Small molecule regulation of Sir2 protein deacetylases

Small molecule regulation of Sir2 protein deacetylases

The Sir2 family of histone/protein deacetylases (sirtuins) is comprised of homologues found across all kingdoms of life. These enzymes catalyse a unique reaction in which NAD+ and acetylated substrate are converted into deacetylated product, nicotinamide, and a novel metabolite O-acetyl ADP-ribose. Although the catalytic mechanism is well conserved across Sir2 family members, sirtuins display differential specificity toward acetylated substrates, which translates into an expanding range of physiological functions. These roles include control of gene expression, cell cycle regulation, apoptosis, metabolism and ageing. The dependence of sirtuin activity on NAD+ has spearheaded investigations into how these enzymes respond to metabolic signals, such as caloric restriction. In addition, NAD+ metabolites and NAD+ salvage pathway enzymes regulate sirtuin activity, supporting a link between deacetylation of target proteins and metabolic pathways. Apart from physiological regulators, forward chemical genetics and high-throughput activity screening has been used to identify sirtuin inhibitors and activators. This review focuses on small molecule regulators that control the activity and functions of this unusual family of protein deacetylases.

The silent information regulator 2 (Sir2) family of proteins (sirtuins) are class III histone/protein deacetylases (HDACs) [1]. Members of this evolutionarily conserved family include five homologues in yeast (ySir2 and Hst1–4) and seven in humans (SIRT1–7) [2,3], with key roles in cellular processes such as gene expression, apoptosis, metabolism and ageing [4]. The founding member, yeast Sir2 (ySir2), was originally identified as a trans-acting factor involved in transcriptional repression of the silent mating type loci in yeast [5]. Now it is well established that ySir2 deacetylase activity is required for silencing at telomeres, rDNA and the silent mating type loci, and for maintaining genome integrity [5,6]. In addition to silencing, Sir2 activity is linked to lifespan extension in yeast [7], worms [8] and flies [9]. SIRT1, the most extensively studied human Sir2 orthologue, localises to the nucleus where it negatively regulates damage-responsive Forkhead transcription factors [10–12] and p53 [13–15], promoting cell survival under stress. SIRT1 also displays tissue-specific roles including skeletal muscle differentiation [16] and fat mobilization in white adipocytes [17]. In contrast to SIRT1, SIRT2, SIRT3 and SIRT5, no NAD+-dependent protein deacetylase activity has been reported for SIRT4, SIRT6 and SIRT7. The possibility remains that SIRT4, 6 and 7 exhibit specificity toward substrates other than those tested or that these proteins catalyse a distinct reaction. In support of the latter, SIRT6 was recently shown to transfer the ADP-ribose moiety of NAD+ and undergo mono-ADP-ribosylation [18].

Unlike class I and II HDACs, which activate a water molecule for direct hydrolysis of the acetyl group [1], class III HDACs require NAD+ as a cosubstrate for the deacetylation reaction [19–22]. NAD+ and the acetylated lysine residue on the substrate react in a 1 : 1 ratio to form deacetylated product, nicotinamide, and a novel metabolite 2′-O-acetyl-ADP ribose (OAADPr) (Fig. 1) [23–26]. The consumption of NAD+ and the generation of OAADPr by class III HDACs probably serve as a link between deacetylation and other physiological processes.

The dependence of sirtuin activity on NAD+ has prompted investigations into how these enzymes might link the cellular energy state to processes such as gene expression, cell cycle regulation, apoptosis and ageing. This review will evaluate recent discoveries concerning the physiological regulation of sirtuins by NAD+ metabolites and by enzymes in the NAD+ salvage pathway. In addition, we will cover the use and efficacy of small molecule inhibitors and activators of sirtuin activity such as sirtinol, splitomicin and resveratrol with particular focus on the ability of these compounds to regulate Sir2-mediated lifespan extension.

The variety of important functions involving Sir2 enzymes underscores the need to understand the mechanisms that regulate their physiological activity. The requirement of NAD+ as a cosubstrate has led to the proposal that either intracellular NAD+ or NADH concentrations or a metabolic parameter such as the NAD+/NADH ratio regulates Sir2 activity (reviewed in [4,29,31]), effectively linking Sir2 activity to the metabolic status of cells. Originally, caloric restriction (CR) in yeast was thought to increase the NAD+ levels, which would increase the activity of ySir2 and promote its role in lifespan extension [32,33]. However, there is little data to support the assertion that global changes in cellular NAD+ and NADH during CR would have a significant impact on ySir2 activity. In yeast grown under aerobic conditions, concentrations of NAD+ and NADH were reported to be approximately 4 mm and 0.2 mm, respectively, yielding an NAD+/NADH ratio of about 20 [34]. Under caloric restriction, a condition that presumably activates Sir2, this ratio fluctuated less than twofold [35], due only to a change in NADH levels. NADH was reported to act as a competitive inhibitor of Sir2 in vitro[35], leading to a conclusion that NADH would compete with NAD+ for binding to Sir2. However, Km values for NAD+ typically fall between 10 and 100 µm, whereas IC50 values for NADH range from 11 to 28 mm[36]. Therefore, it is unlikely that NADH levels would reach high enough concentrations to significantly inhibit Sir2 activity. A dramatic drop in NAD+ levels would be more likely to be a factor in Sir2 regulation, especially if free intracellular NAD+ concentrations were to fall in the low micromolar range. Such instances could occur through activation of NAD+-consuming enzymes such as poly(ADP-ribose) polymerases (PARPs), NAD+ glycohydrolases (NADases), or perhaps mono-ADP-ribosyl transferases [37]. An important caveat to the aforementioned Sir2 studies is the fact that NAD+ and NADH levels were measured from whole cell lysates and the possibility that microdomains of these metabolites exist where ySir functions has not been explored. For instance, NAD+ synthesizing enzymes might be a part of a Sir2-containing complex and these enzymes may channel NAD+ directly to Sir2, creating a microdomain of high NAD+ concentrations specifically accessible to Sir2.

Nicotinamide, a product of the Sir2 deacetylation reaction, is a potent physiological inhibitor of Sir2 enzymes [36,38,39]. In vitro, nicotinamide yields an IC50 of ≈ 120 µm with several Sir2 homologues [36]. Originally, it was believed that nicotinamide bound to an allosteric site and consequently inhibited Sir2 activity [40]. However, it was shown later that nicotinamide inhibition arises from its ability to condense with a high-energy enzyme–ADP ribose–acetyl-lysine intermediate to reverse the reaction, reforming NAD+ and thereby inhibiting product formation [38,39]. Nicotinamide acts as a classical noncompetitive product inhibitor of the forward deacetylation reaction and was shown in vivo to decrease gene silencing, increase rDNA recombination and accelerate ageing in yeast [40]. Because nuclear nicotinamide levels are estimated to be 10–150 µm[41], it is likely that nicotinamide regulates Sir2 activity in vivo.

In summary, we suggest that small molecule regulation of sirtuins involves the cellular balance of NAD+ to nicotinamide, controlled by enzymes involved in NAD+ synthesis or salvage. Small global alterations in NAD+ levels would provide insufficient changes in Sir2 activity, but microdomains of NAD+ produced on location may be an effective regulatory mechanism. We predict that some of these NAD+ synthetic enzymes might be components of sirtuin complexes, channelling NAD+ directly to Sir2 enzymes.

Resveratrol was reported to be a general sirtuin activator; however, recent reports question the validity of that proposal and that resveratrol-dependent lifespan increases are mediated directly by ySir2 activation. Although mammalian SIRT1 appears to be activated by resveratrol treatment, the mechanistic basis for this cellular phenomenon remains to be elucidated.

Small molecule inhibitors (such as splitomicin and sirtinol) were identified based on phenotypic screening for compounds that phenocopy a ySir2 yeast deletion. So far, these compounds only inhibit at the micromolar level, and a full evaluation of their selectivity for other sirtuins has not been determined. Future rational inhibitor design and direct high-throughput screening against all sirtuins, particularly the mammalian homologues, undoubtedly will lead to the development of highly selective and potent inhibitors. These compounds will provide an essential tool to uncover the cellular functions of these enzymes and may lead to therapeutics that target individual sirtuins.

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