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.     

Insights into the sirtuin mechanism from ternary complexes containing NAD+ and acetylated peptide

Sirtuin proteins comprise a unique class of NAD+-dependent protein deacetylases. Although several structures of sirtuins have been determined, the mechanism by which NAD+ cleavage occurs has remained unclear. We report the structures of ternary complexes containing NAD+ and acetylated peptide bound to the bacterial sirtuin Sir2Tm and to a catalytic mutant (Sir2Tm(H116Y)). NAD+ in these structures binds in a conformation different from that seen in previous structures, exposing the alpha face of the nicotinamide ribose to the carbonyl oxygen of the acetyl lysine substrate. The NAD+ conformation is identical in both structures, suggesting that proper coenzyme orientation is not dependent on contacts with the catalytic histidine. We also present the structure of Sir2Tm(H116A) bound to deacteylated peptide and 3'-O-acetyl ADP ribose. Taken together, these structures suggest a mechanism for nicotinamide cleavage in which an invariant phenylalanine plays a central role in promoting formation of the O-alkylamidate reaction intermediate and preventing nicotinamide exchange.     

A link between transcription and intermediary metabolism: a role for Sir2 in the control of acetyl-coenzyme A synthetase

The silent information regulator protein (Sir2) and its homologs (collectively known as sirtuins) are NAD+-dependent deacetylase enzymes involved in chromosome stability, gene silencing and cell aging in eukaryotes and archaea. The discovery that sirtuin-dependent protein deacetylation is a NAD+-consuming reaction established a link with the energy generation systems of the cell. This link to metabolism was recently extended to the post-translational control of the activity of short-chain fatty acyl-coenzyme A (adenosine monophosphate-forming) synthetases in bacteria and yeast. The crystal structure of the Sir protein complexed with a peptide of a protein substrate provided insights into how sirtuins interact with their protein substrates.     

Conserved metabolic regulatory functions of sirtuins

Silent information regulator 2 (Sir2) proteins, or sirtuins, are protein deacetylases/mono-ADP-ribosyltransferases found in organisms ranging from bacteria to humans. Their dependence on nicotinamide adenine dinucleotide (NAD+) links their activity to cellular metabolic status. In bacteria, the sirtuin CobB regulates the metabolic enzyme acetyl-coenzyme A (acetyl-CoA) synthetase. The earliest function of sirtuins therefore may have been regulation of cellular metabolism in response to nutrient availability. Recent findings support the idea that sirtuins play a pivotal role in metabolic control in higher organisms, including mammals. This review surveys evidence for an emerging role of sirtuins as regulators of metabolism in mammals.     

Metabolite of SIR2 reaction modulates TRPM2 ion channel

The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins are protein deacetylases that regulate gene expression, apoptosis, metabolism, and aging. These NAD+-dependent enzymes catalyze a reaction in which the acetyl group from substrate is transferred to the ADP-ribose portion of NAD+ to form deacetylated product, nicotinamide, and the metabolite OAADPr, whose functions remain elusive. Using cell-based assays and RNA interference, we show that puromycin-induced cell death is greatly diminished by nicotinamide (a potent sirtuin inhibitor), and by decreased expression of sirtuins SIRT2 and SIRT3. Furthermore, we demonstrate using channel current recordings and binding assays that OAADPr directly binds to the cytoplasmic domain of TRPM2 and activates the TRPM2 channel. ADP-ribose binds TRPM2 with similarly affinity, whereas NAD+ displays almost negligible binding. These studies provide the first evidence for the potential role of sirtuin-generated OAADPr in TRPM2 channel gating.     

Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP-ribosyltransferase activity.

The yeast Sir2 protein regulates epigenetic gene silencing and as a possible antiaging effect it suppresses recombination of rDNA. Studies involving cobB, a bacterial SIR2-like gene, have suggested it could encode a pyridine nucleotide transferase. Here five human sirtuin cDNAs are characterized. The SIRT1 sequence has the closest homology to the S. cerevisiae Sir2p. The SIRT4 and SIRT5 sirtuins more closely resemble prokaryotic sirtuin sequences. The five human sirtuins are widely expressed in fetal and adult tissues. Recombinant E. coli cobT and cobB proteins each showed a weak NAD-dependent mono-ADP-ribosyltransferase activity using 5, 6-dimethylbenzimidazole as a substrate. Recombinant E. coli cobB and human SIRT2 sirtuin proteins were able to cause radioactivity to be transferred from [32P]NAD to bovine serum albumin (BSA). When a conserved histidine within the human SIRT2 sirtuin was converted to a tyrosine, the mutant recombinant protein was unable to transfer radioactivity from [32P]NAD to BSA. These results suggest that the sirtuins may function via mono-ADP-ribosylation of proteins.     

Sir2 regulation by nicotinamide results from switching between base exchange and deacetylation chemistry

Life span regulation and inhibition of gene silencing in yeast have been linked to nicotinamide effects on Sir2 enzymes. The Sir2 enzymes are NAD(+)-dependent protein deacetylases that influence gene expression by forming deacetylated proteins, nicotinamide and 2'-O-acetyl-ADPR. Nicotinamide is a base-exchange substrate as well as a biologically effective inhibitor. Characterization of the base-exchange reaction reveals that nicotinamide regulates sirtuins by switching between deacetylation and base exchange. Nicotinamide switching is quantitated for the Sir2s from Archeaglobus fulgidus (Sir2Af2), Saccharomyces cerevisiae (Sir2p), and mouse (Sir2alpha). Inhibition of deacetylation was most effective for mouse Sir2 alpha, suggesting species-dependent development of this regulatory mechanism. The Sir2s are proposed to form a relatively stable covalent intermediate between ADPR and the acetyl oxygen of the acetyllysine-protein substrate. During the lifetime of this intermediate, nicotinamide occupation of the catalytic site determines the fate of the covalent complex. Saturation of the nicotinamide site for mouse, yeast, and bacterial Sir2s causes 95, 65, and 21% of the intermediate, respectively, to return to acetylated protein. The fraction of the intermediate committed to deacetylation results from competition between the nicotinamide and the neighboring 2'-hydroxyl group at the opposite stereochemical face. Nicotinamide switching supports the previously proposed Sir2 catalytic mechanism and the existence of a 1'-O-peptidyl-ADPR.Sir2 intermediate. These findings suggest a strategy for increasing Sir2 enzyme catalytic activity in vivo by inhibition of chemical exchange but not deacetylation.     

The structural basis of sirtuin substrate affinity

Sirtuins comprise a family of enzymes that catalyze the deacetylation of acetyllysine side chains in a reaction that consumes NAD+. Although several crystal structures of sirtuins bound to non-native acetyl peptides have been determined, relatively little about how sirtuins discriminate among different substrates is understood. We have carried out a systematic structural and thermodynamic analysis of several peptides bound to a single sirtuin, the Sir2 homologue from Thermatoga maritima (Sir2Tm). We report structures of five different forms of Sir2Tm: two forms bound to the p53 C-terminal tail in the acetylated and unacetylated states, two forms bound to putative acetyl peptide substrates derived from the structured domains of histones H3 and H4, and one form bound to polypropylene glycol (PPG), which resembles the apoenzyme. The structures reveal previously unobserved complementary side chain interactions between Sir2Tm and the first residue N-terminal to the acetyllysine (position -1) and the second residue C-terminal to the acetyllysine (position +2). Isothermal titration calorimetry was used to compare binding constants between wild-type and mutant forms of Sir2Tm and between additional acetyl peptide substrates with substitutions at positions -1 and +2. The results are consistent with a model in which peptide positions -1 and +2 play a significant role in sirtuin substrate binding. This model provides a framework for identifying sirtuin substrates.     

Neuronal protection by sirtuins in Alzheimer's disease

Silent information regulator 2, a member of NAD+-dependent histone deacetylase in yeast, and its homologs in mice and humans, participate in numerous important cell functions, including cell protection and cell cycle regulation. The sirtuin family members are highly conserved evolutionarily, and are predicted to have a role in cell survival. The science of sirtuins is an emerging field and is expected to contribute significantly to the role of sirtuins in healthy aging in humans. The role of sirtuins in neuronal protection has been studied in lower organisms, such as yeast, worms, flies and rodents. Both yeast Sir2 and mammalian sirtuin proteins are up-regulated under calorie-restricted and resveratrol treatments. Increased sirtuin expression protects cells from various insults. Caloric restriction and antioxidant treatments have shown useful effects in mouse models of aging and Alzheimer's disease (AD) and in limited human AD clinical trials. The role sirtuins may play in modifying and protecting neurons in patients with neurodegenerative diseases is still unknown. However, a recent report of Huntington's disease revealed that Sirtuin protects neurons in a Huntington's disease mouse model, suggesting that sirtuins may protect neurons in patients with neurodegenerative diseases, such as AD. In this review, we discuss the possible mechanisms of sirtuins involved in neuronal protection and the potential therapeutic value of sirtuins in healthy aging and AD.     

Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins

Sirtuins (Sir2-like proteins) are present in prokaryotes and eukaryotes. Here, two new human sirtuins (SIRT6 and SIRT7) are found to be similar to a particular subset of insect, nematode, plant, and protozoan sirtuins. Molecular phylogenetic analysis of 60 sirtuin conserved core domain sequences from a diverse array of organisms (including archaeans, bacteria, yeasts, plants, protozoans, and metazoans) shows that eukaryotic Sir2-like proteins group into four main branches designated here as classes I-IV. Prokaryotic sirtuins include members of classes II and III. A fifth class of sirtuin is present in gram positive bacteria and Thermotoga maritima. Saccharomyces cerevisiae has five class I sirtuins. Caenorhabditis elegans and Drosophila melanogaster have sirtuin genes from classes I, II, and IV. The seven human sirtuin genes include all four classes: SIRT1, SIRT2, and SIRT3 are class I, SIRT4 is class II, SIRT5 is class III, and SIRT6 and SIRT7 are class IV.     

Sir2 protein deacetylases: evidence for chemical intermediates and functions of a conserved histidine

Sir2 NAD+-dependent protein deacetylases are implicated in a variety of cellular processes such as apoptosis, gene silencing, life-span regulation, and fatty acid metabolism. Despite this, there have been relatively few investigations into the detailed chemical mechanism. Sir2 proteins (sirtuins) catalyze the chemical conversion of NAD+ and acetylated lysine to nicotinamide, deacetylated lysine, and 2'-O-acetyl-ADP-ribose (OAADPr). In this study, Sir2-catalyzed reactions are shown to transfer an 18O label from the peptide acetyl group to the ribose 1'-position of OAADPr, providing direct evidence for the formation of a covalent alpha-1'-O-alkylamidate, whose existence is further supported by the observed methanolysis of the alpha-1'-O-alkylamidate intermediate to yield beta-1'-O-methyl-ADP-ribose in a Sir2 histidine-to-alanine mutant. This conserved histidine (His-135 in HST2) activates the ribose 2'-hydroxyl for attack on the alpha-1'-O-alkylamidate. The histidine mutant is stalled at the intermediate, allowing water and other alcohols to compete kinetically with the attacking 2'-hydroxyl. Measurement of the pH dependence of kcat and kcat/Km values for both wild-type and histidine-to-alanine mutant enzymes confirms roles of this residue in NAD+ binding and in general-base activation of the 2'-hydroxyl. Also, transfer of an 18O label from water to the carbonyl oxygen of the acetyl group in OAADPr is consistent with water addition to the proposed 1',2'-cyclic intermediate formed after 2'-hydroxyl attack on the alpha-1'-O-alkylamidate. The effect of pH and of solvent viscosity on the kcat values suggests that final product release is rate-limiting in the wild-type enzyme. Implications of this new evidence on the mechanisms of deacetylation and possible ADP-ribosylation catalyzed by Sir2 deacetylases are discussed.     

Structure of the yeast Hst2 protein deacetylase in ternary complex with 2'-O-acetyl ADP ribose and histone peptide

Sir2 proteins are NAD(+)-dependant protein deactylases that have been implicated in playing roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. To define the mechanism of Sir2 activity, we report the 1.5 A crystal structure of the yeast Hst2 (yHst2) Sir2 protein in ternary complex with 2'-O-acetyl ADP ribose and an acetylated histone H4 peptide. The structure captures both ligands meeting within an enclosed tunnel between the small and large domains of the catalytic protein core and permits the assignment of a detailed catalytic mechanism for the Sir2 proteins that is consistent with solution and enzymatic studies. Comparison of the ternary complex with the yHst2/NAD(+) complex, also reported here, and nascent yHst2 structure also reveals that NAD(+) binding accompanies intramolecular loop rearrangement for more stable NAD(+) and acetyl-lysine binding, and that acetyl-lysine peptide binding induces a trimer-monomer protein transition involving nonconserved Sir2 residues.     

Substrate specificity and kinetic mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases

The Silent information regulator 2 (Sir2) family of enzymes consists of NAD(+)-dependent histone/protein deacetylases that tightly couple the hydrolysis of NAD(+) and the deacetylation of an acetylated substrate to form nicotinamide, the deacetylated product, and the novel metabolite O-acetyl-ADP-ribose (OAADPR). In this paper, we analyzed the substrate specificity of the yeast Sir2 (ySir2), the yeast HST2, and the human SIRT2 homologues toward various monoacetylated histone H3 and H4 peptides, determined the basic kinetic mechanism, and resolved individual chemical steps of the Sir2 reaction. Using steady-state kinetic analysis, we have shown that ySir2, HST2, and SIRT2 exhibit varying catalytic efficiencies and display a preference among the monoacetylated peptide substrates. Bisubstrate kinetic analysis indicates that Sir2 enzymes follow a sequential mechanism, where both the acetylated substrate and NAD(+) must bind to form a ternary complex, prior to any catalytic step. Using rapid-kinetic analysis, we have shown that after ternary complex formation, nicotinamide cleavage occurs first, followed by the transfer of the acetyl group from the donor substrate to the ADP-ribose portion of NAD(+) to form OAADPr and the deacetylated product. Product and dead-end inhibition analyses revealed that nicotinamide is the first product released followed by random release of OAADPr and the deacetylated product.