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Structure of the histone deacetylase SIRT2
Abstract
Sir2 is an NAD-dependent histone deacetylase that mediates transcriptional silencing at mating-type loci, telomeres and ribosomal gene clusters, and has a critical role in the determination of life span in yeast and Caenorhabditis elegans. The 1.7 A crystal structure of the 323 amino acid catalytic core of human SIRT2, a homolog of yeast Sir2, reveals an NAD-binding domain, which is a variant of the Rossmann fold, and a smaller domain composed of a helical module and a zinc-binding module. A conserved large groove at the interface of the two domains is the likely site of catalysis based on mutagenesis. Intersecting this large groove, there is a pocket formed by the helical module. The pocket is lined with hydrophobic residues conserved within each of the five Sir2 classes, suggesting that it is a class-specific protein-binding site.
... Figure 6. (A) Superimposition of the SIRT2 apo-structures lacking substrates/reaction products (1J8F [47]: dark blue, 3ZGO [28]: light blue, 3ZGV [28]: dark violet, ADPR in red sticks, and 5D7O [26]: light violet). ADPR is represented as sticks. ...
... Among the SIRT2 apo-conformations, i.e., 1J8F [47], 3ZGO [28], 3ZGV [28], and 5D7O [26], the first two types of experimental data for 1J8F and 3ZGO lacked cofactors and featured RMSD values at the corresponding CA of 0.61 Å. The 3ZGV and 5D7O included ADPR and proved to be highly similar after superimposition as the RMSD value at the corresponding CA atoms was 0.44 Å. ...
... This approach was successfully followed by Tervo et al. [91] in 2004. The research group submitted to MD the available apo-form of SIRT-2 (PDB code 1J8F) [47], and they extracted an artificial enlarged conformation of the target. The binding pocket interaction fields and properties were calculated and used to screen the Maybridge database [92]. ...
Sirtuins (SIRTs) are classified as class III histone deacetylases (HDACs), a family of enzymes that catalyze the removal of acetyl groups from the ε-N-acetyl lysine residues of histone proteins, thus counteracting the activity performed by histone acetyltransferares (HATs). Based on their involvement in different biological pathways, ranging from transcription to metabolism and genome stability, SIRT dysregulation was investigated in many diseases, such as cancer, neurodegenerative disorders, diabetes, and cardiovascular and autoimmune diseases. The elucidation of a consistent number of SIRT–ligand complexes helped to steer the identification of novel and more selective modulators. Due to the high diversity and quantity of the structural data thus far available, we reviewed some of the different ligands and structure-based methods that have recently been used to identify new promising SIRT1/2 modulators. The present review is structured into two sections: the first includes a comprehensive perspective of the successful computational approaches related to the discovery of SIRT1/2 inhibitors (SIRTIs); the second section deals with the most interesting SIRTIs that have recently appeared in the literature (from 2017). The data reported here are collected from different databases (SciFinder, Web of Science, Scopus, Google Scholar, and PubMed) using “SIRT”, “sirtuin”, and “sirtuin inhibitors” as keywords.
... Sirtuins, moreover, often possess other enzymatic activities apart from the deacetylation one, such as deacylation and mono-ADP-ribosylation [2][3][4][5]. Structurally, the seven isoforms share a central catalytic domain of about 270 amino acids, where a Rossman fold and a smaller domain with the NAD +binding module and a zinc-binding one create the enzymatic active site; the seven sirtuins then differ in the N-terminal and C-terminal domains [6]. The crystal structures of the seven sirtuins, chosen on the basis of resolution values (<2.00 Å), are represented in Figure 1. ...
... In particular, the Chembridge database [249] was initially filtered according to lead-like features and to similarity with respect to Cambinol, a known SIRT-2 inhibitor (see Sections 2.2.1 and 2.8.1). The resulting compounds were then docked in the SIRT-2 catalytic pocket (crystal structure in the apo-form, 1J8F [ 6]), and compounds showing a H bond to Gln167 (a key residue) were retained. Compounds with a low logP were prioritized, and the resulting molecules were submitted to in vitro validation. ...
... Most active thiobarbiturates from the initial work were selected, and their fingerprints were used as queries to screen the Chembridge database [249]. The results were filtered according to drug-like properties and docked in the apo-SIRT-2 active site [6] (PDB code 1J8F). Top hits were subjected to additional in silico and in vitro studies, highlighting an ameliorate potency with respect to the previously proposed analogues. ...
Sirtuins are NAD+-dependent deac(et)ylases with different subcellular localization. The sirtuins’ family is composed of seven members, named SIRT-1 to SIRT-7. Their substrates include histones and also an increasing number of different proteins. Sirtuins regulate a wide range of different processes, ranging from transcription to metabolism to genome stability. Thus, their dysregulation has been related to the pathogenesis of different diseases. In this review, we discussed the pharmacological approaches based on sirtuins’ modulators (both inhibitors and activators) that have been attempted in in vitro and/or in in vivo experimental settings, to highlight the therapeutic potential of targeting one/more specific sirtuin isoform(s) in cancer, neurodegenerative disorders and type 2 diabetes. Extensive research has already been performed to identify SIRT-1 and -2 modulators, while compounds targeting the other sirtuins have been less studied so far. Beside sections dedicated to each sirtuin, in the present review we also included sections dedicated to pan-sirtuins’ and to parasitic sirtuins’ modulators. A special focus is dedicated to the sirtuins’ modulators identified by the use of virtual screening.
... The catalytic core has an elongated shape containing two domains, namely, a larger domain (residues 53-89, 146-186, and 241-356) and a smaller domain (residues 90-145 and 187-240) (Finnin et al., 2001). The former is a variant of the Rossmann fold, which is present in many diverse NAD(H)/NADP(H) binding enzymes (Hou et al., 2014). ...
... And the latter is composed of a helical module and a zinc-binding module (Carafa et al., 2012;Karaman et al., 2018). The two domains are connected by a hinge region that consists of four crossovers of the polypeptide chain (Finnin et al., 2001). At the interface of the two domains, the four crossovers and three loops of the large domain form a large groove (Liu Y. M. et al., 2019; Figure 2C). ...
... The larger domain consists of six β strands and eight α helices (North and Verdin, 2004). The β strands form a parallel β sheet, and the α helices pack against the β sheet (Finnin et al., 2001). The Rossmann fold contains various features of a typical NAD+ binding site, such as a conserved Gly-X-Gly sequence important for NAD+ phosphate binding, a charged amino acid residue responsible for ribose group binding, and a small pocket to accommodate an NAD+ molecule (Satoh et al., 2011;Zhang X. et al., 2021; Figure 2C). ...
Neuroinflammatory disorder is a general term that is associated with the progressive loss of neuronal structure or function. At present, the widely studied diseases with neuroinflammatory components are mainly divided into neurodegenerative and neuropsychiatric diseases, namely, Alzheimer’s disease, Parkinson’s disease, depression, stroke, and so on. An appropriate neuroinflammatory response can promote brain homeostasis, while excessive neuroinflammation can inhibit neuronal regeneration and damage the central nervous system. Apart from the symptomatic treatment with cholinesterase inhibitors, antidepressants/anxiolytics, and neuroprotective drugs, the treatment of neuroinflammation is a promising therapeutic method. Sirtuins are a host of class III histone deacetylases, that require nicotinamide adenine dinucleotide for their lysine residue deacetylase activity. The role of sirtuin 2 (SIRT2), one of the sirtuins, in modulating senescence, myelin formation, autophagy, and inflammation has been widely studied. SIRT2 is associated with many neuroinflammatory disorders considering it has deacetylation properties, that regulate the entire immune homeostasis. The aim of this review was to summarize the latest progress in regulating the effects of SIRT2 on immune homeostasis in neuroinflammatory disorders. The overall structure and catalytic properties of SIRT2, the selective inhibitors of SIRT2, the relationship between immune homeostasis and SIRT2, and the multitasking role of SIRT2 in several diseases with neuroinflammatory components were discussed.
... To understand the binding modes of constrained analogs versus non-constrained ones, compounds 19c and 20c were selected for computational docking. Reference compound 7d was first docked into human SIRT2 (PDB: 1j8f) [31]. A top pose showed that compound 7d's nicotinamide moiety (ring C) was projected into the C-site of the NAD + binding pocket and the remaining of the molecule fitted in SIRT2 s substrate binding channel ( Figure 3). ...
... The modeling study was carried out as described previously [29]. The X-ray crystal structure of human SIRT2 (PDB: 1j8f) [31] was used and the energy minimization was performed using OPLS 2005 forcefield. SIRT2 inhibitors were generated by LigPrep and then docked into the Glide grid (20 × 20 × 20 Å) encompassing the active site of SIRT2. ...
SIRT2 is a member of NAD⁺-dependent sirtuins and its inhibition has been proposed as a promising therapeutic approach for treating human diseases, including neurodegenerative diseases, cancer, and infections. Expanding SIRT2 inhibitors based on the 3-aminobenzyloxy nicotinamide core structure, we have synthesized and evaluated constrained analogs and selected stereoisomers. Our structure-activity relationship (SAR) study has revealed that 2,3-constrained (S)-isomers possess enhanced in vitro enzymatic inhibitory activity against SIRT2 and retain excellent selectivity over SIRT1 and SIRT3, provided that a suitable ring A is used. This current study further explores SIRT2 inhibitors based on the 3-aminobenzyloxy nicotinamide scaffold and contributes to the discovery of potent, selective SIRT2 inhibitors that have been actively pursued for their potential therapeutic applications.
... The chemical structure of SIRT2 is fully revealed in 2001, when Finnin et al. (2001) discovered that SIRT2 has a 304-amino acid catalytic core and a 19-residue N-terminal helical extension. The SIRT2 catalytic core has two domains: the larger one is a variant of the Rossmann fold, and the smaller one consists of the zinc-binding and the helical modules (Finnin et al., 2001). ...
... The chemical structure of SIRT2 is fully revealed in 2001, when Finnin et al. (2001) discovered that SIRT2 has a 304-amino acid catalytic core and a 19-residue N-terminal helical extension. The SIRT2 catalytic core has two domains: the larger one is a variant of the Rossmann fold, and the smaller one consists of the zinc-binding and the helical modules (Finnin et al., 2001). At the interface of the two domains, there is a large grove, which includes the NAD binding site and presents the HDAC activity. ...
As a type of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, sirtuin 2 (SIRT2) is predominantly found in the cytoplasm of cells in the central nervous system (CNS), suggesting its potential role in neurological disorders. Though SIRT2 is generally acknowledged to accelerate the development of neurological pathologies, it protects the brain from deterioration in certain circumstances. This review summarized the complex roles SIRT2 plays in the pathophysiology of diverse neurological disorders, compared and analyzed the discrete roles of SIRT2 in different conditions, and provided possible explanations for its paradoxical functions. In the future, the rapid growth in SIRT2 research may clarify its impacts on neurological disorders and develop therapeutic strategies targeting this protein.
... The catalytic core region contains a large domain that is homologous to the Rossmann-fold domain in sequence and structure, with a small Zn 2+ -binding domain and the cofactor binding loop region [23][24][25]. The Rossmann-fold domain has a conserved Gly-X-Gly sequence important for phosphate binding, a pocket to accommodate an NAD + molecule, and charged amino acid residues responsible for ribose group binding, which shows the characteristics of NAD + -binding site [23,26,27]. The Zn 2+ -binding domain is a small but structurally diverse domain that consists of a three-stranded antiparallel β sheet and a variable α helical region, dependent on each sirtuin protein. ...
... The Zn 2+ -binding domain is a small but structurally diverse domain that consists of a three-stranded antiparallel β sheet and a variable α helical region, dependent on each sirtuin protein. The small domain contains the conserved sequence motif Cys-X2-4-Cys-X15-40-Cys-X2-4-Cys, a characteristic Zn 2+ -binding motif [23,27]. The cofactor binding loop region has four loops that link the small and large domains to form the cleft that acts as the enzyme active site. ...
Alternative splicing generates multiple distinct isoforms that increase transcriptome and proteome diversity. There are seven sirtuin genes in humans, each consists of multiple exons that are likely to undergo alternative splicing. Our aim was to characterize the effect of alternative splicing on the sirtuin genes. Here, we report the identification of 23 human sirtuin isoforms, most of which were not previously reported. Five of the sirtuin genes had more than one isoform, whereas sirtuin-6 had nine isoforms. Exon skipping was the main event. Most of the sirtuin isoforms were deficient in parts of the protein domains, including the catalytic domain, the N- or C-terminus, nuclear localization signal or mitochondrial targeting signal. The domain loss caused potential structural changes. Three SIRT1 isoforms had a differential effect on the mitochondrial oxygen consumption rate. Age-related changes in the expression of SIRT1 isoforms were observed in the human heart in fetus, adults, and very old individuals. We also identified 15 sirtuin isoforms in mice. Our data indicate that alternative splicing increases sirtuin gene diversity and may modulate subcellular localization and function, thereby adding complexity to the gene regulation of mitochondrial respiration, metabolism, and cardiac function during maturation and aging.
... In addition, there are 19 residues in the N-terminal extension of SIRT2 from an amphipathic a-helix that has no contacts with the protein, suggesting the N-terminal extension is not essential for SIRT2 catalytic activity, while this amphipathic helixes may have an important contribution to protein-protein interactions in transcriptional regulation. 21 In addition, by comparison of SIRT1, SIRT2 and SIRT3, we observed substantial differences between the loops that can form different hydrophobic binding sites. Uniquely, SIRT2 has a long hydrophobic pocket which is able to accommodate long-chain fatty acyl groups as revealed by crystallographic analyses, 22 and also represent specic structural characteristics for inhibitor development. ...
... As an important member of the SIRT family, SIRT2 is highly homologous to SIRT1 and SIRT3, containing a smaller Zn 2+ binding domain and a relatively larger Rossmann folding domain, and a catalytic core domain (including the substrate binding site and NAD + binding site) formed by the "loop" connecting these two domains. 21 While SIRT2 has a specic, deep hydrophobic site to accommodate long-chain acyl-lysine substrates as well as to recognize selective small-molecule inhibitors. As summarized above, several structurally distinct selective small-molecule SIRT2 inhibitors have been developed, which were demonstrated by crystallographic analyses to bind with the unique hydrophobic site of SIRT2, providing important clues or inspiration idea for future inhibitor design. ...
Sirtuin 2 (SIRT2) is an important and special member of the atypical histone deacetylase Sirtuin (SIRT) family. Due to its extensive catalytic effects, SIRT2 can regulate autophagy, myelination, immunity, inflammation and other physiological processes. Recent evidence revealed that dysregulation of human SIRT2 activity is associated with the pathogenesis and prognosis of cancers, Parkinson's disease and other disorders; thus SIRT2 is a promising target for potential therapeutic intervention. This review presents a systematic summary of nine chemotypes of small-molecule SIRT2 inhibitors, particularly including the discovery and structural optimization strategies, which will be useful for future efforts to develop new inhibitors targeting SIRT2 and associated target proteins.
... Within the last two decades, several sirt1 and sirt2 crystal structures have been solved in both apo and holo forms [31][32][33][34][35][36][37][38][39][40][41][42][43]. Ternary structures of sirt1 and sirt2 in complex with cofactor analogues, peptide-based and structurally diverse inhibitors revealed a high conformational flexibility in the catalytic pocket, especially in the extended C-pocket region. ...
... In the crystal structures of sirt1, it was shown that substrates make H-bond interactions with the backbone of a conserved valine residue (sirt1 numbering Val412) which is crucial for the correct orientation of the acyl-lysine in the active site [44]. In case of sirt2, we first examined the binding interactions of the native ligands including both the peptide substrates, the cofactor fragments and the co-crystallized inhibitors with the protein [31][32][33][34][35][36][37][38][39][40][41][42][43]. In the hit selection process, a special importance was given to compounds that were able to interact with residues Phe234, Phe235, Phe190 and Glu237 in the catalytic pocket. ...
Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylases and have been linked to the pathogenesis of numerous diseases such as HIV, metabolic disorders, neurodegeneration and cancer. Docking of the virtual pan-African natural products library (p-ANAPL), followed by in vitro testing, resulted in the identification of two inhibitors of sirtuin 1, 2 and 3 (sirt1-3). Two bichalcones; rhuschalcone IV (8) and rhuschalcone I (9), previously isolated from the medicinal plant Rhus pyroides, were shown to be active in the in vitro assay, with rhuschalcone I showing the best activity against sirt1, having an IC50 = 40.8 µM. Based on the docking experiments, suggestions for improving the biological activities of the newly identified hit compounds have been provided.
... Sirtuins (Sirts), which are integral members of the atypical class III histone deacetylase (HDAC) superfamily, encompass seven distinguished entities denoted as sirtuin1 to sirtuin7, according to de Ruijter et al. (2003). The mammalian Sirt family, homologous to the yeast silent information regulator 2 (Sir2 protein) and reliant on nicotinamide adenine dinucleotide (NAD)+ for its histone deacetylase activity, regulates diverse biological events such as stress responses, metabolism, and apoptosis, as elucidated by Finnin et al. (2001); North and Verdin (2004); Blander and Guarente (2004); Chalkiadaki and Guarente (2015), and Aventaggiato et al. (2021). The burgeoning literature increasingly reports on the interaction between sirtuins and viral infections, notably highlighted by the findings that sirtuin 1 (Sirt1) suppresses HBV transcription (Belloni et al., 2012). ...
The human sirtuin 2 gene (SIRT2) encodes a full-length Sirt2 protein (i.e., the Sirt2 isoform 1), which primarily functions as a cytoplasmic α-tubulin deacetylase, and which promotes the growth of hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) replication itself, or HBV X (HBx) protein-mediated transcriptional transactivation, enhances Sirt2.1 expression; therefore, Sirt2.1 itself is capable of positively increasing HBV transcription and replication. Sirt2.1 is linked to liver fibrosis and epithelial-to-mesenchymal transition and, consequently, augments the risk of HCC. The Sirt2.1 protein enhances the HBV replication cycle by activating the AKT/glycogen synthase kinase 3 beta (GSK3β)/β-catenin pathway. It also activates the transcription of the viral enhancer I/HBx promoter (EnI/Xp) and enhancer II/HBc promoter (EnII/Cp) by targeting the transcription factor p53. The Sirt2 isoform 2 (Sirt2.2) is mainly localized in the cytoplasm, and the N-terminus is shorter by 37 amino acids than that of Sirt2.1. Despite the truncation of the N-terminal region, Sirt2.2 is still capable of enhancing HBV replication and activating the AKT/GSK3β/β-catenin signaling pathway. The Sirt2 isoform 5 (Sirt2.5) is primarily localized to the nucleus, it lacks a nuclear export signal (NES), and the catalytic domain (CD) is truncated. Upon HBV replication, expression of the Sirt2 isoforms is also enhanced, which further upregulates the HBV replication, and, therefore, supports the vicious cycle of viral replication and progression of the disease. Sirt2 diversely affects HBV replication such that its isoform 1 intensely augments HBV replication and isoform 2 (despite of the truncated N-terminal region) moderately enhances HBV replication. Isoform 5, on the other hand, tends to protect the cell (for smooth long-term continued viral replication) from HBV-induced extreme damage or death via a discrete set of regulatory mechanisms impeding viral mRNAs, the hepatitis B core/capsid protein (HBc), core particles, replicative intermediate (RI) DNAs, and covalently closed circular DNA (cccDNA) levels, and, consequently, limiting HBV replication. In contrast to Sirt2.1 and Sirt 2.2, the Sirt2.5-mediated HBV replication is independent of the AKT/GSK3β/β-catenin signaling cascade. Sirt2.5 is recruited more at cccDNA than the recruitment of Sirt2.1 onto the cccDNA. This recruitment causes the deposition of more histone lysine methyltransferases (HKMTs), including SETDB1, SUV39H1, EZH2, and PR-Set7, along with the respective corresponding transcriptional repressive markers such as H3K9me3, H3K27me3, and H4K20me1 onto the HBV cccDNA. In HBV-replicating cells, Sirt2.5 can also make complexes with PR-Set7 and SETDB1. In addition, Sirt2.5 has the ability to turn off transcription from cccDNA through epigenetic modification via either direct or indirect interaction with HKMTs.
... In 2016, Jing et al. developed thioacyl lysine compounds targeting SIRT that can recognize aliphatic acyl groups for treating especially c-Myc-driven cancers. Of these, TM (thiomyristoyl) (35), with a 14-carbon thioacyl group, was identified as a mechanism-based inhibitor that inhibits SIRT2 with an IC 50 value of 28 nM but inhibits SIRT1 with an IC 50 value of 98 μM and shows no inhibitory potential against SIRT3 even at 200 μM [111]. . JH-T4 is a potent SIRT2 inhibitor, but unlike TM, it can control the lysine fatty acylation levels of the oncoprotein K-Ras4a in vitro and in cells [145]. ...
... Due to a severe orientation preference problem, we did not obtain high-resolution structures of EcSIR2 and EcHerA ( Figures 1A-1C, S1C, and S1D; Table 1). The structure of EcSIR2 at 3.6 Å revealed that EcSIR2 alone assembles as a dodecamer, contrasting the well-studied SIR2 homologs that all exist as monomers 17,18,[20][21][22] (Figures 1A, 1B, and S1D; Table 1). Unexpectedly, EcHerA forms diverse oligomers, including dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, and nonamers ( Figure 1C). ...
SIR2-HerA, a bacterial two-protein anti-phage defense system, induces bacterial death by depleting NAD+ upon phage infection. Biochemical reconstitution of SIR2, HerA, and the SIR2-HerA complex reveals a dynamic assembly process. Unlike other ATPases, HerA can form various oligomers, ranging from dimers to nonamers. When assembled with SIR2, HerA forms a hexamer and converts SIR2 from a nuclease to an NAD+ hydrolase, representing an unexpected regulatory mechanism mediated by protein assembly. Furthermore, high-concentrations of ATP can inhibit NAD+ hydrolysis by the SIR2-HerA complex. Cryo-EM structures of the SIR2-HerA complex reveal a giant supramolecular assembly up to 1 MDa, with SIR2 as a dodecamer and HerA as a hexamer, crucial for anti-phage defense. Unexpectedly, the HerA hexamer resembles a spiral staircase and exhibits helicase activities toward dual-forked DNA. Together, we reveal the supramolecular assembly of SIR2-HerA as a unique mechanism for switching enzymatic activities and bolstering anti-phage defense strategies.
... SIRT3 is a member of the highly conserved sirtuin superfamily (SIRT1-7) and one of three sirtuins localized to the mitochondria where it acts as the primary deacetylase (Onyango et al., 2002;Lombard et al., 2007). Its deacetylase activity is dependent on NAD 1 , linking SIRT3 function to the metabolic status of the cell (Finnin et al., 2001). With a broad range of target proteins involved in antioxidant response, oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and fatty acid oxidation, SIRT3 is therefore postulated to be a critical, redox-sensitive interface between cellular environment and metabolism (Kumar and Lombard, 2015;Yang et al., 2016). ...
Mitochondrial dysfunction is an early event in the pathogenesis of neurological disorders and aging. Sirtuin 3 (SIRT3) regulates mitochondrial function in response to the cellular environment through the reversible deacetylation of proteins involved in metabolism and reactive oxygen species detoxification. As the primary mitochondrial deacetylase, germline, or peripheral tissue-specific deletion of SIRT3 produces mitochondrial hyperacetylation and the accelerated development of age-related diseases. Given the unique metabolic demands of neurons, the role of SIRT3 in the brain is only beginning to emerge. Using mass-spectrometry based acetylomics, high-resolution respirometry, video-EEG, and cognition testing, we report targeted deletion of SIRT3 from select neurons in the cortex and hippocampus produces altered neuronal excitability and metabolic dysfunction in female mice. Targeted deletion of SIRT3 from neuronal helix-loop-helix 1 (NEX)-expressing neurons resulted in mitochondrial hyperacetylation, female-specific superoxide dismutase-2 (SOD2) modification, increased steady-state superoxide levels, metabolic reprogramming, altered neuronal excitability and working spatial memory deficits. Inducible neuronal deletion of SIRT3 likewise produced female-specific deficits in spatial working memory. Together, the data demonstrate that deletion of SIRT3 from forebrain neurons selectively predisposes female mice to deficits in mitochondrial and cognitive function.
SIGNIFICANCE STATEMENT:
Mitochondrial SIRT3 is an enzyme shown to regulate energy metabolism and antioxidant function, by direct deacetylation of proteins. In this study, we show that neuronal SIRT3 deficiency renders female mice selectively vulnerable to impairment in redox and metabolic function, spatial memory, and neuronal excitability. The observed sex-specific effects on cognition and neuronal excitability in female SIRT3 deficient mice suggest that mitochondrial dysfunction may be one factor underlying comorbid neuronal diseases such as Alzheimer’s disease and epilepsy. Furthermore, the data suggest that SIRT3 dysfunction may predispose females to age-related metabolic and cognitive impairment.
... That is why SIRT2 is an emerging drug target for therapeutic intervention 14,27,28 . Like all other sirtuins, SIRT2 has two domain structures, the Rossmann fold domain (RFD) and zinc-binding domain (ZBD) 29,30 . SIRT2 binding pocket is situated between the two domains in a wide hydrophobic groove 31,32 . ...
Sirtuin 2 (SIRT2) is a member of the sirtuin protein family, which includes lysine deacylases that are NAD +-dependent and organize several biological processes. Different forms of cancer have been associated with dysregulation of SIRT2 activity. Hence, identifying potent inhibitors for SIRT2 has piqued considerable attention in the drug discovery community. In the current study, the Natural Products Atlas (NPAtlas) database was mined to hunt potential SIRT2 inhibitors utilizing in silico techniques. Initially, the performance of the employed docking protocol to anticipate ligand-SIRT2 binding mode was assessed according to the accessible experimental data. Based on the predicted docking scores, the most promising NPAtlas molecules were selected and submitted to molecular dynamics (MD) simulations, followed by binding energy computations. Based on the MM-GBSA binding energy estimations over a 200 ns MD course, three NPAtlas compounds, namely NPA009578, NPA006805, and NPA001884, were identified with better ΔG binding towards SIRT2 protein than the native ligand (SirReal2) with values of − 59.9, − 57.4, − 53.5, and − 49.7 kcal/mol, respectively. On the basis of structural and energetic assessments, the identified NPAtlas compounds were confirmed to be steady over a 200 ns MD course. The drug-likeness and pharmacokinetic characteristics of the identified NPAtlas molecules were anticipated, and robust bioavailability was predicted. Conclusively, the current results propose potent inhibitors for SIRT2 deserving more in vitro/in vivo investigation.
... These two domains form a pocket in the middle where NAD and acetylated peptides bind. 2,27 Differences among members of the SIRT protein family were initially attributed to their discrete pattern of subcellular localization. 28 As far as we know, SIRT1 is mainly localized in the nucleus and shuttles to the cytosol under specific circumstances. ...
Sirtuins (SIRTs) are nicotine adenine dinucleotide(+)-dependent histone deacetylases regulating critical signaling pathways in prokaryotes and eukaryotes, and are involved in numerous biological processes. Currently, seven mammalian homologs of yeast Sir2 named SIRT1 to SIRT7 have been identified. Increasing evidence has suggested the vital roles of seven members of the SIRT family in health and disease conditions. Notably, this protein family plays a variety of important roles in cellular biology such as inflammation, metabolism, oxidative stress, and apoptosis, etc., thus, it is considered a potential therapeutic target for different kinds of pathologies including cancer, cardiovascular disease, respiratory disease, and other conditions. Moreover, identification of SIRT modulators and exploring the functions of these different modulators have prompted increased efforts to discover new small molecules, which can modify SIRT activity. Furthermore, several randomized controlled trials have indicated that different interventions might affect the expression of SIRT protein in human samples, and supplementation of SIRT modulators might have diverse impact on physiological function in different participants. In this review, we introduce the history and structure of the SIRT protein family, discuss the molecular mechanisms and biological functions of seven members of the SIRT protein family, elaborate on the regulatory roles of SIRTs in human disease, summarize SIRT inhibitors and activators, and review related clinical studies.
... Sirts and Parps are enzymes that require NAD for their activities (Finnin et al., 2001;Kim et al., 2004). As Sirts and Parps are acetyltransferases and poly(ADP-ribose) polymerases, respectively, lysine acetylation, histone acetylation and poly(ADP-ribosyl)ation (PARylation) levels were examined. ...
Nicotinamide mononucleotide adenylyltransferase (Nmnat) is a class of enzymes consisting of three members (Nmnat1-3). Nmnat1 is in nucleus and associated with Leber congenital amaurosis, a form of early-onset retinal degeneration, while Nmnat2 is in cytoplasm and a well-characterized neuroprotective factor. The differences in their biological roles in the retina are unclear. We performed short hairpin RNA (shRNA)-based loss-of-function analysis of Nmnat2 during mouse retinal development in retinal explant cultures prepared from early (E14.5), middle (E17.5) or late (postnatal day [P]0.5) developmental stages. Nmnat2 has important roles for survival of retinal cells in the early and middle stages of retinal development. Retinal cell death caused by Nmnat2 knockdown could be partially rescued by supplementation with NAD or nicotinamide mononucleotide (NMN). Survival of retinal cells in the late stage of retinal development was unaffected by Nmnat2, but differentiation of Müller glia was controlled by Nmnat2. RNA-Seq analyses showed perturbation of gene expression patterns by shRNAs specific for Nmnat1 or Nmnat2, but Gene Ontology analysis did not provide a rational explanation for the phenotype. This study showed that Nmnat2 has multiple developmental stage-dependent roles during mouse retinal development, which were clearly different from those of Nmnat1, suggesting specific roles for Nmnat1 and Nmnat2.
... Finally, to understand how NAR and HSP interact with SIRT2, we investigated the putative binding mechanism of these two flavanones by computational techniques. SIRT2 structure was fully deciphered in 2001 by Finnin and co-workers [49] who revealed that SIRT2 possesses a catalytic core domain with NAD + -binding capacity as well as N-and C-terminal extensions. The catalytic core contains a variant of the Rossmann fold and a small domain consisting of helical and zinc-binding modules. ...
Acute myeloid leukemia (AML) represents the most alarming hematological disease for adults. Several genetic modifications are known to be pivotal in AML; however, SIRT2 over-expression has attracted the scientific community’s attention as an unfavorable prognostic marker. The plant kingdom is a treasure trove of bioactive principles, with flavonoids standing out among the others. On this line, the aim of this study was to investigate the anti-leukemic properties of the main flavanones of Citrus spp., exploring the potential implication of SIRT2. Naringenin (NAR), hesperetin (HSP), naringin (NRG), and neohesperidin (NHP) inhibited SIRT2 activity in the isolated recombinant enzyme, and more, the combination between NAR and HSP. In monocytic leukemic THP-1 cells, only NAR and HSP induced antiproliferative effects, altering the cell cycle. These effects may be ascribed to SIRT2 inhibition since these flavonoids reduced its gene expression and hampered the deacetylation of p53, known sirtuin substrate, and contextually modulated the expression of the downstream cell cycle regulators p21 and cyclin E1. Additionally, these two flavanones proved to interact with the SIRT2 inhibitory site, as shown by docking simulations. Our results suggest that both NAR and HSP may act as anti-leukemic agents, alone and in combination, via targeting the SIRT2/p53/p21/cyclin E1 pathway, thus encouraging deeper investigations.
... SIRT2 consists of a 304-amino acid catalytic core and a 19 residue N-terminal helical extension. The core is mainly composed of two domains: the larger one is a variant of the Rossmann fold23, which exists in many different NAD (H)/ NADP (H)-binding enzymes, and the smaller one contains a zinc atom (Finnin et al., 2001). Although mainly located in the cytoplasm, under special circumstances SIRT2 can translocate into the nucleus and deacetylase histones. ...
Cardiovascular diseases are a group of diseases with high morbidity and mortality that affect millions of people each year. Vascular calcification (VC) is an active process that involves the mineral deposition of calcium-phosphate complexes. VC is closely related to cardiovascular diseases, such as hypertension, heart failure, and calcific aortic stenosis, and is a type of ectopic calcification that occurs in the vessel walls. The sirtuins (silent mating-type information regulation 2; SIRTs), are a family of histone deacetylases whose function relies on nicotinamide adenine dinucleotide (NAD+). They have non-negligible functions in the regulation of energy metabolism, senescence, apoptosis, and other biological processes. Sirtuins have important effects on bone homeostasis and VC processes that share many similarities with bone formation. Sirtuins have been confirmed to deacetylate a variety of target proteins related to the occurrence and development of VC, thereby affecting the process of VC and providing new possibilities for the prevention and treatment of cardiovascular diseases. To facilitate the understanding of vascular calcification and accelerate the development of cardiovascular drugs, we reviewed and summarized recent research progress on the relationship between different types of sirtuins and VC.
... To assess the potential of SIRT2 to reverse alcoholic liver injury, we developed a recombinant adenoassociated viral vector serotype 8 (AAV8) expressing SIRT2 or its catalytic inactive SIRT2-H187A (H187A) mutant 17,18 under the control of the hepatocyte-specific thyroxin-binding globulin (TBG) promoter (AAV8- , and the percentages of liver tissues with high and low necrosis (e) or fibrosis (f) in SIRT2 low and SIRT2 high groups. Statistical significance was determined by two-tailed Student's t-test (b-d), Pearson's χ 2 test (e, f). ...
Protein acetylation has emerged to play pivotal roles in alcoholic liver disease (ALD). Sirutin 2 (SIRT2) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase involved in the regulation of aging, metabolism, and stress. However, the role of SIRT2 in ALD remains unclear. Here, we report that the SIRT2-mediated deacetylation–deubiquitination switch of CCAAT/enhancer-binding protein beta (C/EBPβ) prevents ALD. Our results showed that hepatic SIRT2 protein expression was negatively correlated with the severity of alcoholic liver injury in ALD patients. Liver-specific SIRT2 deficiency sensitized mice to ALD, whereas transgenic SIRT2 overexpression in hepatocytes significantly prevented ethanol-induced liver injury via normalization of hepatic steatosis, lipid peroxidation, and hepatocyte apoptosis. Mechanistically, we identified C/EBPβ as a critical substrate of SIRT2 implicated in ALD. SIRT2-mediated deacetylation at lysines 102 and 211 decreased C/EBPβ ubiquitination, resulting in enhanced protein stability and subsequently increased transcription of C/EBPβ-target gene LCN2. Importantly, hepatic deacetylated C/EBPβ and LCN2 compensation reversed SIRT2 deletion-induced ALD aggravation in mice. Furthermore, C/EBPβ protein expression was positively correlated with SIRT2 and LCN2 expression in the livers of ALD patients and was inversely correlated with ALD development. Therefore, activating SIRT2-C/EBPβ-LCN2 signaling pathway is a potential therapy for ALD.
... Rapamycin preferentially associates with the beta sheet loop of FKBP12 (Liang et al., 1999). SIRT2 is helical overall in structure, which is highly homologous to that of the FRB domain involved in association with rapamycin-FKBP12 (Finnin et al., 2001). However, the underlying mechanisms still need to be further explored. ...
The mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cellular innate immunity, metabolism, and senescence. FK506-binding protein 12 (FKBP12) inhibits mTOR kinase activity via direct association. The FKBP12-mTOR association can be strengthened by the immunosuppressant rapamycin, but the underlying mechanism remains elusive. We show here that the FKBP12-mTOR association is tightly regulated by an acetylation-deacetylation cycle. FKBP12 is acetylated on the lysine cluster (K45/K48/K53) by CBP in mammalian cells in response to nutrient treatment. Acetyl-FKBP12 associates with CBP acetylated Rheb. Rapamycin recruits SIRT2 with a high affinity for FKBP12 association and deacetylation. SIRT2-deacetylated FKBP12 then switches its association from Rheb to mTOR. Nutrient-activated mTOR phosphorylates IRF3S386 for the antiviral response. In contrast, rapamycin strengthening FKBP12-mTOR association blocks mTOR antiviral activity by recruiting SIRT2 to deacetylate FKBP12. Hence, on/off mTOR activity in response to environmental nutrients relies on FKBP12 acetylation and deacetylation status in mammalian cells.
... ,[100][101] Sirtuins have a conserved catalytic core with varying N-and C-terminal sequences and use NAD + as a cosubstrate to form nicotinamide, adenine diphosphate ribose, acetate, and the deacetylated protein product.100,[102][103] Although there is some functional redundancy, the sirtuins vary in subcellular localization, substrate selectivity, and cellular function. ...
Lysine acetylation regulates thousands of proteins and nearly every cellular process from cell replication to cell death. Dysregulated acetylation has been implicated in diseases including cancer, neurodegenerative disorders, and infectious diseases. For this reason, the enzymes that regulate acetylation, including the histone deacetylases (HDACs), are targeted for drug development, and understanding their biological function is of the utmost importance. Unfortunately, few HDAC-specific substrates have been identified, and how HDACs recognize and select for their substrates, a key aspect of their biological function, is poorly understood. HDAC8, a unique member of class I, is phosphorylated at S39, which affects HDAC8 substrate selectivity in vitro and may be used by the cell to regulate HDAC8 biological function. Measuring HDAC8 phosphomimetic mutant S39E-catalyzed deacetylation of various peptides demonstrates altered HDAC8 activity and importantly substrate selectivity. Structural analyses indicate this alteration is due to changes in the substrate binding pocket and active-site architecture. Moreover, wild-type HDAC8 substrate selectivity is influenced by both substrate sequence and structure in vitro. Comparing HDAC8-catalyzed deacetylation of histone H3 K9ac, K14ac, and K56ac peptides and proteins reveals that protein structure enhances activity from 40- to over 300-fold, and local sequence determines substrate selectivity, particularly in less structured regions. These data support the use of peptide substrates to determine relative activity and to identify HDAC substrates. To expand on these results, HDAC6-catalyzed deacetylation of a library of peptides was tested to develop a structure-based model of HDAC6 activity. The results reveal HDAC6 distinguishes between sequences, catalyzing deacetylation of peptides with kcat/KM values from 10 to 106 M-1s-1. These data demonstrate the usefulness of a prediction model based on peptides. Together, these investigations reveal that phosphorylation, local sequence, and protein structure affect HDAC substrate selectivity and activity in vitro and likely play key roles in the biological function and dysfunction of HDACs in the cell. Finally, development of structure-based models combined with peptide-based experiments can be used to identify HDAC substrate candidates for study in vivo.
... They have conserved catalytic core region consisting of $275 amino acids at the center and is flanked with N-and C-terminal regions varying in length and sequence (Sanders et al., 2010). The crystal structures of sirtuin catalytic core regions are known, and it consists of two domains; a large and structurally homologous Rossmann-fold domain, and a more structurally diverse, smaller, zinc-binding domain (Finnin et al., 2001;Min et al., 2001;Pan et al., 2011). The loops connecting these domains form a cleft, where the co-factor NAD þ and acetyl-lysine containing peptide substrate enter from opposite sides and binds to the enzyme. ...
Sirtuin-6 (SIRT6), class III family of deacetylase regulates several biological functions, including transcriptional repression, telomere maintenance, and DNA repair. It is unique among sirtuin family members with diverse enzymatic functions: mono-ADP-ribosylase, deacetylase and defatty-acylase. The studies so far implicated SIRT6 role in lifespan extension, tumor suppression, and is considered as an attractive drug target for aging-related disease. In this study, we have carried out in silico screening for human SIRT6 modulators using NCI Diversity Set III library, molecular dynamic (MD) simulations to analyze the protein-ligand interaction, and validated their binding-affinity (Kd) using MicroScale Thermophoresis. This study yielded two novel compounds, ((3Z)-3-((4-(dimethylamino)phenyl)methylidene)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)furan-2-one and 5-phenyl-2-(5-phenyl-2,3-dihydro-1,3-benzoxazol-2-yl)-2,3-dihydro-1,3-benzoxazole showing high-affinity interaction for SIRT6. The structural analysis from MD simulation suggests both compounds might act as substrate-analogs or mimic the nicotinamide binding. On considering the uniqueness of SIRT6 substrate binding acyl channel among sirtuin family member, binding of both compounds to the above site suggesting their specificity for SIRT6 isoform. Therefore, it may form the basis for the development of potential modulators for human SIRT6.
Communicated by Ramaswamy H. Sarma
... Therefore, activation and inhibition of Sirt2 have been constantly targeted for small molecule therapeutic development in reference to the biology under analysis [22][23][24][25] . Recent studies have documented the extensive biochemistry of Sirt2 and the crystal structures of the catalytic domain from several human isotopes revealed the various stages of the catalytic cycle [26][27][28][29] . As members of the Sirtuins family vastly shared conserved residues and high structure similarity in the catalytic core, numerous small-molecule isotype-selective inhibitors have been reported 25,[30][31][32] . ...
Sirtuin 2 (Sirt2) nicotinamide adenine dinucleotide-dependent deacetylase enzyme has been reported to alter diverse biological functions in the cells and onset of diseases, including cancer, aging, and neurodegenerative diseases, which implicate the regulation of Sirt2 function as a potential drug target. Available Sirt2 inhibitors or modulators exhibit insufficient specificity and potency, and even partially contradictory Sirt2 effects were described for the available inhibitors. Herein, we applied computational screening and evaluation of FDA-approved drugs for highly selective modulation of Sirt2 activity via a unique inhibitory mechanism as reported earlier for SirReal2 inhibitor. Application of stringent molecular docking results in the identification of 48 FDA-approved drugs as selective putative inhibitors of Sirt2, but only top 10 drugs with docking scores > − 11 kcal/mol were considered in reference to SirReal2 inhibitor for computational analysis. The molecular dynamics simulations and post-simulation analysis of Sirt2-drug complexes revealed substantial stability for Fluphenazine and Nintedanib with Sirt2. Additionally, developed 3D-QSAR-models also support the inhibitory potential of drugs, which exclusively revealed highest activities for Nintedanib (pIC50 ≥ 5.90 µM). Conclusively, screened FDA-approved drugs were advocated as promising agents for Sirt2 inhibition and required in vitro investigation for Sirt2 targeted drug development.
... All isoforms of the sirtuins possess a conserved HDAC domain, and there exists an overlapping activity between them. SIRT2 was the first isoform structure to be uncovered in 2001 (Finnin et al., 2001). However, at present, SIRT1 is the more extensively studied isoform. ...
... All isoforms of the sirtuins possess a conserved HDAC domain, and there exists an overlapping activity between them. SIRT2 was the first isoform structure to be uncovered in 2001 (Finnin et al., 2001). However, at present, SIRT1 is the more extensively studied isoform. ...
... Efforts of numerous groups over the last two decades have led to almost 30 X-ray crystal structures, including several co-crystal structures with ligands and inhibitors. [34][35][36][37][38][39][40][49][50][51][52][53][54][55][56] This has provided insight into the binding mechanism and substrate scope of SIRT2 at the molecular level. In the present work, we have built on this knowledge to develop the most potent and selective SIRT2 inhibitors reported to date. ...
Sirtuin 2 (SIRT2) is a protein deacylase enzyme that removes acetyl groups and longer chain acyl groups from post-translationally modified lysine residues. It affects diverse biological functions in the cell and has been considered a drug target in relation to both neurodegenerative diseases and cancer. Therefore, access to well-characterized and robust tool compounds is essential for the continued investigation of the complex functions of this enzyme. Here, we report a collection of chemical probes that are potent, selective, stable in serum, water-soluble, and inhibit SIRT2-mediated deacetylation and demyristoylation in cells. Compared to the current landscape of SIRT2 inhibitors, this is a unique ensemble of features built into a single compound. We expect the developed chemotypes to find broad application in the interrogation of SIRT2 functions in both healthy and diseased cells, and to provide a foundation for the development of future therapeutics.
... All isoforms of the sirtuins possess a conserved HDAC domain, and there exists an overlapping activity between them. SIRT2 was the first isoform structure to be uncovered in 2001 (Finnin et al., 2001). However, at present, SIRT1 is the more extensively studied isoform. ...
Sirtuins are NAD + dependent histone deacetylases (HDAC) that play a pivotal role in neuroprotection and cellular senescence. SIRT1-7 are different homologs from sirtuins. They play a prominent role in many aspects of physiology and regulate crucial proteins. Modulation of sirtuins can thus be utilized as a therapeutic target for metabolic disorders. Neurological diseases have distinct clinical manifestations but are mainly age-associated and due to loss of protein homeostasis. Sirtuins mediate several life extension pathways and brain functions that may allow therapeutic intervention for age-related diseases. There is compelling evidence to support the fact that SIRT1 and SIRT2 are shuttled between the nucleus and cytoplasm and perform context-dependent functions in neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). In this review, we highlight the regulation of SIRT1 and SIRT2 in various neurological diseases. This study explores the various modulators that regulate the activity of SIRT1 and SIRT2, which may further assist in the treatment of neurodegenerative disease. Moreover, we analyze the structure and function of various small molecules that have potential significance in modulating sirtuins, as well as the technologies that advance the targeted therapy of neurodegenerative disease.
... All isoforms of the sirtuins possess a conserved HDAC domain, and there exists an overlapping activity between them. SIRT2 was the first isoform structure to be uncovered in 2001 (Finnin et al., 2001). However, at present, SIRT1 is the more extensively studied isoform. ...
Sirtuins are NAD + dependent histone deacetylases (HDAC) that play a pivotal role in neuroprotection and cellular senescence. SIRT1-7 are different homologs from sirtuins. They play a prominent role in many aspects of physiology and regulate crucial proteins. Modulation of sirtuins can thus be utilized as a therapeutic target for metabolic disorders. Neurological diseases have distinct clinical manifestations but are mainly age-associated and due to loss of protein homeostasis. Sirtuins mediate several life extension pathways and brain functions that may allow therapeutic intervention for age-related diseases. There is compelling evidence to support the fact that SIRT1 and SIRT2 are shuttled between the nucleus and cytoplasm and perform context-dependent functions in neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). In this review, we highlight the regulation of SIRT1 and SIRT2 in various neurological diseases. This study explores the various modulators that regulate the activity of SIRT1 and SIRT2, which may further assist in the treatment of neurodegenerative disease. Moreover, we analyze the structure and function of various small molecules that have potential significance in modulating sirtuins, as well as the technologies that advance the targeted therapy of neurodegenerative disease.
... Plasmids of , and Sirt6 (1-314) for Escherichia coli expression were generated as previously described (Finnin et al., 2001;Du et al., 2011;Hubbard et al., 2013;Jiang et al., 2013). Plasmids of Sirt3 (102-399) cloned in pTrcHis 2C vector for E. coli expression and full length Sirt3 (wide-type and mutant H248Y) cloned into pcDNA3.1 vector for mammalian cell expression were generous gifts from Dr Eric Verdin (University of California, San Francisco). ...
Posttranslational modifications (PTMs) play a crucial role in a wide range of biological processes. Lysine crotonylation (Kcr) is a newly discovered histone PTM that is enriched at active gene promoters and potential enhancers in mammalian cell genomes. However, the cellular enzymes that regulate the addition and removal of Kcr are unknown, which has hindered further investigation of its cellular functions. Here we used a chemical proteomics approach to comprehensively profile ‘eraser’ enzymes that recognize a lysine-4 crotonylated histone H3 (H3K4Cr) mark. We found that Sirt1, Sirt2, and Sirt3 can catalyze the hydrolysis of lysine crotonylated histone peptides and proteins. More importantly, Sirt3 functions as a decrotonylase to regulate histone Kcr dynamics and gene transcription in living cells. This discovery not only opens opportunities for examining the physiological significance of histone Kcr, but also helps to unravel the unknown cellular mechanisms controlled by Sirt3, that have previously been considered solely as a deacetylase.
Sirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild‐type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 ( h SIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT h SIRT2 HDAC (residues 52–357) forms a homodimer in a concentration‐dependent manner with a dimer–monomer dissociation constant of 8.3 ± 0.5 μM, which was determined by mass spectrometry. The dimer was disrupted into two monomers by binding to the HDAC inhibitors SirReal1 and SirReal2. We also confirmed dimer formation of h SIRT2 HDAC in living cells using a NanoLuc complementation reporter system. Examination of the relationship between dimer formation and deacetylation activity using several mutants of h SIRT2 HDAC revealed that some non‐dimerizing mutants exhibited deacetylation activity for the N‐terminal peptide of histone H3, similar to the wild type. The h SIRT2 HDAC mutant Δ 292–306, which lacks a SIRT2‐specific disordered loop region, was identified to exist as a monomer with slightly reduced deacetylation activity; the X‐ray structure of the mutant Δ 292–306 was almost identical to that of the WT h SIRT2 HDAC bound to an inhibitor. These results indicate that h SIRT2 HDAC forms a dimer, but this is independent of deacetylation activity. Herein, we discuss insights into the dimer formation of h SIRT2 based on our biophysical experimental results.
Sirtuins (SIRTs) belong to the family of nicotine adenine dinucleotide (NAD+)-dependent class III histone deacetylases, which come into play in the regulation of epigenetic processes through the deacetylation of histones and other substrates. The human genome encodes for seven homologs (SIRT1-7), which are localized into the nucleus, cytoplasm, and mitochondria, with different enzymatic activities and regulatory mechanisms. Indeed, SIRTs are involved in different physio-pathological processes responsible for the onset of several human illnesses, such as cardiovascular and neurodegenerative diseases, obesity and diabetes, age-related disorders, and cancer. Nowadays, it is well-known that Citrus fruits, typical of the Mediterranean diet, are an important source of bioactive compounds, such as polyphenols. Among these, flavonoids are recognized as potential agents endowed with a wide range of beneficial properties, including antioxidant, anti-inflammatory, hypolipidemic, and antitumoral ones. On these bases, we offer a comprehensive overview on biological effects exerted by Citrus flavonoids via targeting SIRTs, which acted as modulator of several signaling pathways. According to the reported studies, Citrus flavonoids appear to be promising SIRT modulators in many different pathologies, a role which might be potentially evaluated in future therapies, along with encouraging the study of those SIRT members which still lack proper evidence on their support.
Mammalian members of the lysyl oxidase (LOX) family of proteins carry a copper-dependent monoamine oxidase domain exclusively within the C-terminal region, which catalyzes ε-amine oxidation of lysine residues of various proteins. However, recent studies have demonstrated that in LOX-like (LOXL) 2–4 the C-terminal canonical catalytic domain and N-terminal scavenger receptor cysteine-rich (SRCR)-repeats domain exhibit lysine deacetylation and deacetylimination catalytic activities. Moreover, the N-terminal SRCR-repeats domain is more catalytically active than the C-terminal oxidase domain. Thus, LOX is the third family of lysine deacetylases in addition to histone-deacetylase and sirtuin families. In this review, we discuss how the LOX family targets different cellular proteins for deacetylation and deacetylimination to control the development and metastasis of cancer.
Epigenetic mechanisms are crucial for normal development and maintenance of tissue-specific gene expression patterns in mammals. Impaired epigenetic processes can cause alterations in gene function and malignant cellular transformation. It is now known that epigenetic abnormalities, together with genetic changes, have a role in the onset and progression of cancer, which was once thought to be a genetic disease. Recent developments in the field of cancer epigenetics have demonstrated substantial reprogramming of all elements of the epigenetic machinery in cancer, including DNA methylation, histone modifications, nucleosome positioning, and non-coding RNAs. DNA methyltransferases, histone acetyltransferases, and histone deacetylases are a few examples of epigenetic regulatory enzymes that are involved in epigenetic modification. In recent years, an increasing number of studies have demonstrated that mutations in epigenetic regulatory enzymes occur in various cancer types and are closely associated with the malignant phenotype. Hence, research on inhibitors that target these mutant enzymes has gradually shifted into preclinical and clinical stages. In this chapter, we first discuss the epigenetic regulatory enzymes and then how their mutations are associated with carcinogenesis.
Human sirtuin isoform 2 (SIRT2) is an NAD⁺-dependent enzyme that functions as a lysine deacetylase and defatty-acylase. Here, we report that SIRT2 readily dimerizes in solution and in cells and that dimerization affects its ability to remove different acyl modifications from substrates. Dimerization of recombinant SIRT2 was revealed with analytical size exclusion chromatography and chemical cross-linking. Dimerized SIRT2 dissociates into monomers upon binding long fatty acylated substrates (decanoyl-, dodecanoyl-, and myristoyl-lysine). However, we did not observe dissociation of dimeric SIRT2 in the presence of acetyl-lysine. Analysis of X-ray crystal structures led us to discover a SIRT2 double mutant (Q142A/E340A) that is impaired in its ability to dimerize, which was confirmed with chemical cross-linking and in cells with a split-GFP approach. In enzyme assays, the SIRT2(Q142A/E340A) mutant had normal defatty-acylase activity and impaired deacetylase activity compared with the wild-type protein. These results indicate that dimerization is essential for optimal SIRT2 function as a deacetylase. Moreover, we show that SIRT2 dimers can be dissociated by a deacetylase and defatty-acylase inhibitor, ascorbyl palmitate. Our finding that its oligomeric state can affect the acyl substrate selectivity of SIRT2 is a novel mode of activity regulation by the enzyme that can be altered genetically or pharmacologically.
Lysine lactylation (Kla) is a novel histone post-translational modification discovered in late 2019. Later, HDAC1-3, were identified as the robust Kla erasers. While the Sirtuin family proteins showed weak eraser activities toward Kla, as reported. However, the catalytic mechanisms and physiological functions of HDACs and Sirtuins are not identical. In this study, we observed that SIRT3 exhibits a higher eraser activity against the H4K16la site than the other human Sirtuins. Crystal structures revealed the detailed binding mechanisms between lactyl-lysine peptides and SIRT3. Furthermore, a chemical probe, p-H4K16laAlk, was developed to capture potential Kla erasers from cell lysates. SIRT3 was captured by this probe and detected via proteomic analysis. And another chemical probe, p-H4K16la-NBD, was developed to detect the eraser-Kla delactylation processes directly via fluorescence indication. Our findings and chemical probes provide new directions for further investigating Kla and its roles in gene transcription regulation.
Sirtuin isoform 2 (SIRT2) is one of the seven sirtuin isoforms present in humans, being classified as class III histone deacetylases (HDACs). Based on the high sequence similarity among SIRTs, the identification of isoform selective modulators represents a challenging task, especially for the high conservation observed in the catalytic site. Efforts in rationalizing selectivity based on key residues belonging to the SIRT2 enzyme were accompanied in 2015 by the publication of the first X-ray crystallographic structure of the potent and selective SIRT2 inhibitor SirReal2. The subsequent studies led to different experimental data regarding this protein in complex with further different chemo-types as SIRT2 inhibitors. Herein, we reported preliminary Structure-Based Virtual Screening (SBVS) studies using a commercially available library of compounds to identify novel scaffolds for the design of new SIRT2 inhibitors. Biochemical assays involving five selected compounds allowed us to highlight the most effective chemical features supporting the observed SIRT2 inhibitory ability. This information guided the following in silico evaluation and in vitro testing of further compounds from in-house libraries of pyrazolo-pyrimidine derivatives towards novel SIRT2 inhibitors (1–5). The final results indicated the effectiveness of this scaffold for the design of promising and selective SIRT2 inhibitors, featuring the highest inhibition among the tested compounds, and validating the applied strategy.
The NAD+-dependent lysine deacylase Sirtuin2 (Sirt2) is involved in multiple pathological conditions, including cancer. Targeting Sirt2 has thus received an increased interest for therapeutic purpose, including the ortholog from Schistosoma mansoni (SmSirt2) for the potential treatment of the neglected tropical disease schistosomiasis. We previously identified a 1,2,4-oxadiazole-based scaffold from the screening of the “Kinetobox” library as inhibitor of both human Sirt2 and SmSirt2. Herein, we describe structure-activity studies on 1,2,4-oxadiazole based analogs, which are potent inhibitors of Sirt2 deacetylation and demyristoylation. Docking experiments proposed a substrate competitive and co-factor non-competitive binding mode of inhibition, which could be confirmed in vitro via binding assays and kinetic analysis. Optimized analogs reduced cell viability and inhibited prostate cancer cell migration, in correlation with Sirt2 deacetylase inhibition both in vitro and in cells.The NAD+-dependent lysine deacylase Sirtuin2 (Sirt2) is involved in multiple pathological conditions, including cancer. Targeting Sirt2 has thus received an increased interest for therapeutic purpose, including the ortholog from Schistosoma mansoni (SmSirt2) for the potential treatment of the neglected tropical disease schistosomiasis. We previously identified a 1,2,4-oxadiazole-based scaffold from the screening of the “Kinetobox” library as inhibitor of both human Sirt2 and SmSirt2. Herein, we describe structure-activity studies on 1,2,4-oxadiazole based analogs, which are potent inhibitors of Sirt2 deacetylation and demyristoylation. Docking experiments proposed a substrate competitive and co-factor non-competitive binding mode of inhibition, which could be confirmed in vitro via binding assays and kinetic analysis. Optimized analogs reduced cell viability and inhibited prostate cancer cell migration, in correlation with Sirt2 deacetylase inhibition both in vitro and in cells.
The silent information regulator (sirtuin) is a family of enzymes involved in epigenetic processes with lysine deacetylase activity, having as substrates histones and other proteins. They participate in a wide range of cellular and pathologic processes, such as gene expression, cell division and motility, oxidative-induced stress management, metabolic control and carcinogenesis, among others, thus presenting as interesting therapeutic targets. In this article, the authors describe the inhibitory mechanisms and binding modes of the human sirtuin 2 (hSIRT2) inhibitors, which had their complexes with the enzyme structurally characterized. The results help pave the way for the rational designing of new hSIRT2 inhibitors and the development of novel therapeutic agents targeting this epigenetic enzyme.
Sirtuins (SIRTs 1-7) are a group of histone deacetylase enzymes with a wide range of enzyme activities that target a range of cellular proteins in the nucleus, cytoplasm, and mitochondria for posttranslational modifications by acetylation (SIRT1, 2, 3, and 5) or ADP ribosylation (SIRT4, 6, and 7). A variety of cellular functions, including mitochondrial functions and functions in energy homeostasis, metabolism, cancer, longevity and ageing, are regulated by sirtuins. Compromised sirtuin functions and/or alterations in the expression levels of sirtuins may lead to several pathological conditions and contribute significantly to alterations in metabolic phenotypes as well as oral carcinogenesis. Here, we describe the basic characteristics of seven mammalian sirtuins. This review also emphasizes the key molecular mechanisms of sirtuins in metabolic regulation and discusses the possible relationships of sirtuins with oral cancers. This review will provide novel insight into new therapeutic approaches targeting sirtuins that may potentially lead to effective strategies for combating oral malignancies.
Sirtuins play an important role in signalling pathways associated with various metabolic regulations. They possess mono-ADP-ribosyltransferase or deacylase activity like demalonylase, deacetylase, depalmitoylase, demyristoylase and desuccinylase activity. Sirtuins are histone deacetylases which depends upon nicotinamide adenine dinucleotide (NAD) that deacetylate lysine residues. There are a total of seven human sirtuins that have been identified namely, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7. The subcellular location of mammalian sirtuins, SIRT1, SIRT6, and SIRT7 are in the nucleus; SIRT3, SIRT4, and SIRT5 are in mitochondria, and SIRT2 is in cytoplasm. Structurally sirtuins contains a N-terminal, a C-terminal and a Zn+ binding domain. The sirtuin family has been found to be crucial for maintaining lipid and glucose homeostasis, and also for regulating insulin secretion and sensitivity, DNA repair pathways, neurogenesis, inflammation, and ageing. Based on the literature, sirtuins are overexpressed and play an important role in tumorigenicity in various types of cancer such as non-small cell lung cancer, colorectal cancer, etc. In this review, we have discussed about the different types of human sirtuins along with their structural and functional features. We have also discussed about the various natural and synthetic regulators of sirtuin activities like resveratrol. Our overall study shows that the correct regulation of sirtuins can be a good target for preventing and treating various diseases for improving the human lifespan. To investigate the true therapeutic potential of sirtuin proteins and their efficacy in a variety of pathological diseases, a better knowledge of the link between the structure and function of sirtuin proteins would be necessary.
Sirtuine sind eine evolutionär hoch konservierte Familie der Histon-Deacetylasen. Sie katalysieren die Deacylierung von Acyllysinresten in Substratproteinen durch einen einzigartigen Mechanismus, der NAD+ als Cosubstrat benötigt. Dies macht Sirtuine zu metabolischen Sensoren. Diese Enzyme regulieren Stressreaktionen und Alterungsprozesse. Da sie in altersbedingten und metabolischen Erkrankungen involviert sind, ist die Regulation von Sirtuinen von medizinischem Interesse und besitzt therapeutisches Potenzial. In dieser Arbeit wurden neue Erkenntnisse zur endogenen Regulation von Sirtuinen durch das Protein AROS und zur Sirtuinmodulation durch Kleinmoleküle gewonnen. Das Pflanzenflavonoid Quercetin hat im Gegensatz zur Sirt6-spezifischen Aktvierung eine inhibitorische Wirkung auf die anderen Sirtuinisoformen. Die molekularen Grundlagen der Quercetin-vermittelten Inhibition konnte mit Hilfe einer Kristallstruktur des Sirt2/Quercetin-Komplexes aufgeklärt werden. So inhibiert der Naturstoff die Sirtuinaktivität durch Bindung am Eingang des aktiven Zentrums und daraus resultierender Kompetition mit dem acylierten Substrat. In Sirt6 wird diese Bindungsstelle durch den N-Terminus des Proteins besetzt. Die Sirt2-Quercetinbindungsstelle ist in Sirt6 nicht zugänglich. Quercetin bindet stattdessen in einer Sirt6-spezifischen Bindungstasche. Die Sirt2/Quercetin-Struktur trägt dazu bei, den Sirt6-spezifische Aktivierungseffekt von Quercetin zu erklären und liefert somit Informationen zur Weiterentwicklung von Sirt6-spezifischen, potenten Modulatoren. Die Erkenntnisse der Quercetin-Bindung in Sirt2 könnten zudem zur Entwicklung neuer Quercetin-basierender Inhibitoren beitragen. Neben der Regulation von Sirt6 und Sirt2 steht auch die Aktivierung von Sirt3 im Fokus. Als einziges Sirtuin mit robuster Deacetylaseaktivität im Mitochondrium gilt es als vielversprechendes Ziel zur pharmakologischen Modulation durch Aktivatoren. Aufgrund des Mangels an Sirt3-Aktivatoren wurde durch kristallografisches Screening einer Fragment-Bibliothek nach neuen Modulatoren für Sirt3 gesucht. Auf diese Weise konnten zwei neue Liganden sowie drei bisher unbekannte Liganden-Bindungsstellen für Sirt3 gefunden werden. Aufbauend auf diesen Fragmentgerüsten könnten Kleinmoleküle entwickelt werden, die Potential zur Modulation der Sirt3-Aktivität besitzen. Das Protein AROS wurde zunächst als bisher einziger endogener Aktivator von Sirt1 beschrieben. Dem widersprechend wurde von anderen Autoren eine inhibierende Wirkung von AROS auf die Sirt1-Deacetylaseaktivität festgestellt. Das Verständnis des Regulationsmechanismus von Sirt1 durch AROS könnte zur Entwicklung pharmakologischer Modulatoren beitragen. In dieser Arbeit konnte das Protein AROS zum ersten Mal charakterisiert werden. AROS akkumuliert exprimiert in E. coli in Einschlusskörpern. Daher wurde ein Renaturierungsprotokoll etabliert, welches in löslichem, reinem Protein resultierte. AROS konnte als ein flexibles Protein ohne stabile Tertiärstruktur aber mit Sekundärstrukturelementen wie α Helices charakterisiert werden. Zudem scheint das Protein unter stabilisierenden Bedingungen eine deutlich strukturiertere Konformation einzunehmen. Der Sirt1/AROS-Komplex musste zunächst durch Optimierung des Puffersystems stabilisiert werden. Anschließende Aktivitätstest von Sirt1 und AROS resultierten in einer Inhibition der Sirt1-Deacetylaseaktivität. Für die Isoformen 2,3 und 5 konnte ebenso eine AROS-vermittelte Inhibition gezeigt werden. Diese ist im Vergleich zu Sirt1 jedoch geringer. Als Interaktionsbereich im Sirtuin konnte der generische, katalytische Sirtuinkern identifiziert werden. Zudem lässt sich die höhere inhibitorische Wirkung von AROS auf Sirt1 durch zusätzliche Interaktionen mit der regulatorischen, N-terminalen Domäne von Sirt1 erklären. Für AROS konnte gezeigt werden, dass der mittlere Bereich des Proteins für die Inhibition verantwortlich ist. N- und C-terminale Abschnitte in AROS scheinen die Affinität zum Sirtuin durch zusätzliche Bindungsstellen zu erhöhen bzw. wichtige Bereiche für die Inhibition zu stabilisieren. Titrationen der beiden Sirt1-Substrate resultierten in einer Verringerung der Affinität des acetylierten Peptid-Substrates in Anwesenheit von AROS und liesen daher auf einen Inhibitons-Mechanismus, der auf Kompetiton mit dem acetylierten Peptid-Substrat beruht, schließen. Eine Kristallstruktur von Sirt3 konnte in Komplex mit einem AROS-Peptid gelöst werden. Im aktiven Zentrum des Sirtuins in der Bindungsstelle des acetylierten Lysins befindet sich das Lysin 65 von AROS, was den Kompetitionsmechanismus bestätigt. Unter Verwendung der strukturellen und mechanistischen Daten wurde ein Modell für den Sirt1/AROS-Komplex postuliert, in dem AROS die Bildung eines Enzym-Substrat-Komplexes durch Interaktion mit dem N-Terminus und der katalytischen Domäne von Sirt1 verhindert.
Sirtuin 6 (SIRT6) is an NAD+-dependent protein deacylase and mono-ADP-ribosyltransferase of the sirtuin family with a wide substrate specificity. In vitro and in vivo studies have indicated that SIRT6 overexpression or activation has beneficial effects for cellular processes such as DNA repair, metabolic regulation, and aging. On the other hand, SIRT6 has contrasting roles in cancer, acting either as a tumor suppressor or promoter in a context-specific manner. Given its central role in cellular homeostasis, SIRT6 has emerged as a promising target for the development of small-molecule activators and inhibitors possessing a therapeutic potential in diseases ranging from cancer to age-related disorders. Moreover, specific modulators allow the molecular details of SIRT6 activity to be scrutinized and further validate the enzyme as a pharmacological target. In this Perspective, we summarize the current knowledge about SIRT6 pharmacology and medicinal chemistry and describe the features of the activators and inhibitors identified so far.
Silent information regulator 2 family proteins (sirtuins) are members of a highly conserved family of deacetylases found in organisms ranging from bacteria to human beings, functioning as a molecular chain to transmit cell energy and regulate the cell cycle. Seven members of the human sirtuin family have been identified, which are named as SIRT1–SIRT7. They all have a highly conserved NAD⁺ binding domain and a catalytic domain, and their specific N- and C-terminals enable their substrate specificity. In this study, five sirtuin genes, named SIRT1, SIRT2, SIRT4, SIRT6 and SIRT7, were identified in the Manila clam Ruditapes philippinarum. Phylogenetic analysis showed that the R. philippinarum SIRT proteins could be grouped into three clusters and were closely related to their counterparts in other species of molluscs. The RpSIRT genes were differentially expressed across different developmental stages and at various tissues of the Manila clams. SIRT1 expression increases with the developmental stage, indicating that SIRT1 might play a key role in the early developmental stage of clam through regulating the cell cycle and energy metabolism of clam growth. For the spatial expression patterns of all the five SIRT genes investigated, the expression level of the SIRT1 gene in gonad was the highest, consistent with what was reported in Sparus aurata. In addition, the RpSIRT genes also responded to air exposure and low temperature stresses. After exposure to air, expression levels of the SIRT-1, SIRT-2, SIRT-6 and SIRT-7 genes (except for SIRT4) were increased significantly (P < 0.05). After 24 h of air exposure, expression levels of SIRT1 and SIRT2 in the low temperature group were significantly higher than those in the normal temperature group (P < 0.05), implying that prolonged air exposure might alter the metabolic pattern of clams dominated by aerobic pathway at low temperature. Our results suggest that the Ruditapes philippinarum SIRT genes might play important roles in regulating the level of NAD⁺/NADH in cells, and therefore might regulate the redox pathway of energy metabolism in clams during air exposure, which might eventually improve their antioxidant capacity.
Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme involved in several redox reactions. NAD⁺ also serves as an important substrate for several enzymes associated with DNA repair, secondary messenger signaling, and transcriptional regulation. These NAD⁺-consuming enzymes include poly-ADP-ribose polymerases (PARPs), CD38/157 ectoenzymes, and histone deacetylases known as sirtuins. NAD⁺ levels have been shown to decline during the aging process in several murine models and human clinical studies. NAD⁺ depletion has been associated with deficits in nuclear and mitochondrial function leading to many age-associated pathologies. Maintaining cellular NAD⁺ anabolism by supplementing with NAD⁺ and its related precursors has been reported to attenuate age-related functional defects and improve overall quality of life. Sirtuins are thought to be partly responsible for the beneficial effects of NAD⁺ on the aging phenotype. Thus supplementation with NAD⁺ intermediates may be an efficacious therapeutic intervention to manipulate sirtuin function, improve health span and promote “healthy” aging, bringing hope to our growing aging societies both nationally and abroad.
Recent studies have shown that inhibition of the hSIRT2 enzyme provides favorable effects in neurodegenerative diseases such as Alzheimer's disease. Prenylated xanthone phytochemicals including α-mangostin, β-mangostin and γ-mangostin obtained from Garcinia mangostana, a well-established tropical plant, have been shown experimentally to inhibit sirtuin enzymatic activity. However, the molecular mechanism of this sirtuin inhibition has not been reported. Using comprehensive integrated computational techniques, we provide molecular and timewise dynamical insights into the structural alterations capable of facilitating therapeutically beneficial effects of these phytochemicals at the catalytic core of the hSIRT2 enzyme. Findings revealed the enhanced conformational stability and compactness of the hSIRT2 catalytic core upon binding of γ-mangostin relative to the apoenzyme and better than α-mangostin and β-mangostin. Although thermodynamic calculations revealed favorable binding of all the phytochemicals to the hSIRT2 enzyme, the presence of only hydroxy functional groups on γ-mangostin facilitated the occurrence of additional hydrogen bonds involving Pro115, Phe119, Asn168 and His187 which are absent in α-mangostin- and β-mangostin-bound systems. Per-residue energy contributions showed that van der Waals and more importantly electrostatic interactions are involved in catalytic core stability with Phe96, Tyr104 and Phe235 notably contributing π–π stacking, π–π T shaped and π–sigma interactions. Cumulatively, our study revealed the structural alterations leading to inhibition of hSIRT2 catalysis and findings from this study could be significantly important for the future design and development of sirtuin inhibitors in the management of Alzheimer's disease.
Mammalian cells use a specialized and complex machinery for the removal of altered proteins or dysfunctional organelles. Such machinery is part of a mechanism called autophagy. Moreover, when autophagy is specifically employed for the removal of dysfunctional mitochondria, it is called mitophagy. Autophagy and mitophagy have important physiological implications and roles associated with cellular differentiation, resistance to stresses such as starvation, metabolic control and adaptation to the changing microenvironment. Unfortunately, transformed cancer cells often exploit autophagy and mitophagy for sustaining their metabolic reprogramming and growth to a point that autophagy and mitophagy are recognized as promising targets for ongoing and future antitumoral therapies. Sirtuins are NAD+ dependent deacylases with a fundamental role in sensing and modulating cellular response to external stresses such as nutrients availability and therefore involved in aging, oxidative stress control, inflammation, differentiation and cancer. It is clear, therefore, that autophagy, mitophagy and sirtuins share many common aspects to a point that, recently, sirtuins have been linked to the control of autophagy and mitophagy. In the context of cancer, such a control is obtained by modulating transcription of autophagy and mitophagy genes, by post translational modification of proteins belonging to the autophagy and mitophagy machinery, by controlling ROS production or major metabolic pathways such as Krebs cycle or glutamine metabolism. The present review details current knowledge on the role of sirtuins, autophagy and mitophagy in cancer to then proceed to discuss how sirtuins can control autophagy and mitophagy in cancer cells. Finally, we discuss sirtuins role in the context of tumor progression and metastasis indicating glutamine metabolism as an example of how a concerted activation and/or inhibition of sirtuins in cancer cells can control autophagy and mitophagy by impinging on the metabolism of this fundamental amino acid.
Neuroepigenetics, a new branch of epigenetics, plays an important role in the regulation of gene expression. Neuroepigenetics is associated with holistic neuronal function and helps in formation and maintenance of memory and learning processes. This includes neurodevelopment and neurodegenerative defects in which histone modification enzymes appear to play a crucial role. These modifications, carried out by acetyltransferases and deacetylases, regulate biologic and cellular processes such as apoptosis and autophagy, inflammatory response, mitochondrial dysfunction, cell-cycle progression and oxidative stress. Alterations in acetylation status of histone as well as non-histone substrates lead to transcriptional deregulation. Histone deacetylase decreases acetylation status and causes transcriptional repression of regulatory genes involved in neural plasticity, synaptogenesis, synaptic and neural plasticity, cognition and memory, and neural differentiation. Transcriptional deactivation in the brain results in development of neurodevelopmental and neurodegenerative disorders. Mounting evidence implicates histone deacetylase inhibitors as potential therapeutic targets to combat neurologic disorders. Recent studies have targeted naturally-occurring biomolecules and micro-RNAs to improve cognitive defects and memory. Multi-target drug ligands targeting HDAC have been developed and used in cell-culture and animal-models of neurologic disorders to ameliorate synaptic and cognitive dysfunction. Herein, we focus on the implications of histone deacetylase enzymes in neuropathology, their regulation of brain function and plausible involvement in the pathogenesis of neurologic defects.
The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
Despite its conservation in organisms from bacteria to human and its general requirement for transcriptional silencing in yeast, the function of the Sir2 protein is unknown. Here we show that Sir2 can transfer labeled phosphate from nicotinamide adenine dinucleotide to itself and histones in vitro. A modified form of Sir2, which results from its automodification activity, is specifically recognized by anti-mono-ADP-ribose antibodies, suggesting that Sir2 is an ADP-ribosyltransferase. Mutation of a phylogenetically invariant histidine residue in Sir2 abolishes both its enzymatic activity in vitro and its silencing functions in vivo. However, the mutant protein is associated with chromatin and other silencing factors in a manner similar to wild-type Sir2. These findings suggest that Sir2 contains an ADP-ribosyltransferase activity that is essential for its silencing function.
We demonstrate that in Saccharomyces cerevisiae, the tandem array of ribosomal RNA genes (RDN1) is a target for integration of the Ty1 retrotransposon that results in silencing of Ty1 transcription and transposition. Ty1 elements transpose into random rDNA repeat units and are mitotically stable. In addition, we have found that mutation of several putative modifiers of RDN1 chromatin structure abolishes silencing of Ty1 elements in the rDNA array. Disruption of SIR2, which elevates recombination in RDN1, or TOP1, which increases psoralen accessibility in rDNA, or HTA1-HTB1, which reduces histone H2A-H2B levels and causes localized chromatin perturbations, abolishes transcriptional silencing of Ty1 elements in RDN1. Furthermore, deletion of the gene for the ubiquitin conjugating enzyme Ubc2p, which ubiquitinates histones in vitro, derepresses not only Ty1 transcription but also mitotic recombination in RDN1. On the basis of these results, we propose that a specialized chromatin structure exists in RDN1 that silences transcription of the Ty1 retrotransposon.
Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) has been identified by photoaffinity labeling as a residue within the NAD binding site (S.F. Carroll and R.J. Collier, J. Biol. Chem. 262:8707-8711, 1987). To explore the function of Glu-553 we used oligonucleotide-directed mutagenesis to replace this residue with Asp in cloned ETA and expressed the mutant gene in Escherichia coli K-12. ADP-ribosylation activity of Asp-553 ETA in cell extracts was about 1,800-fold lower and toxicity for mouse L-M929 fibroblasts was at least 10,000-fold lower than that of the wild-type toxin. Extracts containing Asp-553 ETA inhibited the cytotoxicity of authentic ETA on L-M929 fibroblasts, suggesting that the mutant toxin competes for ETA receptors. The results indicate that Glu-553 is crucial for ADP-ribosylation activity and, consequently, cytotoxicity of ETA. Substitution or deletion of this residue may be a route to new ETA vaccines.
Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.
The mating type of haploid yeast (a or alpha) is determined by information present at the MAT locus. Identical copies of a and alpha information are present at distal loci (HMR and HML), but transcription of these copies is repressed by the action, in trans, of four unlinked genes called SIR (silent information regulator). Repression by SIR also requires, in cis, DNA sequences called E which are found to the left of HML and HMR (but not MAT) and are greater than 1 kb from the mating-type gene promoters. SIR control can act on other promoters when they are brought near the E sequence, and thus the SIR gene products act in some general manner to repress transcription. We have determined the DNA sequence of two fragments which complement mutations in the SIR2 and SIR3 genes and show that these contain the structural genes by mapping the cloned sequences onto the yeast chromosome. The SIR2 and SIR3 coding sequences were identified by constructing gene disruptions and using these mutations to replace the normal chromosomal copies. Such null mutants of both SIR2 and SIR3 are defective in the position-effect control of the silent loci but have no other detectable phenotype. We have mapped the 5' and 3' ends of the SIR2 and SIR3 mRNAs and show that their level is unaffected by mutations in any of the four known SIR complementation groups.
We showed earlier that exposing mixtures of NAD and diphtheria toxin fragment A to ultraviolet radiation (253.7 nm) induced the formation of covalently linked protein-ligand photoproducts. Here we report that when [carbonyl-14C]NAD was employed in such procedures, the efficiency of labeling of the protein approached 1 mol/mol, and at least 94% of the incorporated label was associated with a single residue, glutamic acid at position 148. Fragment A photolabeled in this manner was enzymically inactive. The efficiency of photolabeling was much lower (less than 0.2 mol/mol) when NAD radiolabeled in either the adenine moiety or the adenylate phosphate was used, and the label was attached to different site(s) within fragment A. Efficient photochemical transfer of label from [carbonyl-14C]NAD occurred under identical conditions with the nucleotide-free form of whole diphtheria toxin, CRM-45, or activated exotoxin A from Pseudomonas aeruginosa, but not with nucleotide-bound diphtheria toxin, CRM-197, native exotoxin A, or any of several NAD-linked dehydrogenases. On the basis of these and other results we suggest that part or all of the nicotinamide moiety of NAD is efficiently transferred to glutamate-148 of fragment A under the influence of ultraviolet irradiation and that this residue is located within the nicotinamide subsite. This location implies that glutamate-148 is at or near the catalytic center of the toxin. Our data provide direct evidence for the location of the NAD site in an ADP-ribosylating toxin and demonstrate highly efficient and specific photolabeling by [carbonyl-14C]NAD.
Genomic silencing is a fundamental mechanism of transcriptional regulation, yet little is known about conserved mechanisms of silencing. We report here the discovery of four Saccharomyces cerevisiae homologs of the SIR2 silencing gene (HSTs), as well as conservation of this gene family from bacteria to mammals. At least three HST genes can function in silencing; HST1 overexpression restores transcriptional silencing to a sir2 mutant and hst3 hst4 double mutants are defective in telomeric silencing. In addition, HST3 and HST4 together contribute to proper cell cycle progression, radiation resistance, and genomic stability, establishing new connections between silencing and these fundamental cellular processes.
The maintenance of transcriptional silencing at HM mating-type loci and telomeres in yeast requires the SIR2, SIR3, and SIR4 proteins, none of which appear to be DNA-binding proteins. Here we show that SIR3 and SIR4 interact with a carboxy-terminal domain of the silencer, telomere, and UAS-binding protein RAP1. We identified SIR3 and SIR4 in a two-hybrid screen for RAP1-interacting factors and showed that SIR3 interacts both with itself and with SIR4. The interaction between RAP1 and SIR3 can be observed in vitro in the absence of other yeast proteins. Consistent with the notion that native SIR proteins interact with the RAP1 carboxyl terminus, we show that mutation of the endogenous SIR3 and SIR4 genes increases transcriptional activation by LexA/RAP1 hybrids. To test the importance of the RAP1-SIR3 interaction for silencing, we identified mutations in the RAP1 carboxyl terminus that either diminish or abolish this interaction. When introduced into the native RAP1 protein, these mutations cause corresponding defects in silencing at both HMR and telomeres. We propose that RAP1 acts in the initiation of transcriptional silencing by recruiting a complex of SIR proteins to the chromosome via protein-protein interactions. These data are consistent with a model in which SIR3 and SIR4 play a structural role in the maintenance of silent chromatin and indicate that their action is initiated at the silencer itself.
A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.
The SIR genes are determinants of life span in yeast mother cells. Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and alpha mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed.
Yeast Sir2 is a heterochromatin component that silences transcription at silent mating loci, telomeres and the ribosomal DNA, and that also suppresses recombination in the rDNA and extends replicative life span. Mutational studies indicate that lysine 16 in the amino-terminal tail of histone H4 and lysines 9, 14 and 18 in H3 are critically important in silencing, whereas lysines 5, 8 and 12 of H4 have more redundant functions. Lysines 9 and 14 of histone H3 and lysines 5, 8 and 16 of H4 are acetylated in active chromatin and hypoacetylated in silenced chromatin, and overexpression of Sir2 promotes global deacetylation of histones, indicating that Sir2 may be a histone deacetylase. Deacetylation of lysine 16 of H4 is necessary for binding the silencing protein, Sir3. Here we show that yeast and mouse Sir2 proteins are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases, which deacetylate lysines 9 and 14 of H3 and specifically lysine 16 of H4. Our analysis of two SIR2 mutations supports the idea that this deacetylase activity accounts for silencing, recombination suppression and extension of life span in vivo. These findings provide a molecular framework of NAD-dependent histone deacetylation that connects metabolism, genomic silencing and ageing in yeast and, perhaps, in higher eukaryotes.
Silent information regulator (Sir) 2 is a limiting component of the Sir2/3/4 complex, which represses transcription at subtelomeric and HM loci. Sir2p also acts independently of Sir3p and Sir4p to influence chromatin organization in the rDNA locus. Deleted and mutated forms of Sir2p have been tested for their ability to complement and/or to disrupt silencing. The highly conserved C-terminal domain of Sir2p (aa 199-562) is insufficient to restore repression at either telomeric or rDNA reporters in a sir2Delta background and fails to nucleate silencing when targeted to an appropriate reporter gene. However, its expression in an otherwise wild-type strain disrupts telomeric repression. Similarly, a point mutation (P394L) within this conserved core inactivates the full-length protein but renders it dominant negative for all types of silencing. Deletion of aa 1-198 from Sir2(394L) eliminates its dominant negative effect. Thus we define two distinct functional domains in Sir2p, both essential for telomeric and rDNA repression: the conserved core domain found within aa 199-562 and a second domain that encompasses aa 94-198. Immunolocalization and two-hybrid studies show that aa 94-198 are required for the binding of Sir2p to Sir4p and for the targeting of Sir2p to the nucleolus through another ligand. The globular core domain provides an essential silencing function distinct from that of targeting or Sir complex formation that may reflect its reported mono-ADP-ribosyl transferase activity.
Homologs of the chromatin-bound yeast silent information regulator 2 (SIR2) protein are found in organisms from all biological kingdoms. SIR2 itself was originally discovered to influence mating-type control in haploid cells by locus-specific transcriptional silencing. Since then, SIR2 and its homologs have been suggested to play additional roles in suppression of recombination, chromosomal stability, metabolic regulation, meiosis, and aging. Considering the far-ranging nature of these functions, a major experimental goal has been to understand the molecular mechanism(s) by which this family of proteins acts. We report here that members of the SIR2 family catalyze an NAD-nicotinamide exchange reaction that requires the presence of acetylated lysines such as those found in the N termini of histones. Significantly, these enzymes also catalyze histone deacetylation in a reaction that absolutely requires NAD, thereby distinguishing them from previously characterized deacetylases. The enzymes are active on histone substrates that have been acetylated by both chromatin assembly-linked and transcription-related acetyltransferases. Contrary to a recent report, we find no evidence that these proteins ADP-ribosylate histones. Discovery of an intrinsic deacetylation activity for the conserved SIR2 family provides a mechanism for modifying histones and other proteins to regulate transcription and diverse biological processes.
The yeast Sir2 protein, required for transcriptional silencing, has an NAD(+)-dependent histone deacetylase (HDA) activity. Yeast extracts contain a NAD(+)-dependent HDA activity that is eliminated in a yeast strain from which SIR2 and its four homologs have been deleted. This HDA activity is also displayed by purified yeast Sir2p and homologous Archaeal, eubacterial, and human proteins, and depends completely on NAD(+) in all species tested. The yeast NPT1 gene, encoding an important NAD(+) synthesis enzyme, is required for rDNA and telomeric silencing and contributes to silencing of the HM loci. Null mutants in this gene have significantly reduced intracellular NAD(+) concentrations and have phenotypes similar to sir2 null mutants. Surprisingly, yeast from which all five SIR2 homologs have been deleted have relatively normal bulk histone acetylation levels. The evolutionary conservation of this regulated activity suggests that the Sir2 protein family represents a set of effector proteins in an evolutionarily conserved signal transduction pathway that monitors cellular energy and redox states.
Highly purified Aplysia californica ADP-ribosyl cyclase was found to be a multifunctional enzyme. In addition to the known transformation of NAD(+) into cADP-ribose this enzyme is able to catalyse the solvolysis (hydrolysis and methanolysis) of cADP-ribose. This cADP-ribose hydrolase activity, which becomes detectable only at high concentrations of the enzyme, is amplified with analogues such as pyridine adenine dinucleotide, in which the cleavage rate of the pyridinium-ribose bond is much reduced compared with NAD(+). Although the specificity ratio V(max)/K(m) is in favour of NAD(+) by 4 orders of magnitude, this multifunctionality allowed us to propose a 'partitioning' reaction scheme for the Aplysia enzyme, similar to that established previously for mammalian CD38/NAD(+) glycohydrolases. This mechanism involves the formation of a single oxocarbenium-type intermediate that partitions to cADP-ribose and solvolytic products via competing pathways. In favour of this mechanism was the finding that the enzyme also catalysed the hydrolysis of NMN(+), a substrate that cannot undergo cyclization. The major difference between the mammalian and the invertebrate enzymes resides in their relative cyclization/hydrolysis rate-constant ratios, which dictate their respective yields of cADP-ribose (ADP-ribosyl cyclase activity) and ADP-ribose (NAD(+) glycohydrolase activity). For the Aplysia enzyme's catalysed transformation of NAD(+) we favour a mechanism where the formation of cADP-ribose precedes that of ADP-ribose; i.e. macroscopically the invertebrate ADP-ribosyl cyclase conforms to a sequential reaction pathway as a limiting form of the partitioning mechanism.
Calorie restriction extends life-span in a wide variety of organisms. Although it has been suggested that calorie restriction
may work by reducing the levels of reactive oxygen species produced during respiration, the mechanism by which this regimen
slows aging is uncertain. Here, we mimicked calorie restriction in yeast by physiological or genetic means and showed a substantial
extension in life-span. This extension was not observed in strains mutant forSIR2 (which encodes the silencing protein Sir2p) orNPT1 (a gene in a pathway in the synthesis of NAD, the oxidized form of nicotinamide adenine dinucleotide). These findings suggest
that the increased longevity induced by calorie restriction requires the activation of Sir2p by NAD.
The Saccharomyces cerevisiae silencing protein Sir2 is the founding member of a universally conserved family of proteins that have been shown to possess NAD-dependent histone deacetylation and ADP-ribosylation activities. Here we show that histone deacetylation by Sir2 is coupled to cleavage of the high-energy bond that links the ADP-ribose moiety of NAD to nicotinamide. Analysis of the NAD cleavage products revealed the presence of nicotinamide, ADP-ribose, and a third product that appeared to be related to ADP-ribose. With the use of label transfer experiments, we show that the acetyl group in the histone substrate is transferred to this NAD breakdown product during deacetylation, forming a product that we conclude to be O-acetyl-ADP-ribose. Detection of this species strongly argues for obligate coupling of histone deacetylation to NAD breakdown by Sir2. We propose reaction mechanisms that could account for this coupling via acetyl-ADP-ribose formation. The unprecedented coupling of amide bond cleavage to cleavage of a high-energy bond raises the possibility that NAD breakdown by Sir2 plays an important role in silencing that is independent of its requirement for deacetylation.
In Caenorhabditis elegans, mutations that reduce the activity of an insulin-like receptor (daf-2) or a phosphatidylinositol-3-OH kinase (age-1) favour entry into the dauer state during larval development and extend lifespan in adults. Downregulation of this pathway activates a forkhead transcription factor (daf-16), which may regulate targets that promote dauer formation in larvae and stress resistance and longevity in adults. In yeast, the SIR2 gene determines the lifespan of mother cells, and adding an extra copy of SIR2 extends lifespan. Sir2 mediates chromatin silencing through a histone deacetylase activity that depends on NAD (nicotinamide adenine dinucleotide) as a cofactor. We have surveyed the lifespan of C. elegans strains containing duplications of chromosomal regions. Here we report that a duplication containing sir-2.1-the C. elegans gene most homologous to yeast SIR2-confers a lifespan that is extended by up to 50%. Genetic analysis indicates that the sir-2.1 transgene functions upstream of daf-16 in the insulin-like signalling pathway. Our findings suggest that Sir2 proteins may couple longevity to nutrient availability in many eukaryotic organisms.
Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.
The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The programs are written mainly in standard Fortran77. They are from a wide variety of sources but are connected by standard data file formats. The package has been ported to all the major platforms under both Unix and VMS. The suite is distributed by anonymous ftp from Daresbury Laboratory and is widely used throughout the world.
We demonstrate in this work that the surface tension, water-organic solvent, transfer-free energies and the thermodynamics of melting of linear alkanes provide fundamental insights into the nonpolar driving forces for protein folding and protein binding reactions. We first develop a model for the curvature dependence of the hydrophobic effect and find that the macroscopic concept of interfacial free energy is applicable at the molecular level. Application of a well-known relationship involving surface tension and adhesion energies reveals that dispersion forces play little or no net role in hydrophobic interactions; rather, the standard model of disruption of water structure (entropically driven at 25 degrees C) is correct. The hydrophobic interaction is found, in agreement with the classical picture, to provide a major driving force for protein folding. Analysis of the melting behavior of hydrocarbons reveals that close packing of the protein interior makes only a small free energy contribution to folding because the enthalpic gain resulting from increased dispersion interactions (relative to the liquid) is countered by the freezing of side chain motion. The identical effect should occur in association reactions, which may provide an enormous simplification in the evaluation of binding energies. Protein binding reactions, even between nearly planar or concave/convex interfaces, are found to have effective hydrophobicities considerably smaller than the prediction based on macroscopic surface tension. This is due to the formation of a concave collar region that usually accompanies complex formation. This effect may preclude the formation of complexes between convex surfaces.
Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
S. cerevisiae chromosomes end with the telomeric repeat (TG1-3)n. When any of four Pol II genes was placed immediately adjacent to the telomeric repeats, expression of the gene was reversibly repressed as demonstrated by phenotype and mRNA analyses. For example, cells bearing a telomere-linked copy of ADE2 produced predominantly red colonies (a phenotype characteristic of ade2- cells) containing white sectors (characteristic of ADE2+ cells). Repression was due to proximity to the telomere itself since an 81 bp tract of (TG1-3)n positioned downstream of URA3 when URA3 was approximately 20 kb from the end of chromosome VII did not alter expression of the gene. However, this internal tract of (TG1-3)n could spontaneously become telomeric, in which case expression of the URA3 gene was repressed. These data demonstrate that yeast telomeres exert a position effect on the transcription of nearby genes, an effect that is under epigenetic control.
The silent mating loci and chromosomal regions adjacent to telomeres of S. cerevisiae have features similar to heterochromatin of more complex eukaryotes. Transcriptional repression at these sites depends on the silent information regulators SIR3 and SIR4 as well as histones H3 and H4. We show here that the SIR3 and SIR4 proteins interact with specific silencing domains of the H3 and H4 N-termini in vitro. Certain mutations in these factors, which affect their silencing functions in vivo, also disrupt their interactions in vitro. Immunofluorescence studies with antibodies against RAP1 and SIR3 demonstrate that the H3 and H4 N-termini are required for the association of SIR3 with telomeric chromatin and the perinuclear positioning of yeast telomeres. Based on these interactions, we propose a model for heterochromatin-mediated transcriptional silencing in yeast, which may serve as a paradigm for other eukaryotic organisms as well.
Classical nicotinamide adenine dinucleotide (NAD+) binding proteins contain a beta alpha beta alpha beta unit. By comparing 14 such proteins, it is observed that an additional beta strand associates with this unit to form the "core" topology, the minimum structure necessary to bind cofactor. Although the overall topologies of the cofactor binding domains of nicotinamide binding proteins vary, they all contain at least the core topology. The first 30-35 amino acids of the core topology, called the "fingerprint" region, are diagnostic for the presence of a dinucleotide binding fold. There are four characteristics of this fingerprint region: 1) a phosphate binding consensus sequence, GXGXXG, 2) six positions usually occupied by small hydrophobic amino acids, 3) a conserved, negatively charged residue (Glu or Asp) at the end of the second beta strand of the fingerprint region, and 4) a conserved positively charged residue (Arg or Lys) at the beginning of the first beta strand of the fingerprint region. The core topologies of the classical nicotinamide binding proteins overlap well with root mean squared deviations of main chain atoms ranging from 0.7 to 4.7 A. A conserved interaction (found in 8 of the 12 classical nicotinamide binding proteins studied) between the cofactor and the protein is a hydrogen bond between the pyrophosphate oxygen of NAD(P)+ and the carboxy-terminal glycine of the phosphate binding helix, the first alpha helix of the beta alpha beta alpha beta unit. The classical nicotinamide binding proteins all bind their cofactor in the same location and orientation, with the cofactor itself adopting a similar extended conformation in every structure. Although observed less frequently than the classical fold, numerous nonclassical folding patterns are also used by proteins that bind NAD(P)+.
Pertussis toxin from Bordetella pertussis is one of the ADP-ribosylating toxins which are the cytotoxic agents of several infectious diseases. Transition state analogues of these enzymes are expected to be potent inhibitors and may be useful in therapy. Pertussis toxin catalyzes the ADP-ribosylation of a cysteine in the synthetic peptide alphai3C20, corresponding to the C-terminal 20 amino acids of the alpha-subunits of the G-protein Gi3. A family of kinetic isotope effects was determined for the ADP-ribosylation reaction, using 3H-, 14C- and 15N-labeled NAD+ as substrates. Primary kinetic isotope effects were 1.050 +/- 0.006 for [1'N-14C] and 1.021 +/- 0.002 for [1N-15N], the double primary effect of [1'N-14C,1N-15N] was 1.064 +/- 0.002. Secondary kinetic isotope effects were 1.208 +/- 0.014 for [1'N-3H], 1.104 +/- 0.010 for [2'N-3H], 0.989 +/- 0.001 for [4'N-3H], and 1.014 +/- 0.002 for [5'N-3H]. Isotope trapping experiments yielded a commitment factor of 0.01, demonstrating that the observed isotope effects are near intrinsic. Solvent D2O kinetic isotope effects are inverse, consistent with deprotonation of the attacking Cys prior to transition state formation. The transition state structure was determined by a normal mode bond vibrational analysis. The transition state is characterized by a nicotinamide leaving group bond order of 0.14, corresponding to a bond length of 2.06 A. The incoming thiolate nucleophile has a bond order of 0.11, corresponding to 2.47 A. The ribose ring has strong oxocarbenium ion character. Pertussis toxin also catalyzes the slow hydrolysis of NAD+ in the absence of peptides. Comparison of the transition states for NAD+ hydrolysis and for ADP-ribosylation of peptide alphai3C20 indicates that the sulfur nucleophile from the peptide Cys participates more actively as a nucleophile in the reaction than does water in the hydrolytic reaction. Participation of the thiolate anion at the transition state provides partial neutralization of the cationic charge which normally develops at the transition states of N-ribohydrolases and transferases. Thus, the presence of the peptide provides increased SN2 character in a loose transition state which retains oxocarbenium character in the ribose.
Furin is a secretory pathway endoprotease that catalyses the maturation of a strikingly diverse group of proprotein substrates, ranging from growth factors and receptors to pathogen proteins, in multiple compartments within the trans-Golgi network (TGN)/endosomal system. This review focuses on recent developments in the biochemistry and cell biology of the endoprotease, including the mechanism of TGN localization, phosphorylation-dependent regulation of protein traffic, and novel insights into early embryogenesis, extracellular matrix formation and pathogen virulence.
The yeast Sir2 protein regulates epigenetic gene silencing and as a possible antiaging effect it suppresses recombination of rDNA. Studies involving cobB, a bacterial SIR2-like gene, have suggested it could encode a pyridine nucleotide transferase. Here five human sirtuin cDNAs are characterized. The SIRT1 sequence has the closest homology to the S. cerevisiae Sir2p. The SIRT4 and SIRT5 sirtuins more closely resemble prokaryotic sirtuin sequences. The five human sirtuins are widely expressed in fetal and adult tissues. Recombinant E. coli cobT and cobB proteins each showed a weak NAD-dependent mono-ADP-ribosyltransferase activity using 5, 6-dimethylbenzimidazole as a substrate. Recombinant E. coli cobB and human SIRT2 sirtuin proteins were able to cause radioactivity to be transferred from [32P]NAD to bovine serum albumin (BSA). When a conserved histidine within the human SIRT2 sirtuin was converted to a tyrosine, the mutant recombinant protein was unable to transfer radioactivity from [32P]NAD to BSA. These results suggest that the sirtuins may function via mono-ADP-ribosylation of proteins.
Histone deacetylases (HDACs) mediate changes in nucleosome conformation and are important in the regulation of gene expression. HDACs are involved in cell-cycle progression and differentiation, and their deregulation is associated with several cancers. HDAC inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), have anti-tumour effects, as they can inhibit cell growth, induce terminal differentiation and prevent the formation of tumours in mice models, and they are effective in the treatment of promyelocytic leukemia. Here we describe the structure of the histone deacetylase catalytic core, as revealed by the crystal structure of a homologue from the hyperthermophilic bacterium Aquifex aeolicus, that shares 35.2% identity with human HDAC1 over 375 residues, deacetylates histones in vitro and is inhibited by TSA and SAHA. The deacetylase, deacetylase-TSA and deacetylase-SAHA structures reveal an active site consisting of a tubular pocket, a zinc-binding site and two Asp-His charge-relay systems, and establish the mechanism of HDAC inhibition. The residues that make up the active site and contact the inhibitors are conserved across the HDAC family. These structures also suggest a mechanism for the deacetylation reaction and provide a framework for the further development of HDAC inhibitors as antitumour agents.
The nicotinamide nucleotide transhydrogenases (TH) of mitochondria and bacteria are membrane-intercalated proton pumps that transduce substrate binding energy and protonmotive force via protein conformational changes. In mitochondria, TH utilizes protonmotive force to promote direct hydride ion transfer from NADH to NADP, which are bound at the distinct extramembranous domains I and III, respectively. Domain II is the membrane-intercalated domain and contains the enzyme's proton channel. This paper describes the crystal structure of the NADP(H) binding domain III of bovine TH at 1.2 A resolution. The structure reveals that NADP is bound in a manner inverted from that previously observed for nucleotide binding folds. The non-classical binding mode exposes the NADP(H) nicotinamide ring for direct contact with NAD(H) in domain I, in accord with biochemical data. The surface of domain III surrounding the exposed nicotinamide is comprised of conserved residues presumed to form the interface with domain I during hydride ion transfer. Further, an adjacent region contains a number of acidic residues, forming a surface with negative electrostatic potential which may interact with extramembranous loops of domain II. Together, the distinctive surface features allow mechanistic considerations regarding the NADP(H)-promoted conformation changes that are involved in the interactions of domain III with domains I and II for hydride ion transfer and proton translocation.
Sirtuins (Sir2-like proteins) are present in prokaryotes and eukaryotes. Here, two new human sirtuins (SIRT6 and SIRT7) are found to be similar to a particular subset of insect, nematode, plant, and protozoan sirtuins. Molecular phylogenetic analysis of 60 sirtuin conserved core domain sequences from a diverse array of organisms (including archaeans, bacteria, yeasts, plants, protozoans, and metazoans) shows that eukaryotic Sir2-like proteins group into four main branches designated here as classes I-IV. Prokaryotic sirtuins include members of classes II and III. A fifth class of sirtuin is present in gram positive bacteria and Thermotoga maritima. Saccharomyces cerevisiae has five class I sirtuins. Caenorhabditis elegans and Drosophila melanogaster have sirtuin genes from classes I, II, and IV. The seven human sirtuin genes include all four classes: SIRT1, SIRT2, and SIRT3 are class I, SIRT4 is class II, SIRT5 is class III, and SIRT6 and SIRT7 are class IV.
Ubiquitin-protein ligases (E3s) regulate diverse cellular processes by mediating protein ubiquitination. The c-Cbl proto-oncogene is a RING family E3 that recognizes activated receptor tyrosine kinases, promotes their ubiquitination by a ubiquitin-conjugating enzyme (E2) and terminates signaling. The crystal structure of c-Cbl bound to a cognate E2 and a kinase peptide shows how the RING domain recruits the E2. A comparison with a HECT family E3-E2 complex indicates that a common E2 motif is recognized by the two E3 families. The structure reveals a rigid coupling between the peptide binding and the E2 binding domains and a conserved surface channel leading from the peptide to the E2 active site, suggesting that RING E3s may function as scaffolds that position the substrate and the E2 optimally for ubiquitin transfer.
We wish to thank all members of the Allis and Sassone-Corsi labs for exchange and discussion of many of the ideas presented in this review. Also we thank Craig Mizzen for assistance with figures and multiple rounds of proofreading, and Steve Cheung, for critical reading of the manuscript.
Conflicting reports have suggested that the silent information regulator 2 (SIR2) protein family employs NAD(+) to ADP-ribosylate histones [Tanny, J. C., Dowd, G. J., Huang, J., Hilz, H. & Moazed, D. (1999) Cell 99, 735-745; Frye, R. A. (1999) Biochem. Biophys. Res. Commun. 260, 273-279], deacetylate histones [Landry, J., Sutton, A., Tafrov, S. T., Heller, R. C., Stebbins, J., Pillus, L. & Sternglanz, R. (2000) Proc. Natl. Acad. Sci. USA 97, 5807-5811; Smith, J. S., Brachmann, C. B., Celic, I., Kenna, M. A., Muhammad, S., Starai, V. J., Avalos, J. L., Escalante-Semerena, J. C., Grubmeyer, C., Wolberger, C. & Boeke, J. D. (2000) Proc. Natl. Acad. Sci. USA 97, 6658-6663], or both [Imai, S., Armstrong, C. M., Kaeberlein, M. & Guarente, L. (2000) Nature (London) 403, 795-800]. Uncovering the true enzymatic function of SIR2 is critical to the basic understanding of its cellular function. Therefore, we set out to authenticate the reaction products and to determine the intrinsic catalytic mechanism. We provide direct evidence that the efficient histone/protein deacetylase reaction is tightly coupled to the formation of a previously unidentified acetyl-ADP-ribose product (1-O-acetyl-ADP ribose). One molecule of NAD(+) and one molecule of acetyl-lysine are readily catalyzed to one molecule of deacetylated lysine, nicotinamide, and 1-O-acetyl-ADP-ribose. A unique reaction mechanism involving the attack of enzyme-bound acetate or the direct attack of acetyl-lysine on an oxocarbenium ADP-ribose intermediate is proposed. We suggest that the reported histone/protein ADP-ribosyltransferase activity is a low-efficiency side reaction that can be explained through the partial uncoupling of the intrinsic deacetylation and acetate transfer to ADP-ribose.
Nucleosomes play a dynamic role in transcription. The key to this role is the structure of the flexible and charged histone tails that extend from the hydrophobic nucleosome core. These tails are modified by acetylation and deacetylation, phosphorylation, methylation, ubiquitination and ATP-dependent chromatin remodeling, and they regulate functions as diverse as transcription, DNA-dependent chromatin assembly, DNA repair, mitosis and silencing by heterochromatin.
The SIR2 protein family comprises a novel class of nicotinamide-adenine dinucleotide (NAD)-dependent protein deacetylases that function in transcriptional silencing, DNA repair, and life-span extension in Saccharomyces cerevisiae. Two crystal structures of a SIR2 homolog from Archaeoglobus fulgidus complexed with NAD have been determined at 2.1 A and 2.4 A resolutions. The structures reveal that the protein consists of a large domain having a Rossmann fold and a small domain containing a three-stranded zinc ribbon motif. NAD is bound in a pocket between the two domains. A distinct mode of NAD binding and an unusual configuration of the zinc ribbon motif are observed. The structures also provide important insights into the catalytic mechanism of NAD-dependent protein deacetylation by this family of enzymes.
This chapter discusses Raster3D, which is a suite of programs for molecular graphics. Crystallographers were among the first and most avid consumers of graphics workstations. Rapid advances in computer hardware, and particularly in the power of specialized computer graphics boards, have led to successive generations of personal workstations with ever more impressive capabilities for interactive molecular graphics. For many years, it was standard practice in crystallography laboratories to prepare figures by photographing directly from the workstation screen. No matter how beautiful the image on the screen, however, this approach suffers from several intrinsic limitations. Among these is the inherent limitation imposed by the effective resolution of the screen. Use of the graphics hardware in a workstation to generate images for later presentation can also impose other limitations. Designers of workstation hardware must compromise the quality of rendered images to achieve rendering speeds high enough for useful interactive manipulation of three-dimensional objects.
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