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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.
Nat Commun ; 11(1): 3243, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591507

RESUMO

Dysregulation of polyamine metabolism has been linked to the development of colorectal cancer (CRC), but the underlying mechanism is incompletely characterized. Here, we report that spermine synthase (SMS), a polyamine biosynthetic enzyme, is overexpressed in CRC. Targeted disruption of SMS in CRC cells results in spermidine accumulation, which inhibits FOXO3a acetylation and allows subsequent translocation to the nucleus to transcriptionally induce expression of the proapoptotic protein Bim. However, this induction is blunted by MYC-driven expression of miR-19a and miR-19b that repress Bim production. Pharmacological or genetic inhibition of MYC activity in SMS-depleted CRC cells dramatically induces Bim expression and apoptosis and causes tumor regression, but these effects are profoundly attenuated by silencing Bim. These findings uncover a key survival signal in CRC through convergent repression of Bim expression by distinct SMS- and MYC-mediated signaling pathways. Thus, combined inhibition of SMS and MYC signaling may be an effective therapy for CRC.


Assuntos
Proteína 11 Semelhante a Bcl-2/metabolismo , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espermina Sintase/metabolismo , Acetilação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Regulação para Baixo/efeitos dos fármacos , Feminino , Proteína Forkhead Box O3/metabolismo , Deleção de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Poliaminas/metabolismo , Triazóis/farmacologia , Regulação para Cima/efeitos dos fármacos
3.
Nat Commun ; 11(1): 2086, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350249

RESUMO

Gain of function (GOF) DNA binding domain (DBD) mutations of TP53 upregulate chromatin regulatory genes that promote genome-wide histone methylation and acetylation. Here, we therapeutically exploit the oncogenic GOF mechanisms of p53 codon 158 (Arg158) mutation, a DBD mutant found to be prevalent in lung carcinomas. Using high throughput compound screening and combination analyses, we uncover that acetylating mutp53R158G could render cancers susceptible to cisplatin-induced DNA stress. Acetylation of mutp53R158G alters DNA binding motifs and upregulates TRAIP, a RING domain-containing E3 ubiquitin ligase which dephosphorylates IĸB and impedes nuclear translocation of RelA (p65), thus repressing oncogenic nuclear factor kappa-B (NF-ĸB) signaling and inducing apoptosis. Given that this mechanism of cytotoxic vulnerability appears inapt in p53 wild-type (WT) or other hotspot GOF mutp53 cells, our work provides a therapeutic opportunity specific to Arg158-mutp53 tumors utilizing a regimen consisting of DNA-damaging agents and mutp53 acetylators, which is currently being pursued clinically.


Assuntos
Códon/genética , Mutação/genética , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Acetilação/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Epigênese Genética/efeitos dos fármacos , Mutação com Ganho de Função/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos SCID , Modelos Biológicos , Proteínas Mutantes/metabolismo , NF-kappa B/metabolismo , Neoplasias/tratamento farmacológico , Motivos de Nucleotídeos/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/genética , Sulfonamidas/farmacologia , Topotecan/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nucleic Acids Res ; 48(9): 4797-4810, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32246716

RESUMO

Therapeutic targeting of epigenetic modulators offers a novel approach to the treatment of multiple diseases. The cellular consequences of chemical compounds that target epigenetic regulators (epi-drugs) are complex. Epi-drugs affect global cellular phenotypes and cause local changes to gene expression due to alteration of a gene chromatin environment. Despite increasing use in the clinic, the mechanisms responsible for cellular changes are unclear. Specifically, to what degree the effects are a result of cell-wide changes or disease related locus specific effects is unknown. Here we developed a platform to systematically and simultaneously investigate the sensitivity of epi-drugs at hundreds of genomic locations by combining DNA barcoding, unique split-pool encoding, and single cell expression measurements. Internal controls are used to isolate locus specific effects separately from any global consequences these drugs have. Using this platform we discovered wide-spread loci specific sensitivities to epi-drugs for three distinct epi-drugs that target histone deacetylase, DNA methylation and bromodomain proteins. By leveraging ENCODE data on chromatin modification, we identified features of chromatin environments that are most likely to be affected by epi-drugs. The measurements of loci specific epi-drugs sensitivities will pave the way to the development of targeted therapy for personalized medicine.


Assuntos
Epigênese Genética/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Azacitidina/farmacologia , Azepinas/farmacologia , Cromossomos Humanos , Metilação de DNA/efeitos dos fármacos , Genes Reporter , Loci Gênicos , Genômica/métodos , Histonas/metabolismo , Humanos , Células K562 , Análise de Sequência de DNA , Triazóis/farmacologia
5.
Am J Physiol Lung Cell Mol Physiol ; 318(6): L1145-L1157, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32267731

RESUMO

We have demonstrated previously that intracellular transport is impaired in cystic fibrosis (CF) epithelial cells. This impairment is related to both growth and inflammatory regulation in CF cell and animal models. Understanding how transport in CF cells is regulated and identifying means to manipulate that regulation are key to identifying new therapies that can address key CF phenotypes. It was hypothesized that resveratrol could replicate these benefits since it interfaces with multiple pathways identified to affect microtubule regulation in CF. It was found that resveratrol treatment significantly restored intracellular transport as determined by monitoring both cholesterol distribution and the distribution of rab7-positive organelles in CF cells. This restoration of intracellular transport is due to correction of both microtubule formation rates and microtubule acetylation in cultured CF cell models and primary nasal epithelial cells. Mechanistically, the effect of resveratrol on microtubule regulation and intracellular transport was dependent on peroxisome proliferator-activated receptor-γ signaling and its ability to act as a pan-histone deacetylase (HDAC) inhibitor. Resveratrol represents a candidate compound with known anti-inflammatory properties that can restore both microtubule formation and acetylation in CF epithelial cells.


Assuntos
Fibrose Cística/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Espaço Intracelular/metabolismo , Resveratrol/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Acetilação/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Carbazóis/farmacologia , Células Cultivadas , Colesterol/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nariz/patologia , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Resorcinóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirtuínas/metabolismo , Estilbenos/farmacologia , Tubulina (Proteína)/metabolismo
6.
Gene ; 741: 144558, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32165310

RESUMO

The epigenetic regulation of gene expression is controlled by various processes, of which one is histone acetylation. Many proteins control gene expression via histone acetylation. Those proteins include sirtuins (SIRTs) and bromodomain and extraterminal proteins (BETs), which are known to regulate same cellular processes and pathways. The aim of this study was to explore BET inhibitors' effects on SIRT1. Previously we showed that BET inhibitor (+)-JQ1 increases SIRT1 levels, but in the current study we used also other, structurally diverse BET inhibitors, I-BET151 and Pfi-1, and examined their effects on SIRT1 levels in two breast cancer cell lines. The results differed between the inhibitors and also between the cell lines. (+)-JQ1 had opposite effects on SIRT1 levels in the two cell lines, I-BET151 increased the levels in both cell lines, and Pfi-1 had no effect. In conclusion, the effect of structurally diverse BET inhibitors on SIRT1 levels is divergent, and the responses might also be cell type-dependent. These findings are important for all SIRT1 and BET inhibitor-related research, and they show that different BET inhibitors might have important individual effects.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Epigênese Genética , Proteínas/antagonistas & inibidores , Sirtuína 1/genética , Acetilação/efeitos dos fármacos , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Código das Histonas/genética , Humanos , Células MCF-7 , Proteínas/genética
7.
Mol Pharmacol ; 97(4): 259-266, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32005758

RESUMO

Colorectal cancer (CRC) is known to be the third most common cancer disease and the fourth-leading cause of cancer-related deaths worldwide. Bile acid, especially deoxycholic acid and lithocholic acid, were revealed to play an important role during carcinogenesis of CRC. In this study, we found organic solute transporter ß (OSTß), an important subunit of a bile acid export transporter OSTα-OSTß, was noticeably downregulated in CRC. The decline of OSTß expression in CRC was determined by Western blot and real-time polymerase chain reaction (RT-PCR), whereas chromatin immunoprecipitation (ChIP) was used to evaluate the histone acetylation state at the OSTß promoter region in vivo and in vitro. CRC cell lines HT29 and HCT15 were treated with trichostation A (TSA) for the subsequent determination, including RT-PCR, small interfering RNA (siRNA) knockdown, ChIP, and dual-luciferase reporter gene assay, to find out which histone acetyltransferases and deacetylases exactly participated in regulation. We demonstrated that after TSA treatment, OSTß expression increased noticeably because of upregulated H3K27Ac state at OSTß promoter region. We found that stimulating the expression of p300 with CTB (Cholera Toxin B subunit, an activator of p300) and inhibiting p300 expression with C646 (an inhibitor of p300) or siRNA designed for p300 could control OSTß expression through modulating H3K27Ac state at OSTß promoter region. Therefore, downregulated expression of p300 in CRC may cause low expression of OSTß in CRC via epigenetic regulation. Generally, we revealed a novel epigenetic mechanism underlying OSTß repression in CRC, hoping this mechanism would help us to understand and inhibit carcinogenesis of CRC. SIGNIFICANCE STATEMENT: Organic solute transporter ß (OSTß) expression is lower in colon cancer tissues compared with adjacent normal tissues. We revealed the epigenetic mechanisms of it and proved that p300 controls OSTß expression through modulating H3K27Ac state at OSTß promoter region and hence causes low expression of OSTß in colorectal cancer.


Assuntos
Neoplasias Colorretais/genética , Proteína p300 Associada a E1A/metabolismo , Epigênese Genética , Histonas/metabolismo , Proteínas de Membrana Transportadoras/genética , Acetilação/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Toxina da Cólera/farmacologia , Neoplasias Colorretais/patologia , Regulação para Baixo/genética , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
8.
Prostate ; 80(4): 305-318, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31905252

RESUMO

BACKGROUND: Cardiac glycosides, which inhibit Na+ /K+ -ATPase, display inotropic effects for the treatment of congestive heart failure and cardiac arrhythmia. Recent studies have suggested signaling downstream of Na+ /K+ -ATPase action in the regulation of cell proliferation and apoptosis and have revealed the anticancer activity of cardiac glycosides. The study aims to characterize the anticancer potential of ascleposide, a natural cardenolide, and to uncover its primary target and underlying mechanism against human castration-resistant prostate cancer (CRPC). METHODS: Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay, carboxyfluorescein succinimidyl ester staining assay and clonogenic examination. Flow cytometric analysis was used to detect the distribution of cell cycle phase, mitochondrial membrane potential, intracellular Na+ and Ca2+ levels, and reactive oxygen species production. Protein expression was examined using Western blot analysis. Endocytosis of Na+ /K+ -ATPase was determined using confocal immunofluorescence microscopic examination. RESULTS: Ascleposide induced an increase of intracellular Na+ and a potent antiproliferative effect. It also induced a decrease of G1 phase distribution while an increase in both G2/M and apoptotic sub-G1 phases, and downregulated several cell cycle regulator proteins, including cyclins, Cdk, p21, and p27 Cip/Kip proteins, Rb and c-Myc. Ascleposide decreased the expression of antiapoptotic Bcl-2 members (eg, Bcl-2 and Mcl-1) but upregulated proapoptotic member (eg, Bak), leading to a significant loss of mitochondrial membrane potential and activation of both caspase-9 and caspase-3. Ascleposide also dramatically induced tubulin acetylation, leading to inhibition of the catalytic activity of Na+ /K+ -ATPase. Notably, extracellular high K+ (16 mM) significantly blunted ascleposide-mediated effects. Furthermore, ascleposide induced a p38 MAPK-dependent endocytosis of Na+ /K+ -ATPase and downregulated the protein expression of Na+ /K+ -ATPase α1 subunit. CONCLUSION: Ascleposide displays antiproliferative and apoptotic activities dependent on the inhibition of Na+ /K+ -ATPase pumping activity through p38 MAPK-mediated endocytosis of Na+ /K+ -ATPase and downregulation of α1 subunit, which in turn cause tubulin acetylation and cell cycle arrest. Cell apoptosis is ultimately triggered by the activation of caspase cascade attributed to mitochondrial damage through the downregulation of Bcl-2 and Mcl-1 protein expressions while upregulation of Bak protein levels. The data also suggest the potential of ascleposide in anti-CRPC development.


Assuntos
Cardenolídeos/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Humanos , Masculino , Malvaceae/química , Células PC-3 , Extratos Vegetais/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Transdução de Sinais/efeitos dos fármacos
9.
Carbohydr Polym ; 231: 115669, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31888808

RESUMO

This study evaluated the degradation kinetics and structural characteristics of chitin suspension (CS) with a combination of ultrasound and chitinase. Compared with the enzymolysis, the degradation degree of sonoenzymolysis was enhanced to the maximum by 27.93 % at an intensity of 25 W/mL for 20 min. According to degradation kinetics, ultrasound intensified molecular collision rate between chitinase and substrate, thereby increasing the degradation degree. What's more, combined with chitinase, ultrasound intensified the rate of the breakage of glycosidic bond and viscosity-average molecular weight (Mv) decrease, but no obvious change in acetylation degree (DA). Additionally, the intra- or inter-hydrogen bindings were weakened by ultrasound during sonoenzymolysis, leading to a slight decrease in crystalline index and a more ordered structure, which increased the accessibility of the substrate to enzyme. In conclusion, combination of chitinase and ultrasound could enhance the hydrolysis of CS while without changing its primary structure.


Assuntos
Quitina/química , Quitinases/química , Ondas Ultrassônicas , Acetilação/efeitos dos fármacos , Acetilação/efeitos da radiação , Quitina/efeitos da radiação , Quitinases/farmacologia , Hidrogênio/química , Hidrólise/efeitos da radiação , Cinética , Suspensões/química , Suspensões/farmacologia
10.
Int J Radiat Oncol Biol Phys ; 107(1): 212-221, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987970

RESUMO

PURPOSE: Muscle-invasive bladder cancer has a 40% to 60% 5-year survival rate with radical treatment by surgical removal of the bladder or radiation therapy-based bladder preservation techniques, including concurrent chemoradiation. Elderly patients cannot tolerate current chemoradiation therapy regimens and often receive only radiation therapy, which is less effective. We urgently need effective chemotherapy agents for use with radiation therapy combinations that are nontoxic to normal tissues and tolerated by elderly patients. METHODS AND MATERIALS: We have identified histone deacetylase (HDAC) inhibitors as promising agents to study. Pan-HDAC inhibition, using panobinostat, is a good strategy for radiosensitization, but more selective agents may be more useful radiosensitizers in a clinical setting, resulting in fewer systemic side effects. Herein, we study the HDAC class I-selective agent romidepsin, which we predict to have fewer off-target effects than panobinostat while maintaining an effective level of tumor radiosensitization. RESULTS: In vitro effects of romidepsin were assessed by clonogenic assay and showed that romidepsin was effective in the nanomolar range in different bladder cancer cells and radiosensitized these cells. The radiosensitizing effect of romidepsin was confirmed in vivo using superficial xenografts. The drug/irradiation combination treatment resulted in significant tumor growth delay but did not increase the severity of acute (3.75 days) intestinal normal tissue toxicity or late toxicity at 29 weeks. Moreover, we showed that romidepsin treatment impaired both homologous recombination and nonhomologous end joining DNA repair pathways, suggesting that the disruption of DNA repair pathways caused by romidepsin is a key mechanism for its radiosensitizing effect in bladder cancer cells. CONCLUSIONS: This study demonstrates that romidepsin is an effective radiosensitizer in vitro and in vivo and does not increase the acute and late toxicity after ionizing radiation. Romidepsin is already in clinical use for the cutaneous T-cell lymphoma, but a phase 1 clinical trial of romidepsin as a radiosensitizer could be considered in muscle-invasive bladder cancer.


Assuntos
Depsipeptídeos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Radiossensibilizantes/farmacologia , Neoplasias da Bexiga Urinária/patologia , Acetilação/efeitos dos fármacos , Acetilação/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Depsipeptídeos/efeitos adversos , Inibidores de Histona Desacetilases/efeitos adversos , Histonas/metabolismo , Humanos , Órgãos em Risco/efeitos da radiação , Radiossensibilizantes/efeitos adversos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
J Immunol ; 204(5): 1201-1213, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31932499

RESUMO

Vitamin D can modulate the innate and adaptive immune system. Vitamin D deficiency has been associated with various autoimmune diseases. Th9 cells are implicated in the pathogenesis of numerous autoimmune diseases. Thus, we investigated the role of calcitriol (active metabolite of vitamin D) in the regulation of Th9 cell differentiation. In this study, we have unraveled the molecular mechanisms of calcitriol-mediated regulation of Th9 cell differentiation. Calcitriol significantly diminished IL-9 secretion from murine Th9 cells associated with downregulated expression of the Th9-associated transcription factor, PU.1. Ectopic expression of VDR in Th9 cells attenuated the percentage of IL-9-secreting cells. VDR associated with PU.1 in Th9 cells. Using a series of mutations, we were able to dissect the VDR domain involved in the regulation of the Il9 gene. The VDR-PU.1 interaction prevented the accessibility of PU.1 to the Il9 gene promoter, thereby restricting its expression. However, the expression of Foxp3, regulatory T cell-specific transcription factor, was enhanced in the presence of calcitriol in Th9 cells. When Th9 cells are treated with both calcitriol and trichostatin A (histone deacetylase inhibitor), the level of IL-9 reached to the level of wild-type untreated Th9 cells. Calcitriol attenuated specific histone acetylation at the Il9 gene. In contrast, calcitriol enhanced the recruitment of the histone modifier HDAC1 at the Il9 gene promoter. In summary, we have identified that calcitriol blocked the access of PU.1 to the Il9 gene by reducing its expression and associating with it as well as regulated the chromatin of the Il9 gene to regulate expression.


Assuntos
Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/imunologia , Interleucina-9/imunologia , Proteínas Proto-Oncogênicas/imunologia , Linfócitos T Reguladores/imunologia , Transativadores/imunologia , Acetilação/efeitos dos fármacos , Animais , Diferenciação Celular/imunologia , Feminino , Regulação da Expressão Gênica/imunologia , Histonas/imunologia , Camundongos , Regiões Promotoras Genéticas/imunologia , Receptores de Calcitriol/imunologia , Linfócitos T Reguladores/citologia
12.
Psychopharmacology (Berl) ; 237(1): 231-248, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31654083

RESUMO

RATIONALE: Several findings indicate that early-life dysfunction of N-methyl-D-aspartate (NMDA) receptors might cause schizophrenia-like abnormalities in adulthood that might be induced by impairments in epigenetic regulation. OBJECTIVES: In the present study, we investigated whether postnatal blockade of NMDA receptors (within the first 3 weeks of life) by the competitive antagonist CGP 37849 (CGP) might affect some epigenetic markers in the adult medial prefrontal cortex (mPFC). METHODS: Histone H3 phosphorylation at serine 10 (H3S10ph), histone H3 acetylation at lysine 9 or 14 (H3K9ac or H3K14ac, respectively), or expression of histone deacetylase (HDAC) 2, HDAC5, myocyte enhancer factor (MEF) 2D and activity-regulated cytoskeleton-associated protein (Arc) were analysed. Moreover, we also evaluated whether the deacetylase inhibitor sodium butyrate (SB; 1.2 mg/kg, ip) could prevent behavioural and neurochemical changes in the mPFC induced by CGP during memory retrieval in the trace fear conditioning paradigm. RESULTS: The results showed that CGP administration increased the number of H3S10ph nuclei but did not affect H3K9ac and H3K14ac or HDAC2 protein levels. However, CGP administration altered the HDAC5 mRNA and protein levels and increased the mRNA and protein levels of MEF2D. CGP also increased Arc mRNA, which was correlated with an increase in the amount of Arc DNA bound to MEF2D. SB given 2 h after training prevented impairment of the freezing response and disruption of epigenetic markers (H3S10ph, HDAC5, MEF2D) and Arc expression during memory retrieval induced by CGP administration. CONCLUSIONS: The early-life blockade of NMDA receptors impairs some epigenetic regulatory processes in the mPFC that are involved in fear memory formation.


Assuntos
Epigênese Genética/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Medo/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , 2-Amino-5-fosfonovalerato/análogos & derivados , 2-Amino-5-fosfonovalerato/farmacologia , Acetilação/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Medo/fisiologia , Histonas/metabolismo , Masculino , Memória/fisiologia , Córtex Pré-Frontal/metabolismo , Ratos , Esquizofrenia/metabolismo
13.
Nucleic Acids Res ; 48(2): 517-532, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31799598

RESUMO

Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1-9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Acetilação/efeitos dos fármacos , Proteína 9 Associada à CRISPR/genética , Cromatina/genética , Reparo do DNA por Junção de Extremidades/genética , Técnicas de Inativação de Genes , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 2/antagonistas & inibidores , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histonas/química , Histonas/genética , Humanos
14.
Cancer Sci ; 111(1): 112-126, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31675763

RESUMO

Drug repositioning is an emerging approach to developing novel cancer treatments. Vorinostat is a histone deacetylase inhibitor approved for cancer treatment, but it could attenuate its anticancer activity by activating the mTOR pathway. The HMG-CoA reductase inhibitor fluvastatin reportedly activates the mTOR inhibitor AMP-activated protein kinase (AMPK), and we thought that it would potentiate vorinostat's anticancer activity in renal cancer cells. The combination of vorinostat and fluvastatin induced robust apoptosis and inhibited renal cancer growth effectively both in vitro and in vivo. Vorinostat activated the mTOR pathway, as evidenced by the phosphorylation of ribosomal protein S6, and fluvastatin inhibited this phosphorylation by activating AMPK. Fluvastatin also enhanced vorinostat-induced histone acetylation. Furthermore, the combination induced endoplasmic reticulum (ER) stress that was accompanied by aggresome formation. We also found that there was a positive feedback cycle among AMPK activation, histone acetylation, and ER stress induction. This is the first study to report the beneficial combined effect of vorinostat and fluvastatin in cancer cells.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Fluvastatina/farmacologia , Neoplasias Renais/tratamento farmacológico , Vorinostat/farmacologia , Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias Renais/metabolismo , Fosforilação/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
15.
Biochim Biophys Acta Mol Cell Res ; 1867(2): 118564, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31672612

RESUMO

Recently, histone deacetylase inhibitors (HDACi) have become widely used in anti-cancer treatment; however, due to acquired drug resistance and their relatively low specificity, they are largely ineffective against late-stage cancer. Thus, it is critical to elucidate the molecular mechanisms underlying these issues, so as to identify novel therapeutic targets to prevent late-stage cancer progression and resistance acquisition. The present study investigated the Aryl hydrocarbon receptor (AHR), that has been shown to mediate histone acetylation by regulating histone deacetylase (HDAC) activity during HDACi treatment in human gastric-cancer cell lines (i.e. AGS and NCI-N87 cells). The potent HDACi, Aza-PBHA, was thus shown to upregulate AHR expression in both AGS and NCI-N87 cell lines, and to increase histone acetylation levels by facilitating AHR/HDAC interactions. Conversely, AHR knockdown increased HDAC activity. Aza-PBHA also increased PKCα phosphorylation and membrane translocation; however, interestingly, PKCα inhibition reduced the Aza-PBHA-increased AHR and histone acetylation levels, and inhibited the formation of the AHR/HDAC complex, likely upregulating Aza-PBHA-inhibited cell migration. Thus, our results suggest that Aza-PBHA treatment increased AHR levels to suppress HDAC activity, and inhibited cell migration by activating PKCα activation. These findings support the use of drugs to control AHR-related epigenetic regulation as a promising potential method to prevent acquired resistance to cancer treatments.


Assuntos
Movimento Celular/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Proteína Quinase C-alfa/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/química , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Regulação para Cima/efeitos dos fármacos
16.
J Matern Fetal Neonatal Med ; 33(1): 81-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29886761

RESUMO

Objective: Volatile anesthetic such as isoflurane causes widespread neurodegeneration in the developing animal brains and also induces cognitive impairments. Paeonol is a plant-derived phenolic compound possessing numerous bioactive properties. The study investigates the neuroprotective effects of paeonol against isoflurane-induced neurodegeneration and cognitive disturbances in neonatal rats.Methods: Paeonol (50, 100, and 150 mg/kg body weight/day) was given orally to separate groups of neonatal rats from postnatal day 3 (P3) to P21 and were exposed to isoflurane (0.75%; 6 h) on P7.Results: Neuroapoptosis following isoflurane exposure was remarkably reduced by paeonol. Isoflurane-induced elevated cleaved caspase-3, Bad, and Bax expression, were down-regulated on paeonol administration. Paeonol significantly enhanced expression of antiapoptotic proteins (Bcl-2, Bcl-xL, xIAP, c-IAP-1, c-IAP-2, and survivin) and improved acetylation of HK39 and HK412. The expression of histone deacetylases (HDACs)-HDAC2 and HDAC-3 were down-regulated. Isoflurane-induced activation of JNK/p38MAPK signaling and suppressed ERK signaling and were effectively regulated by paeonol. General behavior and freezing responses of the rats were improved. Results of the Morris Water Maze tests revealed improved learning and memory retention on paeonol treatment.Conclusions: Paeonol effectively inhibited neuroapoptosis and improved isoflurane-induced cognitive dysfunctions via regulating histone acetylation and JNK/ERK1/2/p38MAPK signaling pathways.


Assuntos
Acetofenonas/farmacologia , Anestesia/efeitos adversos , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Isoflurano/efeitos adversos , Síndromes Neurotóxicas/prevenção & controle , Acetofenonas/uso terapêutico , Acetilação/efeitos dos fármacos , Anestésicos/efeitos adversos , Animais , Animais Recém-Nascidos , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Histona Acetiltransferases/efeitos dos fármacos , Histonas/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Gravidez , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
Cell Mol Life Sci ; 77(18): 3643-3655, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31873757

RESUMO

Prior studies have established the important role of extracellular signal-regulated kinase 1/2 (ERK1/2) as a mediator of acute kidney injury (AKI). We demonstrated rapid ERK1/2 activation induced renal dysfunction following ischemia/reperfusion (IR)-induced AKI and downregulated the mitochondrial biogenesis (MB) regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in mice. In this study, ERK1/2 regulation of cellular nicotinamide adenine dinucleotide (NAD) and PGC-1α were explored. Inhibition of ERK1/2 activation during AKI in mice using the MEK1/2 inhibitor, trametinib, attenuated renal cortical oxidized NAD (NAD+) depletion. The rate-limiting NAD biosynthesis salvage enzyme, NAMPT, decreased following AKI, and this decrease was prevented by ERK1/2 inhibition. The microRNA miR34a decreased with the inhibition of ERK1/2, leading to increased NAMPT protein. Mice treated with a miR34a mimic prevented increases in NAMPT protein in the renal cortex in the presence of ERK1/2 inhibition. In addition, ERK1/2 activation increased acetylated PGC-1α, the less active form, whereas inhibition of ERK1/2 activation prevented an increase in acetylated PGC-1α after AKI through SIRT1 and NAD+ attenuation. These results implicate IR-induced ERK1/2 activation as an important contributor to the downregulation of both PGC-1α and NAD+ pathways that ultimately decrease cellular metabolism and renal function. Inhibition of ERK1/2 activation prior to the initiation of IR injury attenuated decreases in PGC-1α and NAD+ and prevented kidney dysfunction.


Assuntos
Lesão Renal Aguda/patologia , Citocinas/metabolismo , MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Acetilação/efeitos dos fármacos , Lesão Renal Aguda/metabolismo , Animais , Antagomirs/metabolismo , Creatinina/sangue , Citocinas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Córtex Renal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fosforilação/efeitos dos fármacos , Piridonas/farmacologia , Pirimidinonas/farmacologia , Sirtuína 1/metabolismo
18.
Elife ; 82019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31742554

RESUMO

Because old age is the greatest risk factor for dementia, a successful therapy will require an understanding of the physiological changes that occur in the brain with aging. Here, two structurally distinct Alzheimer's disease (AD) drug candidates, CMS121 and J147, were used to identify a unique molecular pathway that is shared between the aging brain and AD. CMS121 and J147 reduced cognitive decline as well as metabolic and transcriptional markers of aging in the brain when administered to rapidly aging SAMP8 mice. Both compounds preserved mitochondrial homeostasis by regulating acetyl-coenzyme A (acetyl-CoA) metabolism. CMS121 and J147 increased the levels of acetyl-CoA in cell culture and mice via the inhibition of acetyl-CoA carboxylase 1 (ACC1), resulting in neuroprotection and increased acetylation of histone H3K9 in SAMP8 mice, a site linked to memory enhancement. These data show that targeting specific metabolic aspects of the aging brain could result in treatments for dementia.


Assuntos
Envelhecimento/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Mitocôndrias/metabolismo , Acetilcoenzima A/efeitos dos fármacos , Acetilcoenzima A/metabolismo , Acetil-CoA Carboxilase/genética , Acetilação/efeitos dos fármacos , Envelhecimento/patologia , Doença de Alzheimer/fisiopatologia , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Curcumina/análogos & derivados , Curcumina/farmacologia , Humanos , Memória/efeitos dos fármacos , Memória/fisiologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
19.
BMC Cancer ; 19(1): 1106, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727006

RESUMO

BACKGROUND: Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. METHODS: Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay. RESULTS: To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666-1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1) are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10B and resistant 5-8F NPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4-/- mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. CONCLUSION: Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells. The present findings also propose that SIRT2 can be an important biomarker for metastatic and Lapatinib resistant NPC and that targeting the SIRT2-FOXO3 axis may provide novel strategies for treating NPC and for overcoming chemoresistance.


Assuntos
Proteína Forkhead Box O3/genética , Lapatinib/farmacologia , Carcinoma Nasofaríngeo/tratamento farmacológico , Sirtuína 2/genética , Acetilação/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lapatinib/efeitos adversos , Camundongos , Camundongos Knockout , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patologia , Inibidores de Proteínas Quinases/farmacologia
20.
Nucleic Acids Res ; 47(16): 8502-8520, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31616951

RESUMO

Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling enzyme with an emerging role in DNA damage response (DDR), but the underlying mechanism remains largely unknown. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1), a key chromatin-associated enzyme responsible for the synthesis of poly(ADP-ribose) (PAR) polymers in mammalian cells, interacts with and PARylates MORC2 at two residues within its conserved CW-type zinc finger domain. Following DNA damage, PARP1 recruits MORC2 to DNA damage sites and catalyzes MORC2 PARylation, which stimulates its ATPase and chromatin remodeling activities. Mutation of PARylation residues in MORC2 results in reduced cell survival after DNA damage. MORC2, in turn, stabilizes PARP1 through enhancing acetyltransferase NAT10-mediated acetylation of PARP1 at lysine 949, which blocks its ubiquitination at the same residue and subsequent degradation by E3 ubiquitin ligase CHFR. Consequently, depletion of MORC2 or expression of an acetylation-defective PARP1 mutant impairs DNA damage-induced PAR production and PAR-dependent recruitment of DNA repair proteins to DNA lesions, leading to enhanced sensitivity to genotoxic stress. Collectively, these findings uncover a previously unrecognized mechanistic link between MORC2 and PARP1 in the regulation of cellular response to DNA damage.


Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Reparo do DNA , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Acetilação/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Dano ao DNA , Células HEK293 , Humanos , Mutação , Acetiltransferase N-Terminal E/genética , Acetiltransferase N-Terminal E/metabolismo , Proteínas de Neoplasias/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteólise/efeitos dos fármacos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
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