This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bitterman, K. J. Articles by Sinclair, D. A. Search for Related Content PubMed PubMed Citation Articles by Bitterman, K. J. Articles by Sinclair, D. A.

Longevity Regulation in Saccharomyces cerevisiae: Linking Metabolism, Genome Stability, and Heterochromatin

Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

SUMMARY

INTRODUCTION

Yeast as a Model for Aging Research

Definition of Yeast Aging

Replicative versus chronological yeast life span.

REPLICATIVE AGING

Background

Characteristics of Replicatively Aged Cells

Bud scar accumulation.

Increased cell size.

Loss of asymmetry.

Loss of fertility.

Nucleolar fragmentation.

Metabolic changes.

Mechanisms of Yeast Replicative Aging

Background

rDNA stability.

Role of heterochromatin.

Other mechanisms of replicative aging.

CHRONOLOGICAL AGING

Background

Characteristics of Stationary-Phase Cells

Mechanisms

Free-radical theory of aging.

SCH9 and CYR1.

Conservation in Higher Eukaryotes

REGULATION OF REPLICATIVE AGING

Role of Sir2

Overview of Sir2 enzymology.

Evidence from crystal structures.

(i) NAD+-binding site.

(ii) Substrate-binding site.

(iii) Substrate specificity.

Sir2 reaction products.

(i) Nicotinamide.

(ii) O-Acetyl ADP-ribose.

Metabolism, Chromatin, and Longevity

NAD+ synthesis and Sir2 activity.

(i) Kynurenine pathway.

(ii) Salvage synthesis of NAD+.

Calorie restriction, respiration, and NAD+.

Regulation by nicotinamide.

(i) Inhibition of Sir2 by nicotinamide.

(ii) Regulation of nicotinamide levels by Pnc1.

(iii) Nicotinamide and higher organisms.

Rpd3

Glucose Sensing and Calorie Restriction

Background.

HXT and HXK genes.

Snf1 complex.

cAMP signaling and replicative life span.

Retrograde Signaling And Life Span

TELOMERE MAINTENANCE AND PREMATURE-AGING SYNDROMES

Background

Yeast as a Model For Replicative Senescence

Telomeres and Premature-Aging Syndromes

METHODS

Replicative Life Span Analysis

Chronological Life Span Analysis

Isolation of Old Cells

Fluorescence-activated cell sorting.

Magnetic sorting.

Sucrose gradient centrifugation.

Centrifugal elutriation.

Membrane attachment.

Isolation and Analysis of ERCs

CONCLUSIONS AND PERSPECTIVES

ACKNOWLEDGMENTS

REFERENCES

Top Next References

INTRODUCTION

Top Previous Next References

 
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, and for seemingly good reasons. 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, which is 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. Translating these findings to higher organisms remains a major challenge over the next decade. 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.

In the early days of aging research, few researchers suspected the existence of single genes that control aging. This was based in part on the valid assumption that aging was an incredibly complex process that was impacted by hundreds, if not thousands, of genes. Then, genetic studies with model organisms in the 1990s began to uncover numerous single-gene mutations that extend life span (75, 92, 95). Today there are dozens of mutations known to extend life span in model organisms (109) (Table 1). This leads to the question: what had researchers overlooked? The major oversight appears to have been the failure to foresee that organisms have evolved genes, not to promote aging but to promote survival and longevity during times of adversity. Longevity regulation, as it has come to be known, is now generally accepted as a highly adaptive biological trait, especially in a changing environment (94, 100).

A second reason to expect some conservation between yeast and human aging, put simply, is that they have the same fundamental biology. If aging really is due to "wear and tear," and most evidence suggests that it is, then not all biological systems will experience wear and tear at the same rate. Some systems, such as basic metabolic pathways, are relatively robust because they are inherently self-regulating and often contain inbuilt redundancy. The systems that fail first are those whose maintenance is energy demanding and for which the damage is irreversible. A good example of such a system is the genome. The energy required to constantly survey and repair the genome is immense, and the wear and tear often irreversible. Consistent with this idea, as we describe below, a major cause of replicative aging in S. cerevisiae has been shown to arise from the cell's inability to fully maintain the integrity of its genome at its most highly repetitive locus, the ribosomal DNA (rDNA) (180). Although rDNA stability may not play a role in human aging, it is suspected that the cumulative loss of coding or structural DNA (e.g., the repeats found at telomeres) may contribute to aging in humans (33). The important point is that the mechanisms of aging may be related between distant species (but by no means as highly conserved as longevity regulatory pathways) because eukaryotes have the same basic biology and some systems are inherently difficult to maintain over a lifetime.

It is thus of particular concern that some yeast strains used routinely in the field have average life spans that are half that of most wild-type strains (Table 2). Any one of a number of mutations that shorten the life span could have been introduced into laboratory strains over the preceding decades, and researchers would not have detected a difference unless they examined the life span of individual cells. Even very short-lived strains appear to grow at normal rates. One way to ensure that one is studying true aging is to test one's findings with multiple wild-type strains that are not relatively short-lived. This would be an arduous task that most laboratories have yet to embrace.

The alternative measure, "chronological life span," also referred to as "postdiauxic survival," is the length of time a population of yeast cells remains viable in a nondividing state following nutrient deprivation (124). Yeast cells grown in a nutrient-rich medium multiply until all readily utilizable nutrients are exhausted. At this point, cells cease dividing and enter a postdiauxic, hypometabolic state known as stationary phase, where they can remain viable for weeks. In synthetic medium, cells deplete the medium and cease dividing yet retain relatively high metabolism (124). Such cells have a short chronological life span relative to cells in rich medium and are thought to more closely resemble postmitotic cells in multicellular organisms (125).

REPLICATIVE AGING

Top Previous Next References

 

A second important observation came from a genetic screen for long-lived yeast mutants (92). Kennedy, Guarente and colleagues first identified starvation-resistant mutants, having noted that in many other organisms there is a strong correlation between stress resistance and longevity. Rescreening of the stress-resistant mutants for increased longevity led to the isolation of four so-called "youth mutants", uth1 to uth4. Interestingly, three of these have been found to affect the same cellular process: the formation of silent heterochromatin.

In yeast, transcriptional silencing occurs at telomeres, the two mating-type loci (HML and HMR), and rDNA locus RDN1 (127, 142). The establishment of heterochromatin at telomeres and mating-type loci requires the yeast Sir2/3/4 protein complex. Sir2, but not Sir3 or Sir4, also mediates silencing at the rDNA (24, 188). In many strains, the overexpression of Sir2 increases the extent of silencing at both telomeres and rDNA, implying that Sir2 is a limiting component of the silencing apparatus (88, 190).

The most informative allele isolated in the screen for longevity mutants was UTH2-1 (later renamed SIR4-42), which extended replicative life span by 45% (92). This semi-dominant mutation truncated the Sir4 protein, causing the Sir complex to relocalize to the nucleolus (93), thus increasing rDNA silencing (D. A. Sinclair, unpublished results). The other UTH genes were also interesting. The UTH1 gene encodes a SUN domain protein whose expression is greatly induced by oxidative stress and whose deletion results in a global increase in silencing and life span extension (L. Guarente, unpublished results). UTH4, which encodes a Drosophila Pumilio homolog (39), influences the distribution of the Sir complex within the nucleus (54, 93). Deletion of UTH4 reduces life span and decreases rDNA silencing, whereas overexpression of UTH4 has the opposite effect (93). Although the biochemical function of Uth1 and Uth4 remain to be determined, a clear trend emerged from these studies: increased heterochromatin at the yeast rDNA locus correlates with increased longevity.

Bud scar accumulation. When a daughter separates from a mother cell, the boundary between the two cells constricts, leaving behind on the mother cell's surface a circular chitin-containing remnant termed the bud scar (Fig. 1A). Bud scars remain permanently deposited on the surface of the mother cell. As a mother cell goes through successive rounds of cell division, bud scars accumulate on the cell surface, serving as a convenient marker for the number of divisions realized by a single cell. As a cell continues dividing, previous sites of budding are seldom reused. Although it has been hypothesized that the accumulation of bud scars may impose a theoretical upper limit on a cell's replicative potential (25), evidence indicates that it does not result in senescence of wild-type strains. First, most mutations that extend life span appear to have no relation to cell wall biosynthesis. Second, a bud scar typically occupies approximately 1% of the available cell surface, giving a theoretical upper limit of at least 100 divisions, but most laboratory strains have an average life span of 20 to 30 divisions (178) (Table 2). Third, increasing the cell size, and thus increasing the number of available budding sites, does not extend life span (147). Fourth, it is possible to artificially elevate the deposition of chitin by briefly incubating cells containing a temperature-sensitive allele of CDC24 at the nonpermissive temperature (185, 186). At this temperature, cdc24-ts cells arrest in an unbudded state and randomly accumulate cell wall chitin. Elevated chitin deposition does not adversely affect the life span (42), further demonstrating that bud scar accumulation is probably not a cause of yeast aging.

View larger version (52K): [in this window] [in a new window]   FIG. 1. Yeast cell division and bud scar formation. (A) The budding of each daughter cell leaves a ring-shaped deposit, termed the bud scar, on the cell wall of the mother cell. These chitin-containing rings, formed at the neck of buds, can be stained with calcofluor, a fluorescent dye. The exact number of times an individual mother cell has undergone division can thus be determined by counting the number of bud scars present. (B) An aged yeast nucleus has an enlarged and fragmented nucleolus (arrows), unlike the nucleoli of two young cells in the upper left corner.

Loss of asymmetry. As yeast cells age, they grow dramatically larger but continue to give rise to small daughter cells throughout most of their life span. However, very old mother cells tend to produce large, short-lived daughter cells (76, 85, 91). At or near the final division, daughter cells often do not separate from the mother until both cells are similar in size (76, 85). The loss of the asymmetry of age in old cells is intriguing because it provides a strong argument for the ERC model of senescence (discussed below). When old cells become packed with extrachromosomal rDNA circles (ERCs), these molecules "leak" into daughter cells, presumably causing the premature aging seen in these cells (180). The ERC model would also explain why old age is diluted by successive generations, so that the great granddaughter of an old mother, for example, has a normal life span.

Loss of fertility. S. cerevisiae can exist in either a haploid or diploid state. When two fertile haploid cells of opposite mating type encounter one another, they mate to form a diploid zygote. To switch between the two mating types (MATa and MAT), a cell transposes the opposite silent information to the mating-type locus, where it is expressed. Young yeast cells are normally fertile since the two repositories of mating-type information, HMR and HML, remain in a transcriptionally silent state (127). As discussed above, the silent state at HM loci requires the Sir2-Sir3-Sir4 silencing complex. When yeast cells grow old, they become sterile (147), and this phenotype remains one of the most reliable external markers of yeast aging.

Sterility in old cells is caused by a loss of transcriptional silencing at the cryptic mating-type loci, HMRa and HML, resulting in simultaneous expression of both a and information (187). To prove this, HMRa was deleted from a MAT strain. The resulting strain exhibited the same characteristic life span as the wild type but no longer became sterile. In addition to a loss of mating-type silencing, old cells lose silencing near telomeres (97) and the Sir complex relocalizes to the nucleolus in old cells (93).

Nucleolar fragmentation. The nucleolus is a nuclear structure containing the rDNA genes and other components required for ribosome assembly (31, 137). Yeast rDNA, located on chromosome XII, contains 100 to 200 tandemly repeated copies of a 9.1-kb unit. In young yeast cells, the nucleolus forms a crescent-shaped structure retained near the nuclear periphery (168). In old cells, however, the nucleolus becomes enlarged and fragmented into multiple, rounded structures (181). (Fig. 1B). Fragmentation of the nucleolus and relocalization of Sir3 may be a response to the accumulation and aggregation of extrachromosomal rDNA circles (180). It is important to note though, that chromosomal rDNA remains intact in these fragmented nucleoli.

Metabolic changes. The only study to specifically address the question of metabolism in aging yeast cells involved a combination of microanalytical biochemical assays and microarray analyses of mRNA levels (116). The major finding was that yeast cells, as they age, undergo a progressive shift away from glycolysis toward gluconeogenesis and energy storage. This shift is associated with the induction of genes involved in glycogen production, fatty acid degradation, gluconeogenesis, and the glyoxylate cycle. Biochemical profiling of enzymes and metabolic intermediates confirmed the shift to gluconeogenesis. Much of the up-regulation of genes involved in gluconeogenesis appears to be due to the translocation of the Mig1 transcriptional repressor from the nucleus to the cytoplasm as cells age (6) (see below). ATP levels do not decline with age, but there is a 30% drop in the NAD⁺ level between divisions 0 to 1 and 7 to 8. Interestingly, many of these changes are recapitulated in short-lived sip2 cells, which lack the putative repressor of Snf1, a kinase that regulates cellular responses to glucose deprivation. The role of the Snf1/Sip2 pathway in longevity is described in further detail below.

Mortimer and Johnston first noted that daughter cells that budded from old mothers tend to have premature-aging characteristics, including a large size, a slow cell cycle, and a short life span (145). Based on this observation, Egilmez and Jazwinski proposed that yeast aging is caused by a (potentially cytoplasmic) senescence factor that accumulates exponentially as cells divide, reaching a level that eventually kills cells (42). This model is appealing for two reasons. First, it explains how daughters inherit old age from their mothers. When mothers become sufficiently old due to the accumulation of the senescence factor, some of this factor might leak into daughter cells, making them prematurely old themselves. Second, the senescence-factor model explains the dynamics of yeast mortality. All species that experience aging, from yeast to humans, have characteristic rates of mortality that can be described mathematically by the Gompertz-Makeham equation (53). Mortality curves derived by plotting percent survival against age have a characteristic "shoulder," where most individuals remain viable early in life but then viability rapidly declines as the mortality rate increases exponentially. There is also a characteristic "tail," where some lucky individuals seem to experience delayed mortality and remain viable longer than would be expected by extrapolating the curve. These kinetics imply that aging is the sum of two components (182): (i) a stochastic trigger that initiates a process and (ii) an aging process whose negative impact on life span increases exponentially with age. Egilmez and Jazwinski proposed that the triggering event might be the generation of a senescence factor and that aging would result from the exponential accumulation of this factor with each cell division (42). This idea is consistent with the ERC mechanism of yeast aging described below.

Over the past 5 years, researchers have come to the consensus that a primary cause of yeast replicative aging stems from changes within the nucleolus, the distinct nuclear region responsible for rRNA transcription and ribosome assembly. The first clue that nucleolar events were involved in aging came from the aforementioned genetic screen for mutations that increased yeast life span and the subsequent isolation of SIR4 (92). SIR4 encodes a component of the Sir2-Sir3-Sir4 silencing complex that catalyzes the formation of heterochromatin at the silent mating-type (HM) loci and telomeres. The mutation in SIR4 resulted in a C-terminal truncation of the protein, causing it to relocalize from telomeres and HM loci to the nucleolus (93). Although the mechanism by which the Sir4-42 protein increases life span was not immediately clear, the finding focused researchers attention on the nucleolus and the rDNA, eventually leading to the identification of a molecular cause of yeast aging.

rDNA stability. In 1997, Sinclair and Guarente proposed that yeast replicative aging stems from genomic instability at the rDNA locus, RDN1 (180). The yeast rDNA locus is inherently recombinagenic due to its repetitive nature and unidirectional mode of DNA replication. The locus, which comprises 10% of the total yeast genome, consists of 100 to 200 tandemly arrayed 9-kb repeats that encode the rRNAs. Each repeat contains a potential origin of DNA replication, with three ARS consensus sites within each origin. Roughly one of three origins within these repeats fires during each S phase (139). Homologous recombination between adjacent repeats is known to result in the excision of extrachromosomal circular forms of rDNA known as ERCs (Fig. 2). The important aspect of the aging mechanism is that ERCs replicate during S phase but are inefficiently segregated to daughter cells. Because ERCs can double in copy number every S phase, their abundance increases exponentially in mother cells at a rate timed by cell division (180).

View larger version (17K): [in this window] [in a new window]   FIG. 2. Instability of repeated DNA as a cause of replicative aging in S. cerevisiae. The yeast rDNA locus is the most highly repetitive locus in the organism, consisting of 150 tandem 9.1-kb repeats. These repeats are stabilized, in part, by the NAD+-dependent HDAC Sir2. The initiating event is the generation of an ERC by homologous recombination between repeats within the rDNA array on chromosome XII. ERCs have a high probability of replicating and are segregated almost exclusively to the mother cell. They accumulate exponentially in mother cells, resulting in fragmented nucleoli, cessation of cell division, and cellular senescence. Stabilization of the rDNA locus or inhibition of ERC replication extends the life span. Daughters from very old mothers inherit ERCs due to the breakdown in the asymmetry of inheritance, explaining why daughters of old mothers are prematurely old. Ectopic release of an ERC in a young cell accelerates aging. Overexpression of SIR2 or deletion of FOB1 (encoding a replication fork block protein specific to the rDNA) reduces rDNA recombination and ERC formation, and the life span is extended by 30 to 50%. Reprinted from reference 180 with permission.

There is now convincing evidence that in a typical wild-type strain, ERCs are a major cause of aging. First, the ectopic release of an ERC into a virgin cell shortens the life span and causes premature aging, demonstrating that ERCs are sufficient to cause aging (180). Second, genetic manipulations that decrease the formation of ERCs greatly extend life span (2, 6, 36, 114, 117, 152, 180) whereas those that accelerate ERC formation have an opposite effect (88, 152).

Genetic manipulations that extend yeast life span by suppressing ERCs seem to fall into one of two categories. The most common suppress ERC formation by increasing the extent of heterochromatin at the rDNA. Examples of this class include SIR2 and PNC1 overexpression. These are discussed in detail below in the context of environmentally regulated chromatin. The other class includes those that directly suppress homologous recombination between rDNA repeats. A good example of a longevity gene in this class is FOB1 (35). FOB1 encodes a nucleolar protein that is required for a DNA replication fork block immediately downstream of the rDNA origins (86, 102). The fork block is thought to prevent transcription complexes from running head on into moving replication forks and to allow the rDNA locus to expand and contract via recombination. The Fob1-mediated fork block is thought to be responsible for the general instability of the rDNA locus, since strains lacking FOB1 have a 100-fold lower rate of rDNA recombination (118) and have manyfold fewer ERCs than aged-matched wild-type cells (36). As predicted by the ERC model, fob1 mutants live almost twice as long as wild-type controls. Weak mutations in FOB1 also extend the life span but not to the extent of the deletion. Furthermore, ERC levels in these strains correlate with the respective life span extensions. The argument that this life span extension is due to effects on rDNA silencing does not appear to be founded because rDNA silencing in fob1 cells is the same as or less than that in wild-type cells (M. Kaeberlein, personal communication).

Role of heterochromatin. A major mechanism by which yeast cells suppress ERC formation is the packaging of DNA and histones into "silent" heterochromatin. Heterochromatin in yeast occurs at telomeres, HM loci, and the rDNA (142). The formation of heterochromatin at HM loci and telomeres is mediated by the silent information regulatory complex Sir2/3/4 (66, 192). Alternatively, heterochromatin at the rDNA locus is catalyzed by the RENT (for "regulator of nucleolar silencing and telophase exit") complex, which includes Sir2, Net1, Cdc14, and Nan1 (51, 176). Of these proteins, Sir2 is the only factor that is indispensable for silencing at all three silent regions (24, 55, 188). To be precise, "rDNA silencing" is actually a misnomer. Although polymerase II-transcribed marker genes integrated at the rDNA are transcriptionally silenced, transcription of the native rDNA is seemingly unaffected by deletion of SIR2 (166). This suggests that the primary function of heterochromatin at the rDNA locus may be to suppress recombination rather than silence transcription. There have been several reports, though, which demonstrate "natural" silencing of polymerase II-transcribed genes within the rDNA (24, 209).

Other mechanisms of replicative aging. It would be a mistake to assume that there is only one cause of aging for any species, and no doubt other causes of yeast aging will be found. As yet, apart from the ERC mechanism, there is no other known cause of replicative aging, although some strains have been found to age independently of ERC formation (96). One proposed cause of aging is dysregulation of rDNA transcription during the life span of a cell (74). This would also explain the strong correlation between increased rDNA silencing and life span. Another recent finding has uncovered a putative rDNA- and silencing-independent contribution to yeast aging (157). This mechanism appears to be governed by the Slt2 kinase via phosphorylation of Sir3, although the details of how this mechanism influences life span are not yet known.

CHRONOLOGICAL AGING

Top Previous Next References

 

These results have given support to the free-radical theory of aging, first proposed a half a century ago by Harman (64). Subsequent identification of other yeast genes involved in chronological aging, though, has clearly shown that survival in stationary phase requires more than just antioxidant protection. For example, whereas overexpression of SOD1 and SOD2 extends life span by 30%, mutation of specific signaling proteins (discussed below) can increase longevity by as much as 300%. There is no question, though, that free radicals play an important role in this process (see below). At the very least, chronologically aging yeast has proven to be a valuable model for the study of oxidative damage in the postmitotic tissues of higher eukaryotes.

Interestingly, this research has provided novel insights into a process seemingly far removed from yeast biology, i.e., programmed cell death or apoptosis. Expression of the human antiapoptotic Bcl-2 protein in yeast partially reverses defects of sod mutants and increases the survival of wild-type cells (123). These results raised the possibility that components of the apoptotic machinery are present in yeast (reviewed in references 50 and 184). In support of this, the human proapoptotic Bax protein has been shown to promote cell death in yeast, and several putative apoptotic components (including a caspase-like protease) have been recently identified in yeast (130, 131, 133, 172). As suggested by Frohlich and Madeo, the study of old yeast cells that produce free radicals and that may die apoptotically will probably provide clues to similar events involved in mammalian aging (50).

SCH9 and CYR1. The most notable of the above-mentioned discoveries came from a transposon-mediated mutagenesis screen performed by Fabrizio et al. (45). In a search for mutants that were both long-lived and stress resistant, the researchers isolated strains with transposon insertions in two genes, SCH9 and CYR1 (the transposon was in the promoter region of SCH9). Loss-of-function mutations in these genes had dramatic effects on chronological longevity. Mutant sch9 and cyr1 cells survived roughly three- and twofold longer than did the wild type, respectively. Interestingly, these two mutants not only had the greatest extension in chronological life span compared with wild-type cells but also were the only two genes isolated independently from both heat and oxidative stress selections. This supports a model whereby survival in stationary phase and extension of chronological life span depends on development of resistance to multiple forms of stress.

SCH9 encodes a serine/threonine protein kinase, whereas CYR1 encodes adenylate cyclase, required for stimulation of cyclic AMP (cAMP)-dependent protein kinase (PKA) activity (Fig. 3). These proteins appear to function in separate yet parallel signaling pathways, both of which mediate glucose/nutrient signaling, stimulate growth and glycolysis, and function to down-regulate stress resistance, glycogen accumulation, and gluconeogenesis (113, 124). Consistent with a role of cAMP signaling in regulation of chronological aging, deletion of the gene encoding the GTP-binding protein Ras2, an upstream regulator of Cyr1, doubles chronological life span (44) (Fig. 3). Similar to the above mutants, long-lived ras2 cells also display multiple stress resistances, since they are thermotolerant and more resistant to oxidative damage. Furthermore, the life span extension of both cyr1 and ras2 strains requires the stress response transcription factors Msn2 and Msn4. The life span extension in sch9 strains does not require these factors and may act via the protein kinase Rim15 (45). Rim15 in turn functions via the stress response transcription factor Gis1 (153), which binds postdiauxic shift elements found in the promoters of such genes as HSP26, HSP12, and SOD2 (Fig. 3). The above results again highlight the close association between stress response pathways and chronological longevity. Interestingly, mutation of the PKA pathway extends the replicative life span of yeast cells (114), arguing for some degree of overlap between these two distinct measures of aging (see below). It is worth noting that replicative life span extension is independent of Msn2 and Msn4 (114), so that while there may be some commonality in the pathways involved, the downstream mechanisms regulating these two processes are almost certainly distinct (Fig. 3).

View larger version (26K): [in this window] [in a new window]   FIG. 3. Conserved longevity-regulatory pathways in S. cerevisiae, C. elegans, and possibly humans. There are two ways to study longevity in S. cerevisiae: replicative (mitotic) life span and chronological (nonmitotic) life span. Both these approaches have identified yeast genes with functional homologues in a conserved insulin-like signaling longevity pathway. These include SIR2, which encodes an NAD+-dependent deacetylase, RAS, which encodes a GTP-binding protein, and SCH9, which encodes a serine/threonine kinase. These findings are consistent with the idea that longevity regulation is highly adaptive and that components of a primordial pathway have been conserved in a diverse range of species.

These results have been extended to Drosophila, where it has been shown that mutation of components of the fly insulin-IGF-1 pathway can extend life span by 85% (30, 200). These mutants also up-regulate nutrient storage and superoxide dismutase expression, similar to yeast and worms. Yeast Sch9 is also 49% identical to human Akt-1-Akt-2-PKB, involved in insulin and glucose signaling, apoptosis, and cellular proliferation (89) (Fig. 3). Whereas down-regulation of the yeast pathway leads to storage of glycogen, down-regulation of the human insulin-IGF-1 pathway stimulates the storage of fat, each of which is the primary carbon source during starvation for the respective organism (113, 124). The similarity in strategies, pathways, and factors involved in such distantly related organisms has led to the proposal that this common longevity regulatory system arose early in evolution as a way to delay reproduction and increase the chances of survival during times of nutrient limitation (94). This degree of conservation ensures that the study of chronological aging in yeast will continue to shed light on the regulation of, and mechanisms underlying, numerous critical processes in higher eukaryotes.

REGULATION OF REPLICATIVE AGING

Top Previous Next References

 

The role of Sir2 in longevity regulation appears to be conserved. Sir2 homologues can be found in a wide array of organisms, ranging from bacteria to humans, and it has been shown that increased dosage of the Sir2 homologue, sir2.1, can extend the life span of the nematode C. elegans (202). In addition, the nearest human homologue, SIRT1, inhibits apoptosis through deacetylation and negative regulation of p53 (128, 206). These two findings suggest that Sir2 and its homologues play a conserved role in the regulation of survival at both the cellular and organismal levels (62). A recent study with yeast has also implicated Sir2 in the asymmetric inheritance of oxidatively damaged proteins during cell division (1). It was found that carbonylated proteins, which accumulate during aging, are retained in the mother cell during cytokinesis in a Sir2-dependent manner. Although the authors do not claim this process to be a determinant of replicative life span, it probably contributes to the ability of cells to resist oxidative damage and to the overall fitness of daughter cells.

Overview of Sir2 enzymology. Yeast Sir2 is the founding member of class III histone deacetylases (HDACs). Unlike class I or II HDACs, Sir2-like deacetylases are not inhibited by trichostatin A and have the unique characteristic of being NAD⁺ dependent (72, 105, 189, 196). Yeast Sir2 is known to have specificity for lysine 16 of histone H4 and lysines 9 and 14 of histone H3 (72, 105, 189). While many Sir2-like enzymes readily deacetylate histone substrates in vitro, at least two Sir2 homologues, yeast Hst2 and human SIRT2, are localized to the cytoplasm (20, 72, 196, 198). Studies have also demonstrated that human SIRT3 is localized to mitochondria (149, 170), and human SIRT1, a nuclear protein, has recently been shown to target p53 for deacetylation (128, 206). In addition, eubacterial species such as Salmonella lack histones entirely, yet their genomes still encode Sir2 homologues (203). The above results suggest that the Sir2 family of deacetylases may act on a broad range of substrates and that only a subset of these enzymes are likely to target histones for deacetylation in vivo.

Sir2-catalyzed deacetylation is considered an atypical reaction from both thermodynamic and mechanistic standpoints. Although trichostatin A-sensitive HDACs catalyze deacetylation without the need for a cofactor, Sir2 requires NAD⁺, even though deacetylation is a simple and energetically favorable hydrolysis reaction. Furthermore, NAD⁺ is not required catalytically for this reaction but is actually consumed by it. Hydrolysis of the glycosidic bond between the ribose and nicotinamide moieties of NAD⁺ liberates roughly 8.2 kcal of free energy per mol (143). This leads to the question of why the cell would couple cleavage of a high-energy bond in a metabolically valuable molecule to an already exothermic reaction. One possible explanation for these findings is that the NAD⁺ dependence of Sir2 may allow for regulation of its activity through changes in the availability of this cosubstrate (167). In other words, this may allow Sir2-like enzymes to "sense" the energetic and redox states of the cell and adjust their activity accordingly. An additional possibility is that the products formed from the unique Sir2 reaction may initiate a signal transduction pathway or may themselves allow for regulated control of the enzyme (16, 143). These possibilities are discussed in more detail below.

Evidence from crystal structures. Sir2 enzymes couple NAD⁺ hydrolysis to the deacetylation of the amine of lysine residues with a 1:1 stoichiometry. The overall reaction actually consists of two hydrolysis steps, which are thought to be coupled (141). The first step is the cleavage of the high-energy glycosidic bond that joins the ADP-ribose moiety of NAD⁺ to nicotinamide. This is followed by cleavage of the C—N bond between the acetyl group and lysine. Upon cleavage, Sir2 then catalyzes the transfer of the acetyl group to ADP-ribose (189, 196, 198).

Crystal structures of two archaeal Sir2 homologues (Sir2-Af1 and Sir2-Af2), as well as the catalytic core of human SIRT2, have been solved and have begun to shed light on the specifics of the reaction mechanism (9, 27, 46, 141) (Fig. 4). The archaeal structures have been particularly revealing. Both the wild-type and several catalytically deficient varieties of Sir2-Af1 have been crystallized with an (albeit incomplete) NAD⁺ molecule (27, 141). In addition, Avalos et al. have published a crystal structure of Sir2-Af2 bound to an acetylated p53 peptide substrate (9). Conservation previously observed only at the sequence level is immediately apparent when the structures of these distantly related homologues are compared. The conserved core domain of these enzymes forms two regions which can bind an NAD⁺ molecule within a deep pocket between them (Fig. 4A). The larger of these two domains is reminiscent of the Rossmann fold, a motif found in many NAD(H)-NADP(H)-binding proteins (162), whereas the smaller domain coordinates a structural zinc atom.

View larger version (45K): [in this window] [in a new window]   FIG. 4. Crystal structure of the Sir2 deacetylase. (A) Ribbon diagram of Sir2-Af1 complexed with NAD+ (PDB 1ICI based on the structure of Min et al. [141]). The NAD+ molecule and zinc atom are in green. Putative catalytic site B residue His116 is shown as stick model in dark blue. Conserved site C residues Ser24, Asn99, and Asp101 are shown in pink. Gly185, which is located in site A and which is perfectly conserved in all Sir2 family members, is shown in light blue. (B) Surface representation of Sir2-Af1. Blue and red patches show surface electrostatic potential distribution for positively and negatively charged residues, respectively. Amino acid residues 30 to 47 are shown as a ribbon diagram for better visualization of the NAD+-binding pocket. This pocket is spatially divided into three regions, termed sites A, B, and C, which contact different portions of the NAD+ molecule (141). An acetyllysine substrate is proposed to come in close proximity to site B by inserting into a tunnel within the indicated substrate-binding cleft (9). The inhibitor nicotinamide is proposed to bind in the C site (16). Models were generated using Web Lab Viewer Lite software.

Min et al. have suggested that because these residues are on the surface of the NAD⁺-binding pocket, they probably do not contribute to protein stability but may instead be somehow involved in catalysis. They proposed that the first step in Sir2-catalyzed deacetylation may be reminiscent of a serine protease reaction. A conformational change in NAD⁺, due to a rotation around the ester bond joining the adenine-ribose to the pyrophosphate and/or the phosphodiester bonds within the pyrophosphate moiety, would position the nicotinamide in proximity to site C. In this conformation, Ser24 may act as a catalytic base in the cleavage of the glycosidic linkage between the nicotinamide and the ribose (141) (Fig. 4A). Arguing against this model, though, Chang et al. found that mutation of Ser24 in Sir2-Af1 decreases activity by only sixfold, suggesting that this residue is not essential for activity. From an examination of the structures of these mutants, they instead conclude that site C residues play a role in NAD⁺ binding and positioning rather than in catalysis (27). In either case, hydrolysis of the acetyl group from N-acetyllysine in the second step of the reaction seems to require conserved residues in site B. Specifically, His116 (His118 in Sir2-Af2) is proposed to be directly involved in catalyzing this step (27) (Fig. 4A).

(ii) Substrate-binding site. A cleft between the Rossmann fold domain and zinc-binding domain serves as a protein substrate-binding site (9) (Fig 4B). The acetyllysine side chain appears to insert into a tunnel within this cleft, bringing it close to site B of the NAD⁺-binding region. In particular, the structure of Sir2-Af2 bound to a p53 peptide shows the acetyllysine in close proximity to the putative catalytic His118 (His116 in Fig. 4A). This is proposed to make the acetyl group of the acetyllysine a better nucleophile during deacetylation (9). Based on the available data, a current model for the mechanism of deacetylation proposes a nucleophilic attack by the carbonyl oxygen of the N-acetyl group of the substrate on the C-1' of the nicotinamide ribose (167, 199). Since several models detailing the exact chemistry of the two hydrolysis steps have been proposed (9, 27, 141), further investigation is needed to elucidate the precise mechanism in its entirety.

(iii) Substrate specificity. Close analysis of the Sir2-Af2 peptide structure may reveal insights into the substrate specificity of Sir2-like proteins. It appears from this structure that all of the contacts that anchor the substrate to its binding site on the enzyme are simple hydrogen bonds between the two peptide backbones (9). According to the authors, these interactions form an enzyme-substrate ß-sheet which they refer to as a "ß staple," since the substrate appears to link two distinct regions of Sir2-Af2. As Tanny and Moazed point out, this means that any peptide with a stretch of amino acids containing an acetylated lysine and which are flexible enough to form a ß staple can be a putative substrate for Sir2-like enzymes (199). This idea is consistent with the broad specificity displayed by many of these enzymes in vitro. Indeed, Avalos et al. tested the ability of archaeal Sir2-Af1, Sir2-Af2, human SIRT2, and yeast Sir2 to act on both acetylated histones and two monoacetylated p53 peptides. They found that all four enzymes were able to deacetylate each of the four substrates (9). While mutation of several nonconserved residues in this region did alter the specificity of Sir2-Af2, it is likely that in vivo substrate specificity is determined primarily through protein-protein interactions outside the catalytic domain of the enzyme (199).

Sir2 reaction products. (i) Nicotinamide. While a complete understanding of the chemistry underlying the reaction requires further investigation, the products of Sir2-catalyzed deacetylation have been determined with precision. It has been demonstrated that this reaction leads to production of a deacetylated lysine and two additional products in a 1:1:1 molar ratio (104, 196, 198). Cleavage of the glycosidic bond joining the nicotinamide moiety of NAD⁺ to ADP-ribose results in the release of free nicotinamide. Bitterman et al. have shown that nicotinamide is a strong inhibitor of Sir2 and have proposed that it blocks catalysis by occupying site C of the NAD⁺ binding pocket (16). This important regulatory molecule, which is a precursor of nicotinic acid in the cell and a form of vitamin B₃, is discussed in detail below.

(ii) O-Acetyl ADP-ribose. The other reaction product, which results from cleavage of the C—N bond between the acetyl group and lysine followed by transfer of the acetyl to ADP-ribose, has been the focus of considerable speculation. This follows from the fact that this unique reaction leads to the generation of two novel metabolites, namely, a regioisomer of 2'- and 3'-O-acetyl ADP-ribose (73, 167). The initial product of the transfer reaction appears to be the 2' form of this molecule, which quickly converts to 3'-O-acetyl ADP-ribose, eventually forming an equilibrium between the two (27). It has been proposed that Sir2 may initiate a signal transduction cascade through generation of these compounds, since the metabolic instability of these molecules is reminiscent of the initiators of other signaling pathways (167). This is cited as one possible rationale for the consumption of NAD⁺ during deacetylation. In support of this idea, injection of O-acetyl ADP-ribose into living cells causes a delay or block in the cell cycle and maturation of Xenopus oocytes (19). Furthermore, it has recently been demonstrated that ADP-ribose hydrolases may potentially metabolize O-acetyl ADP-ribose in Xenopus cell extracts. Interestingly, though, the predominant activities observed in these cell extracts appear to be those of novel, unidentified enzymes (156). Elucidation of the fate of these small molecules may uncover novel downstream effectors of Sir2-like proteins and shed light on the signaling pathways involved in longevity regulation and other cellular processes.

View larger version (21K): [in this window] [in a new window]   FIG. 5. Pathways for NAD+ and nicotinamide metabolism in S. cerevisiae. In yeast, NAD+ can be recycled from nicotinamide via the NAD+ salvage pathway or synthesized de novo from tryptophan. Nicotinamide generated by Sir2 is converted into nicotinic acid by the nicotinamidase Pnc1 and subsequently into NaMN by Npt1. Nicotinic acid may also enter the pathway exogenously. The formation of desamido-NAD+ (NaAD) is catalyzed by one of two adenylyltransferases encoded by NMA1 and NMA2, and the subsequent formation of NAD+ by the NAD+ synthetase Qns1. Trptophan taken up from the medium is converted into quinolinic acid by Bna1 to Bna5. A quinolinic acid phosphoribosyltransferase, encoded by BNA6/QPT1, catalyzes the subsequent conversion to NaMN, which feeds into the salvage pathway.

(ii) Salvage synthesis of NAD⁺. In yeast, the salvage pathway for NAD⁺ synthesis consists of four steps (Fig. 5). Nicotinamide produced from NAD⁺ cleavage is first converted to nicotinic acid by the nicotinamidase Pnc1 (3, 52). The specific role of this key enzyme in Sir2 regulation is discussed in detail below. Nicotinic acid may also enter the pathway exogenously, and a high-affinity transporter for this compound, Tna1, has recently been identified (120, 165). In either case, nicotinic acid is subsequently converted into NaMN by a phosphoribosyltransferase encoded by NPT1 (2). At this point, the NAD⁺ salvage and the de novo NAD⁺ synthesis pathways converge and NaMN is converted to desamido-NAD⁺ by a nicotinate mononucleotide adenylyltransferase (NaMNAT). In S. cerevisiae, there are two open reading frames (ORFs) with homology to bacterial NaMNAT genes (43, 189), named NMA1 and NMA2 (2). In Salmonella, the final step in the regeneration of NAD⁺ is catalyzed by an NAD⁺ synthetase (71). An as yet uncharacterized yeast ORF, YHR074w/QNS1, is predicted to encode an NAD⁺ synthetase.

The NAD⁺ salvage pathway has been a recent focus of attention because, unlike the de novo pathway, it is critical for Sir2 activity. Cells lacking NPT1 or PNC1, for example, show a loss of silencing at telomeres and the rDNA, reminiscent of a sir2 mutant (165). Furthermore, the replicative life span of a cdc25-10 strain (the genetic mimic of calorie restriction) is no longer extended if NPT1 is absent (114). Conversely, increased dosage of NAD⁺ salvage pathway genes increases silencing, and a single extra copy of the NPT1 gene extends the life span by 60% (2). These results suggest a model whereby up-regulation of NAD⁺ synthesis leads to an increase in cellular NAD⁺ levels and a subsequent stimulation of Sir2 activity. In support of this, NAD⁺ levels are significantly reduced in cells lacking NPT1 but not in those lacking BNA1 or BNA6, consistent with the observed silencing phenotypes of these mutants (165). Interestingly, although overexpression of NPT1 increases silencing and extends life span, NAD⁺ levels are unaltered in these strains (2).

Calorie restriction, respiration, and NAD⁺. As mentioned above, controlled fluctuations in NAD⁺ levels provide an attractive rationale for the cofactor dependence of the Sir2 reaction. But is this the mechanism by which calorie restriction affects Sir2 activity? And if so, how would limiting calorie intake affect a change in cellular NAD⁺ levels? Guarente and colleagues have proposed that this is indeed the mechanism by which Sir2 is regulated, and a recent paper by Lin et al. (115) offers a possible explanation. S. cerevisiae, a facultative anaerobe, can generate energy through either fermentation or the more energy-efficient respiration. When glucose levels are high, cells preferentially utilize fermentation, since energy is in excess. When glucose becomes limiting, though, respiration is preferred because 16 ATP molecules can be produced from a single glucose, compared to only 2 molecules from fermentation (10). Thus, carbon is shunted toward the mitochondrial tricarboxylic acid cycle, increasing electron transport and respiration (155).

By measuring oxygen consumption, Lin et al. showed that yeast grown on low glucose respire at a threefold higher rate. Furthermore, calorie-restricted yeast that cannot respire due to a mutation in their electron transport chain do not live longer than unrestricted cells. In contrast, artificially inducing genes involved in respiration through overexpression of the HAP4 transcription factor causes a Sir2-dependent extension of life span, even under high-glucose conditions (115). The authors propose a model whereby increased respiration leads to increased oxidation of NADH to NAD⁺ in the mitochondria. This change in the NAD⁺/NADH ratio would be transmitted outside the mitochondria, where it would effectively stimulate Sir2 activity.

While this is an attractive hypothesis, an overall increase in the cellular NAD⁺ concentration or in the NAD⁺/NADH ratio has yet to be observed under these conditions. In fact, we and others have been unable to detect increases in NAD⁺ levels in calorie-restricted cells (116) or in genetic mimics of calorie restriction (2), even when the unbound NAD⁺ level was measured (R. M. Anderson, K. J. Bitterman, J. G. Wood, and D. A. Sinclair, unpublished results). Furthermore, and perhaps most striking, is the fact that although cells lacking PNC1 are compromised for Sir2-dependent silencing, their NAD⁺ levels are unaltered (165). It is formally possible that Sir2 is regulated either by nucleus-specific changes in NAD⁺ availability, flux through the salvage pathway, or a different mechanism altogether.

Regulation by nicotinamide. (i) Inhibition of Sir2 by nicotinamide. One intriguing possibility, which may resolve these apparent conflicts, concerns the other product of the Sir2-catalyzed reaction, nicotinamide, produced from the cleavage of the glycosidic bond of NAD⁺. Nicotinamide was recently demonstrated to be a strong inhibitor of yeast Sir2 and human SIRT1 activity, both in vitro and in vivo (