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1.
Int J Mol Sci ; 24(11)2023 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-37298123

RESUMO

Through a comprehensive analysis of the gene expression and dependency in HCC patients and cell lines, LAT1 was identified as the top amino acid transporter candidate supporting HCC tumorigenesis. To assess the suitability of LAT1 as a HCC therapeutic target, we used CRISPR/Cas9 to knockout (KO) LAT1 in the epithelial HCC cell line, Huh7. Knockout of LAT1 diminished its branched chain amino acid (BCAA) transport activity and significantly reduced cell proliferation in Huh7. Consistent with in vitro studies, LAT1 ablation led to suppression of tumor growth in a xenograft model. To elucidate the mechanism underlying the observed inhibition of cell proliferation upon LAT1 KO, we performed RNA-sequencing analysis and investigated the changes in the mTORC1 signaling pathway. LAT1 ablation resulted in a notable reduction in phosphorylation of p70S6K, a downstream target of mTORC1, as well as its substrate S6RP. This reduced cell proliferation and mTORC1 activity were rescued when LAT1 was overexpressed. These findings imply an essential role of LAT1 for maintenance of tumor cell growth and additional therapeutic angles against liver cancer.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Transdução de Sinais , Linhagem Celular , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo
2.
PLoS Biol ; 15(2): e1002597, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28207742

RESUMO

Obesity develops when caloric intake exceeds metabolic needs. Promoting energy expenditure represents an attractive approach in the prevention of this fast-spreading epidemic. Here, we report a novel pharmacological strategy in which a natural compound, narciclasine (ncls), attenuates diet-induced obesity (DIO) in mice by promoting energy expenditure. Moreover, ncls promotes fat clearance from peripheral metabolic tissues, improves blood metabolic parameters in DIO mice, and protects these mice from the loss of voluntary physical activity. Further investigation suggested that ncls achieves these beneficial effects by promoting a shift from glycolytic to oxidative muscle fibers in the DIO mice thereby enhancing mitochondrial respiration and fatty acid oxidation (FAO) in the skeletal muscle. Moreover, ncls strongly activates AMPK signaling specifically in the skeletal muscle. The beneficial effects of ncls treatment in fat clearance and AMPK activation were faithfully reproduced in vitro in cultured murine and human primary myotubes. Mechanistically, ncls increases cellular cAMP concentration and ADP/ATP ratio, which further lead to the activation of AMPK signaling. Blocking AMPK signaling through a specific inhibitor significantly reduces FAO in myotubes. Finally, ncls also enhances mitochondrial membrane potential and reduces the formation of reactive oxygen species in cultured myotubes.


Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Alcaloides de Amaryllidaceae/uso terapêutico , Dieta/efeitos adversos , Músculo Esquelético/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Fenantridinas/farmacologia , Fenantridinas/uso terapêutico , Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Biomarcadores/metabolismo , Respiração Celular/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Dieta Hiperlipídica , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Condicionamento Físico Animal , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
Biochem Biophys Res Commun ; 515(4): 725, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31239030

RESUMO

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The authors have indicated that Fig. 1D data originated from another source not specified in the article. They also indicated image duplication in Fig. 1A and B. The authors of this article would like to apologize to all affected parties.

4.
PLoS Genet ; 12(12): e1006474, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27923061

RESUMO

Increasing energy expenditure through brown adipocyte recruitment is a promising approach to combat obesity. We report here the comprehensive profiling of the epigenome and transcriptome throughout the lineage commitment and differentiation of C3H10T1/2 mesenchymal stem cell line into brown adipocytes. Through direct comparison to datasets from differentiating white adipocytes, we systematically identify stage- and lineage-specific coding genes, lncRNAs and microRNAs. Utilizing chromatin state maps, we also define stage- and lineage-specific enhancers, including super-enhancers, and their associated transcription factor binding motifs and genes. Through these analyses, we found that in brown adipocytes, brown lineage-specific genes are pre-marked by both H3K4me1 and H3K27me3, and the removal of H3K27me3 at the late stage is necessary but not sufficient to promote brown gene expression, while the pre-deposition of H3K4me1 plays an essential role in poising the brown genes for expression in mature brown cells. Moreover, we identify SOX13 as part of a p38 MAPK dependent transcriptional response mediating early brown cell lineage commitment. We also identify and subsequently validate PIM1, SIX1 and RREB1 as novel regulators promoting brown adipogenesis. Finally, we show that SIX1 binds to adipogenic and brown marker genes and interacts with C/EBPα, C/EBPß and EBF2, suggesting their functional cooperation during adipogenesis.


Assuntos
Adipogenia/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Homeodomínio/genética , Obesidade/genética , Tecido Adiposo Marrom/crescimento & desenvolvimento , Tecido Adiposo Marrom/metabolismo , Animais , Autoantígenos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Metabolismo Energético/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais , Camundongos , Obesidade/metabolismo , Obesidade/patologia , RNA Longo não Codificante/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcriptoma/genética
5.
Nucleic Acids Res ; 43(6): e35, 2015 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-25223787

RESUMO

Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (i) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (ii) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (iii) achieved paired-end sequencing of the ChIP-seq libraries; (iv) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq and (v) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies.


Assuntos
Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/genética , Animais , Imunoprecipitação da Cromatina/métodos , DNA/genética , DNA/isolamento & purificação , Camundongos , RNA/genética , RNA/isolamento & purificação , Transcriptoma
6.
Arch Biochem Biophys ; 598: 1-10, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27021582

RESUMO

SIRT1 is a key protein deacetylase that regulates cellular metabolism through lysine deacetylation on both histones and non-histone proteins. Lysine acetylation is a wide-spread post-translational modification found on many regulatory proteins and it plays an essential role in cell signaling, transcription and metabolism. In mice, SIRT1 has known protective functions during high-fat diet but the acetylome regulated by SIRT1 in adipocytes is not completely understood. Here we conducted acetylome analyses in murine adipocytes treated with small-molecule modulators that inhibit or activate the deacetylase activity of SIRT1. We identified a total of 302 acetylated peptides from 78 proteins in this study. From the list of potential SIRT1 targets, we selected seven candidates and further verified that six of them can be deacetylated by SIRT1 in-vitro. Among them, half of the SIRT1 targets are involved in regulating chromatin structure and the other half is involved in RNA processing. Our results provide a resource for further SIRT1 target validation in fat cells and suggest a potential role of SIRT1 in the regulation of chromatin structure and RNA processing, which may possibly extend to other cell types as well.


Assuntos
Adipócitos/metabolismo , Cromatina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , Sirtuína 1/metabolismo , Células 3T3-L1 , Acetilação/efeitos dos fármacos , Animais , Inibidores Enzimáticos/farmacologia , Camundongos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores
7.
Circ Res ; 104(7): 842-50, 2009 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-19229058

RESUMO

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are important pathogenic mechanisms in atherosclerosis and restenosis after vascular injury. In this study, we investigated the effects of beta-lapachone (betaL) (3,4-Dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), which is a potent antitumor agent that stimulates NAD(P)H:quinone oxidoreductase (NQO)1 activity, on neointimal formation in animals given vascular injury and on the proliferation of VSMCs cultured in vitro. betaL significantly reduced the neointimal formation induced by balloon injury. betaL also dose-dependently inhibited the FCS- or platelet-derived growth factor-induced proliferation of VSMCs by inhibiting G(1)/S phase transition. betaL increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase 1 in rat and human VSMCs. Chemical inhibitors of AMPK or dominant-negative AMPK blocked the betaL-induced suppression of cell proliferation and the G(1) cell cycle arrest, in vitro and in vivo. The activation of AMPK in VSMCs by betaL is mediated by LKB1 in the presence of NQO1. Taken together, these results show that betaL inhibits VSMCs proliferation via the NQO1 and LKB1-dependent activation of AMPK. These observations provide the molecular basis that pharmacological stimulation of NQO1 activity is a new therapy for the treatment of vascular restenosis and/or atherosclerosis which are caused by proliferation of VSMCs.


Assuntos
Lesões das Artérias Carótidas/tratamento farmacológico , Estenose das Carótidas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/farmacologia , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Lesões das Artérias Carótidas/enzimologia , Lesões das Artérias Carótidas/patologia , Estenose das Carótidas/enzimologia , Estenose das Carótidas/patologia , Ciclo Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Ativadores de Enzimas/toxicidade , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Hiperplasia , Masculino , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/genética , Naftoquinonas/toxicidade , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína do Retinoblastoma/metabolismo , Prevenção Secundária , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/enzimologia , Túnica Íntima/patologia
8.
Arterioscler Thromb Vasc Biol ; 30(11): 2164-72, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20829507

RESUMO

OBJECTIVE: To explore whether α-lipoic acid (ALA), a naturally occurring antioxidant, inhibits neointimal hyperplasia by inducing apoptosis of vascular smooth muscle cells and to examine its potential effects on reendothelialization and platelet aggregation. METHODS AND RESULTS: Restenosis and late stent thrombosis, caused by neointimal hyperplasia and delayed reendothelialization, are significant clinical problems of balloon angioplasty and drug-eluting stents. ALA treatment strongly induced apoptosis of vascular smooth muscle cells and enhanced the expression and cytoplasmic localization of Nur77, which triggers intrinsic apoptotic events. Small interfering RNA-mediated downregulation of Nur77 diminished this proapoptotic effect of ALA. Moreover, ALA increased p38 mitogen-activated protein kinase phosphorylation, and inhibition of p38 mitogen-activated protein kinase completely blocked ALA-induced vascular smooth muscle cell apoptosis and Nur77 induction and cytoplasmic localization. In balloon-injured rat carotid arteries, ALA enhanced Nur77 expression and increased TUNEL-positive apoptotic cells in the neointima, leading to inhibition of neointimal hyperplasia. This preventive effect of ALA was significantly reduced by infection of an adenovirus encoding Nur77 small hairpin (sh)RNA. Furthermore, ALA reduced basal apoptosis of human aortic endothelial cells and accelerated reendothelialization after balloon injury. ALA also suppressed arachidonic acid-induced platelet aggregation. CONCLUSIONS: ALA could be a promising therapeutic agent to prevent restenosis and late stent thrombosis after angioplasty and drug-eluting stent implantation.


Assuntos
Fármacos Cardiovasculares/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Ácido Tióctico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/lesões , Hiperplasia/prevenção & controle , Masculino , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Ratos , Cicatrização/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Mol Cell Biol ; 27(18): 6506-19, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17636029

RESUMO

The Notch signaling pathway appears to perform an important function in a wide variety of organisms and cell types. In our present study, we provide evidence that UV irradiation-induced Tip60 proteins reduced Notch1 activity to a marked degree. Accumulated UV irradiation-induced Tip60 suppresses Notch1 transcriptional activity via the dissociation of the Notch1-IC-CSL complex. The binding between endogenous Tip60 and Notch1-IC in UV radiation-exposed cells was verified in this study by coimmunoprecipitation. Interestingly, the physical interaction of Tip60 with Notch1-IC occurs to a more profound degree in the presence of CSL but does not exist in a trimeric complex. Using Notch1-IC and Tip60 deletion mutants, we also determined that the N terminus, which harbors the RAM domain and seven ankyrin repeats of Notch1-IC, interacts with the zinc finger and acetyl coenzyme A domains of Tip60. Furthermore, here we report that Notch1-IC is a direct target of the acetyltransferase activity of Tip60. Collectively, our data suggest that Tip60 is an inhibitor of the Notch1 signaling pathway and that Tip60-dependent acetylation of Notch1-IC may be relevant to the mechanism by which Tip60 suppresses Notch1 signaling.


Assuntos
Histona Acetiltransferases/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Acetilação , Animais , Linhagem Celular , Escherichia coli/genética , Deleção de Genes , Genes Reporter , Glutationa Transferase/metabolismo , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Histona Acetiltransferases/efeitos da radiação , Humanos , Rim/citologia , Luciferases/metabolismo , Lisina Acetiltransferase 5 , Camundongos , Modelos Biológicos , Células NIH 3T3 , Testes de Precipitina , Estrutura Terciária de Proteína , Receptor Notch1/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transativadores , Raios Ultravioleta
10.
Biochim Biophys Acta ; 1773(6): 736-46, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17368826

RESUMO

The amyloid beta-precursor protein (APP) and the Notch receptor are both type 1 integral transmembrane proteins, and both are cleaved by presenilin-dependent gamma-secretase activity. In this study, we have demonstrated that the Notch intracellular domain (Notch1-IC) suppresses APP-intracellular domain (AICD)-mediated ROS generation and cell death after being processed by gamma secretase. Notch1-IC physically interacts with AICD, Fe65, and Tip60, thereby disrupting the association of the AICD-Fe65-Tip60 trimeric transcription activator complex in AICD signaling. AICD-Fe65-Tip60 mediated reactive oxygen species generation was found to be suppressed by Notch1-IC. Furthermore, AICD-Fe65-Tip60 was shown to mediate cell death in human neuroblastoma cells, and the overexpression of Notch1-IC inhibited cell death induced by AICD-Fe65-Tip60. Collectively, our findings indicate that Notch1-IC plays the role of a negative regulator in AICD signaling via the disruption of the AICD-Fe65-Tip60 trimeric complex.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Histona Acetiltransferases/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Receptor Notch1/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular Tumoral , Histona Acetiltransferases/genética , Humanos , Lisina Acetiltransferase 5 , Camundongos , Complexos Multiproteicos/genética , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Presenilinas/genética , Presenilinas/metabolismo , Nexinas de Proteases , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptor Notch1/genética , Receptores de Superfície Celular/genética
11.
Free Radic Biol Med ; 45(2): 167-76, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18454945

RESUMO

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species scavenger, but its antioxidant properties have not been completely defined. In this report, we demonstrate that modulation of SAG expression in U937 cells regulates heat shock-induced apoptosis. When we examined the protective role of SAG against heat shock-induced apoptosis with U937 cells transfected with the cDNA for SAG, a clear inverse relationship was observed between the amount of SAG expressed in target cells and their susceptibility to apoptosis. We also observed a significant decrease in the endogenous production of reactive oxygen species and oxidative DNA damage in SAG-overexpressed cells compared to control cells on exposure to heat shock. In addition, transfection of PC3 cells with SAG small interfering RNA markedly decreased the expression of SAG, enhancing the susceptibility of heat shock-induced apoptosis. Taken together, these results indicate that SAG may play an important role in regulating the apoptosis induced by heat shock presumably through maintaining the cellular redox status.


Assuntos
Apoptose/fisiologia , Resposta ao Choque Térmico/fisiologia , Temperatura Alta/efeitos adversos , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Citometria de Fluxo , Humanos , Immunoblotting , Mitocôndrias/patologia , Oxirredução , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
12.
Neurosci Lett ; 413(2): 132-6, 2007 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-17240529

RESUMO

Sensitive to apoptosis gene (SAG), a novel zinc RING finger protein, exhibits anti-apoptotic and antioxidant activity against a variety of redox reagents. In the present study, we have determined that SAG suppresses 1-methyl-4-phenylpyridinium ion (MPP(+))-induced neurotoxicity via the downregulation of ROS generation and c-Jun N-terminal kinase 1 (JNK1) activity. Both transient and constitutively overexpressed SAG were found to inhibit the MPP(+)-induced neurotoxicity of SH-SY5Y neuroblastoma cells. In the SAG-expressing cells, MPP(+) induced ROS generation was suppressed to a significant degree as compared to the cells treated only with MPP(+). MPP(+)-induced JNK1 activation was also determined to be suppressed markedly by SAG. Furthermore, SAG inhibits MEKK1 dependent c-Jun transcription activity in SH-SY5Y cells. Thus, we concluded that SAG is a cellular protective molecule, which appears to function as an antioxidant, suppressing MPP(+)-induced neurotoxicity.


Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/genética , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Regulação da Expressão Gênica/genética , Humanos , MAP Quinase Quinase Quinase 1/metabolismo , Camundongos , Células NIH 3T3 , Neuroblastoma , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurotoxinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Doença de Parkinson/terapia , Ubiquitina-Proteína Ligases
13.
PLoS One ; 12(3): e0174761, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28358928

RESUMO

Adipose tissues constitute an important component of metabolism, the dysfunction of which can cause obesity and type II diabetes. Here we show that differentiation of white and brown adipocytes requires Deleted in Liver Cancer 1 (DLC1), a Rho GTPase Activating Protein (RhoGAP) previously studied for its function in liver cancer. We identified Dlc1 as a super-enhancer associated gene in both white and brown adipocytes through analyzing the genome-wide binding profiles of PPARγ, the master regulator of adipogenesis. We further observed that Dlc1 expression increases during differentiation, and knockdown of Dlc1 by siRNA in white adipocytes reduces the formation of lipid droplets and the expression of fat marker genes. Moreover, knockdown of Dlc1 in brown adipocytes reduces expression of brown fat-specific genes and diminishes mitochondrial respiration. Dlc1-/- knockout mouse embryonic fibroblasts show a complete inability to differentiate into adipocytes, but this phenotype can be rescued by inhibitors of Rho-associated kinase (ROCK) and filamentous actin (F-actin), suggesting the involvement of Rho pathway in DLC1-regulated adipocyte differentiation. Furthermore, PPARγ binds to the promoter of Dlc1 gene to regulate its expression during both white and brown adipocyte differentiation. These results identify DLC1 as an activator of white and brown adipocyte differentiation, and provide a molecular link between PPARγ and Rho pathways.


Assuntos
Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Adipócitos Brancos/citologia , Adipócitos Brancos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adipogenia/genética , Adipogenia/fisiologia , Western Blotting , Calorimetria Indireta , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas Ativadoras de GTPase/genética , Humanos , Consumo de Oxigênio/genética , Consumo de Oxigênio/fisiologia , PPAR gama/genética , PPAR gama/metabolismo , RNA Interferente Pequeno/genética , Proteínas Supressoras de Tumor/genética
14.
Free Radic Res ; 40(11): 1190-7, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17018401

RESUMO

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. The protective role of antioxidant enzymes against singlet oxygen-induced oxidative damage in HL-60 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole and oxlalomalate, specific inhibitors of superoxide dismutase, catalase and NADP+-dependent isocitrate dehydrogenase, respectively. Upon exposure to rose bengal (20 microM)/light (15 min), which generates singlet oxygen, to HL-60 cells, the viability was lower and the lipid peroxidation and oxidative DNA damage were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species as well as the significant decrease in the intracellular GSH level in inhibitor-treated HL-60 cells exposed to singlet oxygen. Upon exposure to rose bengal (3 microM)/light (15 min), which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated HL-60 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against singlet oxygen-induced cell death including necrosis and apoptosis.


Assuntos
Antioxidantes/farmacologia , Inibidores Enzimáticos/farmacologia , Oxigênio/metabolismo , Antioxidantes/metabolismo , Apoptose , Morte Celular , Separação Celular , Dano ao DNA , Glutationa/metabolismo , Células HL-60 , Humanos , Peroxidação de Lipídeos , Necrose , Oxirredução , Estresse Oxidativo , Oxigênio Singlete/metabolismo
15.
PLoS One ; 11(9): e0162528, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27606599

RESUMO

Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies.


Assuntos
Anticorpos/metabolismo , Lisina/metabolismo , Células 3T3-L1 , Acetilação , Motivos de Aminoácidos , Animais , Cromatografia Líquida , Sequência Consenso , Ensaio de Imunoadsorção Enzimática , Ontologia Genética , Células HEK293 , Humanos , Imunoensaio , Masculino , Metaboloma , Camundongos , Peptídeos/química , Peptídeos/metabolismo , Coelhos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
16.
PLoS One ; 10(7): e0133448, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26177453

RESUMO

SIRT1 plays a key role in maintaining metabolic homeostasis in mammals by directly modulating the activities of various transcription factors and metabolic enzymes through lysine deacetylation. White adipose tissue plays a key role in lipid storage and metabolism. To identify novel molecular targets of SIRT1 in fat cells, we used a non-biased proteomic approach. We identified a number of proteins whose acetylation status was significantly affected by SIRT1 modulator treatment in 3T3-L1 adipocytes. Among them, ATP6V1B2, a subunit of the vacuolar (H+)-ATPase, was further shown to be associated with SIRT1 by co-immunoprecipitation assay. Moreover, SIRT1 deacetylates ATP6V1B2 in vitro and in vivo. Taken together, our study demonstrates that ATP6V1B2 is a molecular target of SIRT1 in fat cells and the role of SIRT1 and ATP6V1B2 acetylation in the vacuolar (H+)-ATPase function warrants further investigation.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Sirtuína 1/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Células 3T3-L1 , Acetilação , Animais , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Proteômica
17.
PLoS One ; 10(10): e0140619, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26468954

RESUMO

Lysine acetylation is a post-translational modification found on numerous proteins, a strategy used in cell signaling to change protein activity in response to internal or external cues. Sirtuin 1 (SIRT1) is a central lysine deacetylase involved in a variety of cellular processes including metabolism, apoptosis, and DNA repair. Here we characterize the lysine acetylome in mouse liver, and by using a model of Sirt1-/-knockout mouse, show that SIRT1 regulates the deacetylation of 70 proteins in the liver in-vivo. Amongst these SIRT1-regulated proteins, we find that four RNA-processing proteins and a chromatin-remodeling protein can be deacetylated by SIRT1 directly in-vitro. The discovery that SIRT1 has a potential role in RNA-processing suggests a new layer of regulation in the variety of functions performed by SIRT1.


Assuntos
Fígado/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Sirtuína 1/metabolismo , Acetilação , Animais , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Lisina/metabolismo , Camundongos , Sirtuína 1/genética
18.
Biochimie ; 86(8): 501-7, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15388226

RESUMO

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. When exposed to a singlet oxygen-producing system composed of rose bengal (RB) and visible light, ICDH was susceptible to oxidative modification and damage as indicated by the loss of activity and by the formation of carbonyl groups. The structural alterations of modified enzyme were indicated by the increase in susceptibility to proteases and the change in intrinsic fluorescence spectra. Upon exposure to photoactivated RB, a significant decrease in both cytosolic and mitochondrial ICDH activities was observed in HL-60 cells. The singlet oxygen-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition. When we examined the antioxidant role of cytosolic ICDH against singlet oxygen-induced damage with HL-60 cells transfected with the cDNA for mouse cytosolic ICDH in sense and antisense orientations, a clear inverse relationship was observed between the amount of cytosolic ICDH expressed in target cells and their susceptibility to singlet oxygen-mediated oxidative damage.


Assuntos
Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/metabolismo , NADP/metabolismo , Rosa Bengala/química , Oxigênio Singlete/metabolismo , Oxigênio Singlete/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Células HL-60 , Humanos , Luz , Camundongos , Estresse Oxidativo/efeitos dos fármacos
19.
Free Radic Res ; 37(3): 309-16, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12688426

RESUMO

Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.


Assuntos
Citosol/metabolismo , Desoxiguanosina/análogos & derivados , Isocitrato Desidrogenase/química , NADP/metabolismo , Estresse Oxidativo , Oxigênio/química , Oxigênio/metabolismo , Prolina/análogos & derivados , Oxigênio Singlete , Células 3T3 , 8-Hidroxi-2'-Desoxiguanosina , Animais , Carbono/química , Linhagem Celular , Linhagem Celular Transformada , Sistema Livre de Células , Dano ao DNA , DNA Complementar/metabolismo , Desoxiguanosina/metabolismo , Relação Dose-Resposta a Droga , Corantes Fluorescentes/farmacologia , Vetores Genéticos , Glutationa/metabolismo , Humanos , Immunoblotting , Isocitrato Desidrogenase/metabolismo , Peroxidação de Lipídeos , Azul de Metileno/farmacologia , Camundongos , Microscopia de Fluorescência , Prolina/farmacologia , Proteínas/química , Piridinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Rosa Bengala/farmacologia , Transfecção , Células U937
20.
Free Radic Res ; 36(1): 73-8, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11999705

RESUMO

SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, which is redox responsive and protects mammalian cells from apoptosis, is a metal chelator and a potential reactive oxygen species (ROS) scavenger, but its antioxidant properties have not been completely defined. Here, we show that SAG possesses a potent peroxidase property to decompose hydrogen peroxide in the presence of dithiothreitol (DTT). However, without DTT as a reducing equivalent, SAG was not able to destroy hydrogen peroxide. The peroxidase activity was completely abolished by the reaction of SAG with N-ethylmaleimide (NEM), a chemical modification agent for the sulfhydryl of proteins. These observations suggested that the sulfhydryl of cysteines in SAG could function as strong nucleophiles to destroy hydrogen peroxide. In addition to the peroxidase activity used to remove hydrogen peroxide, SAG also showed t-butylhydroperoxide (t-BOOH) and fatty acid hydroperoxide-selective peroxidase activity.


Assuntos
Peroxidase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Compostos de Sulfidrila/metabolismo , Apoptose , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Etilmaleimida/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Cinética , Espécies Reativas de Oxigênio , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Ubiquitina-Proteína Ligases , Dedos de Zinco , terc-Butil Hidroperóxido/farmacologia
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