RESUMEN
In diverse organisms, calorie restriction slows the pace of ageing and increases maximum lifespan. In the budding yeast Saccharomyces cerevisiae, calorie restriction extends lifespan by increasing the activity of Sir2 (ref. 1), a member of the conserved sirtuin family of NAD(+)-dependent protein deacetylases. Included in this family are SIR-2.1, a Caenorhabditis elegans enzyme that regulates lifespan, and SIRT1, a human deacetylase that promotes cell survival by negatively regulating the p53 tumour suppressor. Here we report the discovery of three classes of small molecules that activate sirtuins. We show that the potent activator resveratrol, a polyphenol found in red wine, lowers the Michaelis constant of SIRT1 for both the acetylated substrate and NAD(+), and increases cell survival by stimulating SIRT1-dependent deacetylation of p53. In yeast, resveratrol mimics calorie restriction by stimulating Sir2, increasing DNA stability and extending lifespan by 70%. We discuss possible evolutionary origins of this phenomenon and suggest new lines of research into the therapeutic use of sirtuin activators.
Asunto(s)
Flavonoides , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/agonistas , Sirtuinas/agonistas , Estilbenos/farmacología , Acetilación/efectos de los fármacos , Restricción Calórica , Catálisis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Cinética , Longevidad/efectos de los fármacos , Fenoles/farmacología , Polímeros/farmacología , Polifenoles , Recombinación Genética/efectos de los fármacos , Resveratrol , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 1 , Sirtuina 2 , Sirtuinas/genética , Sirtuinas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , VinoRESUMEN
When it was first proposed that the budding yeast Saccharomyces cerevisiae might serve as a model for human aging in 1959, the suggestion was met with considerable skepticism. Although yeast had proved a valuable model for understanding basic cellular processes in humans, it was difficult to accept that such a simple unicellular organism could provide information about human aging, one of the most complex of biological phenomena. While it is true that causes of aging are likely to be multifarious, there is a growing realization that all eukaryotes possess surprisingly conserved longevity pathways that govern the pace of aging. This realization has come, in part, from studies of S. cerevisiae, which has emerged as a highly informative and respected model for the study of life span regulation. Genomic instability has been identified as a major cause of aging, and over a dozen longevity genes have now been identified that suppress it. Here we present the key discoveries in the yeast-aging field, regarding both the replicative and chronological measures of life span in this organism. We discuss the implications of these findings not only for mammalian longevity but also for other key aspects of cell biology, including cell survival, the relationship between chromatin structure and genome stability, and the effect of internal and external environments on cellular defense pathways. We focus on the regulation of replicative life span, since recent findings have shed considerable light on the mechanisms controlling this process. We also present the specific methods used to study aging and longevity regulation in S. cerevisiae.
Asunto(s)
Genoma Fúngico , Heterocromatina/fisiología , Longevidad/fisiología , Saccharomyces cerevisiae/fisiología , Regulación Fúngica de la Expresión Génica , Heterocromatina/genética , Histona Desacetilasas/genética , Histona Desacetilasas/fisiología , Longevidad/genética , Modelos Moleculares , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2 , Sirtuinas/genética , Sirtuinas/fisiologíaRESUMEN
Calorie restriction extends lifespan in a broad range of organisms, from yeasts to mammals. Numerous hypotheses have been proposed to explain this phenomenon, including decreased oxidative damage and altered energy metabolism. In Saccharomyces cerevisiae, lifespan extension by calorie restriction requires the NAD+-dependent histone deacetylase, Sir2 (ref. 1). We have recently shown that Sir2 and its closest human homologue SIRT1, a p53 deacetylase, are strongly inhibited by the vitamin B3 precursor nicotinamide. Here we show that increased expression of PNC1 (pyrazinamidase/nicotinamidase 1), which encodes an enzyme that deaminates nicotinamide, is both necessary and sufficient for lifespan extension by calorie restriction and low-intensity stress. We also identify PNC1 as a longevity gene that is responsive to all stimuli that extend lifespan. We provide evidence that nicotinamide depletion is sufficient to activate Sir2 and that this is the mechanism by which PNC1 regulates longevity. We conclude that yeast lifespan extension by calorie restriction is the consequence of an active cellular response to a low-intensity stress and speculate that nicotinamide might regulate critical cellular processes in higher organisms.
Asunto(s)
Restricción Calórica , Niacinamida/metabolismo , Nicotinamidasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Metabolismo Energético , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Respuesta al Choque Térmico , Histona Desacetilasas/metabolismo , Longevidad/genética , Longevidad/fisiología , NAD/metabolismo , Niacina/metabolismo , Nicotinamidasa/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2 , Sirtuinas/metabolismoRESUMEN
The Saccharomyces cerevisiae Sir2 protein is an NAD(+)-dependent histone deacetylase that plays a critical role in transcriptional silencing, genome stability, and longevity. A human homologue of Sir2, SIRT1, regulates the activity of the p53 tumor suppressor and inhibits apoptosis. The Sir2 deacetylation reaction generates two products: O-acetyl-ADP-ribose and nicotinamide, a precursor of nicotinic acid and a form of niacin/vitamin B(3). We show here that nicotinamide strongly inhibits yeast silencing, increases rDNA recombination, and shortens replicative life span to that of a sir2 mutant. Nicotinamide abolishes silencing and leads to an eventual delocalization of Sir2 even in G(1)-arrested cells, demonstrating that silent heterochromatin requires continual Sir2 activity. We show that physiological concentrations of nicotinamide noncompetitively inhibit both Sir2 and SIRT1 in vitro. The degree of inhibition by nicotinamide (IC(50) < 50 microm) is equal to or better than the most effective known synthetic inhibitors of this class of proteins. We propose a model whereby nicotinamide inhibits deacetylation by binding to a conserved pocket adjacent to NAD(+), thereby blocking NAD(+) hydrolysis. We discuss the possibility that nicotinamide is a physiologically relevant regulator of Sir2 enzymes.
Asunto(s)
Envejecimiento/fisiología , Silenciador del Gen , Histona Desacetilasas/metabolismo , Niacinamida/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuinas/metabolismo , ADN Ribosómico/genética , Genes Fúngicos , Proteínas Fluorescentes Verdes , Histona Desacetilasas/genética , Humanos , Esperanza de Vida , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Modelos Biológicos , Estructura Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 1 , Sirtuina 2 , Sirtuinas/genética , Telómero/genéticaRESUMEN
Apoptosis is a key tumor suppression mechanism that can be initiated by activation of the proapoptotic factor Bax. The Ku70 DNA end-joining protein has recently been shown to suppress apoptosis by sequestering Bax from mitochondria. The mechanism by which Bax is regulated remains unknown. Here, we identify eight lysines in Ku70 that are targets for acetylation in vivo. Five of these, K539, K542, K544, K533, and K556, lie in the C-terminal linker domain of Ku70 adjacent to the Bax interaction domain. We show that CBP and PCAF efficiently acetylate K542 in vitro and associate with Ku70 in vivo. Mimicking acetylation of K539 or K542 or treating cells with deacetylase inhibitors abolishes the ability of Ku70 to suppress Bax-mediated apoptosis. We demonstrate that increased acetylation of Ku70 disrupts the Ku70-Bax interaction and coincides with cytoplasmic accumulation of CBP. These results shed light on the role of acetyltransferases as tumor suppressors.
Asunto(s)
Acetiltransferasas/metabolismo , Antígenos Nucleares/metabolismo , Apoptosis/fisiología , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Acetilación , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/genética , Secuencia de Aminoácidos , Animales , Antígenos Nucleares/genética , Sitios de Unión/fisiología , Proteína de Unión a CREB , Cricetinae , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Células HeLa , Histona Acetiltransferasas , Humanos , Autoantígeno Ku , Lisina/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Estructura Terciaria de Proteína/fisiología , Proteínas Proto-Oncogénicas/genética , Homología de Secuencia de Aminoácido , Transactivadores/genética , Proteínas Supresoras de Tumor/genética , Proteína X Asociada a bcl-2RESUMEN
Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span.
Asunto(s)
Núcleo Celular/metabolismo , NAD/metabolismo , Saccharomyces cerevisiae/fisiología , Medios de Cultivo , ADN Ribosómico/metabolismo , Silenciador del Gen , Genes Fúngicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
A major cause of aging is thought to result from the cumulative effects of cell loss over time. In yeast, caloric restriction (CR) delays aging by activating the Sir2 deacetylase. Here we show that expression of mammalian Sir2 (SIRT1) is induced in CR rats as well as in human cells that are treated with serum from these animals. Insulin and insulin-like growth factor 1 (IGF-1) attenuated this response. SIRT1 deacetylates the DNA repair factor Ku70, causing it to sequester the proapoptotic factor Bax away from mitochondria, thereby inhibiting stress-induced apoptotic cell death. Thus, CR could extend life-span by inducing SIRT1 expression and promoting the long-term survival of irreplaceable cells.