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• W2122545253 abstract "•ARTD1 activation-mediated ATP depletion initiates in the mitochondria•ARTD1 activation suppresses glycolysis and oxidative phosphorylation•NAD+ depletion does not affect glycolysis or cellular ATP levels•HK1 activity is inhibited by ARTD1 activation to suppress glycolysis ARTD1 (PARP1) is a key enzyme involved in DNA repair through the synthesis of poly(ADP-ribose) (PAR) in response to strand breaks, and it plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD+ depletion and ATP loss; however, the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we compared the effects of ARTD1 activation and direct NAD+ depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD+ depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics-based PAR interactome after DNA damage and identified hexokinase 1 (HK1) as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing insight into the importance of nucleus-to-mitochondria communication via ARTD1 activation. ARTD1 (PARP1) is a key enzyme involved in DNA repair through the synthesis of poly(ADP-ribose) (PAR) in response to strand breaks, and it plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD+ depletion and ATP loss; however, the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we compared the effects of ARTD1 activation and direct NAD+ depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD+ depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics-based PAR interactome after DNA damage and identified hexokinase 1 (HK1) as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing insight into the importance of nucleus-to-mitochondria communication via ARTD1 activation. The human genome encodes 17 poly(ADP-ribose) polymerase (PARP) or ADP-ribosyltransferase diphtheria toxin-like (ARTD) proteins that are involved in regulating a variety of cellular processes including DNA damage signaling and repair, chromatin remodeling, transcription, epigenetic gene regulation, mitosis, and differentiation (Hassa et al., 2006Hassa P.O. Haenni S.S. Elser M. Hottiger M.O. Nuclear ADP-ribosylation reactions in mammalian cells: where are we today and where are we going?.Microbiol. Mol. Biol. Rev. 2006; 70: 789-829Crossref PubMed Scopus (576) Google Scholar). All of the catalytically active members of the ARTD/PARP family consume NAD+ to catalyze ADP-ribosylation of their target substrates but are classified as mono- or poly-(ADP-ribosyl) transferases depending on their ability to transfer monomers or polymers of ADP-ribose (Hottiger et al., 2010Hottiger M.O. Hassa P.O. Lüscher B. Schüler H. Koch-Nolte F. Toward a unified nomenclature for mammalian ADP-ribosyltransferases.Trends Biochem. Sci. 2010; 35: 208-219Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). In particular, poly(ADP-ribosyl)ation is known for its switch-like effects on acceptor proteins by virtue of its high charge density and steric hindrance. As a consequence, this posttranslational modification can activate or inhibit protein functions, disrupt or promote protein-protein interactions, or facilitate protein subcellular relocalization (Hottiger et al., 2010Hottiger M.O. Hassa P.O. Lüscher B. Schüler H. Koch-Nolte F. Toward a unified nomenclature for mammalian ADP-ribosyltransferases.Trends Biochem. Sci. 2010; 35: 208-219Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1 or PARP1) primarily functions as a key enzyme of the base excision repair (BER) and single-strand break repair (SSBR) pathways (Almeida and Sobol, 2007Almeida K.H. Sobol R.W. A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification.DNA Repair (Amst.). 2007; 6: 695-711Crossref PubMed Scopus (340) Google Scholar). ARTD1 participates in additional DNA repair pathways such as nonhomologous end-joining (NHEJ), nucleotide excision repair (NER), in sensing and repairing DNA double-strand breaks and is suggested to participate in the excision step during mismatch repair (De Vos et al., 2012De Vos M. Schreiber V. Dantzer F. The diverse roles and clinical relevance of PARPs in DNA damage repair: current state of the art.Biochem. Pharmacol. 2012; 84: 137-146Crossref PubMed Scopus (382) Google Scholar). The participation of ARTD1 in these DNA repair pathways depends on its ability to detect and bind to DNA single-strand breaks with high affinity (Langelier et al., 2012Langelier M.F. Planck J.L. Roy S. Pascal J.M. Structural basis for DNA damage-dependent poly(ADP-ribosyl)ation by human PARP-1.Science. 2012; 336: 728-732Crossref PubMed Scopus (427) Google Scholar). In BER, a strand break is a normal repair intermediate, which is formed following hydrolysis of the DNA backbone by an apurinic/apyrimidinic endonuclease, APE1 (Almeida and Sobol, 2007Almeida K.H. Sobol R.W. A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification.DNA Repair (Amst.). 2007; 6: 695-711Crossref PubMed Scopus (340) Google Scholar, Svilar et al., 2011Svilar D. Goellner E.M. Almeida K.H. Sobol R.W. Base excision repair and lesion-dependent subpathways for repair of oxidative DNA damage.Antioxid. Redox Signal. 2011; 14: 2491-2507Crossref PubMed Scopus (195) Google Scholar), triggering ARTD1 activation. Upon activation, ARTD1 synthesizes poly-(ADP-ribose) (PAR) that functions as a mechanism of chromatin decondensation and generates a loading platform for the recruitment of the BER machinery to the lesion site, including proteins such as X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1), poly (ADP-ribose) glycohydrolase (PARG) and DNA polymerase β (Polß; Schreiber et al., 2006Schreiber V. Dantzer F. Ame J.C. de Murcia G. Poly(ADP-ribose): novel functions for an old molecule.Nat. Rev. Mol. Cell Biol. 2006; 7: 517-528Crossref PubMed Scopus (1590) Google Scholar, Svilar et al., 2011Svilar D. Goellner E.M. Almeida K.H. Sobol R.W. Base excision repair and lesion-dependent subpathways for repair of oxidative DNA damage.Antioxid. Redox Signal. 2011; 14: 2491-2507Crossref PubMed Scopus (195) Google Scholar). Successful recruitment of the downstream BER proteins facilitates repair of the strand break, suppressing further ARTD1 activity and PAR synthesis (Masson et al., 1998Masson M. Niedergang C. Schreiber V. Muller S. Menissier-de Murcia J. de Murcia G. XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage.Mol. Cell. Biol. 1998; 18: 3563-3571Crossref PubMed Scopus (833) Google Scholar, Tang et al., 2010Tang J.B. Goellner E.M. Wang X.H. Trivedi R.N. St Croix C.M. Jelezcova E. Svilar D. Brown A.R. Sobol R.W. Bioenergetic metabolites regulate base excision repair-dependent cell death in response to DNA damage.Mol. Cancer Res. 2010; 8: 67-79Crossref PubMed Scopus (56) Google Scholar). Conversely, unrepaired DNA breaks, resulting from excessive genotoxin exposure and/or from DNA repair defects, leads to persistent ARTD1 activation and cell death (Gottipati et al., 2010Gottipati P. Vischioni B. Schultz N. Solomons J. Bryant H.E. Djureinovic T. Issaeva N. Sleeth K. Sharma R.A. Helleday T. Poly(ADP-ribose) polymerase is hyperactivated in homologous recombination-defective cells.Cancer Res. 2010; 70: 5389-5398Crossref PubMed Scopus (179) Google Scholar, Juarez-Salinas et al., 1979Juarez-Salinas H. Sims J.L. Jacobson M.K. Poly(ADP-ribose) levels in carcinogen-treated cells.Nature. 1979; 282: 740-741Crossref PubMed Scopus (256) Google Scholar, Tang et al., 2010Tang J.B. Goellner E.M. Wang X.H. Trivedi R.N. St Croix C.M. Jelezcova E. Svilar D. Brown A.R. Sobol R.W. Bioenergetic metabolites regulate base excision repair-dependent cell death in response to DNA damage.Mol. Cancer Res. 2010; 8: 67-79Crossref PubMed Scopus (56) Google Scholar). Uncontrolled or excessive activation of ARTD1 is responsible for numerous pathological outcomes including streptozotocin-induced pancreatic beta-cell death and the onset of diabetes (Burns and Gold, 2007Burns N. Gold B. The effect of 3-methyladenine DNA glycosylase-mediated DNA repair on the induction of toxicity and diabetes by the beta-cell toxicant streptozotocin.Toxicol. Sci. 2007; 95: 391-400Crossref PubMed Scopus (33) Google Scholar, Masutani et al., 1999Masutani M. Suzuki H. Kamada N. Watanabe M. Ueda O. Nozaki T. Jishage K. Watanabe T. Sugimoto T. Nakagama H. et al.Poly(ADP-ribose) polymerase gene disruption conferred mice resistant to streptozotocin-induced diabetes.Proc. Natl. Acad. Sci. USA. 1999; 96: 2301-2304Crossref PubMed Scopus (263) Google Scholar, Pieper et al., 1999Pieper A.A. Brat D.J. Krug D.K. Watkins C.C. Gupta A. Blackshaw S. Verma A. Wang Z.Q. Snyder S.H. Poly(ADP-ribose) polymerase-deficient mice are protected from streptozotocin-induced diabetes.Proc. Natl. Acad. Sci. USA. 1999; 96: 3059-3064Crossref PubMed Scopus (287) Google Scholar) as well as tissue injury from cerebral and myocardial ischemia (Eliasson et al., 1997Eliasson M.J. Sampei K. Mandir A.S. Hurn P.D. Traystman R.J. Bao J. Pieper A. Wang Z.Q. Dawson T.M. Snyder S.H. Dawson V.L. Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia.Nat. Med. 1997; 3: 1089-1095Crossref PubMed Scopus (951) Google Scholar, Endres et al., 1997Endres M. Wang Z.Q. Namura S. Waeber C. Moskowitz M.A. Ischemic brain injury is mediated by the activation of poly(ADP-ribose)polymerase.J. Cereb. Blood Flow Metab. 1997; 17: 1143-1151Crossref PubMed Scopus (571) Google Scholar, Pieper et al., 2000Pieper A.A. Walles T. Wei G. Clements E.E. Verma A. Snyder S.H. Zweier J.L. Myocardial postischemic injury is reduced by polyADPripose polymerase-1 gene disruption.Mol. Med. 2000; 6: 271-282Crossref PubMed Google Scholar). In these and other mouse model studies, ARTD1 activation-induced tissue injury results from the accumulation of DNA repair intermediates (Calvo et al., 2013Calvo J.A. Moroski-Erkul C.A. Lake A. Eichinger L.W. Shah D. Jhun I. Limsirichai P. Bronson R.T. Christiani D.C. Meira L.B. Samson L.D. Aag DNA glycosylase promotes alkylation-induced tissue damage mediated by Parp1.PLoS Genet. 2013; 9: e1003413Crossref PubMed Scopus (45) Google Scholar). Cell death due to ARTD1 activation was originally suggested to involve energy metabolite (NAD+ and ATP) depletion (Berger, 1985Berger N.A. Poly(ADP-ribose) in the cellular response to DNA damage.Radiat. Res. 1985; 101: 4-15Crossref PubMed Scopus (681) Google Scholar, Berger et al., 1983Berger N.A. Sims J.L. Catino D.M. Berger S.J. Poly(ADP-ribose) polymerase mediates the suicide response to massive DNA damage: studies in normal and DNA-repair defective cells.Int. Symp. Princess Takamatsu Cancer Res. Fund. 1983; 13: 219-226PubMed Google Scholar, Jacobson et al., 1980Jacobson M.K. Levi V. Juarez-Salinas H. Barton R.A. Jacobson E.L. Effect of carcinogenic N-alkyl-N-nitroso compounds on nicotinamide adenine dinucleotide metabolism.Cancer Res. 1980; 40: 1797-1802PubMed Google Scholar). However, the molecular mechanisms underlying ARTD1 hyperactivation-induced ATP depletion and the resulting cell death are unresolved. Confounding this issue, cell type and in particular cellular proliferation status, has yielded widely disparate observations. In astrocytes, cytosolic NAD+ depletion resulting from ARTD1 activation is suggested to trigger a glycolytic block that can be rescued by NAD+ or tricarboxylic acid (TCA) cycle substrates, such as α-ketoglutarate and pyruvate (Ying et al., 2002Ying W. Chen Y. Alano C.C. Swanson R.A. Tricarboxylic acid cycle substrates prevent PARP-mediated death of neurons and astrocytes.J. Cereb. Blood Flow Metab. 2002; 22: 774-779Crossref PubMed Scopus (124) Google Scholar, Ying et al., 2003Ying W. Garnier P. Swanson R.A. NAD+ repletion prevents PARP-1-induced glycolytic blockade and cell death in cultured mouse astrocytes.Biochem. Biophys. Res. Commun. 2003; 308: 809-813Crossref PubMed Scopus (187) Google Scholar). Neuronal cell death from ARTD1 activation is triggered by unregulated PAR synthesis, termed parthanatos (Andrabi et al., 2008Andrabi S.A. Dawson T.M. Dawson V.L. Mitochondrial and nuclear cross talk in cell death: parthanatos.Ann. N Y Acad. Sci. 2008; 1147: 233-241Crossref PubMed Scopus (241) Google Scholar) and has been suggested to play a role in multiple experimental and physiopathological scenarios, including stroke, diabetes, inflammation, and neurodegeneration. Some reports demonstrate a direct link between ARTD1 hyperactivation and mitochondrial dysfunction, notably through the loss of NAD+ that precedes the induction of the mitochondrial depolarization and mitochondria outer membrane permeability transition (Alano et al., 2004Alano C.C. Ying W. Swanson R.A. Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition.J. Biol. Chem. 2004; 279: 18895-18902Crossref PubMed Scopus (327) Google Scholar, Cipriani et al., 2005Cipriani G. Rapizzi E. Vannacci A. Rizzuto R. Moroni F. Chiarugi A. Nuclear poly(ADP-ribose) polymerase-1 rapidly triggers mitochondrial dysfunction.J. Biol. Chem. 2005; 280: 17227-17234Crossref PubMed Scopus (137) Google Scholar). In contrast, in apoptosis-deficient cells, it is suggested that only cells relying on glycolysis are sensitive to DNA damage-mediated ARTD1 hyperactivation and necrotic cell death (Zong et al., 2004Zong W.X. Ditsworth D. Bauer D.E. Wang Z.Q. Thompson C.B. Alkylating DNA damage stimulates a regulated form of necrotic cell death.Genes Dev. 2004; 18: 1272-1282Crossref PubMed Scopus (535) Google Scholar). Herein, we tested the hypothesis that the glycolytic block and loss of ATP induced by DNA damage-induced ARTD1 activation is distinct from the resulting loss of NAD+. In addition, we demonstrate that ARTD1 activation and the resulting synthesis of PAR facilitates the block to glycolysis via regulation/inhibition of PAR binding proteins including the essential glycolytic enzyme hexokinase 1 (HK1). The acute cellular response to DNA alkylation damage is dependent on the expression of the methyl-specific DNA glycosylase MPG (also known as AAG or ANPG; Tang et al., 2010Tang J.B. Goellner E.M. Wang X.H. Trivedi R.N. St Croix C.M. Jelezcova E. Svilar D. Brown A.R. Sobol R.W. Bioenergetic metabolites regulate base excision repair-dependent cell death in response to DNA damage.Mol. Cancer Res. 2010; 8: 67-79Crossref PubMed Scopus (56) Google Scholar) and an imbalance in BER protein expression can lead to an accumulation of toxic DNA repair intermediates (Fu et al., 2012Fu D. Calvo J.A. Samson L.D. Balancing repair and tolerance of DNA damage caused by alkylating agents.Nat. Rev. Cancer. 2012; 12: 104-120Crossref PubMed Scopus (627) Google Scholar). To evaluate the cellular consequences of DNA damage-induced DNA repair intermediates, we used a glioblastoma-derived cell line, LN428, with low levels of MPG and the isogenic derivative LN428/MPG overexpressing MPG (Figures S1A–S1D available online). Upon activation, ARTD1 transfers the ADP-ribosyl unit of NAD+ to synthesize PAR and as a consequence, leads to the rapid loss of total cellular NAD+. ARTD1 activation also leads to the concomitant loss of ATP and the onset of necrosis (Ha and Snyder, 1999Ha H.C. Snyder S.H. Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion.Proc. Natl. Acad. Sci. USA. 1999; 96: 13978-13982Crossref PubMed Scopus (814) Google Scholar, Tang et al., 2010Tang J.B. Goellner E.M. Wang X.H. Trivedi R.N. St Croix C.M. Jelezcova E. Svilar D. Brown A.R. Sobol R.W. Bioenergetic metabolites regulate base excision repair-dependent cell death in response to DNA damage.Mol. Cancer Res. 2010; 8: 67-79Crossref PubMed Scopus (56) Google Scholar). Exposure of LN428/MPG cells to MNNG (5 μM) results in the loss of close to 90% and 70% respectively, of total cellular NAD+ and ATP pools with no measureable metabolite depletion observed in the control LN428 cells at this dose of MNNG (Figures 1A and 1B ). As expected, pretreatment with the PARP-inhibitors ABT-888 or BMN-673 significantly rescues the MNNG-induced NAD+ loss in the LN428/MPG cells (Figure 1C). Importantly, ARTD1 inhibition completely attenuates the MNNG-induced loss of ATP in LN428/MPG cells (Figures 1D and S1K). To further investigate the involvement of ARTD1 in NAD+ and ATP loss, we depleted ARTD1 expression using an shRNA coupled with overexpressed MPG via viral transduction of LN428 cells, as described in the Supplemental Experimental Procedures. We verified ARTD1 knockdown and MPG overexpression by RT-PCR and immunoblot (Figures S1G and S1H). PAR synthesis in LN428/ARTD1-KD/MPG cells is largely impaired (Figure S1H) after MNNG even at 10 μM, as compared to LN428/MPG cells (Figure S1D). Importantly, LN428/ARTD1-KD/MPG cells present no defect in NAD+ nor ATP levels following MNNG treatment, demonstrating a direct role for ARTD1 in the loss of energy metabolites (Figures 1C and 1D; gray bars). ATP is generated in both the cytosol and the mitochondria via glycolysis and oxidative phosphorylation, respectively. However, classical measurements of ATP levels lack subcellular specificity, limiting the conclusions that can be drawn. To monitor changes in ATP levels in different compartments of living cells as a function of MNNG exposure, LN428 and LN428/MPG cells were transiently transfected with FRET-based ATP sensors, specifically targeted to the mitochondria (AT1.03m), cytosol (AT1.03c), or nucleus (AT1.03n), which produce a YFP FRET signal upon ATP binding (Imamura et al., 2009bImamura H. Nhat K.P. Togawa H. Saito K. Iino R. Kato-Yamada Y. Nagai T. Noji H. Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.Proc. Natl. Acad. Sci. USA. 2009; 106: 15651-15656Crossref PubMed Scopus (729) Google Scholar). An increase of the CFP/YFP ratio therefore indicates a loss of ATP bound to the FRET sensor. Expression of the FRET-based ATP sensors showed the expected subcellular localization measured 48 hr post-transfection (Figure S1I) and localization was not affected by DNA damaging agent or PARP inhibitor treatment (not shown). For both the LN428 and LN428/MPG cells, a 10-minute baseline FRET ratio was determined prior to the addition of MNNG (5 μM). A low CFP/YFP ratio was observed prior to treatment suggesting normal baseline ATP levels in all subcellular compartments, consistent with whole cell metabolite measurements (Figure 1B). The different subcellular compartments had slight differences in the absolute starting CFP/YFP ratio and were normalized to 1 at the baseline to compare changes between compartments (Figure S1J). Consistent with the whole cell ATP analysis, the CFP/YFP ratio remains close to the baseline in all three subcellular compartments for up to 60 min after MNNG exposure of the LN428 cells (Figure 1E), indicating that any changes in subcellular ATP due to the minimal PAR formation must be below the sensitivity of the FRET sensors. However, exposure of the LN428/MPG cells to MNNG results in a rapid and dramatic change in the CFP/YFP ratio, in line with the observed loss of total cellular ATP (Figures 1B and 1F) (see Movie S1. Visualization of MNNG-Induced Changes in Cytosolic ATP Levels Using FRET-Based ATP Sensors, Movie S2. Visualization of MNNG-Induced Changes in Mitochondrial ATP Levels Using FRET-Based ATP Sensors, Movie S3. Visualization of MNNG-Induced Changes in Nuclear ATP Levels Using FRET-Based ATP Sensors). Surprisingly, of the three subcellular compartments, an increase in the CFP/YFP ratio (and hence a loss of ATP) was first observed in the mitochondria. The MNNG-induced loss of ATP in the mitochondria began 12 min after the start of MNNG treatment, concomitant with the peak of PAR production (Figures 1F and S1D) followed by a decrease in both the cytosolic and nuclear ATP pools, beginning 24 min after MNNG exposure (Figures 1F and S1I). In the whole cell ATP analysis, the MNNG-induced loss of ATP in the LN428/MPG cells was completely blocked when ARTD1 was inhibited or depleted (Figure 1D). Similarly, pretreatment of LN428/MPG cells with the PARP inhibitor was able to rescue the ATP loss in all three subcellular compartments (Figures S1K and S1L). These results support an ARTD1-dependent signal that provides a means of intracellular communication between the nucleus and the mitochondria in response to genotoxic stress. It has been hypothesized that NAD+ consumption by ARTD1 activation is causative for the rapid depletion of cellular ATP. The kinetics of loss of the mitochondrial, cytosolic, and nuclear ATP pools in response to ARTD1 activation suggests that variations in the total cellular level of NAD+ may signal from the nucleus to the mitochondria by regulating NAD+-dependent enzymes critical for ATP biosynthesis. To examine this, we measured multiple parameters of glycolysis and mitochondrial oxidative phosphorylation in live-cell conditions with the Seahorse XF24 extracellular flux analyzer (SEFA), essentially as described (Qian and Van Houten, 2010Qian W. Van Houten B. Alterations in bioenergetics due to changes in mitochondrial DNA copy number.Methods. 2010; 51: 452-457Crossref PubMed Scopus (102) Google Scholar, Varum et al., 2011Varum S. Rodrigues A.S. Moura M.B. Momcilovic O. Easley 4th, C.A. Ramalho-Santos J. Van Houten B. Schatten G. Energy metabolism in human pluripotent stem cells and their differentiated counterparts.PLoS ONE. 2011; 6: e20914Crossref PubMed Scopus (524) Google Scholar). This real-time, live-cell analysis allows a measure of DNA damage-dependent changes in oxidative phosphorylation (oxygen consumption rate, OCR) and glycolysis (extracellular acidification rate, ECAR). The sequential addition of four metabolic inhibitors: oligomycin, FCCP, 2-deoxyglucose, and rotenone allows the calculation of four critical metabolic parameters: (1) the ATP-coupled OCR; (2) the total mitochondrial reserve capacity (TRC), or maximal respiratory rate; (3) the basal ECAR, which corresponds to the basal glycolytic rate; and (4) the oligomycin-induced ECAR. As expected, the isogenic LN428 and LN428/MPG cell lines have similar basal ECAR and OCR profiles (Figures 2A and 2B ; left), ideal for comparative analysis. Changes in glycolysis and oxidative phosphorylation were then measured in response to MNNG (1 hr, 5 μM) and in combination with ARTD1 knockdown or inhibition by pretreatment with either ABT-888 or BMN-673. In line with earlier reports using mouse astrocytes (Berger, 1985Berger N.A. Poly(ADP-ribose) in the cellular response to DNA damage.Radiat. Res. 1985; 101: 4-15Crossref PubMed Scopus (681) Google Scholar, Ying et al., 2003Ying W. Garnier P. Swanson R.A. NAD+ repletion prevents PARP-1-induced glycolytic blockade and cell death in cultured mouse astrocytes.Biochem. Biophys. Res. Commun. 2003; 308: 809-813Crossref PubMed Scopus (187) Google Scholar), strong ARTD1 activation resulted in the complete loss of glycolysis after MNNG treatment, leading to a decrease of both basal and induced ECAR in LN428/MPG but not in LN428 cells (Figures 2A and S2A). Consistent with our hypothesis that the metabolic defects result from ARTD1 activation, ABT-888 or BMN-673 treatment prior to MNNG exposure leads to a complete rescue of both basal and induced ECAR in LN428/MPG cells (Figures 2C and S2A). Moreover, we show that exposure of LN428/ARTD1-KD/MPG cells to MNNG does not lead to a loss of glycolytic rate as seen in the LN428/MPG cells (Figures 2F and S2D), providing further evidence for the direct involvement of ARTD1 in this defect of cellular metabolism. Consistent with the observed loss of NAD+ and ATP in the LN428/MPG cells in response to MNNG, we found that these cells undergo a complete loss of TRC, with no change to the ATP-coupled OCR (Figure 2B; right). The LN428 cells also present with a partial yet significant loss (50%) of TRC in response to MNNG treatment. To determine if the effects observed in both cell lines was the sole result of ARTD1 activation and NAD+ depletion, the identical analysis was performed with ARTD1 inhibited by pretreatment with ABT-888 or BMN-673. Interestingly, we observed a complete rescue of TRC in LN428 cells upon ARTD1 inhibition (Figures 2E and S2C). These data suggest that even a low level of ARTD1 activation (in the case of LN428 cells and illustrated by the anti-PAR immunoblot; Figure S1D) is able to affect the mitochondrial reserve capacity. ARTD1 inhibition is also able to rescue the mitochondrial reserve capacity defect of MNNG-treated LN428/MPG cells (Figures 2D and S2B). To further investigate the role of ARTD1 in MNNG-induced OCR defects, we submitted LN428/ARTD1-KD and LN428/ARTD1-KD/MPG cells to the same treatment and performed the same analysis. As expected, we show that LN428/ARTD1-KD cells do not present the partial defect observed in LN428 cells after MNNG treatment. Expressing MPG in the LN428/ARTD1-KD cells (LN428/ARTD1-KD/MPG) does not trigger a loss of TRC in response to MNNG treatment such as that seen in the LN428/MPG cells (Figure 2G). These results strongly suggest that cellular oxidative phosphorylation, as measured by oxidative reserve capacity, is extremely sensitive to ARTD1 activation. The same results have been observed in HeLa cells treated with increasing doses of MNNG, demonstrating that the MNNG induced metabolic defects are not glioblastoma specific nor an artifact of MPG overexpression, but applicable to multiple cell types (Figures S2F–S2J). From these data, we hypothesize that ARTD1 activation acts as the initiating signal from the nucleus to mediate suppression of both glycolytic and mitochondrial oxidative phosphorylation activity. One of the strongest phenotypes associated with ARTD1 activation is an acute loss of NAD+, with 90% of the cellular NAD+ content lost within 1 hour (Figure 1A). Therefore, a likely candidate to signal ARTD1 activation in the nucleus to the metabolic machinery in the mitochondria is the change in overall NAD+ content. To test this hypothesis, we directly reduced NAD+ levels in the absence of ARTD1 activation. To inhibit NAD+ biosynthesis, LN428/MPG cells were treated with FK866, a selective small molecule inhibitor of the essential NAD+ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT; Figure 3A; Hasmann and Schemainda, 2003Hasmann M. Schemainda I. FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for induction of tumor cell apoptosis.Cancer Res. 2003; 63: 7436-7442PubMed Google Scholar). After a 24 hr treatment with FK866 (10 nM), the NAD+ pool decreased by approximately 75%, roughly equivalent to the NAD+ loss after ARTD1 activation (Figure 3B). Surprisingly, however, ATP levels remained constant and viability was not affected in LN428/MPG cells after treatment with FK866 (Figures 3C and S3A). Consistent with a role for NAD+ as a cofactor in mitochondrial enzymatic reactions, FK866-treated cells displayed a significant decrease in total mitochondrial reserve capacity (Figures 3D and S3B). Surprisingly, NAD+ depletion by FK866 treatment had no effect on either the basal or induced ECAR, unlike MNNG treatment (Figures 3E and S3C). To demonstrate specificity and selectivity of FK866, treated cells were also supplemented with nicotinamide riboside (NR), a precursor that does not require NAMPT for conversion to NAD+ (Figure 3A). NR was synthesized and purified as described in the Supplemental Experimental Procedures (Figure S4). As expected, NR pretreatment could overcome the NAD+ depletion induced by FK866 (Figure 3F); however, NR did not prevent the loss of NAD+ or ATP after MNNG even at a 10-fold higher dose (Figures 3F and 3G). Consistently, the FK866-induced loss of TRC is completely rescued by NR (Figures 3H and S3D) but NR was unable to rescue the OCR and ECAR defects in LN428/MPG cells treated with MNNG (Figures 3H, 3I, S3D, and S3E). Yet, at low doses of MNNG, NR pretreatment was able to partially rescue NAD+ levels (Figure S3F). Interestingly, this partial rescue was not sufficient to overcome the OCR and ECAR defects (Figures S3G and S3H), suggesting a more complex effect of ARTD1 activation on cellular metabolism. The sensitivity of oxidative phosphorylation but not glycolysis to direct depletion of NAD+, suggests that ARTD1-mediated consumption of NAD+ is a major contributor to the DNA damage-induced loss of oxidative phosphorylation, but inhibition of glycolysis occurs through another unknown mechanism of ARTD1 activation. The nucleo-cytoplasmic translocation of PAR has been extensively described (Andrabi et al., 2006Andrabi S.A. Kim N.S. Yu S.W. Wang H. Koh D.W. Sasaki M. Klaus J.A. Otsuka T. Zhang Z. Koehler R.C. et al.Poly(ADP-ribose) (PAR) polymer is a death signal.Proc. Natl. Acad. Sci. USA. 2006; 103: 18308-18313Crossref PubMed Scopus (520) Google Scholar). As an example, it has been demonstrated that the movement of PAR into the cytosol triggers the release of the mitochondrial protein AIF upon binding of PAR to the AIF PAR-binding motif (PBM; Yu et al., 2006Yu S.W. Andrabi S." @default.
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• W2122545253 date "2014-09-01" @default.
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• W2122545253 title "ARTD1/PARP1 Negatively Regulates Glycolysis by Inhibiting Hexokinase 1 Independent of NAD + Depletion" @default.
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• W2122545253 doi "https://doi.org/10.1016/j.celrep.2014.08.036" @default.
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