John M. Denu

The Human Sir2 Ortholog, SIRT2, Is an NAD + -Dependent Tubulin Deacetylase.

The silent information regulator 2 protein (Sir2p) of Saccharomyces cerevisiae is an NAD-dependent histone deacetylase that plays a critical role in transcriptional silencing. Here, we report that a human ortholog of Sir2p, sirtuin type 2 (SIRT2), is a predominantly cytoplasmic protein that colocalizes with microtubules. SIRT2 deacetylates lysine-40 of alpha-tubulin both in vitro and in vivo. Knockdown of SIRT2 via siRNA results in tubulin hyperacetylation. SIRT2 colocalizes and interacts in vivo with HDAC6, another tubulin deacetylase. Enzymatic analysis of recombinant SIRT2 in comparison to a yeast homolog of Sir2 protein (Hst2p) shows a striking preference of SIRT2 for acetylated tubulin peptide as a substrate relative to acetylated histone H3 peptide. These observations establish SIRT2 as a bona fide tubulin deacetylase.

Mechanism of human SIRT1 activation by resveratrol.

The NAD+-dependent protein deacetylase family, Sir2 (or sirtuins), is important for many cellular processes including gene silencing, regulation of p53, fatty acid metabolism, cell cycle regulation, and life span extension. Resveratrol, a polyphenol found in wines and thought to harbor major health benefits, was reported to be an activator of Sir2 enzymes in vivo and in vitro. In addition, resveratrol was shown to increase life span in three model organisms through a Sir2-dependent pathway. Here, we investigated the molecular basis for Sir2 activation by resveratrol. Among the three enzymes tested (yeast Sir2, human SIRT1, and human SIRT2), only SIRT1 exhibited significant enzyme activation (∼8-fold) using the commercially available Fluor de Lys kit (BioMol). To examine the requirements for resveratrol activation of SIRT1, we synthesized three p53 acetylpeptide substrates either lacking a fluorophore or containing a 7-amino-4-methylcoumarin (p53-AMC) or rhodamine 110 (p53-R110). Although SIRT1 activation was independent of the acetylpeptide sequence, resveratrol activation was completely dependent on the presence of a covalently attached fluorophore. Substrate competition studies indicated that the fluorophore decreased the binding affinity of the peptide, and, in the presence of resveratrol, fluorophore-containing substrates bound more tightly to SIRT1. Using available crystal structures, a model of SIRT1 bound to p53-AMC peptide was constructed. Without resveratrol, the coumarin of p53-AMC peptide is solvent-exposed and makes no significant contacts with SIRT1. We propose that binding of resveratrol to SIRT1 promotes a conformational change that better accommodates the attached coumarin group.

Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation.

Histone acetylation and phosphorylation have separately been suggested to affect chromatin structure and gene expression. Here we report that these two modifications are synergistic. Stimulation of mammalian cells by epidermal growth factor (EGF) results in rapid and sequential phosphorylation and acetylation of H3, and these dimodified H3 molecules are preferentially associated with the EGF-activated c-fos promoter in a MAP kinase-dependent manner. In addition, the prototypical histone acetyltransferase Gcn5 displays an up to 10-fold preference for phosphorylated (Ser-10) H3 over nonphosphorylated H3 as substrate in vitro, suggesting that H3 phosphorylation can affect the efficiency of subsequent acetylation reactions. Together, these results illustrate how the addition of multiple histone modifications may be coupled during the process of gene expression.

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.

A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase

Dual-specificity protein-tyrosine phosphatases (dsPTPases) have been implicated in the inactivation of mitogen-activated protein kinases (MAPKs). We have identified a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, except for the substitution of Gly for Cys in the conserved dsPTPase catalytic loop (HCXXGXXR(S/T)). cDNA subcloning and Northern blot analysis in mouse shows poly(A) hybridization bands of 4.6, 2.4, 1.5, and 1.2 kilobases, with highest abundance in skeletal muscle, testis, and heart. Polymerase chain reaction amplification of reverse-transcribed poly(A) RNA revealed an alternatively spliced form of STYX containing a unique carboxyl terminus. Bacterially expressed STYX is incapable of hydrolyzing Tyr(P)-containing substrates; however, mutation of Gly to Cys (G120C), which structurally mimics the active site of dsPTPases, confers phosphatase activity to this molecule. STYX-G120C mutant hydrolyzes p-nitrophenyl phosphate and dephosphorylates both Tyr(P) and Thr(P) residues of peptide sequences of MAPK homologues. The kinetic parameters of dephosphorylation are similar to human dsPTPase, Vaccinia H1-related, including inhibition by vanadate. We believe this is the first example of a naturally occurring “dominant negative” phosphotyrosine/serine/threonine-binding protein which is structurally related to dsPTPases.

Protein tyrosine phosphatases: mechanisms of catalysis and regulation

Recent structural information suggests that the HC(X)5R active-site motif defines three distinct evolutionary families of phosphatases that employ a common catalytic mechanism. In two instances, regulation of phosphatase activity employs autoinhibitory mechanisms involving either intermolecular or intramolecular interactions, whereby inhibition is mediated by sterically blocking the active-site cleft.

Calorie Restriction and SIRT3 Trigger Global Reprogramming of the Mitochondrial Protein Acetylome

Calorie restriction (CR) extends life span in diverse species. Mitochondria play a key role in CR adaptation; however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR versus control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites-2,193 from mitochondrial proteins-rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.

Crystal structure of the dual specificity protein phosphatase VHR.

Dual specificity protein phosphatases (DSPs) regulate mitogenic signal transduction and control the cell cycle. Here, the crystal structure of a human DSP, vaccinia H1-related phosphatase (or VHR), was determined at 2.1 angstrom resolution. A shallow active site pocket in VHR allows for the hydrolysis of phosphorylated serine, threonine, or tyrosine protein residues, whereas the deeper active site of protein tyrosine phosphatases (PTPs) restricts substrate specificity to only phosphotyrosine. Positively charged crevices near the active site may explain the enzymes preference for substrates with two phosphorylated residues. The VHR structure defines a conserved structural scaffold for both DSPs and PTPs. A “recognition region,” connecting helix α1 to strand β1, may determine differences in substrate specificity between VHR, the PTPs, and other DSPs.

Form and Function in Protein Dephosphorylation

In contrast to the catalytic mechanism employed by PPs, the PTPs proceed through a phosphoenzyme intermediate. The enzymatic reaction involves phosphoryl-cysteine intermediate formation after nucleophilic attack of the phosphorus atom of the substrate by the thiolate anion of cysteine (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996). The reaction can be represented as a two-step chemical process: phosphoryl transfer to the enzyme accompanied by rapid release of dephosphorylated product (denoted by rate constant k(formation) in Equation 1); and hydrolysis of the thiol-phosphate intermediate concomitant with rapid release of phosphate (denoted by rate constant k(hydrolysis) in Equation 1). To form the catalytically competent complex ES, the enzyme binds and reacts with the dianion of phosphate–containing substrate (Figure 3AFigure 3A). On the enzyme an aspartic acid must be protonated and the nucleophilic cysteine must be unprotonated (thiolate anion) for phosphoryl transfer to the enzyme (3xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all References, 20xZhang, Z.-Y. J. Biol. Chem. 1995; 270: 11199–11204Crossref | PubMed | Scopus (86)See all References). In the Michaelis complex (Jia et al. 1995xJia, Z, Barford, D, Flint, A.J, and Tonks, N.K. Science. 1995; 268: 1754–1758Crossref | PubMedSee all ReferencesJia et al. 1995), the three nonbridging oxygens of the phosphoryl group are coordinated by bidentate hydrogen bonds to the guanidinium group of arginine and by the backbone amide N-H groups of the active-site loop (Figure 3AFigure 3A) . Situated directly underneath the phosphoryl group and at the base of the active-site cleft, is the nucleophilic cysteine thiolate anion (shown in green, Figure 3AFigure 3A). The substrate (shown in yellow, Figure 3AFigure 3A) is positioned such that attack of the thiolate is directly in line with the P-O bond and ideally situated for efficient expulsion of the leaving group. To further enhance leaving group expulsion, near the top of the cleft the aspartic acid is positioned to act as a general acid by protonating the leaving group phenolic oxygen. The aspartic acid is found on a separate loop (shown in yellow, Figure 2Figure 2) that was shown to be flexible for both Yersinia PTP (Stuckey et al. 1994xStuckey, J.A, Schubert, H.L, Fauman, E.B, Zhang, Z.-Y, Dixon, J.E, and Saper, M.A. Nature. 1994; 370: 571–575Crossref | PubMed | Scopus (303)See all ReferencesStuckey et al. 1994) and PTP1B (Jia et al. 1995xJia, Z, Barford, D, Flint, A.J, and Tonks, N.K. Science. 1995; 268: 1754–1758Crossref | PubMedSee all ReferencesJia et al. 1995). When the enzyme is complexed with phosphorylated substrates, this loop folds over the active site, bringing the aspartic acid into position for leaving group protonation. In the open conformation, the loop is flipped away from the active site and the aspartic acid is approximately 8–12 A removed from its location in the Michaelis complex (Stuckey et al. 1994xStuckey, J.A, Schubert, H.L, Fauman, E.B, Zhang, Z.-Y, Dixon, J.E, and Saper, M.A. Nature. 1994; 370: 571–575Crossref | PubMed | Scopus (303)See all ReferencesStuckey et al. 1994). It is not yet known whether the analogous loop in VHR is capable of such dramatic movement. In the sulfate-bound complex of VHR, the loop does not cover the active site to the same extent as in the other two PTP crystal structures (Yuvaniyama, et al. 1996xYuvaniyama, J, Denu, J.M, Dixon, J.E, and Saper, M.A. Science. 1996; 272: 1328–1331Crossref | PubMedSee all ReferencesYuvaniyama, et al. 1996).Figure 3Catalytic Mechanism of Protein Tyrosine Phosphatases: Enzyme–Substrate Complex and Phosphoenzyme Intermediate(A) A model of the enzyme-substrate complex of PTP derived from two X-ray crystallographic models: Cys-ser mutant of PTP1B complexed with phosphotyrosine (Jia et al. 1995xJia, Z, Barford, D, Flint, A.J, and Tonks, N.K. Science. 1995; 268: 1754–1758Crossref | PubMedSee all ReferencesJia et al. 1995), and Yersinia PTP complexed with vanadate (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996). The backbone atoms of the active site loop from cysteine to arginine are shown as a ball-and-stick model. The phospho-tyrosine substrate (shown in yellow) is bound to the center of the active-site motif with the protonated general acid (Asp) lying within hydrogen bonding distance of the scissile oxygen of the substrate. The dianion of the phosphoryl group is coordinated by the nitrogens of the arginine side chain and by the amide groups of the active site motif. The thiolate anion of cysteine is poised for nucleophilic attack. Individuals atoms are represented as spheres with the following colors: Cα and carbonyl carbons, gray; oxygens, red; nitrogens, blue; phosphorus, magenta; sulfur, green; and hydrogens, orange (not found in original crystal structures). Dashed lines represent hydrogen bonds.(B) A model of the phosphoenzyme intermediate of PTP derived in a similar manner as (A) with the same atom colors as (A). The phosphorus is covalently bound to the Sγ of the cysteine. A water molecule (Wat) is in a position such that aspartic acid can abstract the proton. Figures were drawn using Molscript and Raster3D.View Large Image | View Hi-Res Image | Download PowerPoint SlideTo explore the transition-state structure for catalysis, heavy atom kinetic isotope effects have been measured with PTP1, Yersinia PTP and VHR (Hengge et al. 1996xHengge, A.C, Denu, J.M, and Dixon, J.E. Biochemistry. 1996; 35: 7084–7092Crossref | PubMed | Scopus (61)See all ReferencesHengge et al. 1996). The kinetic isotope effects indicate that P-O bond cleavage is far advanced and there is little bond formation to the nucleophile cysteine during the transition state. It was also demonstrated that proton transfer from aspartic acid to the bridging oxygen is concomitant with P-O cleavage, such that no charge is developed on the leaving group. Consistent with concomitant proton transfer, leaving group pKa values have little effect on the rate of phosphoryl transfer (20xZhang, Z.-Y. J. Biol. Chem. 1995; 270: 11199–11204Crossref | PubMed | Scopus (86)See all References, 3xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all References). When aspartic acid is replaced by asparagine, phosphopeptides and phosphotyrosine are 100-fold worse than substrates with good leaving groups such as p-nitrophenylphosphate, a commonly employed artificial substrate. Thus, with physiological substrates, proton donation by aspartic acid is more critical for efficient catalysis than with labile artificial substrates.Once the phosphoenzyme intermediate is formed, it must undergo hydrolysis by water to result in the turnover of the enzyme (Figure 3Figure 3). Activation of the water molecule by a general base would be expected to facilitate hydrolysis. Mutagenic studies with VHR have shown that the conserved aspartic acid enhances intermediate hydrolysis (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996), suggesting that the aspartic acid may function as the general base by proton abstraction from a water molecule. Further support for this idea comes from the X-ray structure of Yersinia PTP solved with the inhibitor vanadate covalently bound to cysteine (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996). With trigonal bipyramidal geometry, the vanadate mimics the transition for both chemical steps and underscores the importance of conserved arginine and aspartic acid residues. Similar to the ES complex, two of the equatorial oxygens of vanadate are hydrogen bonded to arginine in a bidendate fashion. The aspartic acid makes a hydrogen bond of 2.8 A to the apical oxygen of vanadate, consistent with the role as general acid/base.The recently solved structures of protein phosphatases have greatly advanced our knowledge of the molecular mechanism of catalysis and substrate specificity. Understanding the molecular events of these essential reactions will lead to a better understanding of the specific physiological functions of the protein phosphatases. Also, a detailed description of the inherent differences between and among PTPs and PPs will undoubtedly augment the search for and design of specific phosphatase inhibitors.Because of reference limitations imposed in minireviews, we wish to acknowledge the important contributions made by other investigators whose work we were unable to cite. Reference to this work can be found within the cited publications of many of the selected reading articles.