Genetic Analysis of Sirtuin Deacetylases in Hyphal Growth of Candida albicans.

Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell's metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here, we studied the roles of three sirtuin deacetylases-Sir2, Hst1, and Hst2-in the hyphal growth of C. albicans We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hypha formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2 Moreover, the expression of hypha-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to the wild type. This regulation of hypha formation was likely dependent on the deacetylase activity of Sir2, as a similar defect in hypha formation was observed when an asparagine known to be required for deacetylation was mutated. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and nonredundant role in hyphal growth of C. albicansIMPORTANCECandida albicans is one of the most common causes of hospital-acquired systemic fungal infections in the United States. It can switch between ovoid yeast and elongated hyphal growth forms in response to environmental cues. This morphological transition is essential for its survival in the host. Thus, identifying regulators involved in this process can lead to new therapies. In this study, we examined the contribution of three regulators called sirtuins (Sir2, Hst1, and Hst2) to the yeast-to-hypha transition of C. albicans We found that loss of Sir2 but not Hst1 or Hst2 hampered hypha formation. Moreover, the defect was caused by the loss of the catalytic activity of Sir2. Our study may lay the groundwork for discovering novel targets for antifungal therapies.

Review: The plant sirtuins.

The sirtuin family of intracellular enzymes are able to catalyze a unique ß-nicotinamide adenine dinucleotide (ß-NAD+)-dependent Nε-acyl-lysine deacylation reaction on histone and non-histone protein substrates. Since 2000, the sirtuin family members have been identified in both prokaryotes and eukaryotes; tremendous accomplishments have also been achieved on the mechanistic and functional (pharmacological) understanding of the sirtuin-catalyzed deacylation reaction. Among the eukaryotic organisms, past research has been focused more on the yeast and mammalian sirtuins than on the plant sirtuins, however, the very presence of sirtuins in various plant species and the functional studies on plant sirtuins published thus far attest to the importance of this particular subfamily of eukaryotic sirtuins in regulating the growth and development of plants and their responses to biotic and abiotic stresses. In this review, an integrated and updated account will be presented on the biochemical, cellular, and functional profiles of all the plant sirtuins identified thus far. It is hoped that this article will also set a stage for expanded efforts in the identification, characterization, and functional interrogation of plant sirtuins; and the development and exploration of their chemical modulators (activators and inhibitors) in plant research and agriculture.

The Role of Sirtuins in Antioxidant and Redox Signaling.

SIGNIFICANCE: Antioxidant and redox signaling (ARS) events are regulated by critical molecules that modulate antioxidants, reactive oxygen species (ROS) or reactive nitrogen species (RNS), and/or oxidative stress within the cell. Imbalances in these molecules can disturb cellular functions to become pathogenic. Sirtuins serve as important regulators of ARS in cells. Recent Advances: Sirtuins (SIRTs 1-7) are a family of nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases with the ability to deacetylate histone and nonhistone targets. Recent studies show that sirtuins modulate the regulation of a variety of cellular processes associated with ARS. SIRT1, SIRT3, and SIRT5 protect the cell from ROS, and SIRT2, SIRT6, and SIRT7 modulate key oxidative stress genes and mechanisms. Interestingly, SIRT4 has been shown to induce ROS production and has antioxidative roles as well. CRITICAL ISSUES: A complete understanding of the roles of sirtuins in redox homeostasis of the cell is very important to understand the normal functioning as well as pathological manifestations. In this review, we have provided a critical discussion on the role of sirtuins in the regulation of ARS. We have also discussed mechanistic interactions among different sirtuins. Indeed, a complete understanding of sirtuin biology could be critical at multiple fronts. FUTURE DIRECTIONS: Sirtuins are emerging to be important in normal mammalian physiology and in a variety of oxidative stress-mediated pathological situations. Studies are needed to dissect the mechanisms of sirtuins in maintaining redox homeostasis. Efforts are also required to assess the targetability of sirtuins in the management of redox-regulated diseases. Antioxid. Redox Signal. 28, 643-661.

Forward and reverse genetics approaches to uncover metabolic aging pathways in Caenorhabditis elegans.

The biological mechanisms of aging have been studied in depth and prominent findings in this field promote the development of new therapies for age-associated disorders. Various model organisms are used for research on aging; among these, the nematode Caenorhabditis elegans has been widely used and has provided valuable knowledge in determining the regulatory mechanisms driving the aging process. Many genes involved in lifespan regulation are associated with metabolic pathways and are influenced by genetic and environmental factors. In line with this, C. elegans provides a promising platform to study such gene by environment interactions, in either a reverse or forward genetics approach. In this review, we discuss longevity mechanisms related to metabolic networks that have been discovered in C. elegans. We also highlight the use of wild populations to study the complex genetic basis of natural variation for quantitative traits that mediate longevity.

Mitochondrial Sirtuin Network Reveals Dynamic SIRT3-Dependent Deacetylation in Response to Membrane Depolarization.

Mitochondrial sirtuins, SIRT3-5, are NAD+-dependent deacylases and ADP-ribosyltransferases that are critical for stress responses. However, a comprehensive understanding of sirtuin targets, regulation of sirtuin activity, and the relationships between sirtuins remains a key challenge in mitochondrial physiology. Here, we employ systematic interaction proteomics to elucidate the mitochondrial sirtuin protein interaction landscape. This work reveals sirtuin interactions with numerous functional modules within mitochondria, identifies candidate sirtuin substrates, and uncovers a fundamental role for sequestration of SIRT3 by ATP synthase in mitochondrial homeostasis. In healthy mitochondria, a pool of SIRT3 binds ATP synthase, but upon matrix pH reduction with concomitant loss of mitochondrial membrane potential, SIRT3 dissociates. This release correlates with rapid deacetylation of matrix proteins, and SIRT3 is required for recovery of membrane potential. In vitro reconstitution experiments, as well as analysis of CRISPR/Cas9-engineered cells, indicate that pH-dependent SIRT3 release requires H135 in the ATP5O subunit of ATP synthase. Our SIRT3-5 interaction network provides a framework for discovering novel biological functions regulated by mitochondrial sirtuins.

Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System.

Sirtuin proteins have a variety of intracellular targets, thereby regulating multiple biological pathways including neurodegeneration. However, relatively little is currently known about the role or expression of the 7 mammalian sirtuins in the central nervous system. Western blotting, PCR and ELISA are the main techniques currently used to measure sirtuin levels. To achieve sufficient sensitivity and selectivity in a multiplex-format, a targeted mass spectrometric assay was developed and validated for the quantification of all seven mammalian sirtuins (SIRT1-7). Quantification of all peptides was by multiple reaction monitoring (MRM) using three mass transitions per protein-specific peptide, two specific peptides for each sirtuin and a stable isotope labelled internal standard. The assay was applied to a variety of samples including cultured brain cells, mammalian brain tissue, CSF and plasma. All sirtuin peptides were detected in the human brain, with SIRT2 being the most abundant. Sirtuins were also detected in human CSF and plasma, and guinea pig and mouse tissues. In conclusion, we have successfully applied MRM mass spectrometry for the detection and quantification of sirtuin proteins in the central nervous system, paving the way for more quantitative and functional studies.

An improved fluorogenic assay for SIRT1, SIRT2, and SIRT3.

Sirtuins are NAD-dependent lysine deacylases that play critical roles in cellular regulation and are implicated in human diseases. Modulators of sirtuins are needed as tools for investigating their biological functions and possible therapeutic applications. However, the discovery of sirtuin modulators is hampered by the lack of efficient sirtuin assays. Here we report an improved fluorogenic assay for SIRT1, SIRT2, and SIRT3 using a new substrate, a myristoyl peptide with a C-terminal aminocoumarin. The new assay has several advantages, including significantly lower substrate concentration needed, increased signal-to-background ratio, and improved Z'-factor. The novel assay thus will expedite high-throughput screening of SIRT1, SIRT2, and SIRT3 modulators.

Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens.

Sirtuins are an ancient family of NAD(+)-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono-ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species.

Sirtuins in vascular diseases: Emerging roles and therapeutic potential.

Silent information regulator-2 (Sir-2) proteins, or sirtuins, are a highly conserved protein family of histone deacetylases that promote longevity by mediating many of the beneficial effects of calorie restriction which extends life span and reduces the incidence of cancer, cardiovascular disease (CVD), and diabetes. Here, we review the role of sirtuins (SIRT1-7) in vascular homeostasis and diseases by providing an update on the latest knowledge about their roles in endothelial damage and vascular repair mechanisms. Among all sirtuins, in the light of the numerous functions reported on SIRT1 in the vascular system, herein we discuss its roles not only in the control of endothelial cells (EC) functionality but also in other cell types beyond EC, including endothelial progenitor cells (EPC), smooth muscle cells (SMC), and immune cells. Furthermore, we also provide an update on the growing field of compounds under clinical evaluation for the modulation of SIRT1 which, at the state of the art, represents the most promising target for the development of novel drugs against CVD, especially when concomitant with type 2 diabetes.

Sirtuin function in aging heart and vessels.

Age is the most important risk factor for metabolic alterations and cardiovascular accidents. Although class III histone deacetylases, alias Sirtuins, have been appealed as "the fountain of youth" their role in longevity control and prevention of aging-associated disease is still under debate. Indeed, several lines of evidence indicate that sirtuin activity is strictly linked to metabolism and dependent on NAD(+) synthesis both often altered as aging progresses. During aging the cardiovascular system is attacked by a variety of environmental stresses, including those determined by high blood glucose and lipid levels, or by the presence of oxidized lipoproteins which, among others, determine important oxidative stress signals. In such a milieu, heart and vessels develop a functional impairment leading to atherosclerosis, ischemia, heart insufficiency and failure. Sirtuins, which are believed to have a positive impact on cardiovascular physiology and physiopathology, are distributed in different subcellular compartments including the nucleus, the cytoplasm and the mitochondria, where they regulate expression and function of a large variety of target genes and proteins. Remarkably, experimental animal models indicate resveratrol, the first natural compound described to positively regulate the activity of sirtuins, as able to protect the endothelium and the heart exposed to a variety of stress agents. This review will focus on the regulation and function of mammalian sirtuins with special attention paid to their role as cardiovascular "defenders" giving indication of their targets of potential relevance for the development of future therapeutics. This article is part of a Special Issue entitled CV Aging.

The sirtuin class of histone deacetylases: regulation and roles in lipid metabolism.

After the completion of the human genome sequence and that from many other organisms, last decade has witnessed a spectacular gain of knowledge on gene functions. These studies provided new insights on the roles of genes in physiology and disease. Nonetheless, the availability of genetically modified models and of "omics" technologies such as next generation sequencing unveiled clear evidences on epigenetic regulation of many cellular functions. At this regard, sirtuins, belonging to class III histone deacetylase family, have emerged as regulators of metabolism as well as other cellular processes and seem ideally suited as targets of future therapeutical interventions. This review deals on general aspects of the biology of sirtuins and focuses on their relevance in lipid metabolism in different tissues, pointing to their exploitation as potential pharmacological targets of compounds that could be used as new therapeutic alternatives in several disorders ranging from type 2 diabetes and obesity to age-related cardiovascular and neurodegenerative diseases.

Fungus-specific sirtuin HstD coordinates secondary metabolism and development through control of LaeA.

The sirtuins are members of the NAD(+)-dependent histone deacetylase family that contribute to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungus-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungus-specific sirtuin HstD/Aspergillus oryzae Hst4 (AoHst4), which is a homolog of Hst4 in A. oryzae yeast. The deletion of all histone deacetylases in A. oryzae demonstrated that the fungus-specific sirtuin HstD/AoHst4 is required for the coordination of fungal development and secondary metabolite production. We also show that the expression of the laeA gene, which is the most studied fungus-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in a ΔhstD strain. Genetic interaction analysis of hstD/Aohst4 and laeA clearly indicated that HstD/AoHst4 works upstream of LaeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungus specific but conserved in the vast family of filamentous fungi. Thus, we conclude that the fungus-specific sirtuin HstD/AoHst4 coordinates fungal development and secondary metabolism via the regulation of LaeA in filamentous fungi.

Altered sirtuin deacetylase gene expression in patients with a mood disorder.

Sirtuins are a family of NAD+-dependent enzymes that regulate cellular functions through deacetylation of various proteins. Although recent reports have suggested an important role of deacetylases (i.e., histone deacetylases) in mood disorders and antidepressant action, the involvement of sirtuins in the pathophysiology of mood disorders is largely unknown. In this study, we aimed to determine whether there are alterations in sirtuin mRNA expression in peripheral white blood cells of patients with a mood disorder. Also, to examine whether the altered sirtuin mRNA expression is state- or trait-dependent, mood disorder patients who were in a remissive state were assessed. We used quantitative real-time polymerase chain reaction to measure the mRNA levels of seven sirtuin isoforms (SIRT1-7) in peripheral white blood cells of patients with major depressive disorder (MDD) or bipolar disorder (BPD) during depressive and remissive states and in normal healthy subjects. The SIRT1, 2 and 6 mRNA levels in MDD and BPD patients decreased significantly in those who were in a depressive state compared to healthy controls, whereas the expression of those mRNAs in both MDD and BPD of patients in a remissive state were comparable to those in healthy controls. Thus, our data suggest that altered SIRT1, 2 and 6 expression is state-dependent and might be associated with the pathogenesis and/or pathophysiology of mood disorders.

Protein deacetylation by sirtuins: delineating a post-translational regulatory program responsive to nutrient and redox stressors.

Lysine acetylation/deacetylation is increasingly being recognized as common post-translational modification that appears to be broadly operational throughout the cell. The functional roles of these modifications, outside of the nucleus, have not been extensively studied. Moreover, as acetyl-CoA donates the acetyl group for acetylation, nutrient availability and energetic status may be pivotal in this modification. Similarly, nutrient limitation is associated with the deacetylation reaction. This modification is orchestrated by a novel family of sirtuin deacetylases that function in a nutrient and redox dependent manner and targets non-histone protein deacetylation. In compartment-specific locations, candidate target proteins undergoing lysine-residue deacetylation are being identified. Through these investigations, the functional role of this post-translational modification is being delineated. We review the sirtuin family proteins, discuss their functional effects on target proteins, and postulate on potential biological programs and disease processes that may be modified by sirtuin-mediated deacetylation of target proteins.

Entamoeba histolytica sirtuin EhSir2a deacetylates tubulin and regulates the number of microtubular assemblies during the cell cycle.

We have discovered four sirtuin genes in Entamoeba histolytica, two of which are similar to eukaryotic sirtuins and two to bacterial and archaeal sirtuins. The eukaryotic sirtuin homologue, EhSir2a, showed NAD(+)-dependent deacetylase activity and was sensitive to class III HDAC inhibitors. Localization of EhSir2a at different cellular sites suggested that this deacetylase could have multiple targets. Using an E. histolytica cDNA library in the yeast two-hybrid genetic screen, we identified several proteins that bound to EhSir2a. These proteins included Eh alpha-tubulin, whose interaction with EhSir2a was validated in E. histolytica. We have shown that EhSir2a deacetylated tubulin and localized with microtubules in E. histolytica. Increased expression levels of EhSir2a in stable transformants led to reduced number of microtubular assemblies in serum synchronized cells. This effect was abrogated by mutations in the deacetylase domain of EhSir2a, showing that EhSir2a deacetylase activity affected the stability and number of microtubular assemblies during the cell cycle of E. histolytica. Our results suggest that epigenetic modification of tubulin by EhSir2a is one of the mechanisms that regulates microtubular assembly in E. histolytica.

Sirtuin/Sir2 phylogeny, evolutionary considerations and structural conservation.

The sirtuins are a protein family named after the first identified member, S. cerevisiae Sir2p. Sirtuins are protein deacetylases whose activity is dependent on NAD(+) as a cosubstrate. They are structurally defined by two central domains that together form a highly conserved catalytic center, which catalyzes the transfer of an acetyl moiety from acetyllysine to NAD(+), yielding nicotinamide, the unique metabolite O-acetyl-ADP-ribose and deacetylated lysine. One or more sirtuins are present in virtually all species from bacteria to mammals. Here we describe a phylogenetic analysis of sirtuins. Based on their phylogenetic relationship, sirtuins can be grouped into over a dozen classes and subclasses. Humans, like most vertebrates, have seven sirtuins: SIRT1-SIRT7. These function in diverse cellular pathways, regulating transcriptional repression, aging, metabolism, DNA damage responses and apoptosis. We show that these seven sirtuins arose early during animal evolution. Conserved residues cluster around the catalytic center of known sirtuin family members.

The conserved role of sirtuins in chromatin regulation.

The members of the Sir2 family, or Sirtuins, have garnered considerable attention because of their key roles as metabolic sensors and mediators of cell survival under stress. Sirtuins may play roles in myriad human pathologies such as cancer, neurological diseases, malaria, leishmaniasis and hormone-related disorders. They are present from prokaryotes to humans and show a high degree of functional diversification that has led to two different enzymatic activities, a wide range of substrates and a highly diversified pattern of cellular localization. Throughout chromatin evolution, Sirtuins have maintained an intimate functional relationship in regulating its structure and function via their targeting of histones, particularly H4K16Ac, as well as other non-histone chromatin proteins. This link permitted fast communication from metabolic fluctuations to chromatin allowing efficient adaptation to environmental stimuli. Therefore, understanding the common path of Sirtuins and chromatin development over the course of evolution might be important for understanding not only the remarkable diversity of functions of these proteins in mammals, but also the path followed by chromatin evolution. Herein is provided an overview of current knowledge of Sirtuin function, from bacteria to humans, including a discussion on its implications for chromatin dynamics, organization and integrity.

Molecular cloning and characterization of porcine sirtuin genes.

The sirtuin family of class III histone deacetylases (HDACs) is named after their homology to yeast silent information regulator 2 (SIR2). SIR2 and its mammalian derivatives (SIRT1-7) play a central role in gene silencing, cell cycle, aging and metabolism. Here we reported cDNA cloning, chromosome mapping,expression and evolutional analysis of sirtuin genes in Sus scrofa (Tongcheng pig). Sequence analysis showed that porcine sirtuin genes contain 7 members designated SIRT1-7. Tissue distribution analysis indicated porcine sirtuin genes ubiquitously expressed but with the highest abundance in brain, spinal cord and genital tissue. In silico and radiation hybrid mapping analysis mapped porcine SIRT1-7 to the chromosomes 14q23,6q11-12, 2q29, 14q19, 7p12, 2q11, and 12p15, respectively. We also isolated and characterized genomic sequence of porcine SIRT1, which spaned a region of 31,834 bp comprising 9 exons ranging in size from 80 bpto 2121 bp. The 5' flanking genomic region preceding an open reading frame of SIRT1 has a TATA box, a small300 bp CpG island and several putative Sp1 and p53 transcription factor binding sites. Moreover, we isolated two novel splicing SIRT6 variants with 346 bp (variant 2) in-frame deletions from lung and 327 bp(variant 3) in-frame deletions from spleen and brain. This is the first systematic report of molecular cloning and characterization of sirtuin genes in pigs, which will be helpful for a better understanding of the physiological role of sirtuin proteins in pigs.

Fluorescence in situ hybridization and chromosomal organization of the sirtuin 4 gene (Sirt4) in the mouse.

The sirtuins (SIRT1-7), also being referred to as class III HDACs, exert NAD-dependent deacetylase and/or ADP-ribosyltransferase activities in various cellular compartments including the cell nucleus, the cytoplasm and the mitochondria. The sirtuins play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. SIRT4 is a mitochondrial protein that lacks deacetylase activities but efficiently works as an ADP-ribosyltransferase. We have isolated and characterized the murine Sirt4 genomic sequence, which spans a region of 12kb and which has one single genomic locus. Determination of the exon-intron splice junctions established that SIRT4 is encoded by 6 exons. The 1648bp murine Sirt4 transcript encodes a 418 aa protein with a predictive molecular weight of 47.3kDa. Fluorescence in situ hybridization analysis identified a single genomic locus for murine Sirt4 gene on chromosome 5F and is neighbored by the PLA2G1B and PXN genes.

Modulation of sirtuins: new targets for antiageing.

Aging is characterized by a progressive deterioration of physiological functions and metabolic processes. Healthy aging remains one of the ideals of modern society. In aging and in diseases associated with the elderly, such as Alzheimer's or Parkinson's, the loss of cells in vital structures or organs may be related to several factors, among which the production of reactive oxygen species (ROS) by mitochondria is a common denominator, one that leads to DNA damage, apoptosis and death. Although a diet rich in antioxidants seems to offer hope in delaying the onset of unhealthy disorders that accompany aging, no clinical treatment as such has yet been developed and anti-aging drugs are still unavailable. It is well established that reducing food intake (caloric restriction) extends the life-span in a wide range of species. The protein implicated in this protective process is the silent information regulator 2 (SIR2, SIRT1 in mammals), an enzyme that belongs to a nicotinamide adenine dinucleotide (NAD)+-dependent protein deacetylases. SIRs regulate gene silencing, DNA repair, rDNA recombination, and ageing, apart from regulating programmed cell death. In this context, increasing SIRT1 has been found to protect cells against amyloid-beta-induced ROS production and DNA damage, thereby reducing apoptotic death in vitro. Moreover, it has been demonstrated that Alzheimer's and Huntington's disease neurons are rescued by the over-expression of SIRT1, induced by either caloric restriction or administration of resveratrol, a potential activator of this enzyme. The therapeutic use of resveratrol (a polyphenol present in red wines) and other related compounds, which utilize SIRT1 pathway modulators, in treating aging-related brain disorders will be discussed in this review. Provided herein are novel new compound related with resveratrol or sirtinol that are able to modulate sirtuin activity that will be tested to treat and/or prevent a wide variety of diseases including, disorders related to aging or neurodegenerative diseases.