Cloning and characterization of two mouse genes with homology to the yeast Sir2 gene

The yeast Sir2 gene encodes a protein (Sir2p) that plays an essential role in silencing regulation at mating-type loci, rDNA, and telomeres. Recent studies have also shown that the protein participates in cell cycle regulation, DNA double-strand break repair, meiotic checkpoint control, and histone deacetylation. Overexpression of wildtype Sir2p in yeast resulted in an extended life span but mutant Sir2p shortened the life span, suggesting its function in aging processes. Sir2p is evolutionarily conserved from prokaryotes to higher eukaryotes. However, its function(s) in mammals remains unknown. To investigate Sir2p function(s) in mice, we cloned and characterized two mouse Sir2-like genes. Our results revealed that the two mouse Sir2-like proteins (mSIR2L2 and mSIR2L3) are most similar to the human Sir2-like proteins SIR2L2 and SIR2L3, respectively. Sir2 core domains are highly conserved in the two proteins and yeast Sir2p; however, the intracellular localizations of both mSIR2L2 and mSIR2L3 differ from that of yeast Sir2p and from one another. The two mouse genes have completely different genomic structures but were mapped on the same chromosome. It seems that the two mouse proteins, though they have Sir2 conserved domains, may function differently than yeast Sir2p.     

Sir2 blocks extreme life-span extension

Sir2 is a conserved deacetylase that modulates life span in yeast, worms, and flies and stress response in mammals. In yeast, Sir2 is required for maintaining replicative life span, and increasing Sir2 dosage can delay replicative aging. We address the role of Sir2 in regulating chronological life span in yeast. Lack of Sir2 along with calorie restriction and/or mutations in the yeast AKT homolog, Sch9, or Ras pathways causes a dramatic chronological life-span extension. Inactivation of Sir2 causes uptake and catabolism of ethanol and upregulation of many stress-resistance and sporulation genes. These changes while sufficient to extend chronological life span in wild-type yeast require severe calorie restriction or additional mutations to extend life span of sir2Delta mutants. Our results demonstrate that effects of SIR2 on chronological life span are opposite to replicatve life span and suggest that the relevant activities of Sir2-like deacetylases may also be complex in higher eukaryotes.     

Sir2-independent life span extension by calorie restriction in yeast

Calorie restriction slows aging and increases life span in many organisms. In yeast, a mechanistic explanation has been proposed whereby calorie restriction slows aging by activating Sir2. Here we report the identification of a Sir2-independent pathway responsible for a majority of the longevity benefit associated with calorie restriction. Deletion of FOB1 and overexpression of SIR2 have been previously found to increase life span by reducing the levels of toxic rDNA circles in aged mother cells. We find that combining calorie restriction with either of these genetic interventions dramatically enhances longevity, resulting in the longest-lived yeast strain reported thus far. Further, calorie restriction results in a greater life span extension in cells lacking both Sir2 and Fob1 than in cells where Sir2 is present. These findings indicate that Sir2 and calorie restriction act in parallel pathways to promote longevity in yeast and, perhaps, higher eukaryotes.     

Mnsod overexpression extends the yeast chronological (G(0)) life span but acts independently of Sir2p histone deacetylase to shorten the replicative life span of dividing cells

Studies in Drosophila and Caenorhabditis elegans have shown increased longevity with the increased free radical scavenging that accompanies overexpression of oxidant-scavenging enzymes. This study used yeast, another model for aging research, to probe the effects of overexpressing the major activity protecting against superoxide generated by the mitochondrial respiratory chain. Manganese superoxide dismutase (MnSOD) overexpression increased chronological life span (optimized survival of stationary (G₀) yeast over time), showing this is a survival ultimately limited by oxidative stress. In contrast, the same overexpression dramatically reduced the replicative life span of dividing cells (the number of daughter buds produced by each newly born mother cell). This reduction in the generational life span by MnSOD overexpression was greater than that generated by loss of the major redox-responsive regulator of the yeast replicative life span, NAD+-dependent Sir2p histone deacetylase. It was also independent of the latter activity. Expression of a mitochondrially targeted green fluorescent protein in the MnSOD overexpressor revealed that the old mother cells of this overexpressor, which had divided for a few generations, were defective in segregation of the mitochondrion from the mother to daughter. Mitochondrial defects are, therefore, the probable reason that MnSOD overexpression shortens replicative life span.     

Telomere Recombination Accelerates Cellular Aging in Saccharomyces cerevisiae

Telomeres are nucleoprotein structures located at the linear ends of eukaryotic chromosomes. Telomere integrity is required for cell proliferation and survival. Although the vast majority of eukaryotic species use telomerase as a primary means for telomere maintenance, a few species can use recombination or retrotransposon-mediated maintenance pathways. Since Saccharomyces cerevisiae can use both telomerase and recombination to replicate telomeres, budding yeast provides a useful system with which to examine the evolutionary advantages of telomerase and recombination in preserving an organism or cell under natural selection. In this study, we examined the life span in telomerase-null, post-senescent type II survivors that have employed homologous recombination to replicate their telomeres. Type II recombination survivors stably maintained chromosomal integrity but exhibited a significantly reduced replicative life span. Normal patterns of cell morphology at the end of a replicative life span and aging-dependent sterility were observed in telomerase-null type II survivors, suggesting the type II survivors aged prematurely in a manner that is phenotypically consistent with that of wild-type senescent cells. The shortened life span of type II survivors was extended by calorie restriction or TOR1 deletion, but not by Fob1p inactivation or Sir2p over-expression. Intriguingly, rDNA recombination was decreased in type II survivors, indicating that the premature aging of type II survivors was not caused by an increase in extra-chromosomal rDNA circle accumulation. Reintroduction of telomerase activity immediately restored the replicative life span of type II survivors despite their heterogeneous telomeres. These results suggest that telomere recombination accelerates cellular aging in telomerase-null type II survivors and that telomerase is likely a superior telomere maintenance pathway in sustaining yeast replicative life span.     

Life span extension and H(2)O(2) resistance elicited by caloric restriction require the peroxiredoxin Tsa1 in Saccharomyces cerevisiae

Caloric restriction (CR) extends the life span of organisms ranging from yeast to primates. Here, we show that the thiol-dependent peroxiredoxin Tsa1 and its partner sulfiredoxin, Srx1, are required for CR to extend the replicative life span of yeast cells. Tsa1 becomes hyperoxidized/inactive during aging, and CR mitigates such oxidation by elevating the levels of Srx1, which is required to reduce/reactivate hyperoxidized Tsa1. CR, by lowering cAMP-PKA activity, enhances Gcn2-dependent SRX1 translation, resulting in increased resistance to H(2)O(2) and life span extension. Moreover, an extra copy of the SRX1 gene is sufficient to extend the life span of cells grown in high glucose concentrations by 20% in?a Tsa1-dependent and Sir2-independent manner. The data demonstrate that Tsa1 is required to ensure yeast longevity and that CR extends yeast life span, in part, by counteracting age-induced hyperoxidation of this peroxiredoxin.     

Localization of Sir2p: the nucleolus as a compartment for silent information regulators.

In wild-type budding yeast strains, the proteins encoded by SIR3, SIR4 and RAP1 co-localize with telomeric DNA in a limited number of foci in interphase nuclei. Immunostaining of Sir2p shows that in addition to a punctate staining that coincides with Rap1 foci, Sir2p localizes to a subdomain of the nucleolus. The presence of Sir2p at both the spacer of the rDNA repeat and at telomeres is confirmed by formaldehyde cross-linking and immunoprecipitation with anti-Sir2p antibodies. In strains lacking Sir4p, Sir3p becomes concentrated in the nucleolus, by a pathway requiring SIR2 and UTH4, a gene that regulates life span in yeast. The unexpected nucleolar localization of Sir2p and Sir3p correlates with observed effects of sir mutations on rDNA stability and yeast longevity, defining a new site of action for silent information regulatory factors.     

Increased life span due to calorie restriction in respiratory-deficient yeast

A model for replicative life span extension by calorie restriction (CR) in yeast has been proposed whereby reduced glucose in the growth medium leads to activation of the NAD⁺–dependent histone deacetylase Sir2. One mechanism proposed for this putative activation of Sir2 is that CR enhances the rate of respiration, in turn leading to altered levels of NAD⁺ or NADH, and ultimately resulting in enhanced Sir2 activity. An alternative mechanism has been proposed in which CR decreases levels of the Sir2 inhibitor nicotinamide through increased expression of the gene coding for nicotinamidase, PNC1. We have previously reported that life span extension by CR is not dependent on Sir2 in the long-lived BY4742 strain background. Here we have determined the requirement for respiration and the effect of nicotinamide levels on life span extension by CR. We find that CR confers robust life span extension in respiratory-deficient cells independent of strain background, and moreover, suppresses the premature mortality associated with loss of mitochondrial DNA in the short-lived PSY316 strain. Addition of nicotinamide to the medium dramatically shortens the life span of wild type cells, due to inhibition of Sir2. However, even in cells lacking both Sir2 and the replication fork block protein Fob1, nicotinamide partially prevents life span extension by CR. These findings (1) demonstrate that respiration is not required for the longevity benefits of CR in yeast, (2) show that nicotinamide inhibits life span extension by CR through a Sir2-independent mechanism, and (3) suggest that CR acts through a conserved, Sir2-independent mechanism in both PSY316 and BY4742.     

Absence of mitochondrial translation control proteins extends life span by activating sirtuin-dependent silencing

Altered mitochondrial functionality can extend organism life span, but the underlying mechanisms are obscure. Here we report that inactivating SOV1, a member of the yeast mitochondrial translation control (MTC) module, causes a robust Sir2-dependent extension of replicative life span in the absence of respiration and without affecting oxidative damage. We found that SOV1 interacts genetically with the cAMP-PKA pathway and the chromatin remodeling apparatus. Consistently, Sov1p-deficient cells displayed reduced cAMP-PKA signaling and an elevated, Sir2p-dependent, genomic silencing. Both increased silencing and life span extension in sov1Δ cells require the PKA/Msn2/4p target Pnc1p, which scavenges nicotinamide, a Sir2p inhibitor. Inactivating other members of the MTC module also resulted in Sir2p-dependent life span extension. The data demonstrate that the nuclear silencing apparatus senses and responds to the absence of MTC proteins and that this response converges with a pathway for life span extension elicited by reducing TOR signaling.     

Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels

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.     

啤酒酵母复制衰老的分子机制

 复制衰老是啤酒酵母衰老形式之一,表现出芽痕累积、细胞体积变大、不对称分裂丧失、不育、核仁脆裂和代谢变化等特征.染色体外rDNA环累积是啤酒酵母复制衰老的重要原因,而组蛋白去乙酰化酶家族成员Sir2蛋白在调节染色体外rDNA环累积、啤酒酵母衰老和寿命方面起到核心作用.作为去乙酰化反应底物的NAD+正性调节Sir2组蛋白去乙酰化酶活性,NAD+代谢产物尼克酰胺对Sir2有负性调节作用,而有尼克酰胺参与的NAD+补救合成途径对于Sir2活性十分重要.目前,已经在人等动物细胞中发现参与这些调节过程的相关蛋白的同源基因,在功能上也表现出一定的相似性.啤酒酵母的衰老机制研究将为人体衰老的认识提供重要线索.     

Involvement of Drosophila Sir2-like genes in the regulation of life span

In Drosophila melanogaster, the Sir2 gene and four Sir2-like genes have been found to be homologous to yeast SIR2 genes. To examine whether the fly Sir2, CG5216, and two Sir2-like genes, CG5085 and CG6284, affect life span, we suppressed their expression using RNAi. Decreased expression of the Sir2 and Sir2-like genes in all cells caused lethality during development. Suppression of the Sir2 in neurons and ubiquitous silencing of the Sir2-like genes shortened life spans. The effects were severer at 28 degrees C than at 25 degrees C. These results suggest that Sir2-like genes as well as Sir2 are involved in the regulation of life span in Drosophila.     

The flavoring agent dihydrocoumarin reverses epigenetic silencing and inhibits sirtuin deacetylases

Sirtuins are a family of phylogenetically conserved nicotinamide adenine dinucleotide-dependent deacetylases that have a firmly established role in aging. Using a simple Saccharomyces cerevisiae yeast heterochromatic derepression assay, we tested a number of environmental chemicals to address the possibility that humans are exposed to sirtuin inhibitors. Here we show that dihydrocoumarin (DHC), a compound found in Melilotus officinalis (sweet clover) that is commonly added to food and cosmetics, disrupted heterochromatic silencing and inhibited yeast Sir2p as well as human SIRT1 deacetylase activity. DHC exposure in the human TK6 lymphoblastoid cell line also caused concentration-dependent increases in p53 acetylation and cytotoxicity. Flow cytometric analysis to detect annexin V binding to phosphatidylserine demonstrated that DHC increased apoptosis more than 3-fold over controls. Thus, DHC inhibits both yeast Sir2p and human SIRT1 deacetylases and increases p53 acetylation and apoptosis, a phenotype associated with senescence and aging. These findings demonstrate that humans are potentially exposed to epigenetic toxicants that inhibit sirtuin deacetylases.