William C. Hallows

Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-Related Hearing Loss under Caloric Restriction

Caloric restriction (CR) extends the life span and health span of a variety of species and slows the progression of age-related hearing loss (AHL), a common age-related disorder associated with oxidative stress. Here, we report that CR reduces oxidative DNA damage in multiple tissues and prevents AHL in wild-type mice but fails to modify these phenotypes in mice lacking the mitochondrial deacetylase Sirt3, a member of the sirtuin family. In response to CR, Sirt3 directly deacetylates and activates mitochondrial isocitrate dehydrogenase 2 (Idh2), leading to increased NADPH levels and an increased ratio of reduced-to-oxidized glutathione in mitochondria. In cultured cells, overexpression of Sirt3 and/or Idh2 increases NADPH levels and protects from oxidative stress-induced cell death. Therefore, our findings identify Sirt3 as an essential player in enhancing the mitochondrial glutathione antioxidant defense system during CR and suggest that Sirt3-dependent mitochondrial adaptations may be a central mechanism of aging retardation in mammals.

Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases

Silent Information Regulator 2 (Sir2) enzymes (or sirtuins) are NAD+-dependent deacetylases that modulate gene silencing, aging and energy metabolism. Previous work has implicated several transcription factors as sirtuin targets. Here, we investigated whether mammalian sirtuins could directly control the activity of metabolic enzymes. We demonstrate that mammalian Acetyl-CoA synthetases (AceCSs) are regulated by reversible acetylation and that sirtuins activate AceCSs by deacetylation. Site-specific acetylation of mouse AceCS1 on Lys-661 was identified by using mass spectrometry and a specific anti-acetyl-AceCS antibody. SIRT1 was the only member of seven human Sir2 homologues capable of deacetylating AceCS1 in cellular coexpression experiments. SIRT1 expression also led to a pronounced increase in AceCS1-dependent fatty-acid synthesis from acetate. Using purified enzymes, only SIRT1 and SIRT3 exhibited high catalytic efficiency against acetylated AceCS1. In mammals, two AceCSs have been identified: cytoplasmic AceCS1 and mitochondrial AceCS2. Because SIRT3 is localized to the mitochondria, we investigated whether AceCS2 also might be regulated by acetylation, and specifically deacetylated by mitochondrial SIRT3. AceCS2 was completely inactivated upon acetylation and was rapidly reactivated by SIRT3 deacetylation. Lys-635 of mouse AceCS2 was identified as the targeted residue. Using reversible acetylation to modulate enzyme activity, we propose a model for the control of AceCS1 by SIRT1 and of AceCS2 by SIRT3.

Mechanisms and Molecular Probes of Sirtuins

Sirtuins are critical regulators of many cellular processes, including insulin secretion, the cell cycle, and apoptosis. Sirtuins are associated with a variety of age-associated diseases such as type II diabetes, obesity, and Alzheimers disease. A thorough understanding of sirtuin chemical mechanisms will aid toward developing novel therapeutics that regulate metabolic disorders and combat associated diseases. In this review, we discuss the unique deacetylase mechanism of sirtuins and how this information might be employed to develop inhibitors and other molecular probes for therapeutic and basic research applications. We also cover physiological regulation of sirtuin activity and how these modes of regulation may be exploited to manipulate sirtuin activity in live cells. Development of molecular probes and drugs that specifically target sirtuins will further understanding of sirtuin biology and potentially afford new treatments of several human diseases.

A continuous microplate assay for sirtuins and nicotinamide-producing enzymes.

Nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases (sirtuins) and other enzymes that produce nicotinamide are integral to many cellular processes. Yet current activity measurements involve expensive and time-consuming assays. Here we present a spectroscopic assay that circumvents many issues of previous methods. This assay permits continuous product monitoring over time, allows determination of steady-state kinetic parameters, and is readily adaptable to high-throughput screening. The methodology uses an enzyme-coupled system in which nicotinamide is converted to nicotinic acid and ammonia by nicotinamidase. The ammonia is transferred to alpha-ketoglutarate via glutamate dehydrogenase, yielding glutamate and the oxidation of NAD(P)H to NAD(P)+, which is measured spectrophotometrically at 340 nm. Using this continuous assay with sirtuin-1 (Sirt1) and the ADP-ribosyl cyclase CD38, the resulting steady-state kinetic parameters are in excellent agreement with values obtained by other published methods. Importantly, this assay permitted determination of k(cat) and K(m) values with the native acetylated substrate acetyl-CoA synthetase-1; measurement of Sirt1, Sirt2, and Sirt3 activities from mammalian cell extracts; and determination of IC(50) values of various Sirt1 inhibitors. This assay is applicable to any nicotinamide-forming enzyme and will be an important tool to address many outstanding questions surrounding their regulation.

Regulation of glycolytic enzyme phosphoglycerate mutase-1 by Sirt1 protein-mediated deacetylation.

Background: Recent proteomic studies suggest that lysine acetylation might regulate many metabolic pathways. Results: NAD+-dependent deacetylase Sirt1 deacetylates phosphoglycerate mutase-1 (PGAM1) and attenuates catalytic activity. Conclusion: Sirt1 directly modulates glycolysis. Significance: These results advance our understanding of the regulatory impact of reversible acetylation on major metabolic pathways. Emerging proteomic evidence suggests that acetylation of metabolic enzymes is a prevalent post-translational modification. In a few recent reports, acetylation down-regulated activity of specific enzymes in fatty acid oxidation, urea cycle, electron transport, and anti-oxidant pathways. Here, we reveal that the glycolytic enzyme phosphoglycerate mutase-1 (PGAM1) is negatively regulated by Sirt1, a member of the NAD+-dependent protein deacetylases. Acetylated PGAM1 displays enhanced activity, although Sirt1-mediated deacetylation reduces activity. Acetylation sites mapped to the C-terminal “cap,” a region previously known to affect catalytic efficiency. Overexpression of a constitutively active variant (acetylated mimic) of PGAM1 stimulated flux through glycolysis. Under glucose restriction, Sirt1 levels dramatically increased, leading to PGAM1 deacetylation and attenuated activity. Previously, Sirt1 has been implicated in the adaptation from glucose to fat burning. This study (i) demonstrates that protein acetylation can stimulate metabolic enzymes, (ii) provides biochemical evidence that glycolysis is modulated by reversible acetylation, and (iii) demonstrates that PGAM1 deacetylation and activity are directly controlled by Sirt1.

Where in the cell is SIRT3?--functional localization of an NAD+-dependent protein deacetylase

Sirtuins are NAD+-dependent enzymes that have been implicated in a wide range of cellular processes, including pathways that affect diabetes, cancer, lifespan and Parkinsons disease. To understand their cellular function in these age-related diseases, identification of sirtuin targets and their subcellular localization is paramount. SIRT3 (sirtuin 3), a human homologue of Sir2 (silent information regulator 2), has been genetically linked to lifespan in the elderly. However, the function and localization of this enzyme has been keenly debated. A number of reports have indicated that SIRT3, upon proteolytic cleavage in the mitochondria, is an active protein deacetylase against a number of mitochondrial targets. In stark contrast, some reports have suggested that full-length SIRT3 exhibits nuclear localization and histone deacetylase activity. Recently, a report comparing SIRT3-/- and SIRT+/+ mice have provided compelling evidence that endogenous SIRT3 is mitochondrial and appears to be responsible for the majority of protein deacetylation in this organelle. In this issue of the Biochemical Journal, Cooper et al. present additional results that address the mitochondrial and nuclear localization of SIRT3. Utilizing fluorescence microscopy and cellular fractionation studies, Cooper et al. have shown that SIRT3 localizes to the mitochondria and is absent in the nucleus. Thus this study provides additional evidence to establish SIRT3 as a proteolytically modified, mitochondrial deacetylase.

SIRT3 substrate specificity determined by peptide arrays and machine learning.

Accumulating evidence suggests that reversible protein acetylation may be a major regulatory mechanism that rivals phosphorylation. With the recent cataloging of thousands of acetylation sites on hundreds of proteins comes the challenge of identifying the acetyltransferases and deacetylases that regulate acetylation levels. Sirtuins are a conserved family of NAD(+)-dependent protein deacetylases that are implicated in genome maintenance, metabolism, cell survival, and lifespan. SIRT3 is the dominant protein deacetylase in mitochondria, and emerging evidence suggests that SIRT3 may control major pathways by deacetylation of central metabolic enzymes. Here, to identify potential SIRT3 substrates, we have developed an unbiased screening strategy that involves a novel acetyl-lysine analogue (thiotrifluoroacetyl-lysine), SPOT-peptide libraries, machine learning, and kinetic validation. SPOT peptide libraries based on known and potential mitochondrial acetyl-lysine sites were screened for SIRT3 binding and then analyzed using machine learning to establish binding trends. These trends were then applied to the mitochondrial proteome as a whole to predict binding affinity of all lysine sites within human mitochondria. Machine learning prediction of SIRT3 binding correlated with steady-state kinetic k(cat)/K(m) values for 24 acetyl-lysine peptides that possessed a broad range of predicted binding. Thus, SPOT peptide-binding screens and machine learning prediction provides an accurate and efficient method to evaluate sirtuin substrate specificity from a relatively small learning set. These analyses suggest potential SIRT3 substrates involved in several metabolic pathways such as the urea cycle, ATP synthesis, and fatty acid oxidation.

Nuclear-cytoplasmic localization of acetyl coenzyme A synthetase-1 in the rat brain

Acetyl coenzyme A synthetase‐1 (AceCS1) catalyzes the synthesis of acetyl coenzyme A from acetate and coenzyme A and is thought to play diverse roles ranging from fatty acid synthesis to gene regulation. By using an affinity‐purified antibody generated against an 18‐mer peptide sequence of AceCS1 and a polyclonal antibody directed against recombinant AceCS1 protein, we examined the expression of AceCS1 in the rat brain. AceCS1 immunoreactivity in the adult rat brain was present predominantly in cell nuclei, with only light to moderate cytoplasmic staining in some neurons, axons, and oligodendrocytes. Some nonneuronal cell nuclei were very strongly immunoreactive, including those of some oligodendrocytes, whereas neuronal nuclei ranged from unstained to moderately stained. Both antibodies stained some neuronal cell bodies and axons, especially in the hindbrain. AceCS1 immunoreactivity was stronger and more widespread in the brains of 18‐day‐old rats than in adults, with increased expression in oligodendrocytes and neurons, including cortical pyramidal cells. Expression of AceCS1 was substantially up‐regulated in neurons throughout the brain after controlled cortical impact injury. The strong AceCS1 expression observed in the nuclei of CNS cells during brain development and after injury is consistent with a role in nuclear histone acetylation and therefore the regulation of chromatin structure and gene expression. The cytoplasmic staining observed in some oligodendrocytes, especially during postnatal brain development, suggests an additional role in CNS lipid synthesis and myelination. Neuronal and axonal localization implicates AceCS1 in cytoplasmic acetylation reactions in some neurons. J. Comp. Neurol. 518:2952–2977, 2010.

Circadian Control of Fatty Acid Elongation by SIRT1 Protein-mediated Deacetylation of Acetyl-coenzyme A Synthetase 1

Background: Circadian clock regulates various aspects of metabolism. Results: Rhythmic acetylation of AceCS1 controls circadian oscillations in acetyl-CoA levels and in fatty acid elongation. Conclusion: A previously unrecognized regulation of acetyl-CoA provides additional evidence to circadian regulation of metabolism. Significance: Understanding of the role of acetyl-CoA in fatty acid elongation might provide therapeutic benefits for treating metabolic diseases and cancer. The circadian clock regulates a wide range of physiological and metabolic processes, and its disruption leads to metabolic disorders such as diabetes and obesity. Accumulating evidence reveals that the circadian clock regulates levels of metabolites that, in turn, may regulate the clock. Here we demonstrate that the circadian clock regulates the intracellular levels of acetyl-CoA by modulating the enzymatic activity of acetyl-CoA Synthetase 1 (AceCS1). Acetylation of AceCS1 controls the activity of the enzyme. We show that acetylation of AceCS1 is cyclic and that its rhythmicity requires a functional circadian clock and the NAD+-dependent deacetylase SIRT1. Cyclic acetylation of AceCS1 contributes to the rhythmicity of acetyl-CoA levels both in vivo and in cultured cells. Down-regulation of AceCS1 causes a significant decrease in the cellular acetyl-CoA pool, leading to reduction in circadian changes in fatty acid elongation. Thus, a nontranscriptional, enzymatic loop is governed by the circadian clock to control acetyl-CoA levels and fatty acid synthesis.

Ure(k)a! Sirtuins Regulate Mitochondria

Increasing evidence suggests that multiple metabolic pathways are regulated by sirtuin-dependent protein deacetylation in the mitochondria. In this issue, Nakagawa et al. (2009) show that the sirtuin SIRT5 deacetylates and activates a mitochondrial enzyme, carbamoyl phosphate synthetase 1, which mediates the first step in the urea cycle.