Background: Sirtuins regulate metabolism, genome maintenance, and stress responses. Results: Long-chain free fatty acids stimulate SIRT6 deacetylase, and sirtuins display distinct but overlapping specificity for diverse acylated peptides. Conclusion: SIRT6 is activated by biologically relevant fatty acids, and long-chain deacylation is a general feature of sirtuins. Significance: Discovery of endogenous, small-molecule activators of SIRT6 demonstrates the therapeutic potential of compounds that promote SIRT6 function.

SIRT6 is a member of the evolutionarily conserved sirtuin family of NAD ؉ -dependent protein deacetylases and functions in genomic stability and transcriptional control of glucose metabolism. Early reports suggested that SIRT6 performs ADPribosylation, whereas more recent studies have suggested that SIRT6 functions mainly as a histone deacetylase. Thus, the molecular functions of SIRT6 remain uncertain. Here, we perform biochemical, kinetic, and structural studies to provide new mechanistic insight into the functions of SIRT6. Utilizing three different assays, we provide biochemical and kinetic evidence that SIRT6-dependent histone deacetylation produces Oacetyl-ADP-ribose but at a rate ϳ1,000 times slower than other highly active sirtuins. To understand the molecular basis for such low deacetylase activity, we solved the first crystal structures of this class IV sirtuin in complex with ADP-ribose and the non-hydrolyzable analog of O-acetyl-ADP-ribose, 2 -N-acetyl-ADP-ribose. The structures revealed unique features of human SIRT6, including a splayed zinc-binding domain and the absence of a helix bundle that in other sirtuin structures connects the zinc-binding motif and Rossmann fold domain. SIRT6 also lacks the conserved, highly flexible, NAD ؉ -binding loop and instead contains a stable single helix. These differences led us to hypothesize that SIRT6, unlike all other studied sirtuins, would be able to bind NAD ؉ in the absence of an acetylated substrate. Indeed, we found that SIRT6 binds NAD ؉ with relatively high affinity (K d ‫؍‬ 27 ؎ 1 M) in the absence of an acetylated substrate. Isothermal titration calorimetry and tryptophan fluorescence binding assays suggested that ADP-ribose and NAD ؉ induce different structural perturbations and that NADH does not bind to SIRT6. Collectively, these new insights imply a unique activating mechanism and/or the possibility that SIRT6 could act as an NAD ؉ metabolite sensor.Sirtuins comprise an ancient and diverse family of nicotinamide adenine dinucleotide (NAD ϩ )-dependent protein deacetylases that are evolutionarily conserved from bacteria to eukaryotes (1). Unlike other classes of histone deacetylases, which utilize an active site Zn 2ϩ and involve direct attack of a water molecule on acetylated lysines, sirtuins transfer the acetyl group from the lysine side chain of a protein or peptide substrate to the co-factor NAD ϩ , generating nicotinamide, 2Ј-Oacetyl-ADP-ribose (OAADPr) 3 (2, 3), and a deacetylated substrate. This unique requirement of NAD ϩ suggests that sirtuins might act as sensors of the cellular metabolic state (4), relaying changes in cellular metabolism to reverse acetylation-mediated pathways, which include transcription, cell cycle progression, genome maintenance, apoptosis, and organism longevity. The founding member of the sirtuin family, yeast Sir2 (silent information regulator 2), has emerged as an important regulator in extending the life span of Saccharomyces cerevisiae (5). In other organisms, such as Caenorhabditis elegans and Drosophila me...

Acylation of lysine is an important protein modification regulating diverse biological processes. It was recently demonstrated that members of the human Sirtuin family are capable of catalyzing long-chain deacylation, in addition to the well-known NAD+-dependent deacetylation activity.1 Here we provide a detailed kinetic and structural analysis that describes the interdependence of NAD+ and acyl-group length for a diverse series of human Sirtuins, SIRT1, SIRT2, SIRT3 and SIRT6. Steady-state and rapid-quench kinetic analyses indicated that differences in NAD+ saturation and susceptibility to nicotinamide inhibition reflect unique kinetic behavior displayed by each Sirtuin and depend on acyl-substrate chain length. Though the rate of nucleophilic attack of the 2′-hydroxyl on the C1′-O-alkylimidate intermediate varies with acyl substrate chain length, this step remains rate-determining for SIRT2 and SIRT3; however for SIRT6, this step is no longer rate-limiting for long-chain substrates. Co-crystallization of SIRT2 with myristoylated peptide and NAD+ yielded a co-complex structure with reaction product 2′-O-myristoyl-ADP-ribose, revealing a latent hydrophobic cavity to accommodate the long chain acyl group, and suggesting a general mechanism for long chain deacylation. Comparing two separately solved co-complex structures containing either a myristoylated peptide or 2′-O-myristoyl-ADP-ribose indicate there are conformational changes at the myristoyl-ribose linkage with minimal structural differences in the enzyme active site. During the deacylation reaction, the fatty acyl group is held in a relatively fixed position. We describe a kinetic and structural model to explain how various Sirtuins display unique acyl-substrate preferences and how different reaction kinetics influence NAD+ dependence. The biological implications are discussed.

Summary Hepatic glucose production (HGP) maintains blood glucose levels during fasting but can also exacerbate diabetic hyperglycemia. HGP is dynamically controlled by a signaling/transcriptional network that regulates the expression/activity of gluconeogenic enzymes. A key mediator of gluconeogenic gene transcription is PGC-1α. PGC-1α’s activation of gluconeogenic gene expression is dependent upon its acetylation state, which is controlled by the acetyltransferase GCN5 and the deacetylase Sirt1. Nevertheless, whether other chromatin modifiers—particularly other sirtuins—can modulate PGC-1α acetylation is currently unknown. Herein we report that Sirt6 strongly controls PGC-1α acetylation. Surprisingly, Sirt6 induces PGC-1α acetylation and suppresses HGP. Sirt6 depletion decreases PGC-1α acetylation and promotes HGP. These acetylation effects are GCN5 dependent: Sirt6 interacts with and modifies GCN5, enhancing GCN5’s activity. Leprdb/Leprdb mice, an obese/diabetic animal model, exhibit reduced Sirt6 levels; ectopic re-expression suppresses gluconeogenic genes and normalizes glycemia. Activation of hepatic Sirt6 may therefore be therapeutically useful for treating insulin-resistant diabetes.

Histone post-translational modifications regulate transcription and other DNA-templated functions. This process is dynamically regulated by specific modifying enzymes whose activities require metabolites that either serve as co-substrates or act as activators/inhibitors. Therefore, metabolism can influence histone modification by changing local concentrations of key metabolites. Physiologically, the epigenetic response to metabolism is important for nutrient sensing and environment adaption. In pathologic states, the connection between metabolism and histone modification mediates epigenetic abnormality in complex disease. In this review, we summarize recent studies of the molecular mechanisms involved in metabolic regulation of histone modifications and discuss their biological significance.

Organogenesis begins with specification of a progenitor cell population, the size of which provides a foundation for the organ's final dimensions. Here, we present a new mechanism for regulating the number of progenitor cells by limiting their density within a competent region. We demonstrate that retinoic acid signaling restricts cardiac specification in the zebrafish embryo. Reduction of retinoic acid signaling causes formation of an excess of cardiomyocytes, via fate transformations that increase cardiac progenitor density within a multipotential zone. Thus, retinoic acid signaling creates a balance between cardiac and noncardiac identities, thereby refining the dimensions of the cardiac progenitor pool.

Sirtuins are a family of NAD؉ -dependent protein deacetylases/deacylases that dynamically regulate transcription, metabolism, and cellular stress response. Their general positive link with improved health span in mammals, potential regulation of pathways mediated by caloric restriction, and growing links to human disease have spurred interest in therapeutics that target their functions. Here, we review the current understanding of the chemistry of catalysis, biological targets, and endogenous regulation of sirtuin activity. We discuss recent efforts to generate small-molecule regulators of sirtuin activity.Accumulating data indicate that lysine acetylation is a prevalent regulatory mechanism of protein function, with thousands of acetylated proteins identified by mass spectrometry (1-3). Sir2 (silent information regulator 2 or sirtuin) protein deacetylases are a class of evolutionarily conserved enzymes that function in critical cellular processes such as transcription, DNA repair, metabolism, and stress resistance (4). Among the major classes of lysine deacetylases, the sirtuins utilize a unique catalytic mechanism that consumes NAD ϩ , providing a direct connection between protein deacetylation and central metabolic pathways. There are seven human sirtuins (SIRT1-7), each with diverse subcellular localization and protein substrates (5). SIRT1-3 display robust deacetylation activity, whereas recent reports implicate SIRT5 as a protein desuccinylase and demalonylase (6). Thus, sirtuins can be considered deacylases. The activities of several other human sirtuins are unsettled. SIRT6 and SIRT7 display weak deacetylase activity in vitro, and SIRT4 was reported to harbor ADP-ribosyltransferase activity (7,8). Structural analysis of the sirtuin family members reveals a conserved catalytic core composed of two subdomains, a Rossmann fold domain at one end and a smaller, more variable zinc-binding domain at the opposite end (Fig. 1). The two domains are connected by several loops that form a binding cleft for the nicotinamide and ribose moieties of NAD ϩ and the acyllysine substrate. Several invariant amino acids are located in the cleft and are responsible for substrate binding and catalysis. The varying hydrophobicity and charge distribution of the acyl-substrate binding cleft allow for varied substrate selectivity among the different human sirtuins (6, 9). Given their regulatory role in transcription, metabolism, and genome maintenance, sirtuins are desirable targets for therapeutic development. In this minireview, we highlight the current molecular understanding of the chemical mechanism, regulation, and substrate selectivity of sirtuins. Unique ChemistrySirtuins catalyze NAD ϩ -dependent deacetylation of acetyllysine, resulting in the production of deacetylated lysine, nicotinamide, and 2Ј-O-acetyl-ADP-ribose (OAADPr) 3 (Fig. 2) (10). Kinetic and biochemical studies revealed that the enzyme binds the acetyllysine substrate prior to NAD ϩ . Nicotinamide is cleaved from NAD ϩ and is the first product released, follo...

Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies.apoptosis | peroxynitrite | PC12 cells