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.     

PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity

Sirtuin1 (SIRT1) deacetylase and poly(ADP-ribose)-polymerase-1 (PARP-1) respond to environmental cues, and both require NAD⁺ cofactor for their enzymatic activities. However, the functional link between environmental/oxidative stress-mediated activation of PARP-1 and SIRT1 through NAD⁺ cofactor availability is not known. We investigated whether NAD⁺ depletion by PARP-1 activation plays a role in environmental stimuli/oxidant-induced reduction in SIRT1 activity. Both H₂O₂ and cigarette smoke (CS) decreased intracellular NAD⁺ levels in vitro in lung epithelial cells and in vivo in lungs of mice exposed to CS. Pharmacological PARP-1 inhibition prevented oxidant-induced NAD⁺ loss and attenuated loss of SIRT1 activity. Oxidants decreased SIRT1 activity in lung epithelial cells; however increasing cellular NAD⁺ cofactor levels by PARP-1 inhibition or NAD⁺ precursors was unable to restore SIRT1 activity. SIRT1 was found to be carbonylated by CS, which was not reversed by PARP-1 inhibition or selective SIRT1 activator. Overall, these data suggest that environmental/oxidant stress-induced SIRT1 down-regulation and PARP-1 activation are independent events despite both enzymes sharing the same cofactor.     

Nicotinamide treatment reduces the levels of histone H3K4 trimethylation in the promoter of the mper1 circadian clock gene and blocks the ability of dexamethasone to induce the acute response

Circadian rhythms, which measure time on a scale of 24 h, are generated by one of the most ubiquitous endogenous mechanisms, the circadian clock. SIRT1, a class III histone deacetylase, and PARP-1, a poly(ADP-ribose) polymerase, are two NAD⁺-dependent enzymes that have been shown to be involved in the regulation of the clock. Here we present evidence that the metabolite nicotinamide, an inhibitor of SIRT1, PARP-1 and mono(ADP-ribosyl) transferases, blocks the ability of dexamethasone to induce the acute response of the circadian clock gene, mper1, while it concomitantly reduces the levels of histone H3 trimethylation of lysine 4 (H3K4me3) in the mper1 promoter. Moreover, application of alternative inhibitors of SIRT1 and ADP-ribosylation did not lead to similar results. Therefore, inhibition of these enzymes does not seem to be the mode by which NAM exerts these effects. These results suggest the presence of a novel mechanism, not previously documented, by which NAM can alter gene expression levels via changes in the histone H3K4 trimethylation state.     

Sirtuin在热量限制延长寿命机制中的研究进展

热量限制(caloric restriction, CR)可以引起细胞、生物体寿命延长和降低衰老相关疾病的发生,其中Sirtuin起着关键作用．Sirtuin将机体能量代谢和基因表达调控相偶联,通过赖氨酸去乙酰化改变蛋白质的活性和稳定性,从而调节衰老进程．酵母中度CR影响其复制寿命和时序寿命,主要依赖于激活Sir2,增加细胞内NAD+/NADH的比例和调节尼克酰胺浓度来实现．类似的机制也存在于秀丽线虫和果蝇中．哺乳动物在CR条件下SIRT1蛋白表达应答性上升,细胞中NAM磷酸基转移酶能够直接影响NAM和NAD+浓度,并影响SIRT1活性．NO表达增加能导致SIRT1上调和线粒体合成增加．SIRT1可能通过改变组蛋白、p53、NES1、FOXO等底物蛋白的乙酰化影响到细胞和个体的衰老．表明不同生物体中的Sirtuin及其同源类似物在CR条件下对衰老进程和寿命都起着非常重要的作用．     

Parp mutations protect from mitochondrial toxicity in Alzheimer’s disease

Alzheimer’s disease is the most common age-related neurodegenerative disorder. Familial forms of Alzheimer’s disease associated with the accumulation of a toxic form of amyloid-β (Aβ) peptides are linked to mitochondrial impairment. The coenzyme nicotinamide adenine dinucleotide (NAD⁺) is essential for both mitochondrial bioenergetics and nuclear DNA repair through NAD⁺-consuming poly (ADP-ribose) polymerases (PARPs). Here we analysed the metabolomic changes in flies overexpressing Aβ and showed a decrease of metabolites associated with nicotinate and nicotinamide metabolism, which is critical for mitochondrial function in neurons. We show that increasing the bioavailability of NAD⁺ protects against Aβ toxicity. Pharmacological supplementation using NAM, a form of vitamin B that acts as a precursor for NAD⁺ or a genetic mutation of PARP rescues mitochondrial defects, protects neurons against degeneration and reduces behavioural impairments in a fly model of Alzheimer’s disease. Next, we looked at links between PARP polymorphisms and vitamin B intake in patients with Alzheimer’s disease. We show that polymorphisms in the human PARP1 gene or the intake of vitamin B are associated with a decrease in the risk and severity of Alzheimer’s disease. We suggest that enhancing the availability of NAD⁺ by either vitamin B supplements or the inhibition of NAD⁺-dependent enzymes such as PARPs are potential therapies for Alzheimer’s disease.Subject terms: Metabolomics, Cell death in the nervous system, Alzheimer''s disease     

Muscle type-specific responses to NAD+ salvage biosynthesis promote muscle function in Caenorhabditis elegans

Salvage biosynthesis of nicotinamide adenine dinucleotide (NAD⁺) from nicotinamide (NAM) lowers NAM levels and replenishes the critical molecule NAD⁺ after it is hydrolyzed. This pathway is emerging as a regulator of multiple biological processes. Here we probe the contribution of the NAM-NAD⁺ salvage pathway to muscle development and function using Caenorhabditis elegans. C. elegans males with mutations in the nicotinamidase pnc-1, which catalyzes the first step of this NAD⁺ salvage pathway, cannot mate due to a spicule muscle defect. Multiple muscle types are impaired in the hermaphrodites, including body wall muscles, pharyngeal muscles and vulval muscles. An active NAD⁺ salvage pathway is required for optimal function of each muscle cell type. However, we found surprising muscle-cell-type specificity in terms of both the timing and relative sensitivity to perturbation of NAD⁺ production or NAM levels. Active NAD⁺ biosynthesis during development is critical for function of the male spicule protractor muscles during adulthood, but these muscles can surprisingly do without salvage biosynthesis in adulthood under the conditions examined. The body wall muscles require ongoing NAD⁺ salvage biosynthesis both during development and adulthood for maximum function. The vulval muscles do not function in the presence of elevated NAM concentrations, but NAM supplementation is only slightly deleterious to body wall muscles during development or upon acute application in adults. Thus, the pathway plays distinct roles in different tissues. As NAM-NAD⁺ biosynthesis also impacts muscle differentiation in vertebrates, we propose that similar complexities may be found among vertebrate muscle cell types.     

Nicotinamide phosphoribosyltransferase/sirtuin 1 pathway is involved in human immunodeficiency virus type 1 Tat‐mediated long terminal repeat transactivation

Tat is a multifunctional transactivator encoded by human immunodeficiency virus type 1 (HIV‐1). Tat transactivating activity is controlled by nicotinamide adenine nucleotide⁺ (NAD⁺)‐dependent deacetylase sirtuin 1 (SIRT1). Nicotinamide phosphoribosyltransferase (Nampt) is a rate‐limiting enzyme in the conversion of nicotinamide into NAD⁺, which is crucial for SIRT1 activation. Thus, the effect of Nampt on Tat‐regulated SIRT activity was studied in Hela‐CD4‐β‐gal (MAGI) cells. We demonstrated that Tat caused NAD⁺ depletion and inhibited Nampt mRNA and protein expression in MAGI cells. Resveratrol reversed Tat‐induced NAD⁺ depletion and inhibition of Nampt mRNA and protein expression. Further investigation revealed that Tat‐induced inhibition of SIRT1 activity was potentiated in Nampt‐knockdown by Nampt siRNA compared to treatment with Tat alone. Nampt siRNA potentiated Tat‐induced HIV‐1 transactivation in MAGI cells. Altogether, these results indicate that Nampt is critical in the regulation of Tat‐induced inhibition of SIRT1 activity and long terminal repeat (LTR) transactivation. Nampt/SIRT1 pathway could be a novel therapeutic tool for the treatment of HIV‐1 infection. J. Cell. Biochem. 110: 1464–1470, 2010. © 2010 Wiley‐Liss, Inc.     

A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases that catalyze the deacetylation of proteins such as histones and p53. A sensitive and convenient fluorometric assay for evaluating the SIRT1 enzymatic activity was developed here. Specifically, the remaining NAD⁺ after the deacetylation was determined by converting NAD⁺ to a highly fluorescent cyclized α-adduct compound. By this assay, we found that nicotinamide, Cu²⁺, and Zn²⁺ antagonize the activity of SIRT1. Resveratrol stimulates the enzymatic activity specifically with 7-amino-4-methylcoumarin (AMC)-labeled acetylated peptide. Epigallocatechin galate (EGCG) inhibits SIRT1 activity with both AMC-labeled and unlabeled peptide. However, a combination of vitamin C with EGCG can reverse the inhibition of EGCG with the unlabeled peptide or stimulate the deacetylation of AMC-labeled peptide by SIRT1. The assay does not require any isotopic material and thus is biologically safe. It can be adapted to a 96-well microplate for high-throughput screening. Notably, the acetylated peptides with or without fluorescent labels may be used in the assay, which facilitates the substrate specificity study of SIRT1 activators or inhibitors in vitro.     

SIRT1 deficiency attenuates MPP+-induced apoptosis in dopaminergic cells

One of the functions mediated by sirtuin 1 (SIRT1), the NAD⁺-dependent protein deacetylase, has been suggested to be neuroprotective since resveratrol, a SIRT1 activator, inhibits 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced cytotoxicity. In this study, we show that SIRT1 siRNA transfection blocks MPP⁺-induced apoptosis in SH-SY5Y cells. The ratio of potential pro-apoptotic BNIP2 to antiapoptotic BCL-xL was attenuated in SIRT1-deficient cells following MPP⁺ treatment. In addition, BNIP2 shRNA-transfected cells showed reduced cleavage of PARP-1, while BNIP2 overexpression intensified the cleavage in MPP⁺-treated SH-SY5Y cells, suggesting that BNIP2 participates in the MPP⁺-induced apoptosis. Overall, these data imply that SIRT1 may mediate MPP⁺-induced cytotoxicity, possibly through the regulation of BNIP2.     

Nicotinamide increases the sensitivity of chronic myeloid leukemia cells to doxorubicin via the inhibition of SIRT1

The NAD-dependent deacetylase Sirtuin 1 (SIRT1) plays a vital role in leukemogenesis. Nicotinamide (NAM) is the principal NAD⁺ precursor and a noncompetitive inhibitor of SIRT1. In our study, we showed that NAM enhanced the sensitivity of chronic myeloid leukemia (CML) to doxorubicin (DOX) via SIRT1. We found that SIRT1 high expression in CML patients was associated with disease progression and drug resistance. Exogenous NAM efficiently repressed the deacetylation activity of SIRT1 and induced the apoptosis of DOX-resistant K562 cells (K562R) in a dose-dependent manner. Notably, the combination of NAM and DOX significantly inhibited tumor cell proliferation and induced cell apoptosis. The knockdown of SIRT1 in K562R cells enhanced NAM+DOX-induced apoptosis. SIRT1 rescue in K562R reduced the NAM+DOX-induced apoptosis. Mechanistically, the combinatory treatment significantly increased the cleavage of caspase-3 and PARP in K562R in vitro and in vivo. These results suggest the potential role of NAM in increasing the sensitivity of CML to DOX via the inhibition of SIRT1.     

Fasting promotes the expression of SIRT1, an NAD+-dependent protein deacetylase, via activation of PPARα in mice

Calorie restriction (CR) extends lifespans in a wide variety of species. CR induces an increase in the NAD⁺/NADH ratio in cells and results in activation of SIRT1, an NAD⁺-dependent protein deacetylase that is thought to be a metabolic master switch linked to the modulation of lifespans. CR also affects the expression of peroxisome proliferator-activated receptors (PPARs). The three subtypes, PPARα, PPARγ, and PPARβ/δ, are expressed in multiple organs. They regulate different physiological functions such as energy metabolism, insulin action and inflammation, and apparently act as important regulators of longevity and aging. SIRT1 has been reported to repress the PPARγ by docking with its co-factors and to promote fat mobilization. However, the correlation between SIRT1 and other PPARs is not fully understood. CR initially induces a fasting-like response. In this study, we investigated how SIRT1 and PPARα correlate in the fasting-induced anti-aging pathways. A 24-h fasting in mice increased mRNA and protein expression of both SIRT1 and PPARα in the livers, where the NAD⁺ levels increased with increasing nicotinamide phosphoribosyltransferase (NAMPT) activity in the NAD⁺ salvage pathway. Treatment of Hepa1-6 cells in a low glucose medium conditions with NAD⁺ or NADH showed that the mRNA expression of both SIRT1 and PPARα can be enhanced by addition of NAD⁺, and decreased by increasing NADH levels. The cell experiments using SIRT1 antagonists and a PPARα agonist suggested that PPARα is a key molecule located upstream from SIRT1, and has a role in regulating SIRT1 gene expression in fasting-induced anti-aging pathways.     

GABA protects pancreatic beta cells against apoptosis by increasing SIRT1 expression and activity

We have previously shown that GABA protects pancreatic islet cells against apoptosis and exerts anti-inflammatory effects. Notably, GABA inhibited the activation of NF-κB in both islet cells and lymphocytes. NF-κB activation is detrimental to beta cells by promoting apoptosis. However, the mechanisms by which GABA mediates these effects are unknown. Because the above-mentioned effects mimic the activity of sirtuin 1 (SIRT1) in beta cells, we investigated whether it is involved. SIRT1 is an NAD⁺-dependent deacetylase that enhances insulin secretion, and counteracts inflammatory signals in beta cells. We found that the incubation of a clonal beta-cell line (rat INS-1) with GABA increased the expression of SIRT1, as did GABA receptor agonists acting on either type A or B receptors. NAD⁺ (an essential cofactor of SIRT1) was also increased. GABA augmented SIRT1 enzymatic activity, which resulted in deacetylation of the p65 component of NF-κB, and this is known to interfere with the activation this pathway. GABA increased insulin production and reduced drug-induced apoptosis, and these actions were reversed by SIRT1 inhibitors. We examined whether SIRT1 is similarly induced in newly isolated human islet cells. Indeed, GABA increased both NAD⁺ and SIRT1 (but not sirtuins 2, 3 and 6). It protected human islet cells against spontaneous apoptosis in culture, and this was negated by a SIRT1 inhibitor. Thus, our findings suggest that major beneficial effects of GABA on beta cells are due to increased SIRT1 and NAD⁺, and point to a new pathway for diabetes therapy.