Sir2 Protein Deacetylases: Evidence for Chemical Intermediates and Functions of a Conserved Histidine

Self Cite

O-Acetyl-ADP-ribose (OAADPr), produced by the Sir2-catalyzed NAD ؉ -dependent histone/protein deacetylase reaction, regulates diverse biological processes. Interconversion between two OAADPr isomers with acetyl attached to the C-2؆ and C-3؆ hydroxyl of ADP-ribose (ADPr) is rapid. We reported earlier that ADP-ribosylhydrolase 3 (ARH3), one of three ARH proteins sharing structural similarities, hydrolyzed OAADPr to ADPr and acetate, and poly(ADPr) to ADPr monomers. ARH1 also hydrolyzed OAADPr and poly(ADPr) as well as ADP-ribose-arginine, with arginine in ␣-anomeric linkage to C-1؆ of ADP-ribose. Because both ARH3-and ARH1-catalyzed reactions involve nucleophilic attacks at the C-1؆ position, it was perplexing that the ARH3 catalytic site would cleave OAADPr at either the 2؆-or 3؆-position, and we postulated the existence of a third isomer, 1؆-OAADPr, in equilibrium with 2؆-and 3؆-isomers. A third isomer, consistent with 1؆-OAADPr, was identified at pH 9.0. Further, ARH3 OAADPr hydrolase activity was greater at pH 9.0 than at neutral pH where 3؆-OAADPr predominated. Consistent with our hypothesis, IC 50 values for ARH3 inhibition by 2؆-and 3؆-N-acetyl-ADPr analogs of OAADPr were significantly higher than that for ADPr. ARH1 also hydrolyzed OAADPr more rapidly at alkaline pH, but cleavage of ADP-ribose-arginine was faster at neutral pH than pH 9.0. ARH3-catalyzed hydrolysis of OAADPr in H 218 O resulted in incorporation of one 18 O into ADP-ribose by mass spectrometric analysis, consistent with cleavage at the C-1؆ position. Together, these data suggest that ARH family members, ARH1 and ARH3, catalyze hydrolysis of the 1؆-O linkage in their structurally diverse substrates.Mono-ADP-ribosylation is a post-translational modification, in which the ADP-ribose (ADPr) 5 moiety of NAD is transferred to an acceptor protein (1). This modification serves as the mechanism by which several bacterial toxins (e.g. Pseudomonas exoenzyme S, cholera toxin, diphtheria toxin) exert their effects on mammalian cells (2, 3). Mammalian cells also produce endogenous ADP-ribosyltransferases that catalyze reactions similar to the bacterial toxins, specifically, the ADPribosylation of arginine residues in proteins (4). In addition, mammalian cells possess hydrolases that cleave the ADPr-protein linkage, releasing ADPr and regenerating the unmodified protein (5, 6). An ADP-ribosyl(arginine) hydrolase, termed ARH1, catalyzes in a stereospecific manner, hydrolysis of the ␣-linkage of arginine-ribose found in ADP-ribosyl(arginine)-protein to ADPr and (arginine)-protein (7, 8), consistent with the regulation of ADP-ribosyl(arginine)-protein levels by opposing activities of transferases and hydrolases, participating in an ADP-ribosylation cycle (4, 9).Three known members (ARH1-3) of the ARH family of proteins are similar in molecular size (ϳ39 kDa) and amino acid sequence (10). As noted above, ARH1 catalyzes the hydrolysis of ADP-ribose-arginine and also hydrolyzes ADP-ribose linkages to guanidine. The reaction is stereospecific, and only the ␣-ano...

Section: Discussionsupporting

confidence: 91%

Hydrolysis of O-Acetyl-ADP-ribose Isomers by ADP-ribosylhydrolase 3

Kasamatsu

Nakao

Smith

et al. 2011

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“…To confirm this model, we examined the interaction between wild-type SIRT1, SIRT1 T522V, or SIRT1 T522D mutants with p53 in cells and in vitro. Consistent with the previous observation that the catalytically inactive SIRT1 H355Y mutant is stalled at the ternary intermediate stage (31), the interaction between SIRT1 and p53 was increased when the SIRT1 HY mutant was expressed in HEK293T SIRT1 RNAi cells (Fig. 7E).…”

Section: Phosphorylation Of Thr 522 Activates Sirt1 By Decreasing Itssupporting

confidence: 92%

DYRK1A and DYRK3 Promote Cell Survival through Phosphorylation and Activation of SIRT1

Guo

Williams

Schug

et al. 2010

215

187

DYRK1A (the dual specificity tyrosine phosphorylation-regulated kinase 1A) plays an important role in body growth and brain physiology. Overexpression of this kinase has been associated with the development of Down syndrome in both human and animal models, whereas single copy loss-of-function of DYRK1A leads to increased apoptosis and decreased brain size. Although more than a dozen of DYRK1A targets have been identified, the molecular basis of its involvement in neuronal development remains unclear. Here we show that DYRK1A and another pro-survival member of the DYRK family, DYRK3, promote cell survival through phosphorylation and activation of SIRT1, an NAD ؉ -dependent protein deacetylase that is essential in a variety of physiological processes including stress response and energy metabolism. DYRK1A and DYRK3 directly phosphorylate SIRT1 at Thr 522 , promoting deacetylation of p53. A SIRT1 phosphorylation mimetic (SIRT1 T522D) displays elevated deacetylase activity, thus inhibiting cell apoptosis. Conversely, a SIRT1 dephosphorylation mimetic (SIRT1 T522V) fails to mediate DYRK-induced deacetylation of p53 and cell survival. We show that knockdown of endogenous DYRK1A and DYRK3 leads to hypophosphorylation of SIRT1, sensitizing cells to DNA damage-induced cell death. We also provide evidence that phosphorylation of Thr 522 activates SIRT1 by promoting product release, thereby increasing its enzymatic turnover. Taken together, our findings provide a novel mechanism by which two anti-apoptotic DYRK members promote cell survival through direct modification of SIRT1. These findings may have important implications in understanding the molecular mechanisms of tumorigenesis, Down syndrome, and aging.

“…N of the acetyl lysine forms a hydrogen bond with the carbonyl oxygen of Val-292, and the acetyl group is sandwiched between His-248 and Phe-180. His-248 is critical for the deacetylation activity of sirtuins (42,(81)(82)(83) and absolutely conserved in SIRT1-7, whereas Phe-180 is not conserved. The methyl group on the acetyl lysine is in van der Waals contact with Ile-291 and Ile-230 (both are functionally conserved in SIRT1-7), and the oxygen atom faces the NAD ϩ binding groove.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structures of Human SIRT3 Displaying Substrate-induced Conformational Changes

Jin

Wei

Jiang

et al. 2009

185

234

SIRT3 is a major mitochondrial NAD؉ -dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into the conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD ؉ . In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD ؉ . These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.Sirtuins are class III histone deacetylases that couple lysine deacetylation with NAD ϩ hydrolysis and are highly conserved in prokaryotes and eukaryotes (1). Mammals possess seven sirtuins, SIRT1-7, that occupy different subcellular compartments such as the nucleus (SIRT1, -6, -7), cytoplasm (SIRT2), and the mitochondria (SIRT3, -4, and -5) (2). They deacetylate lysines not only on histone substrates (3, 4) but also on nonhistone substrates such as p53 tumor suppressor protein (5), Foxo transcription factors (6, 7), PGC-1␣ (8), ␣-tubulin (9), acetyl-CoA synthetases (10 -12), and glutamate dehydrogenase (13). SIRT4 and SIRT6 have been shown to have ADP-ribosyltransferase activity (14 -16). Sirtuins have been reported to play important roles in gene silencing (17), cell cycle regulation (18,19), metabolism (8, 10 -12, 14, 20 -22), apoptosis (5, 23, 24), the lifespan-extension effects of calorie restriction (25,26), and circadian rhythms (27)(28)(29)(30) (50), and SIRT5 (51). Sirtuins contain a conserved enzymatic core with two domains; that is, a large Rossmann fold domain that binds NAD ϩ and a small domain formed by two insertions of the large domain that binds to a zinc atom. The acetylated peptide substrate binds to the cleft between the two domains. Some of the known structures are apo structures with sirtuin protein alone, whereas others are bound to acetylated peptide substrate and/or NAD ϩ and its analogs. These structures revealed the mechanism of action for the deacetylation activity and substrate specificity.SIRT3 localizes in mitochondria (13, 52-54) and is a major mitochondrial deacetylase. Hyperacetylation of mitochondrial proteins have been observed in SIRT3 knock-out mice (13, 55). Several key enzymes involved in energy production in the mitochondria have been identified as SIRT3 substrates. Acetyl-CoA synthetase 2 (AceCS2) 2 converts acetate into acetyl-CoA in the mitochondria. Deacetyla...