SnapShot: Mammalian Sirtuins.

The mammalian sirtuins have emerged as critical regulators of cellular stress resistance, energy metabolism, and tumorigenesis. In some contexts, they delay the onset of age-related diseases and promote a healthy lifespan. The seven mammalian sirtuins, SIRT1-7, share a highly conserved NAD+-binding catalytic core domain although they exhibit distinct expression patterns, catalytic activities, and biological functions. This SnapShot provides an overview of these properties, with an emphasis on their relevance to aging.

Profiling sirtuin activity using Copper-free click chemistry.

The mammalian sirtuins are a group of posttranslational modification enzymes that remove acyl modifications from lysine residues in an NAD+-dependent manner. Although initially proposed as histone deacetylases (HDACs), they are now known to target other cellular enzymes and proteins as well. Sirtuin-catalyzed simple amide hydrolysis has profound biological consequences including suppression of gene expression, promotion of DNA damage repair, and regulation of glucose and lipid metabolism. Human sirtuins have been intensively pursued by both academia and industry as potential therapeutic targets for the treatment of diseases such as cancer and neurodegeneration. To gain a better understanding of their roles in various cellular events, innovative chemical probes are highly sought after. This current study focuses on the development of activity-based chemical probes (ABPs) for the profiling of sirtuin activity in biological samples. Cyclooctyne-containing and azido-containing probes were synthesized to enable the subsequent copper-free "click" conjugation to either a fluorophore or biotin. The two groups of structurally related ABPs demonstrated different labeling efficiency and selectivity: the cyclooctyne-containing probes failed to label recombinant sirtuins to any appreciable level, while the azido-containing ABPs showed good isoform selectivity. The azido-containing ABPs were further analyzed for their ability to label an individual sirtuin isoform in protein mixtures and cell lysates. These biocompatible ABPs allow the study of dynamic cellular protein activity change to become possible.

Developmental programming: intrauterine caloric restriction promotes upregulation of mitochondrial sirtuin with mild effects on oxidative parameters in the ovaries and testes of offspring.

According to the developmental origins of health and disease (DOHaD) hypothesis, changes in the maternal environment are known to reprogram the metabolic response of offspring. Known for its redox modulation, caloric restriction extends the lifespan of some species, which contributes to diminished cellular damage. Little is known about the effects of gestational caloric restriction, in terms of antioxidant parameters and molecular mechanisms of action, on the reproductive organs of offspring. This study assessed the effects of moderate (20%) caloric restriction on redox status parameters, molecular expression of sirtuin (SIRT) 1 and SIRT3 and histopathological markers in the ovaries and testes of adult rats that were subjected to gestational caloric restriction. Although enzyme activity was increased, ovaries from female pups contained high levels of oxidants, whereas testes from male pups had decreased antioxidant enzyme defences, as evidenced by diminished glyoxalase I activity and reduced glutathione content. Expression of SIRT3, a deacetylase enzyme related to cellular bioenergetics, was increased in both ovaries and testes. Previous studies have suggested that, in ovaries, diminished antioxidant metabolism can lead to premature ovarian failure. Unfortunately, there is little information regarding the redox profile in the testis. This study is the first to assess the redox network in both ovaries and testes, suggesting that, although intrauterine caloric restriction improves molecular mechanisms, it has a negative effect on the antioxidant network and redox status of reproductive organs of young adult rats.

Tissue distribution, subcellular localization, and enzymatic activity analysis of human SIRT5 isoforms.

SIRT5 is one of the seven mammalian sirtuins which are NAD+-dependent deacylases. In human beings, SIRT5 gene encodes for four SIRT5 protein isoforms, namely SIRT5iso1, SIRT5iso2, SIRT5iso3, and SIRT5iso4. Previous studies have focused mostly on SIRT5iso1. Characteristics regarding localization, activity and tissue distribution of the other three SIRT5 isoforms remain unclear. In the present study, we characterized these properties of these SIRT5 isoforms. We found that SIRT5iso1-3 were mitochondria-localized, while SIRT5iso4 localized mainly in cytoplasm. SIRT5iso2-4 had little deacylase activity comparing with SIRT5iso1. Although cDNAs of all SIRT5 isoforms were readily detected in multiply tissues according to EST database, proteins of SIRT5iso2-4 were seldom observed in human cell lines. Altogether, we dissected the four isoforms of human SIRT5 protein.

Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis.

Apoptosis and mitochondria dysfunction are key contributors to myocardial ischemia-reperfusion (MI-R) injury. SIRT4, a mitochondrial-localized sirtuin, controls cellular energy metabolism and stress response, and is abundantly present in the heart, however, its role in MI-R injury is not clear. In the current study, we demonstrate that SIRT4 is downregulated in cardiomyocytes both in vitro and in vivo models after MI-R. Functionally, SIRT4 overexpression decreases myocardial infarct size and serum creatine phosphokinase (CPK) level, and vice versa, SIRT4 depletion by siRNA increases myocardial infarct size and serum CPK level. Furthermore, we show that these protective roles of SIRT4 against MI-R injury are associated with preserved mitochondrial function and reduced myocardial apoptosis. Taken together, our findings indicate that SIRT4 ameliorates MI-R injury through regulating mitochondrial function and apoptosis, and suggest that manipulating SIRT4 may be of clinical benefit in MI-R injury.

Nutritional biomarkers: Current view and future perspectives.

There is a poor relationship between nutrient intake and existing nutritional biomarkers due to variety of factors affecting their sensitivity and specificity. To explore the impact of nutrients at molecular level and devising a sensitive biomarker, proteomics is a central technology with sirtuins as one of the most promising nutritional biomarker. Sirtuins (seven mammalian sirtuins reported so far) have been reported to perform protein deacetylases and ADP-ribosyltransferases activity. It is distributed in different cellular compartments thereby controlling several metabolic processes. Sirtuins are oxidized nicotinamide adenine dinucleotide dependent, which implicates a direct effect of the metabolic state of the cell on its activity. Calorie restriction upregulates the mammalian sirtuin protein levels in variety of tissues and organs where it acts upon both histone and nonhistone substrates. Sirtuin senses nutrient availability and impacts gluconeogenesis, glycolysis, and insulin sensitivity. It deacetylates and inhibits the nuclear receptor that activates fat synthesis and adipogenesis in the body, leading to fat loss and bringing favorable cellular and health changes. Sirtuins mediates intracellular response that promotes cell survival, DNA damage repair thereby increasing the cell longitivity. The activation of sirtuins brings a wide spectrum of other health benefits and its activity levels are indicative of nutritional status as well as disease progression in cancer, inflammation, obesity, cardiovascular diseases, and viral infections. There are several foods that activate sirtuin activity and offer significant health benefits by their consumption.

Genetically Encoded Fluorescent Probe for Detecting Sirtuins in Living Cells.

Sirtuins are NAD+ dependent protein deacetylases, which are involved in many biological processes. We now report a novel genetically encoded fluorescent probe (EGFP-K85AcK) that responds to sirtuins in living cells. The probe design exploits a lysyl residue in EGFP that is essential for chromophore maturation, and is also an efficient deacetylation substrate for sirtuins. Analysis of activity in Escherichia coli ΔcobB revealed that the probe can respond to various human sirtuins, including SIRT1, SIRT2, SIRT3 and SIRT5. We also directly monitored SIRT1 and SIRT2 activity in HEK293T cells with an mCherry fusion of EGFP-K85AcK, and showed that this approach can be extended to other fluorescent proteins. Finally, we demonstrate that this approach can be used to examine the activity of sirtuins toward additional lysyl posttranslational modifications, and show that sirtuins can act as erasers of HibK modified proteins.

Using mitochondrial sirtuins as drug targets: disease implications and available compounds.

Sirtuins are an evolutionary conserved family of NAD(+)-dependent protein lysine deacylases. Mammals have seven Sirtuin isoforms, Sirt1-7. They contribute to regulation of metabolism, stress responses, and aging processes, and are considered therapeutic targets for metabolic and aging-related diseases. While initial studies were focused on Sirt1 and 2, recent progress on the mitochondrial Sirtuins Sirt3, 4, and 5 has stimulated research and drug development for these isoforms. Here we review the roles of Sirtuins in regulating mitochondrial functions, with a focus on the mitochondrially located isoforms, and on their contributions to disease pathologies. We further summarize the compounds available for modulating the activity of these Sirtuins, again with a focus on mitochondrial isoforms, and we describe recent results important for the further improvement of compounds. This overview illustrates the potential of mitochondrial Sirtuins as drug targets and summarizes the status, progress, and challenges in developing small molecule compounds modulating their activity.

SIRT6 expression is associated with poor prognosis and chemosensitivity in patients with non-small cell lung cancer.

BACKGROUND: Despite advances in the development of various therapeutic agents, non-small cell lung cancer (NSCLC) is associated with a poor prognosis. To improve the prognosis of patients with NSCLC, new therapeutic targets for overcoming drug resistance are required. The process of autophagy is required to support the tumorigenesis and drug resistance of cancer cells. We investigated the clinical significance of SIRT6, a member of the NAD(+) -dependent deacetylase family, which regulates a variety of cancer-related processes, including autophagy. METHODS: Immunohistochemistry analysis of SIRT6 expression and localization in 98 NSCLC clinical specimens and in vitro analysis using SIRT6-knockout lung carcinoma cell lines were performed. RESULTS: Patients with high cytoplasmic expression and low nuclear expression of SIRT6 (n = 33) had more aggressive cancer, shorter overall survival, and shorter recurrence-free survival than did patients with different SIRT6 expression profiles (P < 0.05). In vitro analysis revealed that SIRT6 knockdown lung adenocarcinoma cell line improved paclitaxel sensitivity (P < 0.05) and reduced the expression levels of both nuclear factor kappaB and autophagy marker Beclin1. CONCLUSION: Our data demonstrated that SIRT6 expression in NSCLC could be a useful prognostic marker and that SIRT6 might represent a novel target gene for predicting sensitivity of chemotherapy in lung adenocarcinoma.

Comparative interactomes of SIRT6 and SIRT7: Implication of functional links to aging.

Sirtuins are NAD(+) -dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 that are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6- and SIRT7-interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming seven and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly, and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co-immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins. All MS data have been deposited in the ProteomeXchange with identifiers PXD000159 and PXD000850 (http://proteomecentral.proteomexchange.org/dataset/PXD000159, http://proteomecentral.proteomexchange.org/dataset/PXD000850).

Adipose tissue and liver expression of SIRT1, 3, and 6 increase after extensive weight loss in morbid obesity.

BACKGROUND & AIMS: Severe obesity is associated with a state of chronic inflammation. Sirtuins (SIRT) are a family of conserved enzymes which are able to affect many metabolic and inflammatory pathways thereby potentially improving health and increasing lifespan. METHODS: We investigated the effect of weight loss on subcutaneous adipose tissue and liver mRNA and immunohistochemical expression of SIRT1, SIRT3, and SIRT6. Twenty-nine severely obese patients undergoing laparoscopic adjustable gastric banding (LAGB) were studied. Tissue samples were collected before and 6months after LAGB surgery. Tissue mRNA expression levels of SIRT1, SIRT3, and SIRT6 were correlated with clinical, biochemical, and histological parameters. In vitro, we studied sirtuin expression in native and stimulated monocytes, adipocytes, and hepatocytes. RESULTS: SIRT1, SIRT3, and SIRT6 mRNA expression was higher in the subcutaneous adipose tissue than in the liver. Weight loss resulted in a significant induction of SIRT1, SIRT3, and SIRT6 expression in the subcutaneous adipose tissue. In the liver, a significant increase after weight loss was observed, particularly for SIRT3 and SIRT6 mRNA expression; immunohistochemically, SIRT1 and SIRT3 expression was upregulated. Endotoxin and tumor necrosis factor-alpha suppressed SIRT1, SIRT3, and SIRT6 expression in human monocytes. The same stimuli suppressed total sirtuin deacetylase activity again, mainly in monocytes and less in adipocytes and hepatocytes. CONCLUSIONS: The relative abundance of adipose tissue mRNA expression of certain sirtuins exceeds its expression in the liver. Extensive weight loss increases sirtuin expression significantly both in adipose tissue and liver, probably as a consequence of reduced inflammation.

Alternate day calorie restriction improves systemic inflammation in a mouse model of sepsis induced by cecal ligation and puncture.

BACKGROUND: Calorie restriction (CR) exerts cytoprotective effects by up-regulating survival factors, such as mammalian target of rapamycin (mTOR), sirtuin, and peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α). These survival factors have well-established roles in attenuating the inflammatory response. However, it is unclear whether CR affects sepsis-related inflammation. The purpose of this study was to determine whether CR affects sepsis-induced inflammation in a cecal ligation and puncture (CLP)-induced mouse model of sepsis. METHODS: Male C57BL/6N mice underwent alternate day calorie restriction or normal feeding for 8 d before CLP-induced sepsis. After induction of sepsis, liver and lung histopathology and serum levels of cytokines and survival factors were assessed. RESULTS: Serum cytokine and high mobility group box protein 1 (HMGB1) levels were lower in animals that underwent alternate day calorie restriction compared with normally-fed mice after CLP. Alternate day calorie restriction also increased levels of sirtuin, PGC-1α, and mTOR. While 80% of mice in the CLP group died within 48 h after undergoing CLP, 50% of mice died in the ACR + CLP group (P < 0.05). CONCLUSION: Alternate day calorie restriction decreased mortality in a mouse model of sepsis. In addition to attenuated organ injury, a significant reduction in cytokine and HMGB1 levels was observed. These findings suggest that alternative day calorie restriction may reduce excessive inflammation.

Histone deacetylase Rpd3 antagonizes Sir2-dependent silent chromatin propagation.

In the eukaryotic genome, transcriptionally silent chromatin tends to propagate along a chromosome and encroach upon adjacent active chromatin. The silencing machinery can be stopped by chromatin boundary elements. We performed a screen in Saccharomyces cerevisiae for proteins that may contribute to the establishment of a chromatin boundary. We found that disruption of histone deacetylase Rpd3p results in defective boundary activity, leading to a Sir-dependent local propagation of transcriptional repression. In rpd3 Delta cells, the amount of Sir2p that was normally found in the nucleolus decreased and the amount of Sir2p found at telomeres and at HM and its adjacent loci increased, leading to an extension of silent chromatin in those areas. In addition, Rpd3p interacted directly with chromatin at boundary regions to deacetylate histone H4 at lysine 5 and at lysine 12. Either the mutation of histone H4 at lysine 5 or a decrease in the histone acetyltransferase (HAT) activity of Esa1p abrogated the silencing phenotype associated with rpd3 mutation, suggesting a novel role for the H4 amino terminus in Rpd3p-mediated heterochromatin boundary regulation. Together, these data provide insight into the molecular mechanisms for the anti-silencing functions of Rpd3p during the formation of heterochromatin boundaries.

Developmentally regulated MAPK pathways modulate heterochromatin in Saccharomyces cerevisiae.

Variegated expression of genes contributes to phenotypic variation within populations of genetically identical cells. Such variation plays a role in development and host pathogen interaction and can be important in adaptation to harsh environments. The expression state of genes placed near telomeres shows a variegated pattern of inheritance due to heterochromatin formation, a phenomenon that is called telomere position effect (TPE). We show that in budding yeast, TPE is controlled by the a1/alpha2 developmental repressor, which dictates developmental decisions in response to environmental changes. Two a1/alpha2 repressed genes, STE5, a MAPK scaffold and HOG1, a stress-activated MAPK, are the targets of this heterochromatin regulation pathway. We provide new evidence that link MAPK signaling and heterochromatin formation in yeast. Our results show that the same components that regulate gene expression states in euchromatic regions regulate heterochromatic expression states and that stress can play a part in turning on or off genes placed in heterochromatic regions.

DeepSIRT: A deep neural network for identification of sirtuin targets and their subcellular localizations.

Sirtuins are a family of proteins that play a key role in regulating a wide range of cellular processes including DNA regulation, metabolism, aging/longevity, cell survival, apoptosis, and stress resistance. Sirtuins are protein deacetylases and include in the class III family of histone deacetylase enzymes (HDACs). The class III HDACs contains seven members of the sirtuin family from SIRT1 to SIRT7. The seven members of the sirtuin family have various substrates and are present in nearly all subcellular localizations including the nucleus, cytoplasm, and mitochondria. In this study, a deep neural network approach using one-dimensional Convolutional Neural Networks (CNN) was proposed to build a prediction model that can accurately identify the outcome of the sirtuin protein by targeting their subcellular localizations. Therefore, the function and localization of sirtuin targets were analyzed and annotated to compartmentalize into distinct subcellular localizations. We further reduced the sequence similarity between protein sequences and three feature extraction methods were applied in datasets. Finally, the proposed method has been tested and compared with various machine-learning algorithms. The proposed method is validated on two independent datasets and showed an average of up to 85.77 % sensitivity, 97.32 % specificity, and 0.82 MCC for seven members of the sirtuin family of proteins.

The Pleiotropic Function of Human Sirtuins as Modulators of Metabolic Pathways and Viral Infections.

Sirtuins (SIRTs) are nicotinamide adenine dinucleotide-dependent histone deacetylases that incorporate complex functions in the mechanisms of cell physiology. Mammals have seven distinct members of the SIRT family (SIRT1-7), which play an important role in a well-maintained network of metabolic pathways that control and adapt the cell to the environment, energy availability and cellular stress. Until recently, very few studies investigated the role of SIRTs in modulating viral infection and progeny. Recent studies have demonstrated that SIRT1 and SIRT2 are promising antiviral targets because of their specific connection to numerous metabolic and regulatory processes affected during infection. In the present review, we summarize some of the recent progress in SIRTs biochemistry and their emerging function as antiviral targets. We also discuss the potential of natural polyphenol-based SIRT modulators to control their functional roles in several diseases including viral infections.

A protein deacetylase SIRT1 is a negative regulator of metalloproteinase-9.

Inappropriate elevation of matrix metalloproteinase-9 (MMP9) is reported to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The object of this study was to identify the molecular mechanism underlying this increase of MMP9 expression, and here we show that oxidative stress-dependent reduction of a protein deacetylase, SIRT1, known as a putative antiaging enzyme, causes elevation of MMP9 expression. A sirtuin inhibitor, splitomycin, and SIRT1 knockdown by RNA interference led an increase in MMP9 expression in human monocytic U937 cells and in primary sputum macrophages, which was detected by RT-PCR, Western blot, activity assay, and zymography. In fact, the SIRT1 level was significantly decreased in peripheral lungs of patients with COPD, and this increase was inversely correlated with MMP9 expression and MMP9 promoter activation detected by a chromatin immunoprecipitation assay. H(2)O(2) reduced SIRT1 expression and activity in U937 cells; furthermore, cigarette smoke exposure also caused reduction of SIRT1 expression in lung tissue of A/J mice, with concomitant elevation of MMP9. Intranasal treatment of a selective and novel SIRT1 small molecule activator, SRT2172, blocked the increase of MMP9 expression in the lung as well as pulmonary neutrophilia and the reduction in exercise tolerance. Thus, SIRT1 is a negative regulator of MMP9 expression, and SIRT1 activation is implicated as a novel therapeutic approach to treating chronic inflammatory diseases, in which MMP9 is abundant.

Statin suppresses sirtuin 6 through miR-495, increasing FoxO1-dependent hepatic gluconeogenesis.

Rationale: Statin, the most widely used medication in lowering cholesterol, is also associated with increased risk of type 2 diabetes, but its molecular basis remains unclear. Methods: Mice were injected intraperitoneally with statins alone or in combination with sirtuin (Sirt) 6 activator, and blood glucose levels were measured. Liver tissues from patients with statin use were analyzed for the expression of Sirt6. Results: Statin treatment up-regulated the hepatic expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, which was prevented by Sirt6 overexpression. Mechanistically, statin directly repressed Sirt6 expression by induction of microRNA (miR)-495, a novel inhibitor of Sirt6. Pathway analysis for predicted target genes of miR-495 recognized forkhead box protein (Fox)O1 as a key downstream signaling of Sirt6. Statin treatment increased the acetylation and protein stability of FoxO1, which was suppressed by Sirt6 overexpression. Inhibiting miR-495 recovered Sirt6 levels, blocking the ability of statin to increase FoxO1 mediated gluconeogenesis, and thus confirming the role of the miR-495/Sirt6/FoxO1 pathway in controlling gluconeogenesis. Moreover, the Sirt6 activator MDL801 prevented gluconeogenesis and hyperglycemia induced by statin in mice. Equally noteworthy was that human liver tissues obtained from statin users showed a significant decrease in Sirt6 protein levels compared to those of non-users. Conclusion: Statin induces miR-495 to suppress Sirt6 expression, which leads to enhancement of FoxO1-mediated hepatic gluconeogenesis. Thus, Sirt6 activation may offer a promising strategy for preventing statin-induced hyperglycemia.

Combating cellular senescence by sirtuins: Implications for atherosclerosis.

Cellular senescence is the permanent cell cycle arrest induced either by chronological ageing or extrinsic stimuli. Recent researches have identified cellular senescence as an important mechanism for atherosclerosis, which is the essential pathophysiological contributor to cardiovascular diseases (CVDs). The sirtuins are a family of cellular deacetylases with fundamental abilities to regulate cellular metabolism and a variety of physiological activities. Previous studies have revealed the anti-ageing functions of sirtuins as the longevity-associated proteins. These advances indicate the potential beneficial functions of sirtuins in atherosclerosis by affecting cellular senescence. Herein, we review the recent findings about sirtuins in regulating atherosclerotic cellular senescence, and discuss the possibility of activating sirtuins as a therapeutic strategy for combating atherosclerosis.

High-throughput assays for sirtuin enzymes: a microfluidic mobility shift assay and a bioluminescence assay.

Silent information regulator or sirtuin (SIRT) enzymes are beta-nicotinamide adenine dinucleotide (oxidized) (NAD(+))-dependent class III histone deacetylases. In this paper, two distinct assays to measure SIRT1 activity are described: a microfluidic mobility shift assay utilizing a fluorophore-labeled peptide substrate and a bioluminescence assay based upon quantitation of remaining NAD(+). The mobility shift assay involves the electrophoretic separation of an N-acetyl-lysine-containing peptide substrate from deacetylated product which bears an additional positive charge. Interference from fluorescent compounds is minimized during screening by direct visualization of separated fluorophore-labeled substrate and product. A preferred peptide substrate for SIRT1 was identified using this assay. The NAD(+) bioluminescence assay couples NAD(+) consumption to the bacterial luciferase-catalyzed oxidation of decanal. This assay does not require synthesis of a labeled peptide and is applicable to sirtuins of any specificity with respect to peptide substrate. The stoichiometry between NAD(+) consumption and peptide deacetylation was shown to be 1:1 by the NAD(+) bioluminescence assay. Kinetic parameters of peptide and NAD(+) cosubstrates and IC(50) values of standard reference inhibitors determined in either assay were similar. With robust Z' values (0.7), both assays are amenable to high-throughput screening.