RESUMEN
Plant homeodomain finger 2 (PHF2) has a role in epigenetic regulation of gene expression by demethylating H3K9-Me2. Several genome-wide studies have demonstrated that the chromosomal region including the PHF2 gene is often deleted in some cancers including colorectal cancer, and this finding encouraged us to investigate the tumor suppressive role of PHF2. As p53 is a critical tumor suppressor in colon cancer, we tested the possibility that PHF2 is an epigenetic regulator of p53. PHF2 was associated with p53, and thereby, promoted p53-driven gene expression in cancer cells under genotoxic stress. PHF2 converted the chromatin that is favorable for transcription by demethylating the repressive H3K9-Me2 mark. In an HCT116 xenograft model, PHF2 was found to be required for the anticancer effects of oxaliplatin and doxorubicin. In PHF2-deficient xenografts, p53 expression was profoundly induced by both drugs, but its downstream product p21 was not, suggesting that p53 cannot be activated in the absence of PHF2. To find clinical evidence about the role of PHF2, we analyzed the expressions of PHF2, p53 and p21 in human colon cancer tissues and adjacent normal tissues from patients. PHF2 was downregulated in cancer tissues and PHF2 correlated with p21 in cancers expressing functional p53. Colon and stomach cancer tissue arrays showed a positive correlation between PHF2 and p21 expressions. Informatics analyses using the Oncomine database also supported our notion that PHF2 is downregulated in colon and stomach cancers. On the basis of these findings, we propose that PHF2 acts as a tumor suppressor in association with p53 in cancer development and ensures p53-mediated cell death in response to chemotherapy.
Asunto(s)
Genes Supresores de Tumor , Proteínas de Homeodominio/fisiología , Neoplasias/genética , Proteína p53 Supresora de Tumor/fisiología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Muerte Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HEK293 , Células Hep G2 , Proteínas de Homeodominio/antagonistas & inhibidores , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , ARN Interferente Pequeño/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Checkpoint kinase 2 (CHK2) kinase is a key mediator in many cellular responses to genotoxic stresses, including ionizing radiation (IR) and topoisomerase inhibitors. Upon IR, CHK2 is activated by ataxia telangiectasia mutated kinase and regulates the S-phase and G1-S checkpoints, apoptosis and DNA repair by phosphorylating downstream target proteins, such as p53 and Brca1. In addition, CHK2 is thought to be a multi-organ cancer susceptibility gene. In this study, we used a tandem affinity purification strategy to identify proteins that interact with CHK2 kinase. Cyclin-dependent kinase 11 (CDK11)(p110) kinase, implicated in pre-mRNA splicing and transcription, was identified as a CHK2-interacting protein. CHK2 kinase phosphorylated CDK11(p110) on serine 737 in vitro. Unexpectedly, CHK2 kinase constitutively phosphorylated CDK11(p110) in a DNA damage-independent manner. At a molecular level, CDK11(p110) phosphorylation was required for homodimerization without affecting its kinase activity. Overexpression of CHK2 promoted pre-mRNA splicing. Conversely, CHK2 depletion decreased endogenous splicing activity. Mutation of the phosphorylation site in CDK11(p110) to alanine abrogated its splicing-activating activity. These results provide the first evidence that CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110).
Asunto(s)
Quinasa de Punto de Control 2/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Precursores del ARN/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , Quinasa de Punto de Control 2/química , Quinasas Ciclina-Dependientes/química , Daño del ADN , Células HEK293 , Células HT29 , Humanos , Fosforilación , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismoRESUMEN
Menin, encoded by the multiple endocrine neoplasia type 1 (MEN1) gene, is a tumor suppressor that leads to multiple endocrine tumors upon loss of its function. Menin functions as a transcriptional activator by tethering MLL complex to mediate histone H3 K4 methylation. It also functions as a repressor. However, the molecular mechanism of how menin contributes to the opposite outcome in gene expression is largely unknown. Here, we investigated the role of menin in the epigenetic regulation of transcription mediated by histone covalent modification. We show that the global methylation level of histone H3 K9, as well as H3 K4, was decreased in Men1(-/-) MEF cells. Consistently, menin was able to interact with the suppressor of variegation 3-9 homolog family protein, SUV39H1, to mediate H3 K9 methylation. This interaction decreased when patient-derived MEN1 mutation was introduced into the SUV39H1-interaction domain. We show that menin mediated different chromatin changes depending on target genes. Chromatin immunoprecipitation studies showed that menin directly associated with the GBX2 promoter and menin-dependent recruitment of SUV39H1 was essential for chromatin remodeling and transcriptional regulation. These results provide a molecular basis of how menin functions as a transcriptional repressor and suggest that menin-dependent integration of H3 K9 methylation might play an important role in preventing tumors.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Ensamble y Desensamble de Cromatina , Epigénesis Genética , Histonas/metabolismo , Lisina/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Transformación Celular Neoplásica/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Células HEK293 , Histonas/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Lisina/genética , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Mutación , Transporte de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
Germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene cause VHL disease, a rare and autosomal-dominant genetic syndrome. Because VHL protein (pVHL) is the master regulator of hypoxia-inducible factor alpha (HIFα), the most prominent feature of VHL disease is the deregulation of HIFα proteins. However, the precise mechanism by which the loss of pVHL function contributes to tumorigenesis is not fully understood. Here, we show that pVHL destabilizes the F-box protein Skp2, a chief component of Skp, Cullin, F-box-containing complex that promotes DNA synthesis in the S phase. The ß-domain of pVHL interacts with Skp2, stimulating proteasome-dependent Skp2 degradation, but the destabilization of Skp2 does not depend on the E3 ubiquitin ligase activity of pVHL. Notably, the generation of DNA damage induces Skp2 degradation, which is attenuated by the suppression of endogenous pVHL expression. One possible mechanism of pVHL-dependent Skp2 degradation entails the antagonizing of Akt-mediated Skp2 phosphorylation, which maintains Skp2 stability. Reintroduction of VHL into VHL-null renal cell carcinoma (RCC) cells decreased Skp2 levels and restored DNA damage-dependent Skp2 degradation. These results identify the tumor suppressor function of pVHL in delaying the S-phase progression to inhibit cell proliferation on DNA damage. Clinically, this report explains as to why Skp2 accumulates abnormally in RCC tissues.
Asunto(s)
Daño del ADN , Proteínas Quinasas Asociadas a Fase-S/química , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Apoptosis , Carcinoma de Células Renales/patología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HEK293 , Humanos , Neoplasias Renales/patología , Cinética , Fosforilación , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fase S , UbiquitinaciónRESUMEN
Many oncolytic viruses are currently being tested as potential cancer therapeutic agents. To be effective, these viruses must replicate and propagate efficiently through the tumor mass. However, it is possible that the hypoxia that characterizes many tumors may be an obstacle to viral therapy because of its inhibition of viral replication and propagation. We, therefore, decided to test how oncolytic reovirus and its target cells respond to hypoxia. We found that reovirus infection suppresses hypoxia inducible factor (HIF)-1alpha protein levels (but not transcript abundance) in colon cancer HCT116 cells under CoCl(2) or hypoxia. Reovirus infection was able to reduce HIF-1alpha levels in both von Hippel Lindau (VHL)-/- renal carcinoma A498 and p53-/- HCT116 cells, indicating that the decrease of HIF-1alpha mediated by reovirus requires neither VHL nor p53 proteins. However, treatment with the inhibitor MG132 restored HIF-1alpha levels, suggesting that reovirus-induced HIF-1alpha decrease needs proteosomal activity. A498 VHL-/- cells with constitutive expression of HIF-1alpha were relatively resistant to reovirus-induced apoptosis when compared with A498 VHL+/+ cells. However, we found that the use of YC-1 to target HIF-1alpha promoted reovirus-induced apoptosis in A498 VHL-/- cells. Accordingly, we propose that reovirus may be used together with YC-1 as a potential therapeutic agent against chemoresistant or radioresistant tumors that are hypoxic and show increased levels of HIF-1alpha.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Virus Oncolíticos/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Regulación hacia Abajo , Células HCT116 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Virus Oncolíticos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Metabolic enzymes have been shown to function as transcriptional regulators. p53, a tumor-suppressive transcription factor, was recently found to regulate energy metabolism. These combined facts raise the possibility that metabolic enzymes may directly regulate p53 function. Here, we discover that nucleocytoplasmic malate dehydrogenase-1 (MDH1) physically associates with p53. Upon glucose deprivation, MDH1 stabilizes and transactivates p53 by binding to p53-responsive elements in the promoter of downstream genes. Knockdown of MDH1 significantly reduces binding of acetylated-p53 and transcription-active histone codes to the promoter upon glucose depletion. MDH1 regulates p53-dependent cell-cycle arrest and apoptosis in response to glucose deprivation, suggesting that MDH1 functions as a transcriptional regulator for a p53-dependent metabolic checkpoint. Our findings provide insight into how metabolism is directly linked to gene expression for controlling cellular events in response to metabolic stress.
Asunto(s)
Malato Deshidrogenasa/metabolismo , Estrés Fisiológico , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis , Ciclo Celular , Línea Celular , Metabolismo Energético , Técnicas de Silenciamiento del Gen , Glucosa/deficiencia , Glucosa/metabolismo , Humanos , Interferencia de ARN , Transcripción Genética , UbiquitinaciónRESUMEN
Mitochondria are essential organelles that are responsible for cellular energy production and cell death in response to various stimuli. Although C-terminal binding protein (CtBP) functions as a metabolic sensor in transcriptional corepressor complex, it is unclear whether CtBP controls gene transcription in response to metabolic stress. In this study, we found that CtBP represses Bcl-2-associated X protein (Bax) transcription in glucose-rich media by binding to the E-box region of the Bax promoter. Glucose withdrawal leads to the dissociation of CtBP from the Bax promoter and significant changes of the histone codes in the Bax promoter. CtBP knockout increases Bax transcription, ablates mitochondrial morphology and reduces mitochondrial activities. Ectopic expression of CtBP or knockdown of Bax in ctbp-knockout cells recovers mitochondrial morphology and function, suggesting that CtBP functions as a metabolic sensor that maintains mitochondrial activities. Our findings provide insights into how the intracellular energy level is reflected into gene transcription involved in mitochondrial morphology and function.
Asunto(s)
Oxidorreductasas de Alcohol/fisiología , Proteínas de Unión al ADN/fisiología , Mitocondrias/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glucosa/deficiencia , Glucosa/farmacología , Células HeLa , Humanos , Ratones , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Proteína X Asociada a bcl-2/genéticaRESUMEN
Cabin1 binds calcineurin and myocyte enhancer factor 2 (MEF2) through its COOH-terminal region. In cell lines, these interactions were shown to inhibit calcineurin activity after T cell receptor (TCR) signaling and transcriptional activation of Nur77 by MEF2. The role of these interactions under physiological conditions was investigated using a mutant mouse strain that expresses a truncated Cabin1 lacking the COOH-terminal calcineurin and MEF2 binding domains. T and B cell development and thymocyte apoptosis were normal in mutant mice. In response to anti-CD3 stimulation, however, mutant T cells expressed significantly higher levels of interleukin (IL)-2, IL-4, IL-9, IL-13, and interferon gamma than wild-type T cells. The enhanced cytokine gene expression was not associated with change in nuclear factor of activated T cells (NF-AT)c or NF-ATp nuclear translocation but was preceded by the induction of a phosphorylated form of MEF2D in mutant T cells. Consistent with the enhanced cytokine expression, mutant mice had elevated levels of serum immunoglobulin (Ig)G1, IgG2b, and IgE and produced more IgG1 in response to a T cell-dependent antigen. These findings suggest that the calcineurin and MEF2 binding domain of Cabin1 is dispensable for thymocyte development and apoptosis, but is required for proper regulation of T cell cytokine expression probably through modulation of MEF2 activity.
Asunto(s)
Calcineurina/fisiología , Citocinas/genética , Proteínas de Unión al ADN/fisiología , Proteínas Nucleares , Fosfoproteínas/fisiología , Linfocitos T/fisiología , Factores de Transcripción/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Apoptosis , Linfocitos B/fisiología , Sitios de Unión , Calcineurina/química , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/clasificación , Péptidos y Proteínas de Señalización Intracelular , Factores de Transcripción MEF2 , Ratones , Factores Reguladores Miogénicos , Factores de Transcripción NFATC , Fosfoproteínas/químicaRESUMEN
Sanglifehrin A belongs to a novel family of immunophilin-binding ligands. Sanglifehrin A is similar to cyclosporin A in that it binds to cyclophilins. Unlike cyclosporin A, however, the cyclophilin-sanglifehrin A complex has no effect on the calcium-dependent protein phosphatase calcineurin. It has been previously shown that sanglifehrin A specifically blocks T cell proliferation in response to interleukin 2 by inhibiting the appearance of cell cycle kinase activity cyclinE-Cdk2. How sanglifehrin A treatment leads to the cell cycle blockade has remained unknown. We report that sanglifehrin A is capable of activating the tumor suppressor gene p53 at the transcription level, leading to up-regulation of p21 that then binds and inhibits the cylcinE-Cdk2 complex. Further analysis of different elements in the p53 promoter showed that sanglifehrin A activates p53 transcription primarily through the activation of the transcription factor NFkappaB by activating IkappaB kinase in a manner that is similar to several genotoxic agents. Unlike other genotoxic drugs, sanglifehrin A does not cause DNA damage, making it a unique natural product that is capable of activating the NFkappaB signaling pathway without affecting DNA.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Ciclofilinas/metabolismo , Inmunosupresores/farmacología , Lactonas/farmacología , Compuestos de Espiro/farmacología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Daño del ADN , Regulación de la Expresión Génica/efectos de los fármacos , Inmunosupresores/metabolismo , Lactonas/metabolismo , Ligandos , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Compuestos de Espiro/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Thapsigargin (TG), which inhibits endoplasmic reticulum-dependent Ca(2 +)-ATPase and thereby increases cytosolic Ca(2 +), has been reported to cause apoptosis in T lymphocytes another cell types. In this study, we investigated the molecular mechanisms that are involved in the apoptosis induced by TG in T cell hybridomas. Exposure to TG results in rapid induction of the orphan steroid receptor, Nur77, accompanied by apoptosis of T cell hybridomas. The expression of Nur77 in response to TG treatment is sensitive to cyclosporin A, implicating that activation of calcineurin is necessary for Nur77 expression. The TG-induced Nur77 expression is also inhibited by overexpression of Cabin1, an endogenous inhibitor of calcineurin and a corepressor of the transcription factor MEF2, suggesting that MEF2 activation is required for Nur77 expression. These results suggest that induction of Nur77 expression and apoptosis by TG are mediated by the same signaling pathways that are involved in T cell receptor-mediated thymocyte apoptosis, including the calcineurin pathway and Cabin1-MEF2 pathway.
Asunto(s)
Apoptosis , Calcineurina/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Fosfoproteínas/metabolismo , Transducción de Señal , Tapsigargina/farmacología , Factores de Transcripción/biosíntesis , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Inhibidores Enzimáticos/metabolismo , Humanos , Células Jurkat , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Tapsigargina/metabolismo , Células Tumorales CultivadasRESUMEN
Nuclear factor of activated T cells (NFAT) plays a key role in T cell activation. The activation of NFAT involves calcium- and calcineurin-dependent dephosphorylation and nuclear translocation from the cytoplasm, a process that is opposed by protein kinases. We show here that the peptidyl prolyl cis-trans isomerase Pin1 interacts specifically with the phosphorylated form of NFAT. The NFAT-Pin1 interaction is mediated through the WW domain of Pin1 and the serine-proline-rich domains of NFAT. Furthermore, binding of Pin1 to NFAT inhibits the calcineurin-mediated dephosphorylation of NFAT in vitro, and overexpression of Pin1 in T cells inhibits calcium-dependent activation of NFAT in vivo. These results suggest a possible role for Pin1 in the regulation of NFAT in T cells.
Asunto(s)
Calcio/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares , Isomerasa de Peptidilprolil/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , cis-trans-Isomerasas/química , Secuencias de Aminoácidos , Western Blotting , Calcineurina/metabolismo , Doxiciclina/farmacología , Glutatión Transferasa/metabolismo , Humanos , Ionomicina/farmacología , Células Jurkat , Activación de Linfocitos , Factores de Transcripción NFATC , Peptidilprolil Isomerasa de Interacción con NIMA , Fosforilación , Plásmidos/metabolismo , Prolina/química , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Serina/química , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética , TransfecciónRESUMEN
T-cell antigen receptor (TCR)-induced thymocyte apoptosis is mediated by calcium-dependent signal transduction pathways leading to the transcriptional activation of members of the Nur77 family. The major calcium- and calcineurin-responsive elements in the Nur77 promoter are binding sites for myocyte enhancer factor-2 (MEF2). It has been shown that nuclear factor of activated T cells (NFAT) interacts with MEF2D and enhances its transcriptional activity, offering a plausible mechanism of activation of MEF2D by calcineurin. We report here that NFATp synergizes with MEF2D to recruit the coactivator p300 for the transcription of Nur77. Surprisingly, the enhancement of transcriptional activity of MEF2D by NFATp does not require its DNA-binding activity, suggesting that NFATp acts as a coactivator for MEF2D. Transient co-expression of p300, MEF2D, NFATp and constitutively active calcineurin is sufficient to recapitulate TCR signaling for the selective induction of the endogenous Nur77 gene. These results implicate NFAT as an important mediator of T-cell apoptosis and suggest that NFAT is capable of integrating the calcineurin signaling pathway and other pathways through direct protein-protein interaction with other transcription factors.
Asunto(s)
Apoptosis , Calcineurina/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Linfocitos T/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Calcineurina/química , Calcio/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Ciclosporina/farmacología , ADN/metabolismo , Proteínas de Unión al ADN/química , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Humanos , Inmunosupresores/farmacología , Interleucina-2/metabolismo , Ionomicina/farmacología , Ionóforos/farmacología , Células Jurkat , Proteínas de Dominio MADS , Factores de Transcripción MEF2 , Modelos Biológicos , Datos de Secuencia Molecular , Factores Reguladores Miogénicos , Factores de Transcripción NFATC , Proteínas Nucleares/química , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Transducción de Señal , Sirolimus/farmacología , Linfocitos T/patología , Tacrolimus/farmacología , Transactivadores/química , Factores de Transcripción/química , Transcripción Genética , Transfección , Células Tumorales Cultivadas , beta-Galactosidasa/metabolismoRESUMEN
TCR signaling leading to thymocyte apoptosis is mediated through the expression of the Nur77 family of orphan nuclear receptors. MEF2 has been shown to be the major transcription factor responsible for calcium-dependent Nur77 transcription. Cabin1 was recently identified as a transcriptional repressor of MEF2, which can be released from MEF2 in a calcium-dependent fashion. The molecular basis of repression of MEF2 by Cabin1, however, has remained unknown. We report that Cabin1 represses MEF2 by two distinct mechanisms. Cabin1 recruits mSin3 and its associated histone deacetylases 1 and 2; Cabin1 also competes with p300 for binding to MEF2. Thus, activation of MEF2 and the consequent transcription of Nur77 are controlled by the association of MEF2 with the histone deacetylases via the calcium-dependent repressor Cabin1.
Asunto(s)
Acetiltransferasas/metabolismo , Apoptosis , Calcineurina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Histona Desacetilasas/metabolismo , Fosfoproteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Linfocitos T/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Sitios de Unión , Unión Competitiva , Calcineurina/genética , Señalización del Calcio , ADN/metabolismo , Histona Acetiltransferasas , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Histona Desacetilasas/genética , Humanos , Células Jurkat , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos , Proteínas Nucleares/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Receptores de Esteroides , Proteínas Represoras/genética , Complejo Correpresor Histona Desacetilasa y Sin3 , Linfocitos T/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
The myocyte enhancer factor 2 (MEF2) consists of a family of transcription factors that play important roles in a number of physiological processes from muscle cell differentiation to neuronal survival and T cell apoptosis. MEF2 has been reported to be associated with several distinct repressors including Cabin1(cain), MEF2-interacting transcriptional repressor (MITR), and HDAC4. It has been previously shown that Cabin1 is associated with MEF2 in a calcium-sensitive manner; activated calmodulin binds to Cabin1 and releases it from MEF2. However, it was not known whether the binding of HDAC4 and MITR to MEF2 is also regulated by calcium. We report that HDAC4 and MITR contain calmodulin-binding domains that overlap with their MEF2-binding domains. Binding of calmodulin to HDAC4 leads to its dissociation from MEF2, relieving MEF2 from the transcriptional repression by HDAC4. Together, HDAC4, MITR, and Cabin1 constitute a family of calcium-sensitive transcriptional repressors of MEF2.
Asunto(s)
Calcio/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Desacetilasas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Calmodulina/metabolismo , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Histona Desacetilasas/genética , Humanos , Células Jurkat , Factores de Transcripción MEF2 , Datos de Secuencia Molecular , Factores Reguladores Miogénicos , Transducción de Señal , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
T cell receptor (TCR)-induced apoptosis of thymocytes is mediated by calcium-dependent expression of the steroid receptors Nur77 and Nor1. Nur77 expression is controlled by the transcription factor myocyte enhancer factor 2 (MEF2), but how MEF2 is activated by calcium signaling is still obscure. Cabin1, a calcineurin inhibitor, was found to regulate MEF2. MEF2 was normally sequestered by Cabin1 in a transcriptionally inactive state. TCR engagement led to an increase in intracellular calcium concentration and the dissociation of MEF2 from Cabin1, as a result of competitive binding of activated calmodulin to Cabin1. The interplay between Cabin1, MEF2, and calmodulin defines a distinct signaling pathway from the TCR to the Nur77 promoter during T cell apoptosis.
Asunto(s)
Apoptosis , Señalización del Calcio , Proteínas de Unión al ADN/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Calcineurina/química , Calcineurina/genética , Calcineurina/metabolismo , Calcineurina/farmacología , Calcio/metabolismo , Calmodulina/metabolismo , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Expresión Génica , Genes Reporteros , Humanos , Células Jurkat , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/farmacología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Lipoamide dehydrogenase was purified around 22-fold relative to the crude extracts of Streptomyces seoulensis with an overall yield of 9. 5%. The enzyme was composed of two identical subunits with a molecular mass of 54 kDa and contained 1 mol of FAD per mol of subunit. The absorption spectra of the enzyme revealed the absorption maxima of flavoprotein at 272, 349, and 457 nm. Catalytically active two-electron reduced lipoamide dehydrogenase was produced by anaerobic reduction with one equivalent of NADH. Addition of excess amount of NADH led to the four-electron reduced lipoamide dehydrogenase. The reaction of the enzyme in the reduction reaction of lipoamide or lipoic acid could be explained by a ping-pong mechanism like many other lipoamide dehydrogenases reported earlier. The enzyme also catalysed the reduction of various quinone compounds with NADH as electron donor via a ping-pong mechanism. The enzyme can catalyse a single electron transfer in case of quinone-reducing process, evidenced by the production of 1, 4-naphthosemiquinone radical anion. The quinone-reducing activity of the enzyme was dramatically inhibited by NAD+, indicating the involvement of four-electron reduced form. The structural gene for the enzyme was cloned using a DNA fragment PCR-amplified with the primers designed from N-terminal and internal amino acid sequences. The deduced amino acid sequence shared striking similarity with those of lipoamide dehydrogenases from prokaryotes and eukaryotes. The gene was named lpd. All tested Streptomyces contained one homologue of the lpd gene, which is consistent with the fact that most organisms contain only one lipoamide dehydrogenase.
Asunto(s)
Dihidrolipoamida Deshidrogenasa/química , Streptomyces/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Clonación Molecular , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Datos de Secuencia Molecular , NAD/farmacología , Naftoquinonas/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Espectrofotometría , Especificidad por SustratoRESUMEN
Calcineurin plays a pivotal role in the T cell receptor (TCR)-mediated signal transduction pathway and serves as a common target for the immunosuppressants FK506 and cyclosporin A. We report the identification of a novel endogenous calcineurin binding protein named Cabin 1 that inhibits calcineurin-mediated signal transduction. The interaction between Cabin 1 and calcineurin is dependent on PKC activation. Overexpression of Cabin 1 or its N-terminal truncation mutants inhibits the transcriptional activation of calcineurin-responsive elements in the interleukin-2 promoter and blocks dephosphorylation of NF-AT upon T cell activation. These results suggest a negative regulatory role for Cabin 1 in calcineurin signaling and provide a possible mechanism of feedback inhibition of TCR signaling through cross-talk between protein kinases and calcineurin.
Asunto(s)
Calcineurina/inmunología , Activación de Linfocitos , Fosfoproteínas/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Calcio/inmunología , Regulación hacia Abajo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Proteína Quinasa C/inmunología , Saccharomyces cerevisiaeRESUMEN
1,2-Diselenolane-3-pentanoic acid, in which the sulfur atoms of alpha-lipoic acid are replaced with selenium, displayed markedly different antioxidant properties when compared to alpha-lipoic acid. 1,2-Diselenolane-3-pentanoic acid was unable to inhibit protein oxidative modification of human low density lipoprotein (LDL) and bovine serum albumin induced by copper ion or hydroxyl radical, whereas alpha-lipoic acid showed significant protection. However, 1,2-diselenolane-3-pentanoic acid was able to inhibit the formation of lipid peroxidation products in LDL after oxidation by copper, while alpha-lipoic acid did not. Hence the diselenium compound exerts its effects in a lipophilic environment whilst lipoic acid exerts its effects in a hydrophilic environment. These differences in antioxidant activities of the two compounds may be explained, at least in part, by their differing partition coefficients.
Asunto(s)
Antioxidantes/farmacología , Cobre/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Lipoproteínas LDL/metabolismo , Compuestos de Organoselenio/farmacología , Ácidos Pentanoicos/farmacología , Antioxidantes/química , Apolipoproteínas B/metabolismo , Depuradores de Radicales Libres/farmacología , Humanos , Estructura Molecular , Naftalenos/farmacología , Compuestos de Organoselenio/química , Oxidación-Reducción , Ácidos Pentanoicos/química , Salicilatos/metabolismo , Ácido Salicílico , Albúmina Sérica/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisRESUMEN
The taxonomic position of an actinomycete strain isolated from Korean soil was examined by a polyphasic approach. The isolate, designated IMSNU-1, was clearly assigned to the genus Streptomyces on the basis of morphological and chemotaxonomic data. The test strain was the subject of a probabilistic identification study using the identification matrices generated by Langham et al. (J. Gen. Microbiol. 135:121-133, 1989) and found to be marginally close to clusters 19 and 39. An almost complete 16S rRNA gene (rDNA) sequence was obtained for the test strain and compared with those of representative streptomycetes. 16S rDNA sequence data not only support the strain's membership in the genus Streptomyces but also provide strong evidence that our isolate is genealogically distant from representatives of clusters 19 and 39, forming a separate phyletic line in a clade encompassed by streptomycetes. It is therefore proposed from the polyphasic evidence that strain IMSNU-1 be classified in the genus Streptomyces as Streptomyces seoulensis sp. nov.
Asunto(s)
Streptomyces/clasificación , Computadores , ADN Bacteriano/genética , ADN Ribosómico/genética , Genes Bacterianos , Corea (Geográfico) , Datos de Secuencia Molecular , Fenotipo , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Microbiología del Suelo , Especificidad de la Especie , Streptomyces/genética , Streptomyces/aislamiento & purificaciónRESUMEN
Two unique isozymes of superoxide dismutase (EC 1.15.1.1) were purified to apparent homogeneity from Streptomyces griseus by a purification procedure consisting of ammonium sulfate precipitation and chromatographies on DEAE Sephacel, Sephacryl S-200, and DEAE 5PW. Superoxide dismutase I was composed of four identical subunits of 13.0 kDa. The absorption spectrum of superoxide dismutase I exhibited absorption bands at 276 and 378 nm and a broad shoulder at 530 nm. The g values of electron paramagnetic resonance spectrum of superoxide dismutase I were g1 = 2.304, g2 = 2.248, and g3 = 2.012 and the resonance centered at g3 = 2.012 was split into triplet, indicating nickel-containing superoxide dismutase. Superoxide dismutase I contained 0.89 g-atom of nickel per mole of 13.0-kDa subunit. Superoxide dismutase II was composed of four identical subunits of 22.0 kDa. The absorption spectrum of superoxide dismutase II showed the featureless absorption band in the range of 300-500 nm. The g values of electron paramagnetic resonance spectrum of superoxide dismutase II were gz = 4.762, gx = 4.072, and gy = 3.742, indicating iron-containing superoxide dismutase. Superoxide dismutase II uniquely contains 0.40 g-atom of iron per mole of monomer as well as 0.43 g-atom of zinc per mole of monomer. The immunological cross-reactivity between two isozymes was not found. Nickel-containing superoxide dismutase was widely distributed within the genus Streptomyces; however, iron- and zinc-containing superoxide dismutase was not found in S. albus and S. longisporoflavus, on the basis of the immunological cross-reactivity.