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Supported by a Leukemia and Lymphoma Society Special Fellowship. To whom correspondence should be addressed: Dept. of Pathology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-3931; Fax: 617-432-1313
* Work was supported in part by The Ellison Foundation, The American Federation for Aging Research, and The Arminese Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Both authors contributed equally to this work. ‖ Supported by a Taplan Fellowship. ¶ Supported by a National Science Foundation scholarship.
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.
Physiological studies and, more recently, DNA array analysis of gene expression patterns have confirmed that aging is a complex biological process. In contrast, genetic studies in model organisms have demonstrated that relatively minor changes to an organism's environment or genetic makeup can dramatically slow the aging process. For example, the life span of many diverse organisms can be greatly extended simply by limiting calorie intake, in a dietary regime known as caloric restriction (
How can simple changes have such profound effects on a complex process such as aging? A picture is emerging in which all eukaryotes possess a surprisingly conserved regulatory system that governs the pace of aging (
). Such a regulatory system may have arisen in evolution to allow organisms to survive in adverse conditions by redirecting resources from growth and reproduction to pathways that provide stress resistance (
One model that has proven particularly useful in the identification of regulatory factors of aging is the budding yeast Saccharomyces cerevisiae. Replicative life span in S. cerevisiae is typically defined as the number of buds or “daughter cells” produced by an individual “mother cell” (
). Mother cells undergo age-dependent changes, including an increase in size, a slowing of the cell cycle, enlargement of the nucleolus, an increase in steady-state NAD+ levels, increased gluconeogenesis and energy storage, and sterility resulting from the loss of silencing at telomeres and mating-type loci (
). Increased chronological life span correlates with increased resistance to heat shock and oxidative stress, suggesting that cumulative damage to cellular components is a major cause of this type of aging (
). This instability gives rise to circular forms of rDNA called extrachromosomal rDNA circles that replicate but fail to segregate to daughter cells. Eventually, extrachromosomal rDNA circles accumulate to over 1000 copies, which are thought to kill cells by titrating essential transcription and/or replication factors (
). Mutations that reduce the activity of the glucose-responsive cAMP (adenosine 3′,5′-monophosphate)-dependent (protein kinase A) pathway extend life span in wild type cells but not in mutant sir2 strains, demonstrating that SIR2 is a key downstream component of the caloric restriction pathway (
In bacteria, NAD+ is synthesized de novo from tryptophan and recycled in four steps from nicotinamide via the NAD+ salvage pathway (see Fig. 5 below). The first step in the bacterial NAD+ salvage pathway, the hydrolysis of nicotinamide to nicotinic acid and ammonia, is catalyzed by the pncA gene product (
). At this point, the NAD+ salvage pathway and the de novoNAD+ pathway converge and NaMN is converted to desamido-NAD+ (NaAD) by a nicotinate mononucleotide adenylyltransferase (NaMNAT). In S. cerevisiae, there are two putative ORFs with homology to bacterial NaMNAT genes,YLR328 (
). One current hypothesis explaining how caloric restriction extends replicative life span is that decreased metabolic activity causes an increase in NAD+ levels, which then stimulate Sir2 activity (reviewed in Campisi (
)). In this study, we tested this theory by examining whether additional copies of NPT1 can promote Sir2-dependent life span extension and whether this correlates with increased NAD+ levels. We show that additional NPT1 extends replicative life span in a SIR2-dependent manner via the caloric restriction pathway. We find that these long-lived strains do not have increased NAD+ levels or altered NAD+/NADH ratios, despite the fact that every SIR2-dependent process we examined was enhanced. Interestingly, increased dosage of SIR2 or NPT1 provides resistance to heat shock, indicating that these genes act in a general manner to promote cell survival.
We find that additional copies of all the salvage pathway genes increase rDNA and telomeric silencing with exception of QNS1. We show that Npt1 and Nma2 are concentrated in the nucleus, raising the possibility that a substantial fraction of NAD+ is recycled within this organelle. We discuss the potential for extending life span in higher organisms by stimulation of the conserved NAD+ salvage pathway.
NPT1 encodes a key component of the yeast salvage pathway that recycles NAD+, a cofactor of Sir2. We have shown that additional copies of NPT1 increase life span by up to 60% in a SIR2-dependent manner. It has been proposed that longevity in yeast may be associated with increased NAD+ levels. However, we have shown that in strains with additional copies of NPT1, steady-state NAD+levels are unaltered. Furthermore, the NAD+/NADH ratios are also similar to wild type cells, indicating that total cellular redox state is not dramatically altered either.
We have also shown that sir2 mutants have wild type NAD+ levels, implying that Sir2 is not a major consumer of NAD+. Nevertheless, by virtue of its ability to convert NAD+ to nicotinamide, Sir2 should be responsive to increased flux through the salvage pathway (Fig. 6). Thus, although steady-state levels of NAD+ remain constant, the turnover of this molecule may be elevated. Localization of GFP-tagged enzymes indicated that at least two of the enzymes in the NAD+ salvage pathway are concentrated in the nucleus. Consistent with this, Nma1 and Nma2 have been shown by high throughput two-hybrid screening to interact with Srp1, a protein that acts as a receptor for nuclear localization sequences (
). It is worth nothing that strains disrupted for either NMA1 or NMA2 are viable, arguing that they may be functionally redundant, given that the conversion of NaMN to NAD+ is apparently essential for viability (
), suggesting that nuclear compartmentalization of the pathway may be a universal property of eukaryotic cells. Having the salvage pathway in proximity to chromatin may allow NAD+ to be rapidly regenerated for silencing proteins. Alternatively, it may permit the coordination of a variety of nuclear activities via the alteration of nuclear NAD+ pools. Testing of these hypotheses will not be a simple task but one that will be greatly assisted by the development of a molecular probe for intracellular NAD+.
In yeast and many metazoans, a number of long-lived mutants display increased stress resistance. However, there are many examples of mutations that extend life span but provide little protection against stress, indicating that this relationship is not straightforward (
). We have shown that additional copies of NPT1 or SIR2 extend life span but do not provide protection against MMS, paraquat, or starvation. Thus, in S. cerevisiae, longevity is not linked to a general increase in stress resistance. The only stress-related phenotype that correlated with longevity was heat-shock resistance. Based on genome-wide analyses of gene expression in sir2Δ strains, it has been proposed that Sir2 regulates genes other than those at the three silent loci (
). If the interpretation is correct, then it is plausible that the heat-shock resistance we observed in 2x NPT1 and 2x SIR2 strains results from Sir2-mediated silencing of genes that suppress heat-shock resistance.
In bacteria, the Npt1 homolog PncB catalyzes a rate-limiting step in the NAD+ salvage pathway (
). In this study we show that additional copies of PNC1, NPT1, NMA1, or NMA2 all increase rDNA and telomeric silencing. The implication is that, in yeast, multiple steps can affect the rate of the pathway. Such a proposal is consistent with Metabolic Control Analysis, a theory based on the observation that flux through most metabolic pathways is controlled by multiple enzymes, rather than by a single rate-liming step (
). Of all the genes in the salvage pathway, only QNS1 had no effect on silencing, suggesting that it is the only enzyme in the pathway limited by substrate availability. This is likely due to the fact that the predicted substrate for Qns1, desamido-NAD+, is the only intermediate that cannot be supplied from a source outside the salvage pathway (see Fig. 6).
In yeast and metazoans there are multiple members of the Sir2 family, many of which have been shown (or are predicted) to be NAD+-dependent deacetylases (
). Our findings show that several SIR2-dependent processes can be enhanced by manipulation of the NAD+ salvage pathway in yeast, and this may hold true for higher organisms. We have identified NPT1 homologs in every genome we have examined, and all possess a highly conserved region around a histidine residue that, in Salmonella, greatly stimulates catalysis when phosphorylated (
). This mode of regulation may permit the design of mutations or small molecules that increase Npt1 activity. Together, our findings show that Npt1 and other members of the salvage pathway are attractive targets for small molecules that may mimic the beneficial effects of caloric restriction.
We thank D. Moazed, J. Smith, C. Grubmeyer, M. Bryk, F. Winston, A. Andalis, and G. Fink for reagents and advice. We also thank S. Luikenhuis for help with manuscript preparation.