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 ).     

Seven sirtuins for seven deadly diseases ofaging

Sirtuins are a class of NAD⁺-dependent deacetylases having beneficial health effects. This extensive review describes the numerous intracellular actions of the seven mammalian sirtuins, their protein targets, intracellular localization, the pathways they modulate, and their role in common diseases of aging. Selective pharmacological targeting of sirtuins is of current interest in helping to alleviate global disease burden. Since all sirtuins are activated by NAD⁺, strategies that boost NAD⁺ in cells are of interest. While most is known about SIRT1, the functions of the six other sirtuins are now emerging. Best known is the involvement of sirtuins in helping cells adapt energy output to match energy requirements. SIRT1 and some of the other sirtuins enhance fat metabolism and modulate mitochondrial respiration to optimize energy harvesting. The AMP kinase/SIRT1–PGC-1α–PPAR axis and mitochondrial sirtuins appear pivotal to maintaining mitochondrial function. Downregulation with aging explains much of the pathophysiology that accumulates with aging. Posttranslational modifications of sirtuins and their substrates affect specificity. Although SIRT1 activation seems not to affect life span, activation of some of the other sirtuins might. Since sirtuins are crucial to pathways that counter the decline in health that accompanies aging, pharmacological agents that boost sirtuin activity have clinical potential in treatment of diabetes, cardiovascular disease, dementia, osteoporosis, arthritis, and other conditions. In cancer, however, SIRT1 inhibitors could have therapeutic value. Nutraceuticals such as resveratrol have a multiplicity of actions besides sirtuin activation. Their net health benefit and relative safety may have originated from the ability of animals to survive environmental changes by utilizing these stress resistance chemicals in the diet during evolution. Each sirtuin forms a key hub to the intracellular pathways affected.     

SIRT3 & SIRT7: Potential Novel Biomarkers for Determining Outcome in Pancreatic Cancer Patients

Purpose

The sirtuin gene family has been linked with tumourigenesis, in both a tumour promoter and suppressor capacity. Information regarding the function of sirtuins in pancreatic cancer is sparse and equivocal. We undertook a novel study investigating SIRT1-7 protein expression in a cohort of pancreatic tumours. The aim of this study was to establish a protein expression profile for SIRT1-7 in pancreatic ductal adenocarcinomas (PDAC) and to determine if there were associations between SIRT1-7 expression, clinico-pathological parameters and patient outcome.

Material and Methods

Immunohistochemical analysis of SIRT1-7 protein levels was undertaken in a tissue micro-array comprising 77 resected PDACs. Statistical analyses determined if SIRT1-7 protein expression was associated with clinical parameters or outcome.

Results

Two sirtuin family members demonstrated significant associations with clinico-pathological parameters and patient outcome. Low level SIRT3 expression in the tumour cytoplasm correlated with more aggressive tumours, and a shorter time to relapse and death, in the absence of chemotherapeutic intervention. Low levels of nuclear SIRT7 expression were also associated with an aggressive tumour phenotype and poorer outcome, as measured by disease-free and disease-specific survival time, 12 months post-diagnosis.

Conclusions

Our data suggests that SIRT3 and SIRT7 possess tumour suppressor properties in the context of pancreatic cancer. SIRT3 may also represent a novel predictive biomarker to determine which patients may or may not respond to chemotherapy. This study opens up an interesting avenue of investigation to potentially identify predictive biomarkers and novel therapeutic targets for pancreatic cancer, a disease that has seen no significant improvement in survival over the past 40 years.     

SIRT4 is the last puzzle of mitochondrial sirtuins

Sirtuins are recently redefined as a family of nicotinamide adenine dinucleotide (NAD)-dependent deacylases. Sirtuins in mammals including human have seven members, which are SIRT1-7. Compared to other sirtuin members, not much study is focused on mitochondrial sirtuins (SIRT3-5). In mitochondrial sirtuins, SIRT4 was the last of less well-understood mitochondrial sirtuins especially for its robust enzymatic activity. This makes SIRT4 become the last puzzle of mitochondrial sirtuins, and thus brings some obstacles for studying SIRT4 biological functions or developing SIRT4 modulators. In this review, we will summarize and discuss the current findings for substrates, biological functions and possible enzymatic activities of SIRT4. The purpose of this review is to facilitate in discovering the robust enzymatic activity of SIRT4 and eventually finish this last puzzle of mitochondrial sirtuins.     

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1^H/H) live shorter and show signs of accelerated aging. As wild‐type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age‐related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1‐7) are a family of NAD⁺‐dependent deacetylases that can delay age‐related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD⁺ and the ability of SIRT2 to maintain lysine‐668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD⁺ precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1^H/H animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD⁺ to delay diseases of aging in mammals is warranted.     

Mitochondrial sirtuins,metabolism, and aging

Maintaining metabolic homeostasis is essential for cellular and organismal health throughout life. Multiple signaling pathways that regulate metabolism also play critical roles in aging, such as PI3K/AKT, mTOR, AMPK, and sirtuins (SIRTs). Among them, sirtuins are known as a protein family with versatile functions, such as metabolic control, epigenetic modification and lifespan extension. Therefore, by understanding how sirtuins regulate metabolic processes, we can start to understand how they slow down or accelerate biological aging from the perspectives of metabolic regulation. Here, we review the biology of SIRT3, SIRT4, and SIRT5, known as the mitochondrial sirtuins due to their localization in the mitochondrial matrix. First, we will discuss canonical pathways that regulate metabolism more broadly and how these are integrated with aging regulation. Then, we will summarize the current knowledge about functional differences between SIRT3, SIRT4, and SIRT5 in metabolic control and integration in signaling networks. Finally, we will discuss how mitochondrial sirtuins regulate processes associated with aging and aging-related diseases.     

SIRT 1 binding with PKM and NSE and modulate their acetylation and activities

SIRT1 (Silent mating type information regulation 2 homolog 1) play a neuroprotective effect through deacetylation target proteins in various neuronal diseases. However, the precise mechanisms remain elusive. In this study, we aim to identify those novel interacting partners of SIRT1 in rat brain tissue. By using a pre-clear GST-Pull down assay followed by the LC-MS/MS analysis, we've identified potential SIRT1's interacting partners, which function annotation by GO and KEGG analysis indicating some metabolic pathways are among the most enriched. Then we confirmed two candidates Enolase-1 (and NSE (Neuron-Specific Enolase) in brain) and PKM (Pyruvate Kinase Muscle) are associated with SIRT1 in brain tissue lysis by co-immunoprecipitation. Furthermore, increase or decrease the SIRT1 enzyme activity by its agonist SRT1720 or antagonist EX527 could significantly affect the acetylation level of endogenous NSE and PKM, SIRT1 overexpression or knock out expreiments also showed the same results as use SIRT1's agonist or antagonist. Moreover, the acetylation changes on NSE or PKM could finally lead to affection on their catalytic activity. Taken together, our findings suggest that the function of SIRT1 binding proteins is enriched in metabolic pathways. NSE and PKM are new SIRT1 binding molecules. SIRT1 may regulate acetylation level of NSE and PKM through deacetylation and further regulate their catalytic activity. Our study provides new evidence for the involvement of SIRT1 in the mechanisms of metabolic regulation in central nervous system.     

Function of SIRT1 in physiology

Sirtuins were originally defined as a family of oxidized nicotinamide adenine nucleotide (NAD⁺)-dependent enzymes that deacetylate lysine residues on various proteins. The sirtuins are remarkably conserved throughout evolution from archae to eukaryotes. They were named after their homology to the Saccharomyces cerevisiae gene silent information regulator 2 (Sir2). The mammalian sirtuins, SIRT1-7, are implicated in a variety of cellular functions ranging from gene silencing, control of the cell cycle and apoptosis, and energy homeostasis. As SIRT1 is a nuclear protein and is the mammalian homolog most highly related to Sir2, it has been the focus of a large number of recent studies. Here we review some of the current data related to SIRT1 and discuss its mode of action and biological role in cellular and organismal models. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 869–876.     

Exogenous NAD Blocks Cardiac Hypertrophic Response via Activation of the SIRT3-LKB1-AMP-activated Kinase Pathway

Since the discovery of NAD-dependent deacetylases, sirtuins, it has been recognized that maintaining intracellular levels of NAD is crucial for the management of stress response of cells. Here we show that agonist-induced cardiac hypertrophy is associated with loss of intracellular levels of NAD, but not exercise-induced physiologic hypertrophy. Exogenous addition of NAD was capable of maintaining intracellular levels of NAD and blocking the agonist-induced cardiac hypertrophic response in vitro as well as in vivo. NAD treatment blocked the activation of pro-hypertrophic Akt1 signaling, and augmented the activity of anti-hypertrophic LKB1-AMPK signaling in the heart, which prevented subsequent induction of mTOR-mediated protein synthesis. By using gene knock-out and transgenic mouse models of SIRT3 and SIRT1, we showed that the anti-hypertrophic effects of exogenous NAD are mediated through activation of SIRT3, but not SIRT1. SIRT3 deacetylates and activates LKB1, thus augmenting the activity of the LKB1-AMPK pathway. These results reveal a novel role of NAD as an inhibitor of cardiac hypertrophic signaling, and suggest that prevention of NAD depletion may be critical in the treatment of cardiac hypertrophy and heart failure.     

Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation

The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol’s anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21^CIP1 and p16^INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21^CIP1 and p16^INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.     

Mapping the in vitro interactome of cardiac sodium (Na+)‐calcium (Ca2+) exchanger 1 (NCX1)

The sodium (Na⁺)‐calcium (Ca²⁺) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca²⁺ homeostasis, serving as the primary mechanism for Ca²⁺ extrusion during relaxation. Dysregulation of NCX1 is observed in end‐stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti‐NCX1 against endogenous NCX1 and (2) anti‐His (where His is histidine) with His‐trigger factor‐NCX1_cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein–protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where “cell communication” and “signal transduction” formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in “cardiovascular disease” which can be explored as novel drug targets in future research.     

Mechanism of Inhibition of the Human Sirtuin Enzyme SIRT3 by Nicotinamide: Computational and Experimental Studies

Sirtuins are key regulators of many cellular functions including cell growth, apoptosis, metabolism, and genetic control of age-related diseases. Sirtuins are themselves regulated by their cofactor nicotinamide adenine dinucleotide (NAD⁺) as well as their reaction product nicotinamide (NAM), the physiological concentrations of which vary during the process of aging. Nicotinamide inhibits sirtuins through the so-called base exchange pathway, wherein rebinding of the reaction product to the enzyme accelerates the reverse reaction. We investigated the mechanism of nicotinamide inhibition of human SIRT3, the major mitochondrial sirtuin deacetylase, in vitro and in silico using experimental kinetic analysis and Molecular Mechanics-Poisson Boltzmann/Generalized Born Surface Area (MM-PB(GB)SA) binding affinity calculations with molecular dynamics sampling. Through experimental kinetic studies, we demonstrate that NAM inhibition of SIRT3 involves apparent competition between the inhibitor and the enzyme cofactor NAD⁺, contrary to the traditional characterization of base exchange as noncompetitive inhibition. We report a model for base exchange inhibition that relates such kinetic properties to physicochemical properties, including the free energies of enzyme-ligand binding, and estimate the latter through the first reported computational binding affinity calculations for SIRT3:NAD⁺, SIRT3:NAM, and analogous complexes for Sir2. The computational results support our kinetic model, establishing foundations for quantitative modeling of NAD⁺/NAM regulation of mammalian sirtuins during aging and the computational design of sirtuin activators that operate through alleviation of base exchange inhibition.     

Activation of the Protein Deacetylase SIRT6 by Long-chain Fatty Acids and Widespread Deacylation by Mammalian Sirtuins

Mammalian sirtuins (SIRT1 through SIRT7) are members of a highly conserved family of NAD⁺-dependent protein deacetylases that function in metabolism, genome maintenance, and stress responses. Emerging evidence suggests that some sirtuins display substrate specificity toward other acyl groups attached to the lysine ϵ-amine. SIRT6 was recently reported to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups. Here we investigated the catalytic ability of all sirtuins to hydrolyze 13 different acyl groups from histone H3 peptides, ranging in carbon length, saturation, and chemical diversity. We find that long-chain deacylation is a general feature of mammalian sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic acid. These results provide new insight into sirtuin function and a means for cellular removal of an expanding list of endogenous lysine modifications. Given that SIRT6 is a poor deacetylase in vitro, but binds and prefers to hydrolyze long-chain acylated peptides, we hypothesize that binding of certain free fatty acids (FFAs) could stimulate deacetylation activity. Indeed, we demonstrate that several biologically relevant FFAs (including myristic, oleic, and linoleic acids) at physiological concentrations induce up to a 35-fold increase in catalytic efficiency of SIRT6 but not SIRT1. The activation mechanism is consistent with fatty acid inducing a conformation that binds acetylated H3 with greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of discovering endogenous, small-molecule activators of SIRT6.     

Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of fasted mice, we found that SIRT3‐targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3‐dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation sites, and greater sensitivity of SIRT3‐targeted sites to chemical acetylation in vitro and fasting‐induced acetylation in vivo, suggest a nonenzymatic mechanism of acetylation. Our data indicate that most mitochondrial acetylation occurs as a low‐level nonenzymatic protein lesion and that SIRT3 functions as a protein repair factor that removes acetylation lesions from lysine residues.     

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.     

A Remarkable Age-Related Increase in SIRT1 Protein Expression against Oxidative Stress in Elderly: SIRT1 Gene Variants and Longevity in Human

Aging is defined as the accumulation of progressive organ dysfunction. Controlling the rate of aging by clarifying the complex pathways has a significant clinical importance. Nowadays, sirtuins have become famous molecules for slowing aging and decreasing age-related disorders. In the present study, we analyzed the SIRT1 gene polymorphisms (rs7895833 A>G, rs7069102 C>G and rs2273773 C>T) and its relation with levels of SIRT1, eNOS, PON-1, cholesterol, TAS, TOS, and OSI to demonstrate the association between genetic variation in SIRT1 and phenotype at different ages in humans. We observed a significant increase in the SIRT1 level in older people and found a significant positive correlation between SIRT1 level and age in the overall studied population. The oldest people carrying AG genotypes for rs7895833 have the highest SIRT1 level suggesting an association between rs7895833 SNP and lifespan longevity. Older people have lower PON-1 levels than those of adults and children which may explain the high levels of SIRT1 protein as a compensatory mechanism for oxidative stress in the elderly. The eNOS protein level was significantly decreased in older people as compared to adults. There was no significant difference in the eNOS level between older people and children. The current study is the first to demonstrate age-related changes in SIRT1 levels in humans and it is important for a much better molecular understanding of the role of the longevity gene SIRT1 and its protein product in aging. It is also the first study presenting the association between SIRT1 expression in older people and rs7895833 in SIRT1 gene.     

Regulation of SIRT1 protein levels by nutrient availability

The mammalian NAD+ dependent deacetylase, SIRT1, was shown to be a key protein in regulating glucose homeostasis, and was implicated in the response to calorie restriction. We show here that levels of SIRT1 increased in response to nutrient deprivation in cultured cells, and in multiple tissues of mice after fasting. The increase in SIRT1 levels was due to stabilization of SIRT1 protein, and not an increase in SIRT1 mRNA. In addition, p53 negatively regulated SIRT1 levels under normal growth conditions and is also required for the elevation of SIRT1 under limited nutrient conditions. These results have important implications on the relationship between sirtuins, nutrient availability and aging.