RESUMEN
The yeast silent information regulator (Sir)2 protein links cellular metabolism and transcriptional silencing through its nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase activity. We report that mitochondria from mammalian cells contain intrinsic NAD-dependent deacetylase activity. This activity is inhibited by the NAD hydrolysis product nicotinamide, but not by trichostatin A, consistent with a class III deacetylase. We identify this deacetylase as the nuclear-encoded human Sir2 homologue hSIRT3, and show that hSIRT3 is located within the mitochondrial matrix. Mitochondrial import of hSIRT3 is dependent on an NH2-terminal amphipathic alpha-helix rich in basic residues. hSIRT3 is proteolytically processed in the mitochondrial matrix to a 28-kD product. This processing can be reconstituted in vitro with recombinant mitochondrial matrix processing peptidase (MPP) and is inhibited by mutation of arginines 99 and 100. The unprocessed form of hSIRT3 is enzymatically inactive and becomes fully activated in vitro after cleavage by MPP. These observations demonstrate the existence of a latent class III deacetylase that becomes catalytically activated upon import into the human mitochondria.
Asunto(s)
Histona Desacetilasas/metabolismo , Metaloendopeptidasas/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , NAD/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Transactivadores/metabolismo , Transporte Biológico , Células Cultivadas , Activación Enzimática , Células HeLa , Humanos , Microscopía Confocal , Péptido Hidrolasas/metabolismo , Sirtuina 1 , Sirtuina 2 , Sirtuina 3 , Sirtuinas , Partículas Submitocóndricas/enzimología , Peptidasa de Procesamiento MitocondrialRESUMEN
Phytate (inositol hexakisphosphate, IP6) is a regulator of intracellular signaling, a highly abundant animal antinutrient, and a phosphate store in plant seeds. Here, we report a requirement for inositol polyphosphate kinases, AtIPK1 and AtIPK2beta, for the later steps of phytate synthesis in Arabidopsis thaliana. Coincident disruption of these kinases nearly ablates seed phytate without accumulation of phytate precursors, increases seed-free phosphate by 10-fold, and has normal seed yield. Additionally, we find a requirement for inositol tetrakisphosphate (IP4)/inositol pentakisphosphate (IP5) 2-kinase activity in phosphate sensing and root hair elongation. Our results define a commercially viable strategy for the genetic engineering of phytate-free grain and provide insights into the role of inositol polyphosphate kinases in phosphate signaling biology.
Asunto(s)
Arabidopsis/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ácido Fítico/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Clonación Molecular , Genes de Plantas , Ingeniería Genética , Mutación , Fenotipo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Ácido Fítico/biosíntesis , Semillas/metabolismo , Transducción de SeñalRESUMEN
Our understanding of pathways leading to antitumor immunity may depend on an undistorted knowledge of the primary antigenic targets of patients' autologous T cell responses. In the melanoma model derived from patient DT, we applied cryopreserved short-term autologous mixed lymphocyte-tumor cell cultures (MLTCs) in combination with an IFN-gamma enzyme-linked immunospot (ELISPOT) assay to cDNA expression screening. We identified three previously unknown peptides processed from melanosomal proteins tyrosinase (presented by HLA-A(*)2601 and -B(*)3801) and gp100 (presented by HLA-B(*)07021) and five neoantigens generated by somatic point mutations in the patient's melanoma. The mutations were found in the genes SIRT2, GPNMB, SNRP116, SNRPD1, and RBAF600. Peptides containing the mutated residues were presented by HLA-A(*)03011, -B(*)07021, and -B(*)3801. Mutation-induced functional impairment was so far demonstrated for SIRT2. Within MLTC responder populations that were independently expanded from the patient's peripheral blood lymphocytes of different years, T cells against mutated epitopes clearly predominated. These results document a high degree of individuality for the cellular antitumor response and support the need for individualizing the monitoring and therapeutic approaches to the primary targets of the autologous T cell response, which may finally lead to a more effective cancer immunotherapy.
Asunto(s)
Antígenos de Neoplasias/genética , Melanoma/inmunología , Mutación Puntual , Linfocitos T/inmunología , Adulto , Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Secuencia de Bases , Técnicas de Cocultivo , Epítopos de Linfocito T , Femenino , Antígenos HLA/inmunología , Humanos , Interferón gamma/análisis , Melanoma/patología , Datos de Secuencia Molecular , Linfocitos T/citología , Células Tumorales CultivadasRESUMEN
Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 (CTIP1 and CTIP2) enhance transcriptional repression mediated by COUP-TF II and have been implicated in hematopoietic cell development and malignancies. CTIP1 and CTIP2 are also sequence-specific DNA-binding proteins that repress transcription through direct, COUP-TF-in-dependent binding to a GC-rich response element. CTIP1- and CTIP2-mediated transcriptional repression is insensitive to trichostatin A, an inhibitor of known class I and II histone deacetylases. However, chromatin immunoprecipitation assays revealed that expression of CTIP2 in mammalian cells resulted in deacetylation of histones H3 and/or H4 that were associated with the promoter region of a reporter gene. CTIP2-mediated transcriptional repression, as well as deacetylation of promoter-associated histones H3/H4 in CTIP2-transfected cells, was reversed by nicotinamide, an inhibitor of class III histone deacetylases such as the mammalian homologs of yeast Silent Information Regulator 2 (Sir2). The human homolog of yeast Sir2, SIRT1, was found to interact directly with CTIP2 and was recruited to the promoter template in a CTIP2-dependent manner. Moreover, SIRT1 enhanced the deacetylation of template-associated histones H3/H4 in CTIP2-transfected cells, and stimulated CTIP2-dependent transcriptional repression. Finally, endogenous SIRT1 and CTIP2 co-purified from Jurkat cell nuclear extracts in the context of a large (1-2 mDa) complex. These findings implicate SIRT1 as a histone H3/H4 deacetylase in mammalian cells and in transcriptional repression mediated by CTIP2.
Asunto(s)
Proteínas Portadoras , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Histona Desacetilasas/fisiología , Proteínas Nucleares , Receptores de Esteroides , Proteínas Represoras/fisiología , Sirtuinas/fisiología , Factores de Transcripción/metabolismo , Factores de Transcripción COUP , Línea Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/química , Inhibidores Enzimáticos/farmacología , Silenciador del Gen , Genes Reporteros , Glutatión Transferasa/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Immunoblotting , Células Jurkat , NAD/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiología , Niacinamida/farmacología , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Sirtuina 1 , Sirtuinas/metabolismo , Transcripción Genética , Transfección , Proteínas Supresoras de TumorRESUMEN
NF-kappaB is responsible for upregulating gene products that control cell survival. In this study, we demonstrate that SIRT1, a nicotinamide adenosine dinucleotide-dependent histone deacetylase, regulates the transcriptional activity of NF-kappaB. SIRT1, the mammalian ortholog of the yeast SIR2 (Silencing Information Regulator) and a member of the Sirtuin family, has been implicated in modulating transcriptional silencing and cell survival. SIRT1 physically interacts with the RelA/p65 subunit of NF-kappaB and inhibits transcription by deacetylating RelA/p65 at lysine 310. Treatment of cells with resveratrol, a small-molecule agonist of Sirtuin activity, potentiates chromatin-associated SIRT1 protein on the cIAP-2 promoter region, an effect that correlates with a loss of NF-kappaB-regulated gene expression and sensitization of cells to TNFalpha-induced apoptosis. While SIRT1 is capable of protecting cells from p53-induced apoptosis, our work provides evidence that SIRT1 activity augments apoptosis in response to TNFalpha by the ability of the deacetylase to inhibit the transactivation potential of the RelA/p65 protein.
Asunto(s)
Supervivencia Celular/fisiología , Histona Desacetilasas/fisiología , FN-kappa B/fisiología , Sirtuinas/fisiología , Transcripción Genética/fisiología , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Humanos , Resveratrol , Sirtuina 1 , Estilbenos/farmacologíaRESUMEN
FOXO transcription factors have important roles in metabolism, cellular proliferation, stress tolerance, and aging. FOXOs are negatively regulated by protein kinase B/c-Akt-mediated phosphorylation. Here we show that FOXO factors are also subject to regulation by reversible acetylation. We provide evidence that the acetyltransferase CREB-binding protein (CBP) binds FOXO resulting in acetylation of FOXO. This acetylation inhibits FOXO transcriptional activity. Binding of CBP and acetylation are induced after treatment of cells with peroxide stress. Deacetylation of FOXOs involves binding of the NAD-dependent deacetylase hSir2(SIRT1). Accordingly, hSir2(SIRT1)-mediated deacetylation precludes FOXO inhibition through acetylation and thereby prolongs FOXO-dependent transcription of stress-regulating genes. These data demonstrate that acetylation functions in a second pathway of negative control for FOXO factors and provides a novel mechanism whereby hSir2(SIRT1) can promote cellular survival and increase lifespan.
Asunto(s)
Histona Desacetilasas/metabolismo , Peróxido de Hidrógeno/metabolismo , Oxidantes/metabolismo , Estrés Oxidativo , Sirtuinas/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Proteína de Unión a CREB , Proteínas de Ciclo Celular , Línea Celular Tumoral , Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Humanos , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Sirtuina 1 , Transactivadores/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.