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Gram-negative intracellular pathogen Vibrio parahaemolyticus manifests its infection through a series of effector proteins released into the host via the type III secretion system. Most of these effector proteins alter signalling pathways of the host to facilitate survival and proliferation of bacteria inside host cells. Here, we report V. parahaemolyticus (serotype O3:K6) infection-induced histone deacetylation in host intestinal epithelial cells, particularly deacetylation of H3K9, H3K56, H3K18 and H4K16 residues. We found a putative NAD+-dependent deacetylase, vp1524 (vpCobB) of V. parahaemolyticus, was overexpressed during infection. Biochemical assays revealed that Vp1524 is a functional NAD+-dependent Sir2 family deacetylase in vitro, which was capable of deacetylating acetylated histones. Furthermore, we observed that vp1524 is expressed and localized to the nuclear periphery of the host cells during infection. Consequently, Vp1524 translocated to nuclear compartments of transfected cells, deacetylated histones, specifically causing deacetylation of those residues (K56, K16, K18) associated with V. parahaemolyticus infection. This infection induced deacetylation resulted in transcriptional repression of several host genes involved in epigenetic regulation, immune response, autophagy etc. Thus, our study shows that a V. parahaemolyticus lysine deacetylase Vp1524 is secreted inside the host cells during infection, modulating host gene expression through histone deacetylation.

To maintain normal function of cartilage tissue normally, the presence of a sufficient amount of type II collagen and aggrecan is essential, and their synthesis is tightly regulated. Therefore, understanding the mechanisms that control the expression of type II collagen and aggrecan would be useful for understanding gene expression changes in diseases such as osteoarthritis. Recently, we have identified two pairs of enhancer elements, termed E1 and E2 in the type II collagen gene and Ea and Eb in the aggrecan gene. However, their different mechanisms of action remained unclear. Thus, the central aim of this study was to clarify the different transcriptional regulation mediated through each enhancer element. To this end, we established different stable reporter cell lines that express a reporter gene under the control of different enhancer elements using a silent reporter system we previously constructed. Using these cell lines, we found that dexamethasone, forskolin, and trichostatin A affect the gene expression of type II collagen and aggrecan via different enhancer elements. Moreover, we clarified that E1 and E2 enhancer activities are regulated through distinct epigenetic modifications by histone deacetylase 10 and sirtuin 6.

Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi, affecting more than 7 million people in the world. Benznidazole and nifurtimox are the only drugs available for treatment and in addition to causing several side effects, are only satisfactory in the acute phase of the disease. Sirtuins are NAD+-dependent deacetylases involved in several biological processes, which have become drug target candidates in various disease settings. T. cruzi presents two sirtuins, one cytosolic (TcSir2rp1) and the latter mitochondrial (TcSir2rp3). Here, we characterized the effects of human sirtuin inhibitors against T. cruzi sirtuins as an initial approach to develop specific parasite inhibitors. We found that, of 33 compounds tested, two inhibited TcSir2rp1 (15 and 17), while other five inhibited TcSir2rp3 (8, 12, 13, 30, and 32), indicating that specific inhibitors can be devised for each one of the enzymes. Furthermore, all inhibiting compounds prevented parasite proliferation in cultured mammalian cells. When combining the most effective inhibitors with benznidazole at least two compounds, 17 and 32, demonstrated synergistic effects. Altogether, these results support the importance of exploring T. cruzi sirtuins as drug targets and provide key elements to develop specific inhibitors for these enzymes as potential targets for Chagas disease treatment.

Diabetes mellitus (DM) is associated with accelerated cognitive decline. However, the mechanism of diabetic cognitive impairment remains poorly understood. In this study, we found that the expression of Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, was downregulated significantly in the hippocampus of streptozotocin (STZ)-induced diabetic cognitive impairment rats. Viral overexpression of hippocampal SIRT1 ameliorated cognitive impairment in diabetic rats, but viral knockdown of hippocampal SIRT1 mimicked the diabetic effect, eliciting the cognitive decline in normal animals. Further study showed that the decreased level of SIRT1 may result in the increase of acetylated tau protein in the hippocampus, which may mediate the development of diabetic cognitive impairment. These results suggest that SIRT1 may be a key epigenetic regulator that guards against the development of diabetic cognitive impairment by deacetylating the tau protein. SIRT1 activator may serve as a new therapeutic approach for the treatment of diabetic cognitive impairment.

Background Mutations in the LAMA2 gene encoding laminin-α2 cause congenital muscular dystrophy Type 1A (MDC1A), a severe recessive disease with no effective treatment. Previous studies have shown that aberrant activation of caspases and cell death through a pathway regulated by BAX and KU70 is a significant contributor to pathogenesis in laminin-α2-deficiency. Objectives To identify mechanisms of pathogenesis in MDC1A. Methods We used immunocytochemical and molecular studies of human myogenic cells and mouse muscles-comparing laminin-α2-deficient vs. healthy controls-to identify mechanisms that regulate pathological activation of caspase in laminin-α2-deficiency. Results In cultures of myogenic cells from MDC1A donors, p53 accumulated in a subset of nuclei and aberrant caspase activation was inhibited by the p53 inhibitor pifithrin-alpha. Also, the p53 target BBC3 (PUMA) was upregulated in both MDC1A myogenic cells and Lama2-/- mouse muscles. In addition, studies with sirtuin inhibitors and SIRT1 overexpression showed that caspase activation in MDC1A myotubes was inversely related to sirtuin deacetylase activity. Caspase activation in laminin-α2-deficiency was, however, not associated with increased phosphorylation of p38 MAPK. Conclusions Aberrant caspase activation in MDC1A cells was mediated both by sirtuin deacetylase activity and by p53. Interventions that inhibit aberrant caspase activation by targeting sirtuin or p53 function could potentially be useful in ameliorating MDC1A.

Sir2 family proteins are highly conserved and catalyze Nicotinamide Adenine Dinucleotide (NAD(+))-dependent protein deacetylation reaction that regulates multiple cellular processes. Little is known about Sir2 family proteins in Giardia. In this research, Sir2 homologs of Giardia were Phylogenetically analyzed. GL50803_10707 (GlSIR2.2) showed strong homology to SIRT1 and was the only parasite SIRT1 homolog being reported to date. Recombinant GlSIR2.2 (rGlSIR2.2) was expressed and purified. The renaturied recombinant protein showed a typical NAD-dependent protein deacetylase activity that could be inhibited by nicotinamide, with IC50 of 4.47 mM rGlSIR2.2 displayed deacetylase activity under varied NAD(+), with Km, kcat and kcat/Km values of 31.71 μM, 1.4 × 10(-3) s(-1), and 4.42 × 10(-5) μM(-1) s(-1). Similarly, the steady-state kinetic parameters with varied ZMAL, yielded Km, kcat and kcat/Km values of 96.89 μM, 4.7 × 10(-3) s(-1), and 4.85 × 10(-5) μM(-1) s(-1). Anti-rGlSIR2.2 serum was used to probe subcellular localization of GlSIR2.2 and strong staining was found predominantly in the nucleus. So we demonstrated that GlSIR2.2 was a SIRT1-like, nuclear-located, NAD(+)-dependent deacetylase. This is the first report of deacetylase activity of Sir2 family protein in Giardia.

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.

Gene duplication promotes the diversification of protein functions in several ways. Ancestral functions can be partitioned between the paralogs, or a new function can arise in one paralog. These processes are generally viewed as unidirectional. However, paralogous proteins often retain related functions and can substitute for one another. Moreover, in the event of gene loss, the remaining paralog might regain ancestral functions that had been shed. To explore this possibility, we focused on the sirtuin deacetylase SIR2 and its homolog HST1 in the CTG clade of yeasts. HST1 has been consistently retained throughout the clade, whereas SIR2 is only present in a subset of species. These NAD+-dependent deacetylases generate condensed chromatin that represses transcription and stabilizes tandemly repeated sequences. By analyzing phylogenetic trees and gene order, we found that a single duplication of the SIR2/HST1 gene occurred, likely prior to the emergence of the CTG clade. This ancient duplication was followed by at least two independent losses of SIR2. Functional characterization of Sir2 and Hst1 in three species revealed that these proteins have not maintained consistent functions since the duplication. In particular, the rDNA locus is deacetylated by Sir2 in Candida albicans, by Hst1 in C. lusitaniae, and by neither paralog in C. parapsilosis. In addition, the subtelomeres in C. albicans are deacetylated by Sir2 rather than by Hst1, which is orthologous to the sirtuin associated with Saccharomyces cerevisiae subtelomeres. These differences in function support the model that sirtuin deacetylases can regain ancestral functions to compensate for gene loss.

Purpose of review: \ud We present a current perspective of epigenetic alterations that can lead to cardiovascular disease (CVD) and the potential of dietary factors to counteract their actions. In addition, we discuss the challenges and opportunities of dietary treatments as epigenetic modifiers for disease prevention and therapy.\ud \ud Recent findings: \ud Recent epigenome-wide association studies along with candidate gene approaches and functional studies in cell culture and animal models have delineated mechanisms through which nutrients, food compounds and dietary patterns may affect the epigenome. Several risk factors for CVD, including adiposity, inflammation and oxidative stress, have been associated with changes in histone acetylation, lower global DNA methylation levels and shorter telomere length. A surplus of macronutrients such as in a high-fat diet or deficiencies of specific nutrients such as folate and other B-vitamins can affect the activity of DNA methyltransferases and histone-modifying enzymes, affecting foetal growth, glucose/lipid metabolism, oxidative stress, inflammation and atherosclerosis. Bioactive compounds such as polyphenols (resveratrol, curcumin) or epigallocatechin may activate deacetylases Sirtuins (SIRTs), histone deacetylases or acetyltransferases and in turn the response of inflammatory mediators. Adherence to cardioprotective dietary patterns, such as the Mediterranean diet (MedDiet), has been associated with altered methylation and expression of genes related to inflammation and immuno-competence.\ud \ud Summary: \ud The mechanisms through which nutrients and dietary patterns may alter the cardiovascular epigenome remain elusive. The research challenge is to determine which of these nutriepigenetic effects are reversible, so that novel findings translate into effective dietary interventions to prevent CVD or its progression.

Heart failure is an aging-associated disease that is the leading cause of death worldwide. Sirtuin family members have been largely studied in the context of aging and aging-associated diseases. Sirtuin 2 (SIRT2) is a cytoplasmic protein in the family of sirtuins that are NAD(+)-dependent class III histone deacetylases. In this work, we studied the role of SIRT2 in regulating nuclear factor of activated T-cells (NFAT) transcription factor and the development of cardiac hypertrophy. Confocal microscopy analysis indicated that SIRT2 is localized in the cytoplasm of cardiomyocytes and SIRT2 levels are reduced during pathological hypertrophy of the heart. SIRT2-deficient mice develop spontaneous pathological cardiac hypertrophy, remodeling, fibrosis, and dysfunction in an age-dependent manner. Moreover, young SIRT2-deficient mice develop exacerbated agonist-induced hypertrophy. In contrast, SIRT2 overexpression attenuated agonist-induced cardiac hypertrophy in cardiomyocytes in a cell-autonomous manner. Mechanistically, SIRT2 binds to and deacetylates NFATc2 transcription factor. SIRT2 deficiency stabilizes NFATc2 and enhances nuclear localization of NFATc2, resulting in increased transcription activity. Our results suggest that inhibition of NFAT rescues the cardiac dysfunction in SIRT2-deficient mice. Thus, our study establishes SIRT2 as a novel endogenous negative regulator of NFAT transcription factor.

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 Nε-Lys acetylation.

Summary 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., Highlights • LRRK2 G2019S iPSC-derived dopaminergic neurons have unique mitochondrial defects • LRRK2 G2019S dopaminergic neurons have increased sirtuin levels but reduced activity • LRRK2 G2019S dopaminergic neurons have reduced NAD+ levels compared with other neurons, In this article, Ebert and colleagues show that iPSC-derived dopaminergic neurons expressing the LRRK2 G2019S mutation exhibit mitochondrial abnormalities, reduced sirtuin activity, and low endogenous NAD+ levels compared with other neuronal subtypes generated from the same patient samples. Therefore, the distinct sirtuin and bioenergetic deficiencies intrinsic to dopaminergic neurons may underlie dopaminergic neuron loss in Parkinson's disease.

Silent information regulator 2 (Sir2) enzymes which catalyze NAD+-dependent protein/histone deacetylation. The mammalian sirtuin family SIRT1, SIRT2, SIRT3 and SIRT6 can regulate oxidative stress. The probiotics (Bifidobacterium longum(B.longum) and Lactobacillus acidophilus(L. acidophilus)) have Sir2 gene family and have antioxidant activity in human body. it remains unknown whether probiotics Sir2 has a direct role in regulating oxidative stress. To this end, we knockout BL-sir2(sir2 B. longum) and LA-sir2(sir2 L.acidophilus) in low oxygen level. The antioxidant activities of two sir2 deficient strains was decreased, while when reintroduction of BL-sir2 and LA-sir2, the antioxidant activities were recoveried. In order to understand the regulation mechanism of probiotics Sir2 oxidation response. Then, we screened 65 acetylated protein, and found that SigH (σ

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.

Melanoma continues to cause more deaths than any other skin cancer, necessitating the development of new avenues of treatment. One promising new opportunity comes in the form of mechanism-based therapeutic targets. We recently reported the overexpression and delocalization of the class III histone deacetylase SIRT1 in melanoma, and demonstrated that its small molecule inhibition via Tenovin-1 decreased cell growth and viability of melanoma cells, possibly by a p53 mediated induction of p21. Here, we support our data using additional SIRT inhibitors, viz. Sirtinol and Ex-527, which suggests possible benefits of concomitantly inhibiting more than one Sirtuin for an effective cancer management strategy. This “Extra View” paper also includes a discussion of our results in the context of similar recent and concurrent studies. Furthermore, we expand upon our findings in an analysis of new research that may link the cellular localization and growth effects of SIRT1 with the PI3K signaling pathway.

Matrix metalloproteinase (MMP) 9 plays an important role in the degradation of the extracellular matrix in fetal membranes, and pathological activation of MMP-9 can lead to preterm birth. In nongestational tissues, modulation of histone deacetylases (HDACs) regulates MMP-9 expression. The aim of this study was to determine whether class I to III HDACs regulate MMP-9 expression and activity in primary amnion cells. Class I and II HDAC regulation of MMP-9 was assessed using the general class I and II HDAC inhibitors (HDACi) trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), the class I HDACi MS-275, and the class II HDACi MC1568. Class III HDAC regulation of MMP-9 was assessed using the SIRT1 activators resveratrol and SRT1720 as well as SIRT1 small interfering RNA (siRNA). Primary amnion epithelial cells were incubated with 1 ng/mL interleukin (IL) 1β in the absence or presence of 0.3 μmol/L TSA, 5 μmol/L SAHA, 2.5 μmol/L MS-275, 2.5 μmol/L MC1568, 50 μmol/L resveratrol, or 10 μmol/L SRT1720 for 20 hours. We found that the class I and II HDACi TSA and SAHA and the class II HDACi MC1568 significantly decreased IL-β-induced MMP-9 gene and pro-MMP-9 expression in primary amnion cells. There was, however, no effect of the class I HDACi MS-275 on IL-β-induced MMP-9 expression. On the other hand, inhibition of class III HDAC SIRT1 using siRNA significantly augmented IL-1β-induced MMP-9, and SIRT1 activation using resveratrol and SRT1720 inhibited IL-1β-induced MMP-9 expression. In summary, class I to III HDACs differentially regulate inflammation-induced MMP-9 expression in primary amnion cells.

SUMMARYChagas disease, which is caused by the parasite Trypanosoma cruzi, affects approximately 7–8 million people in Latin America. The drugs available to treat this disease are ineffective against chronic phase disease and are associated with toxic side effects. Therefore, the development of new compounds that can kill T. cruzi at low concentrations is critically important. Herein, we report the effects of a novel 3-arylideneindolin-2-one that inhibits sirtuins, which are highly conserved proteins that are involved in a variety of physiological processes. The compound KH-TFMDI was tested against the epimastigote, trypomastigote and amastigote forms of T. cruzi, and its effects were evaluated using flow cytometry, light and electron microscopy. KH-TFMDI inhibited the replication of T. cruzi intracellular amastigotes with an IC50 of 0·5±0·2 μm, which is significantly lower than the IC50 of benznidazole. The compound also lysed the highly infectious bloodstream trypomastigotes (BST) with LC50 values of 0·8±0·3 μm at 4 °C and 2·5±1·1 μm at 37 °C. KH-TFMDI inhibited cytokinesis and induced several morphological changes in the parasite, leading to its death by apoptosis and autophagy. This study highlights sirtuins as a potential new target for Chagas disease therapy.

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).

Sirtuins are evolutionary conserved NAD+ dependent acetyl-lysine deacetylases and ADP ribosyltransferases dual-function enzymes involved in the regulation of metabolism and lifespan. Sirtuins represent a promising new class of III NAD dependent histone deacetylases that regulate a number of physiological processes, originally identified in yeast. Sirtuins regulate various normal and abnormal cellular and metabolic processes, including tumorgenesis, neurodegeneration and processes associated with type 2 diabetes and obesity. Several age-related diseases such as Alzheimer’s disease and longevity have also been linked to the functions of sirtuins. Because of these associations, the identification of small molecules sirtuin modulators has been of significant interest. Kathmandu University Medical Journal | Vol.10 | No. 2 | Issue 38 | Apr – June 2012 | Page 77-82 DOI: http://dx.doi.org/10.3126/kumj.v10i2.7350

Sirtuins are NAD

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.

Sirtuins, commonly known as NAD+-dependent class III histone deacetylase enzymes, have been extensively studied to evaluate their potential role in different disease states. Based on the published literature, sirtuins have been implicated in providing a myriad of intrinsic and extrinsic biological effects, which in turn may play an important role in the treatment of various disorders such as type II diabetes, obesity, cancer, aging and different neurodegenerative diseases. In particular, a number of studies have unequivocally supported the idea of sirtuins having therapeutic potential in neurodegenerative diseases such as stroke, ischemic brain injury, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis. To exploit the therapeutic potential of sirtuins, their manipulation in terms of development of small-molecule modulators, inhibitors and analogs has increased dramatically since their inception, in both scientific and industrial worlds. Studies on the structure and catalytic core of sirtuins along with chemical mechanisms and substrate specificity have provided important input into the design and synthesis of sirtuin modulators. To study the role of sirtuins in the biological system, it has become extremely important to understand the molecular and chemical structure of sirtuins. In this review, we have discussed the biological role of sirtuins in various neurodegenerative diseases, and also provided an insight into their chemical structure.

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1α and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1α expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1α expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1α after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1α. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1α or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1α activity after exercise.

Propagation of a species requires periodic cell renewal to avoid clonal extinction. Sexual reproduction and the separation of germ cells from the soma provide a mechanism for such renewal, but are accompanied by an apparently mandatory ageing of the soma. Data obtained during the last decade suggest that a division of labour exists also between cells of vegetatively reproducing unicellular organisms, leading to the establishment of a soma-like and germ-like lineage with distinct fitness and longevity characteristics. This division of labour in both bacteria and yeast entails segregation of damaged and aggregated proteins such that the germ-like lineage is kept free of damage to the detriment of the soma-like lineage. In yeast, this spatial protein quality control (SQC) encompasses a CCT-chaperonin-dependent translocation and merging of cytotoxic protein aggregates. This process is regulated by Sir2, a protein deacetylase that modulates the rate of ageing in organisms ranging from yeast to worms and flies. Recent data also demonstrate that SQC is intimately integrated with the machinery establishing proper cell polarity and that this machinery is required for generating a soma-like and germ-like lineage in yeast. Deciphering the details of the SQC network may increase our understanding of the development of age-related protein folding disorders and shed light on the selective forces that paved the way for polarity and lineage-specific ageing to evolve.

Sirtuins have emerged as important proteins in aging, stress resistance and metabolic regulation. Three sirtuins, SIRT3, 4 and 5, are located within the mitochondrial matrix. SIRT3 and SIRT5 are NAD(+)-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins and yield 2'-O-acetyl-ADP-ribose and nicotinamide. SIRT4 can transfer the ADP-ribose group from NAD(+) onto acceptor proteins. Recent findings reveal that a large fraction of mitochondrial proteins are acetylated and that mitochondrial protein acetylation is modulated by nutritional status. This and the identification of targets for SIRT3, 4 and 5 support the model that mitochondrial sirtuins are metabolic sensors that modulate the activity of metabolic enzymes via protein deacetylation or mono-ADP-ribosylation. Here, we review and discuss recent progress in the study of mitochondrial sirtuins and their targets.

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.

Class III histone deacetylases (sirtuins) play pivotal roles in many cellular processes. They are linked to extended lifespan and to the pathogenesis of cancer and neuronal disorders. We present novel sirtuin inhibitors based on a 6,7-dichloro-2-oxindole scaffold with low micromolar activity. In vitro activity was rationalized by docking studies, and hyperacetylation of sirtuin targets could be demonstrated in cell culture.

Histone deacetylases (HDACs) are enzymes that cleave off acetyl groups from acetyl-lysine residues in histones and various nonhistone proteins. Four different classes of HDACs have been identified in humans so far. Although classes I, II, and IV are zinc-dependent amidohydrolases, class III HDACs depend on nicotinamide adenine dinucleotide (NAD(+)) for their catalytic activity. According to their homology to Sir2p, a yeast histone deacetylase, the class III is also termed sirtuins. Seven members have been described in humans so far. As sirtuins are involved in many physiological and pathological processes, their activity has been associated with the pathogenesis of cancer, HIV, metabolic, or neurological diseases. Herein, we present an overview over sirtuins including their biology, targets, inhibitors, and activators and their potential as new therapeutic agents.

Suramin is a symmetric polyanionic naphthylurea originally used for the treatment of trypanosomiasis and onchocerciasis. Suramin and diverse analogues exhibit a broad range of biological actions in vitro and in vivo, including, among others, antiproliferative and antiviral activity. Suramin derivatives usually target purinergic binding sites. Class III histone deacetylases (sirtuins) are amidohydrolases that require nicotinamide adenine dinucleotide (NAD(+)) as a cofactor for their catalytic mechanism(.) Deacetylation of the target proteins leads to a change in conformation and alters the activity of the proteins in question. Suramin was reported to inhibit human sirtuin 1 (SIRT1). We tested a diverse set of suramin analogues to elucidate the inhibition of the NAD(+)-dependent histone deacetylases SIRT1 and SIRT2 and discovered selective inhibitors of human sirtuins with potency in the two-digit nanomolar range. In addition, the structural requirements for the binding of suramin derivatives to sirtuins were investigated by molecular docking. The recently published X-ray crystal structure of human SIRT5 in complex with suramin and the human SIRT2 structure were used to analyze the interaction mode of the novel suramin derivatives.

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.

Histone deacetylases (HDACs) are involved in the regulation of transcription and their inhibitors are a promising class of new anticancer drugs. We have previously reported Boc(Ac)Lys-AMC, also termed MAL, as a fluorescent substrate for HDACs. Now we present a modification of MAL called Z-MAL that is characterized by an increased rate of conversion by histone deacetylases of classes I and II and the recently discovered sirtuins (histone deacetylases class III). MAL and Z-MAL are the first nonradioactive substrates for class III enzymes. The new substrate Z-MAL allows for shorter assay times in inhibitor screening and is applicable to diverse sources of deacetylase activity even with completely different catalytic mechanisms. Interestingly, MAL shows some relative preference toward class II, indicating that subtype selectivity in small-molecule HDAC substrates might be obtained.

International audience; 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.

In mammals, NAD represents a nodal point for metabolic regulation, and its availability is critical to genome stability. Several NAD-consuming enzymes are induced in various stress conditions and the consequent NAD decline is generally accompanied by the activation of NAD biosynthetic pathways to guarantee NAD homeostasis. In the bacterial world a similar scenario has only recently begun to surface. Here we review the current knowledge on the involvement of NAD homeostasis in bacterial stress response mechanisms. In particular, we focus on the participation of both NAD-consuming enzymes (DNA ligase, mono(ADP-ribosyl) transferase, sirtuins, and RNA 2'-phosphotransferase) and NAD biosynthetic enzymes (both de novo, and recycling enzymes) in the response to DNA/RNA damage. As further supporting evidence for such a link, a genomic context analysis is presented showing several conserved associations between NAD homeostasis and stress responsive genes.

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.

Background In recent years, reversible lysine acylation of proteins has emerged as a major post-translational modification across the cell, and importantly has been shown to regulate many proteins in mitochondria. One key family of deacylase enzymes is the sirtuins, of which SIRT3, SIRT4, and SIRT5 are localised to the mitochondria and regulate acyl modifications in this organelle. Scope of review In this review we discuss the emerging role of lysine acylation in the mitochondrion and summarise the evidence that proposes mitochondrial sirtuins are important players in the modulation of mitochondrial energy metabolism in response to external nutrient cues, via their action as lysine deacylases. We also highlight some key areas of mitochondrial sirtuin biology where future research efforts are required. Major conclusions Lysine deacetylation appears to play some role in regulating mitochondrial metabolism. Recent discoveries of new enzymatic capabilities of mitochondrial sirtuins, including desuccinylation and demalonylation activities, as well as an increasing list of novel protein substrates have identified many new questions regarding the role of mitochondrial sirtuins in the regulation of energy metabolism. General significance Dynamic changes in the regulation of mitochondrial metabolism may have far-reaching consequences for many diseases, and despite promising initial findings in knockout animals and cell models, the role of the mitochondrial sirtuins requires further exploration in this context. This article is part of a Special Issue entitled Frontiers of mitochondrial research.

In many types of tumours, especially pancreatic adenocarcinoma, miR-301a is over-expressed. This over-expression results in negative regulation of the target gene of miR-301a, the nuclear factor-κB (NF-κB) repressing factor (NKRF), increasing the activation of NF-κB and production of NF-κB-responsive pro-inflammatory cytokines such as interleukin-8, interferon-β, nitric oxide synthase 2A and cytochrome oxidase subunit 2 (COX-2). However, in immune cells, mechanisms that regulate miR-301a have not been reported. Similar to tumour cells, Toll-like receptor (TLR) -activated macrophages produce NF-κB-responsive pro-inflammatory cytokines. Therefore, it is of considerable interest to determine whether miR-301a regulates the secretion of cytokines by immune cells. In the present study, we demonstrate that the expression of miR-301a was decreased in TLR-triggered macrophages. Through targeting NKRF, miR-301a affected the activity of NF-κB and the expression of pro-inflammatory genes downstream of NF-κB such as COX-2, prostaglandin E2 and interleukin-6. In addition, when lipopolysaccharide-treated macrophages were simultaneously stimulated with trichostatin A, an inhibitor of histone deacetylases, the expression of miR-301a increased, whereas NKRF and pro-inflammatory cytokine expression decreased. However, further investigation revealed that there was no correlation between the induction of miR-301a and the inhibitory effect of trichostatin A on lipopolysaccharide-induced gene expression in macrophages. In summary, our study indicates a new mechanism by which miR-301a regulates inflammatory cytokine expression in macrophages, which may clarify the regulatory role of microRNAs in immune-mediated inflammatory responses.

In a mouse model of skin repair we found that the class I-IIa histone deacetylase inhibitor trichostatin A accelerated tissue regeneration. Unexpectedly, this effect was suppressed by Sirtinol, a class III histone deacetylase (HDAC) (sirtuin)-selective inhibitor. The role of sirtuins (SIRTs) was then investigated by using resveratrol and a novel SIRT1-2-3 activator, the MC2562 compound we synthesized recently. Both resveratrol and MC2562 were effective in accelerating wound repair. The local administration of natural or synthetic SIRT activators, in fact, significantly accelerated skin regeneration by increasing keratinocyte proliferation. In vitro experiments revealed that the activation of SIRTs stimulated keratinocyte proliferation via endothelial NO synthase phosphorylation and NO production. In this condition, the class I member HDAC2 was found S-nitrosylated on cysteine, a post-transduction modification associated with loss of activity and DNA binding capacity. After deacetylase inhibitor or SIRT activator treatment, ChIP showed, in fact, a significant HDAC2 detachment from the promoter region of insulin growth factor I (IGF-I), fibroblast growth factor 10 (FGF-10), and Epithelial Growth Factor (EGF), which may be the final recipients and effectors of the SIRT-NO-HDAC signaling cascade. Consistently, the effect of SIRT activators was reduced in the presence of NG-nitro-L-arginine methyl ester (L-NAME), a general inhibitor of NO synthesis. In conclusion, the NO-dependent cross-talk among class III and I histone deacetylases suggests an unprecedented signaling pathway important for skin repair.

Cultivation of Chaetomium mollipilium with nicotinamide, a NAD(+)-dependent HDAC inhibitor, stimulated its secondary metabolism, leading to the isolation of structurally diverse new C(13)-polyketides, mollipilin A-E (1-5) as well as two known compounds (6 and 7). Spectroscopic methods, X-ray single crystal diffraction analysis, and VCD elucidated the absolute configurations of structures 1-6, and plausible biosynthetic pathways for 1-7 were proposed based on structural relationships. Mollipilins A (1) and B (2) exhibited moderate growth inhibitory effects on HCT-116 cells.

Seventy-five years after its initial report, lifespan prolongation by caloric restriction (CR) has been confirmed in many animal species. Gathered evidences suggest that the histone deacetylase Sir2/SIRT1 is a key mediator. Moreover, resveratrol (RSV), a plant derived polyphenol, was shown to increase SIRT1 catalytic activity and mimics many aspects of CR in all eukaryotes tested. Therefore, RSV displays great promise in the prevention of illness and delay the aging process.

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.

Histone deacetylases (HDACs) are enzymes that cleave off acetyl groups from acetyl-lysine residues in histones and various nonhistone proteins. Four different classes of HDACs have been identified in humans so far. Although classes I, II, and IV are zinc-dependent amidohydrolases, class III HDACs depend on nicotinamide adenine dinucleotide (NAD(+)) for their catalytic activity. According to their homology to Sir2p, a yeast histone deacetylase, the class III is also termed sirtuins. Seven members have been described in humans so far. As sirtuins are involved in many physiological and pathological processes, their activity has been associated with the pathogenesis of cancer, HIV, metabolic, or neurological diseases. Herein, we present an overview over sirtuins including their biology, targets, inhibitors, and activators and their potential as new therapeutic agents.

The sirtuins are NAD+-dependent histone/protein deacetylases that are similar to Saccharomyces cerevisiae silent information regulator 2 (Sir2). Sirtuins regulate various normal and abnormal cellular and metabolic processes, including tumorigenesis, neurodegeneration, and processes associated with type 2 diabetes and obesity. Several age-related diseases, such as Alzheimer's disease, and longevity have also been linked to the functions of sirtuins. A thorough understanding of the mechanisms of action of the sirtuins may therefore yield novel therapeutic strategies targeting these processes; several small-molecule and naturally occurring inhibitors and activators of these enzymes have been identified. This review describes the mechanisms regulating sirtuin activity, as well as how these modes of regulation may be exploited to manipulate activity in the context of various pathological states (ie, metabolic diseases, cancer and neurodegenerative diseases). The possible metabolic outcomes of the pharmacological manipulation of sirtuins are also discussed.

Small molecules interfering with posttranslational modification of histones are of interest as tools to study epigenetic regulation of gene transcription. Specifically, drugs that interfere with histone deacetylation could be useful to induce differentiation, growth arrest as well as apoptotic cell death in tumor cells. One class of histone deacetylases is known as sirtuins some of which (Saccharomyces cerevisiae Sir2) are for example inhibited by the lactone splitomicin leading to telomeric silencing in yeast. However, splitomicin is only a micromolar inhibitor of yeast Sir2 and does not inhibit human subtypes and the lactone is prone to hydrolytic ring opening. In preliminary SAR-studies, splitomicin analogs lacking this hydrolytically labile ring were described as inactive while the naphthalene moiety could successfully be replaced by smaller aromatic rings in a fragment-like dihydrocoumarin. Here we report the synthesis and biological activity of a series of hydrolytically stable analogs with activity against human SIRT1 and 2. These comparatively small compounds characterized by high ligand efficiency are used as a starting point toward the development of specific inhibitors of histone deacetylases from the class of sirtuins.

Blood vessels deliver nutrients and oxygen to cells and tissues in the body. When blood supply is insufficient new vessels form to meet the metabolic tissue requirements. Several studies have examined the cellular and molecular principles of blood vessel formation, yet little is known about how vessels sense and integrate environmental signals originating from nutrient- and oxygen-deprived tissues to achieve functional vascular patterning. The NAD(+)-dependent deacetylase SIRT1 mediates adaptation to environmental stresses by adjusting cellular responses to the energetic state of the cell and recent studies highlight important functions of SIRT1 in regulating vascular growth, shape, and function. Here, we review the emerging role of SIRT1 as a metabolic sensor coupling energy and oxygen homeostasis to the growth and function of the vasculature.

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.

NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and other proteins to regulate their activity. Identification of potent selective inhibitors would help to elucidate sirtuin biology and could lead to useful therapeutic agents. NAD+ has an adenosine moiety that is also present in the kinase cofactor ATP. Kinase inhibitors based upon adenosine mimesis may thus also target NAD+-dependent enzymes. We present a systematic approach using adenosine mimics from one cofactor class (kinase inhibitors) as a viable method to generate new lead structures in another cofactor class (sirtuin inhibitors). Our findings have broad implications for medicinal chemistry and specifically for sirtuin inhibitor design. Our results also raise a question as to whether selectivity profiling for kinase inhibitors should be limited to ATP-dependent targets.

Histone deacetylases (HDACs) are enzymes that are able to deacetylate lysine side chains in histones and certain non-histone proteins which leads to altered states of conformation and activity for the proteins in question. Three classes of histone deacetylases have been recognized in humans. Class I and II are zinc-dependent amidohydrolases and eleven subtypes have been discovered (HDAC1-11). Class III enzymes depend in their catalysis on NAD(+) and subsequently, O-acetyl ADP ribose and nicotinamide are formed as a consequence of the acetyl transfer. Due to the homology to the yeast histone deacetylase Sir2p the NAD(+)-dependent deacetylases are also termed sirtuins and seven members (Sirt1-7) are known in humans. Sirtuins are found from bacteria to eukaryotes and altogether about 60 isoforms have been characterized in different organisms. Sirtuins have been implicated in the regulation of molecular mechanisms of aging. The overexpression of sirtuin enzymatic activity leads to an increase of lifespan in Saccharomyces cerevisiae and Caenorhabditis elegans that can also be reached by calorie restriction. Sirtuins have been proposed to act as sensors for glucose uptake that respond to the levels of NAD(+) but more complex ways of action have been suggested as well. This article will present the members of the human sirtuin family with their respective functions and review the existing druglike inhibitors and activators of sirtuin activity.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, which belongs to the silent information regulator 2 (Sir2) family of sirtuin histone deacetylases (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 human Sirt1 genomic sequence, which spans a region of 33,660 bp and which has one single genomic locus. Determination of the exon-intron splice junctions established that SIRT1 is encoded by 9 exons ranging in size from 80 bp (exon 6) to 2,120 bp (exon 9). Characterization of the 5' flanking genomic region, which precedes the Sirt1 open reading frame, revealed a CCAAT-box and a number of NF-kappaB and GATA transcription factor binding sites in addition to a small 350 bp CpG island. The 4,107 bp human Sirt1 mRNA has an open reading frame of 2,244 bp and encodes a 747 aa protein with a predictive molecular weight of 81.7 kDa and an isoelectric point of 4.55. Fluorescence in situ hybridization analysis localized the human Sirt1 gene to chromosome 10q21.3.

The yeast transcriptional repressor Sir2p silences gene expression from the telomeric, rDNA, and silent mating-type loci and may play a role in higher order processes such as aging. Sir2p is the founding member of a large family of NAD-dependent deacetylase enzymes, named the sirtuins. These proteins are conserved from prokaryotes to eukaryotes, but most remain uncharacterized, including all seven human sirtuins. A reverse chemical genetic approach would be useful in identifying the biological function of sirtuins in a wide variety of experimental systems, but no cell-permeable small molecule inhibitors of sirtuins have been reported previously. Herein we describe a high throughput, phenotypic screen in cells that led to the discovery of a class of sirtuin inhibitors. All three compounds inhibited yeast Sir2p transcriptional silencing activity in vivo, and yeast Sir2p and human SIRT2 deacetylase activity in vitro. Such specific results demonstrate the utility and robustness of this screening methodology. Structure-activity relationship analysis of the compounds identified a key hydroxy-napthaldehyde moiety that is necessary and sufficient for inhibitory activity. Preliminary studies using one of these compounds suggest that inhibition of sirtuins interferes with body axis formation in Arabidopsis.