RESUMO
Intracellular energy balance is important for cell survival. In eukaryotic cells, the most energy-consuming process is ribosome biosynthesis, which adapts to changes in intracellular energy status. However, the mechanism that links energy status and ribosome biosynthesis is largely unknown. Here, we describe eNoSC, a protein complex that senses energy status and controls rRNA transcription. eNoSC contains Nucleomethylin, which binds histone H3 dimethylated Lys9 in the rDNA locus, in a complex with SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are required for energy-dependent transcriptional repression, suggesting that a change in the NAD(+)/NADH ratio induced by reduction of energy status could activate SIRT1, leading to deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1, thus establishing silent chromatin in the rDNA locus. Furthermore, eNoSC promotes restoration of energy balance by limiting rRNA transcription, thus protecting cells from energy deprivation-dependent apoptosis. These findings provide key insight into the mechanisms of energy homeostasis in cells.
Assuntos
DNA Ribossômico/genética , Metabolismo Energético , Inativação Gênica , Transcrição Gênica , Morte Celular , Linhagem Celular , Nucléolo Celular/metabolismo , Glucose/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , NAD/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Metiltransferases , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas Repressoras/metabolismo , Sirtuína 1 , Sirtuínas/metabolismoRESUMO
UNLABELLED: Liver X receptor (LXR) activation stimulates triglyceride (TG) accumulation in the liver. Several lines of evidence indicate that estradiol-17ß (E2) reduces TG levels in the liver; however, the molecular mechanism underlying the E2 effect remains unclear. Here, we show that administration of E2 attenuated sterol regulatory element-binding protein (SREBP)-1 expression and TG accumulation induced by LXR activation in mouse liver. In estrogen receptor alpha (ERα) knockout (KO) and liver-specific ERα KO mice, E2 did not affect SREBP-1 expression or TG levels. Molecular analysis revealed that ERα is recruited to the SREBP-1c promoter through direct binding to LXR and inhibits coactivator recruitment to LXR in an E2-dependent manner. Our findings demonstrate the existence of a novel liver-dependent mechanism controlling TG accumulation through the nonclassical ER/LXR pathway. To confirm that a nonclassical ER/LXR pathway regulates ERα-dependent inhibition of LXR activation, we screened ERα ligands that were able to repress LXR activation without enhancing ERα transcriptional activity, and, as a result, we identified the phytoestrogen, phloretin. In mice, phloretin showed no estrogenic activity; however, it did reduce SREBP-1 expression and TG levels in liver of mice fed a high-fat diet to an extent similar to that of E2. CONCLUSION: We propose that ER ligands reduce TG levels in the liver by inhibiting LXR activation through a nonclassical pathway. Our results also indicate that the effects of ER on TG accumulation can be distinguished from its estrogenic effects by a specific ER ligand.
Assuntos
Fígado Gorduroso/prevenção & controle , Receptores Nucleares Órfãos/fisiologia , Receptores de Estrogênio/fisiologia , Animais , Dieta Hiperlipídica , Estradiol/farmacologia , Feminino , Ligantes , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C57BL , Receptores Nucleares Órfãos/antagonistas & inibidores , Floretina/farmacologia , Regiões Promotoras Genéticas , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Ativação Transcricional , Triglicerídeos/metabolismoRESUMO
Estrogen receptor alpha (ERα) expression is a risk factor for breast cancer. HDAC inhibitors have been demonstrated to down-regulate ERα expression in ERα-positive breast cancer cell lines, but the molecular mechanisms are poorly understood. Here, we showed that HDAC inhibitors decrease the stability of ERα mRNA, and that knockdown of HDAC3 decreases the stability of ERα mRNA and suppresses estrogen-dependent proliferation of ERα-positive MCF-7 breast cancer cells. In the Oncomine database, expression levels of HDAC3 in ERα-positive tumors are higher than those in ERα-negative tumors, thus suggesting that HDAC3 is necessary for ERα mRNA stability, and is involved in the estrogen-dependent proliferation of ERα-positive tumors.
Assuntos
Neoplasias da Mama/enzimologia , Receptor alfa de Estrogênio/biossíntese , Histona Desacetilases/metabolismo , Estabilidade de RNA , RNA Mensageiro/química , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Feminino , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , HumanosRESUMO
Interleukin-2-inducible T-cell kinase (ITK) plays an important role in T-cell signaling and is considered a promising drug target. As the ATP binding sites of protein kinases are highly conserved, the design of selective kinase inhibitors remains a challenge. Targeting inactive kinase conformations can address the issue of kinase inhibitor selectivity. It is important for selectivity considerations to identify compounds that stabilize inactive conformations from the primary screen hits. Here we screened a library of 390,000 compounds with an ADP-Glo assay using dephosphorylated ITK. After a surface plasmon resonance (SPR) assay was used to filter out promiscuous inhibitors, 105 hits were confirmed. Next, we used a fluorescent biosensor to enable the detection of conformational changes to identify inactive conformation inhibitors. A single-cysteine-substituted ITK mutant was labeled with acrylodan, and fluorescence emission was monitored. Using a fluorescent biosensor assay, we identified 34 inactive conformation inhibitors from SPR hits. Among them, one compound was bound to a site other than the ATP pocket and exhibited excellent selectivity against a kinase panel. Overall, (1) biochemical screening using dephosphorylated kinase, (2) hit confirmation by SPR assay, and (3) fluorescent biosensor assay that can distinguish inactive compounds provide a useful platform and offer opportunities to identify selective kinase inhibitors.
Assuntos
Descoberta de Drogas , Modelos Moleculares , Conformação Proteica , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/química , Técnicas Biossensoriais , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Humanos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Recombinantes , Reprodutibilidade dos Testes , Bibliotecas de Moléculas PequenasRESUMO
Ribosome biosynthesis is a major intracellular energy-consuming process. We previously identified a nucleolar factor, nucleomethylin (NML), which regulates intracellular energy consumption by limiting rRNA transcription. Here, we show that, in livers of obese mice, the recruitment of NML to rRNA gene loci is increased to repress rRNA transcription. To clarify the relationship between obesity and rRNA transcription, we generated NML-null (NML-KO) mice. NML-KO mice show elevated rRNA level, reduced ATP concentration, and reduced lipid accumulation in the liver. Furthermore, in high-fat-diet (HFD)-fed NML-KO mice, hepatic rRNA levels are not decreased. Both weight gain and fat accumulation in HFD-fed NML-KO mice are significantly lower than those in HFD-fed wild-type mice. These findings indicate that rRNA transcriptional activation promotes hepatic energy consumption, which alters hepatic lipid metabolism. Namely, hepatic rRNA transcriptional repression by HFD feeding is essential for energy storage.