia , Hipnóticos e Sedativos/farmacologia , Melanócitos/efeitos dos fármacos , Melanossomas/efeitos dos fármacos , Receptores de GABA-A/genética , Animais , Transporte Biológico/efeitos dos fármacos , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Humanos , Melanócitos/citologia , Melanócitos/metabolismo , Melanossomas/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Receptores de GABA-A/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/metabolismo
12.
Immunity ; 51(3): 535-547.e9, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31519498

RESUMO

Inactivating mutations of the CREBBP and EP300 acetyltransferases are among the most common genetic alterations in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). Here, we examined the relationship between these two enzymes in germinal center (GC) B cells, the normal counterpart of FL and DLBCL, and in lymphomagenesis by using conditional GC-directed deletion mouse models targeting Crebbp or Ep300. We found that CREBBP and EP300 modulate common as well as distinct transcriptional programs implicated in separate anatomic and functional GC compartments. Consistently, deletion of Ep300 but not Crebbp impaired the fitness of GC B cells in vivo. Combined loss of Crebbp and Ep300 completely abrogated GC formation, suggesting that these proteins partially compensate for each other through common transcriptional targets. This synthetic lethal interaction was retained in CREBBP-mutant DLBCL cells and could be pharmacologically targeted with selective small molecule inhibitors of CREBBP and EP300 function. These data provide proof-of-principle for the clinical development of EP300-specific inhibitors in FL and DLBCL.


Assuntos
Linfócitos B/fisiologia , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Epigênese Genética/genética , Centro Germinativo/fisiologia , Linfoma Folicular/etiologia , Linfoma Difuso de Grandes Células B/genética , Acetiltransferases/genética , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Deleção de Sequência/genética , Transcrição Genética/genética
13.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(9): 886-889, 2019 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-31515782

RESUMO

OBJECTIVE: To summarize the clinical characteristics and identify gene mutations of 2 probands with Rubinstein-Taybi syndrome (RSTS). METHODS: Clinical characteristics of 2 probands with Rubinstein-Taybi syndrome were summarized. Genomic DNA was extracted from peripheral blood samples from the patients and their parents. Genomic DNA was subjected to whole exome next generation sequencing. Suspected variants were confirmed by Sanger sequencing. RESULTS: The two patients were characterized by typical facial features, broad thumbs and big toes, intellectual disability, and postnatal growth retardation. Two variants of the CREBBP gene, namely c.3779+1G>A and c.5052_c.5053insT, were respectively identified in the 2 patients. Among these, c.3779+1G>A was a previously known pathological mutation, while c.5052_c.5053insT was unreported previously. Both variants were predicted to be pathological. CONCLUSION: Two cases of Rubinstein-Taybi syndrome were diagnosed, which facilitated the diagnosis and genetic counselling.


Assuntos
Proteína de Ligação a CREB/genética , Síndrome de Rubinstein-Taybi/genética , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fenótipo
14.
Food Funct ; 10(9): 5816-5826, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31463494

RESUMO

Several studies have shown that mushroom polysaccharides enhance the ability of natural killer (NK) cells to recognize cancer cells as foreign and thereby enhance the effectiveness of host immune defence mechanisms. Nevertheless, the use of NK cells in cancer treatment requires finding selective stimulators of their cytotoxicity without disturbing organism homeostasis. Our studies revealed that Cantharellus cibarius polysaccharides present in the CC2a fraction, mainly composed of an O-2 and O-3 branched (1→6)-linked mannan, not only beneficially influenced the viability and proliferation of the human natural killer cells NK92 but also enhanced their anticancer properties against the human lung and colon cancer cells A549 and LS180, and at the same time did not affect the human lung and colon epithelial cells NL20 and CCD841 CoN. Furthermore, the CC2a fraction used alone was also nontoxic to the normal epithelium, while it inhibited the viability of these cancer cells. Nevertheless, the therapeutic potential of NK92 cells was greatly enhanced after coincubation with these polysaccharides and the observed effect was dependent on the CC2a concentrations. The beneficial effect of CC2a on NK92 cells was associated with stimulation of p38 and Erk expression as well as induction of the transcription factor CREB. The discovered beneficial impact of the CC2a fraction on NK92 cells suggested the therapeutic use of the investigated compound especially as an adjuvant. Furthermore, taking into account the abundance of these water soluble mannans in C. cibarius, the results also suggest that an increase in the intake of C. cibarius may promote innate immunity response against cancer through the enhancement of NK cell activity.


Assuntos
Agaricales/química , Antineoplásicos Fitogênicos/farmacologia , Neoplasias do Colo/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Neoplasias Pulmonares/imunologia , Mananas/farmacologia , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/química , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/fisiopatologia , Humanos , Imunidade Inata/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/fisiopatologia , Mananas/química , Extratos Vegetais/química
15.
Artigo em Inglês | MEDLINE | ID: mdl-31465879

RESUMO

The ATF/CREB family of transcription factors represents a large group of basic region-leucine zipper (bZip) proteins that regulate diverse cellular responses. Here we carried out a comprehensive analysis of ATF/CREB family members in 22 representative animal species. The family probably originated from the early diverging metazoan and significantly expanded in vertebrates due to multiple whole genome duplication. Duplicates of atf6 were derived from 2R, and duplicates of creb1, crem, jdp2, creb5, atf4, atf5 and atf7 were products of 3R. We also isolated 21 ATF/CREBs, belonging to 6 subfamilies from Nile tilapia. Based on transcriptome data, most members were found to be dominantly expressed in the head kidney, heart, brain and testis. Some ATF/CREBs displayed sexual dimorphic expression in gonad at 5, 90 and 180 dah (days after hatching), but not at 30 dah. creb1a and atf4a were found to be expressed mainly in phase I and II oocytes of the ovary; while creb1b and atf4b mainly in spermatogenic cells of the testis, indicating divergence of duplicated genes from 3R which suggested neofunctionalization or subfunctionalization in gonad. This is the first genome-wide screening and evolutionary analysis of ATF/CREB family in different animals, particularly in teleosts. The expression analysis of this family in tilapia gonad provided a fundamental clue for understanding their important roles in sex differentiation and gonadal development in teleosts.


Assuntos
Fatores Ativadores da Transcrição/metabolismo , Proteína de Ligação a CREB/metabolismo , Ciclídeos/metabolismo , Evolução Molecular , Gônadas/metabolismo , Fatores Ativadores da Transcrição/genética , Animais , Proteína de Ligação a CREB/genética , Ciclídeos/genética , Feminino , Perfilação da Expressão Gênica , Masculino , Ovário/metabolismo , Testículo/metabolismo
16.
Artigo em Inglês | MEDLINE | ID: mdl-31299884

RESUMO

Altered levels of histone acetylation are associated with changes in chromosomal gene expression. Thus, the specific acetylation of histones bound to plasmid DNA might increase transgene expression. Previously, the expression of the histone acetyltransferase domain of CREB-binding protein fused to the sequence-dependent DNA binding domain of GAL4 (GAL4-HAT) successfully improved reporter gene expression in cultured cells [J. Biosci. Bioengng. 123, 277-280 (2017)]. In this study, the same approach was applied for transgene expression in mice. The activator and reporter plasmid DNAs bearing the genes for GAL4-HAT and Gaussia princeps luciferase, respectively, were co-administered into the mouse liver by hydrodynamics-based tail vein injection, and the Gaussia luciferase activity in serum was measured for two weeks. Unexpectedly, the co-injection of the GAL4-HAT and luciferase plasmid DNAs seemed to decrease, rather than increase, luciferase expression. Moreover, the co-injection apparently reduced the amount of luciferase DNA in the liver. These results indicated that this system is ineffective in vivo and suggested the exclusion of hepatic cells expressing GAL4-HAT.


Assuntos
Histona Acetiltransferases/genética , Plasmídeos , Transgenes , Acetilação , Animais , Proteína de Ligação a CREB/genética , Proteínas de Ligação a DNA/administração & dosagem , Proteínas de Ligação a DNA/genética , Feminino , Genes Reporter , Histona Acetiltransferases/administração & dosagem , Luciferases , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/química , Plasmídeos/genética , Proteínas de Saccharomyces cerevisiae/administração & dosagem , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/administração & dosagem , Fatores de Transcrição/genética
17.
Fish Shellfish Immunol ; 93: 269-277, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31306762

RESUMO

As an isoform of Rho family GTPases, RhoB plays a pivotal role in cytoskeletal organization, cell proliferation, apoptosis and immune response. However, the regulatory mechanisms of RhoB expression in aquatic animals are still unknown. In the present study, we first construct Vibrio anguillarum infection model in S. maximus, including susceptible and resistant individuals. Then the temporal expression of RhoB was detected after V. anguillarum challenge using qRT-PCR and found that RhoB transcripts were significantly induced in the liver, gill and blood despite of differential expression levels and responsive time points. In addition, the mRNA levels of RhoB in resistant individuals were significantly higher than in susceptible ones. The length of 2083 bp sequences of RhoB promoter was cloned and characterized. Moreover, DNA methylation of the RhoB promoter was measured by bisulfite sequencing (BSP) and hypo-methylated was detected in the CpG islands. Three SNPs (-1590, -1575 and -1449) and two haplotypes in the promoter region of RhoB were identified to be associated with V. anguillarum resistance in turbot by association analysis in group 17-R and 17-S. Deletion analysis indicated that these SNPs could negatively mediate the activity of RhoB promoter. Site-directed mutagenesis and qRT-PCR of individuals with different genotypes demonstrated that -1575 T/A polymorphism affected promoter activity. Further study showed that this mutation altered the binding site of the transcription factor CREB. Co-transfection of SmCREB and RhoB promoter was performed in HEK293T cells which confirmed the -1575 allelic differences on transcriptional activity, with the susceptibility allele showing reduced activity. Taken together, our findings implicate that losing of binding of CREB to SmRhoB promoter due to -1575T/A polymorphisms enhances SmRhoB expression in resistant turbot, which provide insights into the effect of SmRhoB expression in response to V. anguillarum infection.


Assuntos
Doenças dos Peixes/imunologia , Linguados/genética , Polimorfismo de Nucleotídeo Único/imunologia , Vibrio/fisiologia , Proteína rhoB de Ligação ao GTP/imunologia , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/veterinária , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Haplótipos/imunologia , Mutação , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Vibrioses/imunologia , Vibrioses/veterinária , Proteína rhoB de Ligação ao GTP/genética
18.
Br J Anaesth ; 123(2): e226-e238, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31253357

RESUMO

BACKGROUND: The long-term use of opioid analgesics is limited by the development of unwanted side-effects, such as tolerance. The molecular mechanisms of morphine anti-nociceptive tolerance are still unclear. The mitochondrial calcium uniporter (MCU) is involved in painful hyperalgesia, but the role of MCU in morphine tolerance has not been uncharacterised. METHODS: Rats received intrathecal injection of morphine for 7 days to induce morphine tolerance. The mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. The effects of an MCU inhibitor, antisense oligodeoxynucleotide against cyclic adenosine monophosphate response element (CRE)-binding protein (CREB) or cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in morphine tolerance were examined. RESULTS: Spinal morphine tolerance was associated with an increased expression of neuronal MCU, phospho-CREB (pCREB), and CPEB1 in the spinal cord dorsal horn. MCU inhibition increased the mechanical threshold and thermal latency, and reduced the accumulation of mitochondrial calcium in morphine tolerance. Intrathecal antisense oligodeoxynucleotide against CREB or CPEB1 restored the anti-nociceptive effects of morphine compared with mismatch oligodeoxynucleotide in von Frey test and hotplate test. Chromatin immunoprecipitation with quantitative PCR assay showed that CREB knockdown reduced the interaction of pCREB with the ccdc109a gene (encoding MCU expression) promoter and decreased the MCU mRNA transcription. RNA immunoprecipitation assay suggested that CPEB1 binds to the MCU mRNA 3' untranslated region. CPEB1 knockdown decreased the expression of MCU protein. CONCLUSIONS: These findings suggest that spinal MCU is regulated by pCREB and CPEB1 in morphine tolerance, and that inhibition of MCU, pCREB, or CPEB1 may be useful in preventing the development of opioid tolerance.


Assuntos
Proteína de Ligação a CREB/genética , Canais de Cálcio/metabolismo , Tolerância a Medicamentos/genética , Morfina/farmacologia , Proteínas de Ligação a RNA/genética , Corno Dorsal da Medula Espinal/metabolismo , Analgésicos Opioides/farmacologia , Animais , Masculino , Modelos Animais , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley
19.
Cancer Res ; 79(15): 3916-3927, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31182547

RESUMO

Regulatory T cells (Treg) are immunosuppressive and negatively impact response to cancer immunotherapies. CREB-binding protein (CBP) and p300 are closely related acetyltransferases and transcriptional coactivators. Here, we evaluate the mechanisms by which CBP/p300 regulate Treg differentiation and the consequences of CBP/p300 loss-of-function mutations in follicular lymphoma. Transcriptional and epigenetic profiling identified a cascade of transcription factors essential for Treg differentiation. Mass spectrometry analysis showed that CBP/p300 acetylates prostacyclin synthase, which regulates Treg differentiation by altering proinflammatory cytokine secretion by T and B cells. Reduced Treg presence in tissues harboring CBP/p300 loss-of-function mutations was observed in follicular lymphoma. Our findings provide novel insights into the regulation of Treg differentiation by CBP/p300, with potential clinical implications on alteration of the immune landscape. SIGNIFICANCE: This study provides insights into the dynamic role of CBP/p300 in the differentiation of Tregs, with potential clinical implications in the alteration of the immune landscape in follicular lymphoma.


Assuntos
Proteína de Ligação a CREB/imunologia , Proteína p300 Associada a E1A/imunologia , Linfoma Folicular/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Acetilação , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteína de Ligação a CREB/antagonistas & inibidores , Proteína de Ligação a CREB/genética , Diferenciação Celular/fisiologia , Regulação para Baixo , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/genética , Histonas/metabolismo , Humanos , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Linfoma Folicular/patologia , Mutação , Pirazóis/farmacologia , Piridinas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Transcrição Genética , Transcriptoma
20.
Nat Commun ; 10(1): 2188, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097695

RESUMO

Although promoter-associated CpG islands have been established as targets of DNA methylation changes in cancer, previous studies suggest that epigenetic dysregulation outside the promoter region may be more closely associated with transcriptional changes. Here we examine DNA methylation, chromatin marks, and transcriptional alterations to define the relationship between transcriptional modulation and spatial changes in chromatin structure. Using human papillomavirus-related oropharyngeal carcinoma as a model, we show aberrant enrichment of repressive H3K9me3 at the transcriptional start site (TSS) with methylation-associated, tumor-specific gene silencing. Further analysis identifies a hypermethylated subtype which shows a functional convergence on MYC targets and association with CREBBP/EP300 mutation. The tumor-specific shift to transcriptional repression associated with DNA methylation at TSSs was confirmed in multiple tumor types. Our data may show a common underlying epigenetic dysregulation in cancer associated with broad enrichment of repressive chromatin marks and aberrant DNA hypermethylation at TSSs in combination with MYC network activation.


Assuntos
Cromatina/metabolismo , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Sítio de Iniciação de Transcrição , Proteína de Ligação a CREB/genética , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Proteína p300 Associada a E1A/genética , Inativação Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Mutação , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/genética
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