RESUMEN
Lipolysis in fat tissue represents a major source of circulating fatty acids. Previously, we have found that lipolysis in adipocytes is controlled by early growth response transcription factor Egr1 that directly inhibits transcription of adipose triglyceride lipase, ATGL (Chakrabarti, P., Kim, J. Y., Singh, M., Shin, Y. K., Kim, J., Kumbrink, J., Wu, Y., Lee, M. J., Kirsch, K. H., Fried, S. K., and Kandror, K. V. (2013) Mol. Cell. Biol. 33, 3659-3666). Here we demonstrate that knockdown of the lipid droplet protein FSP27 (a.k.a. CIDEC) in human adipocytes increases expression of ATGL at the level of transcription, whereas overexpression of FSP27 has the opposite effect. FSP27 suppresses the activity of the ATGL promoter in vitro, and the proximal Egr1 binding site is responsible for this effect. FSP27 co-immunoprecipitates with Egr1 and increases its association with and inhibition of the ATGL promoter. Knockdown of Egr1 attenuates the inhibitory effect of FSP27. These results provide a new model of transcriptional regulation of ATGL.
Asunto(s)
Adipocitos/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Lipasa/metabolismo , Proteínas/metabolismo , Células 3T3-L1 , Adipocitos/citología , Animales , Proteínas Reguladoras de la Apoptosis , Sitios de Unión/genética , Células Cultivadas , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Expresión Génica , Células HEK293 , Humanos , Immunoblotting , Lipasa/genética , Lipólisis/genética , Ratones , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet-associated proteins, and lipases. The discovery of new lipid droplet-associated proteins added complexity to the current model of lipolysis. In this study, we used cultured human adipocytes to demonstrate that fat-specific protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL, also called desnutrin or PNPLA2). We identified a core domain of FSP27, amino acids 120-220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid-induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full-length FSP27 or amino acids 120-220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27-ATGL interactions in regulating lipolysis, triglyceride accumulation, and insulin signaling in human adipocytes.
Asunto(s)
Adipocitos/enzimología , Resistencia a la Insulina , Lipasa/metabolismo , Lipólisis/fisiología , Proteínas/fisiología , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Humanos , Insulina/metabolismo , Insulina/farmacología , Ratones , Ratones Noqueados , Fosforilación , Unión Proteica , Proteínas/genética , Transducción de Señal , Triglicéridos/metabolismoRESUMEN
DNA microarray and genetic studies of Saccharomyces cerevisiae have demonstrated that histone deacetylases (HDACs) are required for transcriptional activation and repression, but the mechanism by which they activate transcription remains poorly understood. We show that two HDACs, RPD3 and HOS2, are required for the activation of DNA damage-inducible genes RNR3 and HUG1. Using mutants specific for the Rpd3L complex, we show that the complex is responsible for regulating RNR3. Furthermore, unlike what was described for the GAL genes, Rpd3L regulates the activation of RNR3 by deacetylating nucleosomes at the promoter, not at the open reading frame. Rpd3 is recruited to the upstream repression sequence of RNR3, which surprisingly does not require Tup1 or Crt1. Chromatin remodeling and TFIID recruitment are largely unaffected in the Deltarpd3/Deltahos2 mutant, but the recruitment of RNA polymerase II is strongly reduced, arguing that Rpd3 and Hos2 regulate later stages in the assembly of the preinitiation complex or facilitate multiple rounds of polymerase recruitment. Furthermore, the histone H4 acetyltransferase Esa1 is required for the activation of RNR3 and HUG1. Thus, reduced or unregulated constitutive histone H4 acetylation is detrimental to promoter activity, suggesting that HDAC-dependent mechanisms are in place to reset promoters to allow high levels of transcription.
Asunto(s)
Daño del ADN , Histona Desacetilasas/metabolismo , Ribonucleósido Difosfato Reductasa/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Activación Transcripcional , Acetilación , Ensamble y Desensamble de Cromatina/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Histonas/metabolismo , Lisina/metabolismo , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Polimerasa II/metabolismo , Proteínas Represoras/metabolismo , Ribonucleósido Difosfato Reductasa/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Recent work with mouse models and human leukemic samples has shown that gain-of-function mutation(s) in Notch1 is a common genetic event in T-cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a gamma-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus, where it forms part of a transcriptional activator complex. To identify Notch1 target genes in leukemia, we developed mouse T-cell leukemic lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel, direct, and critical Notch1 target gene in T-cell leukemia. c-myc mRNA levels are increased in primary mouse T-cell tumors that harbor Notch1 mutations, and Notch1 inhibition decreases c-myc mRNA levels and inhibits leukemic cell growth. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with gamma-secretase inhibitor treatment or Notch1 inhibition. Consistent with these findings, retroviral insertional mutagenesis screening of our T-cell leukemia mouse model revealed common insertions in either notch1 or c-myc genes. These studies define the Notch1 molecular signature in mouse T-ALL and importantly provide mechanistic insight as to how Notch1 contributes to human T-ALL.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia de Células T/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptor Notch1/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Apoptosis/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ciclo Celular/fisiología , Línea Celular Tumoral , Inhibidores Enzimáticos/metabolismo , Perfilación de la Expresión Génica , Humanos , Ratones , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Receptor Notch1/genética , Retroviridae/genética , Retroviridae/metabolismo , Proteína 1 de la Leucemia Linfocítica T AgudaRESUMEN
Stereospecific conversion of hydantoins into their carbamoyl acid derivatives could be achieved by using the enzyme hydantoinase. Specific hydantoinases convert either the D-form or the L-form of the hydantoin and the amino acids responsible for stereospecificity have not been identified. Structural studies on hydantoinases from a few bacterial species were published recently. The structure of a thermostable D-hydantoinase from Bacillus sp. AR9 (bar9HYD) was solved to 2.3 angstroms resolution. The usual modification of carboxylation of the active-site residue Lys150 did not happen in bar9HYD. Two manganese ions were modelled in the active site. Through biochemical studies, it was shown that mercury inhibits the activity of the enzyme. The mercury derivative provided some information about the binding site of the mercuric inhibitors and a possible reason for inhibition is presented.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/química , Bacillus/enzimología , Proteínas Bacterianas/química , Mercurio/metabolismo , Estructura Terciaria de Proteína , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Hidantoínas/metabolismo , Manganeso/química , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Cidea and Cidec play an important role in regulating triglyceride storage in liver and adipose tissue. It is not known if the Cidea and Cidec genes respond to a high fat diet (HFD) or exercise training, two interventions that alter lipid storage. The purpose of the present study was to determine the effect of a HFD and voluntary wheel running (WR) on Cidea and Cidec mRNA and protein expression in adipose tissue and liver of mice. A HFD promoted a significant increase in Cidea and Cidec mRNA levels in adipose tissue and liver. The increase in Cidea and Cidec mRNAs in adipose tissue and liver in response to a HFD was prevented by WR. Similar to the changes in Cidea mRNA, Cidea protein levels in adipose tissue significantly increased in response to a HFD, a process that was, again, prevented by WR. However, in adipose tissue the changes in Cidec mRNA did not correspond to the changes in Cidec protein levels, as a HFD decreased Cidec protein abundance. Interestingly, in adipose tissue Cidea protein expression was significantly related to body weight (R=.725), epididymal adipose tissue (EWAT) mass (R=.475) and insulin resistance (R=.706), whereas Cidec protein expression was inversely related to body weight (R=-.787), EWAT mass (R=-.706), and insulin resistance (R=-.679). Similar to adipose tissue, Cidea protein expression in liver was significantly related to body weight (R=.660), EWAT mass (R=.468), and insulin resistance (R=.599); however, unlike adipose tissue, Cidec protein levels in liver were not related to body weight or EWAT mass and only moderately associated with insulin resistance (R=-.422, P=0.051). Overall, our findings indicate that Cidea is highly associated with adiposity and insulin resistance, whereas Cidec is related to insulin sensitivity. The present study suggests that Cide proteins might play an important functional role in the development of obesity, hepatic steatosis, as well as the pathogenesis of type 2 diabetes.
Asunto(s)
Tejido Adiposo/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Dieta Alta en Grasa/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Condicionamiento Físico Animal , Proteínas/metabolismo , Tejido Adiposo/efectos de los fármacos , Adiposidad/efectos de los fármacos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Insulina/metabolismo , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , PPAR gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Carrera , Factores de Transcripción/genéticaRESUMEN
The Notch receptor family and its ligands (Delta-like and Jagged) have been found deregulated in several human cancers. We and the Aster/Pear group recently identified c-myc as a direct transcriptional target gene of the Notch1 pathway in T cell acute lymphoblastic leukemia (T-ALL). Although the oncogenic roles of c-Myc and Notch1 are established, a direct link between Notch1 and c-Myc had not been demonstrated. Importantly, our work in mouse tal1 tumor cell lines revealed that leukemic growth/survival remains dependent on the Notch1-c-Myc pathway. Studies by the Efstratiadis group provide genetic evidence that the Notch1-c-Myc pathway also contributes to mouse mammary tumorigenesis. Taken together, these studies demonstrate that Notch1 mediates T cell and epithelial cell transformation at least in part by sustaining c-Myc lev.
Asunto(s)
Leucemia de Células T/genética , Proteínas Proto-Oncogénicas c-myc/fisiología , Receptor Notch1/fisiología , Animales , Redes Reguladoras de Genes , Humanos , Modelos Biológicos , Oncogenes/fisiología , Proteínas Proto-Oncogénicas c-myc/genética , Receptor Notch1/genética , Transducción de SeñalRESUMEN
Gene expression requires the recruitment of chromatin remodeling activities and general transcription factors (GTFs) to promoters. Whereas the role of activators in recruiting chromatin remodeling activities has been clearly demonstrated, the contributions of the transcription machinery have not been firmly established. Here we demonstrate that the remodeling of the RNR3 promoter requires a number of GTFs, mediator and RNA polymerase II. We also show that remodeling is dependent upon the SWI/SNF complex, and that TFIID and RNA polymerase II are required for its recruitment to the promoter. In contrast, Gcn5p-dependent histone acetylation occurs independently of TFIID and RNA polymerase II function, and we provide evidence that acetylation increases the extent of nucleosome remodeling, but is not required for SWI/SNF recruitment. Thus, the general transcription machinery can contribute to nucleosome remodeling by mediating the association of SWI/SNF with promoters, thereby revealing a novel pathway for the recruitment of chromatin remodeling activities.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Proteínas Quinasas/metabolismo , Proteínas de Unión al ARN , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Ribonucleótido Reductasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/metabolismo , Transcripción Genética , Levaduras/genética , Acetilación , Cromatina/genética , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histonas/metabolismo , Complejo Mediador , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleótido Reductasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Levaduras/metabolismoRESUMEN
HSP100 protein is an important component of the heat-shock response in diverse organisms. Using specific primers based on cDNA sequence, rice hsp101 gene was PCR-amplified and sequenced. Southern analysis revealed that there appears to be a single gene per haploid genome coding for HSP101 protein in rice. Northern analysis showed that expression of hsp101 transcript is strictly heat-inducible and induction is transient in nature. In the temperature regime tested, 45 degrees C treatment to intact rice seedlings for 2 h showed maximal levels of hsp101 mRNA. Rice full-length hsp101 cDNA complemented yeast mutant disrupted for its own hsp104 gene by insertional mutagenesis, with efficacy that was comparable with Arabidopsis hsp101 cDNA. Electron micrographic evidence suggested that rice hsp101 cDNA in yeast is active in re-solubilizing the stress-induced protein granules in the post-stress recovery period. Rice hsp101 cDNA expression in hsp104 deficient yeast also caused recovery in tolerance against arsenite. Western analyses showed that this protein is expressed more rapidly during the stress period and retained for longer duration in the post-stress recovery period in japonica rice as compared to indica rice types. This is the first report wherein plant HSP100 protein expression is correlated to disappearance of protein granules in the yeast cells and distinct rice type-dependent protein expression patterns are reported.