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(Last modification: 25. October 2010)

Sirtuins: arrangement of pages

• This page: Sirtuins Overview: Structure, Enzyme Activities, Functions; Calorie Restricted Diet

• Sirtuin Activators (for example Resveratrol)

• Sirtuin Inhibitors (Cancer suppression by inhibitors?)

• Sirtuins: Selected References

• Sequences of human SIRT proteins

Sirtuins Overview: Structure, Enzyme Activities, Functions

Note: most of this was extracted from three excellent reviews: Michan and Sinclair (2007), Alcaín and Villalba (2009a, 2009b); a few up to date citations were added.

   The number of publications on sirtuins is huge, and seems to increase daily. It would therefore be futile to attempt here a comprehensive review of the literature. The reviews cited above (all recent) do an excellent job of that, although some of that might already be outdated even at the time of writing this page (November 2009).

   The summary given here will not cover the pysiological roles of sirtuins; that should be the privilege of the specialists in the field. I will simply give an overview of the proteins in humans, the enzyme reactions, and I will briefly dwell on the role of sirtuins in the benefits of calorie restricted diets.

   Sirtuins were first described in yeast, under various names, and even the early results showed already  that the proteins had important functions in metabolism, cell cycle and mating in these 'simple' organisms. The protein discovered first was named Sir2, the abbreviation of 'silent information regulator'. Later studies showed that Sir2 is a member of large family of proteins that occur in all organisms, that is, not only in fungi, but also in bacteria, animals, and plants (Frye, 2000). The members of this large and ancient protein family are now called 'sirtuins'.

   The human sirtuins have attracted considerable interest because of the immense importance of sirtuins for health and longevity that emerged in the last few years. I will attempt to give a very simple overview of these proteins and their enzymatic activities in humans. A few important points are summarized in Table 1.

Table 1.

Human Sirtuins (SIRT): overview of proteins, size, isoforms, enzyme functions, and localization.

[Basic design from Michan and Sinclair (2007), modified].

• Isoforms (schematic), (AA = amino acids)
  SIRT1: isoform b is from an alternate in frame exon in the 5’-coding region and uses a downstream start codon, result:
             isoform b is shorter at the N-terminus
  SIRT2 isoforms: Isoform a contains an additional 37 AA at the N-terminus, by using an alternative start codon
  SIRT3 isoforms: transcript variants from alternative splicing, isoform b is shorter at the N-terminus
  SIRT5 isoforms: transcript variants from alternative splicing, isoform a is longer at the C-terminus

• Accessions of the proteins, with links to the data files
  These data files contain a large number of additional references

• Enzyme functions
  DAC, NAD⁺ Dependent Deacetylase
  ART, Mono-ADP-Ribosyl Transferase

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    Humans contain seven different sirtuins. All share a conserved catalytic core domain of about 275 amino acids, but otherwise they can be rather different in size. If you check up in the databases, the situation is not as simple as described in some reviews: several of the genes can express different protein isoforms, resulting from the use of alternative start codons (SIRT1, SIRT2) or from alternative splicing (SIRT3, SIRT5). The Table contains links to the protein sequences if you want to look directly at the protein files; the data files also contain additional citations that might be interesting to look at.

   Importantly, the cellular localizations are very different: Some are in the nucleus, one may be in both nucleus and cytoplasm (SIRT1), one is mostly in the cytoplasm, but has also been found in the nucleus (SIRT2), and three appear to be exclusively in mitochondria. However, such a strict division should be taken with caution because the distribution may be variable in different tissues/cell types.

   The enzyme functions are also rather interesting: the proteins are either deacetylases (DAC) or mono-ADP-ribosyl transferases (ART) or both. Both are strictly dependent on NAD⁺ .The reactions are summarized in the scheme below (Fig. 1). Apparently the enzyme activities of SIRT7 in vitro are not clear, although it definitely has functions in vivo.  It is noteworthy that most of the studies looking at the physiological roles consider only the DAC activity, and in some reviews the human sirtuins are simply summarized as deacetylases.

Fig. 1.

Enzyme activities of human sirtuins.

These proteins have either deacetylase (DAC) or mono-ADP-ribosyl transferases (ART) activities or both. Both reactions are strictly dependent on NAD⁺. It is not quite clear whether the ART activities are really enzymatic: more...
See Michan and Sinclair (2007) for review (reference at end of page).

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   What is so interesting about these reactions, apart from the unusual biochemistry? It has to do with the fact that they have to do with important regulations in the organisms, and probably the most important are those having to do with health and longevity. At least one of these aspects should be summarized here, without claim of being complete or up to date.

Beneficial effects of calorie restriction: Sirtuins may play a decisive role

   Calorie restriction, i.e. a diet in which an organism receives about 20% (or even 40%) fewer calories than it would consume if given free reign, is the only non-genetic method that is proven to increase the lifespan in all organisms tested: it was demonstrated with Yeast (Saccharomyces cerevisiae) (Jiang et al., 2000), Fruit flies (Drosophila melanogaster) (Mair et al., 2003; Grandison et al., 2009), Nematodes (Caenorhabditis elegans)  (Lakowski and Hekimi, 1998), Crustaceae (Daphnia longispina) (Ingle et al., 1937),  Spiders (Frontinella pyramitela) (Austad, 1989), und Mice (Weindruch and Walford, 1982). Okay, mice are not humans, but recent results demonstrate that this is also true for rhesus monkeys (Colman et al., 2009; Rezzi  et al., 2009), and these are evolutionary not so far away from humans that one would expect basis differences in these mechanisms.

These results suggest that the mechanism might be very old, well conserved, and similar in all organisms.   

   However, other reports claim that the effect of calorie restriction is not universal, but species-specific (Mockett et al., 2006), and they cite the house-fly (Musca domestica) as example that it did not extend lifespan in this species (Cooper et al., 2004).

   A recent study investigated the consequences of calorie restriction in male mice on a much broader level, by analysis of microarray expression data (Estep et al., 2009). The results showed that calorie restriction significantly changed the expression of over 3,000 genes, many between 10- and 50-fold. A general conclusion was that the life-extension effect in males might arise partly from a shift of gene expression to those patterns typical for females which live longer anyway than males (I wonder how readily some people will do what is standard: apply the conclusions to humans?).

   A few recent reviews on calorie restriction and aging are here: more...

   A role for sirtuins was shown first in yeast (Kaeberlein et al., 1999; Lin et al., 2000; Anderson et al., 2003). Essentially the same results were also reported for metazoans. For example, Caenorhabditis elegans (a model organism for developmental studies: Wikipedia) with a chromosome duplication containing a second copy of sir-2.1 (the worm sirtuin most similar to the yeast sir2), lived up to 50% longer than the worms not containing this duplication (Tissenbaum and Guarente, 2001; Wang and Tissenbaum, 2006). The same was demonstrated for Drosophila (Rogina and Helfand, 2004), and there is evidence that this is also true for mammals (see e.g. Cohen et al., 2004; Bordone et al., 2007; Pfluger et al., 2008).

   It should be pointed out, however, that not all scientists agree with the idea that sirtuin functions are always beneficial: they argue, and there is evidence for it, that sirtuins can play both protective and pro-aging roles: reviewed in Longo, 2009.

See also these pages: Sirtuin Activators and Sirtuin Inhibitors.

I would like to add a quote from a commentary paper that was published end of 2008 (Kaeberlein, 2008), with permission of the author:

   So what’s the ‘‘take-home message’’ from all this? Is more SirT1 good, or is less SirT1 good? The answer, as is often the case in biology, is that  there’s no simple answer. Activating SirT1 is probably a good thing in some cells under some conditions and is probably a bad thing in other cells under other conditions. SirT1 activators may be good for diabetes but may cause cancer due to p53 inhibition, SirT1 inhibitors may protect against cancer but cause metabolic disease, and there is evidence supporting the idea that both activators and inhibitors of SirT1 can confer protection against neurodegeneration in different contexts. The one thing that seems clear is that sirtuin activators are unlikely to be a ‘‘magic bullet’’ for aging. A more realistic hope is that, as we continue to unravel the complexities of sirtuin biology, targeted activation or inhibition of SirT1—and perhaps other sirtuins as well—will prove therapeutically useful toward a subset of age-associated diseases. Such an achievement would be a huge step forward in the transition of aging-related science from the laboratory to the clinic, and we eagerly await the next chapter in the unfolding saga that is sirtuin biology.

Have a look at comments in the pages on Sirtuin Activators (more...) and Sirtuin Inhibitors (more...)

Have a look at these pages:

• Sirtuin Activators (for example Resveratrol)

• Sirtuin Inhibitors (Cancer suppression by inhibitors?)

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Note to ADP-ribosylation (ART) activities

   These activities of the sirtuins appears to be a bit of an enigma. For example with the yeast Sir2, the sirtuin prototype: the first reported activity was indeed that of ART (Tanny et al., 1999), long before the discovery of the biologically relevant deacetylase (DAC) activity. The same apparently happened with the mammalian SIRT6: it was first described as ART, without detectable  DAC activity with standard substrates (Liszt et al., 2005), and it was identified only recently as DAC with a very specific substrate, lysine 9 in histone H3 (Michishita et al., 2008; Kawahara et al., 2009).
   There are general doubts whether the ART activity of the sirtuins is truly enzymatic. It was proposed, for example, that it is a non-enzymatic reaction of the 2'-O-acetyl-ADP-ribose liberated by the DAC activity (see for example Smith and Denu, 2006; Kowieski et al., 2008), and there is good evidence that this happens. This can explain the ART activity as a side reaction of the DAC activity, but that obviously runs into problems in cases where there is ART, but no detectable DAC activity.
   The case in question is SIRT4, one of the three mitochondrial sirtuins. Haigis et al. (2006) described that SIRT4 does not display NAD⁺-dependent deacetylase activity but instead uses NAD⁺ to ADP-ribosylate glutamate dehydrogenase (GDH, it desaminates glutamate to oxoglutarate), and the ADP-ribosylation activity was also found by others (Ahuja et al., 2007).
    However, there are some very interesting discussions on the enzyme mechanisms that can explain that: have a look at these publications: Smith et al., 2008; Du et al., 2009. As discussed by them, an imidate intermediate plays a key role in the formation of all reaction products; a simplified scheme is shown in Figure 2. Based on such models, these authors propose that the ADP-ribosylated products are derailments of the imidate in absence of the correct acetylated substrate: it is possible to obtain the intermediate, but it cannot be processed properly.

   Whatever the final conclusion may be: ADP-ribosylation has been known for a long time: more..., and it will be very important to know whether the ART activities of the sirtuins are truly enzymatic and happen in vivo, and if so, whether they have any physiological importance. This information will also be very important if one plans to influence sirtuin activities, e.g. by Sirtuin Activators and Sirtuin Inhibitors.

Fig. 2.

A common intermediate can explain the different activities of sirtuins.

The key intermediate is an imidate (boxed orange). It can be processed into different products, depending on the attacking nucleophiles:

a) deacetylation (boxed black): this is the major reaction in presence of the correct acetylated substrate, under these conditions b) and c) play no significant role;

b) ADP-ribosylation and c) liberation of ADP-ribose: these pathways are always minor in terms of absolute rates, but they can increase if the deacetylation is blocked in any way, e.g. the absence of the right substrate. However, the absolute rates seem to be always very small, when compared with the DAC activity.

See Du et al. (2009) for a more detailed discussion; the scheme shown here is a modified, simplified version of Fig. 9 in that publication.

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 References for the reviews cited on top of the page

• Alcain, F. J., Villalba, J. M., 2009a. Sirtuin activators. Expert Opinion on Therapeutic Patents 19, 403-414.
  Background: Sirtuin 1 - 7 (SIRT1 - 7) are deacetylases that are dependent on NAD⁺ for their activity. SIRT1 down-regulates p53 activity, increasing lifespan, cell survival, and neuroprotection; it also deacetylates peroxisome proliferator-activated receptor-gamma and its coactivator 1α, promoting fat mobilization, increasing mitochondrial size and number, and positively regulating insulin secretion. Sirtuins link nutrient availability and energy metabolism. Calorie restriction, which increases lifespan and is beneficial in age-related disorders, activates sirtuin. Major efforts are thus focused to developing sirtuin activators.
  Objective: After discussing the potential involvement of sirtuins in pathophysiological processes, this review looks at new, synthetic sirtuin activators.
  Conclusions: To date, resveratrol is the most potent natural compound able to activate SIRT1, mimicking the positive effect of calorie restriction. Resveratrol might help in the treatment or prevention of obesity and in preventing the aging-related decline in heart function and neuronal loss. As resveratrol has low bioavailability and interacts with multiple molecular targets, the development of new molecules with better bioavailability and targeting sirtuin at lower concentrations is a promising field of the medicinal chemistry. New SIRT1 activators that are up to 1000 times more effective than resveratrol have recently been identified. These improve the response to insulin and increase the number and activity of mitochondria in obese mice. Human trials with a formulation of resveratrol with improved bioavailability and with a synthetic SIRT1 activator are in progress.
  (Note: In Fig. 3, the structures of fisetin and quercetin lack an H-atom at the single bonded oxygen in the 3-position. Also, although it is formally correct to call them flavones, in the nomenclature used by most plant scientists they are called 'flavonols' because of the -OH group in the 3-position. In Fig. 6, three O-CH₃ groups at the 'top' aromatic ring are missing).
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• Alcain, F. J., Villalba, J. M., 2009b. Sirtuin inhibitors. Expert Opinion on Therapeutic Patents 19, 283-294.
  BACKGROUND: The sirtuin family of deacetylase enzymes comprises seven proteins (SIRT1-7) that are dependent on NAD⁺ for their activity. Three proteins are located in the nucleus, three in the mitochondria and only one is predominantly cytoplasmic. Caloric restriction and oxidative stress generally up-regulate their expression. SIRT1, the orthologue of yeast Sir2, is the mammalian sirtuin that has been most extensively studied to date. Among other targets, SIRT1 down-regulates the activity of the nuclear transcription factor p53, being this related with an increase in lifespan and cell survival associated to stress resistance.
  OBJECTIVE: Because sirtuin modulation could have beneficial effects on several human diseases, there is a growing interest in the discovery and development of small molecules that modify its activity. This review will be focused on sirtuin inhibitors.
  CONCLUSIONS: Several specific inhibitors of SIRT1 have been described. These compounds could be mainly useful for the treatment of cancers by increasing p53 activity that stops the formation of tumours and induces apoptosis. A p53-independent massive induction of apoptosis has been also described for one inhibitor. In addition, a potent and selective SIRT2 inhibitor that ameliorates the alpha-synuclein fibril formation in Parkinson disease has been proposed to treat this kind of neurodegenerative disease.
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• Michan, S., Sinclair, D. A., 2007. Sirtuins in mammals: insights into their biological function. Biochemical Journal 404, 1-13.
     Sirtuins are a conserved family of proteins found in all domains of life. The first known sirtuin, Sir2 (silent information regulator 2) of Saccharomyces cerevisiae, from which the family derives its name, regulates ribosomal DNA recombination, gene silencing, DNA repair, chromosomal stability and longevity. Sir2 homologues also modulate lifespan in worms and flies, and may underlie the beneficial effects of caloric restriction, the only regimen that slows aging and extends lifespan of most classes of organism, including mammals. Sirtuins have gained considerable attention for their impact on mammalian physiology, since they may provide novel targets for treating diseases associated with aging and perhaps extend human lifespan. In this review we describe our current understanding of the biological function of the seven mammalian sirtuins, SIRT1 - 7, and we will also discuss their potential as mediators of caloric restriction and as pharmacological targets to delay and treat human age-related diseases.
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A few recent reviews or articles on calorie restriction and aging

Visits since November 7, 2009

Idaho Web Design Tools

File History:

• 25.10.2010: Addition of a few recent reviews on calorie restriction

• 13.11.2009: Discussion of ART activity

• 03.11.2009: Design of page

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