Sirtuins in mammals: insights into their biological function

DISCOVERY OF THE SIRTUINS

It is a little known fact that the first sirtuin gene, SIR2 from Saccharomyces cerevisiae, was originally known as MAR1 (for mating-type regulator 1). Klar and collegues [1] discovered MAR1 by virtue of a spontaneous mutation that caused sterility by relieving silencing at the mating-type loci HMR and HML. A variety of additional mutations with a sterile phenotype were co-discovered by Jasper Rine, who named the set of four genes responsible SIR (silent information regulator)1–4 [2-4], thereby replacing the MAR nomenclature.

Gottlieb and Esposito [5] demonstrated, 10 years after this initial finding, that SIR2 is the only SIR gene required to suppress recombination between the 100–200 copies of the ribosomal RNA genes repeated in tandem on chromosome XII. By 1991, thanks primarily to work by Gottschling and colleagues [6], SIR2 was also known to be part of the mechanism that silences genes near telomeres. About 2 years later, Braunstein et al. showed that silent regions at telomeres and mating-type loci are associated with histones that are relatively hypoacetylated at the ε-amino group of N-terminal lysine residues [7]. SIR2 overexpression caused substantial histone deacetylation, an additional characteristic that distinguished SIR2 from the other SIR genes.

In 1995, Brachman et al. [8] and Derbyshire et al. [9] discovered four additional S. cerevisiae genes with high homology to SIR2, HST (homologues of SIR2)1–4. None of the four genes were essential, but they all were involved in silencing at the mating-type loci and telomeres, as well as cell-cycle progression and genomic integrity. The finding of SIR2 homologues in yeast and shortly thereafter in organisms ranging from bacteria to plants and mammals, demonstrated SIR2 is a member of a large and ancient family of genes we now refer to as ‘sirtuins’.

SIRTUINS ARE NAD⁺-DEPENDENT DEACETYLASES AND MONO-ADP-RIBOSYL TRANSFERASES

Over the next 2 years a variety of interesting features about Sir2 were discovered: it localizes to the nucleolus [10], it is a component of the Ku-associated apparatus that repairs double-stranded DNA breaks [11], it localizes to DNA breaks in a check-point-dependent manner [12-14] and it silences marker genes and alters chromatin structure at the rDNA (ribosomal DNA) locus [15,16]. The first insight into Sir2 enzymatic activity came from the characterization of CobB, a Salmonella typhimurium protein with nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyl transferase activity. CobB contains a conserved diagnostic feature of the sirtuins: the amino acid sequences GAGISAESGIRTFR and YTQNID (conserved residues are underlined) [17]. By 1999, Roy Frye had identified five of the human SIR2 homologues, SIRT1–5, given the name ‘sirtuins’, and found that SIRT2 could act as an ADP-ribosyl transferase using, as a donor, one of the major nicotinamide nucleotides, NAD⁺. The transfer of an ADP-ribose from radioactively labelled NAD⁺ to BSA, and the loss of catalysis when a highly conserved amino acid in the Sir2 homology domain (H171Y) was mutated, were both good indicators that this family of proteins might act as ADP-ribosyl transferases [18]. Only a few months later, Moazed and colleagues [19] found that the yeast Sir2 had the ability to covalently modify a mixture of histones and itself using NAD⁺ as a donor, via the transfer of ADP-ribose to acceptor amino acids. Accordingly, the analogous mutation in yeast, H364Y, abolished silencing at the mating-type locus, telomeric DNA and rDNA locus. In light of these findings, researchers suspected that ADP-ribosylation of histones by Sir2 interfered with histone acetylation, which explained the higher acetylation and loss of silencing in sir2 mutants.

A few months later, however, two groups showed that Sir2 has histone deacetylase activity that is absolutely dependent on NAD⁺ [20,21]. Acetylated lysine residues at positions 9 and 14 of histone H3 and specifically Lys¹⁶ of histone H4 were found to be in vivo targets, and the H364Y mutation abolished SIR2 deacetylation. Furthermore, a G270A mutant that was defective in ADP-ribosyl tranferase activity, but retained 80 % of its deacetylase activity, proficiently silenced and suppressed rDNA recombination. Thus, Imai et al. [21] concluded that deacetylation rather than mono-ADP-ribosylation was the means by which Sir2 regulated processes in vivo. NAD⁺-dependent deacetylase activity in vitro has also been described for numerous other sirtuins, including bacterial CobB, archeabacterial SIR2-AF (Archaeoglobus fulgidus) and human SIRT1–3 and 5 [22].

By the end of 2000, Landry et al. [23] had described the catalytic mechanism of deacetylation: they found it to be tightly coupled to the generation of a unique acetyl-ADP-ribose metabolite, OAADPr (2′-O-acetyl-ADP-ribose). Hydrolysis of one NAD⁺ molecule produced one molecule of nicotinamide, and one of OAADPr (Figure 1) [24]. The subsequent X-ray crystallographic structures of various sirtuins [including archaeal Sir2Af1 and Sir2Af2, bacterial cobB and Sir2Tm (Thermotoga maritime), human SIRT2 and yeast Hst2; as well as sirtuin core domains in complex with NAD⁺, ADP-ribose, OAADPr or p53 acetyl lysine peptide] have helped us to understand the sirtuin deacetylation mechanism in greater detail (reviewed in [25,26]).

The ADP-ribosyl transferase activity of sirtuins was considered a low efficiency side-reaction due to the partial uncoupling of intrinsic deacetylation and acetate transfer to ADP-ribose [24]. Recently, however, mono-ADP-ribosyl transferase activity has been found to be the main enzymatic activity of at least two of the mammalian sirtuins, SIRT4 and SIRT6 (Figure 1), as discussed below.

In the last 6 years, close to 500 publications have described different aspects of sirtuin biology, ranging from small molecule agonists to finding molecular targets. Functions for almost all of the mammalian sirtuins have now been ascribed, although many more probably await discovery. The requirement for NAD⁺ as a co-substrate suggested that the sirtuins might have evolved as sensors of cellular energy and redox states coupled to the metabolic status of the cell. The generation of OAADPr, a unique by-product of the NAD⁺ dependent deacetylation reaction, raised this molecule as a candidate for sirtuin-triggered signalling pathways, another prediction that has been proven experimentally.

SIRTUINS AND AGING IN NON-MAMMALIAN SPECIES

In 1997, Sinclair and Guarente [27] showed that one of the major causes of aging in S. cerevisiae is the exponential accumulation of ERCs (extrachromosomal rDNA circles). ERCs result from homologous recombination between rDNA repeats, and the introduction of a single ERC into a young cell is sufficient to cause premature aging. In addition, mutation of the SGS1 gene, a homologue of the premature aging disease gene, WRN (Werner syndrome), led to increased rDNA instability and decreased longevity [28]. Consequently, Kaeberlein et al. [29] tested whether extra copies of SIR2 could suppress rDNA recombination and hence extend lifespan. In 1999, they showed that the integration of an extra copy of the SIR2 gene increased the replicative lifespan up to 30 % and that deletion of SIR2, which accelerates ERC formation, shortens lifespan. Interestingly, the replication fork gene (FOB1), which reduces rDNA recombination and ERC formation, largely suppressed the short lifespan of a sir2 mutant. Today, some laboratories study sir2 mutants in the context of a fob1 deletion so that ERCs do not overwhelm the cells before they can be analysed for SIR2-independent longevity pathways.

Metazoans also live longer with extra copies or expression of SIR2, but the mechanism through which SIR2 homologues retard aging remains unknown. Caenorhabditis elegans carrying a chromosome duplication containing sir-2.1, the worm sirtuin most similar to yeast SIR2, exhibits up to a 50 % lifespan increase, and this is strictly dependent on the forkhead transcriptional factor DAF-16, the downstream target of the insulin/IGF (insulin-like growth factor)-1 signalling pathway [30]. Recently, SIR-2.1 was shown to associate with 14-3-3 proteins, which direct its interaction with DAF-16 in an insulin/IGF-1-independent manner under stress conditions [31]. Whether or not this interaction occurs in mammals is unknown, but, given the conservation of the sirtuin, 14-3-3 and Daf16 (abnormal dauer formation 16)/FOXO gene families, it seems very likely. In Drosophila melanogaster, an extra copy of the Sir2 gene (dSir2) expressed ubiquitously causes females and males to live longer by 29 % and 18 % respectively. Likewise, overexpressing dSir2 in the nervous system produced a considerable lifespan extension in both fly genders [32]. Expressing Sir2 specifically in motor neurons did not extend lifespan, however.

THE MAMMALIAN SIRTUINS: SIRT1–7

Although we do not yet know whether SIRT1 or the other mammalian sirtuins regulate health and longevity, knowing the variety of functions of these proteins will certainly give important insights into which biological functions and systems they may influence, and point towards which diseases they may be useful in treating. The mammalian sirtuin family consists of seven members, SIRT1–7. Each sirtuin is characterized by a conserved 275 amino acid catalytic core domain and also by unique additional N-terminal and/or C-terminal sequences of variable length (Figure 2).

Phylogenetic analysis of 60 core domains from different eukaryotes and prokaryotes places the mammalian sirtuins into four different classes (I—IV). SIRT1, SIRT2 and SIRT3 are known as Class I sirtuins, which groups all yeast sirtuins and also at least one of the Sir2-related proteins in most eukaryotes. Class I is divided in three sub-classes: a, b and c. SIRT1 shares the same Class Ia with Sir2 and Hst1 from S. cerevisiae, C. elegans SIR-2.1 and D. melanogaster D.mel1. SIRT2 and SIRT3 reside in Class Ib, together with yeast Hst2, fly D.mel2 and other fungi and protozoa sirtuins. SIRT4 is part of Class II, which also includes sirtuins from bacteria, insects, nematodes, mould fungus and protozoans. SIRT5 is the mammalian member of Class III sirtuins, distributed widely in all prokaryotes either bacteria or archaea. Finally, Class IV contains SIRT6 and SIRT7 in two different sub-classes IVa and IVb respectively; and unlike Class III, sirtuins of this class are not present in prokaryotes, but are broadly distributed in metazoans, plants and vertebrates. Thus sirtuins from Class II and Class III together with Class U (which groups bacterial Sir2 homologues with undifferentiated motifs that are intermediate between classes II and III, and classes I and IV) seem to be the ones that appeared earliest in evolution (Figure 3) [33].

Mammalian sirtuins also differ in their sub-cellular localization. SIRT1, SIRT6 and SIRT7 are predominately in the nucleus (although SIRT1 does have some important cytoplasmic functions as well). In the nucleus a large fraction of SIRT1 is associated with euchromatin, whereas SIRT6 associates with heterochromatin and SIRT7 is found in the nucleolus [34]. The sirtuin that resides most prominently in the cytoplasm is SIRT2 [18,35]. SIRT3, SIRT4 and SIRT5 have been described as mitochondrial sirtuins. In terms of activity, SIRT1 and SIRT5 exhibit robust and weak deacetylase activity respectively [35,36]. SIRT4 and SIRT6 are mono-ADP-ribosyl tranferases [37,38], and both deacetylase and mono-ADP-ribosyl tranferase activities have been detected for SIRT2 and SIRT3 [18,35,39]. No robust activity has been found for SIRT7 as yet (Figure 2).

SIRTUINS MAY UNDERLIE THE BENEFICIAL EFFECTS OF CALORIC RESTRICTION

CR (calorie restriction) is a dietary regimen in which an organism is provided with at least 20 % fewer calories than it would naturally consume ad libitum, while maintaining adequate nutrition [40]. Although it is not correct to say that CR extends the lifespan of every organism tested, it comes close. CR-mediated lifespan extension has been demonstrated in organisms from very different taxa, including yeast (S. cerevisiae) [41], fruit flies (D. melanogaster) [42], nematodes (C. elegans) [43], crustaceans (Daphnia longispina) [44], spiders (Frontinella pyramitela) [45] and rodents [46]. This diversity argues that the mechanisms underlying CR are ancient, relatively simple and well conserved.

CR is the only non-genetic method that consistently increases maximal lifespan in mammals. It does so not by keeping animals in a unhealthy state for a longer time, but by actually retarding age-related deterioration, such as by decreased collagen elasticity, development of insulin resistance, a decline in immune function, neuro-behavioural impairments and by delaying the onset (and incidence) of age-related diseases such as cancer and auto-immune disorders [47]. Although it is unknown whether CR also extends lifespan in primates, ongoing studies in rhesus monkeys (Macaca mulatta) show many benefits from CR, including reduced body weight, lower body fat, decreased blood glucose and lower incidence of diabetes and heart disease. They also show altered hormonal profiles that do not compromise circadian patterns or reproductive timing [48].

A role for sirtuins in mediating CR was first demonstrated in S. cerevisiae. On medium with 0.5 % glucose versus standard 2 % (w/v) glucose, which has been defined as CR for yeast cells [49,50], the replicative lifespan was approx. 30 % longer. Strikingly, this effect is not seen if cells lack the SIR2 gene [32,49,51]. When cells become starved, sirtuin-independent pathways, such as TOR (target of rapamycin), are activated and can also extend lifespan [52]. At least two of the other four yeast sirtuins, Hst1 and Hst2 also participate in the CR response [51], indicating that the entire sirtuin family may control lifespan, perhaps having evolved from a primordial sirtuin that responded to stress and calorie availability. In Drosophila, lifespan extension by dietary restriction also requires the Sir2 gene and overexpression of Sir2 extends lifespan, paralleling the yeast data [32]. These findings have led credence to the hypothesis that sirtuins underlie the beneficial effects of caloric restriction in diverse species, including mammals. Further support for this hypothesis has come from studying the mammalian sirtuins [53], which as we will discuss below, have varied functions that are consistent with the physiological changes observed in rodents and primates on a CR diet.

FUNTIONS OF THE MAMMALIAN SIRTUINS

STACS (SIRTUIN ACTIVATING COMPOUNDS)

The fact that additional SIR2 extends the lifespan of diverse organisms prompted a search for STACs (sirtuin activating compounds) that might extend lifespan in the absence of CR or genetic manipulation. A series of STACs, including resveratrol, a small plant molecule synthesized in response to stress, were identified as activators of mammalian SIRT1 in vitro [132]. Consequently, this polyphenolic compound found naturally in the skin of grapes, and a variety of other plants, has been shown to extend lifespan in organisms of different phyla, such as yeast, worms, flies and fish [133-136]. Resveratrol only extends lifespan when the SIR2 gene is present, i.e. resveratrol has no apparent effect when the SIR2 gene is deleted. Experiments in different mammalian cell lines show that resveratrol modulates a variety of cellular process in a SIRT1-dependent manner, including neuroprotection, tumour suppression, differentiation and inflammation (reviewed in [137-139]).

Two recent studies have addressed the effects of resveratrol in mammals in vivo. Baur et al. [139] showed that, when this polyphenol is administrated in a daily dose of 22.4 mg/kg to middle-aged mice on a high-calorie diet, the animals displayed a shift in their physiology towards that of mice on standard diet, with increased mitochondria, lower blood glucose and insulin, and a hepatic gene expression profile matching that of lean mice, concomitant with increased SIRT1 and PGC1-α activity. Similarly, Lagouge et al. [114] showed that mice fed with a diet that induced obesity supplemented with resveratrol at a dose 20 times higher than the former study (400 mg/kg) were actually leaner than the controls that did not receive resveratrol. They also had increased oxidative-type muscle fibres, enhanced resistance to muscle fatigue, lower blood glucose and insulin, and a greater tolerance to cold. The activities of SIRT1 and PGC-1α were also increased by resveratrol and, even more interestingly, three single nucleotide polymorphisms in the SIRT1 gene of Finnish human subjects were significantly associated with systemic energy expenditure, now implicating SIRT1 as a regulator of energy and metabolic homoeostasis in humans.

PERSPECTIVES

When SIR2 was first discovered in yeast 27 years ago, it could hardly have been anticipated how much interest would be taken in this family of proteins. Several studies over the last 7 years have illuminated our understanding of the role of sirtuins in different organisms. Currently, biological functions ranging from DNA repair to metabolism have been attributed to six of the seven sirtuins found in mammals, with SIRT1 being the most extensively studied.

Arguably, some of the most noteworthy and intriguing discoveries in recent years have been the polymorphisms in human sirtuin genes, which suggest a role for sirtuins in human health and longevity. Allele variants of SIRT1 are associated with energy expenditure and a variant of the SIRT3 gene, specifically in an enhancer within intron 5, correlates with male lifespan beyond 90 years [140].

Many questions remain to be answered, such as whether sirtuins regulate aging in mammals, or if resveratrol effects are indeed SIRT1-dependent. As researchers mine through existing human genome databases and patient records, together with additional studies in conditional knockout and transgenic mice, as well as disease models, these questions may be answered. Finally, all this will give us a far clearer perspective as to whether the sirtuins may provide novel therapies to alleviate age-associated changes, such as diabetes, cancer and cardiovascular disease, and possibly extend healthy human lifespan.