Tankyrase-1 overexpression reduces genotoxin-induced cell death by inhibiting PARP1

Poly(ADP-ribose) polymerases or PARPs are a family of NAD⁺-dependent enzymes that modify themselves and other substrate proteins with ADP-ribose polymers. The founding member PARP1 is localized predominantly in the nucleus and is activated by binding to DNA lesions. Excessive PARP1 activation following genotoxin treatment causes NAD⁺ depletion and cell death, whereas pharmacological PARP1 inhibition protects cells from genotoxicity. This study investigates whether cellular viability and NAD⁺ metabolism are regulated by tankyrase-1, a PARP member localized predominantly in the cytosol. Using a tetracycline-sensitive promoter to regulate tankyrase-1 expression in Madin–Darby canine kidney (MDCK) cells, we found that a 40-fold induction of tankyrase-1 (from 1500 to 60,000 copies per cell) lowers steady-state NAD⁺ levels but does not affect basal cellular viability. Moreover, the induction confers protection against the oxidative agent H₂O₂ and the alkylating agent MNNG, genotoxins that kill cells by activating PARP1. The cytoprotective effect of tankyrase-1 is not due to enhanced scavenging of oxidants or altered expression of Mcl-1, an anti-apoptotic molecule previously shown to be down-regulated by tankyrase-1 in CHO cells. Instead, tankyrase-1 appears to protect cells by preventing genotoxins from activating PARP1-mediated reactions such as PARP1 automodification and NAD⁺ consumption. Our findings therefore indicate a cytoprotective function of tankyrase-1 mediated through altered NAD⁺ homeostasis and inhibition of PARP1 function.     

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     

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.     

NAD+ metabolism: A therapeutic target for age-related metabolic disease

Abstract

Nicotinamide adenine dinucleotide (NAD) is a central metabolic cofactor by virtue of its redox capacity, and as such regulates a wealth of metabolic transformations. However, the identification of the longevity protein silent regulator 2 (Sir2), the founding member of the sirtuin protein family, as being NAD⁺-dependent reignited interest in this metabolite. The sirtuins (SIRT1-7 in mammals) utilize NAD⁺ to deacetylate proteins in different subcellular compartments with a variety of functions, but with a strong convergence on optimizing mitochondrial function. Since cellular NAD⁺ levels are limiting for sirtuin activity, boosting its levels is a powerful means to activate sirtuins as a potential therapy for mitochondrial, often age-related, diseases. Indeed, supplying excess precursors, or blocking its utilization by poly(ADP-ribose) polymerase (PARP) enzymes or CD38/CD157, boosts NAD⁺ levels, activates sirtuins and promotes healthy aging. Here, we discuss the current state of knowledge of NAD⁺ metabolism, primarily in relation to sirtuin function. We highlight how NAD⁺ levels change in diverse physiological conditions, and how this can be employed as a pharmacological strategy.     

Poly(ADP-ribose): PARadigms and PARadoxes

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification (PTM) catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARPs use NAD⁺ as substrate and upon cleaving off nicotinamide they transfer the ADP-ribosyl moiety covalently to suitable acceptor proteins and elongate the chain by adding further ADP-ribose units to create a branched polymer, termed poly(ADP-ribose) (PAR), which is rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). In recent years several key discoveries changed the way we look at the biological roles and mode of operation of PARylation. These paradigm shifts include but are not limited to (1) a single PARP enzyme expanding to a PARP family; (2) DNA-break dependent activation extended to several other DNA dependent and independent PARP-activation mechanisms; (3) one molecular mechanism (covalent PARylation of target proteins) underlying the biological effect of PARPs is now complemented by several other mechanisms such as protein–protein interactions, PAR signaling, modulation of NAD⁺ pools and (4) one principal biological role in DNA damage sensing expanded to numerous, diverse biological functions identifying PARP-1 as a real moonlighting protein. Here we review the most important paradigm shifts in PARylation research and also highlight some of the many controversial issues (or paradoxes) of the field such as (1) the mostly synergistic and not antagonistic biological effects of PARP-1 and PARG; (2) mitochondrial PARylation and PAR decomposition, (3) the cross-talk between PARylation and signaling pathways (protein kinases, phosphatases, calcium) and the (4) divergent roles of PARP/PARylation in longevity and in age-related diseases.     

Tryptophan metabolism and disposition in cancer biology and immunotherapy

Tumours utilise tryptophan (Trp) and its metabolites to promote their growth and evade host defences. They recruit Trp through up-regulation of Trp transporters, and up-regulate key enzymes of Trp degradation and down-regulate others. Thus, Trp 2,3-dioxygenase (TDO2), indoleamine 2,3-dioxygenase 1 (IDO1), IDO2, N′-formylkynurenine formamidase (FAMID) and Kyn aminotransferase 1 (KAT1) are all up-regulated in many cancer types, whereas Kyn monooxygenase (KMO), kynureninase (KYNU), 2-amino-3-carboxymuconic acid-6-semialdehyde decarboxylase (ACMSD) and quinolinate phosphoribosyltransferase (QPRT) are up-regulated in a few, but down-regulated in many, cancers. This results in accumulation of the aryl hydrocarbon receptor (AhR) ligand kynurenic acid and in depriving the host of NAD⁺ by blocking its synthesis from quinolinic acid. The host loses more NAD⁺ by up-regulation of the NAD⁺-consuming poly (ADP-ribose) polymerases (PARPs) and the protein acetylaters SIRTs. The nicotinamide arising from PARP and SIRT activation can be recycled in tumours to NAD⁺ by the up-regulated key enzymes of the salvage pathway. Up-regulation of the Trp transporters SLC1A5 and SLC7A5 is associated mostly with that of TDO2 = FAMID > KAT1 > IDO2 > IDO1. Tumours down-regulate enzymes of serotonin synthesis, thereby removing competition for Trp from the serotonin pathway. Strategies for combating tumoral immune escape could involve inhibition of Trp transport into tumours, inhibition of TDO and IDOs, inhibition of FAMID, inhibition of KAT and KYNU, inhibition of NMPRT and NMNAT, inhibition of the AhR, IL-4I1, PARPs and SIRTs, and by decreasing plasma free Trp availability to tumours by albumin infusion or antilipolytic agents and inhibition of glucocorticoid induction of TDO by glucocorticoid antagonism.     

Evolutionary history of the poly(ADP-ribose) polymerase gene family in eukaryotes

Background  

The Poly(ADP-ribose)polymerase (PARP) superfamily was originally identified as enzymes that catalyze the attachment of ADP-ribose subunits to target proteins using NAD⁺ as a substrate. The family is characterized by the catalytic site, termed the PARP signature. While these proteins can be found in a range of eukaryotes, they have been best studied in mammals. In these organisms, PARPs have key functions in DNA repair, genome integrity and epigenetic regulation. More recently it has been found that proteins within the PARP superfamily have altered catalytic sites, and have mono(ADP-ribose) transferase (mART) activity or are enzymatically inactive. These findings suggest that the PARP signature has a broader range of functions that initially predicted. In this study, we investigate the evolutionary history of PARP genes across the eukaryotes.     

NAD+ ameliorates endotoxin‐induced acute kidney injury in a sirtuin1–dependent manner via GSK‐3β/Nrf2 signalling pathway

Acute kidney injury (AKI) is a substantial worldwide public health concern with no specific and effective therapies in clinic. NAD⁺ is a pivotal determinant of cellular energy metabolism involved in the progression of AKI; however, its mechanism in kidney injury remains poorly understood. Sirtuin 1 (SIRT1) is an NAD⁺‐dependent deacetylase associated with renal protection and acute stress resistance. In this study, we have investigated the role of NAD⁺ in AKI and the potential mechanism(s) involved in its renoprotective effect. NAD⁺ was notably decreased and negatively correlated with kidney dysfunction in AKI, restoring NAD⁺ with NMN significantly ameliorates LPS‐induced oxidative stress and apoptosis and attenuates renal damage. We also found that the protection of NAD⁺ is associated with SIRT1 expressions and performs in a SIRT1‐dependent manner. Inhibition of SIRT1 blunted the protective effect of NAD⁺ and up‐regulated the activity of glycogen synthase kinase‐3β (GSK‐3β) that was concomitant with mitigated Nrf2 nuclear accumulation, thereby exacerbates AKI. These findings suggest that NAD⁺/SIRT1/GSK‐3β/Nrf2 axis is an important mechanism that can protect against AKI which might be a potential therapeutic target for the treatment of AKI.     

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.     

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.     

Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD⁺), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H₂O₂) in the culture medium. Under oxidative stress, the NAD⁺ generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD⁺ reveals an intricate link between metabolism and the processing of genetic information.     

Mitochondrial sirtuins

Sirtuins have emerged as important proteins in aging, stress resistance and metabolic regulation. Three sirtuins, SIRT3, 4 and 5, are located within the mitochondrial matrix. SIRT3 and SIRT5 are NAD⁺-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins and yield 2′-O-acetyl-ADP-ribose and nicotinamide. SIRT4 can transfer the ADP-ribose group from NAD⁺ onto acceptor proteins. Recent findings reveal that a large fraction of mitochondrial proteins are acetylated and that mitochondrial protein acetylation is modulated by nutritional status. This and the identification of targets for SIRT3, 4 and 5 support the model that mitochondrial sirtuins are metabolic sensors that modulate the activity of metabolic enzymes via protein deacetylation or mono-ADP-ribosylation. Here, we review and discuss recent progress in the study of mitochondrial sirtuins and their targets.     

Intra-mitochondrial poly(ADP-ribosyl)ation: Potential role for alpha-ketoglutarate dehydrogenase

Poly(ADP-ribose) polymerase (PARP) is an intracellular enzyme involved in DNA repair and in building poly-ADP-ribose polymers on nuclear proteins using NAD⁺. While the majority of PARP resides in the nucleus, several studies indicated that PARP may also be located in the cytosol or in the mitochondrial matrix. In this study we found several poly-ADP-ribosylated proteins in isolated rat liver mitochondria following hydrogen peroxide (H₂O₂) or nitric oxide donor treatment. Protein poly-ADP-ribosylation was more intense in isolated mitochondria than in whole tissue homogenates and it was not associated with increased nuclear PARP activity. We identified five poly-ADP-ribose (PAR) positive mitochondrial bands by protein mass fingerprinting. All of the identified enzymes exhibited decreased activity or decreased levels following oxidative or nitrosative stress. One of the identified proteins is dihydrolipoamide dehydrogenase (DLDH), a component of the alpha-ketoglutarate dehydrogenase (KGDH) complex, which uses NAD⁺ as a substrate. This raised the possibility that KGDH may have a PARP-like enzymatic activity. The intrinsic PARP activity of KGDH and DLDH was confirmed using a colorimetric PARP assay kit and by the incubation of the recombinant enzymes with H₂O₂. The KGDH enzyme may, therefore, have a novel function as a PARP-like enzyme, which may play a role in regulating intramitochondrial NAD⁺ and poly(ADP-ribose) homeostasis, with possible roles in physiology and pathophysiology.     

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.