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 (ϳ8-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.The silent information regulator 2 (Sir2) family of proteins (sirtuins) are NAD ϩ -dependent histone/protein deacetylases that tightly couple the cleavage of NAD ϩ and deacetylation of protein substrates to form nicotinamide, the deacetylated product, and a novel metabolite, 2Ј-O-acetyl-ADP-ribose (OAADPr) 1(1-7). This family of proteins is evolutionarily conserved, with five homologs in yeast (ySir2 and HST1-4) and seven in humans (SIRT1-7) (8, 9). The founding member of this family, ySir2, is essential for gene silencing at the three silent loci in yeast (10 -19). Besides gene silencing, Sir2 proteins are important for many processes, such as cell cycle regulation (20), fatty acid metabolism (21), and life span extension (22-24). SIRT1, the most extensively studied human homolog, mediates p53-dependent processes (25-27), transcription regulation (28 -31), muscle differentiation (32), adipogenesis (33), protection from axonal degeneration (34), and life span extension (35,36).The importance of Sir2 enzymes in many cellular processes presents the need to understand their regulatory mechanisms. Substrate and product analogs as well as small molecules have been screened for Sir2 regulatory activity. Of all the NAD ϩ -like metabolites and salvage pathway intermediates analyzed for regulatory activities on Sir2 enzymes, onl...
Histone lysine and arginine residues are subject to a wide array of post-translational modifications including methylation, citrullination, acetylation, ubiquitination, and sumoylation. The combinatorial action of these modifications regulates critical DNA processes including replication, repair, and transcription. In addition, enzymes that modify histone lysine and arginine residues have been correlated with a variety of human diseases including arthritis, cancer, heart disease, diabetes, and neurodegenerative disorders. Thus, it is important to fully understand the detailed kinetic and chemical mechanisms of these enzymes. Here, we review recent progress towards determining the mechanisms of histone lysine and arginine modifying enzymes. In particular, the mechanisms of Sadenosyl-methionine (AdoMet) dependent methyltransferases, FAD dependent demethylases, iron dependent demethylases, acetyl-CoA dependent acetyltransferases, zinc dependent deacetylases, NAD + dependent deacetylases, and protein arginine deiminases are covered. Particular attention is paid to the conserved active-site residues necessary for catalysis and the individual chemical steps along the catalytic pathway. When appropriate, areas requiring further work are discussed.
Summary Emerging evidence suggests that protein acetylation is a broad-ranging regulatory mechanism. Here we utilize acetyl-peptide arrays and metabolomic analyses to identify substrates of mitochondrial deacetylase Sirt3. We identified ornithine transcarbamoylase (OTC) from the urea cycle, and enzymes involved in β-oxidation. Metabolomic analyses of fasted mice lacking Sirt3 (sirt3−/−) revealed alterations in β-oxidation and the urea cycle. Biochemical analysis demonstrated that Sirt3 directly deacetylates OTC and stimulates its activity. Mice under caloric restriction (CR) increased Sirt3 protein levels, leading to deacetylation and stimulation of OTC activity. In contrast, sirt3−/− mice failed to deacetylate OTC in response to CR. Inability to stimulate OTC under CR led to a failure to reduce orotic acid levels, a known outcome of OTC deficiency. Thus, Sirt3 directly regulates OTC activity and promotes the urea cycle during CR, and the results suggest that under low energy input, Sirt3 modulates mitochondria by promoting amino-acid catabolism and β-oxidation.
Although it is widely accepted that S-nitrosation occurs in vivo, questions remain regarding S-nitrosation as a signaling mechanism. The chemistry of S-nitrosation includes NO oxidation to N2O3 followed by reaction with thiolates, radical recombination of NO and thiyl radicals, and transition metal catalyzed pathways. Once formed, nitrosothiols can be transferred between small molecule or protein thiols through transnitrosation reactions. The pathways that lead to selective S-nitrosation of only a subset of cellular cysteines remain largely unknown. Selectivity may be conferred through colocalization with NOS isoforms, protein-protein interaction driven transnitrosation reactions, regulation of S-nitrosoglutathione levels, or directed denitrosation of protein nitrosothiols.
Sir2 protein deacetylases (or sirtuins) catalyze NAD+-dependent conversion of epsilon-amino-acetylated lysine residues to deacetylated lysine, nicotinamide, and 2'-O-acetyl-ADP-ribose. Small-molecule modulation of sirtuin activity might treat age-associated diseases, such as type II diabetes, obesity, and neurodegenerative disorders. Here, we have evaluated the mechanisms of sirtuin inhibition of histone peptides containing thioacetyl or mono-, di-, and trifluoroacetyl groups at the epsilon-amino of lysine. Although all substituted peptides yielded inhibition of the deacetylation reaction, the thioacetyl-lysine peptide exhibited exceptionally potent inhibition of sirtuins Sirt1, Sirt2, Sirt3, and Hst2. Using Hst2 as a representative sirtuin, the trifluoroacetyl-lysine peptide displayed competitive inhibition with acetyl-lysine substrate and yielded an inhibition constant (Kis) of 4.8 microM, similar to its Kd value of 3.3 microM. In contrast, inhibition by thioacetyl-lysine peptide yielded an inhibition constant (Kis) of 0.017 microM, 280-fold lower than its Kd value of 4.7 microM. Examination of thioacetyl-lysine peptide as an alternative sirtuin substrate revealed conserved production of deacetylated peptide and 1'-SH-2'-O-acetyl-ADP-ribose. Pre-steady-state and steady-state analysis of the thioacetyl-lysine peptide showed rapid nicotinamide formation (4.5 s-1) but slow overall turnover (0.0024 s-1), indicating that the reaction stalled at an intermediate after nicotinamide formation. Mass spectral analysis yielded a novel species (m/z 1754.3) that is consistent with an ADP-ribose-peptidyl adduct (1'-S-alkylamidate) as the stalled intermediate. Additional experiments involving solvent isotope effects, general base mutational analysis, and density functional calculations are consistent with impaired 2'-hydroxyl attack on the ADP-ribose-peptidyl intermediate. These results have implications for the development of mechanism-based inhibitors of Sir2 deacetylases.
Nitric oxide (NO) produced by NO synthase (NOS) participates in diverse physiological processes such as vasodilation, neurotransmission, and the innate immune response. Mammalian NOS isoforms are homodimers composed of two domains connected by an intervening calmodulin-binding region. The N-terminal oxidase domain binds heme and tetrahydrobiopterin and the arginine substrate. The C-terminal reductase domain binds FAD and FMN and the cosubstrate NADPH. Although several highresolution structures of individual NOS domains have been reported, a structure of a NOS holoenzyme has remained elusive. Determination of the higher-order domain architecture of NOS is essential to elucidate the molecular underpinnings of NO formation. In particular, the pathway of electron transfer from FMN to heme, and the mechanism through which calmodulin activates this electron transfer, are largely unknown. In this report, hydrogendeuterium exchange mass spectrometry was used to map critical NOS interaction surfaces. Direct interactions between the heme domain, the FMN subdomain, and calmodulin were observed. These interaction surfaces were confirmed by kinetic studies of site-specific interface mutants. Integration of the hydrogen-deuterium exchange mass spectrometry results with computational docking resulted in models of the NOS heme and FMN subdomain bound to calmodulin. These models suggest a pathway for electron transfer from FMN to heme and a mechanism for calmodulin activation of this critical step.iNOS | NO signaling | flavin | hemoprotein N itric oxide (NO) has several essential functions in mammalian physiology. NO produced by the neuronal and endothelial nitric oxide synthase isoforms (nNOS and eNOS, respectively) initiates diverse signaling processes including vasodilation, myocardial function, and neurotransmission (1). The eNOS and nNOS isoforms are constitutively expressed and their activity responds to intracellular calcium concentrations. The inducible NOS isoform (iNOS) is transcriptionally controlled and produces NO as a cytotoxin at sites of inflammation or infection. Aberrant NO signaling contributes to a variety of diseases including stroke, hypertension, and neurodegeneration (2).Mammalian NOS isoforms are homodimeric and composed of two principal domains: the N-terminal oxidase domain and C-terminal reductase domain, which are connected by an intervening calmodulin (CaM) binding region (Fig. 1A). The N-terminal oxidase domain contains the heme and tetrahydrobiopterin cofactors and the binding site for the substrate arginine. The reductase domain is further divided into the FMN-binding subdomain and the FAD/NADPH-binding subdomains. This array of cofactors works in concert to catalyze the conversion of arginine to the intermediate N-hydroxyarginine and, ultimately, citrulline and NO. NADPH and oxygen are consumed in the process. During catalysis, electrons are shuttled from the reductase domain of one monomer to the heme domain of the opposite monomer in the homodimer (Fig. 1B) (1, 3). Electron transfer is initiat...
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. In spite of 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, deacetylatedlysine, and 2'-O-acetyl-ADP-ribose (OAADPr). In this study, Sir2-catalyzed reactions are shown to transfer an 18 O-label from the peptide acetyl group to the ribose 1'-position of OAADPr, providing direct evidence for the formation of a covalent α-1'-O-alkylamidate, whose existence is further supported by the observed methanolysis of the α-1'-O-alkylamidate intermediate to yield β-1'-Omethyl-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 α-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 k cat and k cat /K m values for both wild-type and histidine-to-alanine mutant enzymes confirms roles of this residue in NAD + -binding and in generalbase activation of the 2'-hydroxyl. Also, transfer of an 18 O-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 α-1'-O-alkylamidate. The effect of pH and of solvent viscosity on the k cat 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 ADPribosylation catalyzed by Sir2 deacetylases are discussed. Keywords SIR2; sirtuin; Deacetylation; NAD; HistoneThe growing interest in the silent information regulator 2 (Sir2 or sirtuin) family of proteins lies in their involvement in a rapidly expanding list of cellular processes including gene silencing (1,2), cell cycle regulation (3), fatty acid metabolism (4), apoptosis (5-7), and lifespan extension (8)(9)(10). Conserved among all forms of life with five homologs in yeast (ySir2 and HST1−4) and seven in humans (SIRT1−7) (11,12), most sirtuins display NAD + -dependent protein deacetylase activity (13-16). SIRT1 has received the most attention and is reported to deacetylate a growing number of substrates including 18), FOXO proteins † This work was supported by National Institutes of Health grant GM065386 (to J.M.D.) and by National Institutes of Health Biotechnology Training Grant NIH 5 T32 GM08349 (to B.C.S.). This study was also supported by the National Science Foundation grant NSF CHE-9629688 for the NMR spectrometer used. [19][20][21], and HIV Tat protein (22) suggesting its involvement in a broad range of biological processes such as glucose homeostasis, cell survival under stress, and HIV transcription. In contrast to the primarily nuclear SIRT1, SI...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.