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1.
Nat Commun ; 10(1): 2458, 2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31165730

RESUMO

During stress, prompt export of stress-inducible transcripts is critical for cell survival. Here, we characterize a function of the SAGA (Spt-Ada-Gcn5 acetyltransferase) deubiquitylating module (DUBm) in monitoring messenger ribonucleoprotein (mRNP) biogenesis to regulate non-canonical mRNA export of stress-inducible transcripts. Our genetic and biochemical analyses suggest that there is a functional relationship between Sgf73p of DUBm and the essential mRNA export factor, Yra1p. Under physiological conditions, Sgf73p is critical for the proper chromatin localization and RNA binding of Yra1p, while also quality controlling the biogenesis of mRNPs in conjunction with the nuclear exosome exonuclease, Rrp6p. Under environmental stress, when immediate transport of stress-inducible transcripts is imperative, Sgf73p facilitates the bypass of canonical surveillance and promotes the timely export of necessary transcripts. Overall, our results show that the Sgf73p-mediated plasticity of gene expression is important for the ability of cells to tolerate stress and regulate proteostasis to survive under environmental uncertainty.


Assuntos
Adaptação Fisiológica , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Cromatina/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Proteostase , Transporte de RNA , Saccharomyces cerevisiae , Transativadores/metabolismo
3.
Nat Commun ; 10(1): 2314, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127101

RESUMO

Histone methyltransferase MLL4 is centrally involved in transcriptional regulation and is often mutated in human diseases, including cancer and developmental disorders. MLL4 contains a catalytic SET domain that mono-methylates histone H3K4 and seven PHD fingers of unclear function. Here, we identify the PHD6 finger of MLL4 (MLL4-PHD6) as a selective reader of the epigenetic modification H4K16ac. The solution NMR structure of MLL4-PHD6 in complex with a H4K16ac peptide along with binding and mutational analyses reveal unique mechanistic features underlying recognition of H4K16ac. Genomic studies show that one third of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions in vivo and that MLL4 occupancy in a set of genomic targets depends on the acetyltransferase activity of MOF, a H4K16ac-specific acetyltransferase. The recognition of H4K16ac is conserved in the PHD7 finger of paralogous MLL3. Together, our findings reveal a previously uncharacterized acetyllysine reader and suggest that selective targeting of H4K16ac by MLL4 provides a direct functional link between MLL4, MOF and H4K16 acetylation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Dedos de Zinco PHD/fisiologia , Acetilação , Animais , Sítios de Ligação , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Técnicas de Inativação de Genes , Células HEK293 , Histona Acetiltransferases/genética , Histona-Lisina N-Metiltransferase/química , Histonas/química , Humanos , Camundongos Transgênicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
4.
Epigenetics Chromatin ; 12(1): 24, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992049

RESUMO

BACKGROUND: Histone acetylation plays an important role in DNA replication and repair because replicating chromatin is subject to dynamic changes in its structures. However, its precise mechanism remains elusive. In this report, we describe roles of the NuA4 acetyltransferase and histone H4 acetylation in replication fork protection in the fission yeast Schizosaccharomyces pombe. RESULTS: Downregulation of NuA4 subunits renders cells highly sensitive to camptothecin, a compound that induces replication fork breakage. Defects in NuA4 function or mutations in histone H4 acetylation sites lead to impaired recovery of collapsed replication forks and elevated levels of Rad52 DNA repair foci, indicating the role of histone H4 acetylation in DNA replication and fork repair. We also show that Vid21 interacts with the Swi1-Swi3 replication fork protection complex and that Swi1 stabilizes Vid21 and promotes efficient histone H4 acetylation. Furthermore, our genetic analysis demonstrates that loss of Swi1 further sensitizes NuA4 and histone H4 mutant cells to replication fork breakage. CONCLUSION: Considering that Swi1 plays a critical role in replication fork protection, our results indicate that NuA4 and histone H4 acetylation promote repair of broken DNA replication forks.


Assuntos
Replicação do DNA , Histona Acetiltransferases/metabolismo , Acetilação , Camptotecina/toxicidade , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases/genética , Histonas/genética , Histonas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Inibidores da Topoisomerase I/toxicidade
5.
Artif Cells Nanomed Biotechnol ; 47(1): 1207-1215, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30942624

RESUMO

Histone H4 acetylation at lysine 12 (H4K12ac) has been reported to be associated with the poor prognosis of pancreatic cancer. The study intends to study whether H4K12ac participates in regulating the carcinogenic effect of Ras-ERK1/2 on osteosarcoma (OS). The plasmids of pEGFP-N1, pEGFP-RasWT and pEGFP-K-RasG12V/T35S were transfected into MG-63 cells, the protein levels of H4K12ac and ERK1/2 were analyzed by Western blot. Effects of H4K12ac on cell proliferation and migration of MG-63 cells were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide solution (MTT), transwell, soft-agar colony formation and flow cytometry assays. Effect of H4K12ac on the transcription of ERK1/2 downstream genes was analyzed by RT-qPCR and ChIP assays. The involvements of HDAT6, HAT1 and MDM2 in cell proliferation and migration of MG-63 cells were finally studied. We found that H4K12ac was specifically down-regulated by Ras-ERK1/2 activation in MG-63 cells. H4K12ac suppressed Ras-ERK1/2-induced cell viability, colony formation and migration in MG-63 cells. Additionally, HDAC6 silence recovered Ras-ERK1/2-repressed H4K12ac expression, as well as inhibited the carcinogenic effect of Ras-ERK1/2 on MG-63 cells. Besides, down-regulated H4K12ac induced by Ras-ERK1/2 was found to be associated with MDM2-mediated HAT1 degradation. In conclusion, these results testified that Ras-ERK1/2 signalling promoted the development of OS by mediating H4K12ac through MDM2-mediated HAT1 degradation.


Assuntos
Histona Acetiltransferases/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Sistema de Sinalização das MAP Quinases , Osteossarcoma/patologia , Acetilação , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Ativação Enzimática , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transcrição Genética , Proteínas ras/metabolismo
6.
Anal Chim Acta ; 1066: 28-35, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31027532

RESUMO

A novel mushroom-like electrochemical immunoassay for the ultrasensitive detection of histone acetyltransferase activity (HAT p300) has been established on account of the new composite graphene oxide (GO) nanolayer. The immunoassay involves immobilization of substrate peptide onto Au electrode, acetylation in lysine of substrate peptide, and the interaction between acetyl group of lysine and acetyl-antibody (AbAc) of the GO nanolayer. The GO nanolayer comprises large amounts of methylene blue molecules (MB), giving rise to large signal amplification. Only in the presence of HAT p300, an obvious electrochemical signal appears and the peak linear current is proportion to the HAT p300 concentrations ranging from 0.01 to 150 nM with a detection limit of 0.0036 nM. The great enhancement on sensitivity of the proposed mushroom-like immunosensor derives from both the constructed Faraday cage and the extended outer Helmholtz plane (OHP). Further, the immunosensor with excellent sensitivity and selectivity can be applied for the HAT p300 activity detection in Hela cell lysates, serum and urine, hinting an improved and splendid analytical performance. Briefly, this stable, simple and ultrasensitive electrochemical immunoassay has considerable promise for further applications in the HATs-interrelated epigenetic studies and drug development.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Histona Acetiltransferases/análise , Imunoensaio , Eletrodos , Ouro/química , Grafite/química , Células HeLa , Histona Acetiltransferases/metabolismo , Humanos
7.
J Agric Food Chem ; 67(18): 5204-5211, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-30998337

RESUMO

Texture attributes such as firmness and lignification are important for fruit quality. Lignification has been widely studied in model plants and energy crops, but fruit lignification has rarely been investigated, despite having an adverse effect on fruit quality and consumer preference. Chilling-induced loquat fruit lignification that occurs after harvest can be alleviated by heat treatment (HT) applied prior to low temperature storage. Enzyme activity assay showed that HT treatment could retard the low temperature-induced increase in cinnamyl alcohol dehydrogenase (CAD) activity. Transcript analysis and substrate activity assays of recombinant CAD proteins highlighted the key role of EjCAD5 in chilling-induced lignin biosynthesis. A novel homeobox-leucine zipper protein ( EjHAT1) was identified as a negative regulator of EjCAD5. Therefore, the effect of HT treatment on lignification may be partially due to the suppression of the EjCAD5 promoter activity by EjHAT1.


Assuntos
Oxirredutases do Álcool/metabolismo , Eriobotrya/enzimologia , Histona Acetiltransferases/metabolismo , Lignina/biossíntese , Proteínas de Plantas/metabolismo , Oxirredutases do Álcool/genética , Temperatura Baixa , Eriobotrya/genética , Eriobotrya/metabolismo , Frutas/enzimologia , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Histona Acetiltransferases/genética , Temperatura Alta , Proteínas de Plantas/genética , Regiões Promotoras Genéticas
8.
Biomed Pharmacother ; 112: 108741, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30970528

RESUMO

Seaweeds are excellent source of bioactive compounds and seaweed-derived polysaccharides have demonstrated an array of biological effects. Here, we investigated the effect of polysaccharide of Sargassum weizhouense (PSW) on the inflammatory response in porcine circovirus type 2 (PCV2) infected mice and the underlying mechanism was studied according to the histone acetylation. After PCV2 infection, the levels of TNF-α, IL-1ß, IL-6, IL-8, IL-10, MCP-1, COX-1, COX-2 and HAT in both serum and spleen were significantly increased (P <0.05). The mRNA expression of TNF-α, IL-6, IL-10 and NF-κB p65 were elevated in PCV2 infected mice (P <0.05). The HDAC content in both serum and spleen as well the mRNA expression of HDAC1 were greatly decreased (P <0.05). PSW treatment dramatically inhibited the secretions of inflammatory cytokines and HATs, reduced mRNA expression of TNF-α, IL-6, IL-10 and NF-κB p65, but promoted HDAC secretion and mRNA expression of HDAC1 in PCV2-infected mice. The acetylation of both H3 and H4 was significantly up-regulated in PCV2-infected mice, and strongly inhibited by PSW treatment (P <0.01). These results suggested that PCV2 mediate the equilibrium between HATs and HDACs, alternate the histone acetylation and thus DNA packaging, and then activate the transcription of inflammatory cytokines. PSW could inhibit the histone acetylation and the production of inflammatory cytokines, showing excellent potentials in improving the resistance of host against PCV2 infection.


Assuntos
Antivirais/uso terapêutico , Infecções por Circoviridae/tratamento farmacológico , Histonas/metabolismo , Polissacarídeos/uso terapêutico , Sargassum/química , Acetilação , Animais , Antivirais/isolamento & purificação , Infecções por Circoviridae/imunologia , Citocinas/sangue , Feminino , Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Inflamação , Masculino , Camundongos Endogâmicos , Polissacarídeos/isolamento & purificação , Baço/imunologia
9.
Eur J Pharmacol ; 853: 308-315, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30980797

RESUMO

Microbiota produce short chain fatty acids (SCFAs), which are known to maintain gut homeostasis, by the fermentation of dietary fiber in the human colon. Among SCFAs, butyrate has been considered as the most physiologically effective SCFA in colorectal epithelial cells for growth and differentiation. Here we show that the E-type prostanoid 4 (EP4) receptor expression level is regulated by different concentrations of butyrate, but not by other SCFAs, in human colon cancer HCA-7 cells, through sodium-coupled monocarboxylate transporter-1 (SMCT-1)-mediated uptake followed by the activation of histone acetyltransferase: cAMP response element binding protein-binding protein/p300. Of particular interest, the prostanoid EP4 receptors are known to be expressed in normal colorectal crypt epithelial cells and maintain intestinal homeostasis by preserving mucosal integrity, while they are also known to be involved in the early stage of carcinogenesis. Thus, the links between butyrate and the expression of prostanoid EP4 receptors are both important factors for maintaining homeostasis. Based on in silico analysis, almost half of colorectal cancer tissues have lost the expression of SMCT-1 mRNA when compared with healthy corresponding tissues. Therefore, with the collapse of homeostasis systems such as a decrease in the concentration of butyrate in colorectal tissues, or reduced butyrate uptake, there is a possibility of early stage colorectal cancer development; the transformation of normal cells to the cancerous phenotype may be due to the overexpression of prostanoid EP4 receptors followed by excessive cyclooxygenase-2 induction, which are caused by a reduced amount of butyrate and/or its uptake, in/around colorectal epithelial cells.


Assuntos
Butiratos/metabolismo , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/biossíntese , Regulação Neoplásica da Expressão Gênica , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Acetilação , Animais , Linhagem Celular Tumoral , Proliferação de Células , AMP Cíclico/biossíntese , Proteína p300 Associada a E1A/metabolismo , Indução Enzimática , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fragmentos de Peptídeos/metabolismo , Sialoglicoproteínas/metabolismo
10.
Talanta ; 198: 39-44, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30876576

RESUMO

A novel electrogenerated chemiluminescence (ECL) method combining ECL property of silver clusters and hybridization chain reaction (HCR) signal amplification strategy has been designed for the analysis of histone acetyltransferases (HATs) activity and inhibitor evaluation. In this strategy, the substrate peptide of HAT released from the electrode surface due to the charge change based on the acetylated reaction in the presence of HATs, and then the exposed DNA on the electrode initiated the HCR to form the supersandwich DNA sequence, which can adsorb Ag+, and the silver clusters (AgNCs) generated by the electrochemical reduction. The ECL signal generated by AgNCs can be utilized for HATs activities detection. The detection limit of the as-prepared ECL method was 0.49 nM (S/N = 3). The novel ECL method can be used for HATs activity analysis and inhibition in MCF-7 cell lysates which shows high promise in HATs-related clinical diagnostics.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Histona Acetiltransferases/análise , Medições Luminescentes , Hibridização de Ácido Nucleico , Prata/química , Carbono/química , Eletrodos , Histona Acetiltransferases/metabolismo , Humanos , Células MCF-7 , Propriedades de Superfície , Células Tumorais Cultivadas
11.
Nat Commun ; 10(1): 1288, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894545

RESUMO

The TFIIH subunit XPB is involved in combined Xeroderma Pigmentosum and Cockayne syndrome (XP-B/CS). Our analyses reveal that XPB interacts functionally with KAT2A, a histone acetyltransferase (HAT) that belongs to the hSAGA and hATAC complexes. XPB interacts with KAT2A-containing complexes on chromatin and an XP-B/CS mutation specifically elicits KAT2A-mediated large-scale chromatin decondensation. In XP-B/CS cells, the abnormal recruitment of TFIIH and KAT2A to chromatin causes inappropriate acetylation of histone H3K9, leading to aberrant formation of transcription initiation complexes on the promoters of several hundred genes and their subsequent overexpression. Significantly, this cascade of events is similarly sensitive to KAT2A HAT inhibition or to the rescue with wild-type XPB. In agreement, the XP-B/CS mutation increases KAT2A HAT activity in vitro. Our results unveil a tight connection between TFIIH and KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in a cancer-prone DNA repair-deficient disorder.


Assuntos
Cromatina/química , Síndrome de Cockayne/genética , Histona Acetiltransferases/genética , Histonas/metabolismo , Subunidades Proteicas/genética , Fator de Transcrição TFIIH/genética , Xeroderma Pigmentoso/genética , Acetilação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Cromatina/metabolismo , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Histonas/genética , Humanos , Modelos Biológicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Cultura Primária de Células , Subunidades Proteicas/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Transcrição TFIIH/metabolismo , Iniciação da Transcrição Genética , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologia
12.
Nat Commun ; 10(1): 1055, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837475

RESUMO

Lysine acetylation is a reversible posttranslational modification that occurs at thousands of sites on human proteins. However, the stoichiometry of acetylation remains poorly characterized, and is important for understanding acetylation-dependent mechanisms of protein regulation. Here we provide accurate, validated measurements of acetylation stoichiometry at 6829 sites on 2535 proteins in human cervical cancer (HeLa) cells. Most acetylation occurs at very low stoichiometry (median 0.02%), whereas high stoichiometry acetylation (>1%) occurs on nuclear proteins involved in gene transcription and on acetyltransferases. Analysis of acetylation copy numbers show that histones harbor the majority of acetylated lysine residues in human cells. Class I deacetylases target a greater proportion of high stoichiometry acetylation compared to SIRT1 and HDAC6. The acetyltransferases CBP and p300 catalyze a majority (65%) of high stoichiometry acetylation. This resource dataset provides valuable information for evaluating the impact of individual acetylation sites on protein function and for building accurate mechanistic models.


Assuntos
Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Acetilação , Conjuntos de Dados como Assunto , Células HeLa , Histonas/análise , Humanos , Lisina/metabolismo , Proteoma/análise , Proteoma/metabolismo , Software , Estatísticas não Paramétricas
13.
Genes Genet Syst ; 94(1): 51-59, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30905891

RESUMO

Transcription factor II D (TFIID), a multiprotein complex consisting of TATA-binding protein (TBP) and 13-14 TBP-associated factors (Tafs), plays a central role in transcription and regulates nearly all class II genes. The N-terminal domain of Taf1p (TAND) can be divided into two subdomains, TAND1 and TAND2, which bind to the concave and convex surfaces of TBP, respectively. The interaction between TAND and TBP is thought to be regulated by TFIIA, activators and/or DNA during transcriptional activation, as the TAND1-bound form of TBP cannot bind to the TATA box. We previously demonstrated that Drosophila TAND1 binds to TBP with a much stronger affinity than yeast TAND1 and that the expression levels of full-length chimeric Taf1p, whose TAND1 is replaced with the Drosophila counterpart, can be varied in vivo by substituting several methionine residues downstream of TAND2 with alanine residues in various combinations. In this study, we examined the transcriptional activation of the GAL1-lacZ reporter or endogenous genes such as RNR3 or GAL1 in yeast cells expressing various levels of full-length chimeric Taf1p. The results showed that the substitution of TAND1 with the Drosophila counterpart in yeast TFIID weakened the transcriptional activation of GAL1-lacZ and RNR3 but not that of GAL1. These findings strongly support a model in which TBP must be released efficiently from TAND1 within TFIID upon transcriptional activation.


Assuntos
Histona Acetiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Ativação Transcricional , Animais , Drosophila melanogaster , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Domínios Proteicos , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genética
14.
Phytomedicine ; 57: 377-384, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30831486

RESUMO

BACKGROUND: Neurofibromatosis type 1 (NF1) is one of the most common hereditary neurocutaneous disorders. The malignant peripheral nerve sheath tumor (MPNST), transformed from NF1 related plexiform neurofibroma, is a rapidly growing and highly invasive tumor. No effective chemotherapeutic agent is currently available. Calebin-A is a derivative from turmeric Curcuma longa. Given the anti-inflammatory and anticancer potentials of curcumin, whether Calebin-A also had the tumoricidal effect upon MPNST cells is still elusive. PURPOSE: To determine whether Calebin-A has the potential for anti-MPNST effect. METHODS: The MTT and FACS analysis of normal Schwann (HSC) and MPNST cells have been employed to determine the tumoricidal effect of Calebin-A. The expression of the signal pathway molecules was assessed by Western blotting. The CHIP with quantitative PCR assay was performed to quantify the promoter DNA binding to acetylated histone 3 (acetyl H3). The enzyme activities of histone acetyltransferase (HAT) and deacetylase (HDAC) have been evaluated by commercial kits. The measurements of tumor size of the xenograft mouse model were also performed. RESULTS: Calebin-A inhibited the proliferation of MPNST and primary neurofibroma cells in a dose-dependent manner. The flow cytometry analysis of the MPNST cells after treatment of 25 µm of Calebin-A demonstrated an increase of population in the G0/G1 phase but decrease in G2/M phase. Before treatment, the expression of Axl, Tyro3, and acetyl H3 was significantly higher in MPNST cells when compared to HSC. The expression of phosphorylated-AKT, -ERK1/2, survivin, hTERT, and acetyl H3 proteins were reduced after treatment. The CHIP assay shows the promoter DNA copies of survivin (BRIC5) and hTERT genes are significantly reduced post-treatment. The enzyme activity of HAT was significantly reduced, but not that of HDAC. Two HAT inhibitors, epigallocatechin-3-gallate (EGCG) and anacardic acid (AA) have also demonstrated a significant inhibitory effect on MPNST cells. Finally, the measurements of tumor size showed a significant reduction of the xenograft tumors after treatment of Calebin-A. CONCLUSION: Both in vitro and in vivo studies showed Calebin-A could inhibit the proliferation of MPNST with suppression of survivin and hTERT. The reduced expression of these two factors might be through the epigenetic histone modification resulting from the decreased activity of HAT.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Cinamatos/farmacologia , Histona Acetiltransferases/metabolismo , Monoterpenos/farmacologia , Neoplasias da Bainha Neural/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Histona Desacetilases/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Neoplasias da Bainha Neural/enzimologia , Neoplasias da Bainha Neural/patologia , Neurofibroma Plexiforme/patologia , Neurofibromatose 1/patologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Survivina/genética , Survivina/metabolismo , Telomerase/genética , Telomerase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Nat Commun ; 10(1): 733, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760718

RESUMO

A growing emphasis in anticancer drug discovery efforts has been on targeting histone acetylation modulators. Here we comprehensively analyze the genomic alterations of the genes encoding histone acetylation modulator proteins (HAMPs) in the Cancer Genome Atlas cohort and observe that HAMPs have a high frequency of focal copy number alterations and recurrent mutations, whereas transcript fusions of HAMPs are relatively rare genomic events in common adult cancers. Collectively, 86.3% (63/73) of HAMPs have recurrent alterations in at least 1 cancer type and 16 HAMPs, including 9 understudied HAMPs, are identified as putative therapeutic targets across multiple cancer types. For example, the recurrent focal amplification of BRD9 is observed in 9 cancer types and genetic depletion of BRD9 inhibits tumor growth. Our systematic genomic analysis of HAMPs across a large-scale cancer specimen cohort may facilitate the identification and prioritization of potential drug targets and selection of suitable patients for precision treatment.


Assuntos
Genômica/métodos , Histonas/metabolismo , Mutação , Neoplasias/genética , Acetilação , Antineoplásicos/uso terapêutico , Variações do Número de Cópias de DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
J Biol Chem ; 294(16): 6227-6239, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30804216

RESUMO

Gcn5 and sirtuins are highly conserved histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes that were first characterized as regulators of gene expression. Although histone tails are important substrates of these enzymes, they also target many nonhistone proteins that function in diverse biological processes. However, the mechanisms used by these enzymes to choose their nonhistone substrates are unknown. Previously, we used SILAC-based MS to identify novel nonhistone substrates of Gcn5 and sirtuins in yeast and found a shared target consensus sequence. Here, we use a synthetic biology approach to demonstrate that this consensus sequence can direct acetylation and deacetylation targeting by these enzymes in vivo Remarkably, fusion of the sequence to a nonsubstrate confers de novo acetylation that is regulated by both Gcn5 and sirtuins. We exploit this synthetic fusion substrate as a tool to define subunits of the Gcn5-containing SAGA and ADA complexes required for nonhistone protein acetylation. In particular, we find a key role for the Ada2 and Ada3 subunits in regulating acetylations on our fusion substrate. In contrast, other subunits tested were largely dispensable, including those required for SAGA stability. In an extended analysis, defects in proteome-wide acetylation observed in ada3Δ mutants mirror those in ada2Δ mutants. Altogether, our work argues that nonhistone protein acetylation by Gcn5 is determined in part by specific amino acids surrounding target lysines but that even optimal sequences require both Ada2 and Ada3 for robust acetylation. The synthetic fusion substrate we describe can serve as a tool to further dissect the regulation of both Gcn5 and sirtuin activities in vivo.


Assuntos
Histona Acetiltransferases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sirtuínas , Acetilação , Deleção de Genes , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirtuínas/química , Sirtuínas/genética , Sirtuínas/metabolismo , Especificidade por Substrato/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Nat Commun ; 10(1): 625, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30733442

RESUMO

The Elongator complex catalyzes posttranscriptional tRNA modifications by attaching carboxy-methyl (cm5) moieties to uridine bases located in the wobble position. The catalytic subunit Elp3 is highly conserved and harbors two individual subdomains, a radical S-adenosyl methionine (rSAM) and a lysine acetyltransferase (KAT) domain. The details of its modification reaction cycle and particularly the substrate specificity of its KAT domain remain elusive. Here, we present the co-crystal structure of bacterial Elp3 (DmcElp3) bound to an acetyl-CoA analog and compare it to the structure of a monomeric archaeal Elp3 from Methanocaldococcus infernus (MinElp3). Furthermore, we identify crucial active site residues, confirm the importance of the extended N-terminus for substrate recognition and uncover the specific induction of acetyl-CoA hydrolysis by different tRNA species. In summary, our results establish the clinically relevant Elongator subunit as a non-canonical acetyltransferase and genuine tRNA modification enzyme.


Assuntos
Histona Acetiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Domínio Catalítico , Histona Acetiltransferases/química , Methanocaldococcus/metabolismo , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Especificidade por Substrato
18.
J Exp Clin Cancer Res ; 38(1): 47, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30709380

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma is one of the leading causes of cancer-related death worldwide. Immune checkpoint blockade therapy, including anti-PD-1 and anti-PD-L1, is a new therapeutic strategy for cancer treatment but the monotherapy with PD-L1 inhibitors for pancreatic cancer is almost ineffective for pancreatic cancer. Thus, exploring the regulatory mechanism of PD-L1 in cancer cells, especially in pancreatic cancer cells, is one of the key strategies to improving cancer patient response to PD-L1 blockade therapy. Histone acetyltransferase 1(HAT1) is a classic type B histone acetyltransferase and the biological role of HAT1 in pancreatic cancer is unclear. METHODS: The clinical relevance of HAT1 was examined by the GEPIA web tool, Western blotting and immunohistochemistry of pancreatic cancer tissue microarray slides. Tumor cell motility was investigated by MTS assay, colony formation assay and xenografts. The relationship between HAT1 and PD-L1 was examined by Western blot analysis, RT-qPCR and immunohistochemistry. RESULTS: HAT1 was upregulated in PDAC and associated with poor prognosis in PDAC patients. The knockdown of HAT1 decreased the proliferation of pancreatic cancer cells in vivo and in vitro. Strikingly, we showed that HAT1 transcriptionally regulated PD-L1, and this process was mainly mediated by BRD4 in pancreatic cancer. The knockdown of HAT1 improved the efficacy of immune checkpoint blockade by decreasing the PD-L1. CONCLUSIONS: The recognition of HAT1 in regulating tumor cell proliferation and cancer immunity indicated that HAT1 might be employed as a new diagnostic and prognostic marker and a predictive marker for pancreatic cancer therapy, especially in immune checkpoint blockade therapy. Targeting HAT1 highlights a novel therapeutic approach to overcome immune evasion by tumor cells.


Assuntos
Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/imunologia , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/imunologia , Fatores de Transcrição/metabolismo , Animais , Apoptose , Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Estudos de Casos e Controles , Proliferação de Células , Seguimentos , Histona Acetiltransferases/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas Nucleares/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Taxa de Sobrevida , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Nucleic Acids Res ; 47(7): 3383-3394, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30715476

RESUMO

The Gcn5 acetyltransferase functions in multiple acetyltransferase complexes in yeast and metazoans. Yeast Gcn5 is part of the large SAGA (Spt-Ada-Gcn5 acetyltransferase) complex and a smaller ADA acetyltransferase complex. In flies and mammals, Gcn5 (and its homolog pCAF) is part of various versions of the SAGA complex and another large acetyltransferase complex, ATAC (Ada2A containing acetyltransferase complex). However, a complex analogous to the small ADA complex in yeast has never been described in metazoans. Previous studies in Drosophila hinted at the existence of a small complex which contains Ada2b, a partner of Gcn5 in the SAGA complex. Here we have purified and characterized the composition of this complex and show that it is composed of Gcn5, Ada2b, Ada3 and Sgf29. Hence, we have named it the metazoan 'ADA complex'. We demonstrate that the fly ADA complex has histone acetylation activity on histones and nucleosome substrates. Moreover, ChIP-Sequencing experiments identified Ada2b peaks that overlap with another SAGA subunit, Spt3, as well as Ada2b peaks that do not overlap with Spt3 suggesting that the ADA complex binds chromosomal sites independent of the larger SAGA complex.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Histona Acetiltransferases/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/citologia , Histona Acetiltransferases/isolamento & purificação , Complexos Multienzimáticos/isolamento & purificação , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Transativadores/isolamento & purificação , Transativadores/metabolismo
20.
Nucleic Acids Res ; 47(7): 3407-3421, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30753595

RESUMO

The proper tissue-specific regulation of gene expression is essential for development and homeostasis in metazoans. However, the illegitimate expression of normally tissue-restricted genes-like testis- or placenta-specific genes-is frequently observed in tumors; this promotes transformation, but also allows immunotherapy. Two important questions are: how is the expression of these genes controlled in healthy cells? And how is this altered in cancer? To address these questions, we used an unbiased approach to test the ability of 350 distinct genetic or epigenetic perturbations to induce the illegitimate expression of over 40 tissue-restricted genes in primary human cells. We find that almost all of these genes are remarkably resistant to reactivation by a single alteration in signaling pathways or chromatin regulation. However, a few genes differ and are more readily activated; one is the placenta-expressed gene ADAM12, which promotes invasion. Using cellular systems, an animal model, and bioinformatics, we find that a non-canonical but druggable TGF-ß/KAT2A/TAK1 axis controls ADAM12 induction in normal and cancer cells. More broadly, our data show that illegitimate gene expression in cancer is an heterogeneous phenomenon, with a few genes activatable by simple events, and most genes likely requiring a combination of events to become reactivated.


Assuntos
Regulação da Expressão Gênica/genética , Neoplasias/genética , Especificidade de Órgãos/genética , Transcrição Genética/genética , Proteína ADAM12/genética , Proteína ADAM12/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Histona Acetiltransferases/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
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