RESUMO
Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in various cellular biochemical reactions. To date the signaling pathways that regulate NAD(+) metabolism remain unclear due to the dynamic nature and complexity of the NAD(+) metabolic pathways and the difficulty of determining the levels of the interconvertible pyridine nucleotides. Nicotinamide riboside (NmR) is a key pyridine metabolite that is excreted and re-assimilated by yeast and plays important roles in the maintenance of NAD(+) pool. In this study we establish a NmR-specific reporter system and use it to identify yeast mutants with altered NmR/NAD(+) metabolism. We show that the phosphate-responsive signaling (PHO) pathway contributes to control NAD(+) metabolism. Yeast strains with activated PHO pathway show increases in both the release rate and internal concentration of NmR. We further identify Pho8, a PHO-regulated vacuolar phosphatase, as a potential NmR production factor. We also demonstrate that Fun26, a homolog of human ENT (equilibrative nucleoside transporter), localizes to the vacuolar membrane and establishes the size of the vacuolar and cytosolic NmR pools. In addition, the PHO pathway responds to depletion of cellular nicotinic acid mononucleotide (NaMN) and mediates nicotinamide mononucleotide (NMN) catabolism, thereby contributing to both NmR salvage and phosphate acquisition. Therefore, NaMN is a putative molecular link connecting the PHO signaling and NAD(+) metabolic pathways. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes.
Assuntos
NAD/metabolismo , Fosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosfatase Alcalina/metabolismo , Transporte Biológico , Cromatografia Líquida/métodos , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Proteínas Fúngicas/metabolismo , Deleção de Genes , Espectrometria de Massas/métodos , Proteínas de Membrana Transportadoras/química , Microscopia de Fluorescência/métodos , Modelos Genéticos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Vacúolos/metabolismoRESUMO
The Sir2 family proteins (sirtuins) are evolutionally conserved NAD(+) (nicotinamide adenine dinucleotide)-dependent protein deacetylases and ADP-ribosylases, which have been shown to play important roles in the regulation of stress response, gene transcription, cellular metabolism and longevity. Recent studies have also suggested that sirtuins are downstream targets of calorie restriction (CR), which mediate CR-induced beneficial effects including life span extension in an NAD(+)-dependent manner. CR extends life span in many species and has been shown to ameliorate many age-associated disorders such as diabetes and cancers. Understanding the mechanisms of CR as well as the regulation of sirtuins will therefore provide insights into the molecular basis of these age-associated metabolic diseases. This review focuses on discussing advances in studies of sirtuins and NAD(+) metabolism in genetically tractable model system, the budding yeast Saccharomyces cerevisiae. These studies have unraveled key metabolic longevity factors in the CR signaling and NAD(+) biosynthesis pathways, which may also contribute to the regulation of sirtuin activity. Many components of the NAD(+) biosynthesis pathway and CR signaling pathway are conserved in yeast and higher eukaryotes including humans. Therefore, these findings will help elucidate the mechanisms underlying age-associated metabolic disease and perhaps human aging.
Assuntos
Modelos Biológicos , NAD/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Envelhecimento/metabolismo , Animais , Restrição Calórica , Expressão Gênica , Humanos , Longevidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/química , Sirtuína 2/química , Sirtuína 2/metabolismo , Estresse Fisiológico , Telômero/genética , Telômero/metabolismoRESUMO
NAD(+) (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD(+) metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD(+) metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD(+)-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD(+) metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD(+) precursors. Together, our studies provide a molecular basis for how NAD(+) homeostasis factors confer metabolic flexibility.
Assuntos
Niacinamida/análogos & derivados , Saccharomyces cerevisiae/metabolismo , Animais , Restrição Calórica , Genes Fúngicos , Histona Desacetilases/metabolismo , Temperatura Alta/efeitos adversos , Longevidade/fisiologia , Mamíferos , Modelos Biológicos , Mutação , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , NAD/metabolismo , Niacinamida/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Compostos de Piridínio , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2 , Sirtuínas/metabolismo , Estresse Fisiológico , Fatores de TempoRESUMO
Lipid rafts are involved in many cell biology events, yet the molecular mechanisms on how rafts are formed are poorly understood. In this study we probed the possible requirement of reactive oxygen species (ROS) for T-cell receptor (TCR)-induced lipid raft formation. Microscopy and biochemical analyses illustrated that blockage of ROS production, by superoxide dismutase-mimic MnTBAP, significantly reduced partitioning of LAT, phospho-LAT, and PLC-gamma in lipid rafts. Another antioxidant N-acetylcysteine (NAC) displayed a similar suppressive effect on the entry of phospho-LAT into raft microdomains. The involvement of ROS in TCR-mediated raft assembly was observed in T-cell hybridomas, T leukemia cells, and normal T cells. Removal of ROS was accompanied by an attenuated activation of LAT and PKCtheta, with reduced production of IL-2. Consistently, treating T cells with the ROS-producer tert-butyl hydrogen peroxide (TBHP) greatly enhanced membrane raft formation, distribution of phospho-LAT into lipid rafts, and increased IL-2 production. Our results indicate for the first time that ROS contribute to TCR-induced membrane raft formation.
Assuntos
Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/metabolismo , Humanos , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
DNA-damaging reagents may kill tumor cells through the generation of reactive oxygen species (ROS). Cytotoxic reagents may also induce apoptosis of cancer cells in Fas-FADD-dependent manners. In this study, we explored the possible link between these two apparently distinct pathways in T leukemia cell Jurkat. Our results demonstrated that gamma-irradiation, similar to cisplatin, induced apoptosis by triggering Fas aggregation and activating FADD-caspase-8 apoptotic cascade. The absence of caspase-8 or Fas greatly reduced the sensitivity to apoptosis mediated by DNA-damaging agents. In addition, apoptosis induced by cisplatin and gamma-irradiation, but not by Fas, was inhibited by ROS scavengers, including N-acetyl cysteine, MnTBAP, and C60. Importantly, these ROS scavengers effectively prevented the clustering of Fas receptor induced by cisplatin and gamma-irradiation. Our results suggest that cisplatin and gamma-irradiation promote ROS production, which in turn contributes to Fas receptor aggregation and cell death. The novel coupling between ROS and Fas clustering likely plays a significant role in apoptosis triggered by DNA-damaging reagents in Fas-expressing leukemia cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose/efeitos da radiação , Dano ao DNA/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Receptor fas/efeitos da radiação , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Caspase 8 , Caspase 9 , Caspases/metabolismo , Cisplatino/farmacologia , Proteína Ligante Fas , Proteína de Domínio de Morte Associada a Fas , Raios gama , Humanos , Células Jurkat , Glicoproteínas de Membrana/metabolismoRESUMO
Calorie restriction (CR) induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO) production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione) is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX). Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan.
RESUMO
Calorie restriction (CR) extends life span in a wide variety of species. Recent studies suggest that an increase in mitochondrial metabolism mediates CR-induced life span extension. Here we present evidence that Lat1 (dihydrolipoamide acetyltransferase), the E2 component of the mitochondrial pyruvate dehydrogenase complex, is a novel metabolic longevity factor in the CR pathway. Deleting the LAT1 gene abolishes life span extension induced by CR. Overexpressing Lat1 extends life span, and this life span extension is not further increased by CR. Similar to CR, life span extension by Lat1 overexpression largely requires mitochondrial respiration, indicating that mitochondrial metabolism plays an important role in CR. Interestingly, Lat1 overexpression does not require the Sir2 family to extend life span, suggesting that Lat1 mediates a branch of the CR pathway that functions in parallel to the Sir2 family. Lat1 is also a limiting longevity factor in nondividing cells in that overexpressing Lat1 extends cell survival during prolonged culture at stationary phase. Our studies suggest that Lat1 overexpression extends life span by increasing metabolic fitness of the cell. CR may therefore also extend life span and ameliorate age-associated diseases by increasing metabolic fitness through regulating central metabolic enzymes.