In Streptomyces lividans, acetyl-CoA synthetase activity is controlled by O-serine and Nɛ -lysine acetylation.

Protein acetylation is a rapid mechanism for control of protein function. Acetyl-CoA synthetase (AMP-forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK610 ). S608 acetylation rendered SlAcs inactive and non-acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD+ -dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin-dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O-Ser and Nɛ -Lys acetylation.

Decreased Sirtuin Deacetylase Activity in LRRK2 G2019S iPSC-Derived Dopaminergic Neurons.

Mitochondrial changes have long been implicated in the pathogenesis of Parkinson's disease (PD). The glycine to serine mutation (G2019S) in leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause for PD and has been shown to impair mitochondrial function and morphology in multiple model systems. We analyzed mitochondrial function in LRRK2 G2019S induced pluripotent stem cell (iPSC)-derived neurons to determine whether the G2019S mutation elicits similar mitochondrial deficits among central and peripheral nervous system neuron subtypes. LRRK2 G2019S iPSC-derived dopaminergic neuron cultures displayed unique abnormalities in mitochondrial distribution and trafficking, which corresponded to reduced sirtuin deacetylase activity and nicotinamide adenine dinucleotide levels despite increased sirtuin levels. These data indicate that mitochondrial deficits in the context of LRRK2 G2019S are not a global phenomenon and point to distinct sirtuin and bioenergetic deficiencies intrinsic to dopaminergic neurons, which may underlie dopaminergic neuron loss in PD.

Nicotinamide Suppresses the DNA Damage Sensitivity of Saccharomyces cerevisiae Independently of Sirtuin Deacetylases.

Nicotinamide is both a reaction product and an inhibitor of the conserved sirtuin family of deacetylases, which have been implicated in a broad range of cellular functions in eukaryotes from yeast to humans. Phenotypes observed following treatment with nicotinamide are most often assumed to stem from inhibition of one or more of these enzymes. Here, we used this small molecule to inhibit multiple sirtuins at once during treatment with DNA damaging agents in the Saccharomyces cerevisiae model system. Since sirtuins have been previously implicated in the DNA damage response, we were surprised to observe that nicotinamide actually increased the survival of yeast cells exposed to the DNA damage agent MMS. Remarkably, we found that enhanced resistance to MMS in the presence of nicotinamide was independent of all five yeast sirtuins. Enhanced resistance was also independent of the nicotinamide salvage pathway, which uses nicotinamide as a substrate to generate NAD+, and of a DNA damage-induced increase in the salvage enzyme Pnc1 Our data suggest a novel and unexpected function for nicotinamide that has broad implications for its use in the study of sirtuin biology across model systems.

OsSRT1 is involved in rice seed development through regulation of starch metabolism gene expression.

OsSRT1 is a NAD(+)-dependent histone deacetylase, closely related to the human SIRT6 that plays key roles in genome stability and metabolic homeostasis. In this work, we investigated the role of OsSRT1 in rice seed development. Down-regulation of OsSRT1 induced higher expression of Rice Starch Regulator1 (RSR1) and amylases genes in developing seeds, which resulted in a decrease of starch synthesis and an increase of starch degradation, leading to abnormal seed development. ChIP assay showed that OsSRT1 was required to reduce histone H3K9 acetylation on starch metabolism genes and transposons in developing seeds. In addition, OsSRT1 was detected to directly bind to starch metabolism genes such as OsAmy3B, OsAmy3E, OsBmy4, and OsBmy9. Our results suggested that OsSRT1-mediated histone deacetylation is involved in starch accumulation and transposon repression to regulate normal seed development.

High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects.

Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses sirtuin deacetylase 2 (SIRT2) and 6 (SIRT6) expression leading to histone acetylation and gene expression. SIRT down-regulation mediates the teratogenicity of diabetes leading to (NTD) formation. The study provides a mechanistic basis for the development of natural antioxidants and SIRT activators as therapeutics for diabetic embryopathy.

Locus-specific control of DNA resection and suppression of subtelomeric VSG recombination by HAT3 in the African trypanosome.

The African trypanosome, Trypanosoma brucei, is a parasitic protozoan that achieves antigenic variation through DNA-repair processes involving Variant Surface Glycoprotein (VSG) gene rearrangements at subtelomeres. Subtelomeric suppression of DNA repair operates in eukaryotes but little is known about these controls in trypanosomes. Here, we identify a trypanosome histone acetyltransferase (HAT3) and a deacetylase (SIR2rp1) required for efficient RAD51-dependent homologous recombination. HAT3 and SIR2rp1 were required for RAD51-focus assembly and disassembly, respectively, at a chromosome-internal locus and a synthetic defect indicated distinct contributions to DNA repair. Although HAT3 promoted chromosome-internal recombination, it suppressed subtelomeric VSG recombination, and these locus-specific effects were mediated through differential production of ssDNA by DNA resection; HAT3 promoted chromosome-internal resection but suppressed subtelomeric resection. Consistent with the resection defect, HAT3 was specifically required for the G2-checkpoint response at a chromosome-internal locus. HAT3 also promoted resection at a second chromosome-internal locus comprising tandem-duplicated genes. We conclude that HAT3 and SIR2rp1 can facilitate temporally distinct steps in DNA repair. HAT3 promotes ssDNA formation and recombination at chromosome-internal sites but has the opposite effect at a subtelomeric VSG. These locus-specific controls reveal compartmentalization of the T. brucei genome in terms of the DNA-damage response and suppression of antigenic variation by HAT3.

Acetylome analysis reveals diverse functions of lysine acetylation in Mycobacterium tuberculosis.

The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD(+)-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.

The deacetylase Sir2 from the yeast Clavispora lusitaniae lacks the evolutionarily conserved capacity to generate subtelomeric heterochromatin.

Deacetylases of the Sir2 or sirtuin family are thought to regulate life cycle progression and life span in response to nutrient availability. This family has undergone successive rounds of duplication and diversification, enabling the enzymes to perform a wide variety of biological functions. Two evolutionarily conserved functions of yeast Sir2 proteins are the generation of repressive chromatin in subtelomeric domains and the suppression of unbalanced recombination within the tandem rDNA array. Here, we describe the function of the Sir2 ortholog ClHst1 in the yeast Clavispora lusitaniae, an occasional opportunistic pathogen. ClHst1 was localized to the non-transcribed spacer regions of the rDNA repeats and deacetylated histones at these loci, indicating that, like other Sir2 proteins, ClHst1 modulates chromatin structure at the rDNA repeats. However, we found no evidence that ClHst1 associates with subtelomeric regions or impacts gene expression directly. This surprising observation highlights the plasticity of sirtuin function. Related yeast species, including Candida albicans, possess an additional Sir2 family member. Thus, it is likely that the ancestral Candida SIR2/HST1 gene was duplicated and subfunctionalized, such that HST1 retained the capacity to regulate rDNA whereas SIR2 had other functions, perhaps including the generation of subtelomeric chromatin. After subsequent species diversification, the SIR2 paralog was apparently lost in the C. lusitaniae lineage. Thus, C. lusitaniae presents an opportunity to discover how subtelomeric chromatin can be reconfigured.

Sirtuin deacetylases as therapeutic targets in the nervous system.

Sirtuins are a conserved family of deacetylases whose activities are dependent on nicotinamide adenine dinucleotide (NAD+). Sirtuins act in different cellular compartments, such as the nucleus where they deacetylate histones and transcriptional factors, in the cytoplasm where they modulate cytoskeletal and signaling molecules, and in the mitochondria where they engage components of the metabolic machinery. Collectively, they tune metabolic processes to energy availability, and modulate stress responses, protein aggregation, inflammatory processes, and genome stability. As such, they have garnered much interest and have been widely studied in aging and age-related neurodegeneration. In this chapter, we review the identification of sirtuins and their biological targets. We focus on their biological mechanisms of action and how they might be regulated, including via NAD metabolism, transcriptional and posttranscriptional control, and as targets of pharmacological agents. Lastly, we highlight the numerous studies suggesting that sirtuins are efficacious therapeutic targets in neurodegenerative disease and injury.

The rice NAD(+)-dependent histone deacetylase OsSRT1 targets preferentially to stress- and metabolism-related genes and transposable elements.

Histone acetylation/deacetylation is an important chromatin modification for epigenetic regulation of gene expression. Silent information regulation2 (Sir2)-related sirtuins are nicotinamide-adenine dinucleotide (NAD(+))-dependent histone deacetylases (HDAC). The mammalian sirtuin family comprises 7 members (SIRT1-7) that act in different cellular compartments to regulate metabolism and aging. The rice genome contains only two Sir2-related genes: OsSRT1 (or SRT701) and OsSRT2 (orSRT702). OsSRT1 is closely related to the mammalian SIRT6, while OsSRT2 is homologous to SIRT4. Previous work has shown that OsSRT1 is required for the safeguard against genome instability and cell damage in rice plant. In this work we investigated the role of OsSRT1 on genome-wide acetylation of histone H3 lysine 9 (H3K9ac) and studied the genome-wide binding targets of OsSRT1. The study reveals that OsSRT1 binds to loci with relatively low levels of H3K9ac and directly regulates H3K9ac and expression of many genes that are related to stress and metabolism, indicating that OsSRT1 is an important site-specific histone deacetylase for gene regulation in rice. In addition, OsSRT1 is found to also target to several families of transposable elements, suggesting that OsSRT1 is directly involved in transposable element repression.

Cloning, chromosomal characterization and FISH mapping of the NAD(+)-dependent histone deacetylase gene sirtuin 5 in the mouse.

Sirtuin 5 (SIRT5) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, belonging to the silent information regulator 2 (Sir2) family of sirtuin histone deacetylases (sirtuins). The yeast Sir2 protein and its mammalian derivatives are important in epigenetic gene silencing, DNA repair and recombination, cell cycle, microtubule organization and in the regulation of aging. In mammals, 7 sirtuin isoforms have been identified to date of which three (SIRT3, SIRT4 and SIRT5) are localized in the mitochondria, which serve as the center of energy management and the initiation of cellular apoptosis. In the study presented herein, we report the genomic organization and chromosomal localization of the murine sirt5 gene. We have isolated and characterized the murine sirt5 genomic sequence, which spans a region of 24,449 bp and which has one single genomic locus. The murine sirt5 gene consists of 8 exons and encodes a 310-aa protein with a predictive mo-lecular weight of 34.1 kDa and an isoelectric point of 8.90. For the murine sirt5 gene only one single genomic locus has been identified. The gene has been localized to mouse chromosome 13A4 and is flanked by STS-marker 164522 (synonymous WI MRC-RH: 506859).

Genomic organization and localization of the NAD-dependent histone deacetylase gene sirtuin 3 (Sirt3) in the mouse.

Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, which belongs to the Silent information regulator 2 (Sir2) family of histone deacetylases (sirtuin HDACs). The yeast Sir2 protein and its mammalian derivatives play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. We have isolated and characterized the murine Sirt3 genomic sequence, which spans a region of 18,646 bp and which has one single genomic locus. Determination of the exon-intron splice junctions identified murine SIRT3 to be encoded by 7 exons ranging in size from 101 (exon 4) to 420 bp (exon 7). Characterization of the 5' flanking genomic region, which precedes the murine Sirt3 open reading frame, revealed a number of STATx, GATA and SP1 transcription factor binding sites. A CpG island was not detected. The 1,473-bp murine Sirt3 transcript has an open reading frame of 774 bp and encodes a 257-aa protein (cytoplasmic SIRT3) with a predictive molecular weight of 28.8 kDa and an isoelectric point of 5.82. Recently, a 1,406-bp murine SIRT3 splice variant that encodes a 334-aa mitochondrial precursor protein with a molecular weight of 36.6 kDa and an isoelectric point of 7.19 has been described. Fluorescence in situ hybridization analysis identified a single genomic locus for murine Sirt3 gene on chromosome 7F4 and which is neighbored by the Ric8 and PSMD13 genes. Our study brings light and a number of corrections and additions to previous reports on the genomic organization and the genomic sequence of murine Sirt3, which may be of importance in view of studies on potential genetic polymorphisms in relation to cellular respiration, metabolism, aging-related disease and cancer.

Controlling SIRT1 expression by microRNAs in health and metabolic disease.

SIRT1 is a NAD+-dependent deacetylase implicated in longevity and diverse physiological processes. SIRT1, as a key mediator of beneficial effects of caloric restriction, regulates lipid and glucose metabolism by deacetylating metabolic regulators, as well as histones, in response to nutritional deprivation. Here we discuss how SIRT1 levels are regulated by microRNAs (miRs) which are emerging as important metabolic regulators; the recently identified nuclear receptor FXR/SHP cascade pathway that controls the expression of miR-34a and its target SIRT1; and a FXR/SIRT1 positive feedback regulatory loop, which is deregulated in metabolic disease states. The FXR/miR-34a pathway and other miRs controlling SIRT1 may be useful therapeutic targets for age-related diseases, including metabolic disorders.

Genomic comparison of the ants Camponotus floridanus and Harpegnathos saltator.

The organized societies of ants include short-lived worker castes displaying specialized behavior and morphology and long-lived queens dedicated to reproduction. We sequenced and compared the genomes of two socially divergent ant species: Camponotus floridanus and Harpegnathos saltator. Both genomes contained high amounts of CpG, despite the presence of DNA methylation, which in non-Hymenoptera correlates with CpG depletion. Comparison of gene expression in different castes identified up-regulation of telomerase and sirtuin deacetylases in longer-lived H. saltator reproductives, caste-specific expression of microRNAs and SMYD histone methyltransferases, and differential regulation of genes implicated in neuronal function and chemical communication. Our findings provide clues on the molecular differences between castes in these two ants and establish a new experimental model to study epigenetics in aging and behavior.

Arabidopsis putative deacetylase AtSRT2 regulates basal defense by suppressing PAD4, EDS5 and SID2 expression.

The silent information regulator protein (Sir2) and its homologs are NAD(+)-dependent deacetylase enzymes that play important roles in a variety of physiological processes. However, the functions of the Sir2 family in plants are poorly understood. Here, we report that Arabidopsis AtSRT2, a homolog of yeast Sir2, negatively regulates plant basal defense against the pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000). In response to PstDC3000 infection, the expression of AtSRT2 was down-regulated in a salicylic acid (SA)-independent manner. In addition, knock-out of AtSRT2 (srt2) enhanced resistance against PstDC3000 and increased expression of pathogenesis-related gene 1 (PR1). Conversely, overexpression of AtSRT2 resulted in hypersusceptibility to PstDC3000 and impaired PR1 induction. Consistent with this phenotype, expression of PAD4, EDS5 and SID2, three essential genes in the SA biosynthesis pathway, were increased in the srt2 mutant and decreased in AtSRT2-overexpressing plants. Taken together, these results demonstrate that AtSRT2 is a negative regulator of basal defense, possibly by suppressing SA biosynthesis.

Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux.

Lysine acetylation regulates many eukaryotic cellular processes, but its function in prokaryotes is largely unknown. We demonstrated that central metabolism enzymes in Salmonella were acetylated extensively and differentially in response to different carbon sources, concomitantly with changes in cell growth and metabolic flux. The relative activities of key enzymes controlling the direction of glycolysis versus gluconeogenesis and the branching between citrate cycle and glyoxylate bypass were all regulated by acetylation. This modulation is mainly controlled by a pair of lysine acetyltransferase and deacetylase, whose expressions are coordinated with growth status. Reversible acetylation of metabolic enzymes ensure that cells respond environmental changes via promptly sensing cellular energy status and flexibly altering reaction rates or directions. It represents a metabolic regulatory mechanism conserved from bacteria to mammals.

The effect of Plasmodium falciparum Sir2a histone deacetylase on clonal and longitudinal variation in expression of the var family of virulence genes.

Plasmodium falciparum, the most important causative agent of human malaria, undergoes antigenic variation as a means of prolonging infection and ensuring transmission between hosts. Clonal variation is observed in the surface adhesins expressed on infected erythrocytes: primarily in the PfEMP1 adhesin encoded by the large var gene family. The sirtuin PfSIR2A was the first protein discovered to have a major influence on antigenic variation in P. falciparum. In the absence of PfSIR2A, normal silencing of the variantly-expressed var gene family is partially deregulated. To thoroughly investigate the role of PfSIR2A in controlling antigenic variation, multiple independent clones of wildtype and PfSIR2A-knockout (DeltaSir2a) parasites were generated. var gene expression was then measured qualitatively, quantitatively and longitudinally over extended periods in culture. DeltaSir2a parasites were found to activate about 10 specific var genes in every independent clone analyzed. The activated genes were biased towards the upsA, upsBA and upsEvar gene subclasses. The total var transcript level was two to three-fold higher in DeltaSir2a parasites than in wildtype parasites and at least one transcript - encoding the pregnancy malaria adhesin VAR2CSA - was successfully translated and expressed on the infected cell surface. In the absence of PfSIR2A, antigenic switching over time was also diminished, although not abolished. This work expands our understanding of clonal antigenic variation in this important human pathogen and demonstrates a central role for PfSIR2A in regulating both the variant expression of specific var gene subsets and the overall quantity of var gene expression.