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.     

NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos cell death

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD⁺) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD⁺ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD⁺ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD⁺, mediates skin inflammation through parthanatos cell death.

A study of chronic skin inflammation reveals that hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation via parthanatos cell death, identifying NAMPT, PARP1 and AIFM1 as novel therapeutic targets for psoriasis.

Highlights

• Nicotinamide phosphoribosyltransferase (NAMPT) inhibition alleviates inflammation in zebrafish and human organotypic 3D skin models of psoriasis.
• NADPH oxidase–derived reactive oxygen species (ROS) mediate keratinocyte DNA damage and poly(ADP-ribose) polymerase 1 (PARP1) overactivation.
• Inhibition of parthanatos cell death phenocopies the effects of NAMPT inhibition in zebrafish and human psoriasis models.
• NAMPT and poly(ADP-ribose) (PAR) metabolism is altered in psoriasis patients.

     

Skeletal muscle hexokinase: regulation in mammalian hibernation

The perfused rat liver responds in several ways to NAD⁺ infusion (20–100 μM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption and gluconeogenesis are some of the effects that were observed. Extracellular NAD⁺ is also extensively transformed in the liver. The purpose of the present work was to determine the main products of extracellular NAD⁺ transformation under various conditions and to investigate the possible contribution of these products for the metabolic effects of the parent compound. The experiments were done with the isolated perfused rat liver. The NAD⁺ transformation was monitored by HPLC. Confirming previous findings, the single-pass transformation of 100 μM NAD⁺ ranged between 75% at 1.5 min after starting infusion to 95% at 8 min. The most important products of single-pass NAD⁺ transformation appearing in the outflowing perfusate were nicotinamide, ADP-ribose, uric acid, and inosine. The relative proportions of these products presented some variations with the time after initiation of NAD⁺ infusion and the perfusion conditions, but ADP-ribose was always more abundant than uric acid and inosine. Cyclic ADP-ribose (cADP-ribose) as well as adenosine were not detected in the outflowing perfusate. The metabolic effects of ADP-ribose were essentially those already described for NAD⁺. These effects were sensitive to suramin (P2_XY purinergic receptor antagonist) and insensitive to 3,7-dimethyl-1-(2-propargyl)-xanthine (A2 purinergic receptor antagonist). Inosine, a known purinergic A3 agonist, was also active on metabolism, but uric acid and nicotinamide were inactive. It was concluded that the metabolic and hemodynamic effects of extracellular NAD⁺ are caused mainly by interactions with purinergic receptors with a highly significant participation of its main transformation product ADP-ribose.     

Cytoprotective Effects of Nicotinamide Derivatives in Endothelial Cells

Following discovery of NAD⁺-dependent reactions that control gene expression, cytoprotection, and longevity, there has been a renewed therapeutic interest in precursors, such as nicotinamide and its derivatives. We tested 20 analogues of nicotinamide for their ability to protect endothelial cells from peroxynitrite stress and their effect on poly (ADP-ribose) polymerase (PARP) activity. Several nicotinamide derivatives protected endothelial cells from peroxynitrite-induced depletion of cellular NAD⁺ and ATP concentrations, but only some of these compounds inhibited PARP. We conclude that some nicotinamide derivatives provide protection of endothelial cells against peroxynitrite-induced injury independent of inhibition of PARP activity. Preservation of the NAD⁺ pool was a common effect of these compounds.     

Intracellular Ca2+ Chelators Prevent Dna Damage And Protect Hepatoma 1Clc7 Cells From Quinone-Induced Cell Killing

Exposure of hepatoma lclc7 cells to 2,3-drniethoxy-1.4-naphthoquinone (DMNQ) resulted in a sustained elevation of cytosolic Ca²⁺. DNA single strand breaks and cell killing. DNA single strand break formation was prevented when cells were preloaded with either of the intracellular Ca²⁺ chelators. Quin 2 or BAPTA, to buffer the increase in cytosolic Ca²⁺ concentration induced by the quinone. DMNQ caused marked NAD⁺ depletion which was prevented when cells were preincubated with 3-aminobenzamide. an inhibitor of nuclear poly-(ADP-ribose)-synthetase activity. or with either of the two Ca²⁺ chelators. However. 3-aminobenzamide did not protect the hepatoma cells from loss of viability. Our results indicate that quinone-induced DNA damage. NAD⁺ depletion and cell killing are mediated by a sustained elevation of cytosolic Ca²⁺     

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.     

Adaptive changes in NAD+ metabolism in ultraviolet light-irradiated murine lymphoma cells

We have determined the ability of UV_254nm-irradiated murine lymphoma cells to adapt their NAD⁺ metabolism to the increased NAD⁺ consumption for the poly ADP-ribosylation of chromatin proteins. Two murine lymphoma sublines with differential UV-sensitivity and poly(ADP-ribose) turnover were used as a model system. The first subline, designated LY-R is UV_254nm-sensitive and tumorigenic in DBA/2 mice. The second subline, LY-S is UV_254nm-resistant and nontumorigenic. Following treatment of these cells with 2 mM benzamide, an inhibitor of the NAD⁺-utilizing enzyme poly(ADP-ribose) polymerase, NAD⁺ levels slowly increased up to about 160% of control levels after 3 hours. When benzamide was added to these cultures 20 min after UV_254nm irradiation, a dramatic transient increase of NAD⁺ levels was observed within 4 min in LY-R cells and more moderately in LY-S cells. At later times after UV_254nm irradiation, the NAD⁺ levels increased in both sublines reaching up to 200% of the concentrations prior to benzamide treatment. These results demonstrate an adaptative response of NAD⁺ metabolism to UV_254nm irradiation. In parallel, we observed a differential repartitioning of ADP-ribosyl residues between the NAD⁺ and poly(ADP-ribose) pools of LY-R and LY-S cells that correlates with the differential UV sensitivity of these cells.     

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.     

Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.     

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.     

The Early Activation of PI3K Strongly Enhances the Resistance of Cortical Neurons to Hypoxic Injury via the Activation of Downstream Targets of the PI3K Pathway and the Normalization of the Levels of PARP Activity, ATP, and NAD+

Phosphatidylinositol 3-kinase (PI3K) plays several important roles in neuronal survival. Activation of the pathway is essential for the neuroprotective mechanisms of materials that shield neuronal cells from many stressful conditions. However, there have been no reports to date about the effect of the direct activation of the pathway in hypoxic injury of neuronal cells. We investigated whether the direct activation of the PI3K pathway inhibits neuronal cell death induced by hypoxia. Primary cultured cortical neurons (PCCNs) were exposed to hypoxic conditions (less than 1 mol% O₂) and/or treated with PI3K activator. Hypoxia reduced the viability of PCCNs in a time-dependent manner, but treatment with PI3K significantly restored viability in a concentration-dependent manner. Among the signaling proteins involved in the PI3K pathway, those associated with survival, including Akt and glycogen synthase kinase-3β, were decreased shortly after exposure to hypoxia and those associated with cell death, including BAX, apoptosis-induced factor, cytochrome c, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP), were increased. However, treatment with PI3K activator normalized the expression levels of those signaling proteins. PARP activity and levels of ATP and NAD⁺ altered by hypoxia were also normalized with direct PI3K activation. All these findings suggest that direct and early activation is important for protecting neuronal cells from hypoxic injury.     

Poly(ADP-Ribose) Polymerase Inhibition in Oxidant-Stressed Endothelial Cells Prevents Oncosis and Permits Caspase Activation and Apoptosis

Endothelial cells (EC) are subject to oxidative-induced cell death. Activation of poly(ADP-ribose) polymerase (PARP) occurs early in oxidant-induced EC injury and putatively mediates cell death by depleting its substrate, NAD⁺. In this study, the role of PARP in H₂O₂-induced EC death was investigated. EC were exposed to oxidant stress and viability continuously monitored using fluorescent dye exclusion. Inhibition of PARP with 1,5-dihydroxyisoquinoline (DIQ) delayed the time course of oxidant-induced EC death. Concurrent addition of the protein synthesis inhibitor, cycloheximide, or the endonuclease inhibitor, aurintricarboxylic acid, to PARP-inhibited cells further delayed the onset and attenuated the extent of H₂O₂-induced cell lysis, consistent with an active mode of cell death. Caspase-3-like activity, a hallmark of apoptosis, was negligible in oxidant-treated EC alone, however, inhibition of PARP by 3-aminobenzamide or DIQ dramatically increased caspase-3-like activity. Morphological assessment confirmed that the primary mode of death in oxidant-stressed EC was oncosis. However, following PARP inhibition, the cells switched to apoptosis. Since inflammation is associated with oncosis and not apoptosis, the results presented here could explain the beneficial effects seen with PARP inhibition in various in vivo models of oxidant injury and provide a mechanism to manipulate this injury into a state of cell death that could ultimately be controlled.     

Detection of poly(ADP-ribose) synthesis in Drosophila testes upon γ-irradiation

Poly(ADP-ribose) polymerase (PARP) activity is widespread among eukaryotes. Upon DNA damage PARP binds to DNA strand breaks and transfers ADP-ribose residues from NAD⁺ to acceptor proteins and to ADP-ribosyl protein adducts. This leads to branched polymers of protein-coupled poly(ADP-ribose) (pADPr). Because the germline of Drosophila has recently become important in the study of DNA double-strand break repair (DSBR) as opposed to somatic DSBR we tested whether the catalytic activity of PARP can be stimulated by γ-irradiation during Drosophila spermatogenesis. Using antibodies against pADPr we detected a significant increase in PARP activity in male germline cells during spermatogenesis upon γ-irradiation. Different stages of spermatogenesis revealed different subnuclear localization patterns of pADPr. In premeiotic and postmeiotic cells pADPr localized in a pattern overlapping with lamin and topoisomerase II at the nuclear rim. In primary spermatocytes pADPr is associated with three loci corresponding to the chromosomes at the nuclear periphery. Received: 12 October 1998; in revised form: 21 December 1998 / Accepted: 23 December 1998     

Activation of the SNF2 Family ATPase ALC1 by Poly(ADP-ribose) in a Stable ALC1·PARP1·Nucleosome Intermediate

The human ALC1/CHD1L oncogene encodes an SNF2 family ATPase with a macrodomain that binds poly(ADP-ribose) (PAR). We and others previously showed that ALC1 possesses a cryptic ATP-dependent nucleosome remodeling activity that is potently activated in the presence of PARP1 and NAD⁺, its substrate for PAR synthesis. In this work, we dissected the mechanism by which PARP1 and NAD⁺ activate ALC1 nucleosome remodeling. We demonstrate that ALC1 activation depends on the formation of a stable ALC1·PARylated PARP1·nucleosome intermediate. In addition, by exploiting a novel PAR footprinting assay, we obtained evidence that the ALC1 macrodomain remains stably associated with PAR on autoPARylated PARP1 during the course of nucleosome remodeling reactions. Taken together, our findings are consistent with the model that PAR present on PARylated PARP1 acts as an allosteric effector of ALC1 nucleosome remodeling activity.     

Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle

Microorganisms can reduce toxic chromate to less toxic trivalent chromium [Cr(III)]. Besides Cr(OH)₃ precipitates, some soluble organo-Cr(III) complexes are readily formed upon microbial, enzymatic, and chemical reduction of chromate. However, the biotransformation of the organo-Cr(III) complexes has not been characterized. We have previously reported the formation of a nicotinamide adenine dinucleotide (NAD⁺)-Cr(III) complex after enzymatic reduction of chromate. Although the NAD⁺-Cr(III) complex was stable under sterile conditions, microbial cells were identified as precipitates in a non-sterile NAD⁺-Cr(III) solution after extended incubation. The most dominant bacterium PTX1 was isolated and assigned to Leifsonia genus by phylogenetic analysis of 16S rRNA gene sequence. PTX1 grew slowly on NAD⁺ with a doubling time of 17 h, and even more slowly on the NAD⁺-Cr(III) complex with an estimated doubling time of 35 days. The slow growth suggests that PTX1 passively grew on trace NAD⁺ dissociated from the NAD⁺-Cr(III) complex, facilitating further dissociation of the complex and formation of Cr(III) precipitates. Thus, organo-Cr(III) complexes might be an intrinsic link of the chromium biogeochemical cycle; they can be produced during chromate reduction and then further mineralized by microorganisms.     

Conserved water-mediated recognition and dynamics of NAD+ (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10?ns simulation of the IMP–NAD⁺ complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD⁺. Three conserved water molecules (W1, W, and W1′) in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD⁺) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP–NAD⁺-enzyme complexes and their recognition to NAD⁺, some covalent modification at carboxamide group of di-nucleotide (NAD⁺) has been made by substituting the –CONH₂group by –CONHNH₂ (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.     

Development and validation of high-throughput screening assays for poly(ADP-ribose) polymerase-2 inhibitors

Poly(ADP-ribose) polymerase-1 and -2 (PARP1/2) are two key facilitators of DNA repair and are implicated in the pathogenesis of cancers and several chronic diseases. Inhibitors of PARP1/2 have shown powerful therapeutic effects in the treatment of cancer, cerebral ischemia, and inflammation. In addition, evidence from several studies suggests unique functions for PARP2 in genome surveillance, spermatogenesis, adipogenesis, and T cell development, and PARP2-specific inhibitors might have many other applications. To acquire PARP1/2 inhibitors, many high-throughput screening (HTS) assays for PARP1 inhibitors have been developed. However, detailed screening assays for PARP2 inhibitors have not been reported. Herein, three HTS assays for PARP2 inhibitors were developed and validated with reference inhibitors in each case. The results suggest that the HTS assays for PARP2 inhibitors using chemical quantification of NAD⁺, biotin-based quantification of PAR, and ELISA quantification of PAR are sensitive, robust, and cost effective.