Identification of phytoestrogens as sirtuin inhibitor against breast cancer: Multitargeted approach.

Despite progress in diagnosis and treatment strategies, breast cancer remains a primary risk to female health as indicated by second most cancer-deaths globally caused by this cancer. High risk mutation is linked to prognosis of breast cancer. Due to high resistance of breast cancer against current therapies, there is necessity of novel treatment strategies. Sirtuins are signaling proteins belonging to histone deacetylase class III family, known to control several cellular processes. Therefore, targeting sirtuins could be one of the approaches to treat breast cancer. Several plants synthesize phytoestrogens which exhibit structural and physiological similarities to estrogens and have been recognized to possess anticancer activity. In our study, we investigated several phytoestrogens for sirtuin inhibition by conducting molecular docking studies, and in-vitro studies against breast cancer cell lines. In molecular docking studies, we identified coumestrol possessing high binding energy with sirtuin proteins 1-3 as compared to other phytoestrogens. The molecular dynamic studies showed stable interaction of ligand and protein with higher affinity at sirtuin proteins 1-3 binding sites. In cell proliferation assay and colony formation assay using breast cancer cell lines (MCF-7 and MDAMB-231) coumestrol caused significant reduction in cell proliferation and number of colonies formed. Further, the flow cytometric analysis showed that coumestrol induces intracellular reactive oxygen species and the western blot analysis revealed reduction in the level of SIRT-1 expression in breast cancer cell lines. In conclusion, in-silico data and in-vitro studies suggest that the phytoestrogen coumestrol has sirtuin inhibitory activity against breast cancer.

Structural insights into autoinhibition and activation of defense-associated sirtuin protein.

Bacterial defense-associated sirtuin 2 (DSR2) proteins harbor an N-terminal sirtuin (SIR2) domain degrading NAD+. DSR2 from Bacillus subtilis 29R is autoinhibited and unable to hydrolyze NAD+ in the absence of phage infection. A tail tube protein (TTP) of phage SPR activates the DSR2 while a DSR2-inhibiting protein of phage SPbeta, known as DSAD1 (DSR anti-defense 1), inactivates the DSR2. Although DSR2 structures in complexed with TTP and DSAD1, respectively, have been reported recently, the autoinhibition and activation mechanisms remain incompletely understood. Here, we present cryo-electron microscopy structures of the DSR2-NAD+ complex in autoinhibited state and the in vitro assembled DSR2-TFD (TTP tube-forming domain) complex in activated state. The DSR2-NAD+ complex reveals that the autoinhibited DSR2 assembles into an inactive tetramer, binding NAD+ through a distinct pocket situated outside active site. Binding of TFD into cavities within the sensor domains of DSR2 triggers a conformational change in SIR2 regions, activating its NADase activity, whereas the TTP ß-sandwich domain (BSD) is flexible and does not contribute to the activation process. The activated form of DSR2 exists as tetramers and dimers, with the tetramers exhibiting more NADase activity. Overall, our results extend the current understanding of autoinhibition and activation of DSR2 immune proteins.

Effect of quercetin on the protein-substrate interactions in SIRT6: Insight from MD simulations.

SIRT6 is of interest for its promising effect in the treatment of aging-related diseases. Studies have shown quercetin (QUE) and its derivatives have varying degrees of effect on the catalytic effect of SIRT6. In the research, the effect of QUE on the protein-substrate interaction in the SIRT6-mediated mono-ADP ribosylation system was investigated by conventional molecular dynamics (MD) simulations combined with MM/PBSA binding free energy calculations. The results show that QUE can bind stably to SIRT6 with the binding energy of -22.8 kcal/mol and further affect the atomic interaction between SIRT6 and NAD+ (or H3K9), resulting in an increased affinity between SIRT6-NAD+ and decreased SIRT6-H3K9 binding capacity. At the same time, the binding of QUE can also alter some structural characteristics of the protein, with large shifts occurring in the residue regions involving the N-terminal (residues 1-27), Rossmann fold regions (residues 55-92), and ZBD (residues 164-179). Thus, QUE shows great potential as a scaffold for the design of novel potent SIRT6 modulators.

Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide-Activated Sirtuin 7.

The sirtuins are NAD+ -dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18.

Zymograph profiling reveals a divergent evolution of sirtuin that may originate from class III enzymes.

Sirtuins are a group of NAD+-dependent deacylases that conserved in three domains of life and comprehensively involved in the regulation of gene transcription, chromosome segregation, RNA splicing, apoptosis, and aging. Previous studies in mammalian cells have revealed that sirtuins not only exist as multiple copies, but also show distinct deacylase activities in addition to deacetylation. However, the understanding of sirtuin zymographs in other organisms with respect to molecular evolution remains at an early stage. Here, we systematically analyze the sirtuin activities in representative species from archaea, bacteria, and eukaryotes, using both the HPLC assay and a 7-amino-4-methylcoumarin-based fluorogenic method. Global profiling suggests that the deacylase activities of sirtuins could be divided into three categories and reveals undifferentiated zymographs of class III sirtuins, especially for those from bacteria and archaea. Nevertheless, initial differentiation of enzymatic activity was also observed for the class III sirtuins at both paralog and ortholog levels. Further phylogenetic analyses support a divergent evolution of sirtuin that may originate from class III sirtuins. Together, this work demonstrates a comprehensive panorama of sirtuin zymographs and provides new insights into the cellular specific regulation and molecular evolution of sirtuins.

Sirtuin6 and Lipoxin A4 levels are decreased in severe periodontitis.

OBJECTIVE: Sirtuin6 plays an important role in the regulation of inflammation, homeostasis, and apoptosis, and it has anti-inflammatory effects on several diseases. Lipoxin A4 is a pro-resolving lipid mediator of inflammation and inhibits hypoxia-induced apoptosis and oxidative stress. Considering that Lipoxin A4 and Sirtuin6 have protective effects on inflammatory diseases, the aim of this study is to determine the possible roles of these molecules on periodontitis inflammation in saliva and serum and to reveal the relationship of these data with clinical periodontal parameters. MATERIAL AND METHODS: A total of 20 stage III/grade B periodontitis and 20 periodontally healthy subjects were included in this cross-sectional study (all never smokers and systemically healthy). Clinical periodontal parameters (plaque index, probing pocket depth, bleeding on probing, clinical attachment loss) were recorded. Saliva and serum levels of Sirtuin6 and Lipoxin A4 were analyzed by enzyme-linked immunosorbent assay. RESULTS: Serum Sirtuin6 and saliva Lipoxin A4 levels were significantly lower in the periodontitis group than the control group (respectively, p = 0.0098, p = 0.0008). There were negative correlations between all periodontal clinical parameters and saliva Lipoxin A4 level (p < 0.05) and between probing pocket depth, clinical attachment loss, and serum and saliva Sirtuin6 levels (respectively, r = - 0.465 and r = - 0.473, p < 0.05). CONCLUSIONS: Decreased levels of serum Sirtuin6 and saliva Lipoxin A4 in periodontitis patients and their correlation with clinical periodontal parameters suggest that serum Sirtuin6 and saliva Lipoxin A4 may be related with periodontal inflammation. CLINICAL RELEVANCE: Scientific rationale for the study: Sirtuin6 is one of seven members of the family of NAD + dependent protein that played an important role in the regulation of inflammation, energy metabolism, homeostasis, and apoptosis. Sirtuin6 is associated with the pathogenesis of several diseases. Lipoxin A4 is a lipid mediator that inhibits hypoxia-induced apoptosis and oxidative stress, and it has an active role in the resolution of periodontal inflammation. No studies that investigated the potential role Sirtuin6 and its relationship with inflammation resolution and apoptosis mechanisms in severe periodontitis patients. PRINCIPAL FINDINGS: the serum Sirtuin6 and saliva Lipoxin A4 levels were significantly lower and negatively correlated with clinical periodontal parameters in the patients with periodontitis than the healthy controls. PRACTICAL IMPLICATIONS: this study shows that serum Sirtuin6 and saliva Lipoxin A4 may be candidate biomarkers related with periodontal inflammation and estimating to periodontal status. CLINICAL TRIAL REGISTRATION: NCT05417061.

The role of structural biology in the design of sirtuin activators.

Sirtuins are NAD+-dependent protein lysine deacylases and mono-ADP-ribosylases whose activity regulates different pathways, including DNA damage repair, cell survival and metabolism, reactive oxygen species (ROS) detoxification, inflammation, cardiac function, and neuronal signaling. Considering the beneficial effects of specific sirtuin isoforms on health and lifespan, the past two decades have seen a mounting interest in the development of sirtuin activators. The availability of enzyme-activator co-crystal structures has proven significant throughout the years for elucidating the mechanisms of action of activators and designing more potent and selective molecules. In this review, we highlight the most interesting examples of sirtuin activators and provide comprehensive coverage of the role that structural biology played in their discovery and characterization.

LDHA Desuccinylase Sirtuin 5 as A Novel Cancer Metastatic Stimulator in Aggressive Prostate Cancer.

Prostate cancer (PCa) is the most commonly diagnosed genital cancer in men worldwide. Around 80% of the patients who developed advanced PCa suffered from bone metastasis, with a sharp drop in the survival rate. Despite great efforts, the detailed mechanisms underlying castration-resistant PCa (CRPC) remain unclear. Sirtuin 5 (SIRT5), an NAD+-dependent desuccinylase, is hypothesized to be a key regulator of various cancers. However, compared to other SIRTs, the role of SIRT5 in cancer has not been extensively studied. Here, we revealed significantly decreased SIRT5 levels in aggressive PCa cells relative to the PCa stages. The correlation between the decrease in the SIRT5 level and the patient's reduced survival rate was also confirmed. Using quantitative global succinylome analysis, we characterized a significant increase in the succinylation at lysine 118 (K118su) of lactate dehydrogenase A (LDHA), which plays a role in increasing LDH activity. As a substrate of SIRT5, LDHA-K118su significantly increased the migration and invasion of PCa cells and LDH activity in PCa patients. This study reveals the reduction of SIRT5 protein expression and LDHA-K118su as a novel mechanism involved in PCa progression, which could serve as a new target to prevent CPRC progression for PCa treatment.

Arabidopsis thaliana sirtuins control proliferation and glutamate dehydrogenase activity.

Sirtuins are part of a gene family of NAD-dependent deacylases that act on histone and non-histone proteins and control a variety of activities in all living organisms. Their roles are mainly related to energy metabolism and include lifetime regulation, DNA repair, stress resistance, and proliferation. A large amount of knowledge concerning animal sirtuins is available, but data about their plant counterparts are scarce. Plants possess few sirtuins that have, like in animals, a recognized role in stress defense and metabolism regulation. However, engagement in proliferation control, which has been demonstrated for mammalian sirtuins, has not been reported for plant sirtuins so far. In this work, srt1 and srt2 Arabidopsis mutant seedlings have been used to evaluate in vivo the role of sirtuins in cell proliferation and regulation of glutamate dehydrogenase, an enzyme demonstrated to be involved in the control of cell cycle in SIRT4-defective human cells. Moreover, bioinformatic analyses have been performed to elucidate sequence, structure, and function relationships between Arabidopsis sirtuins and between each of them and the closest mammalian homolog. We found that cell proliferation and GDH activity are higher in mutant seedlings, suggesting that both sirtuins exert a physiological inhibitory role in these processes. In addition, mutant seedlings show plant growth and root system improvement, in line with metabolic data. Our data also indicate that utilization of an easy to manipulate organism, such as Arabidopsis plant, can help to shed light on the molecular mechanisms underlying the function of genes present in interkingdom species.

Aryl Fluorosulfate Based Inhibitors That Covalently Target the SIRT5 Lysine Deacylase.

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl ϵ-N-carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture overexpressed SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

In silico screening of small molecule modulators and their binding studies against human sirtuin-6 protein.

Sirtuin-6 (SIRT6), class III family of deacetylase regulates several biological functions, including transcriptional repression, telomere maintenance, and DNA repair. It is unique among sirtuin family members with diverse enzymatic functions: mono-ADP-ribosylase, deacetylase and defatty-acylase. The studies so far implicated SIRT6 role in lifespan extension, tumor suppression, and is considered as an attractive drug target for aging-related disease. In this study, we have carried out in silico screening for human SIRT6 modulators using NCI Diversity Set III library, molecular dynamic (MD) simulations to analyze the protein-ligand interaction, and validated their binding-affinity (Kd) using MicroScale Thermophoresis. This study yielded two novel compounds, ((3Z)-3-((4-(dimethylamino)phenyl)methylidene)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)furan-2-one and 5-phenyl-2-(5-phenyl-2,3-dihydro-1,3-benzoxazol-2-yl)-2,3-dihydro-1,3-benzoxazole showing high-affinity interaction for SIRT6. The structural analysis from MD simulation suggests both compounds might act as substrate-analogs or mimic the nicotinamide binding. On considering the uniqueness of SIRT6 substrate binding acyl channel among sirtuin family member, binding of both compounds to the above site suggesting their specificity for SIRT6 isoform. Therefore, it may form the basis for the development of potential modulators for human SIRT6.Communicated by Ramaswamy H. Sarma.

Roles of SIRT6 in kidney disease: a novel therapeutic target.

SIRT6 is an NAD+ dependent deacetylase that belongs to the mammalian sirtuin family. SIRT6 is mainly located in the nucleus and regulates chromatin remodeling, genome stability, and gene transcription. SIRT6 extensively participates in various physiological activities such as DNA repair, energy metabolism, oxidative stress, inflammation, and fibrosis. In recent years, the role of epigenetics such as acetylation modification in renal disease has gradually received widespread attention. SIRT6 reduces oxidative stress, inflammation, and renal fibrosis, which is of great importance in maintaining cellular homeostasis and delaying the chronic progression of kidney disease. Here, we review the structure and biological function of SIRT6 and summarize the regulatory mechanisms of SIRT6 in kidney disease. Moreover, the role of SIRT6 as a potential therapeutic target for the progression of kidney disease will be discussed. SIRT6 plays an important role in kidney disease. SIRT6 regulates mitochondrial dynamics and mitochondrial biogenesis, induces G2/M cycle arrest, and plays an antioxidant role in nephrotoxicity, IR, obstructive nephropathy, and sepsis-induced AKI. SIRT6 prevents and delays progressive CKD induced by hyperglycemia, kidney senescence, hypertension, and lipid accumulation by regulating mitochondrial biogenesis, and has antioxidant, anti-inflammatory, and antifibrosis effects. Additionally, hypoxia, inflammation, and fibrosis are the main mechanisms of the AKI-to-CKD transition. SIRT6 plays a critical role in the AKI-to-CKD transition and kidney repair through anti-inflammatory, antifibrotic, and mitochondrial quality control mechanisms. AKI Acute kidney injury, CKD Chronic kidney disease.

Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)-based fluorescence quencher.

Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the ε-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted "super-substrates" with catalytic constants of up to 15,000,000 M-1s-1 for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD+ has IC50 values below 200 pM.

Multi-Targeted Molecular Docking, Pharmacokinetics, and Drug-Likeness Evaluation of Okra-Derived Ligand Abscisic Acid Targeting Signaling Proteins Involved in the Development of Diabetes.

Diabetes mellitus is a global threat affecting millions of people of different age groups. In recent years, the development of naturally derived anti-diabetic agents has gained popularity. Okra is a common vegetable containing important bioactive components such as abscisic acid (ABA). ABA, a phytohormone, has been shown to elicit potent anti-diabetic effects in mouse models. Keeping its anti-diabetic potential in mind, in silico study was performed to explore its role in inhibiting proteins relevant to diabetes mellitus- 11ß-hydroxysteroid dehydrogenase (11ß-HSD1), aldose reductase, glucokinase, glutamine-fructose-6-phosphate amidotransferase (GFAT), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and Sirtuin family of NAD(+)-dependent protein deacetylases 6 (SIRT6). A comparative study of the ABA-protein docked complex with already known inhibitors of these proteins relevant to diabetes was compared to explore the inhibitory potential. Calculation of molecular binding energy (ΔG), inhibition constant (pKi), and prediction of pharmacokinetics and pharmacodynamics properties were performed. The molecular docking investigation of ABA with 11-HSD1, GFAT, PPAR-gamma, and SIRT6 revealed considerably low binding energy (ΔG from -8.1 to -7.3 Kcal/mol) and predicted inhibition constant (pKi from 6.01 to 5.21 µM). The ADMET study revealed that ABA is a promising drug candidate without any hazardous effect following all current drug-likeness guidelines such as Lipinski, Ghose, Veber, Egan, and Muegge.

SIRT7: a sentinel of genome stability.

SIRT7 is a class III histone deacetylase that belongs to the sirtuin family. The past two decades have seen numerous breakthroughs in terms of understanding SIRT7 biological function. We now know that this enzyme is involved in diverse cellular processes, ranging from gene regulation to genome stability, ageing and tumorigenesis. Genomic instability is one hallmark of cancer and ageing; it occurs as a result of excessive DNA damage. To counteract such instability, cells have evolved a sophisticated regulated DNA damage response mechanism that restores normal gene function. SIRT7 seems to have a critical role in this response, and it is recruited to sites of DNA damage where it recruits downstream repair factors and directs chromatin regulation. In this review, we provide an overview of the role of SIRT7 in DNA repair and maintaining genome stability. We pay particular attention to the implications of SIRT7 function in cancer and ageing.

Understanding the Function of Mammalian Sirtuins and Protein Lysine Acylation.

Protein lysine acetylation is an important posttranslational modification that regulates numerous biological processes. Targeting lysine acetylation regulatory factors, such as acetyltransferases, deacetylases, and acetyl-lysine recognition domains, has been shown to have potential for treating human diseases, including cancer and neurological diseases. Over the past decade, many other acyl-lysine modifications, such as succinylation, crotonylation, and long-chain fatty acylation, have also been investigated and shown to have interesting biological functions. Here, we provide an overview of the functions of different acyl-lysine modifications in mammals. We focus on lysine acetylation as it is well characterized, and principles learned from acetylation are useful for understanding the functions of other lysine acylations. We pay special attention to the sirtuins, given that the study of sirtuins has provided a great deal of information about the functions of lysine acylation. We emphasize the regulation of sirtuins to illustrate that their regulation enables cells to respond to various signals and stresses.

Screening of SIRT6 inhibitors and activators: A novel activator has an impact on breast cancer cells.

Sirtuin 6 (SIRT6), a member of sirtuin family (SIRT1-7), regulates a variety of cellular processes involved in aging, metabolism, and cancer. Dysregulation of SIRT6 is widely observed in different breast cancer subtypes; however, the role and function of SIRT6 in cancer development remain largely unexplored. The aim of this study was to identify novel compounds targeting SIRT6 which may provide a new approach in development of anti-cancer therapy for breast cancer. Virtual screening was utilized to discover potential compounds targeting SIRT6 for in vitro screening. In addition, novel 1,4-dihydropyridine derivatives were synthetized and further subjected for the screening. The impact of the compounds on the deacetylation activity of SIRT6 was determined with HPLC method. The anti-cancer activities were screened for a panel of breast cancer cells. A set of 1,4-dihydropyridine derivatives was identified as SIRT6 inhibitors. A SIRT6 activating compound, (2,4-dihydroxy-phenyl)-2-oxoethyl 2-(3-methyl-4-oxo-2-phenyl-4H-chromen-8-yl)acetate (later called as 4H-chromen), was discovered and it provided 30-40-fold maximal activation. 4H-chromen was proposed to bind similarly to quercetin and place to previously reported SIRT6 activator sites. 4H-chromen was investigated in various breast cancer cells, and it decreased cell proliferation in all cells as well as arrested cell cycle in triple negative cells. Overall, this study describes a highly potent SIRT6 activator and new inhibitors that represent a novel tool to study the mechanism of SIRT6 function.

Alternative Splicing Increases Sirtuin Gene Family Diversity and Modulates Their Subcellular Localization and Function.

Alternative splicing generates multiple distinct isoforms that increase transcriptome and proteome diversity. There are seven sirtuin genes in humans, each consists of multiple exons that are likely to undergo alternative splicing. Our aim was to characterize the effect of alternative splicing on the sirtuin genes. Here, we report the identification of 23 human sirtuin isoforms, most of which were not previously reported. Five of the sirtuin genes had more than one isoform, whereas sirtuin-6 had nine isoforms. Exon skipping was the main event. Most of the sirtuin isoforms were deficient in parts of the protein domains, including the catalytic domain, the N- or C-terminus, nuclear localization signal or mitochondrial targeting signal. The domain loss caused potential structural changes. Three SIRT1 isoforms had a differential effect on the mitochondrial oxygen consumption rate. Age-related changes in the expression of SIRT1 isoforms were observed in the human heart in fetus, adults, and very old individuals. We also identified 15 sirtuin isoforms in mice. Our data indicate that alternative splicing increases sirtuin gene diversity and may modulate subcellular localization and function, thereby adding complexity to the gene regulation of mitochondrial respiration, metabolism, and cardiac function during maturation and aging.

Development of Second Generation Activity-Based Chemical Probes for Sirtuins.

NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacylases, namely, the sirtuins, are important cell adaptor proteins that alter cell physiology in response to low calorie conditions. They are thought to mediate the beneficial effects of calorie restriction to extend longevity and improve health profiles. Novel chemical probes are highly desired for a better understanding of sirtuin's roles in various biological processes. We developed a group of remarkably simple activity-based chemical probes for the investigation of active sirtuin content in complex native proteomes. These probes harbor a thioacyllysine warhead, a diazirine photoaffinity tag, as well as a terminal alkyne bioorthogonal functional group. Compared to their benzophenone-containing counterparts, these new probes demonstrated improved labeling efficiency and sensitivity, shortened irradiation time, and reduced background signal. They were applied to the labeling of individual recombinant proteins, protein mixtures, and whole cell lysate. These cell permeable small molecule probes also enabled the cellular imaging of sirtuin activity change. Taken together, our study provides new chemical biology tools and future drug discovery strategies for perturbing the activity of different sirtuin isoforms.

Nitro-fatty acids as activators of hSIRT6 deacetylase activity.

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 µm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 µm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.