Inhibiting poly(ADP-ribosylation) improves axon regeneration.

PubMed

Byrne, Alexandra B; McWhirter, Rebecca D; Sekine, Yuichi; Strittmatter, Stephen M; Miller, David M; Hammarlund, Marc

2016-10-04

The ability of a neuron to regenerate its axon after injury depends in part on its intrinsic regenerative potential. Here, we identify novel intrinsic regulators of axon regeneration: poly(ADP-ribose) glycohodrolases (PARGs) and poly(ADP-ribose) polymerases (PARPs). PARGs, which remove poly(ADP-ribose) from proteins, act in injured C. elegans GABA motor neurons to enhance axon regeneration. PARG expression is regulated by DLK signaling, and PARGs mediate DLK function in enhancing axon regeneration. Conversely, PARPs, which add poly(ADP-ribose) to proteins, inhibit axon regeneration of both C. elegans GABA neurons and mammalian cortical neurons. Furthermore, chemical PARP inhibitors improve axon regeneration when administered after injury. Our results indicate that regulation of poly(ADP-ribose) levels is a critical function of the DLK regeneration pathway, that poly-(ADP ribosylation) inhibits axon regeneration across species, and that chemical inhibition of PARPs can elicit axon regeneration.

Inhibition of poly(ADP-ribose) polymerase interferes with Trypanosoma cruzi infection and proliferation of the parasite.

PubMed

Vilchez Larrea, Salomé C; Haikarainen, Teemu; Narwal, Mohit; Schlesinger, Mariana; Venkannagari, Harikanth; Flawiá, Mirtha M; Villamil, Silvia H Fernández; Lehtiö, Lari

2012-01-01

Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.

Inhibiting poly(ADP-ribosylation) improves axon regeneration

PubMed Central

Byrne, Alexandra B; McWhirter, Rebecca D; Sekine, Yuichi; Strittmatter, Stephen M; Miller, David M; Hammarlund, Marc

2016-01-01

The ability of a neuron to regenerate its axon after injury depends in part on its intrinsic regenerative potential. Here, we identify novel intrinsic regulators of axon regeneration: poly(ADP-ribose) glycohodrolases (PARGs) and poly(ADP-ribose) polymerases (PARPs). PARGs, which remove poly(ADP-ribose) from proteins, act in injured C. elegans GABA motor neurons to enhance axon regeneration. PARG expression is regulated by DLK signaling, and PARGs mediate DLK function in enhancing axon regeneration. Conversely, PARPs, which add poly(ADP-ribose) to proteins, inhibit axon regeneration of both C. elegans GABA neurons and mammalian cortical neurons. Furthermore, chemical PARP inhibitors improve axon regeneration when administered after injury. Our results indicate that regulation of poly(ADP-ribose) levels is a critical function of the DLK regeneration pathway, that poly-(ADP ribosylation) inhibits axon regeneration across species, and that chemical inhibition of PARPs can elicit axon regeneration. DOI: http://dx.doi.org/10.7554/eLife.12734.001 PMID:27697151

The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases.

PubMed

Hassa, Paul O; Hottiger, Michael O

2008-01-01

Poly-ADP-ribose metabolism plays a mayor role in a wide range of biological processes, such as maintenance of genomic stability, transcriptional regulation, energy metabolism and cell death. Poly-ADP-ribose polymerases (PARPs) are an ancient family of enzymes, as evidenced by the poly-ADP-ribosylating activities reported in dinoflagellates and archaebacteria and by the identification of Parp-like genes in eubacterial and archaeabacterial genomes. Six genes encoding "bona fide" PARP enzymes have been identified in mammalians: PARP1, PARP2, PARP3, PARP4/vPARP, PARP5/Tankyrases-1 and PARP6/Tankyrases-2. The best studied of these enzymes PARP1 plays a primary role in the process of poly-ADP-ribosylation. PARP1-mediated poly-ADP-ribosylation has been implicated in the pathogenesis of cancer, inflammatory and neurodegenerative disorders. This review will summarize the novel findings and concepts for PARP enzymes and their poly-ADP-ribosylation activity in the regulation of physiological and pathophysiological processes. A special focus is placed on the proposed molecular mechanisms involved in these processes, such as signaling, regulation of telomere dynamics, remodeling of chromatin structure and transcriptional regulation. A potential functional cross talk between PARP family members and other NAD+-consuming enzymes is discussed.

The 193-Kd Vault Protein, Vparp, Is a Novel Poly(Adp-Ribose) Polymerase

PubMed Central

Kickhoefer, Valerie A.; Siva, Amara C.; Kedersha, Nancy L.; Inman, Elisabeth M.; Ruland, Cristina; Streuli, Michel; Rome, Leonard H.

1999-01-01

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of ∼350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle. PMID:10477748

Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis.

PubMed

Li, Mo; Bian, Chunjing; Yu, Xiaochun

2014-01-01

Poly(ADP-ribosyl)ation is an unique posttranslational modification and required for spindle assembly and function during mitosis. However, the molecular mechanism of poly(ADP-ribose) (PAR) in mitosis remains elusive. Here, we show the evidence that PAR is recognized by ECT2, a key guanine nucleotide exchange factor in mitosis. The BRCT domain of ECT2 directly binds to PAR both in vitro and in vivo. We further found that α-tubulin is PARylated during mitosis. PARylation of α-tubulin is recognized by ECT2 and recruits ECT2 to mitotic spindle for completing mitosis. Taken together, our study reveals a novel mechanism by which PAR regulates mitosis.

Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis.

PubMed

Andrabi, Shaida A; Umanah, George K E; Chang, Calvin; Stevens, Daniel A; Karuppagounder, Senthilkumar S; Gagné, Jean-Philippe; Poirier, Guy G; Dawson, Valina L; Dawson, Ted M

2014-07-15

Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated "parthanatos" in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD(+) and energetic collapse, which have been thought to be caused by the consumption of cellular NAD(+) by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD(+) depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD(+) depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1-mediated mitochondrial dysfunction. Depleting neurons of NAD(+) with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.

Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription.

PubMed

Bütepage, Mareike; Preisinger, Christian; von Kriegsheim, Alexander; Scheufen, Anja; Lausberg, Eva; Li, Jinyu; Kappes, Ferdinand; Feederle, Regina; Ernst, Sabrina; Eckei, Laura; Krieg, Sarah; Müller-Newen, Gerhard; Rossetti, Giulia; Feijs, Karla L H; Verheugd, Patricia; Lüscher, Bernhard

2018-04-30

Macrodomains are conserved protein folds associated with ADP-ribose binding and turnover. ADP-ribosylation is a posttranslational modification catalyzed primarily by ARTD (aka PARP) enzymes in cells. ARTDs transfer either single or multiple ADP-ribose units to substrates, resulting in mono- or poly-ADP-ribosylation. TARG1/C6orf130 is a macrodomain protein that hydrolyzes mono-ADP-ribosylation and interacts with poly-ADP-ribose chains. Interactome analyses revealed that TARG1 binds strongly to ribosomes and proteins associated with rRNA processing and ribosomal assembly factors. TARG1 localized to transcriptionally active nucleoli, which occurred independently of ADP-ribose binding. TARG1 shuttled continuously between nucleoli and nucleoplasm. In response to DNA damage, which activates ARTD1/2 (PARP1/2) and promotes synthesis of poly-ADP-ribose chains, TARG1 re-localized to the nucleoplasm. This was dependent on the ability of TARG1 to bind to poly-ADP-ribose. These findings are consistent with the observed ability of TARG1 to competitively interact with RNA and PAR chains. We propose a nucleolar role of TARG1 in ribosome assembly or quality control that is stalled when TARG1 is re-located to sites of DNA damage.

Regulatory Control of Breast Tumor Cell Poly (ADP-Ribose) Polymerase

DTIC Science & Technology

2002-08-01

DNA replication complex (designated the DNA synthesome) from a variety of non-malignant and malignant tumor cells including breast cancer cells. We have shown that poly(ADP-ribose) polymerase PARP is among the components of the DNA synthesome. The transformation of a non-malignant human breast cell to a malignant state was accompanied by a significant alteration in the 2-D PAGE profile of specific protein components of the DNA synthesome (such as PCNA) together with a 6-8 decrease in the replication fidelity of the DNA

CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation.

PubMed

Smith, Rebecca; Sellou, Hafida; Chapuis, Catherine; Huet, Sébastien; Timinszky, Gyula

2018-05-04

One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. This early relaxation is triggered by poly(ADP-ribosyl)ation by PARP1 in addition to ATP-dependent chromatin remodeling. CHD4 recruits to DNA breaks in a PAR-dependent manner, although it lacks any recognizable PAR-binding domain, and has the ability to relax chromatin structure. However, its role in chromatin relaxation at the site of DNA damage has not been explored. Using a live cell fluorescence three-hybrid assay, we demonstrate that the recruitment of CHD4 to DNA damage, while being poly(ADP-ribosyl)ation-dependent, is not through binding poly(ADP-ribose). Additionally, we show that CHD3 is recruited to DNA breaks in the same manner as CHD4 and that both CHD3 and CHD4 play active roles in chromatin remodeling at DNA breaks. Together, our findings reveal a two-step mechanism for DNA damage induced chromatin relaxation in which PARP1 and the PAR-binding remodeler activities of Alc1/CHD1L induce an initial chromatin relaxation phase that promotes the subsequent recruitment of CHD3 and CHD4 via binding to DNA for further chromatin remodeling at DNA breaks.

Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

PubMed Central

Pellegrino, Stefania; Altmeyer, Matthias

2016-01-01

Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

Approche morphologique de la fragmentation de l'ADN radio-induite par immunomarquage anti-poly (ADP-ribose) polymérase (PARP) : étude de cultures d'oligodendrogliomes

NASA Astrophysics Data System (ADS)

Varlet, P.; Beuvon, F.; Cervera, P.; Averbeck, D.; Daumas-Duport, C.

1998-04-01

Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme encompassing two zinc finger motifs which specifically binds to radiation induced DNA strand breaks. We develop a new immuno-labelling of poly ADP-ribose which coupled together with the immunodetection of cells in cycle with MIB1, permits to detect and quantify the DNA fragmentation induced by radiations (Cesium137). This method, applied to organotypical cultures of human oligodendroglioma, submitted to radiation, a dose dependant nuclear signal. This one increased significantly in the presence of a radiosensitizer like iododeoxyuridine (IUDR 5 g/ml). This poly ADP-ribose immunodetection can be useful, to detect furtherly the individual radiosensitivity of human glioma. Les protéases “ICE-like" ou caspases, sont les homologues humaines du produit du gène ced-3 du ver Caenorhabditis elegans et sont activées lors des étapes précoces de l'apoptose. L'objectif de ce travail vise à déterminer dans quelle mesure l'inhibition de l'une d'entre elles, la caspase-3 est susceptible de modifier la sensibilité des cellules vis-à-vis de l'apoptose radioinduite. Des lymphocytes spléniques murins irradiés en présence de Ac-DVED-CHO un inhibiteur spécifique de la caspase-3 présentent un taux de particules hypodiploïdes radioinduites bien inférieur à celui des contrôles et une diminution drastique de la fragmentation internucléosomale de l'ADN. Toutefois, ni l'externalisation des phospholipides anioniques, autre marqueur spécifique de l'apoptose, ni la viabilité ne sont affectées.

Protein Poly(ADP-ribosyl)ation Regulates Arabidopsis Immune Gene Expression and Defense Responses

PubMed Central

Feng, Baomin; Liu, Chenglong; de Oliveira, Marcos V. V.; Intorne, Aline C.; Li, Bo; Babilonia, Kevin; de Souza Filho, Gonçalo A.; Shan, Libo; He, Ping

2015-01-01

Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose) glycohydrolase 1 (atparg1) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R) in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks. PMID:25569773

Molecular Bases of Catalysis and ADP-Ribose Preference of Human Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase and Conversion by Mutagenesis to a Preferential Cyclic ADP-Ribose Phosphohydrolase

PubMed Central

Cabezas, Alicia; Ribeiro, João Meireles; Rodrigues, Joaquim Rui; López-Villamizar, Iralis; Fernández, Ascensión; Canales, José; Pinto, Rosa María; Costas, María Jesús; Cameselle, José Carlos

2015-01-01

Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose K m and unchanged k cat of F37A-ADPRibase-Mn, while the K m values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type. PMID:25692488

Crosstalk between poly(ADP-ribose) polymerase and sirtuin enzymes

PubMed Central

Cantó, Carles; Sauve, Anthony A.; Bai, Peter

2013-01-01

Poly(ADP-ribose) polymerases (PARPs) are NAD+ dependent enzymes that were identified as DNA repair proteins, however, today it seems clear that PARPs are responsible for a plethora of biological functions. Sirtuins (SIRTs) are NAD+-dependent deacetylase enzymes involved in the same biological processes as PARPs raising the question whether PARP and SIRT enzymes may interact with each other in physiological and pathophysiological conditions. Hereby we review the current understanding of the SIRT-PARP interplay in regard to the biochemical nature of the interaction (competition for the common NAD+ substrate, mutual posttranslational modifications and direct transcriptional effects) and the physiological, or pathophysiological consequences of the interactions (metabolic events, oxidative stress response, genomic stability and ageing). Finally, we give an overview of the possibilities of pharmacological intervention to modulate PARP and SIRT enzymes either directly, or through modulating NAD+ homeostasis. PMID:23357756

Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation

PubMed Central

Hodgson, Andrea; Wier, Eric M.; Wen, Matthew G.; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y.

2016-01-01

The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production. PMID:27635653

Structural basis for lack of ADP-ribosyltransferase activity in poly(ADP-ribose) polymerase-13/zinc finger antiviral protein.

PubMed

Karlberg, Tobias; Klepsch, Mirjam; Thorsell, Ann-Gerd; Andersson, C David; Linusson, Anna; Schüler, Herwig

2015-03-20

The mammalian poly(ADP-ribose) polymerase (PARP) family includes ADP-ribosyltransferases with diphtheria toxin homology (ARTD). Most members have mono-ADP-ribosyltransferase activity. PARP13/ARTD13, also called zinc finger antiviral protein, has roles in viral immunity and microRNA-mediated stress responses. PARP13 features a divergent PARP homology domain missing a PARP consensus sequence motif; the domain has enigmatic functions and apparently lacks catalytic activity. We used x-ray crystallography, molecular dynamics simulations, and biochemical analyses to investigate the structural requirements for ADP-ribosyltransferase activity in human PARP13 and two of its functional partners in stress granules: PARP12/ARTD12, and PARP15/BAL3/ARTD7. The crystal structure of the PARP homology domain of PARP13 shows obstruction of the canonical active site, precluding NAD(+) binding. Molecular dynamics simulations indicate that this closed cleft conformation is maintained in solution. Introducing consensus side chains in PARP13 did not result in 3-aminobenzamide binding, but in further closure of the site. Three-dimensional alignment of the PARP homology domains of PARP13, PARP12, and PARP15 illustrates placement of PARP13 residues that deviate from the PARP family consensus. Introducing either one of two of these side chains into the corresponding positions in PARP15 abolished PARP15 ADP-ribosyltransferase activity. Taken together, our results show that PARP13 lacks the structural requirements for ADP-ribosyltransferase activity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of poly(ADP-ribose) polymerase prevents allergen-induced asthma-like reaction in sensitized Guinea pigs.

PubMed

Suzuki, Ylenia; Masini, Emanuela; Mazzocca, Cosimo; Cuzzocrea, Salvatore; Ciampa, Anna; Suzuki, Hisanori; Bani, Daniele

2004-12-01

Poly(ADP-ribose) polymerase (PARP) plays an important role in tissue injury in conditions associated with oxidative stress and inflammation. Because asthma is a chronic inflammatory disorder of the airways, we designed the present experimental study to evaluate the effects of PARP inhibition on allergen-induced asthma-like reaction in ovalbumin-sensitized guinea pigs. Cough and dyspnea in response to ovalbumin aerosol were absent in naive guinea pigs, whereas they became severe in the sensitized animals. In the latter ones, ovalbumin aerosol also induced a rapid increase in PARP activity, bronchiolar constriction, pulmonary air space inflation, mast cell degranulation, poly(ADP-ribose) and nitrotyrosine immunostaining, myeloperoxidase activity, and malondialdehyde in lung tissue, as well as a rise in the amounts of nitrites and tumor necrosis factor-alpha in bronchoalveolar lavage fluid. Pretreatment with the PARP inhibitors 3-aminobenzamide (10 mg/kg b.wt.) or 5-aminoisoquinolinone (0.5 mg/kg b.wt.) given i.p. 3 h before ovalbumin challenge significantly reduced the severity of cough and the occurrence of dyspnea and delayed the onset of respiratory abnormalities. Both PARP inhibitors were also able to prevent the above morphological and biochemical changes of lung tissue or bronchoalveolar lavage fluid induced by ovalbumin challenge. Conversely, p-aminobenzoic acid, the inactive analog of 3-aminobenzamide, had no effects.

Poly(ADP-ribose) polymerase-1 (Parp-1)-deficient mice demonstrate abnormal antibody responses

PubMed Central

Ambrose, Helen E; Willimott, Shaun; Beswick, Richard W; Dantzer, Françoise; de Murcia, Josiane Ménissier; Yelamos, José; Wagner, Simon D

2009-01-01

Poly(ADP-ribosylation) of acceptor proteins is an epigenetic modification involved in DNA strand break repair, recombination and transcription. Here we provide evidence for the involvement of poly(ADP-ribose) polymerase-1 (Parp-1) in antibody responses. Parp-1−/− mice had increased numbers of T cells and normal numbers of total B cells. Marginal zone B cells were mildly reduced in number, and numbers of follicular B cells were preserved. There were abnormal levels of basal immunoglobulins, with reduced levels of immunoglobulin G2a (IgG2a) and increased levels of IgA and IgG2b. Analysis of specific antibody responses showed that T cell-independent responses were normal but T cell-dependent responses were markedly reduced. Germinal centres were normal in size and number. In vitro purified B cells from Parp-1−/− mice proliferated normally and showed normal IgM secretion, decreased switching to IgG2a but increased IgA secretion. Collectively our results demonstrate that Parp-1 has essential roles in normal T cell-dependent antibody responses and the regulation of isotype expression. We speculate that Parp-1 forms a component of the protein complex involved in resolving the DNA double-strand breaks that occur during class switch recombination. PMID:18778284

Poly(ADP-ribose) polymerase-deficient mice are protected from streptozotocin-induced diabetes

PubMed Central

Pieper, Andrew A.; Brat, Daniel J.; Krug, David K.; Watkins, Crystal C.; Gupta, Alok; Blackshaw, Seth; Verma, Ajay; Wang, Zhao-Qi; Snyder, Solomon H.

1999-01-01

Streptozotocin (STZ) selectively destroys insulin-producing beta islet cells of the pancreas providing a model of type I diabetes. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme whose overactivation by DNA strand breaks depletes its substrate NAD+ and then ATP, leading to cellular death from energy depletion. We demonstrate DNA damage and a major activation of PARP in pancreatic islets of STZ-treated mice. These mice display a 500% increase in blood glucose and major pancreatic islet damage. In mice with homozygous targeted deletion of PARP (PARP −/−), blood glucose and pancreatic islet structure are normal, indicating virtually total protection from STZ diabetes. Partial protection occurs in PARP +/− animals. Thus, PARP activation may participate in the pathophysiology of type I diabetes, for which PARP inhibitors might afford therapeutic benefit. PMID:10077636

ExpandplusCrystal Structures of Poly(ADP-ribose) Polymerase-1 (PARP-1) Zinc Fingers Bound to DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

M Langelier; J Planck; S Roy

2011-12-31

Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNAmore » interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.« less

Tocotrienols promote apoptosis in human breast cancer cells by inducing poly(ADP-ribose) polymerase cleavage and inhibiting nuclear factor kappa-B activity.

PubMed

Loganathan, R; Selvaduray, K R; Nesaretnam, K; Radhakrishnan, A K

2013-04-01

Tocotrienols and tocopherols are members of the vitamin E family, with similar structures; however, only tocotrienols have been reported to achieve potent anti-cancer effects. The study described here has evaluated anti-cancer activity of vitamin E to elucidate mechanisms of cell death, using human breast cancer cells. Anti-cancer activity of a tocotrienol-rich fraction (TRF) and a tocotrienol-enriched fraction (TEF) isolated from palm oil, as well as pure vitamin E analogues (α-tocopherol, α-, δ- and γ-tocotrienols) were studied using highly aggressive triple negative MDA-MB-231 cells and oestrogen-dependent MCF-7 cells, both of human breast cancer cell lines. Cell population growth was evaluated using a Coulter particle counter. Cell death mechanism, poly(ADP-ribose) polymerase cleavage and levels of NF-κB were determined using commercial ELISA kits. Tocotrienols exerted potent anti-proliferative effects on both types of cell by inducing apoptosis, the underlying mechanism of cell death being ascertained using respective IC50 concentrations of all test compounds. There was marked induction of apoptosis in both cell lines by tocotrienols compared to treatment with Paclitaxel, which was used as positive control. This activity was found to be associated with cleavage of poly(ADP-ribose) polymerase (a DNA repair protein), demonstrating involvement of the apoptotic cell death signalling pathway. Tocotrienols also inhibited expression of nuclear factor kappa-B (NF-κB), which in turn can increase sensitivity of cancer cells to apoptosis. Tocotrienols induced anti-proliferative and apoptotic effects in association with DNA fragmentation, poly(ADP-ribose) polymerase cleavage and NF-κB inhibition in the two human breast cancer cell lines. © 2013 Blackwell Publishing Ltd.

DNA Damage Repair and the Emerging Role of Poly(ADP-ribose) Polymerase Inhibition in Cancer Therapeutics.

PubMed

Rabenau, Karen; Hofstatter, Erin

2016-07-01

As a result of improved understanding of DNA repair mechanisms, poly(ADP-ribose) polymerase inhibitors (PARPi) are increasingly recognized to play an important therapeutic role in the treatment of cancer. The aim of this article is to provide a review of PARPi function in DNA damage repair and synthetic lethality and to demonstrate how these mechanisms can be exploited to provide new PARPi-based therapies to patients with solid tumors. Literature from a range of sources, including PubMed and MEDLINE, were searched to identify recent reports regarding DNA damage repair and PARPi. DNA damage repair is central to cellular viability. The family of poly(ADP-ribose) polymerase proteins play multiple intracellular roles in DNA repair, but function primarily in the resolution of repair of single-strand DNA breaks. Insights through the discovery of germline BRCA1/2 mutations led to the understanding of synthetic lethality and the potential therapeutic role of PARPi in the treatment of cancer. Further understanding of DNA damage repair and the concept of BRCA-like tumors have catalyzed PARPi clinical investigation in multiple oncologic settings. PARPi hold great promise in the treatment of solid tumors, both as monotherapy and in combination with other cancer therapeutics. Multiple PARPi clinical trials are currently underway. Further understanding of aberrant DNA repair mechanisms in the germline and in the tumor genome will allow clinicians and researchers to apply PARPi most strategically in the era of personalized medicine. Copyright © 2016 Elsevier HS Journals, Inc. All rights reserved.

Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors.

PubMed

Thorsell, Ann-Gerd; Ekblad, Torun; Karlberg, Tobias; Löw, Mirjam; Pinto, Ana Filipa; Trésaugues, Lionel; Moche, Martin; Cohen, Michael S; Schüler, Herwig

2017-02-23

Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

The nuclear protein PH5P of the inter-alpha-inhibitor superfamily: a missing link between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and a novel actor of DNA repair?

PubMed

Jean, L; Risler, J L; Nagase, T; Coulouarn, C; Nomura, N; Salier, J P

1999-03-05

Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair.

Nickel(II) affects poly(ADP-ribose) polymerase-mediated DNA repair in normal and cancer cells.

PubMed

Wozniak, Katarzyna; Czechowska, Agnieszka; Blasiak, Janusz

2006-01-01

Nickel(II) can be genotoxic, but the mechanism of its genotoxicity is not fully understood and the process of DNA repair may be considered as its potential target. We studied the effect of nickel chloride on the poly(ADP-ribose) polymerase (PARP)-mediated repair of DNA damaged by gamma-radiation and idarubicin with the alkaline comet assay in normal and cancer cells. Our results indicate that nickel chloride at very low, non-cytotoxic concentration of 1 microM can affect PARP-mediated DNA repair of lesions evoked by idarubicin and gamma-radiation. We also suggest that in the quiescent lymphocytes treated with gamma-radiation, nickel(II) could interfere with DNA repair process independent of PARP.

Poly(ADP-Ribose) Polymerase 1 (PARP-1) Regulates Ribosomal Biogenesis in Drosophila Nucleoli

PubMed Central

Boamah, Ernest K.; Kotova, Elena; Garabedian, Mikael; Jarnik, Michael; Tulin, Alexei V.

2012-01-01

Poly(ADP-ribose) polymerase 1 (PARP1), a nuclear protein, utilizes NAD to synthesize poly(AD-Pribose) (pADPr), resulting in both automodification and the modification of acceptor proteins. Substantial amounts of PARP1 and pADPr (up to 50%) are localized to the nucleolus, a subnuclear organelle known as a region for ribosome biogenesis and maturation. At present, the functional significance of PARP1 protein inside the nucleolus remains unclear. Using PARP1 mutants, we investigated the function of PARP1, pADPr, and PARP1-interacting proteins in the maintenance of nucleolus structure and functions. Our analysis shows that disruption of PARP1 enzymatic activity caused nucleolar disintegration and aberrant localization of nucleolar-specific proteins. Additionally, PARP1 mutants have increased accumulation of rRNA intermediates and a decrease in ribosome levels. Together, our data suggests that PARP1 enzymatic activity is required for targeting nucleolar proteins to the proximity of precursor rRNA; hence, PARP1 controls precursor rRNA processing, post-transcriptional modification, and pre-ribosome assembly. Based on these findings, we propose a model that explains how PARP1 activity impacts nucleolar functions and, consequently, ribosomal biogenesis. PMID:22242017

Mitochondrial poly(ADP-ribose) polymerase: The Wizard of Oz at work.

PubMed

Brunyanszki, Attila; Szczesny, Bartosz; Virág, László; Szabo, Csaba

2016-11-01

Among multiple members of the poly(ADP-ribose) polymerase (PARP) family, PARP1 accounts for the majority of PARP activity in mammalian cells. Although PARP1 is predominantly localized to the nucleus, and its nuclear regulatory roles are most commonly studied and are the best characterized, several lines of data demonstrate that PARP1 is also present in the mitochondria, and suggest that mitochondrial PARP (mtPARP) plays an important role in the regulation of various cellular functions in health and disease. The goal of the current article is to review the experimental evidence for the mitochondrial localization of PARP1 and its intra-mitochondrial functions, with focus on cellular bioenergetics, mitochondrial DNA repair and mitochondrial dysfunction. In addition, we also propose a working model for the interaction of mitochondrial and nuclear PARP during oxidant-induced cell death. MtPARP is similar to the Wizard of Oz in the sense that it is enigmatic, it has been elusive for a long time and it remains difficult to be interrogated. mtPARP - at least in some cell types - works incessantly "behind the curtains" as an orchestrator of many important cellular functions. Copyright © 2016 Elsevier Inc. All rights reserved.

Poly ADP-Ribose Polymerase Inhibition Ameliorates Hind Limb Ischemia Reperfusion Injury in a Murine Model of Type 2 Diabetes

PubMed Central

Long, Chandler A.; Boloum, Valy; Albadawi, Hassan; Tsai, Shirling; Yoo, Hyung-Jin; Oklu, Rahmi; Goldman, Mitchell H.; Watkins, Michael T.

2013-01-01

Introduction Diabetes is known to increase poly-ADP-ribose-polymerase (PARP) activity and posttranslational poly-ADP-ribosylation of several regulatory proteins involved in inflammation and energy metabolism. These experiments test the hypothesis that PARP inhibition will modulate hind limb ischemia reperfusion (IR) in a mouse model of type-II diabetes; ameliorate the ribosylation and the activity/transnuclear localization of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Methods db/db mice underwent 1.5hrs of hind limb ischemia followed by 1, 7, or 24hrs reperfusion. The treatment group received the PARP inhibitor PJ34 (PJ34) over a 24hrs period; the untreated group received Lactated ringer’s (LR) at the same time points. IR muscles were analyzed for indices of PARP activity, fiber injury, metabolic activity, inflammation, GAPDH activity /intracellular localization and poly-ADP-ribosylation of GAPDH. Results PARP activity was significantly lower in the PJ34 treated groups compared to the LR group at 7 and 24 hours reperfusion. There was significantly less muscle fiber injury in the PJ34 treated group compared to LR treated mice at 24 hrs reperfusion. PJ34 lowered levels of select proinflammatory molecules at 7hrs and 24hrs IR. There were significant increases in metabolic activity only at 24 hours IR in the PJ34 group, which temporally correlated with increase in GAPDH activity, decreased GAPDH poly ADP-ribosylation and nuclear translocation of GAPDH. Conclusions PJ34 reduced PARP activity, GAPDH ribosylation, GAPDH translocation, ameliorated muscle fiber injury, and increased metabolic activity following hind limb IR injury in a murine model of type-II diabetes. PARP inhibition might be a therapeutic strategy following IR in diabetic humans. PMID:23549425

Multiple receptor conformation docking, dock pose clustering and 3D QSAR studies on human poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors.

PubMed

Fatima, Sabiha; Jatavath, Mohan Babu; Bathini, Raju; Sivan, Sree Kanth; Manga, Vijjulatha

2014-10-01

Poly(ADP-ribose) polymerase-1 (PARP-1) functions as a DNA damage sensor and signaling molecule. It plays a vital role in the repair of DNA strand breaks induced by radiation and chemotherapeutic drugs; inhibitors of this enzyme have the potential to improve cancer chemotherapy or radiotherapy. Three-dimensional quantitative structure activity relationship (3D QSAR) models were developed using comparative molecular field analysis, comparative molecular similarity indices analysis and docking studies. A set of 88 molecules were docked into the active site of six X-ray crystal structures of poly(ADP-ribose)polymerase-1 (PARP-1), by a procedure called multiple receptor conformation docking (MRCD), in order to improve the 3D QSAR models through the analysis of binding conformations. The docked poses were clustered to obtain the best receptor binding conformation. These dock poses from clustering were used for 3D QSAR analysis. Based on MRCD and QSAR information, some key features have been identified that explain the observed variance in the activity. Two receptor-based QSAR models were generated; these models showed good internal and external statistical reliability that is evident from the [Formula: see text], [Formula: see text] and [Formula: see text]. The identified key features enabled us to design new PARP-1 inhibitors.

Paternal poly (ADP-ribose) metabolism modulates retention of inheritable sperm histones and early embryonic gene expression.

PubMed

Ihara, Motomasa; Meyer-Ficca, Mirella L; Leu, N Adrian; Rao, Shilpa; Li, Fan; Gregory, Brian D; Zalenskaya, Irina A; Schultz, Richard M; Meyer, Ralph G

2014-05-01

To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo.

Paternal Poly (ADP-ribose) Metabolism Modulates Retention of Inheritable Sperm Histones and Early Embryonic Gene Expression

PubMed Central

Leu, N. Adrian; Rao, Shilpa; Li, Fan; Gregory, Brian D.; Zalenskaya, Irina A.; Schultz, Richard M.; Meyer, Ralph G.

2014-01-01

To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo. PMID:24810616

The inhibition of nitric oxide-activated poly(ADP-ribose) synthetase attenuates transsynaptic alteration of spinal cord dorsal horn neurons and neuropathic pain in the rat.

PubMed

Mao, J; Price, D D; Zhu, J; Lu, J; Mayer, D J

1997-09-01

Transsynaptic alteration of spinal cord dorsal horn neurons characterized by hyperchromatosis of cytoplasm and nucleoplasm (so-called 'dark' neurons) occurs in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the common sciatic nerve. The incidence of dark neurons in CCI rats has been proposed to be mediated by glutamate-induced neurotoxicity. In the present study, we examined whether the inhibition of the nitric oxide (NO)-activated poly(ADP-ribose) synthetase (PARS), a nuclear enzyme critical to glutamate-induced neurotoxicity, would both reduce the incidence of dark neurons and attenuate behavioral manifestations of neuropathic pain in CCI rats. Dark neurons were observed bilaterally (with ipsilateral predominance) within the spinal cord dorsal horn, particularly in laminae I-II, of rats 8 days after unilateral sciatic nerve ligation as compared to sham operated rats. The number of dark neurons in the dorsal horn was dose-dependently reduced in CCI rats receiving once daily intrathecal (i.t.) treatment with the PARS inhibitor benzamide (200 or 400 nmol, but not 100 nmol benzamide or saline) for 7 days. Consistent with the histological improvement, thermal hyperalgesia, mechanical hyperalgesia, and low threshold mechano-allodynia also were reliably reduced in CCI rats treated with either 200 or 400 nmol benzamide. Neither dark neurons nor neuropathic pain behaviors were reliably affected by i.t. administration of either 800 nmol novobiocin (a mono(ADP-ribose) synthetase) or 800 nmol benzoic acid (the backbone structure of benzamide), indicating a selective effect of benzamide. Intrathecal treatment with an NO synthase inhibitor NG-nitro-L-arginine methyl ester (40 nmol, but not its inactive D-isomer) utilizing the same benzamide treatment regimen resulted in similar reductions of both dark neurons and neuropathic pain behaviors in CCI rats. These results provide, for the first time, in vivo evidence indicating that benzamide is

Poly(ADP-ribosylation) is present in murine sciatic nerve fibers and is altered in a Charcot-Marie-Tooth-1E neurodegenerative model

PubMed Central

Romeo Cardeillac, Carlos J.; Cal Castillo, Karina B.; Vilchez Larrea, Salomé C.; Sotelo Sosa, José R.; Folle Ungo, Gustavo A.; Fernández Villamil, Silvia H.

2017-01-01

Background Poly-ADP-ribose (PAR) is a polymer synthesized by poly-ADP-ribose polymerases (PARPs) as a postranslational protein modification and catabolized mainly by poly-ADP-ribose glycohydrolase (PARG). In spite of the existence of cytoplasmic PARPs and PARG, research has been focused on nuclear PARPs and PAR, demonstrating roles in the maintenance of chromatin architecture and the participation in DNA damage responses and transcriptional regulation. We have recently detected non-nuclear PAR structurally and functionally associated to the E-cadherin rich zonula adherens and the actin cytoskeleton of VERO epithelial cells. Myelinating Schwann cells (SC) are stabilized by E-cadherin rich autotypic adherens junctions (AJ). We wondered whether PAR would map to these regions. Besides, we have demonstrated an altered microfilament pattern in peripheral nerves of Trembler-J (Tr-J) model of CMT1-E. We hypothesized that cytoplasmic PAR would accompany such modified F-actin pattern. Methods Wild-type (WT) and Tr-J mice sciatic nerves cryosections were subjected to immunohistofluorescence with anti-PAR antibodies (including antibody validation), F-actin detection with a phalloidin probe and DAPI/DNA counterstaining. Confocal image stacks were subjected to a colocalization highlighter and to semi-quantitative image analysis. Results We have shown for the first time the presence of PAR in sciatic nerves. Cytoplasmic PAR colocalized with F-actin at non-compact myelin regions in WT nerves. Moreover, in Tr-J, cytoplasmic PAR was augmented in close correlation with actin. In addition, nuclear PAR was detected in WT SC and was moderately increased in Tr-J SC. Discussion The presence of PAR associated to non-compact myelin regions (which constitute E-cadherin rich autotypic AJ/actin anchorage regions) and the co-alterations experienced by PAR and the actin cytoskeleton in epithelium and nerves, suggest that PAR may be a constitutive component of AJ/actin anchorage regions. Is PAR

The dual role of poly(ADP-ribose) polymerase-1 in modulating parthanatos and autophagy under oxidative stress in rat cochlear marginal cells of the stria vascularis.

PubMed

Jiang, Hong-Yan; Yang, Yang; Zhang, Yuan-Yuan; Xie, Zhen; Zhao, Xue-Yan; Sun, Yu; Kong, Wei-Jia

2018-04-01

Oxidative stress is reported to regulate several apoptotic and necrotic cell death pathways in auditory tissues. Poly(ADP-ribose) polymerase-1 (PARP-1) can be activated under oxidative stress, which is the hallmark of parthanatos. Autophagy, which serves either a pro-survival or pro-death function, can also be stimulated by oxidative stress, but the role of autophagy and its relationship with parthanatos underlying this activation in the inner ear remains unknown. In this study, we established an oxidative stress model in vitro by glucose oxidase/glucose (GO/G), which could continuously generate low concentrations of H 2 O 2 to mimic continuous exposure to H 2 O 2 in physiological conditions, for investigation of oxidative stress-induced cell death mechanisms and the regulatory role of PARP-1 in this process. We observed that GO/G induced stria marginal cells (MCs) death via upregulation of PARP-1 expression, accumulation of polyADP-ribose (PAR) polymers, decline of mitochondrial membrane potential (MMP) and nuclear translocation of apoptosis-inducing factor (AIF), which all are biochemical features of parthanatos. PARP-1 knockdown rescued GO/G-induced MCs death, as well as abrogated downstream molecular events of PARP-1 activation. In addition, we demonstrated that GO/G stimulated autophagy and PARP-1 knockdown suppressed GO/G-induced autophagy in MCs. Interestingly, autophagy suppression by 3-Methyladenine (3-MA) accelerated GO/G-induced parthanatos, indicating a pro-survival function of autophagy in GO/G-induced MCs death. Taken together, these data suggested that PARP-1 played dual roles by modulating parthanatos and autophagy in oxidative stress-induced MCs death, which may be considered as a promising therapeutic target for ameliorating oxidative stress-related hearing disorders. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Administration of poly(ADP-ribose) polymerase inhibitor into bronchial artery attenuates pulmonary pathophysiology after smoke inhalation and burn in an ovine model.

PubMed

Hamahata, Atsumori; Enkhbaatar, Perenlei; Lange, Matthias; Yamaki, Takashi; Sakurai, Hiroyuki; Shimoda, Katsumi; Nakazawa, Hiroaki; Traber, Lillian D; Traber, Daniel L

2012-12-01

Poly(ADP-ribose) polymerase (PARP) is well known to be an enzyme that repairs damaged DNA and also induces cell death when overactivated. It has been reported that PARP plays a significant role in burn and smoke inhalation injury, and the pathophysiology is thought to be localized in the airway during early stages of activation. Therefore, we hypothesized that local inhibition of PARP in the airway by direct delivery of low dose PJ-34 [poly(ADP-ribose) polymerase inhibitor] into the bronchial artery would attenuate burn and smoke-induced acute lung injury. The bronchial artery in sheep was cannulated in preparation for surgery. After a 5-7 day recovery period, sheep were administered a burn and inhalation injury. Adult female sheep (n=19) were divided into four groups following the injury: (1) PJ-34 group A: 1h post-injury, PJ-34 (0.003mg/kg/h, 2mL/h) was continuously injected into the bronchial artery, n=5; (2) PJ-34 group B: 1h post-injury, PJ-34 (0.03mg/kg/h, 2mL/h) was continuously injected into bronchial artery, n=4; (3) CONTROL GROUP: 1h post-injury, an equivalent amount of saline was injected into the bronchial artery, n=5; (4) Sham group: no injury, no treatment, same operation and anesthesia, n=5. After injury, all animals were placed on a ventilator and fluid resuscitated equally. Pulmonary function as evaluated by measurement of blood gas analysis, pulmonary mechanics, and pulmonary transvascular fluid flux was severely deteriorated in the control group. However, the above changes were markedly attenuated by PJ-34 infusion into the bronchial artery (P/F ratio at 24h: PJ-34 group A 398±40*, PJ-34 group B 438±41*†‡, Control 365±58*, Sham 547±47; * vs. sham [p<0.05], † vs. control [p<0.05], ‡ vs. PJ-34 group A [p<0.05]). Our data strongly suggest that local airway production of poly(ADP-ribose) polymerase contributes to pulmonary dysfunction following smoke inhalation and burn. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

PubMed Central

Kozaki, Tatsuya; Komano, Jun; Kanbayashi, Daiki; Takahama, Michihiro; Misawa, Takuma; Satoh, Takashi; Takeuchi, Osamu; Kawai, Taro; Shimizu, Shigeomi; Matsuura, Yoshiharu; Akira, Shizuo; Saitoh, Tatsuya

2017-01-01

The innate immune system senses RNA viruses by pattern recognition receptors (PRRs) and protects the host from virus infection. PRRs mediate the production of immune modulatory factors and direct the elimination of RNA viruses. Here, we show a unique PRR that mediates antiviral response. Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP ribose) polymerase (TIPARP), a Cysteine3 Histidine (CCCH)-type zinc finger-containing protein, binds to Sindbis virus (SINV) RNA via its zinc finger domain and recruits an exosome to induce viral RNA degradation. TIPARP typically localizes in the nucleus, but it accumulates in the cytoplasm after SINV infection, allowing targeting of cytoplasmic SINV RNA. Redistribution of TIPARP is induced by reactive oxygen species (ROS)-dependent oxidization of the nuclear pore that affects cytoplasmic-nuclear transport. BCL2-associated X protein (BAX) and BCL2 antagonist/killer 1 (BAK1), B-cell leukemia/lymphoma 2 (BCL2) family members, mediate mitochondrial damage to generate ROS after SINV infection. Thus, TIPARP is a viral RNA-sensing PRR that mediates antiviral responses triggered by BAX- and BAK1-dependent mitochondrial damage. PMID:28213497

Analysis of Poly(ADP-Ribose) Polymerases in Arabidopsis Telomere Biology

PubMed Central

Townley, Jennifer M.; Shippen, Dorothy E.

2014-01-01

Maintaining the length of the telomere tract at chromosome ends is a complex process vital to normal cell division. Telomere length is controlled through the action of telomerase as well as a cadre of telomere-associated proteins that facilitate replication of the chromosome end and protect it from eliciting a DNA damage response. In vertebrates, multiple poly(ADP-ribose) polymerases (PARPs) have been implicated in the regulation of telomere length, telomerase activity and chromosome end protection. Here we investigate the role of PARPs in plant telomere biology. We analyzed Arabidopsis thaliana mutants null for PARP1 and PARP2 as well as plants treated with the PARP competitive inhibitor 3-AB. Plants deficient in PARP were hypersensitive to genotoxic stress, and expression of PARP1 and PARP2 mRNA was elevated in response to MMS or zeocin treatment or by the loss of telomerase. Additionally, PARP1 mRNA was induced in parp2 mutants, and conversely, PARP2 mRNA was induced in parp1 mutants. PARP3 mRNA, by contrast, was elevated in both parp1 and parp2 mutants, but not in seedlings treated with 3-AB or zeocin. PARP mutants and 3-AB treated plants displayed robust telomerase activity, no significant changes in telomere length, and no end-to-end chromosome fusions. Although there remains a possibility that PARPs play a role in Arabidopsis telomere biology, these findings argue that the contribution is a minor one. PMID:24551184

PIASy Mediates SUMO-2/3 Conjugation of Poly(ADP-ribose) Polymerase 1 (PARP1) on Mitotic Chromosomes*

PubMed Central

Ryu, Hyunju; Al-Ani, Gada; Deckert, Katelyn; Kirkpatrick, Donald; Gygi, Steven P.; Dasso, Mary; Azuma, Yoshiaki

2010-01-01

PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins. PMID:20228053

Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chassé, Maggie H.; Muthurajan, Uma M.; Clark, Nicholas J.

Poly (ADP-Ribose) Polymerase I (PARP-1) is a first responder to DNA damage and participates in the regulation of gene expression. The interaction of PARP-1 with chromatin and DNA is complex and involves at least two different modes of interaction. In its enzymatically inactive state, PARP-1 binds native chromatin with similar affinity as it binds free DNA ends. Automodification of PARP-1 affects interaction with chromatin and DNA to different extents. Here we describe a series of biochemical and biophysical techniques to quantify and dissect the different binding modes of PARP-1 with its various substrates. The techniques listed here allow for highmore » throughput and quantitative measurements of the interaction of different PARP-1 constructs (inactive and automodified) with chromatin and DNA damage models.« less

Pharmacological Inhibition of Poly(ADP-Ribose) Polymerases Improves Fitness and Mitochondrial Function in Skeletal Muscle

PubMed Central

Pirinen, Eija; Canto, Carles; Jo, Young-Suk; Morato, Laia; Zhang, Hongbo; Menzies, Keir; Williams, Evan G.; Mouchiroud, Laurent; Moullan, Norman; Hagberg, Carolina; Li, Wei; Timmers, Silvie; Imhof, Ralph; Verbeek, Jef; Pujol, Aurora; van Loon, Barbara; Viscomi, Carlo; Zeviani, Massimo; Schrauwen, Patrick; Sauve, Anthony; Schoonjans, Kristina; Auwerx, Johan

2014-01-01

SUMMARY We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function. PMID:24814482

Differential Role of Poly(ADP-ribose) polymerase in D. discoideum growth and development

PubMed Central

2011-01-01

Background Poly(ADP-ribose) polymerase is evolutionarily conserved as a responder to various forms of stress. Though PARP's role in cell death is well addressed, its role in development and multicellularity is still an enigma. We have previously reported the role of PARP in oxidative stress induced delayed development of D. discoideum. Results In the current study we highlight the involvement of PARP during D. discoideum development. Oxidative stress affects expression of aca and cAR1 thus affecting aggregation. Although parp expression is not affected during oxidative stress but it is involved during normal development as confirmed by our PARP down-regulation studies. Constitutive PARP down-regulation resulted in blocked development while no effect was observed on D. discoideum growth. Interestingly, stage specific PARP down-regulation arrested development at the slug stage. Conclusion These results emphasize that PARP is essential for complex differentiation and its function may be linked to multicellularity. This is the first report where the involvement of PARP during normal multicellular development in D. discoideum, an ancient eukaryote, is established which could be of evolutionary significance. Thus our study adds one more role to the multitasking function of PARP. PMID:21385463

Poly(ADP-ribose) polymerase inhibition reveals a potential mechanism to promote neuroprotection and treat neuropathic pain.

PubMed

Komirishetty, Prashanth; Areti, Aparna; Gogoi, Ranadeep; Sistla, Ramakrishna; Kumar, Ashutosh

2016-10-01

Neuropathic pain is triggered by the lesions to peripheral nerves which alter their structure and function. Neuroprotective approaches that limit the pathological changes and improve the behavioral outcome have been well explained in different experimental models of neuropathy but translation of such strategies to clinics has been disappointing. Experimental evidences revealed the role of free radicals, especially peroxynitrite after the nerve injury. They provoke oxidative DNA damage and consequent over-activation of the poly(ADP-ribose) polymerase (PARP) upregulates pro-inflammatory pathways, causing bioenergetic crisis and neuronal death. Along with these changes, it causes mitochondrial dysfunction leading to neuronal apoptosis. In related preclinical studies agents that neutralize the free radicals and pharmacological inhibitors of PARP have shown benefits in treating experimental neuropathy. This article reviews the involvement of PARP over-activation in trauma induced neuropathy and therapeutic significance of PARP inhibitors in the experimental neuropathy and neuropathic pain.

[Role of hydrogen gas in regulating of poly (ADP-ribose) polymerase-1 dependent cell death in rat Schwann cells].

PubMed

Yu, Yang; Jiao, Yang; Li, Bo; Ma, Xiaoye; Yang, Tao; Xie, Keliang; Yu, Yonghao

2016-08-01

To investigate the protective effects and underlying molecular mechanisms of hydrogen (H2) on high glucose-induced poly (ADP-ribose) polymerase-1 (PARP-1) dependent cell death (PARthanatos) in primary rat Schwann cells. Cultured primary rat Schwann cells were randomly divided into five groups: blank control group (C group), H2 control group (H2 group), high osmotic control group (M group), high glucose treatment group (HG group), and H2 treatment group (HG+H2 group). The cells in H2 group and HG+H2 group were cultured with saturated hydrogen-rich medium containing 0.6 mmol/L of H2, and those in three control groups were cultured with low sugar DMEM medium containing 5.6 mmol/L of sugar, and the cells in HG and HG+H2 groups were given 44.4 mmol/L of glucose in addition (the medium containing 50 mmol/L of glucose), the cells in C group and H2 group were given the same volume of normal saline, and the cells in M group were given the same volume of mannitol. Cytotoxicity was evaluated using lactate dehydrogenase (LDH) release rate assays after treatment for 48 hours in each group. The contents of peroxynitrite (ONOO(-)) and 8-hydroxy-2-deoxyguanosine (8-OHdG) reflecting oxidative stress injury and DNA damage were detected by enzyme linked immunosorbent assay (ELISA). Poly (ADP-ribose) (PAR) protein expression was analyzed by Western Blot, and immunofluorescence staining was used to determine the nuclear translocation of the apoptosis-inducing factor (AIF). The cytotoxicity in HG and HG+H2 groups was significantly increased as compared with that of C group [LDH release rate: (61.40±2.89)%, (42.80±2.32)% vs. (9.92±0.38)%, both P < 0.01], the levels of ONOO(-) and 8-OHdG were markedly elevated [ONOO(-) (ng/L): 853.58±51.00, 553.11±38.66 vs. 113.56±14.22; 8-OHdG (ng/L): 1?177.37±60.97, 732.06±54.29 vs. 419.67±28.77, all P < 0.01], and the PAR protein expression was up-regulated (A value: 0.603±0.028, 0.441±0.010 vs. 0.324±0.021, both P < 0.01). The cytotoxicity

Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

PubMed Central

Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye

2015-01-01

Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. PMID:26627003

Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

PubMed

Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

2016-01-15

Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. Copyright © 2015 Elsevier Inc. All rights reserved.

Poly(ADP-Ribose) Polymerase-1: A Novel Therapeutic Target in Necrotizing Enterocolitis

PubMed Central

Giannone, Peter J.; Alcamo, Alicia A.; Schanbacher, Brandon L.; Nankervis, Craig A.; Besner, Gail E.; Bauer, John A.

2011-01-01

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of infancy, afflicting 11% of infants born 22–28 weeks gestational age. Both inflammation and oxidation may be involved in NEC pathogenesis through reactive nitrogen species production, protein oxidation and DNA damage. Poly(ADP-ribose) polymerase-1 (PARP-1) is a critical enzyme activated to facilitate DNA repair using nicotinamide adenine dinucleotide (NAD+) as a substrate. However, in the presence of severe oxidative stress and DNA damage, PARP-1 over-activation may ensue, depleting cells of NAD+ and ATP, killing them by metabolic catastrophe. Here we tested the hypothesis that NO dysregulation in intestinal epithelial cells during NEC leads to marked PARP-1 expression and that administration of a PARP-1 inhibitor (nicotinamide) attenuates intestinal injury in a newborn rat model of NEC. In this model, 56% of control pups developed NEC (any stage), versus 14% of pups receiving nicotinamide. Forty-four percent of control pups developed high-grade NEC (grades 3–4), whereas only 7% of pups receiving nicotinamide developed high-grade NEC. Nicotinamide treatment protects pups against intestinal injury incurred in the newborn rat NEC model. We speculate that PARP-1 over-activation in NEC may drive mucosal cell death in this disease and that PARP-1 may be a novel therapeutic target in NEC. PMID:21399558

Poly ADP-ribose polymerase-1 as a potential therapeutic target in Merkel cell carcinoma.

PubMed

Ferrarotto, Renata; Cardnell, Robert; Su, Shirley; Diao, Lixia; Eterovic, A Karina; Prieto, Victor; Morrisson, William H; Wang, Jing; Kies, Merrill S; Glisson, Bonnie S; Byers, Lauren Averett; Bell, Diana

2018-03-23

Patients with metastatic Merkel cell carcinoma are treated similarly to small cell lung cancer (SCLC). Poly ADP-ribose polymerase-1 (PARP1) is overexpressed in SCLC and response to PARP inhibitors have been reported in patients with SCLC. Our study explores PARP as a therapeutic target in Merkel cell carcinoma. We evaluated PARP1 expression and Merkel cell polyomavirus (MCPyV) in 19 patients with Merkel cell carcinoma. Target exome-sequencing was performed in 14 samples. Sensitivity to olaparib was tested in 4 Merkel cell carcinoma cell lines. Most Merkel cell carcinomas (74%) express PARP1 at high levels. Mutations in DNA-damage repair genes were identified in 9 samples (64%), occurred exclusively in head neck primaries, and correlated with TP53/RB1 mutations. The TP53/RB1 mutations were more frequent in MCPyV-negative tumors. Sensitivity to olaparib was seen in the Merkel cell carcinoma line with highest PARP1 expression. Based on PARP1 overexpression, DNA-damage repair gene mutations, platinum sensitivity, and activity of olaparib in a Merkel cell carcinoma line, clinical trials with PARP inhibitors are warranted in Merkel cell carcinoma. © 2018 Wiley Periodicals, Inc.

PARPs and ADP-Ribosylation: 50 Years … and Counting.

PubMed

Kraus, W Lee

2015-06-18

Over 50 years ago, the discovery of poly(ADP-ribose) (PAR) set a new field of science in motion-the field of poly(ADP-ribosyl) transferases (PARPs) and ADP-ribosylation. The field is still flourishing today. The diversity of biological processes now known to require PARPs and ADP-ribosylation was practically unimaginable even two decades ago. From an initial focus on DNA damage detection and repair in response to genotoxic stresses, the field has expanded to include the regulation of chromatin structure, gene expression, and RNA processing in a wide range of biological systems, including reproduction, development, aging, stem cells, inflammation, metabolism, and cancer. This special focus issue of Molecular Cell includes a collection of three Reviews, three Perspectives, and a SnapShot, which together summarize the current state of the field and suggest where it may be headed. Copyright © 2015 Elsevier Inc. All rights reserved.

Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate cellular protection through NF-kB-dependent signaling

PubMed Central

Castri, Paola; Lee, Yang-ja; Ponzio, Todd; Maric, Dragan; Spatz, Maria; Bembry, Joliet; Hallenbeck, John

2014-01-01

Poly(ADP-ribose) polymerase-1 (PARP-1) and its cleavage products regulate cell viability and NF-kB activity when expressed in neurons. PARP-1 cleavage generates a 24kDa (PARP-124) and an 89kDa fragment (PARP-189). Compared to WT (PARP-1WT), the expression of an uncleavable PARP-1 (PARP-1UNCL) or of PARP-124 conferred protection from oxygen/glucose deprivation (OGD) or OGD/restoration of oxygen and glucose (ROG) damage in vitro, whereas expression of PARP-189 was cytotoxic. Viability experiments were performed in SH-SY5Y, a human neuroblastoma cell line, as well as in rat primary cortical neurons. Following OGD, the higher viability in the presence of PARP-1UNCL or PARP-124 was not accompanied with decreased formation of poly(ADP-riboses) or higher NAD levels. PARP-1 is a known cofactor for NF-kB, hence we investigated whether PARP-1 cleavage influences the inflammatory response. All PARP-1 constructs mimicked PARP-1WT in regards to induction of NF-kB translocation into the nucleus and its increased activation during ischemic challenge. However, expression of PARP-189 construct induced significantly higher NF-kB activity than PARP-1WT; and the same was true for NF-kB-dependent iNOS promoter binding activity. At a protein level, PARP-1UNCL and PARP-124 decreased iNOS (and lower levels of iNOS transcript) and COX-2, and increased Bcl-xL. The increased levels of NF-kB and iNOS transcriptional activities, seen with cytotoxic PARP-189, were accompanied by higher protein expression of COX-2 and iNOS (and higher levels of iNOS transcript) and lower protein expression of Bcl-xL. Taken together, these findings suggest that PARP-1 cleavage products may regulate cellular viability and inflammatory responses in opposing ways during in vitro models of “ischemia”. PMID:24333653

Small interfering RNA mediated Poly (ADP-ribose) Polymerase-1 inhibition upregulates the heat shock response in a murine fibroblast cell line

PubMed Central

2011-01-01

Poly (ADP-ribose) polymerase-1 (PARP-1) is a highly conserved multifunctional enzyme, and its catalytic activity is stimulated by DNA breaks. The activation of PARP-1 and subsequent depletion of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) contributes to significant cytotoxicity in inflammation of various etiologies. On the contrary, induction of heat shock response and production of heat shock protein 70 (HSP-70) is a cytoprotective defense mechanism in inflammation. Recent data suggests that PARP-1 modulates the expression of a number of cellular proteins at the transcriptional level. In this study, small interfering RNA (siRNA) mediated PARP-1 knockdown in murine wild-type fibroblasts augmented heat shock response as compared to untreated cells (as evaluated by quantitative analysis of HSP-70 mRNA and HSP-70 protein expression). These events were associated with increased DNA binding of the heat shock factor-1 (HSF-1), the major transcription factor of the heat shock response. Co-immunoprecipitation experiments in nuclear extracts of the wild type cells demonstrated that PARP-1directly interacted with HSF-1. These data demonstrate that, in wild type fibroblasts, PARP-1 plays a pivotal role in modulating the heat shock response both through direct interaction with HSF-1 and poly (ADP-ribosylation). PMID:21345219

Radiosensitization of Pancreatic Cancer Cells In Vitro and In Vivo through Poly (ADP-ribose) Polymerase Inhibition with ABT-888.

PubMed

Tuli, Richard; Surmak, Andrew J; Reyes, Juvenal; Armour, Michael; Hacker-Prietz, Amy; Wong, John; DeWeese, Theodore L; Herman, Joseph M

2014-05-13

To determine whether poly (ADP-ribose) polymerase-1/2 (PARP-1/2) inhibition enhances radiation-induced cytotoxicity of pancreatic adenocarcinoma in vitro and in vivo, and the mechanism by which this occurs. Pancreatic carcinoma cells were treated with ABT-888, radiation, or both. In vitro cell viability, apoptosis, and PARP activity were measured. Orthotopic xenografts were generated in athymic mice and treated with ABT-888 (25mg/kg), radiation (5Gy), both, or no treatment. Mice were monitored with bioluminescence imaging. In vitro, treatment with ABT-888 and radiation led to higher rates of cell death after 8days (P < .01). Co-treatment with 5Gy and 1, 10 or 100μmol/l of ABT-888 led to dose enhancement factors of 1.29, 1.41 and 2.36, respectively. Caspase activity was not significantly increased when treated with ABT-888 (10 μmol/l) alone (1.28-fold, P = .08), but became significant when radiation was added (2.03-fold, P < .01). PARP activity increased post-radiation and was abrogated following co-treatment with ABT-888. In vivo, treatment with ABT-888, radiation or both led to tumor growth inhibition (TGI) of 8, 30 and 39days, and survival at 60days of 0%, 0% and 40%, respectively. ABT-888 with radiation significantly enhanced tumor response in vitro and in vivo. ABT-888 inhibited PAR protein polymerization resulting in dose-dependent feedback up-regulation of PARP and p-ATM suggesting increased DNA damage. This translated into enhancement in TGI and survival with radiation in vivo. In vitro PAR levels correlated with levels of tumor apoptosis suggesting potential as a predictive biomarker. These data are being used to support a Phase I study in locally advanced pancreatic cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Ethanol-induced changes in Poly (ADP ribose) Polymerase and neuronal developmental gene expression

PubMed Central

Gavin, David P.; Kusumo, Handojo; Sharma, Rajiv P.; Guizzetti, Marina

2016-01-01

Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that elevated PARP enzymatic activity reduced PPARγ promoter binding, and this corresponded to decreased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression. PMID:27497606

Nuclear ADP-Ribosylation Reactions in Mammalian Cells: Where Are We Today and Where Are We Going?

PubMed Central

Hassa, Paul O.; Haenni, Sandra S.; Elser, Michael; Hottiger, Michael O.

2006-01-01

Since poly-ADP ribose was discovered over 40 years ago, there has been significant progress in research into the biology of mono- and poly-ADP-ribosylation reactions. During the last decade, it became clear that ADP-ribosylation reactions play important roles in a wide range of physiological and pathophysiological processes, including inter- and intracellular signaling, transcriptional regulation, DNA repair pathways and maintenance of genomic stability, telomere dynamics, cell differentiation and proliferation, and necrosis and apoptosis. ADP-ribosylation reactions are phylogenetically ancient and can be classified into four major groups: mono-ADP-ribosylation, poly-ADP-ribosylation, ADP-ribose cyclization, and formation of O-acetyl-ADP-ribose. In the human genome, more than 30 different genes coding for enzymes associated with distinct ADP-ribosylation activities have been identified. This review highlights the recent advances in the rapidly growing field of nuclear mono-ADP-ribosylation and poly-ADP-ribosylation reactions and the distinct ADP-ribosylating enzyme families involved in these processes, including the proposed family of novel poly-ADP-ribose polymerase-like mono-ADP-ribose transferases and the potential mono-ADP-ribosylation activities of the sirtuin family of NAD+-dependent histone deacetylases. A special focus is placed on the known roles of distinct mono- and poly-ADP-ribosylation reactions in physiological processes, such as mitosis, cellular differentiation and proliferation, telomere dynamics, and aging, as well as “programmed necrosis” (i.e., high-mobility-group protein B1 release) and apoptosis (i.e., apoptosis-inducing factor shuttling). The proposed molecular mechanisms involved in these processes, such as signaling, chromatin modification (i.e., “histone code”), and remodeling of chromatin structure (i.e., DNA damage response, transcriptional regulation, and insulator function), are described. A potential cross talk between nuclear

Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

PubMed

Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

2009-12-15

Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

Toward a unified nomenclature for mammalian ADP-ribosyltransferases.

PubMed

Hottiger, Michael O; Hassa, Paul O; Lüscher, Bernhard; Schüler, Herwig; Koch-Nolte, Friedrich

2010-04-01

ADP-ribosylation is a post-translational modification of proteins catalyzed by ADP-ribosyltransferases. It comprises the transfer of the ADP-ribose moiety from NAD+ to specific amino acid residues on substrate proteins or to ADP-ribose itself. Currently, 22 human genes encoding proteins that possess an ADP-ribosyltransferase catalytic domain are known. Recent structural and enzymological evidence of poly(ADP-ribose)polymerase (PARP) family members demonstrate that earlier proposed names and classifications of these proteins are no longer accurate. Here we summarize these new findings and propose a new consensus nomenclature for all ADP-ribosyltransferases (ARTs) based on the catalyzed reaction and on structural features. A unified nomenclature would facilitate communication between researchers both inside and outside the ADP-ribosylation field. 2009 Elsevier Ltd. All rights reserved.

Ethanol-induced changes in poly (ADP ribose) polymerase and neuronal developmental gene expression.

PubMed

Gavin, David P; Kusumo, Handojo; Sharma, Rajiv P; Guizzetti, Marina

2016-11-01

Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that inhibition of PARP enzymatic activity increased PPARγ promoter binding, and this corresponded to increased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression. Published by Elsevier Ltd.

Ets-1 interacts through a similar binding interface with Ku70 and Poly (ADP-Ribose) Polymerase-1.

PubMed

Choul-Li, Souhaila; Legrand, Arnaud J; Vicogne, Dorothée; Villeret, Vincent; Aumercier, Marc

2018-06-18

The Ets-1 transcription factor plays an important role in various physiological and pathological processes. These diverse roles of Ets-1 are likely to depend on its interaction proteins. We have previously showed that Ets-1 interacted with DNA-dependent protein kinase (DNA-PK) complex including its regulatory subunits, Ku70 and Ku86 and with poly (ADP-ribose) polymerase-1 (PARP-1). In this study, the binding domains for the interaction between Ets-1 and these proteins were reported. We demonstrated that the interaction of Ets-1 with DNA-PK was mediated through the Ku70 subunit and was mapped to the C-terminal region of Ets-1 and the C-terminal part of Ku70 including SAP domain. The interactive domains between Ets-1 and PARP-1 have been mapped to the C-terminal region of Ets-1 and the BRCA1 carboxy-terminal (BRCT) domain of PARP-1. The results presented in this study may advance our understanding of the functional link between Ets-1 and its interaction partners, DNA-PK and PARP-1.

Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda

Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; < 10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein.more » Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. - Highlights: • Low micromolar concentration of uranium inhibits polymerase-1 (PARP-1) activity. • Uranium causes zinc loss from multiple DNA repair proteins. • Uranium enhances retention of DNA damage caused by ultraviolet radiation. • Zinc reverses the effects of uranium on PARP activity and DNA damage repair.« less

Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abdelali, Ala

Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional proteinmore » 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers

Excitotoxicity in the Lung: N-Methyl-D-Aspartate-Induced, Nitric Oxide-Dependent, Pulmonary Edema is Attenuated by Vasoactive Intestinal Peptide and by Inhibitors of Poly(ADP-Ribose) Polymerase

NASA Astrophysics Data System (ADS)

Said, Sami I.; Berisha, Hasan I.; Pakbaz, Hedayatollah

1996-05-01

Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the ``adult respiratory distress syndrome,'' and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase.

Hydrofluoric Acid-Based Derivatization Strategy To Profile PARP-1 ADP-Ribosylation by LC-MS/MS.

PubMed

Gagné, Jean-Philippe; Langelier, Marie-France; Pascal, John M; Poirier, Guy G

2018-06-11

Despite significant advances in the development of mass spectrometry-based methods for the identification of protein ADP-ribosylation, current protocols suffer from several drawbacks that preclude their widespread applicability. Given the intrinsic heterogeneous nature of poly(ADP-ribose), a number of strategies have been developed to generate simple derivatives for effective interrogation of protein databases and site-specific localization of the modified residues. Currently, the generation of spectral signatures indicative of ADP-ribosylation rely on chemical or enzymatic conversion of the modification to a single mass increment. Still, limitations arise from the lability of the poly(ADP-ribose) remnant during tandem mass spectrometry, the varying susceptibilities of different ADP-ribose-protein bonds to chemical hydrolysis, or the context dependence of enzyme-catalyzed reactions. Here, we present a chemical-based derivatization method applicable to the confident identification of site-specific ADP-ribosylation by conventional mass spectrometry on any targeted amino acid residue. Using PARP-1 as a model protein, we report that treatment of ADP-ribosylated peptides with hydrofluoric acid generates a specific +132 Da mass signature that corresponds to the decomposition of mono- and poly(ADP-ribosylated) peptides into ribose adducts as a consequence of the cleavage of the phosphorus-oxygen bonds.

A novel and selective poly (ADP-ribose) polymerase inhibitor ameliorates chemotherapy-induced painful neuropathy.

PubMed

Ta, Lauren E; Schmelzer, James D; Bieber, Allan J; Loprinzi, Charles L; Sieck, Gary C; Brederson, Jill D; Low, Philip A; Windebank, Anthony J

2013-01-01

Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose) polymerase (PARP) inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888) would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice. An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p.) injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment. Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy.

Poly(ADP-ribose) polymerase-1 regulates microglia mediated decrease of endothelial tight junction integrity.

PubMed

Mehrabadi, Abbas Rezaeian; Korolainen, Minna A; Odero, Gary; Miller, Donald W; Kauppinen, Tiina M

2017-09-01

Alzheimer's disease pathology includes, beside neuronal damage, reactive gliosis and reduced blood-brain barrier (BBB) integrity. Microglia are intimately associated with the BBB and upon AD pathology, pro-inflammatory responses of microglia could contribute to BBB damage. To study whether microglia can directly affect BBB integrity, the effects of amyloid beta (Aβ) -stimulated primary murine microglia on co-cultured mouse brain endothelial cells (bEnd3) and murine astrocyte cultures were assessed. We also assessed whether microglial phenotype modulation via poly(ADP-ribose) polymerase-1 (PARP-1) inhibition/ablation can reverse microglial impact on these BBB forming cells. Unstimulated microglia promoted expression of tight junction proteins (TJPs), zonula ocluden-1 (ZO-1) and occludin in co-cultured endothelia cells, whereas Aβ-stimulated microglia reduced endothelial expression of ZO-1 and occludin. Astrocytes co-cultured with microglia showed elevated glial fibrillary acidic protein (GFAP) expression, which was further increased if microglia had been stimulated with Aβ. Aβ induced microglial release of nitric oxide (NO) and tumour necrosis factor alpha (TNFα), which resulted in reduced endothelial expression of TJPs and increased paracellular permeability. Microglial PARP-1 inhibition attenuated these Aβ-induced events. These findings demonstrate that PARP-1 mediated microglial responses (NO and TNFα) can directly reduce BBB integrity by promoting TJP degradation, increasing endothelial cell permeability and inducing astrogliosis. PARP-1 as a modulator of microglial phenotype can prevent microglial BBB damaging events, and thus is a potential therapeutic target. Copyright © 2017 Elsevier Ltd. All rights reserved.

Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gebhard, Catherine; Staehli, Barbara E.; Zurich Center for Integrative Human Physiology

2011-11-04

Highlights: Black-Right-Pointing-Pointer The nuclear enzyme PARP-1 is a downstream effector of oxidative stress. Black-Right-Pointing-Pointer PARP-1 protects from oxidative stress induced endothelial dysfunction. Black-Right-Pointing-Pointer This effect is mediated through inhibition of vasoconstrictor prostanoid production. Black-Right-Pointing-Pointer Thus, PARP-1 may play a protective role as antioxidant defense mechanism. -- Abstract: Background: Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress. Methods: PARP-1(-/-) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings weremore » suspended in organ chambers for isometric tension recording to analyze vascular function. Results: PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(-/-), but not PARP-1(+/+) mice (p < 0.0001). Maximal relaxation was 45% in PQ treated PARP-1(-/-) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, L-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(-/-) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(-/-) mice (p < 0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(-/-) mice (p < 0.05 vs. PQ treated PARP-1(+/+). Conclusion: PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.« less

Involvement of cytosolic NAD+ glycohydrolase in cyclic ADP-ribose metabolism.

PubMed

Matsumura, N; Tanuma, S

1998-12-18

The NAD+ glycohydrolase homogeneously purified from bovine brain cytosol was found to catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). Although the formation of cADPR from NAD+ does not exceed about 2% of the reaction products, the cyclase activity is clearly evidenced by its conversion of NGD+ to cyclic GDP-ribose (cGDPR), which cannot be hydrolyzed to GDPR. Importantly, a steep increase in cADPR hydrolytic activity was observed at cADPR concentrations above 60 microM, which could be reproduced on a Hill curve with a Hill coefficient of 2. Thus, the allosteric binding of cADPR to the NAD+ glycohydrolase (E) molecule promotes the hydrolysis of cADPR. These results suggest that NAD+ hydrolysis to ADPR and nicotinamide catalyzed by the NAD+ glycohydrolase occurs through the formation of a cADPR. E. cADP-ribosyl complex. The low production of cADPR by NAD+ glycohydrolase compared with invertebrate ADP-ribosyl cyclase is believed to be attributable to the fast hydrolysis of cADPR by the allosteric effect of cADPR bound to the same enzyme that produces it. Copyright 1998 Academic Press.

The structure of human ADP-ribosylhydrolase 3 (ARH3) provides insights into the reversibility of protein ADP-ribosylation

PubMed Central

Mueller-Dieckmann, Christoph; Kernstock, Stefan; Lisurek, Michael; von Kries, Jens Peter; Haag, Friedrich; Weiss, Manfred S.; Koch-Nolte, Friedrich

2006-01-01

Posttranslational modifications are used by cells from all kingdoms of life to control enzymatic activity and to regulate protein function. For many cellular processes, including DNA repair, spindle function, and apoptosis, reversible mono- and polyADP-ribosylation constitutes a very important regulatory mechanism. Moreover, many pathogenic bacteria secrete toxins which ADP-ribosylate human proteins, causing diseases such as whooping cough, cholera, and diphtheria. Whereas the 3D structures of numerous ADP-ribosylating toxins and related mammalian enzymes have been elucidated, virtually nothing is known about the structure of protein de-ADP-ribosylating enzymes. Here, we report the 3Dstructure of human ADP-ribosylhydrolase 3 (hARH3). The molecular architecture of hARH3 constitutes the archetype of an all-α-helical protein fold and provides insights into the reversibility of protein ADP-ribosylation. Two magnesium ions flanked by highly conserved amino acids pinpoint the active-site crevice. Recombinant hARH3 binds free ADP-ribose with micromolar affinity and efficiently de-ADP-ribosylates poly- but not monoADP-ribosylated proteins. Docking experiments indicate a possible binding mode for ADP-ribose polymers and suggest a reaction mechanism. Our results underscore the importance of endogenous ADP-ribosylation cycles and provide a basis for structure-based design of ADP-ribosylhydrolase inhibitors. PMID:17015823

The KRAS Promoter Responds to Myc-associated Zinc Finger and Poly(ADP-ribose) Polymerase 1 Proteins, Which Recognize a Critical Quadruplex-forming GA-element*

PubMed Central

Cogoi, Susanna; Paramasivam, Manikandan; Membrino, Alexandro; Yokoyama, Kazunari K.; Xodo, Luigi E.

2010-01-01

The murine KRAS promoter contains a G-rich nuclease hypersensitive element (GA-element) upstream of the transcription start site that is essential for transcription. Pulldown and chromatin immunoprecipitation assays demonstrate that this GA-element is bound by the Myc-associated zinc finger (MAZ) and poly(ADP-ribose) polymerase 1 (PARP-1) proteins. These proteins are crucial for transcription, because when they are knocked down by short hairpin RNA, transcription is down-regulated. This is also the case when the poly(ADP-ribosyl)ation activity of PARP-1 is inhibited by 3,4-dihydro-5-[4-(1-piperidinyl) butoxyl]-1(2H) isoquinolinone. We found that MAZ specifically binds to the duplex and quadruplex conformations of the GA-element, whereas PARP-1 shows specificity only for the G-quadruplex. On the basis of fluorescence resonance energy transfer melting and polymerase stop assays we saw that MAZ stabilizes the KRAS quadruplex. When the capacity of folding in the GA-element is abrogated by specific G → T or G → A point mutations, KRAS transcription is down-regulated. Conversely, guanidine-modified phthalocyanines, which specifically interact with and stabilize the KRAS G-quadruplex, push the promoter activity up to more than double. Collectively, our data support a transcription mechanism for murine KRAS that involves MAZ, PARP-1 and duplex-quadruplex conformational changes in the promoter GA-element. PMID:20457603

New PARP targets for cancer therapy

PubMed Central

Vyas, Sejal; Chang, Paul

2015-01-01

Poly(ADP-ribose) polymerases (PARPs) modify target proteins post-translationally with poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR) using NAD+ as substrate. The best-studied PARPs generate PAR modifications and include PARP1 and the tankyrase PARP5a, both of which are targets for cancer therapy with inhibitors in either clinical trials or preclinical development. There are 15 additional PARPs, the majority of which modify proteins with MAR, and their biology is less well understood. Recent data identify potentially cancer relevant functions for these PARPs, indicating that we need to understand more about these PARPs in order to target them effectively. PMID:24898058

Role of poly-(ADP-ribose) synthetase in lipopolysaccharide-induced vascular failure and acute lung injury in pigs.

PubMed

Albertini, M; Clement, M G; Lafortuna, C L; Caniatti, M; Magder, S; Abdulmalek, K; Hussain, S N

2000-06-01

To assess the contribution of poly (adenosine 5'-diphosphate ribose) synthetase (PARS) to the development of bacterial lipopolysaccharide (LPS)-induced acute lung injury and vascular failure in pigs. Four groups of anesthetized, paralyzed, and mechanically ventilated domestic white pigs. Group 1 served as control, whereas Escherichia coli LPS (20 microg/kg/h) was continuously infused in group 2. Group 3 received 20 mg/kg injection of 3-aminobenzamide (a selective inhibitor of PARS activity) 15 minutes before LPS infusion. Only 3-aminobenzamide and not LPS was injected in group 4. All animals were examined for 180 minutes. Systemic and pulmonary hemodynamics and lung mechanics were measured during the experimental period. Lung wet/dry ratio, bronchoalveolar lavage (BAL) protein levels and cell counts and lung nitrotyrosine (footprint of peroxynitrite) immunostaining were also measured in a few animals. LPS infusion evoked a progressive decline in systemic arterial pressure, a small increase in cardiac output, and biphasic elevation of pulmonary arterial pressure. Lung compliance declined progressively, whereas lung and total respiratory resistance rose significantly after LPS infusion. Prominent nitrotyrosine immunostaining was detected around small airways and pulmonary endothelium of LPS-infused animals. No significant changes in lung wet/dry ratio and BAL protein levels and cell counts were produced by LPS infusion. Pretreatment with 3-aminobenzamide did not alter the systemic and pulmonary hemodynamic responses to LPS infusion but eliminated the rise in pulmonary and total respiratory resistance. We concluded that PARS activation plays an important role in the changes of lung mechanics associated with LPS-induced acute lung injury but had no role in vascular failure.

Metabolic responses induced by DNA damage and poly (ADP-ribose) polymerase (PARP) inhibition in MCF-7 cells

PubMed Central

Bhute, Vijesh J.; Palecek, Sean P.

2015-01-01

Genomic instability is one of the hallmarks of cancer. Several chemotherapeutic drugs and radiotherapy induce DNA damage to prevent cancer cell replication. Cells in turn activate different DNA damage response (DDR) pathways to either repair the damage or induce cell death. These DDR pathways also elicit metabolic alterations which can play a significant role in the proper functioning of the cells. The understanding of these metabolic effects resulting from different types of DNA damage and repair mechanisms is currently lacking. In this study, we used NMR metabolomics to identify metabolic pathways which are altered in response to different DNA damaging agents. By comparing the metabolic responses in MCF-7 cells, we identified the activation of poly (ADP-ribose) polymerase (PARP) in methyl methanesulfonate (MMS)-induced DNA damage. PARP activation led to a significant depletion of NAD+. PARP inhibition using veliparib (ABT-888) was able to successfully restore the NAD+ levels in MMS-treated cells. In addition, double strand break induction by MMS and veliparib exhibited similar metabolic responses as zeocin, suggesting an application of metabolomics to classify the types of DNA damage responses. This prediction was validated by studying the metabolic responses elicited by radiation. Our findings indicate that cancer cell metabolic responses depend on the type of DNA damage responses and can also be used to classify the type of DNA damage. PMID:26478723

Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism.

PubMed

Rotin, Lianne E; Gronda, Marcela; MacLean, Neil; Hurren, Rose; Wang, XiaoMing; Lin, Feng-Hsu; Wrana, Jeff; Datti, Alessandro; Barber, Dwayne L; Minden, Mark D; Slassi, Malik; Schimmer, Aaron D

2016-01-19

Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease.

Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide.

PubMed Central

Schraufstatter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

1986-01-01

To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis. PMID:2937805

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53.

PubMed

Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

2015-11-02

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway.

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

PubMed Central

Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

2015-01-01

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway. PMID:26522181

Activation of Poly(ADP-Ribose)Polymerase in rat hepatocytes does not contribute to their cell death by oxidative stress.

PubMed

Latour, I; Leunda-Casi, A; Denef, J F; Buc Calderon, P

2000-01-10

Oxidative stress induced by tert-butyl hydroperoxide (tBOOH) in freshly isolated rat hepatocytes caused DNA damage and loss of membrane integrity. Such DNA lesions are likely to be single strand breaks since neither caryolysis nor chromatine condensation was seen in electron micrographs from tBOOH-treated cells. In addition, pulsed field gel electrophoresis of genomic DNA from both control and tBOOH-treated hepatocytes showed similar profiles, indicating the absence of internucleosomal DNA cleavage, a classical reflection of apoptotic endonuclease activity. The activation of the repair enzyme poly(ADP-ribose)polymerase (PARP) following DNA damage by tBOOH induced a dramatic drop in both NAD(+) and ATP. The inhibition of PARP by 3-aminobenzamide enhanced DNA damage by tBOOH, restored NAD(+) and ATP levels, but did not result in better survival against cell killing by tBOOH. The lack of the protective effect of PARP inhibitor, therefore, does not implicate PARP in the mechanism of tBOOH-induced cytotoxicity. Electron micrographs also show no mitochondrial swelling in cells under oxidative stress, but such organelles were mainly located around the nucleus, a picture already observed in autoschizis, a new suggested kind of cell death which shows both apoptotic and necrotic morphological characteristics. Copyright 2000 Academic Press.

Tankyrase 2 Poly(ADP-Ribose) Polymerase Domain-Deleted Mice Exhibit Growth Defects but Have Normal Telomere Length and Capping

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsiao, Susan J; Poitras, Marc; Cook, Brandoch

Regulation of telomere length maintenance and capping are a critical cell functions in both normal and tumor cells. Tankyrase 2 (Tnks2) is a poly(ADP-ribose) polymerase (PARP) that has been shown to modify itself and TRF1, a telomere-binding protein. We show here by overexpression studies that tankyrase 2, like its closely related homolog tankyrase 1, can function as a positive regulator of telomere length in human cells, dependent on its catalytic PARP activity. To study the role of Tnks2 in vivo, we generated mice with the Tnks2 PARP domain deleted. These mice are viable and fertile but display a growth retardationmore » phenotype. Telomere analysis by quantitative fluorescence in situ hybridization (FISH), flow-FISH, and restriction fragment analysis showed no change in telomere length or telomere capping in these mice. To determine the requirement foTnks2 in long-term maintenance of telomeres, we generated embryonic stem cells with the Tnks2 PARP domain deleted and observed no change, even upon prolonged growth, in telomere length or telomere capping. Together these results suggest that Tnkjs2 has a role in normal growth and development but is not essential for telomere length maintenance or telomere capping in mice.« less

Arsenite-induced ROS/RNS generation causes zinc loss and inhibits the activity of poly(ADP-ribose) polymerase-1.

PubMed

Wang, Feng; Zhou, Xixi; Liu, Wenlan; Sun, Xi; Chen, Chen; Hudson, Laurie G; Jian Liu, Ke

2013-08-01

Arsenic enhances the genotoxicity of other carcinogenic agents such as ultraviolet radiation and benzo[a]pyrene. Recent reports suggest that inhibition of DNA repair is an important aspect of arsenic cocarcinogenesis, and DNA repair proteins such as poly(ADP ribose) polymerase (PARP)-1 are direct molecular targets of arsenic. Although arsenic has been shown to generate reactive oxygen/nitrogen species (ROS/RNS), little is known about the role of arsenic-induced ROS/RNS in the mechanism underlying arsenic inhibition of DNA repair. We report herein that arsenite-generated ROS/RNS inhibits PARP-1 activity in cells. Cellular exposure to arsenite, as well as hydrogen peroxide and NONOate (nitric oxide donor), decreased PARP-1 zinc content, enzymatic activity, and PARP-1 DNA binding. Furthermore, the effects of arsenite on PARP-1 activity, DNA binding, and zinc content were partially reversed by the antioxidant ascorbic acid, catalase, and the NOS inhibitor, aminoguanidine. Most importantly, arsenite incubation with purified PARP-1 protein in vitro did not alter PARP-1 activity or DNA-binding ability, whereas hydrogen peroxide or NONOate retained PARP-1 inhibitory activity. These results strongly suggest that cellular generation of ROS/RNS plays an important role in arsenite inhibition of PARP-1 activity, leading to the loss of PARP-1 DNA-binding ability and enzymatic activity. Copyright © 2013 Elsevier Inc. All rights reserved.

Adipose tissue NAD+-homeostasis, sirtuins and poly(ADP-ribose) polymerases -important players in mitochondrial metabolism and metabolic health.

PubMed

Jokinen, Riikka; Pirnes-Karhu, Sini; Pietiläinen, Kirsi H; Pirinen, Eija

2017-08-01

Obesity, a chronic state of energy overload, is characterized by adipose tissue dysfunction that is considered to be the major driver for obesity associated metabolic complications. The reasons for adipose tissue dysfunction are incompletely understood, but one potential contributing factor is adipose tissue mitochondrial dysfunction. Derangements of adipose tissue mitochondrial biogenesis and pathways associate with obesity and metabolic diseases. Mitochondria are central organelles in energy metabolism through their role in energy derivation through catabolic oxidative reactions. The mitochondrial processes are dependent on the proper NAD + /NADH redox balance and NAD + is essential for reactions catalyzed by the key regulators of mitochondrial metabolism, sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs). Notably, obesity is associated with disturbed adipose tissue NAD + homeostasis and the balance of SIRT and PARP activities. In this review we aim to summarize existing literature on the maintenance of intracellular NAD + pools and the function of SIRTs and PARPs in adipose tissue during normal and obese conditions, with the purpose of comprehending their potential role in mitochondrial derangements and obesity associated metabolic complications. Understanding the molecular mechanisms that are the root cause of the adipose tissue mitochondrial derangements is crucial for developing new effective strategies to reverse obesity associated metabolic complications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Poly-(ADP-ribose)-polymerase inhibitors as radiosensitizers: a systematic review of pre-clinical and clinical human studies.

PubMed

Lesueur, Paul; Chevalier, François; Austry, Jean-Baptiste; Waissi, Waisse; Burckel, Hélène; Noël, Georges; Habrand, Jean-Louis; Saintigny, Yannick; Joly, Florence

2017-09-15

Poly-(ADP-Ribose)-Polymerase (PARP) inhibitors are becoming important actors of anti-neoplasic agents landscape, with recent but narrow FDA's approvals for ovarian BRCA mutated cancers and prostatic cancer. Nevertheless, PARP inhibitors are also promising drugs for combined treatments particularly with radiotherapy. More than seven PARP inhibitors have been currently developed. Central Role of PARP in DNA repair, makes consider PARP inhibitor as potential radiosensitizers, especially for tumors with DNA repair defects, such as BRCA mutation, because of synthetic lethality. Furthermore the replication-dependent activity of PARP inhibitor helps to maintain the differential effect between tumoral and healthy tissues. Inhibition of chromatin remodeling, G2/M arrest, vasodilatory effect induced by PARP inhibitor, also participate to their radio-sensitization effect. Here, after highlighting mechanisms of PARP inhibitors radiosensitization we methodically searched PubMed, Google Scholar, Cochrane Databases and meeting proceedings for human pre-clinical and clinical studies that evaluated PARP inhibitor radiosensitizing effect. Enhancement ratio, when available, was systematically reported. Sixty four studies finally met our selection criteria and were included in the analysis. Only three pre-clinical studies didn't find any radiosensitizing effect. Median enhancement ratio vary from 1,3 for prostate tumors to 1,5 for lung cancers. Nine phase I or II trials assessed safety data. PARP inhibitors are promising radiosensitizers, but need more clinical investigation. The next ten years will be determining for judging their real potential.

Imidazoquinolinone, imidazopyridine, and isoquinolindione derivatives as novel and potent inhibitors of the poly(ADP-ribose) polymerase (PARP): a comparison with standard PARP inhibitors.

PubMed

Eltze, Tobias; Boer, Rainer; Wagner, Thomas; Weinbrenner, Steffen; McDonald, Michelle C; Thiemermann, Christoph; Bürkle, Alexander; Klein, Thomas

2008-12-01

We have identified three novel structures for inhibitors of the poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA and implicated in DNA repair, apoptosis, organ dysfunction or necrosis. 2-[4-(5-Methyl-1H-imidazol-4-yl)-piperidin-1-yl]-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK49187), 2-(4-pyridin-2-yl-phenyl)-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK236864), 6-chloro-8-hydroxy-2,3-dimethyl-imidazo-[1,2-alpha]-pyridine (BYK20370), and 4-(1-methyl-1H-pyrrol-2-ylmethylene)-4H-isoquinolin-1,3-dione (BYK204165) inhibited cell-free recombinant human PARP-1 with pIC(50) values of 8.36, 7.81, 6.40, and 7.35 (pK(i) 7.97, 7.43, 5.90, and 7.05), and murine PARP-2 with pIC(50) values of 7.50, 7.55, 5.71, and 5.38, respectively. BYK49187, BYK236864, and BYK20370 displayed no selectivity for PARP-1/2, whereas BYK204165 displayed 100-fold selectivity for PARP-1. The IC(50) values for inhibition of poly(ADP-ribose) synthesis in human lung epithelial A549 and cervical carcinoma C4I cells as well in rat cardiac myoblast H9c2 cells after PARP activation by H(2)O(2) were highly significantly correlated with those at cell-free PARP-1 (r(2) = 0.89-0.96, P < 0.001) but less with those at PARP-2 (r(2) = 0.78-0.84, P < 0.01). The infarct size caused by coronary artery occlusion and reperfusion in the anesthetized rat was reduced by 22% (P < 0.05) by treatment with BYK49187 (3 mg/kg i.v. bolus and 3 mg/kg/h i.v. during 2-h reperfusion), whereas the weaker PARP inhibitors, BYK236864 and BYK20370, were not cardioprotective. In conclusion, the imidazoquinolinone BYK49187 is a potent inhibitor of human PARP-1 activity in cell-free and cellular assays in vitro and reduces myocardial infarct size in vivo. The isoquinolindione BYK204165 was found to be 100-fold more selective for PARP-1. Thus, both compounds might be novel and valuable tools for investigating PARP-1-mediated effects.

ADP-ribosylation of proteins: Enzymology and biological significance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Althaus, F.R.; Richter, C.

1987-01-01

This book presents an overview of the molecular and biological consequences of the posttranslational modification of proteins with ADP-ribose monomers and polymers. Part one focuses on chromatin-associated poly ADP-ribosylation reactions which have evolved in higher eukaryotes as modulators of chromatin functions. The significance of poly ADP-ribosylation in DNA repair, carcinogenesis, and gene expression during terminal differentiation is discussed. Part two reviews mono ADP-ribosylation reactions which are catalyzed by prokaryotic and eukaryotic enzymes. Consideration is given to the action of bacterial toxins, such as cholera toxin, pertussis toxin, and diphtheria toxin. These toxins have emerged as tools for the molecular probingmore » of proteins involved in signal transduction and protein biosynthesis.« less

Sulforaphane inhibits damage-induced poly (ADP-ribosyl)ation via direct interaction of its cellular metabolites with PARP-1.

PubMed

Piberger, Ann Liza; Keil, Claudia; Platz, Stefanie; Rohn, Sascha; Hartwig, Andrea

2015-11-01

The isothiocyanate sulforaphane, a major breakdown product of the broccoli glucosinolate glucoraphanin, has frequently been proposed to exert anticarcinogenic properties. Potential underlying mechanisms include a zinc release from Kelch-like ECH-associated protein 1 followed by the induction of detoxifying enzymes. This suggests that sulforaphane may also interfere with other zinc-binding proteins, e.g. those essential for DNA repair. Therefore, we explored the impact of sulforaphane on poly (ADP-ribose)polymerase-1 (PARP-1), poly (ADP-ribosyl)ation (PARylation), and DNA single-strand break repair (SSBR) in cell culture. Immunofluorescence analyses showed that sulforaphane diminished H2 O2 -induced PARylation in HeLa S3 cells starting from 15 μM despite increased lesion induction under these conditions. Subcellular experiments quantifying the damage-induced incorporation of (32) P-ADP-ribose by PARP-1 displayed no direct impact of sulforaphane itself, but cellular metabolites, namely the glutathione conjugates of sulforaphane and its interconversion product erucin, reduced PARP-1 activity concentration dependently. Interestingly, this sulforaphane metabolite-induced PARP-1 inhibition was prevented by thiol compounds. PARP-1 is a stimulating factor for DNA SSBR-rate and we further demonstrated that 25 μM sulforaphane also delayed the rejoining of H2 O2 -induced DNA strand breaks, although this might be partly due to increased lesion frequencies. Sulforaphane interferes with damage-induced PARylation and SSBR, which implies a sulforaphane-dependent impairment of genomic stability. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair

PubMed Central

Robu, Mihaela; Shah, Rashmi G.; Purohit, Nupur K.; Zhou, Pengbo; Naegeli, Hanspeter

2017-01-01

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV–DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1–XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV–DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins. PMID:28760956

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

PubMed

Robu, Mihaela; Shah, Rashmi G; Purohit, Nupur K; Zhou, Pengbo; Naegeli, Hanspeter; Shah, Girish M

2017-08-15

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism

PubMed Central

Rotin, Lianne E.; Gronda, Marcela; MacLean, Neil; Hurren, Rose; Wang, XiaoMing; Lin, Feng-Hsu; Wrana, Jeff; Datti, Alessandro; Barber, Dwayne L.; Minden, Mark D.; Slassi, Malik; Schimmer, Aaron D.

2016-01-01

Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease. PMID:26624983

Minocycline attenuates streptomycin-induced cochlear hair cell death by inhibiting protein nitration and poly (ADP-ribose) polymerase activation.

PubMed

Wang, Ping; Li, Haonan; Yu, Shuyuan; Jin, Peng; Hassan, Abdurahman; Du, Bo

2017-08-24

This study aimed to elucidate the protective effect of minocycline against streptomycin-induced damage of cochlear hair cells and its mechanism. Cochlear membranes were isolated from newborn Wistar rats and randomly divided into control, 500μmol/L streptomycin, 100μmol/L minocycline, and streptomycin and minocycline treatment groups. Hair cell survival was analyzed by detecting the expression of 3-nitrotyrosine (3-NT) in cochlear hair cells by immunofluorescence and an enzyme-linked immunosorbent assay. Expression of 3-NT and inducible nitric oxide synthase (iNOS), and poly (ADP-Ribose) polymerase (PARP) and caspase-3 activation were evaluated by western blotting. The results demonstrated hair cell loss at 24h after streptomycin treatment. No change was found in supporting cells of the cochleae. Minocycline pretreatment improved hair cell survival and significantly reduced the expression of iNOS and 3-NT in cochlear tissues compared with the streptomycin treatment group. PARP and caspase-3 activation was increased in the streptomycin treatment group compared with the control group, and pretreatment with minocycline decreased cleaved PARP and activated caspase-3 expression. Minocycline protected cochlear hair cells from injury caused by streptomycin in vitro. The mechanism underlying the protective effect may be associated with the inhibition of excessive formation of nitric oxide, reduction of the nitration stress reaction, and inhibition of PARP and caspase-3 activation in cochlear hair cells. Combined minocycline therapy can be applied to patients requiring streptomycin treatment. Copyright © 2017. Published by Elsevier B.V.

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases.

PubMed

Simpson, L L; Stiles, B G; Zepeda, H; Wilkins, T D

1989-01-01

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins.

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases.

PubMed Central

Simpson, L L; Stiles, B G; Zepeda, H; Wilkins, T D

1989-01-01

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins. Images PMID:2521214

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle.

PubMed

Franco, L; Bruzzone, S; Song, P; Guida, L; Zocchi, E; Walseth, T F; Crimi, E; Usai, C; De Flora, A; Brusasco, V

2001-01-01

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.

The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2.

PubMed

Croy, Heather E; Fuller, Caitlyn N; Giannotti, Jemma; Robinson, Paige; Foley, Andrew V A; Yamulla, Robert J; Cosgriff, Sean; Greaves, Bradford D; von Kleeck, Ryan A; An, Hyun Hyung; Powers, Catherine M; Tran, Julie K; Tocker, Aaron M; Jacob, Kimberly D; Davis, Beckley K; Roberts, David M

2016-06-10

Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein β-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the β-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate β-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases β-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Influence of MLH1 on colon cancer sensitivity to poly(ADP-ribose) polymerase inhibitor combined with irinotecan.

PubMed

Tentori, Lucio; Leonetti, Carlo; Muzi, Alessia; Dorio, Annalisa Susanna; Porru, Manuela; Dolci, Susanna; Campolo, Federica; Vernole, Patrizia; Lacal, Pedro Miguel; Praz, Françoise; Graziani, Grazia

2013-07-01

Poly(ADP-ribose) polymerase inhibitors (PARPi) are currently evaluated in clinical trials in combination with topoisomerase I (Top1) inhibitors against a variety of cancers, including colon carcinoma. Since the mismatch repair component MLH1 is defective in 10-15% of colorectal cancers we have investigated whether MLH1 affects response to the Top1 inhibitor irinotecan, alone or in combination with PARPi. To this end, the colon cancer cell lines HCT116, carrying MLH1 mutations on chromosome 3 and HCT116 in which the wild-type MLH1 gene was replaced via chromosomal transfer (HCT116+3) or by transfection of the corresponding MLH1 cDNA (HCT116 1-2) were used. HCT116 cells or HCT116+3 cells stably silenced for PARP-1 expression were also analysed. The results of in vitro and in vivo experiments indicated that MLH1, together with low levels of Top1, contributed to colon cancer resistance to irinotecan. In the MLH1-proficient cells SN-38, the active metabolite of irinotecan, induced lower levels of DNA damage than in MLH1-deficient cells, as shown by the weaker induction of γ-H2AX and p53 phosphorylation. The presence of MLH1 contributed to induce of prompt Chk1 phosphorylation, restoring G2/M cell cycle checkpoint and repair of DNA damage. On the contrary, in the absence of MLH1, HCT116 cells showed minor Chk1 phosphorylation and underwent apoptosis. Remarkably, inhibition of PARP function by PARPi or by PARP-1 gene silencing always increased the antitumor activity of irinotecan, even in the presence of low PARP-1 expression.

The role of ADP-ribosylation in regulating DNA interstrand crosslink repair

PubMed Central

Gunn, Alasdair R.; Banos-Pinero, Benito; Paschke, Peggy; Sanchez-Pulido, Luis; Ariza, Antonio; Day, Joseph; Emrich, Mehera; Leys, David; Ponting, Chris P.

2016-01-01

ABSTRACT ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here, we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches were used to identify a protein that contains a permutated macrodomain (which we call aprataxin/APLF-and-PNKP-like protein; APL). Structural analysis reveals that this permutated macrodomain retains features associated with ADP-ribose interactions and that APL is capable of binding poly(ADP-ribose) through this macrodomain. APL is enriched in chromatin in response to cisplatin treatment, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2− cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells owing to redundant signalling by the double-strand break (DSB)-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify that NHEJ can function to resolve this form of DNA damage in the absence of Adprt2. PMID:27587838

Poly(ADP-ribose) polymerase inhibitors sensitize cancer cells to death receptor-mediated apoptosis by enhancing death receptor expression.

PubMed

Meng, X Wei; Koh, Brian D; Zhang, Jin-San; Flatten, Karen S; Schneider, Paula A; Billadeau, Daniel D; Hess, Allan D; Smith, B Douglas; Karp, Judith E; Kaufmann, Scott H

2014-07-25

Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation.

Poly(ADP-ribose) Polymerase Inhibitors Sensitize Cancer Cells to Death Receptor-mediated Apoptosis by Enhancing Death Receptor Expression*

PubMed Central

Meng, X. Wei; Koh, Brian D.; Zhang, Jin-San; Flatten, Karen S.; Schneider, Paula A.; Billadeau, Daniel D.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Kaufmann, Scott H.

2014-01-01

Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation. PMID:24895135

The nuclear protein Poly(ADP-ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana.

PubMed

Rissel, D; Losch, J; Peiter, E

2014-11-01

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP-ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear-localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3-1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col-0 wild-type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Inhibition of poly(ADP-ribose) polymerase 1 protects against acute myeloid leukemia by suppressing the myeloproliferative leukemia virus oncogene

PubMed Central

Wang, Lingbo; Cai, Weili; Zhang, Wei; Chen, Xueying; Dong, Wenqian; Tang, Dongqi; Zhang, Yun; Ji, Chunyan; Zhang, Mingxiang

2015-01-01

An abnormal expression of poly(ADP-ribose) polymerase 1 (PARP-1) has been described in many tumors. PARP-1 promotes tumorigenesis and cancer progression by acting on different molecular pathways. PARP-1 inhibitors can be used with radiotherapy or chemotherapy to enhance the susceptibility of tumor cells to the treatment. However, the specific mechanism of PARP-1 in acute myeloid leukemia (AML) remains unknown. Our study showed that expression of PARP-1 was upregulated in AML patients. PARP-1 inhibition slowed AML cell proliferation, arrested the cell cycle, induced apoptosis in vitro and improved AML prognosis in vivo. Mechanistically, microarray assay of AML cells with loss of PARP-1 function revealed that the myeloproliferative leukemia virus oncogene (MPL) was significantly downregulated. In human AML samples, MPL expression was increased, and gain-of-function and loss-of-function analysis demonstrated that MPL promoted cell growth. Moreover, PARP-1 and MPL expression were positively correlated in AML samples, and their overexpression was associated with an unfavorable prognosis. Furthermore, PARP-1 and MPL consistently acted on Akt and ERK1/2 pathways, and the anti-proliferative and pro-apoptotic function observed with PARP-1 inhibition were reversed in part via MPL activation upon thrombopoietin stimulation or gene overexpression. These data highlight the important function of PARP-1 in the progression of AML, which suggest PARP-1 as a potential target for AML treatment. PMID:26314963

Minocycline blocks asthma-associated inflammation in part by interfering with the T cell receptor-nuclear factor κB-GATA-3-IL-4 axis without a prominent effect on poly(ADP-ribose) polymerase.

PubMed

Naura, Amarjit S; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C; Jordan, Joaquin; Catling, Andrew D; Rezk, Bashir M; Abd Elmageed, Zakaria Y; Pyakurel, Kusma; Tarhuni, Abdelmetalab F; Abughazleh, Mohammad Q; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C; Boulares, A Hamid

2013-01-18

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

Minocycline Blocks Asthma-associated Inflammation in Part by Interfering with the T Cell Receptor-Nuclear Factor κB-GATA-3-IL-4 Axis without a Prominent Effect on Poly(ADP-ribose) Polymerase*

PubMed Central

Naura, Amarjit S.; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C.; Jordan, Joaquin; Catling, Andrew D.; Rezk, Bashir M.; Elmageed, Zakaria Y. Abd; Pyakurel, Kusma; Tarhuni, Abdelmetalab F.; Abughazleh, Mohammad Q.; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C.; Boulares, A. Hamid

2013-01-01

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N′-nitro-N-nitroso-guanidine-treated mice or H2O2-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production. PMID:23184953

Poly(ADP-ribose) polymerase inhibition combined with irradiation: A dual treatment concept to prevent neointimal hyperplasia after endarterectomy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beller, Carsten J.; Kosse, Jens; Radovits, Tamas

2006-11-01

Purpose: In a rat model of endarterectomy we investigated the potential role of the peroxynitrite-poly(ADP-ribose) polymerase (PARP) pathway in neointima formation and the effects of irradiation, pharmacologic inhibition of PARP, or combined pharmacologic inhibition of PARP and irradiation on vascular remodeling. Methods and Materials: Carotid endarterectomy was performed by incision of the left carotid artery with removal of intima in Sprague-Dawley rats. Six groups were studied: sham-operated rats (n = 10), control endarterectomized rats (n = 10), or endarterectomized rats irradiated with 15 Gy (n = 10), or treated with PARP inhibitor, INO-1001 (5 mg/kg/day) (n = 10), or withmore » combined treatment with INO-1001 and irradiation with 5 Gy (n = 10) or with 15 Gy (n = 10). After 21 days, neointima formation and vascular remodeling were assessed. Results: Neointima formation after endarterectomy was inhibited by postoperative irradiation with 15 Gy and was attenuated by PARP inhibition. However, in parallel to inhibition of neointimal hyperplasia, activation of the peroxynitrite-PARP pathway in the outer vessel wall layers was triggered by postoperative irradiation. Combined pharmacologic PARP inhibition and irradiation with 15 Gy significantly reduced both neointimal hyperplasia and activation of the peroxynitrite-PARP pathway in the outer vessel wall layers. Combination of PARP inhibition and irradiation with 5 Gy was less effective than both PARP inhibition or irradiation with 15 Gy alone. Conclusions: We conclude, that combined PARP inhibition and irradiation with 15 Gy may be a new dual strategy for prevention of restenosis after surgical vessel reconstruction: combining the strong antiproliferative effect of irradiation and ameliorating irradiation-induced side effects caused by excessive PARP activation.« less

Replication-Dependent Radiosensitization of Human Glioma Cells by Inhibition of Poly(ADP-Ribose) Polymerase: Mechanisms and Therapeutic Potential

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dungey, Fiona A.; Loeser, Dana A.; Chalmers, Anthony J.

2008-11-15

Purpose: Current treatments for glioblastoma multiforme are inadequate and limited by the radiation sensitivity of normal brain. Because glioblastoma multiforme are rapidly proliferating tumors within nondividing normal tissue, the therapeutic ratio might be enhanced by combining radiotherapy with a replication-specific radiosensitizer. KU-0059436 (AZD2281) is a potent and nontoxic inhibitor of poly(ADP-ribose) polymerase-1 (PARP-1) undergoing a Phase II clinical trial as a single agent. Methods and Materials: Based on previous observations that the radiosensitizing effects of PARP inhibition are more pronounced in dividing cells, we investigated the mechanisms underlying radiosensitization of human glioma cells by KU-0059436, evaluating the replication dependence ofmore » this effect and its therapeutic potential. Results: KU-0059436 increased the radiosensitivity of four human glioma cell lines (T98G, U373-MG, UVW, and U87-MG). Radiosensitization was enhanced in populations synchronized in S phase and abrogated by concomitant exposure to aphidicolin. Sensitization was further enhanced when the inhibitor was combined with a fractionated radiation schedule. KU-0059436 delayed repair of radiation-induced DNA breaks and was associated with a replication-dependent increase in {gamma}H2AX and Rad51 foci. Conclusion: The results of our study have shown that KU-0059436 increases radiosensitivity in a replication-dependent manner that is enhanced by fractionation. A mechanism is proposed whereby PARP inhibition increases the incidence of collapsed replication forks after ionizing radiation, generating persistent DNA double-strand breaks. These observations indicate that KU-0059436 is likely to enhance the therapeutic ratio achieved by radiotherapy in the treatment of glioblastoma multiforme. A Phase I clinical trial is in development.« less

Poly (ADP-Ribose) Polymerase-1 (PARP-1) Induction by Cocaine Is Post-Transcriptionally Regulated by miR-125b

PubMed Central

Dash, Sabyasachi; Balasubramaniam, Muthukumar; Godino, Arthur; Villalta, Fernando; Calipari, Erin S.; Dash, Chandravanu

2017-01-01

Abstract Cocaine exposure alters gene expression in the brain via methylation and acetylation of histones along with methylation of DNA. Recently, poly (ADP-ribose) polymerase-1 (PARP-1) catalyzed PARylation has been reported as an important regulator of cocaine-mediated gene expression. In this study, we report that the cellular microRNA “miR-125b” plays a key role for cocaine-induced PARP-1 expression. Acute and chronic cocaine exposure resulted in the downregulation of miR-125b concurrent with upregulation of PARP-1 in dopaminergic neuronal cells and nucleus accumbens (NAc) of mice but not in the medial prefrontal cortex (PFC) or ventral tegmental area (VTA). In silico analysis predicted a binding site of miR-125b in a conserved 3’-untranslated region (3’UTR) of the PARP-1 mRNA. Knockdown and overexpression studies showed that miR-125b levels negatively correlate with PARP-1 protein expression. Luciferase reporter assay using a vector containing the 3’UTR of PARP-1 mRNA confirmed regulation of PARP-1 by miR-125b. Specific nucleotide mutations within the binding site abrogated miR-125b’s regulatory effect on PARP-1 3’UTR. Finally, we established that downregulation of miR-125b and concurrent upregulation of PARP-1 is dependent on binding of cocaine to the dopamine transporter (DAT). Collectively, these results identify miR-125b as a post-transcriptional regulator of PARP-1 expression and establish a novel mechanism underlying the molecular effects of cocaine action. PMID:28828398

PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes

PubMed Central

Gupte, Rebecca; Liu, Ziying; Kraus, W. Lee

2017-01-01

The discovery of poly(ADP-ribose) >50 years ago opened a new field, leading the way for the discovery of the poly(ADP-ribose) polymerase (PARP) family of enzymes and the ADP-ribosylation reactions that they catalyze. Although the field was initially focused primarily on the biochemistry and molecular biology of PARP-1 in DNA damage detection and repair, the mechanistic and functional understanding of the role of PARPs in different biological processes has grown considerably of late. This has been accompanied by a shift of focus from enzymology to a search for substrates as well as the first attempts to determine the functional consequences of site-specific ADP-ribosylation on those substrates. Supporting these advances is a host of methodological approaches from chemical biology, proteomics, genomics, cell biology, and genetics that have propelled new discoveries in the field. New findings on the diverse roles of PARPs in chromatin regulation, transcription, RNA biology, and DNA repair have been complemented by recent advances that link ADP-ribosylation to stress responses, metabolism, viral infections, and cancer. These studies have begun to reveal the promising ways in which PARPs may be targeted therapeutically for the treatment of disease. In this review, we discuss these topics and relate them to the future directions of the field. PMID:28202539

Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

PubMed

Verrax, Julien; Vanbever, Stéphanie; Stockis, Julie; Taper, Henryk; Calderon, Pedro Buc

2007-03-15

Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells. (c) 2006 Wiley-Liss, Inc.

Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aoyagi-Scharber, Mika, E-mail: maoyagi@bmrn.com; Gardberg, Anna S.; Yip, Bryan K.

2014-08-29

BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers.more » In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity.« less

MSH3 mediates sensitization of colorectal cancer cells to cisplatin, oxaliplatin, and a poly(ADP-ribose) polymerase inhibitor.

PubMed

Takahashi, Masanobu; Koi, Minoru; Balaguer, Francesc; Boland, C Richard; Goel, Ajay

2011-04-08

The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2, forms the MutSβ heteroduplex, which interacts with interstrand cross-links (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. In this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a Tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of phosphorylated histone H2AX and 53BP1 after oxaliplatin treatment in comparison with MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSBs). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited a synergistic effect compared with either treatment individually. Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay the foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency.

MSH3 Mediates Sensitization of Colorectal Cancer Cells to Cisplatin, Oxaliplatin, and a Poly(ADP-ribose) Polymerase Inhibitor*

PubMed Central

Takahashi, Masanobu; Koi, Minoru; Balaguer, Francesc; Boland, C. Richard; Goel, Ajay

2011-01-01

The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2, forms the MutSβ heteroduplex, which interacts with interstrand cross-links (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. In this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a Tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of phosphorylated histone H2AX and 53BP1 after oxaliplatin treatment in comparison with MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSBs). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited a synergistic effect compared with either treatment individually. Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay the foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency. PMID:21285347

Identification of Poly(ADP-Ribose) Polymerase as a Transcriptional Coactivator of the Human T-Cell Leukemia Virus Type 1 Tax Protein

PubMed Central

Anderson, Mark G.; Scoggin, Kirsten E. S.; Simbulan-Rosenthal, Cynthia M.; Steadman, Jennifer A.

2000-01-01

Human T-cell leukemia virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose activity is believed to contribute significantly to cellular transformation. Tax stimulates transcription from the proviral promoter as well as from promoters for a variety of cellular genes. The mechanism through which Tax communicates to the general transcription factors and RNA polymerase II has not been completely determined. We investigated whether Tax could function directly through the general transcription factors and RNA polymerase II or if other intermediary factors or coactivators were required. Our results show that a system consisting of purified recombinant TFIIA, TFIIB, TFIIE, TFIIF, CREB, and Tax, along with highly purified RNA polymerase II, affinity-purified epitope-tagged TFIID, and semipurified TFIIH, supports basal transcription of the HTLV-1 promoter but is not responsive to Tax. Two additional activities were required for Tax to stimulate transcription. We demonstrate that one of these activities is poly(ADP-ribose) polymerase (PARP), a molecule that has been previously identified to be the transcriptional coactivator PC1. PARP functions as a coactivator in our assays at molar concentrations approximately equal to those of the DNA and equal to or less than those of the transcription factors in the assay. We further demonstrate that PARP stimulates Tax-activated transcription in vivo, demonstrating that this biochemical approach has functionally identified a novel target for the retroviral transcriptional activator Tax. PMID:10666246

Poly[ADP-ribose] polymerase-1 expression is related to cold ischemia, acute tubular necrosis, and delayed renal function in kidney transplantation.

PubMed

O'Valle, Francisco; Del Moral, Raimundo G M; Benítez, María del Carmén; Martín-Oliva, David; Gómez-Morales, Mercedes; Aguilar, David; Aneiros-Fernández, José; Hernández-Cortés, Pedro; Osuna, Antonio; Moreso, Francesc; Serón, Daniel; Oliver, Francisco J; Del Moral, Raimundo G

2009-09-28

Cold ischemia time especially impacts on outcomes of expanded-criteria donor (ECD) transplantation. Ischemia-reperfusion (IR) injury produces excessive poly[ADP-Ribose] Polymerase-1 (PARP-1) activation. The present study explored the hypothesis that increased tubular expression of PARP-1 contributes to delayed renal function in suboptimal ECD kidney allografts and in non-ECD allografts that develop posttransplant acute tubular necrosis (ATN). Nuclear PARP-1 immunohistochemical expression was studied in 326 paraffin-embedded renal allograft biopsies (193 with different degrees of ATN and 133 controls) and in murine Parp-1 knockout model of IR injury. PARP-1 expression showed a significant relationship with cold ischemia time (r coefficient = 0.603), time to effective diuresis (r = 0.770), serum creatinine levels at biopsy (r = 0.649), and degree of ATN (r = 0.810) (p = 0.001, Pearson test). In the murine IR model, western blot showed an increase in PARP-1 that was blocked by Parp-1 inhibitor. Immunohistochemical study of PARP-1 in kidney allograft biopsies would allow early detection of possible delayed renal function, and the administration of PARP-1 inhibitors may offer a therapeutic option to reduce damage from IR in donor kidneys by preventing or minimizing ATN. In summary, these results suggest a pivotal role for PARP-1 in the ATN of renal transplantation. We propose the immunohistochemical assessment of PARP-1 in kidney allograft biopsies for early detection of a possible delayed renal function.

Effects of 7-ketocholesterol on the activity of endothelial poly(ADP-ribose) polymerase and on endothelium-dependent relaxant function.

PubMed

Kiss, Levente; Chen, Min; Gero, Domokos; Módis, Katalin; Lacza, Zsombor; Szabó, Csaba

2006-12-01

Oxidative and nitrosative stress play an important role in the development of endothelial vascular dysfunction during early atherosclerosis. Oxidative stress activates the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in endothelial cells. In patients with atherosclerosis the level of oxidized LDL in the plasma is elevated. In oxidized LDL various oxysterols have been identified, such as 7-ketocholesterol (7K). 7K has been shown to induce PARP activation in microglial cells. The aim of the current study was to clarify the effects of 7K on the activity of endothelial PARP and on the endothelium-dependent relaxant function of blood vessels. We treated human umbilical vein endothelial (HUVEC) cells with 2-16 microg/ml 7K as well as vascular rings harvested from BALB/c mouse thoracic aorta with 90 microg/ml 7K for 2 h. A group of mice was treated with 7K subcutaneously for 1 week (10 mg/kg/day). We also conducted in vitro and in vivo experiments using pretreatment with buthionine sulphoximine (BSO), a glutathione-lowering agent. The activity of PARP was calculated by measurement of tritiated NAD incorporation. The activity of PARP increased significantly in 7K-treated HUVEC cells. After BSO pretreatment, this increase was higher. Isolated vascular rings demonstrated no change in endothelium-dependent relaxant function after 2 h of incubation with 7K, even after BSO pretreatment. In vivo treatment with 7K for 1 week had no effect on the relaxant function. Our experimental results suggest that although 7-ketocholesterol can activate PARP enzyme in endothelial cells, it is not sufficient on its own to cause impairment in the endothelium-dependent vascular reactivity.

Methotrexate induces poly(ADP-ribose) polymerase-dependent, caspase 3-independent apoptosis in subsets of proliferating CD4+ T cells.

PubMed

Nielsen, C H; Albertsen, L; Bendtzen, K; Baslund, B

2007-05-01

The mechanism of action of methotrexate (MTX) in autoimmune diseases (AID) is unclear. A pro-apoptotic effect has been demonstrated in mitogen-stimulated peripheral blood mononuclear cells (PBMC), but studies employing conventional antigens have disputed a pro-apoptotic effect. CD4+ T helper (Th) cells play a significant role in most AID. We therefore examined directly, by flow cytometry, the uptake of MTX by the T helper (Th) cells stimulated for 6 days with Candida albicans (CA) or tetanus toxoid (TT), and its consequences with respect to induction of apoptosis. While none of the resting Th cells took up MTX, nearly all the dividing Th cells did, and this abrogated further cell division. Among dividing Th cells, MTX induced an approximately sixfold increase over baseline levels in the proportion of apoptotic cells. This proportion could be reverted to baseline by the addition of folic acid. Exposure of CA-stimulated PBMC to MTX significantly increased their level of cleaved poly(ADP-ribose) polymerase (PARP), and a similar tendency was observed in TT-stimulated cells. Unlike CA and TT, the mitogen phytohaemagglutinin (PHA) induced proliferation of both CD4- and CD4+ T cells, and induced apoptosis in both undivided and divided Th cells. PHA-induced apoptosis involved activation of caspase-3 and the anti-apoptotic protein Bcl-2 in addition to PARP cleavage, suggesting that PHA induces apoptosis via different pathways than CA and TT. We suggest that the latter are more representative of stimulation with self-antigens in AID, and that a pro-apoptotic effect of MTX on self-antigen-stimulated Th cells contributes to the effect of MTX in the treatment of AID.

Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

PubMed Central

Lauretti, Elisabetta; Hulse, Michael; Siciliano, Micheal; Lupey-Green, Lena N.; Abraham, Aaron; Skorski, Tomasz; Tempera, Italo

2018-01-01

The enzyme Poly(ADP-ribose) polymerase 1 (PARP1) plays a very important role in the DNA damage response, but its role in numerous aspects is not fully understood. We recently showed that in the absence of DNA damage, PARP1 regulates the expression of the chromatin-modifying enzyme EZH2. Work from other groups has shown that EZH2 participates in the DNA damage response. These combined data suggest that EZH2 could be a target of PARP1 in both untreated and genotoxic agent-treated conditions. In this work we tested the hypothesis that, in response to DNA damage, PARP1 regulates EZH2 activity. Here we report that PARP1 regulates EZH2 activity after DNA damage. In particular, we find that EZH2 is a direct target of PARP1 upon induction of alkylating and UV-induced DNA damage in cells and in vitro. PARylation of EZH2 inhibits EZH2 histone methyltransferase (H3K27me) enzymatic activity. We observed in cells that the induction of PARP1 activity by DNA alkylating agents decreases the association of EZH2 with chromatin, and PARylation of histone H3 reduces EZH2 affinity for its target histone H3. Our findings establish that PARP1 and PARylation are important regulators of EZH2 function and link EZH2-mediated heterochromatin formation, DNA damage and PARylation. These findings may also have clinical implications, as they suggest that inhibitors of EZH2 can improve anti-tumor effects of PARP1 inhibitors in BRCA1/2-deficient cancers. PMID:29535829

ADP-ribosyl-N₃: A Versatile Precursor for Divergent Syntheses of ADP-ribosylated Compounds.

PubMed

Li, Lingjun; Li, Qianqian; Ding, Shengqiang; Xin, Pengyang; Zhang, Yuqin; Huang, Shenlong; Zhang, Guisheng

2017-08-14

Adenosine diphosphate-ribose (ADP-ribose) and its derivatives play important roles in a series of complex physiological procedures. The design and synthesis of artificial ADP-ribosylated compounds is an efficient way to develop valuable chemical biology tools and discover new drug candidates. However, the synthesis of ADP-ribosylated compounds is currently difficult due to structural complexity, easily broken pyrophosphate bond and high hydrophilicity. In this paper, ADP-ribosyl-N₃ was designed and synthesized for the first time. With ADP-ribosyl-N₃ as the key precursor, a divergent post-modification strategy was developed to prepare structurally diverse ADP-ribosylated compounds including novel nucleotides and peptides bearing ADP-ribosyl moieties.

Enzymatic synthesis and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with ADP-ribosyl cyclase activity.

PubMed

Graeff, R M; Walseth, T F; Fryxell, K; Branton, W D; Lee, H C

1994-12-02

Cyclic nucleotides such as cAMP and cGMP are second messengers subserving various signaling pathways. Cyclic ADP-ribose (cADPR), a recently discovered member of the family, is derived from NAD+ and is a mediator of Ca2+ mobilization in various cellular systems. The synthesis and degradation of cADPR are, respectively, catalyzed by ADP-ribosyl cyclase and cADPR hydrolase. CD38, a differentiation antigen of B lymphocytes, has recently been shown to be a bifunctional enzyme catalyzing both the formation and hydrolysis of cADPR. The overall reaction catalyzed by CD38 is the formation of ADP-ribose and nicotinamide from NAD+, identical to that catalyzed by NADase. The difficulties in detecting the formation of cADPR have led to frequent identification of CD38 as a classical NADase. In this study, we show that both ADP-ribosyl cyclase and CD38, but not NADase, can cyclize nicotinamide guanine dinucleotide (NGD+) producing a new nucleotide. Analyses by high performance liquid chromatography and mass spectroscopy indicate the product is cyclic GDP-ribose (cGDPR) with a structure similar to cADPR except with guanine replacing adenine. Compared to cADPR, cGDPR is a more stable compound showing 2.8 times more resistance to heat-induced hydrolysis. These results are consistent with a catalytic scheme for CD38 where the cyclization of the substrate precedes the hydrolytic reaction. Spectroscopic analyses show that cGDPR is fluorescent and has an absorption spectrum different from both NGD+ and GDPR, providing a very convenient way for monitoring its enzymatic formation. The use of NGD+ as substrate for assaying the cyclization reaction was found to be applicable to pure enzymes as well as crude tissue extracts making it a useful diagnostic tool for distinguishing CD38-like enzymes from degradative NADases.

Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors

PubMed Central

Bruzzone, Santina; Kunerth, Svenja; Zocchi, Elena; De Flora, Antonio; Guse, Andreas H.

2003-01-01

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38− cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 ± 5.2 and 50.5 ± 8.0 pmol/mg protein). P2Y receptor stimulation of CD38− cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave. PMID:14623867

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de; Wagner, Silvia; Buerkle, Alexander

Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin didmore » not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.« less

Poly-ADP-ribose polymerase inhibition enhances ischemic and diabetic wound healing by promoting angiogenesis.

PubMed

Zhou, Xin; Patel, Darshan; Sen, Sabyasachi; Shanmugam, Victoria; Sidawy, Anton; Mishra, Lopa; Nguyen, Bao-Ngoc

2017-04-01

Chronic nonhealing wounds are a major health problem for patients in the United States and worldwide. Diabetes and ischemia are two major risk factors behind impaired healing of chronic lower extremity wounds. Poly-ADP-ribose polymerase (PARP) is found to be overactivated with both ischemic and diabetic conditions. This study seeks a better understanding of the role of PARP in ischemic and diabetic wound healing, with a specific focus on angiogenesis and vasculogenesis. Ischemic and diabetic wounds were created in FVB/NJ mice and an in vitro scratch wound model. PARP inhibitor PJ34 was delivered to the animals at 10 mg/kg/d through implanted osmotic pumps or added to the culture medium, respectively. Animal wound healing was assessed by daily digital photographs. Animal wound tissues, peripheral blood, and bone marrow cells were collected at different time points for further analysis with Western blot and flow cytometry. Scratch wound migration and invasion angiogenesis assays were performed using human umbilical vein endothelial cells (HUVECs). Measurements were reported as mean ± standard deviation. Continuous measurements were compared by t-test. P < .05 was considered statistically significant. A significant increase in PARP activity was observed under ischemic and diabetic conditions that correlated with delayed wound healing and slower HUVEC migration. The beneficial effect of PARP inhibition with PJ34 on ischemic and diabetic wound healing was observed in both animal and in vitro models. In the animal model, the percentage of wound healing was significantly enhanced from 43% ± 6% to 71% ± 9% (P < .05) by day 7 with the addition of PJ34. PARP inhibition promoted angiogenesis at the ischemic and diabetic wound beds as evidenced by significantly higher levels of endothelial cell markers (vascular endothelial growth factor receptor 2 [VEGFR2] and endothelial nitric oxide synthase) in mice treated with PJ34 compared with controls. Flow cytometry

Protective actions of PJ34, a poly(ADP-ribose)polymerase inhibitor, on the blood-brain barrier after traumatic brain injury in mice.

PubMed

Tao, X; Chen, X; Hao, S; Hou, Z; Lu, T; Sun, M; Liu, B

2015-04-16

Poly(ADP-ribose) polymerase (PARP) is activated by oxidative stress and plays an important role in traumatic brain injury (TBI). The objective of this study was to investigate whether PARP activation participated in the blood-brain barrier (BBB) disruption and edema formation in a mouse model of controlled cortical impact (CCI). N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) (10 mg/kg), a selective PARP inhibitor, was administered intraperitoneally at 5 min and 8 h after experimental CCI. After 6 h and 24 h of CCI, the permeability of the cortical BBB was determined after Evans Blue administration. The water content of the brain was also measured. Treatment with PJ34 markedly attenuated the permeability of the BBB and decreased the brain edema at 6 h and 24 h after CCI. Our data showed the up-regulation of nuclear factor-κB in cytosolic fractions and nuclear fractions in the injured cortex, and these changes were reversed by PJ34. Moreover, PJ34 significantly lessened the activities of myeloperoxidase and the levels of matrix metalloproteinase-9, enhanced the levels of occludin, laminin, collagen IV and integrin β1, reduced neurological deficits, decreased the contusion volume, and attenuated the necrotic and apoptotic neuronal cell death. These data suggest the protective effects of PJ34 on BBB integrity and cell death during acute TBI. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li

2013-02-01

Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently ofmore » its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.« less

Combining poly(ADP-ribose) polymerase 1 (PARP-1) inhibition and radiation in Ewing sarcoma results in lethal DNA damage

PubMed Central

Lee, Hae-June; Yoon, Changhwan; Schmidt, Benjamin; Park, Do Joong; Zhang, Alexia Y.; Erkizan, Hayriye V.; Toretsky, Jeffrey A.; Kirsch, David G.; Yoon, Sam S.

2013-01-01

Ewing sarcomas (ES) harbor a chromosomal translocation that fuses the EWS gene to an ETS transcription factor, most commonly FLI1. The EWS-FLI1 fusion acts in a positive feedback loop to maintain expression of poly(ADP-ribose) polymerase 1 (PARP-1), which is involved in repair of DNA damage. Here, we examine the effects of PARP-1 inhibition and radiation therapy (RT) on ES. In proliferation assays, the ES cell lines RD-ES and SK-N-MC were much more sensitive than non-ES cell lines to the PARP-1 inhibitor olaparib (Ola) (IC50 0.5–1 uM vs >5 uM) and to radiation (IC50 2–4 Gy vs >6 Gy). PARP-1 inhibition with shRNA or Ola sensitized ES cells but not non-ES cells to RT in both proliferation and colony formation assays. Using the Comet assay, radiation of ES cells with Ola, compared to without Ola, resulted in more DNA damage at 1 hr (mean tail moment 36–54 vs. 26–28) and sustained DNA damage at 24 hr (24–29 vs. 6–8). This DNA damage led to a 2.9–4.0 fold increase in apoptosis and a 1.6–2.4 fold increase in cell death. The effect of PARP-1 inhibition and RT on ES cells was lost when EWS-FLI1 was silenced by shRNA. A small dose of RT (4 Gy), when combined with PARP-1 inhibition, stopped growth of SK-N-MC flank tumors xenografts. In conclusion, PARP-1 inhibition in ES amplifies the level and duration of DNA damage caused by RT leading to synergistic increases in apoptosis and cell death in a EWS-FLI1 dependent manner. PMID:23966622

Novel poly (ADP-ribose) polymerase inhibitor, AZD2281, enhances radiosensitivity of both normoxic and hypoxic esophageal squamous cancer cells.

PubMed

Zhan, L; Qin, Q; Lu, J; Liu, J; Zhu, H; Yang, X; Zhang, C; Xu, L; Liu, Z; Cai, J; Ma, J; Dai, S; Tao, G; Cheng, H; Sun, X

2016-04-01

Radiotherapy plays an important role in the treatment of esophageal squamous cell carcinoma (ESCC). However, the outcome of radiotherapy in ESCC remains unsatisfactory because esophageal squamous cancer cells, particularly those under hypoxic condition, exhibit radioresistance. The aim of this study was to determine whether or not AZD2281, a potent poly (ADP-ribose) polymerase (PARP) inhibitor, could enhance the radiation sensitivity of two ESCC cell lines, namely ECA109 and TE13. The radiosensitizing effect of AZD2281 was evaluated on the basis of cell death, clonogenic survival and tumor xenograft progression. AZD2281 alone was slightly toxic to ESCC cell lines. Apoptosis was increased and clonogenic survival was decreased in both cell lines when AZD2281 was combined with ionizing radiation (IR) under normoxic condition. AZD2281 enhanced IR-induced apoptosis to a more significant level under chronic hypoxic condition (0.2% O(2), 48 hour) than under normoxic condition. AZD2281 also slightly enhanced clonogenic cell death under chronic hypoxic condition compared with that under normoxic condition. This result could be associated with increased radiation-induced DNA double-strand breaks (DSB), decreased DSB repair and increased apoptosis of ESCC cells. Furthermore, homologous recombination (HR) protein Rad51 expression and focus formation were decreased in ESCC cells exposed to moderate chronic hypoxic condition (0.2% O(2), 48 hour); this result indicated that chronic hypoxic ESCC cells were HR deficient, possibly causing contextual synthetic lethality with PARP inhibitor in radiation sensitization. AZD2281 was also a radiation sensitizer in ESCC tumor xenograft models. Hence, in vitro and in vivo findings provide evidence that AZD2281 potently sensitizes ESCC cells to X-ray irradiation. The selective cell killing of HR-defective hypoxic cells contributes to radiosensitization by PARP inhibitor in ESCC cells under hypoxic condition. © 2015 International Society for

Poly[ADP-Ribose] Polymerase-1 Expression Is Related To Cold Ischemia, Acute Tubular Necrosis, and Delayed Renal Function In Kidney Transplantation

PubMed Central

O'Valle, Francisco; Del Moral, Raimundo G. M.; Benítez, María del Carmén; Martín-Oliva, David; Gómez-Morales, Mercedes; Aguilar, David; Aneiros-Fernández, José; Hernández-Cortés, Pedro; Osuna, Antonio; Moreso, Francesc; Serón, Daniel; Oliver, Francisco J.; Del Moral, Raimundo G.

2009-01-01

Cold ischemia time especially impacts on outcomes of expanded-criteria donor (ECD) transplantation. Ischemia-reperfusion (IR) injury produces excessive poly[ADP-Ribose] Polymerase-1 (PARP-1) activation. The present study explored the hypothesis that increased tubular expression of PARP-1 contributes to delayed renal function in suboptimal ECD kidney allografts and in non-ECD allografts that develop posttransplant acute tubular necrosis (ATN). Materials and Methods Nuclear PARP-1 immunohistochemical expression was studied in 326 paraffin-embedded renal allograft biopsies (193 with different degrees of ATN and 133 controls) and in murine Parp-1 knockout model of IR injury. Results PARP-1 expression showed a significant relationship with cold ischemia time (r coefficient = 0.603), time to effective diuresis (r = 0.770), serum creatinine levels at biopsy (r = 0.649), and degree of ATN (r = 0.810) (p = 0.001, Pearson test). In the murine IR model, western blot showed an increase in PARP-1 that was blocked by Parp-1 inhibitor. Immunohistochemical study of PARP-1 in kidney allograft biopsies would allow early detection of possible delayed renal function, and the administration of PARP-1 inhibitors may offer a therapeutic option to reduce damage from IR in donor kidneys by preventing or minimizing ATN. In summary, these results suggest a pivotal role for PARP-1 in the ATN of renal transplantation. We propose the immunohistochemical assessment of PARP-1 in kidney allograft biopsies for early detection of a possible delayed renal function. PMID:19784367

The Septic Shock-associated IL-10 -1082 A>G Polymorphism Mediates Allele-specific Transcription via Poly ADP-ribose Polymerase 1 in Macrophages Engulfing Apoptotic Cells

PubMed Central

Kang, Xiaoyan; Kim, Ha-Jeong; Ramirez, Michelle; Salameh, Sarah; Ma, Xiaojing

2013-01-01

The biallelic Interleukin-10 single nucleotide polymorphism (SNP) at -1082 of the promoter region linked to individual variation in cytokine inducibility has been strongly implicated in several pathological conditions including the development of, and outcomes in, septic shock during pneumococcal infection, acute respiratory distress syndrome, and cardiac dysfunction. However, the molecular basis of the SNP-mediated variable IL-10 production levels has not been explored. Here we report that the -1082G>A alleles in the promoter region of the human IL-10 gene physically interact with a nuclear protein in an allele-specific manner that results in different levels of IL-10 transcription. This protein has been identified as poly ADP-ribose polymerase 1 (PARP-1). We show that PARP-1 acts as a transcription repressor, and its DNA-binding activity is strongly regulated in macrophages that engulf apoptotic cells but not stimulated with lippopolysaccharides. These findings unveil a novel role of PARP-1 in the regulation of IL-10 production in an allele-dependent way, which determines individual susceptibility to sepsis-induced inflammatory pathology and the immunological sequelae in a physiological process where clearance of infection-induced apoptotic cells by professional phagocytes triggers the cytokine synthesis. PMID:20181890

Design, Synthesis, and Chemical and Biological Properties of Cyclic ADP-4-Thioribose as a Stable Equivalent of Cyclic ADP-Ribose.

PubMed

Tsuzuki, Takayoshi; Takano, Satoshi; Sakaguchi, Natsumi; Kudoh, Takashi; Murayama, Takashi; Sakurai, Takashi; Hashii, Minako; Higashida, Haruhiro; Weber, Karin; Guse, Andreas H; Kameda, Tomoshi; Hirokawa, Takatsugu; Kumaki, Yasuhiro; Arisawa, Mitsuhiro; Potter, Barry V L; Shuto, Satoshi

2014-01-01

Here we describe the successful synthesis of cyclic ADP-4-thioribose (cADPtR, 3 ), designed as a stable mimic of cyclic ADP-ribose (cADPR, 1 ), a Ca 2+ -mobilizing second messenger, in which the key N1-β-thioribosyladenosine structure was stereoselectively constructed by condensation between the imidazole nucleoside derivative 8 and the 4-thioribosylamine 7 via equilibrium in 7 between the α-anomer ( 7α ) and the β-anomer ( 7β ) during the reaction course. cADPtR is, unlike cADPR, chemically and biologically stable, while it effectively mobilizes intracellular Ca 2+ like cADPR in various biological systems, such as sea urchin homogenate, NG108-15 neuronal cells, and Jurkat T-lymphocytes. Thus, cADPtR is a stable equivalent of cADPR, which can be useful as a biological tool for investigating cADPR-mediated Ca 2+ -mobilizing pathways.

TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion

PubMed Central

Togashi, Kazuya; Hara, Yuji; Tominaga, Tomoko; Higashi, Tomohiro; Konishi, Yasunobu; Mori, Yasuo; Tominaga, Makoto

2006-01-01

There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (>35°C) apparently via direct heat-evoked channel gating. β-NAD+- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25°C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca2+ and insulin release, which is KATP channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca2+ entry into pancreatic β-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion. PMID:16601673

Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization.

PubMed

La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca; Caligo, Maria Adelaide; Galli, Alvaro

2015-04-01

The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the deubiquitination enzyme gene OTU1, the nuclear pore protein POM152 and the SNT1 that encodes for the Set3C subunit of the histone deacetylase complex. In these strains the PARP-1 level was roughly the same as in the wild type. PARP-1 localized in the nucleus more in the snt1Δ than in the wild type strain; after UV radiation, PARP-1 localized in the nucleus more in hho1 and pom152 deletion strains than in the wild type indicating that these functions may have a role on regulating PARP-1 level and activity in the nucleus. Copyright © 2015 Elsevier B.V. All rights reserved.

The ARTT motif and a unified structural understanding of substraterecognition in ADP ribosylating bacterial toxins and eukaryotic ADPribosyltransferases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, S.; Tainer, J.A.

2001-08-01

ADP-ribosylation is a widely occurring and biologically critical covalent chemical modification process in pathogenic mechanisms, intracellular signaling systems, DNA repair, and cell division. The reaction is catalyzed by ADP-ribosyltransferases, which transfer the ADP-ribose moiety of NAD to a target protein with nicotinamide release. A family of bacterial toxins and eukaryotic enzymes has been termed the mono-ADP-ribosyltransferases, in distinction to the poly-ADP-ribosyltransferases, which catalyze the addition of multiple ADP-ribose groups to the carboxyl terminus of eukaryotic nucleoproteins. Despite the limited primary sequence homology among the different ADP-ribosyltransferases, a central cleft bearing NAD-binding pocket formed by the two perpendicular b-sheet core hasmore » been remarkably conserved between bacterial toxins and eukaryotic mono- and poly-ADP-ribosyltransferases. The majority of bacterial toxins and eukaryotic mono-ADP-ribosyltransferases are characterized by conserved His and catalytic Glu residues. In contrast, Diphtheria toxin, Pseudomonas exotoxin A, and eukaryotic poly-ADP-ribosyltransferases are characterized by conserved Arg and catalytic Glu residues. The NAD-binding core of a binary toxin and a C3-like toxin family identified an ARTT motif (ADP-ribosylating turn-turn motif) that is implicated in substrate specificity and recognition by structural and mutagenic studies. Here we apply structure-based sequence alignment and comparative structural analyses of all known structures of ADP-ribosyltransfeases to suggest that this ARTT motif is functionally important in many ADP-ribosylating enzymes that bear a NAD binding cleft as characterized by conserved Arg and catalytic Glu residues. Overall, structure-based sequence analysis reveals common core structures and conserved active sites of ADP-ribosyltransferases to support similar NAD binding mechanisms but differing mechanisms of target protein binding via sequence variations within

Unifying mechanism for Aplysia ADP-ribosyl cyclase and CD38/NAD(+) glycohydrolases.

PubMed Central

Cakir-Kiefer, C; Muller-Steffner, H; Schuber, F

2000-01-01

Highly purified Aplysia californica ADP-ribosyl cyclase was found to be a multifunctional enzyme. In addition to the known transformation of NAD(+) into cADP-ribose this enzyme is able to catalyse the solvolysis (hydrolysis and methanolysis) of cADP-ribose. This cADP-ribose hydrolase activity, which becomes detectable only at high concentrations of the enzyme, is amplified with analogues such as pyridine adenine dinucleotide, in which the cleavage rate of the pyridinium-ribose bond is much reduced compared with NAD(+). Although the specificity ratio V(max)/K(m) is in favour of NAD(+) by 4 orders of magnitude, this multifunctionality allowed us to propose a 'partitioning' reaction scheme for the Aplysia enzyme, similar to that established previously for mammalian CD38/NAD(+) glycohydrolases. This mechanism involves the formation of a single oxocarbenium-type intermediate that partitions to cADP-ribose and solvolytic products via competing pathways. In favour of this mechanism was the finding that the enzyme also catalysed the hydrolysis of NMN(+), a substrate that cannot undergo cyclization. The major difference between the mammalian and the invertebrate enzymes resides in their relative cyclization/hydrolysis rate-constant ratios, which dictate their respective yields of cADP-ribose (ADP-ribosyl cyclase activity) and ADP-ribose (NAD(+) glycohydrolase activity). For the Aplysia enzyme's catalysed transformation of NAD(+) we favour a mechanism where the formation of cADP-ribose precedes that of ADP-ribose; i.e. macroscopically the invertebrate ADP-ribosyl cyclase conforms to a sequential reaction pathway as a limiting form of the partitioning mechanism. PMID:10861229

Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxiainducible factor-1alpha expression via Poly(ADP-ribose) polymerase -1

PubMed Central

Pan, Rong; Chen, Chen; Liu, Wenlan; Liu, Ke Jian

2013-01-01

Aim Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study testes the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Methods Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-hour hypoxic treatment. Results Although 3-hr hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc concentration dependent manner. Exposure of astrocytes to hypoxia for 3-hr remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Conclusions Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. PMID:23582235

Upregulation of Poly (ADP-Ribose) Polymerase-1 (PARP1) in Triple-Negative Breast Cancer and Other Primary Human Tumor Types

PubMed Central

Ossovskaya, Valeria; Koo, Ingrid Chou; Kaldjian, Eric P.; Alvares, Christopher; Sherman, Barry M.

2010-01-01

Poly (ADP-ribose) polymerase-1 (PARP1) is a key facilitator of DNA repair and is implicated in pathways of tumorigenesis. PARP inhibitors have gained recent attention as rationally designed therapeutics for the treatment of several malignancies, particularly those associated with dysfunctional DNA repair pathways, including triple-negative breast cancer (TNBC). We investigated the PARP1 gene expression profile in surgical samples from more than 8,000 primary malignant and normal human tissues. PARP1 expression was found to be significantly increased in several malignant tissues, including those isolated from patients with breast, uterine, lung, ovarian, and skin cancers, and non-Hodgkin’s lymphoma. Within breast infiltrating ductal carcinoma (IDC) samples tested, mean PARP1 expression was significantly higher relative to normal breast tissue, with over 30% of IDC samples demonstrating upregulation of PARP1, compared with 2.9% of normal tissues. Because of known DNA repair defects, including BRCA1 dysfunction, associated with TNBC, exploration of PARP1 expression in breast cancers related to expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) led to the observation that negative expression of any of the 3 receptors was associated with upregulation of PARP1 expression, compared with receptor-positive tissues. To validate these observations, an independent set of breast adenocarcinomas was evaluated and demonstrated >2-fold upregulation of PARP1 in approximately 70% of primary breast adenocarcinomas, including TNBC, compared with syngeneic nonmalignant breast tissues. Immunohistochemistry (IHC) showed that upregulation of the PARP1 gene was consistent with increased protein expression in TNBC. These analyses suggest a potential biological role for PARP1 in several distinct malignancies, including TNBC. Further investigation of PARP1 as a biomarker for the therapeutic activity of PARP inhibitor

Pharmacological inhibition of poly(ADP-ribose) polymerase-1 modulates resistance of human glioblastoma stem cells to temozolomide

PubMed Central

2014-01-01

Background Chemoresistance of glioblastoma multiforme (GBM) has been attributed to the presence within the tumor of cancer stem cells (GSCs). The standard therapy for GBM consists of surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ). However, TMZ efficacy is limited by O6-methylguanine-DNA-methyltransferase (MGMT) and Mismatch Repair (MMR) functions. Strategies to counteract TMZ resistance include its combination with poly(ADP-ribose) polymerase inhibitors (PARPi), which hamper the repair of N-methylpurines. PARPi are also investigated as monotherapy for tumors with deficiency of homologous recombination (HR). We have investigated whether PARPi may restore GSC sensitivity to TMZ or may be effective as monotherapy. Methods Ten human GSC lines were assayed for MMR proteins, MGMT and PARP-1 expression/activity, MGMT promoter methylation and sensitivity to TMZ or PARPi, alone and in combination. Since PTEN defects are frequently detected in GBM and may cause HR dysfunction, PTEN expression was also analyzed. The statistical analysis of the differences in drug sensitivity among the cell lines was performed using the ANOVA and Bonferroni’s post-test or the non-parametric Kruskal-Wallis analysis and Dunn’s post-test for multiple comparisons. Synergism between TMZ and PARPi was analyzed by the median-effect method of Chou and Talalay. Correlation analyses were done using the Spearman’s rank test. Results All GSCs were MMR-proficient and resistance to TMZ was mainly associated with high MGMT activity or low proliferation rate. MGMT promoter hypermethylation of GSCs correlated both with low MGMT activity/expression (Spearman’s test, P = 0.004 and P = 0.01) and with longer overall survival of GBM patients (P = 0.02). Sensitivity of each GSC line to PARPi as single agent did not correlate with PARP-1 or PTEN expression. Notably, PARPi and TMZ combination exerted synergistic antitumor effects in eight out of ten GSC lines and

Sensitization to radiation and alkylating agents by inhibitors of poly(ADP-ribose) polymerase is enhanced in cells deficient in DNA double-strand break repair.

PubMed

Löser, Dana A; Shibata, Atsushi; Shibata, Akiko K; Woodbine, Lisa J; Jeggo, Penny A; Chalmers, Anthony J

2010-06-01

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

Modulation of farnesoid X receptor results in post-translational modification of poly (ADP-ribose) polymerase 1 in the liver

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Yan; Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS; Li, Guodong

2013-01-15

The farnesoid X receptor (FXR) is a bile acid-activated transcription factor belonging to the nuclear receptor superfamily. FXR deficiency in mice results in cholestasis, metabolic disorders, and tumorigenesis in liver and intestine. FXR is known to contribute to pathogenesis by regulating gene transcription; however, changes in the post-transcriptional modification of proteins associated with FXR modulation have not been determined. In the current study, proteomic analysis of the livers of wild-type (WT) and FXR knockout (FXR-KO) mice treated with a FXR synthetic ligand or vehicle was performed. The results identified five proteins as novel FXR targets. Since FXR deficiency in micemore » leads to liver tumorigenesis, poly (ADP-ribose) polymerase family, member 1 (Parp1) that is important for DNA repair, was validated in the current study by quantitative real-time PCR, and 1- and 2-dimensional gel electrophoresis/western blot. The results showed that Parp1 mRNA levels were not altered by FXR genetic status or by agonist treatment. However, total Parp1 protein levels were increased in FXR-KO mice as early as 3 month old. Interestingly, total Parp1 protein levels were increased in WT mice in an age-dependent manner (from 3 to 18 months), but not in FXR-KO mice. Finally, activation of FXR in WT mice resulted in reduction of phosporylated Parp1 protein in the liver without affecting total Parp1 protein levels. In conclusion, this study reveals that FXR genetic status and agonist treatment affects basal levels and phosphorylation state of Parp1, respectively. These alterations, in turn, may be associated with the hepatobiliary alterations observed in FXR-KO mice and participate in FXR agonist-induced protection in the liver. -- Highlights: ► Proteomic analysis identified novel FXR targets. ► FXR modification altered post-translational modification of the Parp1 protein. ► Altered Parp1 function may contribute to mechanisms of FXR regulation of liver functions.« less

Cross talk between poly(ADP-ribose) polymerase 1 methylation and oxidative stress involved in the toxic effect of anatase titanium dioxide nanoparticles.

PubMed

Bai, Wenlin; Chen, Yujiao; Gao, Ai

2015-01-01

Given the tremendous growth in the application of titanium dioxide nanoparticles (TNPs), concerns about the potential health hazards of TNPs to humans have been raised. Poly(ADP-ribose) polymerase 1 (PARP-1), a highly conserved DNA-binding protein, is involved in many molecular and cellular processes. Limited data demonstrated that certain nanomaterials induced the aberrant hypermethylation of PARP-1. However, the mechanism involved in TNP-induced PARP-1 abnormal methylation has not been studied. A549 cells were incubated with anatase TNPs (22.1 nm) for 24 hours pretreatment with or without methyltransferase inhibitor 5-aza-2'-deoxycytidine and the reactive oxygen species (ROS) scavenger α-lipoic acid to assess the possible role of methylation and ROS in the toxic effect of TNPs. After TNPs characterization, a battery of assays was performed to evaluate the toxic effect of TNPs, PARP-1 methylation status, and oxidative damage. Results showed that TNPs decreased the cell viability in a dose-dependent manner, in accordance with the increase of lactate dehydrogenase activity, which indicated membrane damage of cells. Similar to the high level of PARP-1 methylation, the generation of ROS was significantly increased after exposure to TNPs for 24 hours. Furthermore, α-lipoic acid decreased TNP-induced ROS generation and then attenuated TNP-triggered PARP-1 hypermethylation. Meanwhile, 5-aza-2'-deoxycytidine simultaneously decreased the ROS generation induced by TNPs, resulting in the decline of PARP-1 methylation. In summary, TNPs triggered the aberrant hypermethylation of the PARP-1 promoter and there was a cross talk between oxidative stress and PARP-1 methylation in the toxic effect of TNPs.

Poly(ADP-Ribose) Polymerase-1 and DNA-Dependent Protein Kinase Have Equivalent Roles in Double Strand Break Repair Following Ionizing Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Jody; Smith, Graeme; Curtin, Nicola J., E-mail: n.j.curtin@ncl.ac.u

2009-12-01

Purpose: Radiation-induced DNA double strand breaks (DSBs) are predominantly repaired by nonhomologous end joining (NHEJ), involving DNA-dependent protein kinase (DNA-PK). Poly(ADP-ribose) polymerase-1 (PARP-1), well characterized for its role in single strand break repair, may also facilitate DSB repair. We investigated the activation of these enzymes by differing DNA ends and their interaction in the cellular response to ionizing radiation (IR). Methods and Materials: The effect of PARP and DNA-PK inhibitors (KU-0058684 and NU7441) on repair of IR-induced DSBs was investigated in DNA-PK and PARP-1 proficient and deficient cells by measuring gammaH2AX foci and neutral comets. Complementary in vitro enzyme kineticsmore » assays demonstrated the affinities of DNA-PK and PARP-1 for DSBs with varying DNA termini. Results: DNA-PK and PARP-1 both promoted the fast phase of resolution of IR-induced DSBs in cells. Inactivation of both enzymes was not additive, suggesting that PARP-1 and DNA-PK cooperate within the same pathway to promote DSB repair. The affinities of the two enzymes for oligonucleotides with blunt, 3' GGG or 5' GGG overhanging termini were similar and overlapping (K{sub dapp} = 2.6-6.4nM for DNA-PK; 1.7-4.5nM for PARP-1). DNA-PK showed a slightly greater affinity for overhanging DNA and was significantly more efficient when activated by a 5' GGG overhang. PARP-1 had a preference for blunt-ended DNA and required a separate factor for efficient stimulation by a 5' GGG overhang. Conclusion: DNA-PK and PARP-1 are both required in a pathway facilitating the fast phase of DNA DSB repair.« less

Anti-tumor activity of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in cultured endometrial carcinoma cells

PubMed Central

2014-01-01

Background PTEN inactivation is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in nucleus. Poly (ADP-ribose) polymerase (PARP) plays key roles in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in endometrial cancer cell lines. Methods The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN on the sensitivity to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN + (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and γH2AX, and induction of cleaved PARP. The effects of siRNA to PTEN were analyzed in cells with wild-type PTEN. Results The SF50 values were 100 nM or less in four (25%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in four (25%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 746 ± 838 nM; Wild-type [n = 4]: 215 ± 85 nM, p = 0.26 by Student’s t test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and γH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN + cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN + cells. In addition, knocking down PTEN by siRNA did not alter the sensitivity to olaparib in 2 cell lines with wild-type PTEN. Conclusions Our results suggest that olaparib, a PARP

Kinetic competence of the cADP-ribose-CD38 complex as an intermediate in the CD38/NAD+ glycohydrolase-catalysed reactions: implication for CD38 signalling.

PubMed Central

Cakir-Kiefer, C; Muller-Steffner, H; Oppenheimer, N; Schuber, F

2001-01-01

CD38/NAD(+) glycohydrolase is a type II transmembrane glycoprotein widely used to study T- and B-cell activation and differentiation. CD38 is endowed with two different activities: it is a signal transduction molecule and an ectoenzyme that converts NAD(+) into ADP-ribose (NAD(+) glycohydrolase activity) and small proportions of cADP-ribose (cADPR; ADP-ribosyl cyclase activity), a calcium-mobilizing metabolite, which, ultimately, can also be hydrolysed (cADPR hydrolase activity). The relationship between these two properties, and strikingly the requirement for signalling in the formation of free or enzyme-complexed cADPR, is still ill-defined. In the present study we wanted to test whether the CD38-cADPR complex is kinetically competent in the conversion of NAD(+) into the reaction product ADP-ribose. In principle, such a complex could be invoked for cross-talk, via conformational changes, with neighbouring partner(s) of CD38 thus triggering the signalling phenomena. Analysis of the kinetic parameters measured for the CD38/NAD(+) glycohydrolase-catalysed hydrolysis of 2'-deoxy-2'-aminoribo-NAD(+) and ADP-cyclo[N1,C1']-2'-deoxy-2'-aminoribose (slowly hydrolysable analogues of NAD(+) and cADPR respectively) ruled out that the CD38-cADPR complex can accumulate under steady-state conditions. This was borne out by simulation of the prevalent kinetic mechanism of CD38, which involve the partitioning of a common E.ADP-ribosyl intermediate in the formation of the enzyme-catalysed reaction products. Using this mechanism, microscopic rate conditions were found which transform a NAD(+) glycohydrolase into an ADP-ribosyl cyclase. Altogether, the present work shows that if the cross-talk with a partner depends on a conformational change of CD38, this is most probably not attributable to the formation of the CD38-cADPR complex. In line with recent results on the conformational change triggered by CD38 ligands [Berthelier, Laboureau, Boulla, Schuber and Deterre (2000) Eur. J

Discovery of potent 2,4-difluoro-linker poly(ADP-ribose) polymerase 1 inhibitors with enhanced water solubility and in vivo anticancer efficacy.

PubMed

Chen, Wen-Hua; Song, Shan-Shan; Qi, Ming-Hui; Huan, Xia-Juan; Wang, Ying-Qing; Jiang, Hualiang; Ding, Jian; Ren, Guo-Bin; Miao, Ze-Hong; Li, Jian

2017-11-01

Poly (ADP-ribose) polymerase 1 (PARP1) is overexpressed in a variety of cancers, especially in breast and ovarian cancers; tumor cells that are deficient in breast cancer gene 1/2 (BRCA1/2) are highly sensitive to PARP1 inhibition. In this study, we identified a series of 2,4-difluorophenyl-linker analogs (15-55) derived from olaparib as novel PARP1 inhibitors. Four potent analogs 17, 43, 47, and 50 (IC 50 =2.2-4.4 nmol/L) effectively inhibited the proliferation of Chinese hamster lung fibroblast V-C8 cells (IC 50 =3.2-37.6 nmol/L) in vitro, and showed specificity toward BRCA-deficient cells (SI=40-510). The corresponding hydrochloride salts 56 and 57 (based on 43 and 47) were highly water soluble in pH=1.0 buffered salt solutions (1628.2 μg/mL, 2652.5 μg/mL). In a BRCA1-mutated xenograft model, oral administration of compound 56 (30 mg·kg -1 ·d -1 , for 21 d) exhibited more prominent tumor growth inhibition (96.6%) compared with the same dose of olaparib (56.3%); in a BRCA2-mutated xenograft model, oral administration of analog 43 (10 mg·kg -1 ·d -1 , for 28 d) significantly inhibited tumor growth (69.0%) and had no negative effects on the body weights. Additionally, compound 56 exhibited good oral bioavailability (F=32.2%), similar to that of olaparib (F=45.4%). Furthermore, the free base 43 of the hydrochloride salt 56 exhibited minimal hERG inhibition activity (IC 50 =6.64 μmol/L). Collectively, these data demonstrate that compound 56 may be an excellent drug candidate for the treatment of cancer, particularly BRCA-deficient tumors.

The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

PubMed Central

Rom, Slava; Reichenbach, Nancy L.; Dykstra, Holly; Persidsky, Yuri

2015-01-01

Multifactorial mechanisms comprising countless cellular factors and virus-encoded transactivators regulate the transcription of HIV-1 (HIV). Since poly(ADP-ribose) polymerase 1 (PARP-1) regulates numerous genes through its interaction with various transcription factors, inhibition of PARP-1 has surfaced recently as a powerful anti-inflammatory tool. We suggest a novel tactic to diminish HIV replication via PARP-1 inhibition in an in vitro model system, exploiting human primary monocyte-derived macrophages (MDM). PARP-1 inhibition was capable to lessen HIV replication in MDM by 60–80% after 7 days infection. Tat, tumor necrosis factor α (TNFα), and phorbol 12-myristate 13-acetate (PMA) are known triggers of the Long Terminal Repeat (LTR), which can switch virus replication. Tat overexpression in MDM transfected with an LTR reporter plasmid resulted in a 4.2-fold increase in LTR activation; PARP inhibition caused 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85–95%). PARP inhibition in MDM exhibited 90% diminution in NFκB activity (known to mediate TNFα- and PMA-induced HIV LTR activation). Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These discoveries suggest that inactivation of PARP suppresses HIV replication in MDM by via attenuation of LTR activation, NFκB suppression and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide an effective approach to management of HIV infection. PMID:26379653

The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity.

PubMed

Rom, Slava; Reichenbach, Nancy L; Dykstra, Holly; Persidsky, Yuri

2015-01-01

Multifactorial mechanisms comprising countless cellular factors and virus-encoded transactivators regulate the transcription of HIV-1 (HIV). Since poly(ADP-ribose) polymerase 1 (PARP-1) regulates numerous genes through its interaction with various transcription factors, inhibition of PARP-1 has surfaced recently as a powerful anti-inflammatory tool. We suggest a novel tactic to diminish HIV replication via PARP-1 inhibition in an in vitro model system, exploiting human primary monocyte-derived macrophages (MDM). PARP-1 inhibition was capable to lessen HIV replication in MDM by 60-80% after 7 days infection. Tat, tumor necrosis factor α (TNFα), and phorbol 12-myristate 13-acetate (PMA) are known triggers of the Long Terminal Repeat (LTR), which can switch virus replication. Tat overexpression in MDM transfected with an LTR reporter plasmid resulted in a 4.2-fold increase in LTR activation; PARP inhibition caused 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85-95%). PARP inhibition in MDM exhibited 90% diminution in NFκB activity (known to mediate TNFα- and PMA-induced HIV LTR activation). Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These discoveries suggest that inactivation of PARP suppresses HIV replication in MDM by via attenuation of LTR activation, NFκB suppression and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide an effective approach to management of HIV infection.

Metabolic Enhancer Piracetam Attenuates the Translocation of Mitochondrion-Specific Proteins of Caspase-Independent Pathway, Poly [ADP-Ribose] Polymerase 1 Up-regulation and Oxidative DNA Fragmentation.

PubMed

Verma, Dinesh Kumar; Gupta, Sonam; Biswas, Joyshree; Joshi, Neeraj; Sivarama Raju, K; Wahajuddin, Mu; Singh, Sarika

2018-03-12

Piracetam, a nootropic drug, has been clinically used for decades; however, its mechanism of action still remains enigmatic. The present study was undertaken to evaluate the role of mitochondrion-specific factors of caspase-independent pathway like apoptotic-inducing factor (AIF) and endonuclease-G (endo-G) in piracetam-induced neuroprotection. N2A cells treated with lipopolysaccharide (LPS) exhibited significant cytotoxicity, impaired mitochondrial activity, and reactive oxygen species generation which was significantly attenuated with piracetam co-treatment. Cells co-treated with LPS and piracetam exhibited significant uptake of piracetam in comparison to only piracetam-treated cells as estimated by liquid chromatography-mass spectrometry (LC-MSMS). LPS treatment caused significant translocation of AIF and endonuclease-G in neuronal N2A cells which were significantly attenuated with piracetam co-treatment. Significant over-expression of proinflammatory cytokines was also observed after treatment of LPS to cells which was inhibited with piracetam co-treatment demonstrating its anti-inflammatory property. LPS-treated cells exhibited significant oxidative DNA fragmentation and poly [ADP-ribose] polymerase-1 (PARP-1) up-regulation in nucleus, both of which were attenuated with piracetam treatment. Antioxidant melatonin but not z-VAD offered the inhibited LPS-induced DNA fragmentation indicating the involvement of oxidative DNA fragmentation. Further, we did not observe the altered caspase-3 level after LPS treatment initially while at a later time point, significantly augmented level of caspase-3 was observed which was not inhibited with piracetam treatment. In total, our findings indicate the interference of piracetam in mitochondrion-mediated caspase-independent pathway, as well as its anti-inflammatory and antioxidative properties. Graphical Abstract Graphical abstract indicating the novel interference of metabolic enhancer piracetam (P) in neuronal death

Modulation of the poly (ADP-ribose) polymerase inhibitor response and DNA recombination in breast cancer cells by drugs affecting endogenous wild-type p53.

PubMed

Ireno, Ivanildce Cristiane; Wiehe, Rahel Stephanie; Stahl, Andreea Iulia; Hampp, Stephanie; Aydin, Sevtap; Troester, Melissa A; Selivanova, Galina; Wiesmüller, Lisa

2014-10-01

Synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) and homologous recombination (HR) repair pathways have been exploited for the development of novel mono- and combination cancer therapies. The tumor suppressor p53 was demonstrated to exhibit indirect and direct regulatory activities in DNA repair, particularly in DNA double-strand break (DSB)-induced and replication-associated HR. In this study, we tested a potential influence of the p53 status on the response to PARP inhibition, which is known to cause replication stress. Silencing endogenous or inducibly expressing p53 we found a protective effect of p53 on PARP inhibitor (PARPi)-mediated cytotoxicities. This effect was specific for wild-type versus mutant p53 and observed in cancer but not in non-transformed cell lines. Enhanced cytotoxicities after treatment with the p53-inhibitory drug Pifithrinα further supported p53-mediated resistance to PARP inhibition. Surprisingly, we equally observed increased PARPi sensitivity in the presence of the p53-activating compound Nutlin-3. As a common denominator, both drug responses correlated with decreased HR activities: Pifithrinα downregulated spontaneous HR resulting in damage accumulation. Nutlin-3 induced a decrease of DSB-induced HR, which was accompanied by a severe drop in RAD51 protein levels. Thus, we revealed a novel link between PARPi responsiveness and p53-controlled HR activities. These data expand the concept of cell and stress type-dependent healer and killer functions of wild-type p53 in response to cancer therapeutic treatment. Our findings have implications for the individualized design of cancer therapies using PARPi and the potentially combined use of p53-modulatory drugs. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Inhibition of poly(ADP-ribose) polymerase-1 alters expression of mitochondria-related genes in PC12 cells: relevance to mitochondrial homeostasis in neurodegenerative disorders.

PubMed

Czapski, Grzegorz A; Cieślik, Magdalena; Wencel, Przemysław L; Wójtowicz, Sylwia; Strosznajder, Robert P; Strosznajder, Joanna B

2018-02-01

Alzheimer's disease (AD) is characterized by the release of amyloid beta peptides (Aβ) in the form of monomers/oligomers which may lead to oxidative stress, mitochondria dysfunction, synaptic loss, neuroinflammation and, in consequence, to overactivation of poly(ADP-ribose) polymerase-1 (PARP-1). However, Aβ peptides are also released in the brain ischemia, traumatic injury and in inflammatory response. PARP-1 is suggested to be a promising target in therapy of neurodegenerative disorders. We investigated the impact of PARP-1 inhibition on transcription of mitochondria-related genes in PC12 cells. Moreover, the effect of PARP-1 inhibitor (PJ34) on cells subjected to Aβ oligomers (AβO) - evoked stress was analyzed. Our data demonstrated that inhibition of PARP-1 in PC12 cells enhanced the transcription of genes for antioxidative enzymes (Sod1, Gpx1, Gpx4), activated genes regulating mitochondrial fission/fusion (Mfn1, Mfn2, Dnm1l, Opa1, Fis1), subunits of ETC complexes (mt-Nd1, Sdha, mt-Cytb) and modulated expression of several TFs, enhanced Foxo1 and decreased Nrf1, Stat6, Nfkb1. AβO elevated free radicals concentration, decreased mitochondria membrane potential (MMP) and cell viability after 24h. Gene transcription was not affected by AβO after 24h, but was significantly downregulated after 96h. In AβO stress, PJ34 exerted stimulatory effect on expression of several genes (Gpx1, Gpx4, Opa1, Mfn2, Fis1 and Sdha), decreased transcription of numerous TFs (Nrf1, Tfam, Stat3, Stat6, Trp53, Nfkb1) and prevented oxidative stress. Our results indicated that PARP-1 inhibition significantly enhanced transcription of genes involved in antioxidative defense and in regulation of mitochondria function, but was not able to ameliorate cells viability affected by Aβ. Copyright © 2017 Elsevier B.V. All rights reserved.

Discovery and Structure–Activity Relationship of Novel 2,3-Dihydrobenzofuran-7-carboxamide and 2,3-Dihydrobenzofuran-3(2H)-one-7-carboxamide Derivatives as Poly(ADP-ribose)polymerase-1 Inhibitors

PubMed Central

2015-01-01

Novel substituted 2,3-dihydrobenzofuran-7-carboxamide (DHBF-7-carboxamide) and 2,3-dihydrobenzofuran-3(2H)-one-7-carboxamide (DHBF-3-one-7-carboxamide) derivatives were synthesized and evaluated as inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1). A structure-based design strategy resulted in lead compound 3 (DHBF-7-carboxamide; IC50 = 9.45 μM). To facilitate synthetically feasible derivatives, an alternative core was designed, DHBF-3-one-7-carboxamide (36, IC50 = 16.2 μM). The electrophilic 2-position of this scaffold was accessible for extended modifications. Substituted benzylidene derivatives at the 2-position were found to be the most potent, with 3′,4′-dihydroxybenzylidene 58 (IC50 = 0.531 μM) showing a 30-fold improvement in potency. Various heterocycles attached at the 4′-hydroxyl/4′-amino of the benzylidene moiety resulted in significant improvement in inhibition of PARP-1 activity (e.g., compounds 66–68, 70, 72, and 73; IC50 values from 0.718 to 0.079 μM). Compound 66 showed selective cytotoxicity in BRCA2-deficient DT40 cells. Crystal structures of three inhibitors (compounds (−)-13c, 59, and 65) bound to a multidomain PARP-1 structure were obtained, providing insights into further development of these inhibitors. PMID:24922587

Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2.

PubMed

Kowieski, Terri M; Lee, Susan; Denu, John M

2008-02-29

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.

The road to survival goes through PARG.

PubMed

Koh, David W; Dawson, Valina L; Dawson, Ted M

2005-03-01

Unlike poly(ADP-ribose) polymerase-1 (PARP-1), poly(ADP-ribose) glycohydrolase (PARG) has long been a difficult protein to study. However, the complete absence of PARG activity was recently characterized in mice via disruption of the murine PARG gene. As expected, PARG is critical for the maintenance of steady-state poly(ADP-ribose) levels. But surprisingly, the disruption of PARG led to embryonic lethality and increased susceptibility to mild cell stress. Therefore, the protective role of PARG and its involvement in development indicate that these roads to viability go through PARG.

Inhibiting poly ADP-ribosylation increases fatty acid oxidation and protects against fatty liver disease.

PubMed

Gariani, Karim; Ryu, Dongryeol; Menzies, Keir J; Yi, Hyon-Seung; Stein, Sokrates; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Katsyuba, Elena; Jha, Pooja; Vijgen, Sandrine; Rubbia-Brandt, Laura; Kim, Yong Kyung; Kim, Jung Tae; Kim, Koon Soon; Shong, Minho; Schoonjans, Kristina; Auwerx, Johan

2017-01-01

To date, no pharmacological therapy has been approved for non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to evaluate the therapeutic potential of poly ADP-ribose polymerase (PARP) inhibitors in mouse models of NAFLD. As poly ADP-ribosylation (PARylation) of proteins by PARPs consumes nicotinamide adenine dinucleotide (NAD + ), we hypothesized that overactivation of PARPs drives NAD + depletion in NAFLD. Therefore, we assessed the effectiveness of PARP inhibition to replenish NAD + and activate NAD + -dependent sirtuins, hence improving hepatic fatty acid oxidation. To do this, we examined the preventive and therapeutic benefits of the PARP inhibitor (PARPi), olaparib, in different models of NAFLD. The induction of NAFLD in C57BL/6J mice using a high-fat high-sucrose (HFHS)-diet increased PARylation of proteins by PARPs. As such, increased PARylation was associated with reduced NAD + levels and mitochondrial function and content, which was concurrent with elevated hepatic lipid content. HFHS diet supplemented with PARPi reversed NAFLD through repletion of NAD + , increasing mitochondrial biogenesis and β-oxidation in liver. Furthermore, PARPi reduced reactive oxygen species, endoplasmic reticulum stress and fibrosis. The benefits of PARPi treatment were confirmed in mice fed with a methionine- and choline-deficient diet and in mice with lipopolysaccharide-induced hepatitis; PARP activation was attenuated and the development of hepatic injury was delayed in both models. Using Sirt1 hep-/- mice, the beneficial effects of a PARPi-supplemented HFHS diet were found to be Sirt1-dependent. Our study provides a novel and practical pharmacological approach for treating NAFLD, fueling optimism for potential clinical studies. Non-alcoholic fatty liver disease (NAFLD) is now considered to be the most common liver disease in the Western world and has no approved pharmacological therapy. PARP inhibitors given as a treatment in two different mouse

Serine is the major residue for ADP-ribosylation upon DNA damage

PubMed Central

Dauben, Helen

2018-01-01

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that synthesise ADP-ribosylation (ADPr), a reversible modification of proteins that regulates many different cellular processes. Several mammalian PARPs are known to regulate the DNA damage response, but it is not clear which amino acids in proteins are the primary ADPr targets. Previously, we reported that ARH3 reverses the newly discovered type of ADPr (ADPr on serine residues; Ser-ADPr) and developed tools to analyse this modification (Fontana et al., 2017). Here, we show that Ser-ADPr represents the major fraction of ADPr synthesised after DNA damage in mammalian cells and that globally Ser-ADPr is dependent on HPF1, PARP1 and ARH3. In the absence of HPF1, glutamate/aspartate becomes the main target residues for ADPr. Furthermore, we describe a method for site-specific validation of serine ADP-ribosylated substrates in cells. Our study establishes serine as the primary form of ADPr in DNA damage signalling. PMID:29480802

Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

PubMed

Ohkura, Naganari; Nagamura, Yuko; Tsukada, Toshihiko

2008-10-15

In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins. (c) 2008 Wiley-Liss, Inc.

Studies of locomotor network neuroprotection by the selective poly(ADP-ribose) polymerase-1 inhibitor PJ-34 against excitotoxic injury to the rat spinal cord in vitro.

PubMed

Nasrabady, Sara E; Kuzhandaivel, Anujaianthi; Nistri, Andrea

2011-06-01

Delayed neuronal destruction after acute spinal injury is attributed to excitotoxicity mediated by hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1) that induces 'parthanatos', namely a non-apoptotic cell death mechanism. With an in vitro model of excitotoxicity, we have previously observed parthanatos of rat spinal cord locomotor networks to be decreased by a broad spectrum PARP-1 inhibitor. The present study investigated whether the selective PARP-1 inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acetamide.HCl (PJ-34) not only protected networks from kainate-evoked excitotoxicity, but also prevented loss of locomotor patterns recorded as fictive locomotion from lumbar (L) ventral roots (VRs) 24 h later. PJ-34 (60 μm) blocked PARP-1 activation and preserved dorsal, central and ventral gray matter with maintained reflex activity even after a large dose of kainate. Fictive locomotion could not, however, be restored by either electrical stimulation or bath-applied neurochemicals (N-methyl-D-aspartate plus 5-hydroxytryptamine). A low kainate concentration induced less histological damage that was widely prevented by PJ-34. Nonetheless, fictive locomotion was observed in just over 50% of preparations whose histological profile did not differ (except for the dorsal horn) from those lacking such a rhythm. Our data show that inhibition of PARP-1 could amply preserve spinal network histology after excitotoxicity, with return of locomotor patterns only when the excitotoxic stimulus was moderate. These results demonstrated divergence between histological and functional outcome, implying a narrow borderline between loss of fictive locomotion and neuronal preservation. Our data suggest that either damage of a few unidentified neurons or functional network inhibition was critical for ensuring locomotor cycles. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Loss of the Mono-ADP-ribosyltransferase, Tiparp, Increases Sensitivity to Dioxin-induced Steatohepatitis and Lethality*

PubMed Central

Ahmed, Shaimaa; Bott, Debbie; Gomez, Alvin; Tamblyn, Laura; Rasheed, Adil; Cho, Tiffany; MacPherson, Laura; Sugamori, Kim S.; Yang, Yang; Grant, Denis M.; Cummins, Carolyn L.; Matthews, Jason

2015-01-01

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD). Dioxin causes a range of toxic responses, including hepatic damage, steatohepatitis, and a lethal wasting syndrome; however, the mechanisms are still unknown. Here, we show that the loss of TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), an ADP-ribosyltransferase and AHR repressor, increases sensitivity to dioxin-induced toxicity, steatohepatitis, and lethality. Tiparp−/− mice given a single injection of 100 μg/kg dioxin did not survive beyond day 5; all Tiparp+/+ mice survived the 30-day treatment. Dioxin-treated Tiparp−/− mice exhibited increased liver steatosis and hepatotoxicity. Tiparp ADP-ribosylated AHR but not its dimerization partner, the AHR nuclear translocator, and the repressive effects of TIPARP on AHR were reversed by the macrodomain containing mono-ADP-ribosylase MACROD1 but not MACROD2. These results reveal previously unidentified roles for Tiparp, MacroD1, and ADP-ribosylation in AHR-mediated steatohepatitis and lethality in response to dioxin. PMID:25975270

Parthanatos, a messenger of death.

PubMed

David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

2009-01-01

Poly-ADP-ribose polymerase-1 (PARP-1)'s roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 overactivation underlies cell death in models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into understanding mechanisms downstream of PARP-1 overactivation. Recent evidence shows that poly-ADP ribose (PAR) polymer itself can act as a cell death effector downstream of PARP-1. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will present evidence and questions raised by these recent findings, and summarize the proposed mechanisms by which PARP-1 overactivation kills. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 overactivation.

Activity-based assay for human mono-ADP-ribosyltransferases ARTD7/PARP15 and ARTD10/PARP10 aimed at screening and profiling inhibitors.

PubMed

Venkannagari, Harikanth; Fallarero, Adyary; Feijs, Karla L H; Lüscher, Bernhard; Lehtiö, Lari

2013-05-13

Poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin like ADP-ribosyl transferases (ARTDs) are enzymes that catalyze the covalent modification of proteins by attachment of ADP-ribose units to the target amino acid residues or to the growing chain of ADP-ribose. A subclass of the ARTD superfamily consists of mono-ADP-ribosyl transferases that are thought to modify themselves and other substrate proteins by covalently adding only a single ADP-ribose moiety to the target. Many of the ARTD enzymes are either established or potential drug targets and a functional activity assay for them will be a valuable tool to identify selective inhibitors for each enzyme. Existing assays are not directly applicable for screening of inhibitors due to the different nature of the reaction and different target molecules. We modified and applied a fluorescence-based assay previously described for PARP1/ARTD1 and tankyrase/ARTD5 for screening of PARP10/ARTD10 and PARP15/ARTD7 inhibitors. The assay measures the amount of NAD(+) present after chemically converting it to a fluorescent analog. We demonstrate that by using an excess of a recombinant acceptor protein the performance of the activity-based assay is excellent for screening of compound libraries. The assay is homogenous and cost effective, making it possible to test relatively large compound libraries. This method can be used to screen inhibitors of mono-ARTDs and profile inhibitors of the enzyme class. The assay was optimized for ARTD10 and ARTD7, but it can be directly applied to other mono-ARTDs of the ARTD superfamily. Profiling of known ARTD inhibitors against ARTD10 and ARTD7 in a validatory screening identified the best inhibitors with submicromolar potencies. Only few of the tested ARTD inhibitors were potent, implicating that there is a need to screen new compound scaffolds. This is needed to create small molecules that could serve as biological probes and potential starting points for drug discovery projects against

Role of peroxynitrite and poly (ADP-ribosyl) synthetase activation in cardiovascular derangement induced by zymosan in the rat.

PubMed

Cuzzocrea, S; Zingarelli, B; Caputi, A P

1998-01-01

Peritoneal administration of zymosan in the rat induced a severe inflammatory process characterised by an increase in the plasma levels of nitrite and nitrate, stable metabolites of nitric oxide (NO) and in the levels of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, at 18 hours zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, in the aorta of zymosan-shocked rats. In ex vivo experiments, thoracic aorta rings of zymosan-treated rats showed a reduced contraction to noradrenaline and reduced responsiveness to the relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). Treatment of zymosan-shocked rats with 3-aminobenzamide or Nicotinamide, inhibitors of poly ADP-ribosil synthetase (PARS) activity reduced the production of peroxynitrite and significantly prevented the cardiovascular dysfunction. Our data suggest that peroxynitrite and PARS activation play a role in the zymosan-induced cardiovascular derangements in the rat.

Positive transcriptional regulation of the human micro opioid receptor gene by poly(ADP-ribose) polymerase-1 and increase of its DNA binding affinity based on polymorphism of G-172 -> T.

PubMed

Ono, Takeshi; Kaneda, Toshio; Muto, Akihiro; Yoshida, Tadashi

2009-07-24

Micro opioid receptor (MOR) agonists such as morphine are applied widely in clinical practice as pain therapy. The effects of morphine through MOR, such as analgesia and development of tolerance and dependence, are influenced by individual specificity. Recently, we analyzed single nucleotide polymorphisms on the human MOR gene to investigate the factors that contribute to individual specificity. In process of single nucleotide polymorphisms analysis, we found that specific nuclear proteins bound to G(-172) --> T region in exon 1 in MOR gene, and its affinity to DNA was increased by base substitution from G(-172) to T(-172). The isolated protein was identified by mass spectrometry and was confirmed by Western blotting to be poly(ADP-ribose) polymerase-1 (PARP-1). The overexpressed PARP-1 bound to G(-172) --> T and enhanced the transcription of reporter vectors containing G(-172) and T(-172). Furthermore, PARP-1 inhibitor (benzamide) decreased PARP-1 binding to G(-172) --> T without affecting mRNA or protein expression level of PARP-1 and down-regulated the subsequent MOR gene expression in SH-SY5Y cells. Moreover, we found that tumor necrosis factor-alpha enhanced MOR gene expression as well as increased PARP-1 binding to the G(-172) --> T region and G(-172) --> T-dependent transcription in SH-SY5Y cells. These effects were also inhibited by benzamide. In this study, our data suggest that PARP-1 positively regulates MOR gene transcription via G(-172) --> T, which might influence individual specificity in therapeutic opioid effects.

Electrophoretic characterization of the Mammalian nuclear matrix proteome, nuclear envelope, nucleoli and covalently bound ADP-ribose polymers: potential applications to cancer.

PubMed

Aranda, Xavier G; Racho, Ronald G; Pacheco-Rodríguez, Gustavo; Alvarez-González, Rafael

2014-01-01

Nucleic acid metabolism is biochemically compartmentalized to the nucleus. Thus, it is necessary to define the proteome of the various macromolecular structures within this organelle. We isolated the nuclear matrix (NM) fraction from rat liver by sequential centrifugation steps at 13,000 rpm, staggered between endogenous nuclease treatment for 2 h at 37°C, followed by high-salt (H.S.; 2.0 M NaCl) and non-ionic detergent extractions (0.1%- or 1.0% Triton X-100) to eliminate the bulk of chromosomal DNA/RNA, histone proteins and the nuclear envelope (NE). Integrity of the NM and NE structures was confirmed by electron microscopy. Next, we analyzed the NM proteome on a 20% polyacrylamide gel using the PhastSystem. We observed the absence of histone proteins and the characteristic presence of the lamins by Coomassie blue staining. By contrast, upon silver staining, following electrophoretic separation with a Tris-Borate-EDTA buffer, we observed the NM-associated nucleic RNA and protein-free ADP-ribose polymers. While polymers are found in much lower concentration than RNA in NM, they were purified by affinity chromatography on boronate resin prior to electrophoresis. We observed the electrophoretic resolution of free ADP-ribose chains (5-25 units) by silver staining. The significance of our observations to cancer studies and carcinogenesis is discussed. Copyright© 2014, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shirai, Hidenori; Fujimori, Hiroaki; Gunji, Akemi

Highlights: •Parg{sup −/−} ES cells were more sensitive to γ-irradiation than Parp-1{sup −/−} ES cells. •Parg{sup −/−} cells were more sensitive to carbon-ion irradiation than Parp-1{sup −/−} cells. •Parg{sup −/−} cells showed defects in DSB repair after carbon-ion irradiation. •PAR accumulation was enhanced after carbon-ion irradiation compared to γ-irradiation. -- Abstract: Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg{sup −/−} and poly(ADP-ribose) polymerase-1 deficient (Parp-1{sup −/−}) ES cells were used and responsesmore » to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg{sup −/−} cells were more sensitive to γ-irradiation than Parp-1{sup −/−} cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg{sup −/−} cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg{sup −/−} ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1{sup −/−} cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg{sup −/−} ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/μm) and Fe-ion irradiation (200 keV/μm) were also examined. Parg{sup −/−} cells were more sensitive to LET 70 keV/μm carbon-ion irradiation than Parp-1{sup −/−} cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24 h. The induction level of p53 phophorylation at

MSH3 expression does not influence the sensitivity of colon cancer HCT116 cell line to oxaliplatin and poly(ADP-ribose) polymerase (PARP) inhibitor as monotherapy or in combination.

PubMed

Tentori, Lucio; Muzi, Alessia; Dorio, Annalisa Susanna; Dolci, Susanna; Campolo, Federica; Vernole, Patrizia; Lacal, Pedro Miguel; Praz, Françoise; Graziani, Grazia

2013-07-01

Defective expression of the mismatch repair protein MSH3 is frequently detected in colon cancer, and down-regulation of its expression was found to decrease sensitivity to platinum compounds or poly(ADP-ribose) polymerase inhibitors (PARPi) monotherapy. We have investigated whether MSH3 transfection in MSH3-deficient colon cancer cells confers resistance to oxaliplatin or PARPi and whether their combination restores chemosensitivity. MSH3-deficient/MLH1-proficient colon cancer HCT116(MLH1) cells were transfected with the MSH3 cDNA cloned into the pcDNA3.1(-) vector. MSH3/MLH1-deficient HCT116, carrying MLH1 and MSH3 mutations on chromosome 3 and 5, respectively, and HCT116 in which wild-type MLH1 (HCT116+3), MSH3 (HCT116+5) or both genes (HCT116+3+5) were introduced by chromosome transfer were also tested. Sensitivity to oxaliplatin and to PARPi was evaluated by analysis of clonogenic survival, cell proliferation, apoptosis and cell cycle. MSH3 transfection in HCT116 cells did not confer resistance to oxaliplatin or PARPi monotherapy. MSH3-proficient HCT116+5 or HCT116+3+5 cells, which were more resistant to oxaliplatin and PARPi in comparison with their MSH3-deficient counterparts, expressed higher levels of the nucleotide excision repair ERCC1 and XPF proteins, involved in the resistance to platinum compounds, and lower PARP-1 levels. In all cases, PARPi increased sensitivity to oxaliplatin. Restoring of MSH3 expression by cDNA transfection, rather than by chromosome transfer, did not affect colon cancer sensitivity to oxaliplatin or PARPi monotherapy; PARP-1 levels seemed to be more crucial for the outcome of PARPi monotherapy.

[The severity of gestational diabetes mellitus affects microvascular dysfunction measured three years after pregnancy that may be related to increased oxidative stress].

PubMed

Horváth, Eszter Mária; Mágenheim, Rita; Domján, Beatrix Annamária; Ferencz, Viktória; Tänczer, Tímea; Szabó, Eszter; Benkő, Rita; Szabó, Csaba; Tabák, Ádám; Somogyi, Anikó

2015-11-22

Oxidative-nitrative stress and poly(ADP-ribose) polymerase activation observed in gestational diabetes may play role in the increased cardiovascular risk in later life. The present study aimed to examine the influence of the severity of previous gestational diabetes (insulin need) on vascular function three years after delivery. Furthermore, the authors investigated the relation of vascular function with oxidative-nitrative stress and poly(ADP-ribose) polymerase activation. Macrovascular function was measured by applanation tonometry; microvascular reactivity was assessed by provocation tests during Laser-Doppler flowmetry in 40 women who had gestational diabetes 3 years before the study. Oxidative-nitrative stress and poly(ADP-ribose) polymerase activity in blood components were determined by colorimetry and immunohistochemistry. Three years after insulin treated gestational diabetes impaired microvascular function and increased oxidative stress was observed compared to mild cases. The severity of previous gestational diabetes affects microvascular dysfunction that is accompanied by elevated oxidative stress. Nitrative stress and poly(ADP-ribose) polymerase activity correlates with certain vascular factors not related to the severity of the disease.

Bok Is Not Pro-Apoptotic But Suppresses Poly ADP-Ribose Polymerase-Dependent Cell Death Pathways and Protects against Excitotoxic and Seizure-Induced Neuronal Injury.

PubMed

D'Orsi, Beatrice; Engel, Tobias; Pfeiffer, Shona; Nandi, Saheli; Kaufmann, Thomas; Henshall, David C; Prehn, Jochen H M

2016-04-20

Bok (Bcl-2-related ovarian killer) is a Bcl-2 family member that, because of its predicted structural homology to Bax and Bak, has been proposed to be a pro-apoptotic protein. In this study, we demonstrate that Bok is highly expressed in neurons of the mouse brain but that bok was not required for staurosporine-, proteasome inhibition-, or excitotoxicity-induced apoptosis of cultured cortical neurons. On the contrary, we found that bok-deficient neurons were more sensitive to oxygen/glucose deprivation-induced injury in vitro and seizure-induced neuronal injury in vivo Deletion of bok also increased staurosporine-, excitotoxicity-, and oxygen/glucose deprivation-induced cell death in bax-deficient neurons. Single-cell imaging demonstrated that bok-deficient neurons failed to maintain their neuronal Ca(2+)homeostasis in response to an excitotoxic stimulus; this was accompanied by a prolonged deregulation of mitochondrial bioenergetics.bok deficiency led to a specific reduction in neuronal Mcl-1 protein levels, and deregulation of both mitochondrial bioenergetics and Ca(2+)homeostasis was rescued by Mcl-1 overexpression. Detailed analysis of cell death pathways demonstrated the activation of poly ADP-ribose polymerase-dependent cell death in bok-deficient neurons. Collectively, our data demonstrate that Bok acts as a neuroprotective factor rather than a pro-death effector during Ca(2+)- and seizure-induced neuronal injury in vitro and in vivo Bcl-2 proteins are essential regulators of the mitochondrial apoptosis pathway. The Bcl-2 protein Bok is highly expressed in the CNS. Because of its sequence similarity to Bax and Bak, Bok has long been considered part of the pro-apoptotic Bax-like subfamily, but no studies have yet been performed in neurons to test this hypothesis. Our study provides important new insights into the functional role of Bok during neuronal apoptosis and specifically in the setting of Ca(2+)- and seizure-mediated neuronal injury. We show that Bok

A BRCA1 deficient-like signature is enriched in breast cancer brain metastases and predicts DNA damage-induced poly (ADP-ribose) polymerase inhibitor sensitivity.

PubMed

McMullin, Ryan P; Wittner, Ben S; Yang, Chuanwei; Denton-Schneider, Benjamin R; Hicks, Daniel; Singavarapu, Raj; Moulis, Sharon; Lee, Jeongeun; Akbari, Mohammad R; Narod, Steven A; Aldape, Kenneth D; Steeg, Patricia S; Ramaswamy, Sridhar; Sgroi, Dennis C

2014-03-14

There is an unmet clinical need for biomarkers to identify breast cancer patients at an increased risk of developing brain metastases. The objective is to identify gene signatures and biological pathways associated with human epidermal growth factor receptor 2-positive (HER2+) brain metastasis. We combined laser capture microdissection and gene expression microarrays to analyze malignant epithelium from HER2+ breast cancer brain metastases with that from HER2+ nonmetastatic primary tumors. Differential gene expression was performed including gene set enrichment analysis (GSEA) using publicly available breast cancer gene expression data sets. In a cohort of HER2+ breast cancer brain metastases, we identified a gene expression signature that anti-correlates with overexpression of BRCA1. Sequence analysis of the HER2+ brain metastases revealed no pathogenic mutations of BRCA1, and therefore the aforementioned signature was designated BRCA1 Deficient-Like (BD-L). Evaluation of an independent cohort of breast cancer metastases demonstrated that BD-L values are significantly higher in brain metastases as compared to other metastatic sites. Although the BD-L signature is present in all subtypes of breast cancer, it is significantly higher in BRCA1 mutant primary tumors as compared with sporadic breast tumors. Additionally, BD-L signature values are significantly higher in HER2-/ER- primary tumors as compared with HER2+/ER + and HER2-/ER + tumors. The BD-L signature correlates with breast cancer cell line pharmacologic response to a combination of poly (ADP-ribose) polymerase (PARP) inhibitor and temozolomide, and the signature outperformed four published gene signatures of BRCA1/2 deficiency. A BD-L signature is enriched in HER2+ breast cancer brain metastases without pathogenic BRCA1 mutations. Unexpectedly, elevated BD-L values are found in a subset of primary tumors across all breast cancer subtypes. Evaluation of pharmacological sensitivity in breast cancer

A BRCA1 deficient-like signature is enriched in breast cancer brain metastases and predicts DNA damage-induced poly (ADP-ribose) polymerase inhibitor sensitivity

PubMed Central

2014-01-01

Introduction There is an unmet clinical need for biomarkers to identify breast cancer patients at an increased risk of developing brain metastases. The objective is to identify gene signatures and biological pathways associated with human epidermal growth factor receptor 2-positive (HER2+) brain metastasis. Methods We combined laser capture microdissection and gene expression microarrays to analyze malignant epithelium from HER2+ breast cancer brain metastases with that from HER2+ nonmetastatic primary tumors. Differential gene expression was performed including gene set enrichment analysis (GSEA) using publicly available breast cancer gene expression data sets. Results In a cohort of HER2+ breast cancer brain metastases, we identified a gene expression signature that anti-correlates with overexpression of BRCA1. Sequence analysis of the HER2+ brain metastases revealed no pathogenic mutations of BRCA1, and therefore the aforementioned signature was designated BRCA1 Deficient-Like (BD-L). Evaluation of an independent cohort of breast cancer metastases demonstrated that BD-L values are significantly higher in brain metastases as compared to other metastatic sites. Although the BD-L signature is present in all subtypes of breast cancer, it is significantly higher in BRCA1 mutant primary tumors as compared with sporadic breast tumors. Additionally, BD-L signature values are significantly higher in HER2-/ER- primary tumors as compared with HER2+/ER + and HER2-/ER + tumors. The BD-L signature correlates with breast cancer cell line pharmacologic response to a combination of poly (ADP-ribose) polymerase (PARP) inhibitor and temozolomide, and the signature outperformed four published gene signatures of BRCA1/2 deficiency. Conclusions A BD-L signature is enriched in HER2+ breast cancer brain metastases without pathogenic BRCA1 mutations. Unexpectedly, elevated BD-L values are found in a subset of primary tumors across all breast cancer subtypes. Evaluation of

Concurrent targeting of nitrosative stress-PARP pathway corrects functional, behavioral and biochemical deficits in experimental diabetic neuropathy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Negi, Geeta; Kumar, Ashutosh; Sharma, Shyam S., E-mail: sssharma@niper.ac.in

2010-01-01

Peroxynitrite mediated nitrosative stress, an indisputable initiator of DNA damage and overactivation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated after sensing DNA damage, are two crucial pathogenetic mechanisms in diabetic neuropathy. The intent of the present study was to investigate the effect of combination of a peroxynitrite decomposition catalyst (PDC), FeTMPyP and a PARP inhibitor, 4-ANI against diabetic peripheral neuropathy. The end points of evaluation of the study included motor nerve conduction velocity (MNCV) and nerve blood flow (NBF) for evaluating nerve functions; thermal hyperalgesia and mechanical allodynia for assessing nociceptive alterations, malondialdehyde and peroxynitrite levels to detect oxidativemore » stress-nitrosative stress; NAD concentration in sciatic nerve to assess overactivation of PARP. Additionally immunohistochemical studies for nitrotyrosine and Poly(ADP-ribose) (PAR) was also performed. Treatment with the combination of FeTMPyP and 4-ANI led to significant improvement in nerve functions and pain parameters and also attenuated the oxidative-nitrosative stress markers. Further, the combination also reduced the overactivation of PARP as evident from increased NAD levels and decreased PAR immunopositivity in sciatic nerve microsections. Thus, it can be concluded that treatment with the combination of a PDC and PARP inhibitor attenuates alteration in peripheral nerves in diabetic neuropathy (DN).« less

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila.

PubMed

Ji, Yingbiao; Tulin, Alexei V

2016-10-01

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates the posttranscriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germ line stem cell maintenance and oocyte localization. The mRNA targets regulated by Hrp38 are mostly unknown. We identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. We found that Hrp38 binds to the 3' untranslated region (UTR) of Nanos mRNA, which contains a translation control element. We have demonstrated that translation of the luciferase reporter bearing the Nanos 3' UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. Our data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to the Nanos 3' UTR, increasing the translation in vivo and in vitro hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. We conclude that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila

PubMed Central

Ji, Yingbiao

2016-01-01

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates the posttranscriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germ line stem cell maintenance and oocyte localization. The mRNA targets regulated by Hrp38 are mostly unknown. We identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. We found that Hrp38 binds to the 3′ untranslated region (UTR) of Nanos mRNA, which contains a translation control element. We have demonstrated that translation of the luciferase reporter bearing the Nanos 3′ UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. Our data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to the Nanos 3′ UTR, increasing the translation in vivo and in vitro. hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. We conclude that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis. PMID:27402862

NAD+ depletion or PAR polymer formation: which plays the role of executioner in ischaemic cell death?

PubMed

Siegel, C; McCullough, L D

2011-09-01

Multiple cell death pathways are activated in cerebral ischaemia. Much of the initial injury, especially in the core of the infarct where cerebral blood flow is severely reduced, is necrotic and secondary to severe energy failure. However, there is considerable evidence that delayed cell death continues for several days, primarily in the penumbral region. As reperfusion therapies grow in number and effectiveness, restoration of blood flow early after injury may lead to a shift towards apoptosis. It is important to elucidate what are the key mediators of apoptotic cell death after stroke, as inhibition of apoptosis may have therapeutic implications. There are two well described pathways that lead to apoptotic cell death; the caspase pathway and the more recently described caspase-independent pathway triggered by poly-ADP-ribose polymers (PARP) activation. Caspase-induced cell death is initiated by release of mitochondrial cytochrome c, formation of the cytosolic apoptosome, and activation of endonucleases leading to a multitude of small randomly cleaved DNA fragments. In contrast caspase-independent cell death is secondary to activation of apoptosis inducing factor (AIF). Mitochondrial AIF translocates to the nucleus, where it induces peripheral chromatin condensation, as well as characteristic high-molecular-weight (50 kbp) DNA fragmentation. Although caspase-independent cell death has been recognized for some time and is known to contribute to ischaemic injury, the upstream triggering events leading to activation of this pathway remain unclear. The two major theories are that ischaemia leads to nicotinamide adenine dinucleotide (NAD+) depletion and subsequent energy failure, or alternatively that cell death is directly triggered by a pro-apoptotic factor produced by activation of the DNA repair enzyme PARP. PARP activation is robust in the ischaemic brain producing variable lengths of poly-ADP-ribose (PAR) polymers as byproducts of PARP activation. PAR polymers

Effects of Site-Directed Mutagenesis of Escherichia coli Heat-Labile Enterotoxin on ADP-Ribosyltransferase Activity and Interaction with ADP-Ribosylation Factors

PubMed Central

A. Stevens, Linda; Moss, Joel; Vaughan, Martha; Pizza, Mariagrazia; Rappuoli, Rino

1999-01-01

Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsα, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction. PMID:9864224

Studying Catabolism of Protein ADP-Ribosylation.

PubMed

Palazzo, Luca; James, Dominic I; Waddell, Ian D; Ahel, Ivan

2017-01-01

Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD + ) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.

A systematic analysis of the PARP protein family identifies new functions critical for cell physiology

PubMed Central

Vyas, Sejal; Chesarone-Cataldo, Melissa; Todorova, Tanya; Huang, Yun-Han; Chang, Paul

2013-01-01

The poly(ADP-ribose) polymerase (PARP) family of proteins use NAD+ as their substrate to modify acceptor proteins with adenosine diphosphate-ribose (ADPr) modifications. The function of most PARPs under physiological conditions is unknown. Here, to better understand this protein family, we systematically analyze the cell cycle localization of each PARP and of poly(ADP-ribose), a product of PARP activity, then identify the knock-down phenotype of each protein and perform secondary assays to elucidate function. We show that most PARPs are cytoplasmic, identify cell cycle differences in the ratio of nuclear to cytoplasmic poly(ADP-ribose), and identify four phenotypic classes of PARP function. These include the regulation of membrane structures, cell viability, cell division, and the actin cytoskeleton. Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function. In total, we show that PARP proteins are critical regulators of eukaryotic physiology. PMID:23917125

Interdependent genotoxic mechanisms of monomethylarsonous acid: Role of ROS-induced DNA damage and poly(ADP-ribose) polymerase-1 inhibition in the malignant transformation of urothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wnek, Shawn M.; Kuhlman, Christopher L.; Camarillo, Jeannie M.

2011-11-15

Exposure of human bladder urothelial cells (UROtsa) to 50 nM of the arsenic metabolite, monomethylarsonous acid (MMA{sup III}), for 12 weeks results in irreversible malignant transformation. The ability of continuous, low-level MMA{sup III} exposure to cause an increase in genotoxic potential by inhibiting repair processes necessary to maintain genomic stability is unknown. Following genomic insult within cellular systems poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger protein, is rapidly activated and recruited to sites of DNA strand breaks. When UROtsa cells are continuously exposed to 50 nM MMA{sup III}, PARP-1 activity does not increase despite the increase in MMA{sup III}-induced DNA single-strandmore » breaks through 12 weeks of exposure. When UROtsa cells are removed from continuous MMA{sup III} exposure (2 weeks), PARP-1 activity increases coinciding with a subsequent decrease in DNA damage levels. Paradoxically, PARP-1 mRNA expression and protein levels are elevated in the presence of continuous MMA{sup III} indicating a possible mechanism to compensate for the inhibition of PARP-1 activity in the presence of MMA{sup III}. The zinc finger domains of PARP-1 contain vicinal sulfhydryl groups which may act as a potential site for MMA{sup III} to bind, displace zinc ion, and render PARP-1 inactive. Mass spectrometry analysis demonstrates the ability of MMA{sup III} to bind a synthetic peptide representing the zinc-finger domain of PARP-1, and displace zinc from the peptide in a dose-dependent manner. In the presence of continuous MMA{sup III} exposure, continuous 4-week zinc supplementation restored PARP-1 activity levels and reduced the genotoxicity associated with MMA{sup III}. Zinc supplementation did not produce an overall increase in PARP-1 protein levels, decrease the levels of MMA{sup III}-induced reactive oxygen species, or alter Cu-Zn superoxide dismutase levels. Overall, these results present two potential interdependent mechanisms in which

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scaife, R.M.; Wilson, L.; Purich, D.L.

1992-01-14

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extentmore » of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.« less

Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

PubMed

Long, Aaron; Klimova, Nina; Kristian, Tibor

2017-10-01

NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

A Phase 1 trial of the poly(ADP-ribose) polymerase inhibitor olaparib (AZD2281) in combination with the anti-angiogenic cediranib (AZD2171) in recurrent epithelial ovarian or triple-negative breast cancer.

PubMed

Liu, Joyce F; Tolaney, Sara M; Birrer, Michael; Fleming, Gini F; Buss, Mary K; Dahlberg, Suzanne E; Lee, Hang; Whalen, Christin; Tyburski, Karin; Winer, Eric; Ivy, Percy; Matulonis, Ursula A

2013-09-01

Poly(ADP-ribose) polymerase (PARP)-inhibitors and anti-angiogenics have activity in recurrent ovarian and breast cancer; however, the effect of combined therapy against PARP and angiogenesis in this population has not been reported. We investigated the toxicities and recommended phase 2 dosing (RP2D) of the combination of cediranib, a multitargeted inhibitor of vascular endothelial growth factor receptor (VEGFR)-1/2/3 and olaparib, a PARP-inhibitor (NCT01116648). Cediranib tablets once daily and olaparib capsules twice daily were administered orally in a standard 3+3 dose escalation design. Patients with recurrent ovarian or metastatic triple-negative breast cancer were eligible. Patients had measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 or met Gynecologic Cancer InterGroup (GCIG) CA125 criteria. No prior PARP-inhibitors or anti-angiogenics in the recurrent setting were allowed. 28 patients (20 ovarian, 8 breast) enrolled to 4 dose levels. 2 dose limiting toxicities (DLTs) (1 grade 4 neutropenia ≥ 4 days; 1 grade 4 thrombocytopenia) occurred at the highest dose level (cediranib 30 mg daily; olaparib 400 mg twice daily [BID]). The RP2D was cediranib 30 mg daily and olaparib 200 mg BID. Grade 3 or higher toxicities occurred in 75% of patients, and included grade 3 hypertension (25%) and grade 3 fatigue (18%). One grade 3 bowel obstruction occurred. The overall response rate (ORR) in the 18 RECIST-evaluable ovarian cancer patients was 44%, with a clinical benefit rate (ORR plus stable disease (SD) > 24 weeks) of 61%. None of the seven evaluable breast cancer patients achieved clinical response; two patients had stable disease for > 24 weeks. The combination of cediranib and olaparib has haematologic DLTs and anticipated class toxicities, with promising evidence of activity in ovarian cancer patients. Copyright © 2013 Elsevier Ltd. All rights reserved.

Parthanatos, a messenger of death

PubMed Central

David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

2015-01-01

Poly-ADP-ribose polymerase-1 (PARP-1)'s multiple roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include, but are not limited to DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its active role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 over activation underlies cell death in experimental models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into designing PARP-1 inhibitors and understanding mechanisms downstream of PARP-1 over activation. PARP-1 overactivation may kill by depleting cellular energy through nicotinamide adenine dinucleotide (NAD+) consumption, and by releasing the cell death effector apoptosis-inducing factor (AIF). Unexpectedly, recent evidence shows that poly-ADP ribose (PAR) polymer itself, and not the consumption of NAD+ is the source of cytotoxicity. Thus, PAR polymer acts as a cell death effector downstream of PARP-1-mediated cell death signaling. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will summarize the proposed mechanisms by which PARP-1 overactivation kills. We will present evidence for parthanatos, and the questions raised by these recent findings. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 over activation. PMID:19273119

Automodification of PARP and fatty acid-based membrane lipidome as a promising integrated biomarker panel in molecular medicine.

PubMed

Bianchi, Anna Rita; Ferreri, Carla; Ruggiero, Simona; Deplano, Simone; Sunda, Valentina; Galloro, Giuseppe; Formisano, Cesare; Mennella, Maria Rosaria Faraone

2016-01-01

Establishing by statistical analyses whether the analyses of auto-modified poly(ADP-ribose)polymerase and erythrocyte membrane fatty acid composition (Fat Profile(®)), separately or in tandem, help monitoring the physio-pathology of the cell, and correlate with diseases, if present. Ninety five subjects were interviewed and analyzed blindly. Blood lymphocytes and erythrocytes were prepared to assay poly(ADP-ribose)polymerase automodification and fatty acid based membrane lipidome, respectively. Poly(ADP-ribose)polymerase automodification levels confirmed their correlation with DNA damage extent, and allowed monitoring disease activity, upon surgical/therapeutic treatment. Membrane lipidome profiles showed lipid unbalance mainly linked to inflammatory states. Statistically both tests were separately significant, and correlated each other within some pathologies. In the laboratory routine, both tests, separately or in tandem, might be a preliminary and helpful step to investigate the occurrence of a given disease. Their combination represents a promising integrated panel for sensible, noninvasive and routine health monitoring.

Glycolytic rate and lymphomagenesis depend on PARP14, an ADP ribosyltransferase of the B aggressive lymphoma (BAL) family.

PubMed

Cho, Sung Hoon; Ahn, Annie K; Bhargava, Prerna; Lee, Chih-Hao; Eischen, Christine M; McGuinness, Owen; Boothby, Mark

2011-09-20

Poly(ADP-ribose)polymerase (PARP)14--a member of the B aggressive lymphoma (BAL) family of macrodomain-containing PARPs--is an ADP ribosyltransferase that interacts with Stat6, enhances induction of certain genes by IL-4, and is expressed in B lymphocytes. We now show that IL-4 enhancement of glycolysis in B cells requires PARP14 and that this process is central to a role of PARP14 in IL-4-induced survival. Thus, enhancements of AMP-activated protein kinase activity restored both IL-4-induced glycolytic activity in Parp14(-/-) B cells and prosurvival signaling by this cytokine. Suppression of apoptosis is central to B-lymphoid oncogenesis, and elevated macro-PARP expression has been correlated with lymphoma aggressiveness. Strikingly, PARP14 deficiency delayed B lymphomagenesis and reversed the block to B-cell maturation driven by the Myc oncogene. Collectively, these findings reveal links between a mammalian ADP ribosyltransferase, cytokine-regulated metabolic activity, and apoptosis; show that PARP14 influences Myc-induced oncogenesis; and suggest that the PARP14-dependent capacity to increase cellular metabolic rates may be an important determinant of lymphoma pathobiology.

Novel bacterial ADP-ribosylating toxins: structure and function

PubMed Central

Simon, Nathan C.; Aktories, Klaus; Barbieri, Joseph T.

2018-01-01

Preface Bacterial ADP-ribosyltransferase toxins (bARTTs) transfer ADP-ribose to eukaryotic proteins to promote bacterial pathogenesis. In this review we use prototype bARTTs, such as diphtheria and pertussis toxins, as references for the characterization of several new bARTTs from human, insect, and plant pathogens, which were identified recently through bioinformatic analyses. Several of these toxins, including Cholix toxin from Vibrio cholerae, SpyA from Streptococcus pyogenes, HopU1 from Pseudomonas syringae, and the Tcc toxins from Photorhabdus luminescens, ADP-ribosylate novel substrates and possess unique organizations, which distinguish them from the reference toxins. The characterization of these toxins extends our appreciation for the variety of structure-function properties possessed by bARTTs and their roles in bacterial pathogenesis. PMID:25023120

Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jorgensen, Rene; Purdy, Alexandra E.; Fieldhouse, Robert J.

2008-07-15

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 {+-} 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 {+-} 2 {mu}g/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity againstmore » ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-{angstrom} resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.« less

ADP-ribosylation of membrane components by pertussis and cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ribeiro-Neto, F.A.P.; Mattera, F.; Hildebrandt, J.D.

1985-01-01

Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (N/sub s/) and inhibitory (N/sub i/) regulatory components of adenylyl cyclase. Cholera toxin acts on N/sub s/ by ADP-ribosylating its ..cap alpha../sub s/ subunit; pertussis toxin acts on N/sub i/ by ADP-ribosylating its ..cap alpha..; subunit. By using (/sup 32/P)NAD/sup +/ and determining the transfer of its (/sup 32/P)ADP-ribose moiety to membrane components, it is possible to obtain information on N/sub s/ and N/sub i/. A set of protocols is presented that can be used to study simultaneously and comparatively the susceptibility of N/submore » s/ and N/sub i/ to be ADP-ribosylated by cholera and pertussis toxin.« less

Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

PubMed

Krastev, Dragomir B; Pettitt, Stephen J; Campbell, James; Song, Feifei; Tanos, Barbara E; Stoynov, Stoyno S; Ashworth, Alan; Lord, Christopher J

2018-05-22

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Komissarova, Elena V.; Rossman, Toby G., E-mail: toby.rossman@nyumc.or

2010-03-15

Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21{sup WAF1/CIP1} expression, normally a block to cell cycle progression after DNA damage,more » is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 muM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 muM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.« less

Redesign of Schistosoma mansoni NAD+ catabolizing enzyme : the active site H103W mutation restores ADP-ribosyl cyclase activity†

PubMed Central

Kuhn, Isabelle; Kellenberger, Esther; Rognan, Didier; Lund, Frances E.; Muller-Steffner, Hélène; Schuber, Francis

2008-01-01

Schistosoma mansoni NAD(P)+ catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes which are involved in the production of metabolites that elicit Ca2+ mobilization, SmNACE is virtually unable to transform NAD+ into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced in SmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability of SmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change we have replaced His103 with a tryptophan. The H103W mutation in SmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products, i.e., a value larger than observed for other members of this family such as CD38. Introduction of a Trp103 residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provide new insight into the catalytic mechanism of SmNACE. Both residue side chains share similar roles in the nicotinamide-ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge however in the evolution of this intermediate; His103 provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR. PMID:17002287

Nucleosides and nucleotides. 192. Toward the total synthesis of cyclic ADP-carbocyclic-ribose. Formation of the intramolecular pyrophosphate linkage by a conformation-restriction strategy in a syn-form using a halogen substitution at the 8-position of the adenine ring.

PubMed

Sumita, Y; Shirato, M; Ueno, Y; Matsuda, A; Shuto, S

2000-01-01

The synthesis of cyclic ADP-carbocyclic-ribose (2), as a stable mimic for cyclic ADP-ribose, was investigated. Construction of the 18-membered backbone structure was successfully achieved by condensation of the two phosphate groups of 19, possibly due to restriction of the conformation of the substrate in a syn-form using an 8-chloro substituent at the adenine moiety. SN2 reactions between an optically active carbocyclic unit 8, which was constructed by a previously developed method, and 8-bromo-N6-trichloroacetyl-2',3'-O-isopropylideneadenosine 9c gave N-1-carbocyclic derivative, which was deprotected to give 5'-5"-diol derivatives 18. When 18 was treated with POCl3 in PO(OEt)3, the bromo group at the 8-position was replaced to give N-1-carbocyclic-8-chloroadenosine 5',5"-diphosphate derivative 19 in 43% yield. Treatment of 19 with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride gave the desired intramolecular condensation product 20 in 10% yield. This is the first chemical construction of the 18-membered backbone structure containing an intramolecular pyrophosphate linkage of a cADPR-related compound with an adenine base.

PARP-1 may be involved in hydroquinone-induced apoptosis by poly ADP-ribosylation of ZO-2

PubMed Central

Liu, Jiaxian; Yuan, Qian; Ling, Xiaoxuan; Tan, Qiang; Liang, Hairong; Chen, Jialong; Lin, Lianzai; Xiao, Yongmei; Chen, Wen; Liu, Linhua; Tang, Huanwen

2017-01-01

Hydroquinone (HQ), a major reactive metabolite of benzene, contributes to benzene-induced leukemia. The molecular mechanisms that underlie this activity remain to be elucidated. Poly ADP-ribosylation (PARylation) is a type of reversible posttranslational modification that is performed by enzymes in the PAR polymerase (PARP) family and mediates different biological processes, including apoptosis. Zona occludens 2 (ZO-2) is a tight junction scaffold protein, which is involved in cell proliferation and apoptosis. The present study investigated the activity and mechanisms regulated by PARP-1 during HQ-induced apoptosis using TK6 lymphoblastoid cells and PARP-1-silenced TK6 cells. The results revealed that exposure to 10 µM HQ for 72 h induced apoptosis in TK6 cells and that apoptosis was attenuated in PARP-1-silenced TK6 cells. In cells treated with HQ, inhibition of PARP-1 increased the expression of B cell leukemia/lymphoma 2 (Bcl-2), increased ATP production and reduced reactive oxygen species (ROS) production relative to the levels observed in cells treated with HQ alone. Co-localization of ZO-2 and PAR (or PARP-1 protein) was determined using immunofluorescence confocal microscopy. The findings of the present study revealed that ZO-2 was PARylated via an interaction with PARP-1, which was consistent with an analysis of protein expression that was performed using western blot analysis, which determined that ZO-2 protein expression was upregulated in HQ-treated control cells and downregulated in HQ-treated PARP-1-silenced TK6 cells. These findings indicated that prolonged exposure to a low dose of HQ induced TK6 cells to undergo apoptosis, whereas inhibiting PARP-1 attenuates cellular apoptosis by activating Bcl-2 and energy-saving processes and reducing ROS. The present study determined that PARP-1 was involved in HQ-induced apoptosis by PARylation of ZO-2. PMID:28983606

Automodification of PARP-1 mediates its tight binding to the nuclear matrix

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zaalishvili, Giorgi, E-mail: giozaal@gmail.com; Margiani, Dina; Kutalia, Ketevan

2010-02-26

Poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that catalyzes the NAD{sup +}-dependent addition of ADP-ribose polymers on a variety of nuclear proteins, has been shown to be associated with the nuclear matrix. As yet, the properties and conditions of this association are unclear. Here, we show the existence of two PARP-1 pools associated with the nuclear matrix of rat liver and the ability of PARP-1 automodification to facilitate its binding to the nuclear matrix.

Beneficial effects of n-acetylcysteine on ischaemic brain injury

PubMed Central

Cuzzocrea, Salvatore; Mazzon, Emanuela; Costantino, Giuseppina; Serraino, Ivana; Dugo, Laura; Calabrò, Giusy; Cucinotta, Giovanni; De Sarro, Angela; Caputi, A P

2000-01-01

Nitric oxide (NO), peroxynitrite, formed from NO and superoxide anion, poly (ADP-ribole) synthetase have been implicated as mediators of neuronal damage following focal ischaemia. Here we have investigated the effects of n-acetylcysteine (NAC) treatment in Mongolian gerbils subjected to cerebral ischaemia.Treatment of gerbils with NAC (20 mg kg−1 30 min before reperfusion and 1, 2 and 6 h after reperfusion) reduced the formation of post-ischaemic brain oedema, evaluated by water content.NAC also attenuated the increase in the brain levels of malondialdehyde (MDA) and the increase in the hippocampus of myeloperoxidase (MPO) caused by cerebral ischaemia.Positive staining for nitrotyrosine was found in the hippocampus in Mongolian gerbils subjected to cerebral ischaemia. Hippocampus tissue sections from Mongolian gerbils subjected to cerebral ischaemia also showed positive staining for poly (ADP-ribose) synthetase (PARS). The degree of staining for nitrotyrosine and for PARS were markedly reduced in tissue sections obtained from animals that received NAC.NAC treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischaemia and reperfusion.Histological observations of the pyramidal layer of CA1 showed a reduction of neuronal loss in animals that received NAC.These results show that NAC improves brain injury induced by transient cerebral ischaemia. PMID:10903958

Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca2+ signaling in ventricular myocytes.

PubMed

Park, Seon-Ah; Hong, Bing-Zhe; Ha, Ki-Chan; Kim, Uh-Hyun; Han, Myung-Kwan; Kwak, Yong-Geun

2017-06-02

Cyclic ADP-ribose (cADPR) releases Ca 2+ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca 2+ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca 2+ ([Ca 2+ ] i ) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca 2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca 2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

CD38-dependent ADP-ribosyl cyclase activity in developing and adult mouse brain.

PubMed Central

Ceni, Claire; Pochon, Nathalie; Brun, Virginie; Muller-Steffner, Hélène; Andrieux, Annie; Grunwald, Didier; Schuber, Francis; De Waard, Michel; Lund, Frances; Villaz, Michel; Moutin, Marie-Jo

2003-01-01

CD38 is a transmembrane glycoprotein that is expressed in many tissues throughout the body. In addition to its major NAD+-glycohydrolase activity, CD38 is also able to synthesize cyclic ADP-ribose, an endogenous calcium-regulating molecule, from NAD+. In the present study, we have compared ADP-ribosyl cyclase and NAD+-glycohydrolase activities in protein extracts of brains from developing and adult wild-type and Cd38 -/- mice. In extracts from wild-type brain, cyclase activity was detected spectrofluorimetrically, using nicotinamide-guanine dinucleotide as a substrate (GDP-ribosyl cyclase activity), as early as embryonic day 15. The level of cyclase activity was similar in the neonate brain (postnatal day 1) and then increased greatly in the adult brain. Using [14C]NAD+ as a substrate and HPLC analysis, we found that ADP-ribose is the major product formed in the brain at all developmental stages. Under the same experimental conditions, neither NAD+-glycohydrolase nor GDP-ribosyl cyclase activity could be detected in extracts of brains from developing or adult Cd38 -/- mice, demonstrating that CD38 is the predominant constitutive enzyme endowed with these activities in brain at all developmental stages. The activity measurements correlated with the level of CD38 transcripts present in the brains of developing and adult wild-type mice. Using confocal microscopy we showed, in primary cultures of hippocampal cells, that CD38 is expressed by both neurons and glial cells, and is enriched in neuronal perikarya. Intracellular NAD+-glycohydrolase activity was measured in hippocampal cell cultures, and CD38-dependent cyclase activity was higher in brain fractions enriched in intracellular membranes. Taken together, these results lead us to speculate that CD38 might have an intracellular location in neural cells in addition to its plasma membrane location, and may play an important role in intracellular cyclic ADP-ribose-mediated calcium signalling in brain tissue. PMID

ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion

PubMed Central

Fouquerel, Elise; Goellner, Eva M.; Yu, Zhongxun; Gagné, Jean-Philippe; de Moura, Michelle Barbi; Feinstein, Tim; Wheeler, David; Redpath, Philip; Li, Jianfeng; Romero, Guillermo; Migaud, Marie; Van Houten, Bennett; Poirier, Guy G.; Sobol, Robert W.

2014-01-01

Summary ARTD1 (PARP1) is a key enzyme involved in DNA repair by synthesizing poly(ADP-ribose) (PAR) in response to strand breaks and 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 directly 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 new insight on the importance of nucleus to mitochondria communication via ARTD1 activation. PMID:25220464

PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage

PubMed Central

Rulten, Stuart L.; Rotheray, Amy; Green, Ryan L.; Grundy, Gabrielle J.; Moore, Duncan A. Q.; Gómez-Herreros, Fernando; Hafezparast, Majid; Caldecott, Keith W

2014-01-01

Amyotrophic lateral sclerosis (ALS) is associated with progressive degeneration of motor neurons. Several of the genes associated with this disease encode proteins involved in RNA processing, including fused-in-sarcoma/translocated-in-sarcoma (FUS/TLS). FUS is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins that bind thousands of pre-mRNAs and can regulate their splicing. Here, we have examined the possibility that FUS is also a component of the cellular response to DNA damage. We show that both GFP-tagged and endogenous FUS re-localize to sites of oxidative DNA damage induced by UVA laser, and that FUS recruitment is greatly reduced or ablated by an inhibitor of poly (ADP-ribose) polymerase activity. Consistent with this, we show that recombinant FUS binds directly to poly (ADP-ribose) in vitro, and that both GFP-tagged and endogenous FUS fail to accumulate at sites of UVA laser induced damage in cells lacking poly (ADP-ribose) polymerase-1. Finally, we show that GFP-FUSR521G, harbouring a mutation that is associated with ALS, exhibits reduced ability to accumulate at sites of UVA laser-induced DNA damage. Together, these data suggest that FUS is a component of the cellular response to DNA damage, and that defects in this response may contribute to ALS. PMID:24049082

Different Principles of ADP-Ribose-Mediated Activation and Opposite Roles of the NUDT9 Homology Domain in the TRPM2 Orthologs of Man and Sea Anemone

PubMed Central

Kühn, Frank; Kühn, Cornelia; Lückhoff, Andreas

2017-01-01

A decisive element in the human cation channel TRPM2 is a region in its cytosolic C-terminus named NUDT9H because of its homology to the NUDT9 enzyme, a pyrophosphatase degrading ADP-ribose (ADPR). In hTRPM2, however, the NUDT9H domain has lost its enzymatic activity but serves as a binding domain for ADPR. As consequence of binding, gating of the channel is initiated. Since ADPR is produced after oxidative DNA damage, hTRPM2 mediates Ca2+ influx in response to oxidative stress which may lead to cell death. In the genome of the sea anemone Nematostella vectensis (nv), a preferred model organism for the evolution of key bilaterian features, a TRPM2 ortholog has been identified that contains a NUDT9H domain as well. Heterologous expression of nvTRPM2 in HEK-293 cells reveals a cation channel with many close similarities to the human counterpart. Most notably, nvTRPM2 is activated by ADPR, and Ca2+ is a co-agonist. However, the intramolecular mechanisms of ADPR gating as well as the role of NUDT9H are strikingly different in the two species. Whereas already subtle changes of NUDT9H abolish ADPR gating in hTRPM2, the region can be completely removed from nvTRPM2 without loss of responses to ADPR. An alternative ADPR binding site seems to be present but has not yet been characterized. The ADP-ribose pyrophosphatase (ADPRase) function of nvNUDT9H has been preserved but can be abolished by numerous genetic manipulations. All these manipulations create channels that are sensitive to hydrogen peroxide which fails to induce channel activity in wild-type nvTRPM2. Therefore, the function of NUDT9H in nvTRPM2 is the degradation of ADPR, thereby reducing agonist concentration in the presence of oxidative stress. Thus, the two TRPM2 orthologs have evolved divergently but nevertheless gained analogous functional properties, i.e., gating by ADPR with Ca2+ as co-factor. Opposite roles are played by the respective NUDT9H domains, either binding of ADPR and mediating channel activity

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

Key role of an ADP - ribose - dependent transcriptional regulator of NAD metabolism for fitness and virulence of Pseudomonas aeruginosa.

PubMed

Okon, Elza; Dethlefsen, Sarah; Pelnikevich, Anna; Barneveld, Andrea van; Munder, Antje; Tümmler, Burkhard

2017-01-01

NAD is an essential co-factor of redox reactions and metabolic conversions of NAD-dependent enzymes. NAD biosynthesis in the opportunistic pathogen Pseudomonas aeruginosa has yet not been experimentally explored. The in silico search for orthologs in the P. aeruginosa PAO1 genome identified the operon pncA - pncB1-nadE (PA4918-PA4920) to encode the nicotinamidase, nicotinate phosporibosyltransferase and Nad synthase of salvage pathway I. The functional role of the preceding genes PA4917 and PA4916 was resolved by the characterization of recombinant protein. PA4917 turned out to encode the nicotinate mononucleotide adenylyltransferase NadD2 and PA4916 was determined to encode the transcriptional repressor NrtR that binds to an intergenic sequence between nadD2 and pncA. Complex formation between the catalytically inactive Nudix protein NrtR and its DNA binding site was suppressed by the antirepressor ADP-ribose. NrtR plasposon mutagenesis abrogated virulence of P. aeruginosa TBCF10839 in a murine acute airway infection model and constrained its metabolite profile. When grown together with other isogenic plasposon mutants, the nrtR knock-out was most compromised in competitive fitness to persist in nutrient-rich medium in vitro or murine airways in vivo. This example demonstrates how tightly metabolism and virulence can be intertwined by key elements of metabolic control. Copyright © 2016 Elsevier GmbH. All rights reserved.

Rucaparib

MedlinePlus

... Rucaparib is in a class of medications called poly (ADP-ribose) polymerase (PARP) inhibitors. It works by ... not go away: nausea vomiting constipation diarrhea stomach pain loss of appetite bad taste in the mouth ...

Niraparib

MedlinePlus

... Niraparib is in a class of medications called poly (ADP-ribose) polymerase (PARP) inhibitors. It works by ... sores in the mouth loss of appetite back pain headache dizziness changes in taste difficulty falling asleep ...

Autophagy requires poly(adp-ribosyl)ation-dependent AMPK nuclear export

PubMed Central

Rodríguez-Vargas, José M; Rodríguez, María I; Majuelos-Melguizo, Jara; García-Diaz, Ángel; González-Flores, Ariannys; López-Rivas, Abelardo; Virág, László; Illuzzi, Giuditta; Schreiber, Valerie; Dantzer, Françoise; Oliver, F Javier

2016-01-01

AMPK is a central energy sensor linking extracellular milieu fluctuations with the autophagic machinery. In the current study we uncover that Poly(ADP-ribosyl)ation (PARylation), a post-translational modification (PTM) of proteins, accounts for the spatial and temporal regulation of autophagy by modulating AMPK subcellular localisation and activation. More particularly, we show that the minority AMPK pool needs to be exported to the cytosol in a PARylation-dependent manner for optimal induction of autophagy, including ULK1 phosphorylation and mTORC1 inactivation. PARP-1 forms a molecular complex with AMPK in the nucleus in non-starved cells. In response to nutrient deprivation, PARP-1 catalysed PARylation, induced the dissociation of the PARP-1/AMPK complex and the export of free PARylated nuclear AMPK to the cytoplasm to activate autophagy. PARP inhibition, its silencing or the expression of PARylation-deficient AMPK mutants prevented not only the AMPK nuclear-cytosolic export but also affected the activation of the cytosolic AMPK pool and autophagosome formation. These results demonstrate that PARylation of AMPK is a key early signal to efficiently convey extracellular nutrient perturbations with downstream events needed for the cell to optimize autophagic commitment before autophagosome formation. PMID:27689873

NIH study uncovers new mechanism of action for class of chemotherapy drugs

Cancer.gov

NIH researchers have discovered a significant new mechanism of action for a class of chemotherapy drugs known as poly (ADP-ribose) polymerase inhibitors, or PARP inhibitors. They have also identified differences in the toxic capabilities of three drugs in

In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes*

PubMed Central

Clifton, Matthew C.; Simon, Michael J.; Erramilli, Satchal K.; Zhang, Huide; Zaitseva, Jelena; Hermodson, Mark A.; Stauffacher, Cynthia V.

2015-01-01

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg2+. We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg2+, and VO4); a RbsAC complex in the presence of ADP and Mg2+; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters. PMID:25533465

Effects of Sulfur Mustard on Intracellular Calcium and Synthesis of Basement Membrane Zone Proteins in Human Skin

DTIC Science & Technology

1993-08-04

development of blisters. They postulated that DNA single strand breaks (SSB) due to spontaneous or enzymatically induced depurination of alkylated DNA ... bases activate the chromosomal enzyme poly(ADP-ribose)transferase (PADPRT). The latter would deplete cellular NAD÷ pools. In literature, this

Neer Award 2016: reduced muscle degeneration and decreased fatty infiltration after rotator cuff tear in a poly(ADP-ribose) polymerase 1 (PARP-1) knock-out mouse model.

PubMed

Kuenzler, Michael B; Nuss, Katja; Karol, Agnieszka; Schär, Michael O; Hottiger, Michael; Raniga, Sumit; Kenkel, David; von Rechenberg, Brigitte; Zumstein, Matthias A

2017-05-01

Disturbed muscular architecture, atrophy, and fatty infiltration remain irreversible in chronic rotator cuff tears even after repair. Poly (adenosine 5'-diphosphate-ribose) polymerase 1 (PARP-1) is a key regulator of inflammation, apoptosis, muscle atrophy, muscle regeneration, and adipocyte development. We hypothesized that the absence of PARP-1 would lead to a reduction in damage to the muscle subsequent to combined tenotomy and neurectomy in a PARP-1 knockout (KO) mouse model. PARP-1 KO and wild-type C57BL/6 (WT group) mice were analyzed at 1, 6, and 12 weeks (total n = 84). In all mice, the supraspinatus and infraspinatus muscles of the left shoulder were detached and denervated. Macroscopic analysis, magnetic resonance imaging, gene expression analysis, immunohistochemistry, and histology were used to assess the differences in PARP-1 KO and WT mice. The muscles in the PARP-1 KO group had significantly less retraction, atrophy, and fatty infiltration after 12 weeks than in the WT group. Gene expression of inflammatory, apoptotic, adipogenic, and muscular atrophy genes was significantly decreased in PARP-1 KO mice in the first 6 weeks. Absence of PARP-1 leads to a reduction in muscular architectural damage, early inflammation, apoptosis, atrophy, and fatty infiltration after combined tenotomy and neurectomy of the rotator cuff muscle. Although the macroscopic reaction to injury is similar in the first 6 weeks, the ability of the muscles to regenerate was much greater in the PARP-1 KO group, leading to a near-normalization of the muscle after 12 weeks. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

Quantification of diphtheria toxin mediated ADP-ribosylation in a solid-phase assay.

PubMed

Bachran, Christopher; Sutherland, Mark; Bachran, Diana; Fuchs, Hendrik

2007-09-01

Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADP-ribosylation by DT. Solid phase-bound his-tag eEF2 is ADP-ribosylated by toxins using biotinylated NAD(+) as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti-his-tag antibody using an LAS-1000 System. The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase. The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT.

Identifying Determinants of PARP Inhibitor Sensitivity in Ovarian Cancer

DTIC Science & Technology

2015-10-01

such as those lacking functional BRCA1 are highly sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Ovarian cancer patients that harbored...Principal Investigator (Last, first, middle): Johnson, Neil  Dr. Johnson’s mentor, Dr. Jeffrey Boyd, left Fox Chase for Florida International

Comparison of the acid-base properties of ribose and 2'-deoxyribose nucleotides.

PubMed

Mucha, Ariel; Knobloch, Bernd; Jezowska-Bojczuk, Małgorzata; Kozłowski, Henryk; Sigel, Roland K O

2008-01-01

The extent to which the replacement of a ribose unit by a 2'-deoxyribose unit influences the acid-base properties of nucleotides has not hitherto been determined in detail. In this study, by potentiometric pH titrations in aqueous solution, we have measured the acidity constants of the 5'-di- and 5'-triphosphates of 2'-deoxyguanosine [i.e., of H(2)(dGDP)(-) and H(2)(dGTP)(2-)] as well as of the 5'-mono-, 5'-di-, and 5'-triphosphates of 2'-deoxyadenosine [i.e., of H(2)(dAMP)(+/-), H(2)(dADP)(-), and H(2)(dATP)(2-)]. These 12 acidity constants (of the 56 that are listed) are compared with those of the corresponding ribose derivatives (published data) measured under the same experimental conditions. The results show that all protonation sites in the 2'-deoxynucleotides are more basic than those in their ribose counterparts. The influence of the 2'-OH group is dependent on the number of 5'-phosphate groups as well as on the nature of the purine nucleobase. The basicity of N7 in guanine nucleotides is most significantly enhanced (by about 0.2 pK units), while the effect on the phosphate groups and the N1H or N1H(+) sites is less pronounced but clearly present. In addition, (1)H NMR chemical shift change studies in dependence on pD in D(2)O have been carried out for the dAMP, dADP, and dATP systems, which confirmed the results from the potentiometric pH titrations and showed the nucleotides to be in their anti conformations. Overall, our results are not only of relevance for metal ion binding to nucleotides or nucleic acids, but also constitute an exact basis for the calculation, determination, and understanding of perturbed pK(a) values in DNAzymes and ribozymes, as needed for the delineation of acid-base mechanisms in catalysis.

Combination strategy of PARP inhibitor with antioxidant prevent bioenergetic deficits and inflammatory changes in CCI-induced neuropathy.

PubMed

Komirishetty, Prashanth; Areti, Aparna; Gogoi, Ranadeep; Sistla, Ramakrishna; Kumar, Ashutosh

2017-02-01

Neuropathic pain, a debilitating pain condition and the underlying pathogenic mechanisms are complex and interwoven amongst each other and still there is scant information available regarding therapies which promise to treat the condition. Evidence indicate that oxidative/nitrosative stress induced poly (ADP-ribose) polymerase (PARP) overactivation initiate neuroinflammation and bioenergetic crisis culminating into neurodegenerative changes following nerve injury. Hence, we investigated the therapeutic effect of combining an antioxidant, quercetin and a PARP inhibitor, 4-amino 1, 8-naphthalimide (4-ANI) on the hallmark deficits induced by chronic constriction injury (CCI) of sciatic nerve in rats. Quercetin (25 mg/kg, p.o.) and 4-ANI (3 mg/kg, p.o.) were administered either alone or in combination for 14 days to examine sciatic functional index, allodynia and hyperalgesia using walking track analysis, Von Frey, acetone spray and hot plate tests respectively. Malondialdehyde, nitrite and glutathione levels were estimated to detect oxidative/nitrosative stress; mitochondrial membrane potential and cytochrome c oxidase activity to assess mitochondrial function; NAD & ATP levels to examine the bioenergetic status and levels of inflammatory markers were evaluated in ipsilateral sciatic nerve. Quercetin and 4-ANI alone improved the pain behaviour and biochemical alterations but the combination therapy demonstrated an appreciable reversal of CCI-induced changes. Nitrotyrosine and Poly ADP-Ribose (PAR) immunopositivity was decreased and nuclear factor erythroid 2-related factor (Nrf-2) levels were increased significantly in micro-sections of the sciatic nerve and dorsal root ganglion (DRG) of treatment group. These results suggest that simultaneous inhibition of oxidative stress-PARP activation cascade may potentially be useful strategies for management of trauma induced neuropathic pain. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular mechanism of the short-term cardiotoxicity caused by 2',3'-dideoxycytidine (ddC): modulation of reactive oxygen species levels and ADP-ribosylation reactions.

PubMed

Skuta, G; Fischer, G M; Janaky, T; Kele, Z; Szabo, P; Tozser, J; Sumegi, B

1999-12-15

The short-term cardiac side effects of 2',3'-dideoxycytidine (ddC, zalcitabine) were studied in rats in order to understand the biochemical events contributing to the development of ddC-induced cardiomyopathy. In developing animals, ddC treatment provoked a surprisingly rapid appearance of cardiac malfunctions characterized by prolonged RR, PR, and QT intervals and J point depression. The energy metabolism in the heart was compromised, characterized by a decreased creatine phosphate/creatine ratio (from 2.05 normal value to 0.75) and a decreased free ATP/ADP ratio (from 332 normal value to 121). The activity of respiratory complexes (NADH: cytochrome c oxidoreductase and cytochrome oxidase) also decreased significantly. Southern blot and polymerase chain reaction analysis did not show deletions or a decrease in the quantity of mitochondrial DNA (mtDNA) deriving from ddC-treated rat hearts, indicating that under our experimental conditions, ddC-induced heart abnormalities were not the direct consequence of mtDNA-related damage. The ddC treatment of rats significantly increased the formation of reactive oxygen species (ROS) in heart and skeletal muscle as determined by the oxidation of non-fluorescent dihydrorhodamine123 to fluorescent rhodamine123 and the oxidation of cellular proteins determined from protein carbonyl content. An activation of the nuclear poly-(ADP-ribose) polymerase (EC 2.4.2.30) and an increase in the mono-ADP-ribosylation of glucose-regulated protein and desmin were observed in the cardiac tissue from ddC-treated animals. A decrease in the quantity of heat shock protein (HSP)70s was also detected, while the level of HSP25 and HSP60 remained unchanged. Surprisingly, ddC treatment induced a skeletal muscle-specific decrease in the quantity of three proteins, one of which was identified by N-terminal sequencing as myoglobin, and another by tandem mass spectrometer sequencing as triosephosphate isomerase (EC 5.3.1.1). These data show that the short

Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

PubMed Central

Rank, Lisa; Veith, Sebastian; Gwosch, Eva C.; Demgenski, Janine; Ganz, Magdalena; Jongmans, Marjolijn C.; Vogel, Christopher; Fischbach, Arthur; Buerger, Stefanie; Fischer, Jan M.F.; Zubel, Tabea; Stier, Anna; Renner, Christina; Schmalz, Michael; Beneke, Sascha; Groettrup, Marcus; Kuiper, Roland P.; Bürkle, Alexander; Ferrando-May, Elisa; Mangerich, Aswin

2016-01-01

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure–function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1–PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\\L713F expression triggered apoptosis, whereas PARP1\\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure–function relationships of natural and artificial PARP1 variants. PMID:27694308

The apoptotic effects of silibinin on MDA-MB-231 and MCF-7 human breast carcinoma cells.

PubMed

Bayram, D; Çetin, E S; Kara, M; Özgöçmen, M; Candan, I A

2017-06-01

Silibinin is a bioactive flavonolignan extracted from milk thistle, known as Silybum marianum. Silibinin exerts strong antiproliferative, proapoptotic, and anti-inflammatory effects. Many studies have shown that silibinin inhibits experimentally induced malignancies of the liver, prostate, skin, and colon as well as promotes inhibition of the proliferation of cancer cell lines in vitro. This study aimed to investigate the effects of silibinin on the human breast carcinoma cell lines MDA-MB-231 and MCF-7 in monolayer and spheroid cultures. The MDA-MB-231 and MCF-7 cell lines were cultured in both monolayer and spheroid cultures. Cells were treated with silibinin at 24, 48, and 72 h of incubation. The 5-bromo-2'-deoxyuridine labeling index was used to determine the cells of the synthesis phase. Poly-ADP-ribose-polimerase immunohistochemical staining and the terminal deoxynucleotidyl transferase dUTP nick and labeling assay were used to determine the death of cells in both the monolayer and spheroid cultures. An half maximal inhibitory concentration dose of silibinin in MDA-MB-231 and MCF-7 cells was 100 µM/mL at 24, 48, and 72 h of incubation. Terminal deoxynucleotidyl transferase dUTP nick and labeling positive cells and active poly-ADP-ribose-polimerase were detected after treatment with silibinin in both the monolayer and spheroid cultures. The dead cell count was higher in the MDA-MB-231 and MCF-7 cell lines with silibinin applied than in the controls. Our study demonstrated that silibinin applications enhanced terminal deoxynucleotidyl transferase dUTP nick and labeling positive cells and active poly-ADP-ribose-polimerase in comparison to the control in both the monolayer and spheroid cultures.

Cloning of the poly(ADP-ribose) Gene from Rat Liver.

DTIC Science & Technology

1986-09-24

Levinson, Ph.D. (Cetus Corp., Berkeley). 5. Amino acid analysis done in UCSF Bioanal. Lab. TABLE OF CONTENTS Page METHOD I...TABLE I ............. ............................... ... 12 Proteolytic degradation, isolation of peptide and amino acid sequences...technique developed for enzyme quantitation in biological materials. The amino- acid sequence of the enzyme has so far been determined because the amino

Regulatory Control of Breast Tumor Cell Poly (ADP-Ribose) Polymerase

DTIC Science & Technology

2004-08-01

polyethylene glycol precipitation, ion exchange chromatography, and density gradient sedimentation (Malkas et al., 1990; Applegren et al., 1995; Coll et...jtl of 25 mM NH4HCO 3/50% acetonitrile were added and the tubes were mixed for 35-40 min on a low setting using a microtube mixer. The pale blue...these isoforms. Proteins identified in these spots are shown in table 1: Spot #* Predominant Protein MCF-10A 1 Heat Shock Protein 90 cc (hsp-90 ct) 2

Novel Mechanisms of PARP Inhibitor Resistance in BRCA1-Deficient Breast Cancers

DTIC Science & Technology

2014-12-01

Zou L Molecular Cell. 2014 Jan   23;53(2):235-­‐46.  PMID: 24332808     Inventions, Patents, and Licenses: Nothing to report Reportable... Carmichael , J. (2010). Oral poly(ADP-ribose) polymerase inhibitor olaparib in   11   patients with BRCA1 or BRCA2 mutations and advanced breast cancer

Metabolism Dealing with Thermal Degradation of NAD+ in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

PubMed

Hachisuka, Shin-Ichi; Sato, Takaaki; Atomi, Haruyuki

2017-10-01

NAD + is an important cofactor for enzymatic oxidation reactions in all living organisms, including (hyper)thermophiles. However, NAD + is susceptible to thermal degradation at high temperatures. It can thus be expected that (hyper)thermophiles harbor mechanisms that maintain in vivo NAD + concentrations and possibly remove and/or reuse undesirable degradation products of NAD + Here we confirmed that at 85°C, thermal degradation of NAD + results mostly in the generation of nicotinamide and ADP-ribose, the latter known to display toxicity by spontaneously linking to proteins. The hyperthermophilic archaeon Thermococcus kodakarensis possesses a putative ADP-ribose pyrophosphatase (ADPR-PPase) encoded by the TK2284 gene. ADPR-PPase hydrolyzes ADP-ribose to ribose 5-phosphate (R5P) and AMP. The purified recombinant TK2284 protein exhibited activity toward ADP-ribose as well as ADP-glucose. Kinetic analyses revealed a much higher catalytic efficiency toward ADP-ribose, suggesting that ADP-ribose was the physiological substrate. To gain insight into the physiological function of TK2284, a TK2284 gene disruption strain was constructed and examined. Incubation of NAD + in the cell extract of the mutant strain at 85°C resulted in higher ADP-ribose accumulation and lower AMP production compared with those in experiments with the host strain cell extract. The mutant strain also exhibited lower cell yield and specific growth rates in a synthetic amino acid medium compared with those of the host strain. The results obtained here suggest that the ADPR-PPase in T. kodakarensis is responsible for the cleavage of ADP-ribose to R5P and AMP, providing a means to utilize the otherwise dead-end product of NAD + breakdown. IMPORTANCE Hyperthermophilic microorganisms living under high temperature conditions should have mechanisms that deal with the degradation of thermolabile molecules. NAD + is an important cofactor for enzymatic oxidation reactions and is susceptible to thermal

Transcriptional Control by PARP-1: Chromatin Modulation, Enhancer-binding, Coregulation, and Insulation

PubMed Central

Kraus, W. Lee

2008-01-01

Summary The regulation of gene expression requires a wide array of protein factors that can modulate chromatin structure, act at enhancers, function as transcriptional coregulators, or regulate insulator function. Poly(ADP-ribose) polymerase-1 (PARP-1), an abundant and ubiquitous nuclear enzyme that catalyzes the NAD+-dependent addition of ADP-ribose polymers on a variety of nuclear proteins, has been implicated in all of these functions. Recent biochemical, genomic, proteomic, and cell-based studies have highlighted the role of PARP-1 in each of these processes and provided new insights about the molecular mechanisms governing PARP-1-dependent regulation of gene expression. In addition, these studies have demonstrated how PARP-1 functions as an integral part of cellular signaling pathways that culminate in gene regulatory outcomes. PMID:18450439

Green tea polyphenol extract attenuates lung injury in experimental model of carrageenan-induced pleurisy in mice

PubMed Central

Di Paola, Rosanna; Mazzon, Emanuela; Muià, Carmelo; Genovese, Tiziana; Menegazzi, Marta; Zaffini, Raffaela; Suzuki, Hisanory; Cuzzocrea, Salvatore

2005-01-01

Here we investigate the effects of the green tea extract in an animal model of acute inflammation, carrageenan-induced pleurisy. We report here that green tea extract (given at 25 mg/kg i.p. bolus 1 h prior to carrageenan), exerts potent anti-inflammatory effects in an animal model of acute inflammation in vivo. Injection of carrageenan (2%) into the pleural cavity of mice elicited an acute inflammatory response characterized by fluid accumulation in the pleural cavity that contained many neutrophils (PMNs), an infiltration of PMNs in lung tissues and increased production of nitrite/nitrate, tumour necrosis factor alpha. All parameters of inflammation were attenuated by green tea extract treatment. Furthermore, carrageenan induced an up-regulation of the adhesion molecule ICAM-1, as well as nitrotyrosine and poly (ADP-ribose) synthetase (PARS) formation, as determined by immunohistochemical analysis of lung tissues. Staining for the ICAM-1, nitrotyrosine, and PARS was reduced by green tea extract. Our results clearly demonstrate that treatment with green tea extract exerts a protective effect and offers a novel therapeutic approach for the management of lung injury. PMID:15987519

DDB2 promotes chromatin decondensation at UV-induced DNA damage

PubMed Central

Lindh, Michael; Acs, Klara; Vrouwe, Mischa G.; Pines, Alex; van Attikum, Haico; Mullenders, Leon H.

2012-01-01

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A–RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)–dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]–ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains. PMID:22492724

Zinc release contributes to hypoglycemia-induced neuronal death.

PubMed

Suh, Sang Won; Garnier, Philippe; Aoyama, Koji; Chen, Yongmei; Swanson, Raymond A

2004-08-01

Neurons exposed to zinc exhibit activation of poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme that normally participates in DNA repair but promotes cell death when extensively activated. Endogenous, vesicular zinc in brain is released to the extracellular space under conditions causing neuronal depolarization. Here, we used a rat model of insulin-induced hypoglycemia to assess the role of zinc release in PARP-1 activation and neuronal death after severe hypoglycemia. Zinc staining with N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) showed depletion of presynaptic vesicular zinc from hippocampal mossy fiber terminals and accumulation of weakly bound zinc in hippocampal CA1 cell bodies after severe hypoglycemia. Intracerebroventricular injection of the zinc chelator calcium ethylene-diamine tetraacetic acid (CaEDTA) blocked the zinc accumulation and significantly reduced hypoglycemia-induced neuronal death. CaEDTA also attenuated the accumulation of poly(ADP-ribose), the enzymatic product of PARP-1, in hippocampal neurons. These results suggest that zinc translocation is an intermediary step linking hypoglycemia to PARP-1 activation and neuronal death.

Analyzing structure-function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells.

PubMed

Rank, Lisa; Veith, Sebastian; Gwosch, Eva C; Demgenski, Janine; Ganz, Magdalena; Jongmans, Marjolijn C; Vogel, Christopher; Fischbach, Arthur; Buerger, Stefanie; Fischer, Jan M F; Zubel, Tabea; Stier, Anna; Renner, Christina; Schmalz, Michael; Beneke, Sascha; Groettrup, Marcus; Kuiper, Roland P; Bürkle, Alexander; Ferrando-May, Elisa; Mangerich, Aswin

2016-12-01

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure-function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1-PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\\L713F expression triggered apoptosis, whereas PARP1\\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure-function relationships of natural and artificial PARP1 variants. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Induction of apoptosis in human promyelocytic leukemia HL-60 cells by Ampelopsis cantoniensis crude extract.

PubMed

Tan, Tzu-Wei; Tsai, Huei-Yann; Chen, Yuh-Fung; Chung, Jing-Gung

2004-01-01

The crude extract of Ampelopsis cantoniensis induced apoptosis in human promyelocytic leukemia HL-60 cells and this induction was investigated by flow cytometric analysis, DNA gel electrophoresis and poly (ADP-ribose) fluorescence staining. The results demonstrated that this extract induced dose-dependent cytotoxicity and apoptosis. The level of active caspase-3 was increased after treatment with the crude extract for 24 hours.

DNA-independent PARP-1 activation by phosphorylated ERK2 increases Elk1 activity: a link to histone acetylation.

PubMed

Cohen-Armon, Malka; Visochek, Leonid; Rozensal, Dana; Kalal, Adi; Geistrikh, Ilona; Klein, Rodika; Bendetz-Nezer, Sarit; Yao, Zhong; Seger, Rony

2007-01-26

PolyADP-ribose polymerases (PARPs) catalyze a posttranslational modification of nuclear proteins by polyADP-ribosylation. The catalytic activity of the abundant nuclear protein PARP-1 is stimulated by DNA strand breaks, and PARP-1 activation is required for initiation of DNA repair. Here we show that PARP-1 also acts within extracellular signal-regulated kinase (ERK) signaling cascade that mediates growth and differentiation. The findings reveal an alternative mode of PARP-1 activation, which does not involve binding to DNA or DNA damage. In a cell-free system, recombinant PARP-1 was intensively activated and thereby polyADP-ribosylated by a direct interaction with phosphorylated ERK2, and the activated PARP-1 dramatically increased ERK2-catalyzed phosphorylation of the transcription factor Elk1. In cortical neurons treated with nerve growth factors and in stimulated cardiomyocytes, PARP-1 activation enhanced ERK-induced Elk1-phosphorylation, core histone acetylation, and transcription of the Elk1-target gene c-fos. These findings constitute evidence for PARP-1 activity within the ERK signal-transduction pathway.

Structural basis of detection and signaling of DNA single-strand breaks by human PARP-1

DOE PAGES

Eustermann, Sebastian; Wu, Wing -Fung; Langelier, Marie -France; ...

2015-11-25

Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformabilitymore » of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Finally, our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.« less

PARP1 expression, activity and ex vivo sensitivity to the PARP inhibitor, talazoparib (BMN 673), in chronic lymphocytic leukaemia

PubMed Central

Herriott, Ashleigh; Tudhope, Susan J.; Junge, Gesa; Rodrigues, Natalie; Patterson, Miranda J.; Woodhouse, Laura; Lunec, John; Hunter, Jill E.; Mulligan, Evan A.; Cole, Michael; Allinson, Lisa M.; Wallis, Jonathan P.; Marshall, Scott; Wang, Evelyn; Curtin, Nicola J.; Willmore, Elaine

2015-01-01

In chronic lymphocytic leukemia (CLL), mutation and loss of p53 and ATM abrogate DNA damage signalling and predict poorer response and shorter survival. We hypothesised that poly (ADP-ribose) polymerase (PARP) activity, which is crucial for repair of DNA breaks induced by oxidative stress or chemotherapy, may be an additional predictive biomarker and a target for therapy with PARP inhibitors. We measured PARP activity in 109 patient-derived CLL samples, which varied widely (192 – 190052 pmol PAR/106 cells) compared to that seen in healthy volunteer lymphocytes (2451 – 7519 pmol PAR/106 cells). PARP activity was associated with PARP1 protein expression and endogenous PAR levels. PARP activity was not associated with p53 or ATM loss, Binet stage, IGHV mutational status or survival, but correlated with Bcl-2 and Rel A (an NF-kB subunit). Levels of 8-hydroxy-2′-deoxyguanosine in DNA (a marker of oxidative damage) were not associated with PAR levels or PARP activity. The potent PARP inhibitor, talazoparib (BMN 673), inhibited CD40L-stimulated proliferation of CLL cells at nM concentrations, independently of Binet stage or p53/ATM function. PARP activity is highly variable in CLL and correlates with stress-induced proteins. Proliferating CLL cells (including those with p53 or ATM loss) are highly sensitive to the PARP inhibitor talazoparib. PMID:26539646

PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression.

PubMed

Augustin, Angélique; Spenlehauer, Catherine; Dumond, Hélène; Ménissier-De Murcia, Josiane; Piel, Matthieu; Schmit, Anne-Catherine; Apiou, Françoise; Vonesch, Jean-Luc; Kock, Michael; Bornens, Michel; De Murcia, Gilbert

2003-04-15

A novel member of the poly(ADP-ribose) polymerase (PARP) family, hPARP-3, is identified here as a core component of the centrosome. hPARP-3 is preferentially localized to the daughter centriole throughout the cell cycle. The N-terminal domain (54 amino acids) of hPARP-3 is responsible for its centrosomal localization. Full-length hPAPR-3 (540 amino acids, with an apparent mass of 67 kDa) synthesizes ADP-ribose polymers during its automodification. Overexpression of hPARP-3 or its N-terminal domain does not influence centrosomal duplication or amplification but interferes with the G1/S cell cycle progression. PARP-1 also resides for part of the cell cycle in the centrosome and interacts with hPARP-3. The presence of both PARP-1 and PARP-3 at the centrosome may link the DNA damage surveillance network to the mitotic fidelity checkpoint.

BRCA2 Mutation as a Possible Cause of Poor Response to 177Lu-PSMA Therapy.

PubMed

Ahmadzadehfar, Hojjat; Gaertner, Florian; Lossin, Philipp S; Schwarz, Bettina; Essler, Markus

2018-05-14

We present the case of a 66-year-old man with castration-resistant prostate cancer, with an increasing prostate-specific antigen level, and a progressive disease during Lu-PSMA radionuclide therapy. Because the patient had a BRCA2 mutation, poly-ADP ribose polymerase inhibitor therapy was started. The patient showed a dramatic subjective and biological response to this therapy with a progression-free survival of 5 months.

PARP inhibition: PARP1 and beyond

PubMed Central

Rouleau, Michèle; Patel, Anand; Hendzel, Michael J.; Kaufmann, Scott H.; Poirier, Guy G.

2010-01-01

Recent findings have thrust poly(ADP-ribose) polymerases (PARPs) into the limelight as potential chemotherapeutic targets. To provide a framework for understanding these recent observations, we review what is known about the structures and functions of the family of PARP enzymes, and then outline a series of questions that should be addressed to guide the rational development of PARP inhibitors as anticancer agents. PMID:20200537

Cancer Risks Associated With Inherited Mutations in Ovarian Cancer Susceptibility Genes Beyond BRCA1 and BRCA2

DTIC Science & Technology

2017-05-01

TERMS Ovarian cancer, drug resistance, rucaparib, phase 2, DNA repair, homologous recombination, nonhomologous end-joining (NHEJ), poly(ADP-ribose...tissues from AA patients with OC. This should add 50 AA OC patients. We are also requesting anonymized DNA from AA OC patients who participated on...extracts DNA and creates library pretps for DNA sequencing. He performs Sanger sequencing validations. Funding Support: Has there been a change

ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair.

PubMed

Tsuda, Masataka; Cho, Kosai; Ooka, Masato; Shimizu, Naoto; Watanabe, Reiko; Yasui, Akira; Nakazawa, Yuka; Ogi, Tomoo; Harada, Hiroshi; Agama, Keli; Nakamura, Jun; Asada, Ryuta; Fujiike, Haruna; Sakuma, Tetsushi; Yamamoto, Takashi; Murai, Junko; Hiraoka, Masahiro; Koike, Kaoru; Pommier, Yves; Takeda, Shunichi; Hirota, Kouji

2017-01-01

ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1's ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1's role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.

PubMed

Han, S; Arvai, A S; Clancy, S B; Tainer, J A

2001-01-05

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors

Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria.

PubMed

Nocek, Boguslaw; Kochinyan, Samvel; Proudfoot, Michael; Brown, Greg; Evdokimova, Elena; Osipiuk, Jerzy; Edwards, Aled M; Savchenko, Alexei; Joachimiak, Andrzej; Yakunin, Alexander F

2008-11-18

Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer alpha/beta/alpha sandwich fold with an alpha-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.

Understanding D-Ribose and Mitochondrial Function.

PubMed

Mahoney, Diane E; Hiebert, John B; Thimmesch, Amanda; Pierce, John T; Vacek, James L; Clancy, Richard L; Sauer, Andrew J; Pierce, Janet D

2018-01-01

Mitochondria are important organelles referred to as cellular powerhouses for their unique properties of cellular energy production. With many pathologic conditions and aging, mitochondrial function declines, and there is a reduction in the production of adenosine triphosphate. The energy carrying molecule generated by cellular respiration and by pentose phosphate pathway, an alternative pathway of glucose metabolism. D-ribose is a naturally occurring monosaccharide found in the cells and particularly in the mitochondria is essential in energy production. Without sufficient energy, cells cannot maintain integrity and function. Supplemental D-ribose has been shown to improve cellular processes when there is mitochondrial dysfunction. When individuals take supplemental D-ribose, it can bypass part of the pentose pathway to produce D-ribose-5-phosphate for the production of energy. In this article, we review how energy is produced by cellular respiration, the pentose pathway, and the use of supplemental D-ribose.

Oxaliplatin triggers necrosis as well as apoptosis in gastric cancer SGC-7901 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Ping; Zhu, Xueping; Jin, Wei

Intrinsic apoptotic pathway is considered to be responsible for cell death induced by platinum anticancer drugs. While in this study, we found that, necrosis is an indispensable pathway besides apoptosis in oxaliplatin-treated gastric cancer SGC-7901 cells. Upon exposure to oxaliplatin, both apoptotic and necrotic features were observed. The majority of dead cells were double positive for Annexin V and propidium iodide (PI). Moreover, mitochondrial membrane potential collapsed and caspase cascades were activated. However, ultrastructural changes under transmission electron microscope, coupled with the release of cellular contents, demonstrated the rupture of the plasma membrane. Oxaliplatin administration did not stimulate reactive oxygenmore » species (ROS) production and autophagy, but elevated the protein level of Bmf. In addition, receptor interacting protein 1 (RIP1), but not receptor interacting protein 3 (RIP3) and its downstream components participated in this death process. Necrostatin-1 (Nec-1) blocked oxaliplatin-induced cell death nearly completely, whereas z-VAD-fmk could partially suppress cell death. Oxaliplatin treatment resulted in poly(ADP-ribose) polymerase-1 (PARP-1) overactivation, as indicated by the increase of poly(ADP-ribose) (PAR), which led to NAD{sup +} and ATP depletion. PARP-1 inhibitor, olaparib, could significantly block oxaliplatin-induced cell death, thus confirming that PARP-1 activation is mainly responsible for the cytotoxicity of oxaliplatin. Phosphorylation of H2AX at Ser139 and translocalization of apoptosis-inducing factor (AIF) are critical for this death process. Taken together, these results indicate that oxaliplatin can bypass canonical cell death pathways to kill gastric cancer cells, which may be of therapeutic advantage in the treatment of gastric cancer. - Highlights: • Oxaliplatin induces apoptotic and necrotic cell death. • Nec-1 can inhibit oxaliplatin-induced cell death nearly completely. • RIP3 and its

Deciphering the Role of Alternative nonhomologous End Joining (Alt NHEJ) DNA Repair in Breast Cancer

DTIC Science & Technology

2016-10-01

carrying mutations in homology-directed repair genes. Here we report that PolQ inhibition can be used to increase the efficiency of CRISPR targeting...Telomeres. • Poly-ADP-ribose polymerase 1, PARP1. • Chromosomal translocation. • Chromosomal aberrancies. • Chromosomal fusions. • CRISPR ...showed that PolQ promotes A-NHEJ while suppresses HR and I have analyzed the impact of PolQ on CRISPR targeting when HR is required to

[Design, synthesis and biological evaluation of novel para-substituted 1-benzyl-quinazoline-2, 4 (1H, 3H)-diones as human PARP-1 inhibitors].

PubMed

Yao, Hai-Ping; Zhu, Zhi-Xiang; Ji, Ming; Chen, Xiao-Guang; Xu, Bai-Ling

2014-04-01

Poly(ADP-ribose) polymerase-1 (PARP-1) has emerged as a promising anticancer drug target due to its key role in the DNA repair process. It can polymerize ADP-ribose units on its substrate proteins which are involved in the regulation of DNA repair. In this work, a novel series of para-substituted 1-benzyl-quinazoline-2, 4 (1H, 3H)-diones was designed and synthesized, and the inhibitory activities against PARP-1 of compounds 7a-7e, 8a-8f, 9a-9c and 10a-10c were evaluated. Of all the tested compounds, nine compounds displayed inhibitory activities with IC50 values ranging from 4.6 to 39.2 micromol x L(-1). In order to predict the binding modes of the potent molecules, molecular docking was performed using CDOCKER algorithm, and that will facilitate to further develop more potent PARP-1 inhibitors with a quinazolinedione scaffold.

Behavior of adsorbed Poly-A onto sodium montmorillonite

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palomino-Aquino, Nayeli; Negrón-Mendoza, Alicia, E-mail: negron@nucleares.unam.mx

2015-07-23

The adsorption of Poly-A (a polynucleotide consisting of adenine, ribose and a phosphate group), onto a clay mineral, was studied to investigate the extent of adsorption, the site of binding, and the capacity of the clay to protect Poly-A, while it is adsorbed onto the clay, from external sources of energy. The results showed that Poly-A presented a high percentage of adsorption at the edges of the clay and that the survival of the polynucleotide was superior to irradiating the polymer in the absence of the clay.

Diabetic Neuropathy and Oxidative Stress: Therapeutic Perspectives

PubMed Central

Hosseini, Asieh; Abdollahi, Mohammad

2013-01-01

Diabetic neuropathy (DN) is a widespread disabling disorder comprising peripheral nerves' damage. DN develops on a background of hyperglycemia and an entangled metabolic imbalance, mainly oxidative stress. The majority of related pathways like polyol, advanced glycation end products, poly-ADP-ribose polymerase, hexosamine, and protein kinase c all originated from initial oxidative stress. To date, no absolute cure for DN has been defined; although some drugs are conventionally used, much more can be found if all pathophysiological links with oxidative stress would be taken into account. In this paper, although current therapies for DN have been reviewed, we have mainly focused on the links between DN and oxidative stress and therapies on the horizon, such as inhibitors of protein kinase C, aldose reductase, and advanced glycation. With reference to oxidative stress and the related pathways, the following new drugs are under study such as taurine, acetyl-L-carnitine, alpha lipoic acid, protein kinase C inhibitor (ruboxistaurin), aldose reductase inhibitors (fidarestat, epalrestat, ranirestat), advanced glycation end product inhibitors (benfotiamine, aspirin, aminoguanidine), the hexosamine pathway inhibitor (benfotiamine), inhibitor of poly ADP-ribose polymerase (nicotinamide), and angiotensin-converting enzyme inhibitor (trandolapril). The development of modern drugs to treat DN is a real challenge and needs intensive long-term comparative trials. PMID:23738033

Antioxidant and Anti-Inflammatory Effects in RAW264.7 Macrophages of Malvidin, a Major Red Wine Polyphenol

PubMed Central

Bognar, Eszter; Sarszegi, Zsolt; Szabo, Aliz; Debreceni, Balazs; Kalman, Nikoletta; Tucsek, Zsuzsanna; Sumegi, Balazs; Gallyas, Ferenc

2013-01-01

Background Red wine polyphenols can prevent cardiovascular and inflammatory diseases. Resveratrol, the most extensively studied constituent, is unlikely to solely account for these beneficial effects because of its rather low abundance and bioavailability. Malvidin is far the most abundant polyphenol in red wine; however, very limited data are available about its effect on inflammatory processes and kinase signaling pathways. Methods & Findings The present study was carried out by using RAW 264.7 macrophages stimulated by bacterial lipopolysaccharide in the presence and absence of malvidin. From the cells, activation of nuclear factor-kappaB, mitogen-activated protein kinase, protein kinase B/Akt and poly ADP-ribose polymerase, reactive oxygen species production, mitogen-activated protein kinase phosphatase-1 expression and mitochondrial depolarization were determined. We found that malvidin attenuated lipopolysaccharide-induced nuclear factor-kappaB, poly ADP-ribose polymerase and mitogen-activated protein kinase activation, reactive oxygen species production and mitochondrial depolarization, while upregulated the compensatory processes; mitogen-activated protein kinase phosphatase-1 expression and Akt activation. Conclusions These effects of malvidin may explain the previous findings and at least partially account for the positive effects of moderate red wine consumption on inflammation-mediated chronic maladies such as obesity, diabetes, hypertension and cardiovascular disease. PMID:23755222

Lethality in PARP-1/Ku80 double mutant mice reveals physiologicalsynergy during early embryogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henrie, Melinda S.; Kurimasa, Akihiro; Burma, Sandeep

2002-09-24

Ku is an abundant heterodimeric nuclear protein, consisting of 70-kDa and 86-kDa tightly associated subunits that comprise the DNA binding component of DNA-dependent protein kinase. Poly(ADP)ribose polymerase-1 (PARP-1) is a 113-kDa protein that catalyzes the synthesis of poly(ADP-ribose) on target proteins. Both Ku and PARP-1 recognize and bind to DNA ends. Ku functions in the non-homologous end joining (NHEJ) repair pathway whereas PARP-1 functions in the single strand break repair and base excision repair (BER) pathways. Recent studies have revealed that PARP-1 and Ku80 interact in vitro. To determine whether the association of PARP-1 and Ku80 has any physiological significancemore » or synergistic function in vivo, mice lacking both PARP-1 and Ku80 were generated. The resulting offspring died during embryonic development displaying abnormalities around the gastrulation stage. In addition, PARP-1-/-Ku80-/- cultured blastocysts had an increased level of apoptosis. These data suggest that the functions of both Ku80 and PARP-1 are essential for normal embryogenesis and that a loss of genomic integrity leading to cell death through apoptosis is likely the cause of the embryonic lethality observed in these mice.« less

Timing of developmental reduction in epithelial glutathione redox potential is associated with increased epithelial proliferation in the immature murine intestine.

PubMed

Reid, Graham K; Berardinelli, Andrew J; Ray, Laurie; Jackson, Arena R; Neish, Andrew S; Hansen, Jason M; Denning, Patricia W

2017-08-01

BackgroundThe intracellular redox potential of the glutathione (GSH)/glutathione disulfide (GSSG) couple regulates cellular processes. In vitro studies indicate that a reduced GSH/GSSG redox potential favors proliferation, whereas a more oxidized redox potential favors differentiation. Intestinal growth depends upon an appropriate balance between the two. However, how the ontogeny of intestinal epithelial cellular (IEC) GSH/GSSG redox regulates these processes in the developing intestine has not been fully characterized in vivo.MethodsOntogeny of intestinal GSH redox potential and growth were measured in neonatal mice.ResultsWe show that IEC GSH/GSSG redox potential becomes increasingly reduced (primarily driven by increased GSH concentration) over the first 3 weeks of life. Increased intracellular GSH has been shown to drive proliferation through increased poly-ADP-ribose polymerase (PARP) activity. We show that increasing IEC poly-ADP-ribose chains can be measured over the first 3 weeks of life, indicating an increase in IEC PARP activity. These changes are accompanied by increased intestinal growth and IEC proliferation as assessed by villus height/crypt depth, intestinal length, and Ki67 staining.ConclusionUnderstanding how IEC GSH/GSSG redox potential is developmentally regulated may provide insight into how premature human intestinal redox states can be manipulated to optimize intestinal growth and adaptation.

Mechanistic Links between PARP, NAD, and Brain Inflammation after TBI

DTIC Science & Technology

2014-10-01

metabolite which we have in prior studies shown to also suppress poly(ADP-ribose) polymerase activity and inflammatory responses) and ketogenic diet . CtBP1/2...knockout mice will be generated to test a specific mechanisms by which ketogenic diet can have anti-inflammatory effects. For all studies, outcome...inflammatory responses. (3) Ketogenic diet , begun 12 hours after TBI. CtBP1/2 knockout mice will be generated to test a specific mechanisms by which

Ribose in the heart.

PubMed

Herrick, James; St Cyr, John

2008-01-01

Every cell needs energy, i.e., adenosine triphosphate (ATP), to carry out its function. Decreased oxygen levels, decreased blood flow, and other stressful conditions can drastically effect the intracellular concentrations of these energy compounds. Skeletal muscle, unlike the heart, can address this drop in ATP by employing the myokinase reaction, ultimately producing ATP with a subsequent elevation in adenosine monophosphate (AMP). Ribose, a naturally occurring 5-carbon monosaccharide, is a key component of RNA, DNA (which has deoxyribose), acetyl coenzyme A, and ATP. Each cell produces its own ribose, involved in the pentose phosphate pathway (PPP), to aid in ATP production. States of ischemia and/or hypoxia can severely lower levels of cellular energy compounds in the heart, with an associated compromise in cellular processes, ultimately reflected in altered function. Ribose appears to provide a solution to the problem in replenishing the depressed ATP levels and improving functional status of patients afflicted with cardiovascular diseases.

Identification and structural characterization of O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate in yeast methionine initiator tRNA.

PubMed Central

Keith, G; Glasser, A L; Desgrès, J; Kuo, K C; Gehrke, C W

1990-01-01

We report in this paper on the complete structure determination of the modified nucleotide A*, now called Ar(p), that was previously identified in yeast methionine initiator tRNA as an isomeric form of O-ribosyl-adenosine bearing an additional phosphoryl-monoester group on its ribose2 moiety. By using the chemical procedure of periodate oxidation and subsequent beta-elimination with cyclohexylamine on mono- and dinucleotides containing Ar(p), we characterized the location of the phosphate group on the C-5" of the ribose2 moiety, and the linkage between the two riboses as a (1"----2')-glycosidic bond. Since the structural difference between phosphatase treated Ar(p) and authentic O-alpha-ribosyl-(1"----2')-adenosine from poly(ADP-Ribose) was previously assigned to an isomeric difference in the ribose2-ribose1 linkage, the (1"----2')-glycosidic bond of Ar(p) was deduced to have a beta-spatial configuration. Thus, final chemical structure for Ar(p) at the position 64 in yeast initiator tRNA(Met) has been established as O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate. This nucleotide is linked by a 3',5'-phosphodiester bond to G at the position 65. PMID:2235481

Identification and structural characterization of O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate in yeast methionine initiator tRNA.

PubMed

Keith, G; Glasser, A L; Desgrès, J; Kuo, K C; Gehrke, C W

1990-10-25

We report in this paper on the complete structure determination of the modified nucleotide A*, now called Ar(p), that was previously identified in yeast methionine initiator tRNA as an isomeric form of O-ribosyl-adenosine bearing an additional phosphoryl-monoester group on its ribose2 moiety. By using the chemical procedure of periodate oxidation and subsequent beta-elimination with cyclohexylamine on mono- and dinucleotides containing Ar(p), we characterized the location of the phosphate group on the C-5" of the ribose2 moiety, and the linkage between the two riboses as a (1"----2')-glycosidic bond. Since the structural difference between phosphatase treated Ar(p) and authentic O-alpha-ribosyl-(1"----2')-adenosine from poly(ADP-Ribose) was previously assigned to an isomeric difference in the ribose2-ribose1 linkage, the (1"----2')-glycosidic bond of Ar(p) was deduced to have a beta-spatial configuration. Thus, final chemical structure for Ar(p) at the position 64 in yeast initiator tRNA(Met) has been established as O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate. This nucleotide is linked by a 3',5'-phosphodiester bond to G at the position 65.

Unfolding of core nucleosomes by PARP-1 revealed by spFRET microscopy

PubMed Central

Sultanov, Daniel C.; Gerasimova, Nadezhda S.; Kudryashova, Kseniya S.; Maluchenko, Natalya V.; Kotova, Elena Y.; Langelier, Marie-France; Pascal, John M.; Kirpichnikov, Mikhail P.; Feofanov, Alexey V.; Studitsky, Vasily M.

2017-01-01

DNA accessibility to various protein complexes is essential for various processes in the cell and is affected by nucleosome structure and dynamics. Protein factor PARP-1 (poly(ADP-ribose)polymerase 1) increases the accessibility of DNA in chromatin to repair proteins and transcriptional machinery, but the mechanism and extent of this chromatin reorganization are unknown. Here we report on the effects of PARP-1 on single nucleosomes revealed by spFRET (single-particle Förster Resonance Energy Transfer) microscopy. PARP-1 binding to a double-strand break in the vicinity of a nucleosome results in a significant increase of the distance between the adjacent gyres of nucleosomal DNA. This partial uncoiling of the entire nucleosomal DNA occurs without apparent loss of histones and is reversed after poly(ADP)-ribosylation of PARP-1. Thus PARP-1-nucleosome interactions result in reversible, partial uncoiling of the entire nucleosomal DNA. PMID:28804761

Structural Implications for Selective Targeting of PARPs.

PubMed

Steffen, Jamin D; Brody, Jonathan R; Armen, Roger S; Pascal, John M

2013-12-20

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD(+) as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed "synthetic lethality." In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients.

Structural Implications for Selective Targeting of PARPs

PubMed Central

Steffen, Jamin D.; Brody, Jonathan R.; Armen, Roger S.; Pascal, John M.

2013-01-01

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD+ as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed “synthetic lethality.” In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients. PMID:24392349

A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors.

PubMed

Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C; Westbrook, Thomas F; Harper, J Wade; Elledge, Stephen J

2015-06-09

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Possible involvement of 12-lipoxygenase activation in glucose-deprivation/reload-treated neurons.

PubMed

Nagasawa, Kazuki; Kakuda, Taichi; Higashi, Youichirou; Fujimoto, Sadaki

2007-12-18

The aim of this study was to clarify whether 12-lipoxygenase (12-LOX) activation was involved in reactive oxygen species (ROS) generation, extensive poly(ADP-ribose) polymerase (PARP) activation and neuronal death induced by glucose-deprivation, followed by glucose-reload (GD/R). The decrease of neuronal viability and accumulation of poly(ADP-ribose) induced by GD/R were prevented 3-aminobenzamide, a representative PARP inhibitor, demonstrating this treatment protocol caused the same oxidative stress with the previously reported one. The PARP activation, ROS generation and decrease of neuron viability induced by GD/R treatment were almost completely abolished by an extracellular zinc chelator, CaEDTA. p47(phox), a cytosolic component of NADPH oxidase was translocated the membrane fraction by GD/R, indicating its activation, but it did not generate detectable ROS. Surprisingly, pharmacological inhibition of NADPH oxidase with apocynin and AEBSF further decreased the decreased neuron viability induced by GD/R. On the other hand, AA861, a 12-LOX inhibitor, prevented ROS generation and decrease of neuron viability caused by GD/R. Interestingly, an antioxidant, N-acetyl-l-cysteine rescued the neurons from GD/R-induced oxidative stress, implying effectiveness of antioxidant administration. These findings suggested that activation of 12-LOX, but not NADPH oxidase, following to zinc release might play an important role in ROS generation and decrease of viability in GD/R-treated neurons.

Characterization of hMTr1, a Human Cap1 2′-O-Ribose Methyltransferase*

PubMed Central

Bélanger, François; Stepinski, Janusz; Darzynkiewicz, Edward; Pelletier, Jerry

2010-01-01

Cellular eukaryotic mRNAs are capped at their 5′ ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly(A) addition, nucleocytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap, called cap0, higher eukaryotes are methylated at the 2′-O-ribose of the first or the first and second transcribed nucleotides, called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyzes specific methylation of the 2′-O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA-mediated knockdown of hMTr1 in HeLa cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo. PMID:20713356

Molecular Toxicology of Chromatin: The Role of Poly (ADP-Ribose) in Gene Control

DTIC Science & Technology

1985-12-15

remaining aý minor camponent. There- fore, in order to minimize this minor product we include potassium phosphate (p1 6.6) in an amount equivalent to the... phosphate (pH 6.6) followed by 0.005 ml of a 0.10 M solution of sodium 3 H]-borohydride in 0.10 M potassium hydroxide. The resultant solution, which...AMP. Five hundred nmol of oxidized AMP was mixed with 5000 nmol of borohydride in 4.5 mM potassium phosphate , pH 9, in a final volume of 1100 Ul

Molecular Toxicology of Chromatin: The Role of Poly(ADP-Ribose) in Gene Control.

DTIC Science & Technology

1985-02-01

04 4 0V 0 r4. $) 4 0n U) Ln 𔃺 V W 0 Ř (u %D -T -a Ai j4 W0 .. 04 04 0 04rd 412% I cb 01 41 410 41 .j A . . to 2.4 Ř -A 411 0 2 0 -r 0. 04 0A CAt e4...reported previously (1). The selection procedures used for NNSF * were compared with fibrosarcoma . Ieionmyosarcoui and synovial sarcoma cells. V...tumor incidence of human fibrosarcoma , lelomyosarcoma and synovial sarcoma cells in nude mice was identical with that of UV-transformed human

Activation of Poly(ADP-Ribose) Polymerase by Sulfur Mustard in Hela Cell Cultures

DTIC Science & Technology

1993-05-13

i O : DUTiC-TID INTRODUCTION Sulfur mustard ( 2,2’-dichlorodiethyl sulfide or HD) is a bifunctional alkylating agent which reacts with a wide variety...of biological molecules. It is a strong alkylating agent of purine bases in DNA (Kohn 1983). Early studies strongly implicate DNA as a principal...cells have previously demonstrated stimulation of PADPRP activity following exposure to a monofunctional alkylating agent , methylnitrosourea (MNU

Tankyrase1-mediated poly(ADP-ribosyl)ation of TRF1 maintains cell survival after telomeric DNA damage

PubMed Central

Yang, Lu; Sun, Luxi; Teng, Yaqun; Chen, Hao; Gao, Ying; Levine, Arthur S.; Nakajima, Satoshi

2017-01-01

Abstract Oxidative DNA damage triggers telomere erosion and cellular senescence. However, how repair is initiated at telomeres is largely unknown. Here, we found unlike PARP1-mediated Poly-ADP-Ribosylation (PARylation) at genomic damage sites, PARylation at telomeres is mainly dependent on tankyrase1 (TNKS1). TNKS1 is recruited to damaged telomeres via its interaction with TRF1, which subsequently facilitates the PARylation of TRF1 after damage. TNKS inhibition abolishes the recruitment of the repair proteins XRCC1 and polymerase β at damaged telomeres, while the PARP1/2 inhibitor only has such an effect at non-telomeric damage sites. The ANK domain of TNKS1 is essential for the telomeric damage response and TRF1 interaction. Mutation of the tankyrase-binding motif (TBM) on TRF1 (13R/18G to AA) disrupts its interaction with TNKS1 concomitant recruitment of TNKS1 and repair proteins after damage. Either TNKS1 inhibition or TBM mutated TRF1 expression markedly sensitizes cells to telomere oxidative damage as well as XRCC1 inhibition. Together, our data reveal a novel role of TNKS1 in facilitating SSBR at damaged telomeres through PARylation of TRF1, thereby protecting genome stability and cell viability. PMID:28160604

Identification of Determinants Required for Agonistic and Inverse Agonistic Ligand Properties at the ADP Receptor P2Y12

PubMed Central

Schmidt, Philipp; Ritscher, Lars; Dong, Elizabeth N.; Hermsdorf, Thomas; Cöster, Maxi; Wittkopf, Doreen; Meiler, Jens

2013-01-01

The ADP receptor P2Y12 belongs to the superfamily of G protein–coupled receptors (GPCRs), and its activation triggers platelet aggregation. Therefore, potent antagonists, such as clopidogrel, are of high clinical relevance in prophylaxis and treatment of thromboembolic events. P2Y12 displays an elevated basal activity in vitro, and as such, inverse agonists may be therapeutically beneficial compared with antagonists. Only a few inverse agonists of P2Y12 have been described. To expand this limited chemical space and improve understanding of structural determinants of inverse agonist-receptor interaction, this study screened a purine compound library for lead structures using wild-type (WT) human P2Y12 and 28 constitutively active mutants. Results showed that ATP and ATP derivatives are agonists at P2Y12. The potency at P2Y12 was 2-(methylthio)-ADP > 2-(methylthio)-ATP > ADP > ATP. Determinants required for agonistic ligand activity were identified. Molecular docking studies revealed a binding pocket for the ATP derivatives that is bordered by transmembrane helices 3, 5, 6, and 7 in human P2Y12, with Y105, E188, R256, Y259, and K280 playing a particularly important role in ligand interaction. N-Methyl-anthraniloyl modification at the 3′-OH of the 2′-deoxyribose leads to ligands (mant-deoxy-ATP [dATP], mant-deoxy-ADP) with inverse agonist activity. Inverse agonist activity of mant-dATP was found at the WT human P2Y12 and half of the constitutive active P2Y12 mutants. This study showed that, in addition to ADP and ATP, other ATP derivatives are not only ligands of P2Y12 but also agonists. Modification of the ribose within ATP can result in inverse activity of ATP-derived ligands. PMID:23093496

[The effect of 3-aminobenzamide on the mitotic cycle of Chinese hamster cells cultured on a medium with 5-bromodeoxyuridine following ionizing radiation action].

PubMed

Kirillova, T V; Rozanov, Iu M; Spivak, I M

1992-01-01

A specific inhibitor of poly(ADP-ribose)polymerase-3-aminobenzamide (6 mM) has been shown to: 1) reduce survival of non-irradiated CHO-K1 cells, cultivated in medium containing 5-bromodeoxyuridine (10 mkM, BDU cells), and increase their radiosensitivity; 2) induce G2 delay in BDU cells while progressing through the cell cycle as analysed by the DNA flow cytometry; 3) increase to a great degree G2 delay in X-irradiated BDU cells. 3-Aminobenzamide is primarily effective when it is present during the first or two first cell cycles after the initial addition of BDU. The above data confirm the involvement, presumably an indirect one, of ADP-ribosylation in the DNA repair through affecting the chromatin structure.

Influence of nucleotide modifications at the C2' position on the Hoogsteen base-paired parallel-stranded duplex of poly(A) RNA.

PubMed

Copp, William; Denisov, Alexey Y; Xie, Jingwei; Noronha, Anne M; Liczner, Christopher; Safaee, Nozhat; Wilds, Christopher J; Gehring, Kalle

2017-09-29

Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2'-deoxyribose, 2'-O-methyl-ribose, 2'-deoxy-2'-fluoro-ribose, arabinose and 2'-deoxy-2'-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2' modifications gave a variety of effects. Arabinose and 2'-deoxy-2'-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2'-O-methyl and 2'-deoxy-2'-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Binary actin-ADP-ribosylating toxins and their use as molecular Trojan horses for drug delivery into eukaryotic cells.

PubMed

Barth, Holger; Stiles, Bradley G

2008-01-01

Binary bacterial toxins are unique AB-type toxins, composed of two non-linked proteins that act as a binding/translocation component and an enzyme component. All known actin-ADP-ribosylating toxins from clostridia possess this binary structure. This toxin family is comprised of the prototypical Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, Clostridium difficile CDT, and Clostridium spiroforme toxin. Once in the cytosol of host cells, these toxins transfer an ADP-ribose moiety from nicotinamide-adenosine-dinucleotide onto G-actin that then leads to depolymerization of actin filaments. In recent years much progress has been made towards understanding the cellular uptake mechanism of binary actin-ADP-ribosylating toxins, and in particular that of C2 toxin. Both components act in a precisely concerted manner to intoxicate eukaryotic cells. The binding/translocation (B-) component forms a complex with the enzyme (A-) component and mediates toxin binding to a cell-surface receptor. Following receptor-mediated endocytosis, the enzyme component escapes from acidic endosomes into the cytosol. Acidification of endosomes triggers pore formation by the binding/translocation component in endosomal membranes and the enzyme component subsequently translocates through the pore. This step requires a host cell chaperone, Hsp90. Due to their unique structure, binary toxins are naturally "tailor made" for transporting foreign proteins into the cytosol of host cells. Several highly specific and cell-permeable recombinant fusion proteins have been designed and successfully used in experimental cell research. This review will focus on the recent progress in studying binary actin ADP-ribosylating toxins as highly effective virulence factors and innovative tools for cell physiology as well as pharmacology.

Synergistic inhibition of Streptococcal biofilm by ribose and xylitol.

PubMed

Lee, Heon-Jin; Kim, Se Chul; Kim, Jinkyung; Do, Aejin; Han, Se Yeong; Lee, Bhumgey David; Lee, Hyun Ho; Lee, Min Chan; Lee, So Hui; Oh, Taejun; Park, Sangbin; Hong, Su-Hyung

2015-02-01

Streptococcus mutans and Streptococcus sobrinus are the major causative agents of human dental caries. Therefore, the removal or inhibition of these streptococcal biofilms is essential for dental caries prevention. In the present study, we evaluated the effects of ribose treatment alone or in combination with xylitol on streptococcal biofilm formation for both species. Furthermore, we examined the expression of genes responsible for dextran-dependent aggregation (DDAG). In addition, we investigated whether ribose affects the biofilm formation of xylitol-insensitive streptococci, which results from long-term exposure to xylitol. The viability of streptococci biofilms formed in a 24-well polystyrene plate was quantified by fluorescent staining with the LIVE/DEAD bacterial viability and counting kit, which was followed by fluorescence activated cell sorting analysis. The effects of ribose and/or xylitol on the mRNA expression of DDAG-responsible genes, gbpC and dblB, was evaluated by RT-qPCR. Our data showed that ribose and other pentose molecules significantly inhibited streptococcal biofilm formation and the expression of DDAG-responsible genes. In addition, co-treatment with ribose and xylitol decreased streptococcal biofilm formation to a further extent than ribose or xylitol treatment alone in both streptococcal species. Furthermore, ribose attenuated the increase of xylitol-insensitive streptococcal biofilm, which results in the reduced difference of biofilm formation between S. mutans that are sensitive and insensitive to xylitol. These data suggest that pentose may be used as an additive for teeth-protective materials or in sweets. Furthermore, ribose co-treatment with xylitol might help to increase the anti-cariogenic efficacy of xylitol. Copyright © 2014 Elsevier Ltd. All rights reserved.

Long-lasting neuroprotection and neurological improvement in stroke models with new, potent and brain permeable inhibitors of poly(ADP-ribose) polymerase

PubMed Central

Moroni, F; Cozzi, A; Chiarugi, A; Formentini, L; Camaioni, E; Pellegrini-Giampietro, DE; Chen, Y; Liang, S; Zaleska, MM; Gonzales, C; Wood, A; Pellicciari, R

2012-01-01

BACKGROUND AND PURPOSES Thienyl-isoquinolone (TIQ-A) is a relatively potent PARP inhibitor able to reduce post-ischaemic neuronal death in vitro. Here we have studied, in different stroke models in vivo, the neuroprotective properties of DAMTIQ and HYDAMTIQ, two TIQ-A derivatives able to reach the brain and to inhibit PARP-1 and PARP-2. EXPERIMENTAL APPROACH Studies were carried out in (i) transient (2 h) middle cerebral artery occlusion (tMCAO), (ii) permanent MCAO (pMCAO) and (iii) electrocoagulation of the distal portion of MCA in conjunction with transient (90 min) bilateral carotid occlusion (focal cortical ischaemia). KEY RESULTS In male rats with tMCAO, HYDAMTIQ (0.1–10 mg·kg−1) injected i.p. three times, starting 4 h after MCAO, reduced infarct volumes by up to 70%, reduced the loss of body weight by up to 60% and attenuated the neurological impairment by up to 40%. In age-matched female rats, HYDAMTIQ also reduced brain damage. Protection, however, was less pronounced than in the male rats. In animals with pMCAO, HYDAMTIQ administered 30 min after MCAO reduced infarct volumes by approximately 40%. In animals with focal cortical ischaemia, HYDAMTIQ treatment decreased post-ischaemic accumulation of PAR (the product of PARP activity) and the presence of OX42-positive inflammatory cells in the ischaemic cortex. It also reduced sensorimotor deficits for up to 90 days after MCAO. CONCLUSION AND IMPLICATIONS Our results show that HYDAMTIQ is a potent PARP inhibitor that conferred robust neuroprotection and long-lasting improvement of post-stroke neurological deficits. PMID:21913897

Influence of nucleotide modifications at the C2’ position on the Hoogsteen base-paired parallel-stranded duplex of poly(A) RNA

PubMed Central

Copp, William; Denisov, Alexey Y.; Xie, Jingwei; Noronha, Anne M.; Liczner, Christopher; Safaee, Nozhat

2017-01-01

Abstract Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2′-deoxyribose, 2′-O-methyl-ribose, 2′-deoxy-2′-fluoro-ribose, arabinose and 2′-deoxy-2′-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2’ modifications gave a variety of effects. Arabinose and 2′-deoxy-2′-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2′-O-methyl and 2′-deoxy-2′-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability. PMID:28973475

Lifelong endurance training attenuates age-related genotoxic stress in human skeletal muscle.

PubMed

Cobley, James N; Sakellariou, George K; Murray, Scott; Waldron, Sarah; Gregson, Warren; Burniston, Jatin G; Morton, James P; Iwanejko, Lesley A; Close, Graeme L

2013-07-12

The aim of the present study was to determine the influence of age and habitual activity level, at rest and following a single bout of high-intensity exercise, on the levels of three proteins poly(ADP-ribose) polymerase-1 (PARP-1), cleaved-PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), involved in the DNA repair and cell death responses to stress and genotoxic insults. Muscle biopsies were obtained from the vastus lateralis of young trained (22 ± 3 years, n = 6), young untrained (24 ± 4 years, n = 6), old trained (64 ± 3 years, n = 6) and old untrained (65 ± 6 years, n = 6) healthy males before, immediately after and three days following a high-intensity interval exercise bout. PARP-1, which catalyzes poly(ADP-ribosyl)ation of proteins and DNA in response to a range of intrinsic and extrinsic stresses, was increased at baseline in old trained and old untrained compared with young trained and young untrained participants (P ≤ 0.05). Following exercise, PARP-1 levels remained unchanged in young trained participants, in contrast to old trained and old untrained where levels decreased and young untrained where levels increased (P ≤ 0.05). Interestingly, baseline levels of the cleaved PARP-1, a marker of apoptosis, and PARG, responsible for polymer degradation, were both significantly elevated in old untrained compared with old trained, young trained and young untrained (P ≤ 0.05). Despite this baseline difference in PARG, there was no change in any group following exercise. There was a non-significant statistical trend (P = 0.072) towards increased cleaved-PARP-1 expression post-exercise in younger but not old persons, regardless of training status. Collectively, these results show that exercise slows the progression towards a chronically stressed state but has no impact on the age-related attenuated response to acute exercise. Our findings provide valuable insight into how habitual exercise training could protect skeletal muscle from chronic damage to

Rates of Decomposition of Ribose and other Sugars: Implications for Chemical Evolution

NASA Technical Reports Server (NTRS)

Larralde, Rosa; Robertson, Michael P.; Miller, Stanley L.

1995-01-01

The existence of the RNA world, in which RNA acted as a catalyst as well as an informational macromolecule, assumes a large prebiotic source of ribose or the existence of pre-RNA molecules with backbones different from ribose-phosphate. The generally accepted prebiotic synthesis of ribose, the formose reaction, yields numerous sugars without any selectivity. Even if there were a selective synthesis of ribose, there is still the problem of stability. Sugars are known to be unstable in strong acid or base, but there are few data for neutral solutions. Therefore, we have measured the rate of decomposition of ribose between pH 4 and pH 8 from 40 C to 120 C. The ribose half-lives are very short (73 min at pH 7.0 and 100 C and 44 years at pH 7.0 and 0 C). The other aldopentoses and aldohexoses have half-lives within an order of magnitude of these values, as do 2-deoxyribose, ribose 5-phosphate, and ribose 2,4bisphosphate. These results suggest that the backbone of the first genetic material could not have contained ribose or other sugars because of their instability.

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

PubMed Central

Nikiforov, Andrey; Dölle, Christian; Niere, Marc; Ziegler, Mathias

2011-01-01

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule. PMID:21504897

A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex.

PubMed

Kusov, Yuri; Tan, Jinzhi; Alvarez, Enrique; Enjuanes, Luis; Hilgenfeld, Rolf

2015-10-01

The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity. Copyright © 2015 Elsevier Inc. All rights reserved.

A new glycation product ‘norpronyl-lysine,’ and direct characterization of cross linking and other glycation adducts: NMR of model compounds and collagen

PubMed Central

Bullock, Peter T. B.; Reid, David G.; Ying Chow, W.; Lau, Wendy P. W.; Duer, Melinda J.

2014-01-01

NMR is ideal for characterizing non-enzymatic protein glycation, including AGEs (advanced glycation endproducts) underlying tissue pathologies in diabetes and ageing. Ribose, R5P (ribose-5-phosphate) and ADPR (ADP-ribose), could be significant and underinvestigated biological glycating agents especially in chronic inflammation. Using [U-13C]ribose we have identified a novel glycoxidation adduct, 5-deoxy-5-desmethylpronyl-lysine, ‘norpronyl-lysine’, as well as numerous free ketones, acids and amino group reaction products. Glycation by R5P and ADPR proceeds rapidly with R5P generating a brown precipitate with PLL (poly-L-lysine) within hours. ssNMR (solid-state NMR) 13C–13C COSY identifies several crosslinking adducts such as the newly identified norpronyl-lysine, in situ, from the glycating reaction of 13C5-ribose with collagen. The same adducts are also identifiable after reaction of collagen with R5P. We also demonstrate for the first time bio-amine (spermidine, N-acetyl lysine, PLL) catalysed ribose 2-epimerization to arabinose at physiological pH. This work raises the prospect of advancing understanding of the mechanisms and consequences of glycation in actual tissues, in vitro or even ex vivo, using NMR isotope-labelled glycating agents, without analyses requiring chemical or enzymatic degradations, or prior assumptions about glycation products. PMID:27919030

Differential Effect of Zoledronic Acid on Human Vascular Smooth Muscle Cells

PubMed Central

Albadawi, Hassan; Haurani, Mounir J.; Oklu, Rahmi; Trubiano, Jordan P.; Laub, Peter J.; Yoo, Hyung-Jin; Watkins, Michael T.

2012-01-01

Introduction The activation of human vascular smooth muscle cell proliferation, adhesion and migration is essential for intimal hyperplasia formation. These experiments were designed to test whether Zoledronic Acid (ZA) would modulate indices of human smooth muscle cell activation, exert differential effects on proliferating vs. quiescent cells and determine whether these effects were dependent on GTPase binding proteins prenylation. ZA was chosen for testing in these experiments because it is clinically used in humans with cancer, and has been shown to modulate rat smooth muscle cell proliferation and migration. Methods Human aortic smooth muscle cells (HASMC) were cultured under either proliferating or growth arrest (quiescent) conditions in the presence or absence of ZA for 48 hours, whereupon the effect of ZA on HASMC proliferation, cellular viability, metabolic activity and membrane integrity were compared. In addition, the effect of ZA on adhesion and migration were assessed in proliferating cells. The effect of increased concentration of ZA on the mevalonate pathway and genomic/cellular stress related poly ADP Ribose polymerase (PARP) enzyme activity were assessed using the relative prenylation of Rap-1A/B protein and the formation of poly ADP- ribosylated proteins (PAR) respectively. Results There was a dose dependent inhibition of cellular proliferation, adhesion and migration following ZA treatment. ZA treatment decreased indices of cellular viability and significantly increased membrane injury in proliferating vs. quiescent cells. This was correlated with the appearance of unprenylated Rap-1A protein and dose dependent down regulation of PARP activity. Conclusions These data suggest that ZA is effective in inhibiting HASMC proliferation, adhesion and migration which coincide with the appearance of unprenylated RAP-1A/B protein, thereby suggesting that the mevalonate pathway may play a role in the inhibition of HASMC activation. PMID:23164362

Mitochondrial Free [Ca2+] Increases during ATP/ADP Antiport and ADP Phosphorylation: Exploration of Mechanisms

PubMed Central

Haumann, Johan; Dash, Ranjan K.; Stowe, David F.; Boelens, Age D.; Beard, Daniel A.; Camara, Amadou K.S.

2010-01-01

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca2+] ([Ca2+]m) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca2+]m is affected by H2PO4− (Pi), Mg2+, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca2+]m, membrane potential, redox state, matrix volume, pHm, and O2 consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg2+ and Pi. Energized mitochondria showed a dose-dependent increase in [Ca2+]m after adding CaCl2 equivalent to 20, 114, and 485 nM extramatrix free [Ca2+] ([Ca2+]e); this uptake was attenuated at higher buffer Mg2+. Adding ADP transiently increased [Ca2+]m up to twofold. The ADP effect on increasing [Ca2+]m could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg2+ or Pi. Oligomycin largely reduced the increase in [Ca2+]m by ADP compared to control, and [Ca2+]m did not return to baseline. Carboxyatractyloside prevented the ADP-induced [Ca2+]m increase. Adding CaCl2 had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca2+]e. These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca2+]m largely due to the interaction of matrix Ca2+ with ATP, ADP, Pi, and cation buffering proteins in the matrix. PMID:20712982

Trial watch – inhibiting PARP enzymes for anticancer therapy

PubMed Central

Sistigu, Antonella; Manic, Gwenola; Obrist, Florine; Vitale, Ilio

2016-01-01

ABSTRACT Poly(ADP-ribose) polymerases (PARPs) are a members of family of enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) and/or mono(ADP-ribosyl)ation (MARylation), two post-translational protein modifications involved in crucial cellular processes including (but not limited to) the DNA damage response (DDR). PARP1, the most abundant family member, is a nuclear protein that is activated upon sensing distinct types of DNA damage and contributes to their resolution by PARylating multiple DDR players. Recent evidence suggests that, along with DDR, activated PARP1 mediates a series of prosurvival and proapoptotic processes aimed at preserving genomic stability. Despite this potential oncosuppressive role, upregulation and/or overactivation of PARP1 or other PARP enzymes has been reported in a variety of human neoplasms. Over the last few decades, several pharmacologic inhibitors of PARP1 and PARP2 have been assessed in preclinical and clinical studies showing potent antineoplastic activity, particularly against homologous recombination (HR)-deficient ovarian and breast cancers. In this Trial Watch, we describe the impact of PARP enzymes and PARylation in cancer, discuss the mechanism of cancer cell killing by PARP1 inactivation, and summarize the results of recent clinical studies aimed at evaluating the safety and therapeutic profile of PARP inhibitors in cancer patients. PMID:27308587

Hyperthermal (1-100 eV) nitrogen ion scattering damage to D-ribose and 2-deoxy-D-ribose films.

PubMed

Deng, Zongwu; Bald, Ilko; Illenberger, Eugen; Huels, Michael A

2007-10-14

Highly charged heavy ion traversal of a biological medium can produce energetic secondary fragment ions. These fragment ions can in turn cause collisional and reactive scattering damage to DNA. Here we report hyperthermal (1-100 eV) scattering of one such fragment ion (N(+)) from biologically relevant sugar molecules D-ribose and 2-deoxy-D-ribose condensed on polycrystalline Pt substrate. The results indicate that N(+) ion scattering at kinetic energies down to 10 eV induces effective decomposition of both sugar molecules and leads to the desorption of abundant cation and anion fragments. Use of isotope-labeled molecules (5-(13)C D-ribose and 1-D D-ribose) partly reveals some site specificity of the fragment origin. Several scattering reactions are also observed. Both ionic and neutral nitrogen atoms abstract carbon from the molecules to form CN(-) anion at energies down to approximately 5 eV. N(+) ions also abstract hydrogen from hydroxyl groups of the molecules to form NH(-) and NH(2) (-) anions. A fraction of OO(-) fragments abstract hydrogen to form OH(-). The formation of H(3)O(+) ions also involves hydrogen abstraction as well as intramolecular proton transfer. These findings suggest a variety of severe damaging pathways to DNA molecules which occur on the picosecond time scale following heavy ion irradiation of a cell, and prior to the late diffusion-limited homogeneous chemical processes.

45 CFR 95.621 - ADP reviews.

Code of Federal Regulations, 2011 CFR

2011-10-01

... use; (C) Software and data security; (D) Telecommunications security; (E) Personnel security; (F... Federal review. (f) ADP System Security Requirements and Review Process—(1) ADP System Security Requirement. State agencies are responsible for the security of all ADP projects under development, and...

Hyperthermal (1-100 eV) nitrogen ion scattering damage to D-ribose and 2-deoxy-D-ribose films

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng Zongwu; Bald, Ilko; Illenberger, Eugen

2007-10-14

Highly charged heavy ion traversal of a biological medium can produce energetic secondary fragment ions. These fragment ions can in turn cause collisional and reactive scattering damage to DNA. Here we report hyperthermal (1-100 eV) scattering of one such fragment ion (N{sup +}) from biologically relevant sugar molecules D-ribose and 2-deoxy-D-ribose condensed on polycrystalline Pt substrate. The results indicate that N{sup +} ion scattering at kinetic energies down to 10 eV induces effective decomposition of both sugar molecules and leads to the desorption of abundant cation and anion fragments. Use of isotope-labeled molecules (5-{sup 13}C D-ribose and 1-D D-ribose) partlymore » reveals some site specificity of the fragment origin. Several scattering reactions are also observed. Both ionic and neutral nitrogen atoms abstract carbon from the molecules to form CN{sup -} anion at energies down to {approx}5 eV. N{sup +} ions also abstract hydrogen from hydroxyl groups of the molecules to form NH{sup -} and NH{sub 2}{sup -} anions. A fraction of O/O{sup -} fragments abstract hydrogen to form OH{sup -}. The formation of H{sub 3}O{sup +} ions also involves hydrogen abstraction as well as intramolecular proton transfer. These findings suggest a variety of severe damaging pathways to DNA molecules which occur on the picosecond time scale following heavy ion irradiation of a cell, and prior to the late diffusion-limited homogeneous chemical processes.« less

26 CFR 1.401(k)-2 - ADP test.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 5 2010-04-01 2010-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual deferral percentage (ADP) test—(1) In general—(i) ADP test formula. A cash or deferred arrangement satisfies the ADP...

Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

PubMed Central

Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G.

2013-01-01

Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. PMID:23523584

The Juxtaposition of Ribose Hydroxyl Groups: The Root of Biological Catalysis and the RNA World?

NASA Astrophysics Data System (ADS)

Bernhardt, Harold S.

2015-06-01

We normally think of enzymes as being proteins; however, the RNA world hypothesis suggests that the earliest biological catalysts may have been composed of RNA. One of the oldest surviving RNA enzymes we are aware of is the peptidyl transferase centre (PTC) of the large ribosomal RNA, which joins amino acids together to form proteins. Recent evidence indicates that the enzymatic activity of the PTC is principally due to ribose 2 '-OHs. Many other reactions catalyzed by RNA and/or in which RNA is a substrate similarly utilize ribose 2 '-OHs, including phosphoryl transfer reactions that involve the cleavage and/or ligation of the ribose-phosphate backbone. It has recently been proposed by Yakhnin (2013) that phosphoryl transfer reactions were important in the prebiotic chemical evolution of RNA, by enabling macromolecules composed of polyols joined by phosphodiester linkages to undergo recombination reactions, with the reaction energy supplied by the phosphodiester bond itself. The almost unique juxtaposition of the ribose 2'-hydroxyl and 3'-oxygen in ribose-containing polymers such as RNA, which gives ribose the ability to catalyze such reactions, may have been an important factor in the selection of ribose as a component of the first biopolymer. In addition, the juxtaposition of hydroxyl groups in free ribose: (i) allows coordination of borate ions, which could have provided significant and preferential stabilization of ribose in a prebiotic environment; and (ii) enhances the rate of permeation by ribose into a variety of lipid membrane systems, possibly favouring its incorporation into early metabolic pathways and an ancestral ribose-phosphate polymer. Somewhat more speculatively, hydrogen bonds formed by juxtaposed ribose hydroxyl groups may have stabilized an ancestral ribose-phosphate polymer against degradation (Bernhardt and Sandwick 2014). I propose that the almost unique juxtaposition of ribose hydroxyl groups constitutes the root of both biological

A critical role for topoisomerase IIb and DNA double strand breaks in transcription

PubMed Central

Calderwood, Stuart K.

2016-01-01

ABSTRACT Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb. PMID:27100743

A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

PubMed

Calderwood, Stuart K

2016-05-26

Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

Overexpression of dominant negative PARP interferes with tumor formation of HeLa cells in nude mice: evidence for increased tumor cell apoptosis in vivo.

PubMed

Hans, M A; Müller, M; Meyer-Ficca, M; Bürkle, A; Küpper, J H

1999-11-25

Poly(ADP-ribose) polymerase (PARP4) catalyzes the formation of ADP-ribose polymers covalently attached to proteins by using NAD+ as substrate. PARP is strongly activated by DNA single- or double-strand breaks and is thought to be involved in cellular responses to DNA damage. We characterized a dominant negative PARP mutant, i.e. the DNA-binding domain of this enzyme, whose overexpression in cells leads to increased genetic instability following DNA damage. In order to study whether PARP activity is also implicated in the process of tumorigenesis, we generated stably transfected HeLa cell clones with constitutive overexpression of dominant negative PARP and investigated tumor formation of these clones in nude mice. We found that inhibition of PARP activity dramatically reduces tumor forming ability of HeLa cells. Moreover, we provide strong evidence that the observed reduction in tumor forming ability is due to increased tumor cell apoptosis in vivo. Viewed together, our data and those from other groups show that inhibition of PARP enzyme activity interferes with DNA base excision repair and leads to increased genetic instability and recombination but, on the other hand, can sensitize cells to apoptotic stimuli and by this mechanism may prevent tumor formation.

E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Turner, Roberta L.; Wilkinson, John C., E-mail: john.wilkinson@ndsu.edu; Ornelles, David A., E-mail: ornelles@wakehealth.edu

2014-05-15

Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4ORF3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose)more » polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4ORF3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. - Highlights: • E1B-55K or E4orf3 prevents nuclear fragmentation. • Nuclear fragmentation requires AIF and PARP-1 activity. • Adenovirus DNA replication activates PARP-1. • E1B-55K or E4orf3 proteins alter the distribution of PAR.« less

Selective derivatization and sequestration of ribose from a prebiotic mix.

PubMed

Springsteen, Greg; Joyce, Gerald F

2004-08-11

Observations regarding the catalytic potential of RNA and the role of RNA in biology have formed the basis for the "RNA world" hypothesis, which suggests that a genetic system based on self-replicating polyribonucleotides preceded modern biology. However, attempts to devise a realistic prebiotic synthesis of nucleic acids from simple starting materials have been plagued by problems of poor chemical selectivity, lack of stereo- and regiospecificity, and similar rates of formation and degradation of some of the key intermediates. For example, ribose would have been only a small component of a highly complex mix of sugars resulting from the condensation of formaldehyde in a prebiotic world. In addition, ribose is more reactive and degrades more rapidly compared with most other monosaccharides. This study demonstrates an approach for the preferential sequestration of ribose relative to other sugars that takes advantage of its greater reactivity. Cyanamide reacts especially rapidly with ribose to form a stable bicyclic adduct. This product crystallizes spontaneously in aqueous solution, whereas the corresponding products derived from threose, galactose, glucose, mannose, and each of the other pentoses do not. Furthermore, when employing a racemic mixture of d- and l-ribose, enantiomerically twinned crystals are formed that contain discrete homochiral domains.

26 CFR 1.401(k)-2 - ADP test.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 5 2013-04-01 2013-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual deferral percentage (ADP) test—(1) In general—(i) ADP test formula. A cash or deferred arrangement...

26 CFR 1.401(k)-2 - ADP test.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 5 2014-04-01 2014-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual deferral percentage (ADP) test—(1) In general—(i) ADP test formula. A cash or deferred arrangement...

ADP--A Must in the Secondary School

ERIC Educational Resources Information Center

Majernik, John A.

1974-01-01

The rationale for including automated data processing (ADP) in secondary schools is given. ADP instruction: prepares students for data processing employment and for advanced ADP study, aids all students preparing for business careers, aids students in choosing a career, provides consumer information, and adds realism to other classroom…

PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity.

PubMed

Feng, Baomin; Ma, Shisong; Chen, Sixue; Zhu, Ning; Zhang, Shuxin; Yu, Bin; Yu, Yu; Le, Brandon; Chen, Xuemei; Dinesh-Kumar, Savithramma P; Shan, Libo; He, Ping

2016-12-01

Protein poly(ADP-ribosyl)ation (PARylation) primarily catalyzed by poly(ADP-ribose) polymerases (PARPs) plays a crucial role in controlling various cellular responses. However, PARylation targets and their functions remain largely elusive. Here, we deployed an Arabidopsis protein microarray coupled with in vitro PARylation assays to globally identify PARylation targets in plants. Consistent with the essential role of PARylation in plant immunity, the forkhead-associated (FHA) domain protein DAWDLE (DDL), one of PARP2 targets, positively regulates plant defense to both adapted and non-adapted pathogens. Arabidopsis PARP2 interacts with and PARylates DDL, which was enhanced upon treatment of bacterial flagellin. Mass spectrometry and mutagenesis analysis identified multiple PARylation sites of DDL by PARP2. Genetic complementation assays indicate that DDL PARylation is required for its function in plant immunity. In contrast, DDL PARylation appears to be dispensable for its previously reported function in plant development partially mediated by the regulation of microRNA biogenesis. Our study uncovers many previously unknown PARylation targets and points to the distinct functions of DDL in plant immunity and development mediated by protein PARylation and small RNA biogenesis, respectively. © 2016 The Authors.

Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogues of oxidized nicotinamide adenine dinucleotide.

PubMed

Slama, J T; Simmons, A M

1989-09-19

Analogues of oxidized nicotinamide adenine dinucleotide (NAD+) in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ have recently been synthesized [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183]. Carbocyclic NAD+ analogues have been shown to inhibit NAD glycohydrolases and ADP-ribosyl transferases such as cholera toxin A subunit. In this study, the diastereomeric mixture of dinucleotides was separated, and the inhibitory capacity of each of the purified diastereomers was defined. The NAD+ analogue in which the D-dihydroxycyclopentane is substituted for the D-ribose is designated carba-NAD and was demonstrated to be a poor inhibitor of the Bungarus fasciatus venom NAD glycohydrolase. The diastereomeric dinucleotide pseudo-carbocyclic-NAD (psi-carba-NAD), containing L-dihydroxycyclopentane in place of the D-ribose of NAD+, was shown, however, to be a potent competitive inhibitor of the venom NAD glycohydrolase with an inhibitor dissociation constant (Ki) of 35 microM. This was surprising since psi-carba-NAD contains the carbocyclic analogue of the unnatural L-ribotide and was therefore expected to be a biologically inactive diastereomer. psi-Carba-NAD also competitively inhibited the insoluble brain NAD glycohydrolase from cow (Ki = 6.7 microM) and sheep (Ki = 31 microM) enzyme against which carba-NAD is ineffective. Sensitivity to psi-carba-NAD was found to parallel sensitivity to inhibition by isonicotinic acid hydrazide, another NADase inhibitor. psi-Carba-NAD is neither a substrate for nor an inhibitor of alcohol dehydrogenase, whereas carba-NAD is an efficient dehydrogenase substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Defeating EpCAM(+) liver cancer stem cells by targeting chromatin remodeling enzyme CHD4 in human hepatocellular carcinoma.

PubMed

Nio, Kouki; Yamashita, Taro; Okada, Hikari; Kondo, Mitsumasa; Hayashi, Takehiro; Hara, Yasumasa; Nomura, Yoshimoto; Zeng, Sha Sha; Yoshida, Mariko; Hayashi, Tomoyuki; Sunagozaka, Hajime; Oishi, Naoki; Honda, Masao; Kaneko, Shuichi

2015-11-01

Hepatocellular carcinoma is composed of a subset of cells with enhanced tumorigenicity and chemoresistance that are called cancer stem (or stem-like) cells. We explored the role of chromodomain-helicase-DNA-binding protein 4, which is encoded by the CHD4 gene and is known to epigenetically control gene regulation and DNA damage responses in EpCAM(+) liver cancer stem cells. Gene and protein expression profiles were determined by microarray and immunohistochemistry in 245 and 144 hepatocellular carcinoma patients, respectively. The relationship between gene/protein expression and prognosis was examined. The functional role of CHD4 was evaluated in primary hepatocellular carcinoma cells and in cell lines in vitro and in vivo. CHD4 was abundantly expressed in EpCAM(+) hepatocellular carcinoma with expression of hepatic stem cell markers and poor prognosis in two independent cohorts. In cell lines, CHD4 knockdown increased chemosensitivity and CHD4 overexpression induced epirubicin chemoresistance. To inhibit the functions of CHD4 that are mediated through histone deacetylase and poly (ADP-ribose) polymerase, we evaluated the effect of the histone deacetylase inhibitor suberohydroxamic acid and the poly (ADP-ribose) polymerase inhibitor AG-014699. Treatment with either suberohydroxamic acid or AG-014699 reduced the number of EpCAM(+) liver cancer stem cells in vitro, and suberohydroxamic acid and AG-014699 in combination successfully inhibited tumor growth in a mouse xenograft model. CHD4 plays a pivotal role in chemoresistance and the maintenance of stemness in liver cancer stem cells and is therefore a good target for the eradication of hepatocellular carcinoma. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

The potential regulatory roles of NAD(+) and its metabolism in autophagy.

PubMed

Zhang, Dong-Xia; Zhang, Jia-Ping; Hu, Jiong-Yu; Huang, Yue-Sheng

2016-04-01

(Macro)autophagy mediates the bulk degradation of defective organelles, long-lived proteins and protein aggregates in lysosomes and plays a critical role in cellular and tissue homeostasis. Defective autophagy processes have been found to contribute to a variety of metabolic diseases. However, the regulatory mechanisms of autophagy are not fully understood. Increasing data indicate that nicotinamide adenine nucleotide (NAD(+)) homeostasis correlates intimately with autophagy. NAD(+) is a ubiquitous coenzyme that functions primarily as an electron carrier of oxidoreductase in multiple redox reactions. Both NAD(+) homeostasis and its metabolism are thought to play critical roles in regulating autophagy. In this review, we discuss how the regulation of NAD(+) and its metabolism can influence autophagy. We focus on the regulation of NAD(+)/NADH homeostasis and the effects of NAD(+) consumption by poly(ADP-ribose) (PAR) polymerase-1 (PARP-1), NAD(+)-dependent deacetylation by sirtuins and NAD(+) metabolites on autophagy processes and the underlying mechanisms. Future studies should provide more direct evidence for the regulation of autophagy processes by NAD(+). A better understanding of the critical roles of NAD(+) and its metabolites on autophagy will shed light on the complexity of autophagy regulation, which is essential for the discovery of new therapeutic tools for autophagy-related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Cyclophilin B protects SH-SY5Y human neuroblastoma cells against MPP(+)-induced neurotoxicity via JNK pathway.

PubMed

Oh, Yoojung; Jeong, Kwon; Kim, Kiyoon; Lee, Young-Seok; Jeong, Suyun; Kim, Sung Soo; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug; Choe, Wonchae

2016-09-23

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. PD involves a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyidine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) inhibit the complex I of the mitochondrial electron transport chain, and have been widely used to construct PD models. Cyclophilin B (CypB) is an endoplasmic reticulum protein that binds to cyclosporine A as a cyclophilin family member. CypB has peptidyl-prolyl cis-trans isomerase (PPIase) activity. We investigated the protective effects of overexpressed CypB on MPP+-induced neurocytotoxicity in SH-SY5Y human neuroblastoma cells. Overexpressed CypB decreased MPP(+)-induced oxidative stress through the modulation of antioxidant enzymes including manganese superoxide dismutase and catalase, and prevented neurocytotoxicity via mitogen-activated protein kinase, especially the c-Jun N-terminal kinase pathway. In addition, CypB inhibited the activation of MPP(+)-induced the pro-apoptotic molecules poly (ADP-ribose) polymerase, Bax, and Bcl-2, and attenuated MPP(+)-induced mitochondrial dysfunction. The data suggest that overexpressed CypB protects neuronal cells from MPP+-induced dopaminergic neuronal cell death. Copyright © 2016 Elsevier Inc. All rights reserved.

Perspective on the pipeline of drugs being developed with modulation of DNA damage as a target.

PubMed

Plummer, Ruth

2010-09-15

Inhibitors of various elements of the DNA repair pathways have entered clinical development or are in late preclinical stages of drug development. It was initially considered that agents targeting DNA repair would act to overcome tumor resistance to chemotherapy and radiotherapy. More recent data have shown that targeting DNA repair pathways can be effective in selected tumors via a synthetically lethal route, with single agent activity having been shown with poly-ADP ribose polymerase (PARP) inhibitors. An increased understanding of the biology and interaction of the DNA repair pathways also means that rational combination of DNA repair inhibitors may also give great benefit in the clinic. ©2010 AACR.

The apoptotic effect of somatostatin analogue SMS 201-995 on human lymphocytes.

PubMed

Lattuada, D; Casnici, C; Venuto, A; Marelli, O

2002-12-01

The antiproliferative effect of a synthetic octapeptide, somatostatin analogue SMS 201-995 (SMS), and its capacity to bind were evaluated on human peripheral blood lymphocytes (PBL) activated by phytohemoagglutinin (PHA). We then addressed our work to investigate if SMS inhibits PHA activation of PBL by a cytostatic rather than a cytotoxic mechanism. Consequently, we studied the cell cycle distribution and the activation of caspase-3, measuring the presence of the cleavage product of poly(ADP-ribose) polymerases (PARP), and we evaluated the presence of apoptotic DNA by using a monoclonal antibody specific for the single-stranded regions of DNA. All our results indicate that SMS induces apoptosis in activated lymphocytes.

Analytical method development for directed enzyme evolution research: a high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers.

PubMed

Sun, Baoguo; Miller, Gregory; Lee, Wan Yee; Ho, Kelvin; Crowe, Michael A; Partridge, Leslie

2013-01-04

Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality L-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a β-cyclobond 2000 analytical column separated ribose and ribitol in 2.3 min, a C(18) guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4 min which provided capability of screening 4×96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25 mM tetraborate with 5 mM β-cyclodextrin was used as electrolyte. 0.35%of D-ribose in L-ribose can be detected which can be translated into 99.3% ee of L-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement. Copyright © 2012 Elsevier B.V. All rights reserved.

Mechanical stiffness of TMJ condylar cartilage increases after artificial aging by ribose.

PubMed

Mirahmadi, Fereshteh; Koolstra, Jan Harm; Lobbezoo, Frank; van Lenthe, G Harry; Ghazanfari, Samaneh; Snabel, Jessica; Stoop, Reinout; Everts, Vincent

2018-03-01

Aging is accompanied by a series of changes in mature tissues that influence their properties and functions. Collagen, as one of the main extracellular components of cartilage, becomes highly crosslinked during aging. In this study, the aim was to examine whether a correlation exists between collagen crosslinking induced by artificial aging and mechanical properties of the temporomandibular joint (TMJ) condyle. To evaluate this hypothesis, collagen crosslinks were induced using ribose incubation. Porcine TMJ condyles were incubated for 7 days with different concentrations of ribose. The compressive modulus and stiffness ratio (incubated versus control) was determined after loading. Glycosaminoglycan and collagen content, and the number of crosslinks were analyzed. Tissue structure was visualized by microscopy using different staining methods. Concomitant with an increasing concentration of ribose, an increase of collagen crosslinks was found. The number of crosslinks increased almost 50 fold after incubation with the highest concentration of ribose. Simultaneously, the stiffness ratio of the samples showed a significant increase after incubation with the ribose. Pearson correlation analyses showed a significant positive correlation between the overall stiffness ratio and the crosslink level; the higher the number of crosslinks the higher the stiffness. The present model, in which ribose was used to mimic certain aspects of age-related changes, can be employed as an in vitro model to study age-related mechanical changes in the TMJ condyle. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mobilization of Ca2+ by Cyclic ADP-Ribose from the Endoplasmic Reticulum of Cauliflower Florets1

PubMed Central

Navazio, Lorella; Mariani, Paola; Sanders, Dale

2001-01-01

The NAD+ metabolite cADP-Rib (cADPR) elevates cytosolic free Ca2+ in plants and thereby plays a central role in signal transduction pathways evoked by the drought and stress hormone abscisic acid. cADPR is known to mobilize Ca2+ from the large vacuole of mature cells. To determine whether additional sites for cADPR-gated Ca2+ release reside in plant cells, microsomes from cauliflower (Brassica oleracea) inflorescences were subfractionated on sucrose density gradients, and the distribution of cADPR-elicited Ca2+ release was monitored. cADPR-gated Ca2+ release was detected in the heavy-density fractions associated with rough endoplasmic reticulum (ER). cADPR-dependent Ca2+ release co-migrated with two ER markers, calnexin and antimycin A-insensitive NADH-cytochrome c reductase activity. To investigate the possibility that contaminating plasma membrane in the ER-rich fractions was responsible for the observed release, plasma membrane vesicles were purified by aqueous two-phase partitioning, everted with Brij-58, and loaded with Ca2+: These vesicles failed to respond to cADPR. Ca2+ release evoked by cADPR at the ER was fully inhibited by ruthenium red and 8-NH2-cADPR, a specific antagonist of cADPR-gated Ca2+ release in animal cells. The presence of a Ca2+ release pathway activated by cADPR at higher plant ER reinforces the notion that, alongside the vacuole, the ER participates in Ca2+ signaling. PMID:11299392

CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells.

PubMed

Skvarova Kramarzova, Karolina; Osborn, Mark J; Webber, Beau R; DeFeo, Anthony P; McElroy, Amber N; Kim, Chong Jai; Tolar, Jakub

2017-06-14

Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 ( FANCD1 ) primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was employed to target and correct a FANCD1 gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase). FANCD1 function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.

26 CFR 1.401(k)-2 - ADP test.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 5 2012-04-01 2011-04-01 true ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual...(k)(3)(F), the ADP test is performed under the plan (determined without regard to disaggregation...

26 CFR 1.401(k)-2 - ADP test.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 5 2011-04-01 2011-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual...(k)(3)(F), the ADP test is performed under the plan (determined without regard to disaggregation...

Enantioselective synthesis of tetrafluorinated ribose and fructose.

PubMed

Linclau, Bruno; Boydell, A James; Timofte, Roxana S; Brown, Kylie J; Vinader, Victoria; Weymouth-Wilson, Alexander C

2009-02-21

A perfluoroalkylidene lithium mediated cyclisation approach for the enantioselective synthesis of a tetrafluorinated aldose (ribose) and of a tetrafluorinated ketose (fructose), both in the furanose and in the pyranose form, is described.

Co-targeting deoxyribonucleic acid-dependent protein kinase and poly(adenosine diphosphate-ribose) polymerase-1 promotes accelerated senescence of irradiated cancer cells.

PubMed

Azad, Arun; Bukczynska, Patricia; Jackson, Susan; Haupt, Ygal; Haput, Ygal; Cullinane, Carleen; McArthur, Grant A; Solomon, Benjamin

2014-02-01

To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination. Copyright © 2014. Published by Elsevier Inc.

Methods for the determination of intracellular levels of ribose phosphates.

PubMed

Camici, Marcella; Tozzi, Maria Grazia; Ipata, Piero Luigi

2006-10-31

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway or stem from the phosphorolytic cleavage of the N-glycosidic bond of ribonucleosides. The two major pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, can be readily interconverted by phosphopentomutase. Ribose-5-phosphate is also the direct precursor of 5-phosphoribosyl-1-pyrophosphate, which is used for both de novo and salvage synthesis of nucleotides. On the other hand, the phosphorolysis of deoxyribonucleosides is the major source of deoxyribose phosphates. While the destiny of the nucleobase stemming from nucleoside phosphorolysis has been extensively investigated, the fate of the sugar moiety has been somehow neglected. However, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. Nevertheless, many aspects of pentose phosphate metabolism, and the possible involvement of these compounds in a number of cellular processes still remain obscure. The comprehension of the role played by pentose phosphates may be greatly facilitated by the knowledge of their steady-state intracellular levels and of their changes in response to variations of intra- and extracellular signals.

45 CFR 95.621 - ADP reviews.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false ADP reviews. 95.621 Section 95.621 Public Welfare....621 ADP reviews. The Department will conduct periodic onsite surveys and reviews of State and local... the Department and State or local agencies prior to conducting such surveys or reviews, which may...

The benefits of ribose in cardiovascular disease.

PubMed

Pauly, D F; Johnson, C; St Cyr, J A

2003-02-01

Cardiovascular disease still ranks as the leading cause of death in men and women. Adults have tried to lower their risk of cardiovascular disease by improving their diet, quitting smoking, controlling blood pressure and exercising regularly. Additionally, many adults have turned to nutriceutical or natural products. Myocardial ischemia, produces a depression in myocardial tissue levels of high energy compounds, along with a compromise in myocardial function. Ribose, a naturally occurring sugar, has been extensively investigated, both in animal and clinical studies, as an agent to enhance the recovery of these depressed energy compounds. Results of these studies have been promising in enhancing the recovery of these energy molecules along with an improvement in myocardial function. Therefore, ribose should be considered as a potential agent in the treatment of ischemic cardiovascular disease.

NAD+ and vitamin B3: from metabolism to therapies.

PubMed

Sauve, Anthony A

2008-03-01

The role of NAD(+) metabolism in health and disease is of increased interest as the use of niacin (nicotinic acid) has emerged as a major therapy for treatment of hyperlipidemias and with the recognition that nicotinamide can protect tissues and NAD(+) metabolism in a variety of disease states, including ischemia/reperfusion. In addition, a growing body of evidence supports the view that NAD(+) metabolism regulates important biological effects, including lifespan. NAD(+) exerts potent effects through the poly(ADP-ribose) polymerases, mono-ADP-ribosyltransferases, and the recently characterized sirtuin enzymes. These enzymes catalyze protein modifications, such as ADP-ribosylation and deacetylation, leading to changes in protein function. These enzymes regulate apoptosis, DNA repair, stress resistance, metabolism, and endocrine signaling, suggesting that these enzymes and/or NAD(+) metabolism could be targeted for therapeutic benefit. This review considers current knowledge of NAD(+) metabolism in humans and microbes, including new insights into mechanisms that regulate NAD(+) biosynthetic pathways, current use of nicotinamide and nicotinic acid as pharmacological agents, and opportunities for drug design that are directed at modulation of NAD(+) biosynthesis for treatment of human disorders and infections.

Structure-function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa.

PubMed

Yates, Susan P; Taylor, Patricia L; Jørgensen, René; Ferraris, Dana; Zhang, Jie; Andersen, Gregers R; Merrill, A Rod

2005-02-01

The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD+ to its protein substrate. A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD+ was investigated for their inhibition of the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC50 values. Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible. One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with an inhibition constant K(i) of 140 nM. We also report the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor. The 2.1 A (1 A=0.1 nm) resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side-chain oxygen of Gln-485, a member of a proposed catalytic loop. Structural comparison of this inhibitor complex with diphtheria toxin (a mono-ADPRT) and with PARPs [poly(ADP-ribose) polymerases] shows similarity of the catalytic residues; however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP. The present study provides insight into the important features required for inhibitors that mimic NAD+ and their binding to the mono-ADPRT family of toxins.

Adenovirus Death Protein (ADP) Is Required for Lytic Infection of Human Lymphocytes

PubMed Central

Murali, V. K.; Ornelles, D. A.; Gooding, L. R.; Wilms, H. T.; Huang, W.; Tollefson, A. E.; Wold, W. S. M.

2014-01-01

The adenovirus death protein (ADP) is expressed at late times during a lytic infection of species C adenoviruses. ADP promotes the release of progeny virus by accelerating the lysis and death of the host cell. Since some human lymphocytes survive while maintaining a persistent infection with species C adenovirus, we compared ADP expression in these cells with ADP expression in lymphocytes that proceed with a lytic infection. Levels of ADP were low in KE37 and BJAB cells, which support a persistent infection. In contrast, levels of ADP mRNA and protein were higher in Jurkat cells, which proceed with a lytic infection. Epithelial cells infected with an ADP-overexpressing virus died more quickly than epithelial cells infected with an ADP-deleted virus. However, KE37, and BJAB cells remained viable after infection with the ADP-overexpressing virus. Although the levels of ADP mRNA increased in KE37 and BJAB cells infected with the ADP-overexpressing virus, the fraction of cells with detectable ADP was unchanged, suggesting that the control of ADP expression differs between epithelial and lymphocytic cells. When infected with an ADP-deleted adenovirus, Jurkat cells survived and maintained viral DNA for greater than 1 month. These findings are consistent with the notion that the level of ADP expression determines whether lymphocytic cells proceed with a lytic or a persistent adenovirus infection. PMID:24198418

Synthesis and SAR of novel tricyclic quinoxalinone inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyashiro, Julie; Woods, Keith W.; Park, Chang H.

2010-09-03

Based on screening hit 1, a series of tricyclic quinoxalinones have been designed and evaluated for inhibition of PARP-1. Substitutions at the 7- and 8-positions of the quinoxalinone ring led to a number of compounds with good enzymatic and cellular potency. The tricyclic quinoxalinone class is sensitive to modifications of both the amine substituent and the tricyclic core. The synthesis and structure-activity relationship studies are presented.

D-ribose--an additive with caffeine.

PubMed

Herrick, Jim; Shecterle, L M; St Cyr, J A

2009-05-01

Caffeine acts as a stimulant, in which approximately 90% of people in the United States consume daily. Besides its beneficial effects, many individuals have experienced unpleasant reactions following the consumption of caffeine: such as insomnia, an increase in heart rate, feelings of nervousness, headaches, occasional lightheadedness, a state of "jitters," and a potential "crash" state following its metabolism. Researchers have proposed mechanisms responsible for caffeine's interactions, which include its blocking capacity of adenosine receptors, its role with the pituitary gland, increasing levels of dopamine, and its role with the intracellular release of calcium from the sarcoplasmic reticulum, which is dependent on intracellular adenosine triphosphate levels. Specific substrates have been investigated to lessen the undesirable effects of caffeine and still preserve its stimulatory benefits. The results of these investigations have produced no positive consensus. However, D-ribose, an important pentose carbohydrate in the energy molecule of adenosine triphosphate, as well as our genetic code and other cellular processes, could offer such a solution to this problem. D-ribose could potentially aid in maintaining or potentially lowering extra-cellular adenosine concentrations, aid in the flux of intracellular calcium, aid in intracellular energy production, and potentially lessen the perceived "crash" state felt by many. Every cell requires adequate levels of energy to maintain its integrity and function. Caffeine has the potential to task this energy equilibrium. D-ribose with caffeine may be the substrate to aid in the potential intracellular energy demand, aid in lessening the perceived unpleasant side effects of caffeine, and still preserving the desired benefits of this stimulant consumed by all of us daily.

Effects of oral administration of caffeine and D-ribose on mental fatigue.

PubMed

Ataka, Suzuka; Tanaka, Masaaki; Nozaki, Satoshi; Mizuma, Hiroshi; Mizuno, Kei; Tahara, Tsuyoshi; Sugino, Tomohiro; Shirai, Tomoko; Kajimoto, Yoshitaka; Kuratsune, Hirohiko; Kajimoto, Osami; Watanabe, Yasuyoshi

2008-03-01

We examined the effects of administering two different candidate antifatigue substances, caffeine and D-ribose, on mental fatigue. In a double-blinded, placebo-controlled, three-way crossover design, 17 healthy volunteers were randomized to oral caffeine (200 mg/d), D-ribose (2000 mg/d), or placebo for 8 d. As fatigue-inducing mental tasks, subjects performed a 30-min Uchida-Kraepelin psychodiagnostic test and a 30-min advanced trail-making test on four occasions. During the tasks, the task performance of the caffeine group was better than that of the placebo group. However, after the fatigue-inducing tasks, although subjective perception of fatigue, motivation, or sleepiness was not significantly different, plasma branched-chain amino acid levels in the caffeine group were lower than those of the placebo group. Administration of D-ribose had no effect. Because plasma branched-chain amino acid levels are decreased by mental fatigue, these results suggest that administration of caffeine improved task performance through the enhancement of central nervous system activity without increasing the sensation of fatigue. However, further decreases in branched-chain amino acid levels indicate that caffeine might promote deeper fatigue than placebo. Unfortunately, research subsequent to our study design has shown that D-ribose dosing higher than we used is needed to see a clinical effect and therefore no conclusions can be made from this study as to the efficacy of D-ribose.

Oligonucleotide microarray analysis of apoptosis induced by 15-methoxypinusolidic acid in microglial BV2 cells

PubMed Central

Choi, Y; Lim, SY; Jeong, HS; Koo, KA; Sung, SH; Kim, YC

2009-01-01

Background and purpose: We conducted a genome wide gene expression analysis to explore the biological aspects of 15-methoxypinusolidic acid (15-MPA) isolated from Biota orientalis and tried to confirm the suitability of 15-MPA as a therapeutic candidate for CNS injuries focusing on microglia. Experimental approach: Murine microglial BV2 cells were treated with 15-MPA, and their transcriptome was analysed by using oligonucleotide microarrays. Genes differentially expressed upon 15-MPA treatment were selected for RT-PCR (reverse transcription-polymerase chain reaction) analysis to confirm the gene expression. Inhibition of cell proliferation and induction of apoptosis by 15-MPA were examined by bromodeoxyuridine assay, Western blot analysis of poly-ADP-ribose polymerase and flow cytometry. Key results: A total of 514 genes were differentially expressed by 15-MPA treatment. Biological pathway analysis revealed that 15-MPA induced significant changes in expression of genes in the cell cycle pathway. Genes involved in growth arrest and DNA damage [gadd45α, gadd45γ and ddit3 (DNA damage-inducible transcript 3)] and cyclin-dependent kinase inhibitor (cdkn2b) were up-regulated, whereas genes involved in cell cycle progression (ccnd1, ccnd3 and ccne1), DNA replication (mcm4, orc1l and cdc6) and cell proliferation (fos and jun) were down-regulated. RT-PCR analysis for representative genes confirmed the expression levels. 15-MPA significantly reduced bromodeoxyuridine incorporation, increased poly-ADP-ribose polymerase cleavage and the number of apoptotic cells, indicating that 15-MPA induces apoptosis in BV2 cells. Conclusion and implications: 15-MPA induced apoptosis in murine microglial cells, presumably via inhibition of the cell cycle progression. As microglial activation is detrimental in CNS injuries, these data suggest a strong therapeutic potential of 15-MPA. PMID:19466985

Cytoprotective dibenzoylmethane derivatives protect cells from oxidative stress-induced necrotic cell death.

PubMed

Hegedűs, Csaba; Lakatos, Petra; Kiss-Szikszai, Attila; Patonay, Tamás; Gergely, Szabolcs; Gregus, Andrea; Bai, Péter; Haskó, György; Szabó, Éva; Virág, László

2013-06-01

Screening of a small in-house library of 1863 compounds identified 29 compounds that protected Jurkat cells from hydrogen peroxide-induced cytotoxicity. From the cytoprotective compounds eleven proved to possess antioxidant activity (ABTS radical scavenger effect) and two were found to inhibit poly(ADP-ribosyl)ation (PARylation), a cytotoxic pathway operating in severely injured cells. Four cytoprotective dibenzoylmethane (DBM) derivatives were investigated in more detail as they did not scavenge hydrogen peroxide nor did they inhibit PARylation. These compounds protected cells from necrotic cell death while caspase activation, a parameter of apoptotic cell death was not affected. Hydrogen peroxide activated extracellular signal regulated kinase (ERK1/2) and p38 MAP kinases but not c-Jun N-terminal kinase (JNK). The cytoprotective DBMs suppressed the activation of Erk1/2 but not that of p38. Cytoprotection was confirmed in another cell type (A549 lung epithelial cells), indicating that the cytoprotective effect is not cell type specific. In conclusion we identified DBM analogs as a novel class of cytoprotective compounds inhibiting ERK1/2 kinase and protecting from necrotic cell death by a mechanism independent of poly(ADP-ribose) polymerase inhibition. Copyright © 2013 Elsevier Ltd. All rights reserved.

Functional genomic analysis of drug sensitivity pathways to guide adjuvant strategies in breast cancer

PubMed Central

Swanton, Charles; Szallasi, Zoltan; Brenton, James D; Downward, Julian

2008-01-01

The widespread introduction of high throughput RNA interference screening technology has revealed tumour drug sensitivity pathways to common cytotoxics such as paclitaxel, doxorubicin and 5-fluorouracil, targeted agents such as trastuzumab and inhibitors of AKT and Poly(ADP-ribose) polymerase (PARP) as well as endocrine therapies such as tamoxifen. Given the limited power of microarray signatures to predict therapeutic response in associative studies of small clinical trial cohorts, the use of functional genomic data combined with expression or sequence analysis of genes and microRNAs implicated in drug response in human tumours may provide a more robust method to guide adjuvant treatment strategies in breast cancer that are transferable across different expression platforms and patient cohorts. PMID:18986507

Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach.

PubMed

Anumala, Upendra Rao; Waaler, Jo; Nkizinkiko, Yves; Ignatev, Alexander; Lazarow, Katina; Lindemann, Peter; Olsen, Petter Angell; Murthy, Sudarshan; Obaji, Ezeogo; Majouga, Alexander G; Leonov, Sergey; von Kries, Jens Peter; Lehtiö, Lari; Krauss, Stefan; Nazaré, Marc

2017-12-28

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC 50 values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.

Recent advances in targeting DNA repair pathways for the treatment of ovarian cancer and their clinical relevance.

PubMed

Oda, Katsutoshi; Tanikawa, Michihiro; Sone, Kenbun; Mori-Uchino, Mayuyo; Osuga, Yutaka; Fujii, Tomoyuki

2017-08-01

Poly (ADP-ribose) polymerase (PARP) inhibitors have attracted much attention as one of the major molecular-targeted therapeutics for inhibiting DNA damage response. The PARP inhibitor, olaparib, has been clinically applied for treating certain recurrent ovarian cancer patients with BRCA1/2 mutations in Europe and the United States. It was also designated on 24 March 2017 as an orphan drug in Japan for similar clinical indications. In this review, we discuss (i) the prevalence of BRCA1/2 mutations in ovarian cancer, (ii) clinical trials of PARP inhibitors in ovarian cancer, (iii) genetic counseling for hereditary breast and ovarian cancer patients, and (iv) non-BRCA genes that may be associated with homologous recombination deficiency.

Identification of a receptor for ADP on blood platelets by photoaffinity labelling.

PubMed Central

Cristalli, G; Mills, D C

1993-01-01

The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5'-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase. Images Figure 5 PMID:8387782

ADP Analysis project for the Human Resources Management Division

NASA Technical Reports Server (NTRS)

Tureman, Robert L., Jr.

1993-01-01

The ADP (Automated Data Processing) Analysis Project was conducted for the Human Resources Management Division (HRMD) of NASA's Langley Research Center. The three major areas of work in the project were computer support, automated inventory analysis, and an ADP study for the Division. The goal of the computer support work was to determine automation needs of Division personnel and help them solve computing problems. The goal of automated inventory analysis was to find a way to analyze installed software and usage on a Macintosh. Finally, the ADP functional systems study for the Division was designed to assess future HRMD needs concerning ADP organization and activities.

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

PubMed Central

Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

2006-01-01

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

Preferential uptake of ribose by primitive cells might explain why RNA was favored over its analogs

NASA Astrophysics Data System (ADS)

Pohorille, Andrew; Wei, Chenyu

perme-ation, even though it is non-negligibly populated in aqueous solution. The differences in free energy barrier between ribose and arabinose or xylose are due to stronger, highly cooperative, intramolecular interactions between consecutive exocyclic hydroxyl groups, which are stable in non-polar media, but rare in water. Most recently, we extended calculations of permeations to ribonucleosides and their anomers. We determined that, in contrast to sugars, permeation of membranes to these species is nearly identical. This is because sugars of nucleotides exist in the furanose rather than pyranose form. In this form intermolecular interactions between hydroxyl groups are not nearly as efficient for sterical reasons. Our results contribute to the discussion about autotrophic vs. heterotrophic origins of life. Chemical reactions inside protobiological vesicle required supply of organic material from the environment. What was the inventory of organics that must have been delivered to primitive cells is still being debated. According to the autotrophic hypothesis, ancestors of cells pro-duced complex organic molecules from simple substrates. In contrast, the heterotrophic model implies that protocells were able to utilize complex organics delivered from external sources. A possibility of sufficiently efficient uptake of molecules needed to build biopolymers provides an important argument supporting the heterotrophic hypothesis [3]. Viewed in the context of the "RNA world" hypothesis [4], which states that RNA molecules were the first biological poly-mers and acted as both catalysts of biochemical reactions and information storage systems, our results demonstrate that, in the absence of sophisticated mechanisms available to contemporary organisms for achieving selectivity during synthesis and transmembrane transport, preferential uptake of ribose by primitive cells might have provided a kinetic mechanism that favored its selective incorporation into nucleic acids and

The Relative Reactivity of Deoxyribose and Ribose: Did DNA Come Before RNA?

NASA Technical Reports Server (NTRS)

Dworkin, Jason P.; Miller, Stanley L.

1995-01-01

If it is assumed that there was a precursor to the ribose-phosphate backbone of RNA in the preRNA world (such as peptide nucleic acid), then the entry of various sugars into the genetic material may be related to the stability and non-enzymatic reactivity of the aldose. The rate of decomposition of 2-deoxyribose has been determined to be 1/3 that of ribose. In addition we have measured the amount of free aldehyde by H-1 and C-13 NMR and find that it has approximately 0.15% free aldehyde compared to 0.05% for ribose at 25 C. This suggests that deoxyribose would be significantly more reactive with early bases in the absence of enzymes. This is confirmed by urazole and deoxyribose reacting to form the deoxynucleoside 45 times faster as 25 C than urazole reacts with ribose to form the Ribonucleoside. Urazole is a potential precursor of uracil and is a plausible prebiotic compound which reacts with aldoses to form nucleosides. Thus the non-enzymatic reactivity of deoxyribose would favor its early use over ribose until enzymes could change the relative reactivities. Most of the reasons that RNA is presumed to have come before DNA are extrapolations back from contemporary metabolism (e.g. the abundance of ribose based coenzymes, the biosynthesis of histidine, deoxyribonucleotides are synthesized from ribonucleotides, etc.). It is very difficult to reconstruct biochemical pathways much before the last common ancestor, and it is even more difficult to do more than guess at the biochemistry of very early self-replicating systems. Thus we believe that these reasons are not compelling and that the non-enzymatic chemistry may be more important than enzymatic pathways for constructing the earliest of biochemical pathways. While the RNA world has been discussed at great length, there has not been an exploration of the transition out of the RNA world. We have constructed many possible schemes of genetic takeover events from preRNA to modern DNA, RNA, protein system which could

Efficient biosynthesis of d-ribose using a novel co-feeding strategy in Bacillus subtilis without acid formation.

PubMed

Cheng, J; Zhuang, W; Li, N N; Tang, C L; Ying, H J

2017-01-01

Normally, low d-ribose production was identified as responsible for plenty of acid formation by Bacillus subtilis due to its carbon overflow. An approach of co-feeding glucose and sodium citrate is developed here and had been proved to be useful in d-ribose production. This strategy is critical because it affects the cell concentration, the productivity of d-ribose and, especially, the formation of by-products such as acetoin, lactate and acetate. d-ribose production was increased by 59·6% from 71·06 to 113·41 g l -1 without acid formation by co-feeding 2·22 g l -1  h -1 glucose and 0·036 g l -1  h -1 sodium citrate to a 60 g l -1 glucose reaction system. Actually, the cell density was also enhanced from 11·51 to 13·84 g l -1 . These parameters revealed the importance of optimization and modelling of the d-ribose production process. Not only could zero acid formation was achieved over a wide range of co-feeding rate by reducing glycolytic flux drastically but also the cell density and d-ribose yield were elevated by increasing the hexose monophosphate pathway flux. Bacillus subtilis usually produce d-ribose accompanied by plenty of organic acids when glucose is used as a carbon source, which is considered to be a consequence of mismatched glycolytic and tricarboxylic acid cycle capacities. This is the first study to provide high-efficiency biosynthesis of d-ribose without organic acid formation in B. subtilis, which would be lower than the cost of separation and purification. The strain transketolase-deficient B. subtilis CGMCC 3720 can be potentially applied to the production of d-ribose in industry. © 2016 The Society for Applied Microbiology.

ADP-ribosyl cyclases regulate early development of the sea urchin.

PubMed

Ramakrishnan, Latha; Uhlinger, Kevin; Dale, Leslie; Hamdoun, Amro; Patel, Sandip

2016-06-01

ADP-ribosyl cyclases are multifunctional enzymes involved in the metabolism of nucleotide derivatives necessary for Ca 2+ signalling such as cADPR and NAADP. Although Ca 2+ signalling is a critical regulator of early development, little is known of the role of ADP-ribosyl cyclases during embryogenesis. Here we analyze the expression, activity and function of ADP-ribosyl cyclases in the embryo of the sea urchin - a key organism for study of both Ca 2+ signalling and embryonic development. ADP-ribosyl cyclase isoforms (SpARC1-4) showed unique changes in expression during early development. These changes were associated with an increase in the ratio of cADPR:NAADP production. Over-expression of SpARC4 (a preferential cyclase) disrupted gastrulation. Our data highlight the importance of ADP-ribosyl cyclases during embryogenesis.

Zinc is released by cultured astrocytes as a gliotransmitter under hypoosmotic stress-loaded conditions and regulates microglial activity.

PubMed

Segawa, Shohei; Nishiura, Takeshi; Furuta, Takahiro; Ohsato, Yuki; Tani, Misaki; Nishida, Kentaro; Nagasawa, Kazuki

2014-01-17

Astrocytes contribute to the maintenance of brain homeostasis via the release of gliotransmitters such as ATP and glutamate. Here we examined whether zinc was released from astrocytes under stress-loaded conditions, and was involved in the regulation of microglial activity as a gliotransmitter. Hypoosmotic stress was loaded to astrocytes using balanced salt solution prepared to 214-314 mOsmol/L, and then intra- and extra-cellular zinc levels were assessed using Newport Green DCF diacetate (NG) and ICP-MS, respectively. Microglial activation by the astrocytic supernatant was assessed by their morphological changes and poly(ADP-ribose) (PAR) polymer accumulation. Exposure of astrocytes to hypoosmotic buffer, increased the extracellular ATP level in osmolarity-dependent manners, indicating a load of hypoosmotic stress. In hypoosmotic stress-loaded astrocytes, there were apparent increases in the intra- and extra-cellular zinc levels. Incubation of microglia in the astrocytic conditioned medium transformed them into the activated "amoeboid" form and induced PAR formation. Administration of an extracellular zinc chelator, CaEDTA, to the astrocytic conditioned medium almost completely prevented the microglial activation. Treatment of astrocytes with an intracellular zinc chelator, TPEN, suppressed the hypoosmotic stress-increased intracellular, but not the extracellular, zinc level, and the increase in the intracellular zinc level was blocked partially by a nitric oxide synthase inhibitor, but not by CaEDTA, indicating that the mechanisms underlying the increases in the intra- and extra-cellular zinc levels might be different. These findings suggest that under hypoosmotic stress-loaded conditions, zinc is released from astrocytes and then plays a primary role in microglial activation as a gliotransmitter. Copyright © 2013 Elsevier Inc. All rights reserved.

Thermal Behavior of d-Ribose Adsorbed on Silica: Effect of Inorganic Salt Coadsorption and Significance for Prebiotic Chemistry.

PubMed

Akouche, Mariame; Jaber, Maguy; Zins, Emilie-Laure; Maurel, Marie-Christine; Lambert, Jean-Francois; Georgelin, Thomas

2016-10-24

Understanding ribose reactivity is a crucial step in the "RNA world" scenario because this molecule is a component of all extant nucleotides that make up RNA. In solution, ribose is unstable and susceptible to thermal destruction. We examined how ribose behaves upon thermal activation when adsorbed on silica, either alone or with the coadsorption of inorganic salts (MgCl 2 , CaCl 2 , SrCl 2 , CuCl 2 , FeCl 2 , FeCl 3 , ZnCl 2 ). A combination of 13 C NMR, in situ IR, and TGA analyses revealed a variety of phenomena. When adsorbed alone, ribose remains stable up to 150 °C, at which point ring opening is observed, together with minor oxidation to a lactone. All the metal salts studied showed specific interactions with ribose after dehydration, resulting in the formation of polydentate metal ion complexes. Anomeric equilibria were affected, generally favoring ribofuranoses. Zn 2+ stabilized ribose up to higher temperatures than bare silica (180 to 200 °C). Most other cations had an adverse effect on ribose stability, with ring opening already upon drying at 70 °C. In addition, alkaline earth cations catalyzed the dehydration of ribose to furfural and, to variable degrees, its further decarbonylation to furan. Transition-metal ions with open d-shells took part in redox reactions with ribose, either as reagents or as catalysts. These results allow the likelihood of prebiotic chemistry scenarios to be evaluated, and may also be of interest for the valorization of biomass-derived carbohydrates by heterogeneous catalysis. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ribose mediated crosslinking of collagen-hydroxyapatite hybrid scaffolds for bone tissue regeneration using biomimetic strategies.

PubMed

Krishnakumar, Gopal Shankar; Gostynska, Natalia; Campodoni, Elisabetta; Dapporto, Massimiliano; Montesi, Monica; Panseri, Silvia; Tampieri, Anna; Kon, Elizaveta; Marcacci, Maurilio; Sprio, Simone; Sandri, Monica

2017-08-01

This study explores for the first time the application of ribose as a highly biocompatible agent for the crosslinking of hybrid mineralized constructs, obtained by bio-inspired mineralization of self-assembling Type I collagen matrix with magnesium-doped-hydroxyapatite nanophase, towards a biomimetic mineralized 3D scaffolds (MgHA/Coll) with excellent compositional and structural mimicry of bone tissue. To this aim, two different crosslinking mechanisms in terms of pre-ribose glycation (before freeze drying) and post-ribose glycation (after freeze drying) were investigated. The obtained results explicate that with controlled freeze-drying, highly anisotropic porous structures with opportune macro-micro porosity are obtained. The physical-chemical features of the scaffolds characterized by XRD, FTIR, ICP and TGA demonstrated structural mimicry analogous to the native bone. The influence of ribose greatly assisted in decreasing solubility and increased enzymatic resistivity of the scaffolds. In addition, enhanced mechanical behaviour in response to compressive forces was achieved. Preliminary cell culture experiments reported good cytocompatibility with extensive cell adhesion, proliferation and colonization. Overall, scaffolds developed by pre-ribose glycation process are preferred, as the related crosslinking technique is more facile and robust to obtain functional scaffolds. As a proof of concept, we have demonstrated that ribose crosslinking is cost-effective, safe and functionally effective. This study also offers new insights and opportunities in developing promising scaffolds for bone tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

The route of non-enzymic and enzymic breakdown of 5-phosphoribosyl 1-pyrophosphate to ribose 1-phosphate.

PubMed Central

Trembacz, H; Jezewska, M M

1990-01-01

Spontaneous decomposition of 5-phosphoribosyl 1-pyrophosphate at pH 5.5 was established to occur as follows: 5-Phosphoribosyl 1-pyrophosphate----5-phosphoribosyl 1,2-(cyclic)phosphate----ribose 1-phosphate----ribose Enzymic degradation of 5-phosphoribosyl 1-pyrophosphate by alkaline phosphatase from calf intestine and by acid phosphatases from potato and Aspergillus niger was found to proceed according to this pathway within the pH range 2.5-7.4 with accumulation of ribose 1-phosphate. In the case of alkaline phosphatase, Mg2+ ions inhibit the pyrophosphorolysis of 5-phosphoribosyl 1-pyrophosphate and stimulate the hydrolysis of ribose 1-phosphate. PMID:1700897

Poly (ADP-ribose) polymerase inhibitor CEP-8983 synergizes with bendamustine in chronic lymphocytic leukemia cells in vitro

PubMed Central

Dilley, Robert L.; Poh, Weijie; Gladstone, Douglas E.; Herman, James G.; Showel, Margaret M.; Karp, Judith E.; McDevitt, Michael A.; Pratz, Keith W.

2014-01-01

DNA repair aberrations and associated chromosomal instability is a feature of chronic lymphocytic leukemia (CLL). To evaluate if DNA repair insufficiencies are related to methylation changes, we examined the methylation of nine promoter regions of DNA repair proteins by bisulfide sequencing in 26 CLL primary samples and performed quantitative PCR on a subset of samples to examine BRCA1 expression. We also investigated if changes in cytogenetic or expression level of DNA repair proteins led to changes in sensitivity to a novel PARP inhibitor, CEP-8983, alone and in combination with bendamustine. No changes in promoter methylation were identified in BRCA1, BRCA2, FANC-C, FANC-F, FANC-L, ATM, MGMT, hMLH1 and H2AX except for two cases of minor BRCA1 hypermethylation. CLL samples appeared to have reduced BRCA1 mRNA expression uniformly in comparison to non-malignant lymphocytes irrespective of promoter hypermethylation. CEP-8983 displayed single agent cytotoxicity and the combination with bendamustine demonstrated synergistic cytotoxicity in the majority of CLL samples. These results were consistent across cytogenetic subgroups, including 17p deleted and previously treated patients. Our results provide rationale for further exploration of the combination of a PARP inhibitor and DNA damaging agents as a novel therapeutic strategy in CLL. PMID:24439051

Poly(ADP-ribose) polymerase inhibitor CEP-8983 synergizes with bendamustine in chronic lymphocytic leukemia cells in vitro.

PubMed

Dilley, Robert L; Poh, Weijie; Gladstone, Douglas E; Herman, James G; Showel, Margaret M; Karp, Judith E; McDevitt, Michael A; Pratz, Keith W

2014-03-01

DNA repair aberrations and associated chromosomal instability is a feature of chronic lymphocytic leukemia (CLL). To evaluate if DNA repair insufficiencies are related to methylation changes, we examined the methylation of nine promoter regions of DNA repair proteins by bisulfide sequencing in 26 CLL primary samples and performed quantitative PCR on a subset of samples to examine BRCA1 expression. We also investigated if changes in cytogenetic or expression level of DNA repair proteins led to changes in sensitivity to a novel PARP inhibitor, CEP-8983, alone and in combination with bendamustine. No changes in promoter methylation were identified in BRCA1, BRCA2, FANC-C, FANC-F, FANC-L, ATM, MGMT, hMLH1 and H2AX except for two cases of minor BRCA1 hypermethylation. CLL samples appeared to have reduced BRCA1 mRNA expression uniformly in comparison to non-malignant lymphocytes irrespective of promoter hypermethylation. CEP-8983 displayed single agent cytotoxicity and the combination with bendamustine demonstrated synergistic cytotoxicity in the majority of CLL samples. These results were consistent across cytogenetic subgroups, including 17p deleted and previously treated patients. Our results provide rationale for further exploration of the combination of a PARP inhibitor and DNA damaging agents as a novel therapeutic strategy in CLL. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of Novel Cytoplasmic PARP in the Brain of Octopus vulgaris

PubMed Central

DE LISA, EMILIA; DE MAIO, ANNA; MOROZ, LEONID L.; MOCCIA, FRANCESCO; MENNELLA, MARIA ROSARIA FARAONE; DI COSMO, ANNA

2014-01-01

Recent investigation has focused on the participation of the poly (ADP-ribose) polymerase (PARP) reaction in the invertebrate central nervous system (CNS) during the process of long-term memory (LTM). In this paper, we characterize, localize, and assign a possible role to a cytoplasmic PARP in the brain of Octopus vulgaris. PARP activity was assayed in optic lobes, supraesophageal mass, and optic nerves. The highest levels of enzyme were found in the cytoplasmic fraction. Hyper-activation of the enzyme was detected in Octopus brain after visual discrimination training. Finally, cytoplasmic PARP was found to inhibit Octopus vulgaris actin polymerization. We propose that the cytoplasmic PARP plays a role in vivo to induce the cytoskeletonal reorganization that occurs during learning-induced neuronal plasticity. PMID:22815366

The dynamic regulation of NAD metabolism in mitochondria

PubMed Central

Stein, Liana Roberts; Imai, Shin-ichiro

2012-01-01

Mitochondria are intracellular powerhouses that produce ATP and carry out diverse functions for cellular energy metabolism. While the maintenance of an optimal NAD/NADH ratio is essential for mitochondrial function, it has recently become apparent that the maintenance of the mitochondrial NAD pool also has critical importance. The biosynthesis, transport, and catabolism of NAD and its key intermediates play an important role in the regulation of NAD-consuming mediators, such as sirtuins, poly-ADP-ribose polymerases, and CD38/157 ectoenzymes, in intra- and extracellular compartments. Mitochondrial NAD biosynthesis is also modulated in response to nutritional and environmental stimuli. In this article, we discuss this dynamic regulation of NAD metabolism in mitochondria to shed light on the intimate connection between NAD and mitochondrial function. PMID:22819213

Mechanisms of the cytopathic action of actin-ADP-ribosylating toxins.

PubMed

Aktories, K; Wegner, A

1992-10-01

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.

Structural insights into SAM domain-mediated tankyrase oligomerization.

PubMed

DaRosa, Paul A; Ovchinnikov, Sergey; Xu, Wenqing; Klevit, Rachel E

2016-09-01

Tankyrase 1 (TNKS1; a.k.a. ARTD5) and tankyrase 2 (TNKS2; a.k.a ARTD6) are highly homologous poly(ADP-ribose) polymerases (PARPs) that function in a wide variety of cellular processes including Wnt signaling, Src signaling, Akt signaling, Glut4 vesicle translocation, telomere length regulation, and centriole and spindle pole maturation. Tankyrase proteins include a sterile alpha motif (SAM) domain that undergoes oligomerization in vitro and in vivo. However, the SAM domains of TNKS1 and TNKS2 have not been structurally characterized and the mode of oligomerization is not yet defined. Here we model the SAM domain-mediated oligomerization of tankyrase. The structural model, supported by mutagenesis and NMR analysis, demonstrates a helical, homotypic head-to-tail polymer that facilitates TNKS self-association. Furthermore, we show that TNKS1 and TNKS2 can form (TNKS1 SAM-TNKS2 SAM) hetero-oligomeric structures mediated by their SAM domains. Though wild-type tankyrase proteins have very low solubility, model-based mutations of the SAM oligomerization interface residues allowed us to obtain soluble TNKS proteins. These structural insights will be invaluable for the functional and biophysical characterization of TNKS1/2, including the role of TNKS oligomerization in protein poly(ADP-ribosyl)ation (PARylation) and PARylation-dependent ubiquitylation. © 2016 The Protein Society.

Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins.

PubMed

Aktories, K

1994-09-01

The actin cytoskeleton appears to be as the cellular target of various clostridial ADP-ribosyltransferases which have been described during recent years. Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium spiroforme toxin ADP-ribosylate actin monomers and inhibit actin polymerization. Clostridium botulinum exoenzyme C3 and Clostridium limosum exoenzyme ADP-ribosylate the low-molecular-mass GTP-binding proteins of the Rho family, which participate in the regulation of the actin cytoskeleton. ADP-ribosylation inactivates the regulatory Rho proteins and disturbs the organization of the actin cytoskeleton.

FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

PubMed Central

Marshall, Kathryn E.; Robinson, Errol W.; Hengel, Shawna M.; Paša-Tolić, Ljiljana; Roesijadi, Guritno

2012-01-01

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation. PMID:22470473

FRET imaging of diatoms expressing a biosilica-localized ribose sensor.

PubMed

Marshall, Kathryn E; Robinson, Errol W; Hengel, Shawna M; Paša-Tolić, Ljiljana; Roesijadi, Guritno

2012-01-01

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation.

Polymeric mixed micelles loaded mitoxantrone for overcoming multidrug resistance in breast cancer via photodynamic therapy

PubMed Central

Zhao, Yiqiao; Yu, Hua; Zhou, Haiyu; Chen, Meiwan

2017-01-01

Mitoxantrone (MIT) is an anticancer agent with photosensitive properties that is commonly used in various cancers. Multidrug resistance (MDR) effect has been an obstacle to using MIT for cancer therapy. Photochemical internalization, on account of photodynamic therapy, has been applied to improve the therapeutic effect of cancers with MDR effect. In this study, an MIT-poly(ε-caprolactone)-pluronic F68-poly(ε-caprolactone)/poly(d,l-lactide-co-glycolide)–poly(ethylene glycol)–poly(d,l-lactide-co-glycolide) (MIT-PFP/PPP) mixed micelles system was applied to reverse the effect of MDR in MCF-7/ADR cells via photochemical reaction when exposed to near-infrared light. MIT-PFP/PPP mixed micelles showed effective interaction with near-infrared light at the wavelength of 660 nm and exerted great cytotoxicity in MCF-7/ADR cells with irradiation. Furthermore, MIT-PFP/PPP mixed micelles could improve reactive oxygen species (ROS) levels, decrease P-glycoprotein activity, and increase the cellular uptake of drugs with improved intracellular drug concentrations, which induced cell apoptosis in MCF-7/ADR cells under irradiation, despite MDR effect, as indicated by the increased level of cleaved poly ADP-ribose polymerase. These findings suggested that MIT-PFP/PPP mixed micelles may become a promising strategy to effectively reverse the MDR effect via photodynamic therapy in breast cancer. PMID:28919756

Growth and gas production of a novel obligatory heterofermentative Cheddar cheese nonstarter lactobacilli species on ribose and galactose.

PubMed

Ortakci, Fatih; Broadbent, Jeffery R; Oberg, Craig J; McMahon, Donald J

2015-06-01

An obligatory heterofermentative lactic acid bacterium, Lactobacillus wasatchii sp. nov., isolated from gassy Cheddar cheese was studied for growth, gas formation, salt tolerance, and survival against pasteurization treatments at 63°C and 72°C. Initially, Lb. wasatchii was thought to use only ribose as a sugar source and we were interested in whether it could also utilize galactose. We conducted experiments to determine the rate and extent of growth and gas production in carbohydrate-restricted (CR) de Man, Rogosa, and Sharpe (MRS) medium under anaerobic conditions with various combinations of ribose and galactose at 12, 23, and 37°C, with 23°C being the optimum growth temperature of Lb. wasatchii among the 3 temperatures studied. When Lb. wasatchii was grown on ribose (0.1, 0.5, and 1%), maximum specific growth rates (µmax) within each temperature were similar. When galactose was the only sugar, compared with ribose, µmax was 2 to 4 times lower. At all temperatures, the highest final cell densities (optical density at 640 nm) of Lb. wasatchii were achieved in CR-MRS plus 1% ribose, 0.5% ribose and 0.5% galactose, or 1% ribose and 1% galactose. Similar µmax values and final cell densities were achieved when 50% of the ribose in CR-MRS was substituted with galactose. Such enhanced utilization of galactose in the presence of ribose to support bacterial growth has not previously been reported. It appears that Lb. wasatchii co-metabolizes ribose and galactose, utilizing ribose for energy and galactose for other functions such as cell wall biosynthesis. Co-utilization of both sugars could be an adaptation mechanism of Lb. wasatchii to the cheese environment to efficiently ferment available sugars for maximizing metabolism and growth. As expected, gas formation by the heterofermenter was observed only when galactose was present in the medium. Growth experiments with MRS plus 1.5% ribose at pH 5.2 or 6.5 with 0, 1, 2, 3, 4, or 5% NaCl revealed that Lb. wasatchii is

Calcium modulates the ATP and ADP hydrolysis in human placental mitochondria.

PubMed

Martínez, Federico; Uribe, Aida; Espinosa-García, M Teresa; Flores-Herrera, Oscar; García-Pérez, Cecilia; Milán, Rebeca

2002-08-01

This study evaluated the effect of Ca2+ on the extramitochondrial hydrolysis of ATP and ADP by the extramitochondrial ATPase in isolated mitochondria and submitochondrial particles (SMPs) from human term placenta. The effect of different oxidizable substrates on the hydrolysis of ATP and ADP in the presence of sucrose or K+ was evaluated. Ca2+ increased phosphate release from ATP and ADP, but this stimulation showed different behavior depending on the oxidizable substrate present in the incubation media. Ca2+ stimulated the hydrolysis of ATP and ADP in the presence of sucrose. However, Ca2+ did not stimulate the hydrolysis of ADP in the medium containing K+. Ca2+ showed inhibition depending on the respiratory substrate. This study suggests that the energetic state of mitochondria controls the extramitochondrial ATPase activity, which is modulated by Ca2+ and respiratory substrates.

Dual functional bioactive-peptide, AIMP1-derived peptide (AdP), for anti-aging.

PubMed

Kim, Jina; Kang, Sujin; Kwon, HanJin; Moon, HoSang; Park, Min Chul

2018-06-19

Human skin aging is caused by several factors, such as UV irradiation, stress, hormone, and pollution. Wrinkle formation and skin pigmentation are representative features of skin aging. Although EGF and arbutin are used as anti-wrinkle and skin whitening agents, respectively, they have adverse effects on skin. When more cosmeceutical ingredients are added to cosmetic product, adverse effects are also accumulated. For these reasons, multifunctional and safe cosmetic ingredients are in demand. The aim of the present study is to investigate the novel anti-aging agents, AIMP1-derived peptide (AdP, INCI name: sh-oligopeptide-5/sh-oligopeptide SP) for cosmetic products. To assess the anti-wrinkle effect of AdP, collagen type I synthesis and fibroblast proliferation were determined on human fibroblasts. The anti-wrinkle effect of AdP was examined by ELISA and cell titer glo assay. To assess the whitening, melanin content and tyrosinase activity were determined on melanocytes. The whitening effect of AdP was examined by melanin measurement and enzyme activity assay. The safety of AdP was determined by cytotoxicity and immunogenicity, CCK-8 and TNF-α ELISA assay, respectively. AdP treatment induced the collagen type I synthesis and fibroblast proliferation. Also, AdP treatment inhibited melanin synthesis by regulating tyrosinase activity. The anti-aging effect of AdP is more potent than EGF and albutin. AdP did not show adverse effects. These results show that AdP can be dual functional and safe cosmeceutical agent to prevent skin aging. © 2018 Wiley Periodicals, Inc.

Neuroprotective and antioxidant activities of bamboo salt soy sauce against H2O2-induced oxidative stress in rat cortical neurons.

PubMed

Jeong, Jong Hee; Noh, Min-Young; Choi, Jae-Hyeok; Lee, Haiwon; Kim, Seung Hyun

2016-04-01

Bamboo salt (BS) and soy sauce (SS) are traditional foods in Asia, which contain antioxidants that have cytoprotective effects on the body. The majority of SS products contain high levels of common salt, consumption of which has been associated with numerous detrimental effects on the body. However, BS may be considered a healthier substitute to common salt. The present study hypothesized that SS made from BS, known as bamboo salt soy sauce (BSSS), may possess enhanced cytoprotective properties; this was evaluated using a hydrogen peroxide (H 2 O 2 )-induced neuronal cell death rat model. Rat neuronal cells were pretreated with various concentrations (0.001, 0.01, 0.1, 1 and 10%) of BSSS, traditional soy sauce (TRSS) and brewed soy sauce (BRSS), and were subsequently exposed to H 2 O 2 (100 µM). The viability of neuronal cells, and the occurrence of DNA fragmentation, was subsequently examined. Pretreatment of neuronal cells with TRSS and BRSS reduced cell viability in a concentration-dependent manner, whereas neuronal cells pretreated with BSSS exhibited increased cell viability, as compared with non-treated neuronal cells. Furthermore, neuronal cells pretreated with 0.01% BSSS exhibited the greatest increase in viability. Exposure of neuronal cells to H 2 O 2 significantly increased the levels of reactive oxygen species (ROS), B-cell lymphoma 2-associated X protein, poly (ADP-ribose), cleaved poly (ADP-ribose) polymerase, cytochrome c , apoptosis-inducing factor, cleaved caspase-9 and cleaved caspase-3, in all cases. Pretreatment of neuronal cells with BSSS significantly reduced the levels of ROS generated by H 2 O 2 , and increased the levels of phosphorylated AKT and phosphorylated glycogen synthase kinase-3β. Furthermore, the observed effects of BSSS could be blocked by administration of 10 µM LY294002, a phosphatidylinositol 3-kinase inhibitor. The results of the present study suggested that BSSS may exert positive neuroprotective effects against H 2 O 2

Effect of an ADP analog on isometric force and ATPase activity of active muscle fibers.

PubMed

Karatzaferi, Christina; Myburgh, Kathryn H; Chinn, Marc K; Franks-Skiba, Kathleen; Cooke, Roger

2003-04-01

The role played by ADP in modulating cross-bridge function has been difficult to study, because it is hard to buffer ADP concentration in skinned muscle preparations. To solve this, we used an analog of ADP, spin-labeled ADP (SL-ADP). SL-ADP binds tightly to myosin but is a very poor substrate for creatine kinase or pyruvate kinase. Thus ATP can be regenerated, allowing well-defined concentrations of both ATP and SL-ADP. We measured isometric ATPase rate and isometric tension as a function of both [SL-ADP], 0.1-2 mM, and [ATP], 0.05-0.5 mM, in skinned rabbit psoas muscle, simulating fresh or fatigued states. Saturating levels of SL-ADP increased isometric tension (by P'), the absolute value of P' being nearly constant, approximately 0.04 N/mm(2), in variable ATP levels, pH 7. Tension decreased (50-60%) at pH 6, but upon addition of SL-ADP, P' was still approximately 0.04 N/mm(2). The ATPase was inhibited competitively by SL-ADP with an inhibition constant, K(i), of approximately 240 and 280 microM at pH 7 and 6, respectively. Isometric force and ATPase activity could both be fit by a simple model of cross-bridge kinetics.

ADP's ABCs of Training

ERIC Educational Resources Information Center

Weinstein, Margery

2010-01-01

When a company's core competence is processing data, it is sometimes easy to lose sight of the obvious--the information right under its nose. In the case of Automatic Data Processing, Inc. (ADP), a business outsourcing company specializing in human resources, payroll, tax, and benefits administrations solutions, that is not a problem. Through…

PARP13 and RNA regulation in immunity and cancer

PubMed Central

Todorova, Tanya; Bock, Florian; Chang, Paul

2015-01-01

Posttranscriptional regulation of RNA is an important mechanism for activating and resolving cellular stress responses. Poly(ADP-ribose) Polymerase-13 (PARP13), also known as ZC3HAV1 and Zinc-finger Antiviral Protein (ZAP), is an RNA-binding protein that regulates the stability, and translation of specific mRNAs, and modulates the miRNA silencing pathway to globally impact miRNA targets. These functions of PARP13 are important components of the cellular response to stress. In addition, the ability of PARP13 to restrict oncogenic viruses and to repress the pro-survival cytokine receptor TRAILR4 suggests that it can be protective against malignant transformation and cancer development. The relevance of PARP13 to human health and disease make it a promising therapeutic target. PMID:25851173

Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Haibing; Department of Ophthalmology, Anhui Provincial Hospital, Hefei; Jia Weiping

2008-05-02

Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might representmore » a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent.« less

A review on PARP1 inhibitors: Pharmacophore modeling, virtual and biological screening studies to identify novel PARP1 inhibitors.

PubMed

Singh, Sardar Shamshair; Sarma, Jagarlapudi A R P; Narasu, Lakshmi; Dayam, Raveendra; Xu, Shili; Neamati, Nouri

2014-01-01

A tremendous research on Poly (ADP-ribose) polymerase (PARP) pertaining to cancer and ischemia is in very rapid progress. PARP's are a specific class of enzymes that repairs the damaged DNA. Recent findings suggest also that PARP-1 is the most abundantly expressed nuclear enzyme which involves in various therapeutic areas like inflammation, stroke, cardiac ischemia, cancer and diabetes. The current review describes the overview on clinical candidates of PARP1 and its current status in clinical trials. This paper also covers identification of potent PARP1 inhibitors using structure and ligand based pharmacophore models. Finally 36 potential hits were identified from the virtual screening of pharmacophore models and screened for PARP1 activity. 15 actives were identified as potent PARP1 inhibitors and further optimization of these analogues are in progress.

ADP-ribosylation of transducin by pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watkins, P.A.; Burns, D.L.; Kanaho, Y.

1985-11-05

Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is (TSP)ADP-ribosylated by pertussis toxin and (TSP)NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1more » molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and TS kDa. The amino terminus of both 38- and TS-kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The TS-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of (TSP)ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased (TSP)ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed (TSP)ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma.« less

The anti-inflammatory and antioxidant effects of bergamot juice extract (BJe) in an experimental model of inflammatory bowel disease.

PubMed

Impellizzeri, Daniela; Bruschetta, Giuseppe; Di Paola, Rosanna; Ahmad, Akbar; Campolo, Michela; Cuzzocrea, Salvatore; Esposito, Emanuela; Navarra, Michele

2015-12-01

The beneficial properties of the flavonoid fraction of bergamot juice (BJe) have been raising interest and have been the subject of recent studies, considering the potentiality of its health promoting substances. Flavonoids have demonstrated radical-scavenging and anti-inflammatory activities. The aim of the present study was to examine the effects of BJe in mice subjected to experimental colitis. Colitis was induced in mice by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). BJe was administered daily orally (at 5, 10 and 20 mg/kg). Four days after DNBS administration, colon nuclear factor NF-κB translocation and MAP kinase phospho-JNK activation were increased as well as cytokine production such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Neutrophil infiltration, by myeloperoxidase (MPO) activity, in the mucosa was associated with up-regulation of adhesion molecules (ICAM-1 and P-selectin). Immunohistochemistry for nitrotyrosine and poly ADP-ribose (PAR) also showed an intense staining in the inflamed colon. Treatment with BJe decreased the appearance of diarrhea and body weight loss. This was associated with a reduction in colonic MPO activity. BJe reduced nuclear NF-κB translocation, p-JNK activation, the pro-inflammatory cytokines release, the appearance of nitrotyrosine and PAR in the colon and reduced the up-regulation of ICAM-1 and P-selectin. In addition, colon inflammation was also associated with apoptotic damage. Treatment with BJe caused a decrease of pro-apoptotic Bax expression and an increase of anti-apoptotic Bcl-2 expression. The results of this study suggested that administration of BJe induced, partly specified, anti-inflammatory mechanisms, which potentially may be beneficial for the treatment of IBD in humans. Copyright © 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

ε Subunit of Bacillus subtilis F1-ATPase Relieves MgADP Inhibition

PubMed Central

Mizumoto, Junya; Kikuchi, Yuka; Nakanishi, Yo-Hei; Mouri, Naoto; Cai, Anrong; Ohta, Tokushiro; Haruyama, Takamitsu; Kato-Yamada, Yasuyuki

2013-01-01

MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF1. We conclude that the ε subunit relieves BF1 from MgADP inhibition. PMID:23967352

Synergistic role of ADP and Ca2+ in diastolic myocardial stiffness

PubMed Central

Sequeira, Vasco; Najafi, Aref; McConnell, Mark; Fowler, Ewan D; Bollen, Ilse A E; Wüst, Rob C I; dos Remedios, Cris; Helmes, Michiel; White, Ed; Stienen, Ger J M; Tardiff, Jil; Kuster, Diederik W D; van der Velden, Jolanda

2015-01-01

Abstract Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca2+] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca2+ handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca2+] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca2+ in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca2+ both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca2+] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca2+ overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca2+]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca2+, and thereby increase myocardial stiffness. Key points Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin

Poly(ADP-ribose)polymerases are involved in microhomology mediated back-up non-homologous end joining in Arabidopsis thaliana.

PubMed

Jia, Qi; den Dulk-Ras, Amke; Shen, Hexi; Hooykaas, Paul J J; de Pater, Sylvia

2013-07-01

Besides the KU-dependent classical non-homologous end-joining (C-NHEJ) pathway, an alternative NHEJ pathway first identified in mammalian systems, which is often called the back-up NHEJ (B-NHEJ) pathway, was also found in plants. In mammalian systems PARP was found to be one of the essential components in B-NHEJ. Here we investigated whether PARP1 and PARP2 were also involved in B-NHEJ in Arabidopsis. To this end Arabidopsis parp1, parp2 and parp1parp2 (p1p2) mutants were isolated and functionally characterized. The p1p2 double mutant was crossed with the C-NHEJ ku80 mutant resulting in the parp1parp2ku80 (p1p2k80) triple mutant. As expected, because of their role in single strand break repair (SSBR) and base excision repair (BER), the p1p2 and p1p2k80 mutants were shown to be sensitive to treatment with the DNA damaging agent MMS. End-joining assays in cell-free leaf protein extracts of the different mutants using linear DNA substrates with different ends reflecting a variety of double strand breaks were performed. The results showed that compatible 5'-overhangs were accurately joined in all mutants, that KU80 protected the ends preventing the formation of large deletions and that PARP proteins were involved in microhomology mediated end joining (MMEJ), one of the characteristics of B-NHEJ.

ADP1 Affects Plant Architecture by Regulating Local Auxin Biosynthesis

PubMed Central

Li, Shibai; Qin, Genji; Novák, Ondřej; Pěnčík, Aleš; Ljung, Karin; Aoyama, Takashi; Liu, Jingjing; Murphy, Angus; Gu, Hongya; Tsuge, Tomohiko; Qu, Li-Jia

2014-01-01

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs. PMID:24391508

ADP1 affects plant architecture by regulating local auxin biosynthesis.

PubMed

Li, Ruixi; Li, Jieru; Li, Shibai; Qin, Genji; Novák, Ondřej; Pěnčík, Aleš; Ljung, Karin; Aoyama, Takashi; Liu, Jingjing; Murphy, Angus; Gu, Hongya; Tsuge, Tomohiko; Qu, Li-Jia

2014-01-01

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

PubMed

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

PARP10 (ARTD10) modulates mitochondrial function

PubMed Central

Nagy, Lilla; Vida, András; Kis, Gréta; Brunyánszki, Attila; Antal, Miklós; Lüscher, Bernhard; Bai, Péter

2018-01-01

Poly(ADP-ribose) polymerase (PARP)10 is a PARP family member that performs mono-ADP-ribosylation of target proteins. Recent studies have linked PARP10 to metabolic processes and metabolic regulators that prompted us to assess whether PARP10 influences mitochondrial oxidative metabolism. The depletion of PARP10 by specific shRNAs increased mitochondrial oxidative capacity in cellular models of breast, cervical, colorectal and exocrine pancreas cancer. Upon silencing of PARP10, mitochondrial superoxide production decreased in line with increased expression of antioxidant genes pointing out lower oxidative stress upon PARP10 silencing. Improved mitochondrial oxidative capacity coincided with increased AMPK activation. The silencing of PARP10 in MCF7 and CaCo2 cells decreased the proliferation rate that correlated with increased expression of anti-Warburg enzymes (Foxo1, PGC-1α, IDH2 and fumarase). By analyzing an online database we showed that lower PARP10 expression increases survival in gastric cancer. Furthermore, PARP10 expression decreased upon fasting, a condition that is characterized by increases in mitochondrial biogenesis. Finally, lower PARP10 expression is associated with increased fatty acid oxidation. PMID:29293500

Defense ADP Acquisition Study.

DTIC Science & Technology

1981-11-30

Logistics ALS - Advanced Logistics System AMP - ADPS Master Plan ANSI - American National Standards Institute APR - Agency Procurement Request ASD(C...Computers IRM - Information Resources Management ISO - International Standards Organization L LCC - Life Cycle Costs LCM - Life Cycle Management LE...man- agement in the process * Lack of a mission orientation . Lack of systems management and life cycle perspectives * Lack of effective leadership

Monitoring of the ADP/ATP Ratio by Induced Circularly Polarised Europium Luminescence.

PubMed

Shuvaev, Sergey; Fox, Mark A; Parker, David

2018-06-18

A series of three europium complexes bearing picolyl amine moieties was found to possess differing binding affinities towards Zn 2+ and three nucleotides: AMP, ADP, and ATP. A large increase in the total emission intensity was observed upon binding Zn 2+ , followed by signal amplification upon the addition of nucleotides. The resulting adducts possessed strong induced circularly polarised emission, with ADP and ATP signals of opposite sign. Model DFT geometries of the adducts suggest the Δ diastereoisomer is preferred for ATP and the Λ isomer for ADP/AMP. This change in sign allows the ADP/ATP (or AMP/ATP) ratio to be assessed by monitoring changes in the emission dissymmetry factor, g em . © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of ADP on slow-twitch muscle fibres of the rat: implications for muscle fatigue.

PubMed

Macdonald, W A; Stephenson, D G

2006-05-15

Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 microm to 1.0 mm). The effect of ADP on sarcoplasmic reticulum (SR) Ca2+-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca2+-content allowed determination of the effective rates of the SR Ca2+-pump and of the SR Ca2+-leak. The SR Ca2+-pump rate, estimated at pCa (-log10[Ca2+]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 microm, with no further alteration when the [ADP] was increased to 1.0 mm. The SR Ca2+-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 microm, but was increased by 26% when the [ADP] was elevated to 1.0 mm. This ADP-induced SR Ca2+-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca2+-pump and not the SR Ca2+-release channel. The decrease in SR Ca2+-pump rate and SR Ca2+-leak rate when [ADP] was increased led to a 40% decrease in SR Ca2+-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres.

Effect of ADP on slow-twitch muscle fibres of the rat: implications for muscle fatigue

PubMed Central

Macdonald, W A; Stephenson, D G

2006-01-01

Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 μm to 1.0 mm). The effect of ADP on sarcoplasmic reticulum (SR) Ca2+-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca2+-content allowed determination of the effective rates of the SR Ca2+-pump and of the SR Ca2+-leak. The SR Ca2+-pump rate, estimated at pCa (−log10[Ca2+]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 μm, with no further alteration when the [ADP] was increased to 1.0 mm. The SR Ca2+-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 μm, but was increased by 26% when the [ADP] was elevated to 1.0 mm. This ADP-induced SR Ca2+-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca2+-pump and not the SR Ca2+-release channel. The decrease in SR Ca2+-pump rate and SR Ca2+-leak rate when [ADP] was increased led to a 40% decrease in SR Ca2+-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres. PMID:16556653

Fracture resistance behaviour of gamma-irradiation sterilized cortical bone protected with a ribose pre-treatment

NASA Astrophysics Data System (ADS)

Woodside, Carman Mitchell

Structural bone allograft reconstructions are often implemented to repair large skeletal defects. To ensure the biological safety of the patient, allograft material is routinely sterilized with gamma-irradiation prior to implantation. The sterilization process damages the tissue, specifically the collagen protein network, leading to severe losses in the mechanical properties of the bone. Our lab has begun developing a ribose pre-treatment that can protect bone from these harmful effects. The goals of the present study were to develop a method to measure the fracture toughness of bone, an important clinical failure mode, and implement it to determine the effectiveness of the ribose pre-treatment on fracture toughness. We have shown that the ribose pre-treatment is successful at protecting some of the original fracture toughness of sterilized bone, and that the connectivity of the collagen network is an important contributor to the fracture resistance of bone.

Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

PubMed Central

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-01-01

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx

PubMed Central

Suárez, Gabriel A.; Renda, Brian A.; Dasgupta, Aurko

2017-01-01

ABSTRACT The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase (dinP) and a DNA damage response regulator (umuDAb [the umuD gene of A. baylyi]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx.

PubMed

Suárez, Gabriel A; Renda, Brian A; Dasgupta, Aurko; Barrick, Jeffrey E

2017-09-01

The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS 1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS 1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS 1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase ( dinP ) and a DNA damage response regulator ( umuD Ab [the umuD gene of A. baylyi ]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves

Glycosidation of Methanol with Ribose: An Interdisciplinary Undergraduate Laboratory Experiment

ERIC Educational Resources Information Center

Simon, Erin; Cook, Katie; Pritchard, Meredith R.; Stripe, Wayne; Bruch, Martha; Bendinskas, Kestutis

2010-01-01

This exercise provides students hands-on experience with the topics of glycosidation, hemiacetal and acetal formation, proton nuclear magnetic resonance ([superscript 1]H NMR) spectroscopy, and kinetic and thermodynamic product formation. In this laboratory experiment, the methyl acetal of ribose is synthesized, and the kinetic and thermodynamic…

Consumer beware: a systematic assessment of potential bias in the lay electronic media to examine the portrayal of "PARP" inhibitors for cancer treatment.

PubMed

Coleman, Shawnta; Peethambaram, Prema P; Jatoi, Aminah

2011-09-01

This study examined how the lay electronic media covers poly-ADP-ribose polymerase, or "PARP," inhibitors, a class of cancer agents currently under clinical investigation. Of 771 internet links, 51 targeted the lay public. Independent review by two investigators yielded the following categorizations: 36 (71%) were "overly positive", 15 (29%) "neutral", and none "overly negative". "Overly positive" articles used: (l) overstated benefit, (2) included quotations from enthusiastic scientists, and (3) discussed single or small patient subsets. They used such phrases as "the holy grail of cancer research", "the most exciting development in cancer research in a decade or more…. it could save thousands of lives", and "we were surprised and delighted…. it's the kind of thing you don't really think will happen". Healthcare providers should be aware of the foregoing when discussing PARP inhibitors-and perhaps other novel therapies-with cancer patients.

Neurodegeneration and Sensorimotor Deficits in the Mouse Model of Traumatic Brain Injury

PubMed Central

Bhowmick, Saurav; D‘Mello, Veera; Ponery, Nizmi; Abdul-Muneer, P. M.

2018-01-01

Traumatic brain injury (TBI) can result in persistent sensorimotor and cognitive deficits, which occur through a cascade of deleterious pathophysiological events over time. In this study, we investigated the hypothesis that neurodegeneration caused by TBI leads to impairments in sensorimotor function. TBI induces the activation of the caspase-3 enzyme, which triggers cell apoptosis in an in vivo model of fluid percussion injury (FPI). We analyzed caspase-3 mediated apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and poly (ADP-ribose) polymerase (PARP) and annexin V western blotting. We correlated the neurodegeneration with sensorimotor deficits by conducting the animal behavioral tests including grid walk, balance beam, the inverted screen test, and the climb test. Our study demonstrated that the excess cell death or neurodegeneration correlated with the neuronal dysfunction and sensorimotor impairments associated with TBI. PMID:29316623

Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-like Cell Death via Intrinsic Reactive Oxygen Cascades

PubMed Central

Yoon, Jonghee; Ryu, Seung-wook; Lee, Seunghee; Choi, Chulhee

2015-01-01

High-intensity femtosecond lasers have recently been used to irreversibly disrupt nanoscale structures, such as intracellular organelles, and to modify biological functions in a reversible manner: so-called nanosurgery and biophotomodulation. Femtosecond laser pulses above the threshold intensity sufficient for reversible biophotomodulation can cause irreversible changes in the irradiated cell, eventually leading to cell death. Here, we demonstrated that cytosolic irradiation with a femtosecond laser produced intrinsic cascades of reactive oxygen species (ROS), which led to rapid apoptosis-like cell death via a caspase and poly (ADP-ribose) polymerase 1 (PARP-1) signaling pathway. We further showed that cells with enhanced mitochondrial fusion activity are more resilient to laser-induced stress compared to those with enforced mitochondrial fission. Taken together, these findings provide fundamental insight into how optical stimulation intervenes in intrinsic cellular signaling pathways and functions. PMID:25648455

Cytosolic irradiation of femtosecond laser induces mitochondria-dependent apoptosis-like cell death via intrinsic reactive oxygen cascades.

PubMed

Yoon, Jonghee; Ryu, Seung-Wook; Lee, Seunghee; Choi, Chulhee

2015-02-04

High-intensity femtosecond lasers have recently been used to irreversibly disrupt nanoscale structures, such as intracellular organelles, and to modify biological functions in a reversible manner: so-called nanosurgery and biophotomodulation. Femtosecond laser pulses above the threshold intensity sufficient for reversible biophotomodulation can cause irreversible changes in the irradiated cell, eventually leading to cell death. Here, we demonstrated that cytosolic irradiation with a femtosecond laser produced intrinsic cascades of reactive oxygen species (ROS), which led to rapid apoptosis-like cell death via a caspase and poly (ADP-ribose) polymerase 1 (PARP-1) signaling pathway. We further showed that cells with enhanced mitochondrial fusion activity are more resilient to laser-induced stress compared to those with enforced mitochondrial fission. Taken together, these findings provide fundamental insight into how optical stimulation intervenes in intrinsic cellular signaling pathways and functions.

Load-dependent ADP binding to myosins V and VI: Implications for subunit coordination and function

PubMed Central

Oguchi, Yusuke; Mikhailenko, Sergey V.; Ohki, Takashi; Olivares, Adrian O.; De La Cruz, Enrique M.; Ishiwata, Shin'ichi

2008-01-01

Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in cells, taking multiple steps in a “hand-over-hand” fashion. The catalytic cycles of both myosins are limited by ADP dissociation, which is considered a key step in the walking mechanism of these motors. Here, we demonstrate that external loads applied to individual actomyosin V or VI bonds asymmetrically affect ADP affinity, such that ADP binds weaker under loads assisting motility. Model-based analysis reveals that forward and backward loads modulate the kinetics of ADP binding to both myosins, although the effect is less pronounced for myosin VI. ADP dissociation is modestly accelerated by forward loads and inhibited by backward loads. Loads applied in either direction slow ADP binding to myosin V but accelerate binding to myosin VI. We calculate that the intramolecular load generated during processive stepping is ≈2 pN for both myosin V and myosin VI. The distinct load dependence of ADP binding allows these motors to perform different cellular functions. PMID:18509050

Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex

PubMed Central

Tsurumura, Toshiharu; Tsumori, Yayoi; Qiu, Hao; Oda, Masataka; Sakurai, Jun; Nagahama, Masahiro; Tsuge, Hideaki

2013-01-01

Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD+ analog βTAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD+-bound form (NAD+-Ia-actin) and the ADP ribosylated form [Ia-ADP ribosylated (ADPR)-actin] remain unclear. Accidentally, we found that ethylene glycol as cryo-protectant inhibits ADP ribosylation and crystallized the NAD+-Ia-actin complex. Here we report high-resolution structures of NAD+-Ia-actin and Ia-ADPR-actin obtained by soaking apo-Ia-actin crystal with NAD+ under different conditions. The structures of NAD+-Ia-actin and Ia-ADPR-actin represent the pre- and postreaction states, respectively. By assigning the βTAD-Ia-actin structure to the transition state, the strain-alleviation model of ADP ribosylation, which we proposed previously, is experimentally confirmed and improved. Moreover, this reaction mechanism appears to be applicable not only to Ia but also to other ADP ribosyltransferases. PMID:23382240

ADP Regulates SNF1, the Saccharomyces cerevisiae Homolog of AMP-Activated Protein Kinase

PubMed Central

Mayer, Faith V.; Heath, Richard; Underwood, Elizabeth; Sanders, Matthew J.; Carmena, David; McCartney, Rhonda R.; Leiper, Fiona C.; Xiao, Bing; Jing, Chun; Walker, Philip A.; Haire, Lesley F.; Ogrodowicz, Roksana; Martin, Stephen R.; Schmidt, Martin C.; Gamblin, Steven J.; Carling, David

2011-01-01

Summary The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK. PMID:22019086

Time course and strain dependence of ADP release during contraction of permeabilized skeletal muscle fibers.

PubMed

West, Timothy G; Hild, Gabor; Siththanandan, Verl B; Webb, Martin R; Corrie, John E T; Ferenczi, Michael A

2009-04-22

A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P approximately NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12 degrees C of 10(5) M(-1) s(-1). This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P approximately NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa(2+) of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 +/- 0.6 microM and 4.7 +/- 1.5 microM at 12 and 20 degrees C, respectively. The rates of P approximately NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s(-1) at 12 and 20 degrees C, respectively. The time courses of isometric force and P approximately NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.P(i) and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3-6 s(-1)) in the cross-bridge turnover during isometric contraction. At 12 degrees C, the A.M.ADP.P(i) and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20 degrees C, the force-bearing A.M.ADP.P(i) state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of

Diadenosine Homodinucleotide Products of ADP-ribosyl Cyclases Behave as Modulators of the Purinergic Receptor P2X7*

PubMed Central

Bruzzone, Santina; Basile, Giovanna; Chothi, Madhu Parakkottil; Nobbio, Lucilla; Usai, Cesare; Jacchetti, Emanuela; Schenone, Angelo; Guse, Andreas H.; Di Virgilio, Francesco; De Flora, Antonio; Zocchi, Elena

2010-01-01

ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5′,5′"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509–14514). P24, but not P18, proved to increase the intracellular Ca2+ concentration ([Ca2+]i) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca2+ through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca2+ influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC50 of ∼1 μm. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca2+]i has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146–23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca2+]i to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A. PMID:20439466

Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

ERIC Educational Resources Information Center

Jewett, Kathy; Sandwick, Roger K.

2011-01-01

The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives.

PubMed

Plesniak, Leigh; Horiuchi, Yuki; Sem, Daniel; Meinenger, David; Stiles, Linda; Shaffer, Jennifer; Jennings, Patricia A; Adams, Joseph A

2002-11-26

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.

Polyethylene glycol-functionalized poly (Lactic Acid-co-Glycolic Acid) and graphene oxide nanoparticles induce pro-inflammatory and apoptotic responses in Candida albicans-infected vaginal epithelial cells.

PubMed

Wagner, R Doug; Johnson, Shemedia J; Danielsen, Zhixia Yan; Lim, Jin-Hee; Mudalige, Thilak; Linder, Sean

2017-01-01

Mucous-penetrating nanoparticles consisting of poly lactic acid-co-glycolic acid (PLGA)-polyethylene glycol (PEG) could improve targeting of microbicidal drugs for sexually transmitted diseases by intravaginal inoculation. Nanoparticles can induce inflammatory responses, which may exacerbate the inflammation that occurs in the vaginal tracts of women with yeast infections. This study evaluated the effects of these drug-delivery nanoparticles on VK2(E6/E7) vaginal epithelial cell proinflammatory responses to Candida albicans yeast infections. Vaginal epithelial cell monolayers were infected with C. albicans and exposed to 100 μg/ml 49.5 nm PLGA-PEG nanospheres or 20 μg/ml 1.1 x 500 nm PEG-functionalized graphene oxide (GO-PEG) sheets. The cells were assessed for changes in mRNA and protein expression of inflammation-related genes by RT-qPCR and physiological markers of cell stress using high content analysis and flow cytometry. C. albicans exposure suppressed apoptotic gene expression, but induced oxidative stress in the cells. The nanomaterials induced cytotoxicity and programmed cell death responses alone and with C. albicans. PLGA-PEG nanoparticles induced mRNA expression of apoptosis-related genes and induced poly (ADP-ribose) polymerase (PARP) cleavage, increased BAX/BCL2 ratios, and chromatin condensation indicative of apoptosis. They also induced autophagy, endoplasmic reticulum stress, and DNA damage. They caused the cells to excrete inflammatory recruitment molecules chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin-1α (IL1A), interleukin-1β (IL1B), calprotectin (S100A8), and tumor necrosis factor α (TNF). GO-PEG nanoparticles induced expression of necrosis-related genes and cytotoxicity. They reduced autophagy and endoplasmic reticulum stress, and apoptotic gene expression responses. The results show that stealth nanoparticle drug-delivery vehicles may cause intracellular damage to vaginal epithelial cells by several mechanisms and that their use

Polyethylene glycol-functionalized poly (Lactic Acid-co-Glycolic Acid) and graphene oxide nanoparticles induce pro-inflammatory and apoptotic responses in Candida albicans-infected vaginal epithelial cells

PubMed Central

Johnson, Shemedia J.; Danielsen, Zhixia Yan; Lim, Jin-Hee; Mudalige, Thilak; Linder, Sean

2017-01-01

Mucous-penetrating nanoparticles consisting of poly lactic acid-co-glycolic acid (PLGA)-polyethylene glycol (PEG) could improve targeting of microbicidal drugs for sexually transmitted diseases by intravaginal inoculation. Nanoparticles can induce inflammatory responses, which may exacerbate the inflammation that occurs in the vaginal tracts of women with yeast infections. This study evaluated the effects of these drug-delivery nanoparticles on VK2(E6/E7) vaginal epithelial cell proinflammatory responses to Candida albicans yeast infections. Vaginal epithelial cell monolayers were infected with C. albicans and exposed to 100 μg/ml 49.5 nm PLGA-PEG nanospheres or 20 μg/ml 1.1 x 500 nm PEG-functionalized graphene oxide (GO-PEG) sheets. The cells were assessed for changes in mRNA and protein expression of inflammation-related genes by RT-qPCR and physiological markers of cell stress using high content analysis and flow cytometry. C. albicans exposure suppressed apoptotic gene expression, but induced oxidative stress in the cells. The nanomaterials induced cytotoxicity and programmed cell death responses alone and with C. albicans. PLGA-PEG nanoparticles induced mRNA expression of apoptosis-related genes and induced poly (ADP-ribose) polymerase (PARP) cleavage, increased BAX/BCL2 ratios, and chromatin condensation indicative of apoptosis. They also induced autophagy, endoplasmic reticulum stress, and DNA damage. They caused the cells to excrete inflammatory recruitment molecules chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin-1α (IL1A), interleukin-1β (IL1B), calprotectin (S100A8), and tumor necrosis factor α (TNF). GO-PEG nanoparticles induced expression of necrosis-related genes and cytotoxicity. They reduced autophagy and endoplasmic reticulum stress, and apoptotic gene expression responses. The results show that stealth nanoparticle drug-delivery vehicles may cause intracellular damage to vaginal epithelial cells by several mechanisms and that their use

Combinatorial Study of a Novel Poly (ADP-ribose) Polymerase Inhibitor and an HDAC Inhibitor, SAHA, in Leukemic Cell Lines.

PubMed

Hegde, Mahesh; Mantelingu, Kempegowda; Pandey, Monica; Pavankumar, Chottanahalli S; Rangappa, Kanchugarakoppal S; Raghavan, Sathees C

2016-10-01

Cancer is a multifactorial disease, which makes it difficult to cure. Since more than one defective cellular component is often involved during oncogenesis, combination therapy is gaining prominence in the field of cancer therapeutics. The purpose of this study was to investigate the combinatorial effects of a novel PARP inhibitor, P10, and HDAC inhibitor, SAHA, in leukemic cells. Combinatorial effects of P10 and SAHA were tested using propidium iodide staining in different leukemic cells. Further, flowcytometry-based assays such as calcein-AM/ethidium homodimer staining, annexin-FITC/PI staining, and JC-1 staining were carried out to elucidate the mechanism of cell death. In addition, cell-cycle analysis, immunocytochemistry studies, and western blotting analysis were conducted to check the combinatorial effect in Nalm6 cells. Propidium iodide staining showed that P10 in combination with SAHA induced cell death in Nalm6 cells, in which PARP expression and activity is high with a combination index of <0.2. Annexin-FITC/PI staining, JC-1 staining, and other biochemical assays revealed that P10 in combination with SAHA induced apoptosis by causing a change in mitochondrial membrane potential in >65 % cells. Importantly, combinatorial treatment induced S phase arrest in 40-45 % cells due to DNA damage and plausible replicative stress. Finally, we demonstrated that treatment with P10 led to DNA strand breaks, which were further potentiated by SAHA (p < 0.01), leading to activation of apoptosis and increased cell death in PARP-positive leukemic cells. Our study reveals that coadministration of PARP inhibitor with SAHA could be used as a combination therapy against leukemic cells that possess high levels of intrinsic PARP activity.

Analytical Design Package (ADP2): A computer aided engineering tool for aircraft transparency design

NASA Technical Reports Server (NTRS)

Wuerer, J. E.; Gran, M.; Held, T. W.

1994-01-01

The Analytical Design Package (ADP2) is being developed as a part of the Air Force Frameless Transparency Program (FTP). ADP2 is an integrated design tool consisting of existing analysis codes and Computer Aided Engineering (CAE) software. The objective of the ADP2 is to develop and confirm an integrated design methodology for frameless transparencies, related aircraft interfaces, and their corresponding tooling. The application of this methodology will generate high confidence for achieving a qualified part prior to mold fabrication. ADP2 is a customized integration of analysis codes, CAE software, and material databases. The primary CAE integration tool for the ADP2 is P3/PATRAN, a commercial-off-the-shelf (COTS) software tool. The open architecture of P3/PATRAN allows customized installations with different applications modules for specific site requirements. Integration of material databases allows the engineer to select a material, and those material properties are automatically called into the relevant analysis code. The ADP2 materials database will be composed of four independent schemas: CAE Design, Processing, Testing, and Logistics Support. The design of ADP2 places major emphasis on the seamless integration of CAE and analysis modules with a single intuitive graphical interface. This tool is being designed to serve and be used by an entire project team, i.e., analysts, designers, materials experts, and managers. The final version of the software will be delivered to the Air Force in Jan. 1994. The Analytical Design Package (ADP2) will then be ready for transfer to industry. The package will be capable of a wide range of design and manufacturing applications.

An Ancient Fingerprint Indicates the Common Ancestry of Rossmann-Fold Enzymes Utilizing Different Ribose-Based Cofactors

PubMed Central

Laurino, Paola; Tóth-Petróczy, Ágnes; Meana-Pañeda, Rubén; Lin, Wei; Truhlar, Donald G.; Tawfik, Dan S.

2016-01-01

Nucleoside-based cofactors are presumed to have preceded proteins. The Rossmann fold is one of the most ancient and functionally diverse protein folds, and most Rossmann enzymes utilize nucleoside-based cofactors. We analyzed an omnipresent Rossmann ribose-binding interaction: a carboxylate side chain at the tip of the second β-strand (β2-Asp/Glu). We identified a canonical motif, defined by the β2-topology and unique geometry. The latter relates to the interaction being bidentate (both ribose hydroxyls interacting with the carboxylate oxygens), to the angle between the carboxylate and the ribose, and to the ribose’s ring configuration. We found that this canonical motif exhibits hallmarks of divergence rather than convergence. It is uniquely found in Rossmann enzymes that use different cofactors, primarily SAM (S-adenosyl methionine), NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide). Ribose-carboxylate bidentate interactions in other folds are not only rare but also have a different topology and geometry. We further show that the canonical geometry is not dictated by a physical constraint—geometries found in noncanonical interactions have similar calculated bond energies. Overall, these data indicate the divergence of several major Rossmann-fold enzyme classes, with different cofactors and catalytic chemistries, from a common pre-LUCA (last universal common ancestor) ancestor that possessed the β2-Asp/Glu motif. PMID:26938925

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation

PubMed Central

Ryu, Dongryeol; Zhang, Hongbo; Ropelle, Eduardo R.; Sorrentino, Vincenzo; Mázala, Davi A. G.; Mouchiroud, Laurent; Marshall, Philip L.; Campbell, Matthew D.; Ali, Amir Safi; Knowels, Gary M.; Bellemin, Stéphanie; Iyer, Shama R.; Wang, Xu; Gariani, Karim; Sauve, Anthony A.; Cantó, Carles; Conley, Kevin E.; Walter, Ludivine; Lovering, Richard M.; Chin, Eva R.; Jasmin, Bernard J.; Marcinek, David J.; Menzies, Keir J.; Auwerx, Johan

2017-01-01

Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene’s muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5’-diphosphate (ADP)–ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr−/− mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures. PMID:27798264

Proteomic Analysis of the Downstream Signaling Network of PARP1.

PubMed

Zhen, Yuanli; Yu, Yonghao

2018-01-30

Poly-ADP-ribosylation (PARylation) is a protein posttranslational modification (PTM) that is critically involved in many biological processes that are linked to cell stress responses. It is catalyzed by a class of enzymes known as poly-ADP-ribose polymerases (PARPs). In particular, PARP1 is a nuclear protein that is activated upon sensing nicked DNA. Once activated, PARP1 is responsible for the synthesis of a large number of PARylated proteins and initiation of the DNA damage response mechanisms. This observation provided the rationale for developing PARP1 inhibitors for the treatment of human malignancies. Indeed, three PARP1 inhibitors (Olaparib, Rucaparib, and Niraparib) have recently been approved by the Food and Drug Administration for the treatment of ovarian cancer. Moreover, in 2017, both Olaparib and Niraparib have also been approved for the treatment of fallopian tube cancer and primary peritoneal cancer. Despite this very exciting progress in the clinic, the basic signaling mechanism that connects PARP1 to a diverse array of biological processes is still poorly understood. This is, in large part, due to the inherent technical difficulty associated with the analysis of protein PARylation, which is a low-abundance, labile, and heterogeneous PTM. The study of PARylation has been greatly facilitated by the recent advances in mass spectrometry-based proteomic technologies tailored to the analysis of this modification. In this Perspective, we discuss these breakthroughs, including their technical development, and applications that provide a global view of the many biological processes regulated by this important protein modification.

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation.

PubMed

Ryu, Dongryeol; Zhang, Hongbo; Ropelle, Eduardo R; Sorrentino, Vincenzo; Mázala, Davi A G; Mouchiroud, Laurent; Marshall, Philip L; Campbell, Matthew D; Ali, Amir Safi; Knowels, Gary M; Bellemin, Stéphanie; Iyer, Shama R; Wang, Xu; Gariani, Karim; Sauve, Anthony A; Cantó, Carles; Conley, Kevin E; Walter, Ludivine; Lovering, Richard M; Chin, Eva R; Jasmin, Bernard J; Marcinek, David J; Menzies, Keir J; Auwerx, Johan

2016-10-19

Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD + ) synthesis, consistent with a potential role for the essential cofactor NAD + in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD + and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD + levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD + biosynthesis. Replenishing NAD + stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr -/- mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD + repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD + may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures. Copyright © 2016, American Association for the Advancement of Science.

A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

PubMed

Patel, Manisha J; Akhani, Rekha C; Patel, Arti T; Dedania, Samir R; Patel, Darshan H

2017-02-01

l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency. Copyright © 2016 Elsevier Inc. All rights reserved.

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

PubMed Central

Suszynski, T.M.; Wildey, G.M.; Falde, E.J.; Cline, G.W.; Maynard, K. Stewart; Ko, N.; Sotiris, J.; Naji, A.; Hering, B.J.; Papas, K.K.

2009-01-01

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio. PMID:18374063

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, M.; Allison, W.S.

1986-05-05

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with (/sup 3/H)ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. (/sup 3/H)ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with (/sup 3/H)ADP in 30more » min with a Kd of 30 microM. (/sup 3/H)ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of (/sup 3/H)ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. (/sup 3/H)ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits.« less

Daily Supplementation of D-ribose Shows No Therapeutic Benefits in the MHC-I Transgenic Mouse Model of Inflammatory Myositis

PubMed Central

Coley, William; Rayavarapu, Sree; van der Meulen, Jack H.; Duba, Ayyappa S.; Nagaraju, Kanneboyina

2013-01-01

Background Current treatments for idiopathic inflammatory myopathies (collectively called myositis) focus on the suppression of an autoimmune inflammatory response within the skeletal muscle. However, it has been observed that there is a poor correlation between the successful suppression of muscle inflammation and an improvement in muscle function. Some evidence in the literature suggests that metabolic abnormalities in the skeletal muscle underlie the weakness that continues despite successful immunosuppression. We have previously shown that decreased expression of a purine nucleotide cycle enzyme, adenosine monophosphate deaminase (AMPD1), leads to muscle weakness in a mouse model of myositis and may provide a mechanistic basis for muscle weakness. One of the downstream metabolites of this pathway, D-ribose, has been reported to alleviate symptoms of myalgia in patients with a congenital loss of AMPD1. Therefore, we hypothesized that supplementing exogenous D-ribose would improve muscle function in the mouse model of myositis. We treated normal and myositis mice with daily doses of D-ribose (4 mg/kg) over a 6-week time period and assessed its effects using a battery of behavioral, functional, histological and molecular measures. Results Treatment with D-ribose was found to have no statistically significant effects on body weight, grip strength, open field behavioral activity, maximal and specific forces of EDL, soleus muscles, or histological features. Histological and gene expression analysis indicated that muscle tissues remained inflamed despite treatment. Gene expression analysis also suggested that low levels of the ribokinase enzyme in the skeletal muscle might prevent skeletal muscle tissue from effectively utilizing D-ribose. Conclusions Treatment with daily oral doses of D-ribose showed no significant effect on either disease progression or muscle function in the mouse model of myositis. PMID:23785461

Daily supplementation of D-ribose shows no therapeutic benefits in the MHC-I transgenic mouse model of inflammatory myositis.

PubMed

Coley, William; Rayavarapu, Sree; van der Meulen, Jack H; Duba, Ayyappa S; Nagaraju, Kanneboyina

2013-01-01

Current treatments for idiopathic inflammatory myopathies (collectively called myositis) focus on the suppression of an autoimmune inflammatory response within the skeletal muscle. However, it has been observed that there is a poor correlation between the successful suppression of muscle inflammation and an improvement in muscle function. Some evidence in the literature suggests that metabolic abnormalities in the skeletal muscle underlie the weakness that continues despite successful immunosuppression. We have previously shown that decreased expression of a purine nucleotide cycle enzyme, adenosine monophosphate deaminase (AMPD1), leads to muscle weakness in a mouse model of myositis and may provide a mechanistic basis for muscle weakness. One of the downstream metabolites of this pathway, D-ribose, has been reported to alleviate symptoms of myalgia in patients with a congenital loss of AMPD1. Therefore, we hypothesized that supplementing exogenous D-ribose would improve muscle function in the mouse model of myositis. We treated normal and myositis mice with daily doses of D-ribose (4 mg/kg) over a 6-week time period and assessed its effects using a battery of behavioral, functional, histological and molecular measures. Treatment with D-ribose was found to have no statistically significant effects on body weight, grip strength, open field behavioral activity, maximal and specific forces of EDL, soleus muscles, or histological features. Histological and gene expression analysis indicated that muscle tissues remained inflamed despite treatment. Gene expression analysis also suggested that low levels of the ribokinase enzyme in the skeletal muscle might prevent skeletal muscle tissue from effectively utilizing D-ribose. Treatment with daily oral doses of D-ribose showed no significant effect on either disease progression or muscle function in the mouse model of myositis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagy, Edit, E-mail: edit.nagy@karolinska.se; Department of Cardiology, Karolinska University Hospital, Stockholm; Caidahl, Kenneth

Highlights: Black-Right-Pointing-Pointer Oxidative stress has been implicated in the pathomechanism of calcific aortic valve stenosis. Black-Right-Pointing-Pointer We assessed the transcript levels for PARP-1 (poly(ADP-ribose) polymerase), acts as a DNA damage nick sensor in stenotic valves. Black-Right-Pointing-Pointer Early stage of diseased tricuspid valves exhibited higher mRNA levels for PARP-1 compared to bicuspid valves. Black-Right-Pointing-Pointer The mRNA levels for PARP-1 inversely correlated with the clinical stenosis severity in tricuspid valves. Black-Right-Pointing-Pointer Our data demonstrated that DNA damage pathways might be associated with stenosis severity only in tricuspid valves. -- Abstract: Oxidative stress may contribute to the hemodynamic progression of aortic valve stenosis,more » and is associated with activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) 1. The aim of the present study was to assess the transcriptional profile and the topological distribution of PARP-1 in human aortic valves, and its relation to the stenosis severity. Human stenotic aortic valves were obtained from 46 patients undergoing aortic valve replacement surgery and used for mRNA extraction followed by quantitative real-time PCR to correlate the PARP-1 expression levels with the non invasive hemodynamic parameters quantifying the stenosis severity. Primary isolated valvular interstitial cells (VICs) were used to explore the effects of cytokines and leukotriene C{sub 4} (LTC{sub 4}) on valvular PARP-1 expression. The thickened areas of stenotic valves with tricuspid morphology expressed significantly higher levels of PARP-1 mRNA compared with the corresponding part of bicuspid valves (0.501 vs 0.243, P = 0.01). Furthermore, the quantitative gene expression levels of PARP-1 were inversely correlated with the aortic valve area (AVA) (r = -0.46, P = 0.0469) and AVA indexed for body surface area (BSA) (r = -0.498; P = 0.0298) only in tricuspid aortic

Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor

PubMed Central

Suzuki, Kano; Mizutani, Kenji; Maruyama, Shintaro; Shimono, Kazumi; Imai, Fabiana L.; Muneyuki, Eiro; Kakinuma, Yoshimi; Ishizuka-Katsura, Yoshiko; Shirouzu, Mikako; Yokoyama, Shigeyuki; Yamato, Ichiro; Murata, Takeshi

2016-01-01

V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V1 crystals in ADP. In the presence of 20 μM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model. PMID:27807367

Role of PARP activity in lung cancer-induced cachexia: Effects on muscle oxidative stress, proteolysis, anabolic markers, and phenotype.

PubMed

Chacon-Cabrera, Alba; Mateu-Jimenez, Mercè; Langohr, Klaus; Fermoselle, Clara; García-Arumí, Elena; Andreu, Antoni L; Yelamos, Jose; Barreiro, Esther

2017-12-01

Strategies to treat cachexia are still at its infancy. Enhanced muscle protein breakdown and ubiquitin-proteasome system are common features of cachexia associated with chronic conditions including lung cancer (LC). Poly(ADP-ribose) polymerases (PARP), which play a major role in chromatin structure regulation, also underlie maintenance of muscle metabolism and body composition. We hypothesized that protein catabolism, proteolytic markers, muscle fiber phenotype, and muscle anabolism may improve in respiratory and limb muscles of LC-cachectic Parp-1-deficient (Parp-1 -/- ) and Parp-2 -/- mice. In diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing mice (wild type, Parp-1 -/- , and Parp-2 -/- ), PARP activity (ADP-ribose polymers, pADPr), redox balance, muscle fiber phenotype, apoptotic nuclei, tyrosine release, protein ubiquitination, muscle-specific E3 ligases, NF-κB signaling pathway, markers of muscle anabolism (Akt, mTOR, p70S6K, and mitochondrial DNA) were evaluated along with body and muscle weights, and limb muscle force. Compared to wild type cachectic animals, in both respiratory and limb muscles of Parp-1 -/- and Parp-2 -/- cachectic mice: cancer induced-muscle wasting characterized by increased PARP activity, protein oxidation, tyrosine release, and ubiquitin-proteasome system (total protein ubiquitination, atrogin-1, and 20S proteasome C8 subunit) were blunted, the reduction in contractile myosin and atrophy of the fibers was attenuated, while no effects were seen in other structural features (inflammatory cells, internal or apoptotic nuclei), and markers of muscle anabolism partly improved. Activation of either PARP-1 or -2 is likely to play a role in muscle protein catabolism via oxidative stress, NF-κB signaling, and enhanced proteasomal degradation in cancer-induced cachexia. Therapeutic potential of PARP activity inhibition deserves attention. © 2017 Wiley Periodicals, Inc.

Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

PubMed Central

Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

2016-01-01

DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

Born to run: control of transcription elongation by RNA polymerase II.

PubMed

Chen, Fei Xavier; Smith, Edwin R; Shilatifard, Ali

2018-05-08

The dynamic regulation of transcription elongation by RNA polymerase II (Pol II) is an integral part of the implementation of gene expression programmes during development. In most metazoans, the majority of transcribed genes exhibit transient pausing of Pol II at promoter-proximal regions, and the release of Pol II into gene bodies is controlled by many regulatory factors that respond to environmental and developmental cues. Misregulation of the elongation stage of transcription is implicated in cancer and other human diseases, suggesting that mechanistic understanding of transcription elongation control is therapeutically relevant. In this Review, we discuss the features, establishment and maintenance of Pol II pausing, the transition into productive elongation, the control of transcription elongation by enhancers and by factors of other cellular processes, such as topoisomerases and poly(ADP-ribose) polymerases (PARPs), and the potential of therapeutic targeting of the elongation stage of transcription by Pol II.

Targeting the unmet medical need: the Abbott Laboratories oncology approach.

PubMed

Carlson, Dawn M; Steinberg, Joyce L; Gordon, Gary

2005-09-01

While significant advances in the treatment of cancer occured during the last half of the twentieth century, parallel decreases in overall cancer death rates were not observed. Cancer therapy remains an area of significant unmet medical need. Abbott's oncology research programs are focused on pioneering trageted, less toxic therapies, aimed at different aspects of tumor growth and development. Oncology drugs in development at Abbott target several mechanisms of cancer progression by interfering with multiple processes necessary for tumor growth: recruitment of a blood supply, cell proliferation, and the development of metastases. They include a selective endothelin A-receptor antagonist (atrasentan/Xinlay), 3 angiogenesis inhibitors (ABT 510, a thrombospondin mimetic: ABT-869, a multitargeted receptor tyrosine kinase inhibitor; and ABT 828, recombinant human plasminogen kringle 5), a cell proliferation inhibitor (ABT-751, an antimitotic agent), an apoptosis inducer (ABT 737, a Bcl-2 family inhibitor), and a poly(ADP-ribose)polymerase inhibitor.

Regulatory role of calpain in neuronal death

PubMed Central

Cheng, Si-ying; Wang, Shu-chao; Lei, Ming; Wang, Zhen; Xiong, Kun

2018-01-01

Calpains are a group of calcium-dependent proteases that are over activated by increased intracellular calcium levels under pathological conditions. A wide range of substrates that regulate necrotic, apoptotic and autophagic pathways are affected by calpain. Calpain plays a very important role in neuronal death and various neurological disorders. This review introduces recent research progress related to the regulatory mechanisms of calpain in neuronal death. Various neuronal programmed death pathways including apoptosis, autophagy and regulated necrosis can be divided into receptor interacting protein-dependent necroptosis, mitochondrial permeability transition-dependent necrosis, pyroptosis and poly (ADP-ribose) polymerase 1-mediated parthanatos. Calpains cleave series of key substrates that may lead to cell death or participate in cell death. Regarding the investigation of calpain-mediated programed cell death, it is necessary to identify specific inhibitors that inhibit calpain mediated neuronal death and nervous system diseases. PMID:29623944

[Hereditary breast and ovarian cancers].

PubMed

Gevensleben, H; Serçe, N; Büttner, R

2010-10-01

Hereditary factors are responsible for 5-10% of all breast cancers and 10% of all ovarian cancer cases and are predominantly caused by mutations in the high risk genes BRCA1 and BRCA2 (BRCA: breast cancer). Additional moderate and low penetrance gene variants are currently being analyzed via whole genome association studies. Interdisciplinary counseling, quality managed genetic testing and intensified prevention efforts in specialized medical centres are essential for members of high risk families considering the high prevalence of malignant tumors and the early age of onset. Furthermore, the identification of BRCA-deficient carcinomas is of particular clinical interest, especially regarding new specific therapeutic options, e.g. treatment with poly (ADP-ribose) polymerase (PARP) inhibitors. There are presently no valid surrogate markers verifying the association of BRCA1/BRC2 in tumors. However, breast cancers harboring pathogenic BRCA1 mutations in particular display specific histopathological features.

Phloretin induces apoptosis in H-Ras MCF10A human breast tumor cells through the activation of p53 via JNK and p38 mitogen-activated protein kinase signaling.

PubMed

Kim, Mi-Sung; Kwon, Jung Yeon; Kang, Nam Joo; Lee, Ki Won; Lee, Hyong Joo

2009-08-01

Mutations in Ras play a critical role in the development of human cancers, including breast cancer. We investigated the possible antiproliferative effects of the naturally occurring dihydrochalcone phloretin [2',4',6'-trihydroxy-3-(4-hydroxyphenyl)-propiophenone] on H-Ras-transformed MCF10A human breast epithelial (H-Ras MCF10A) cells. Phloretin suppressed H-Ras MCF10A cell proliferation in a dose-dependent manner and induced nuclear condensation in the cells, indicating that phloretin-induced cell death occurs mainly via the induction of apoptosis. Prominent upregulation of p53 and Bax and cleavage of poly (ADP)-ribose polymerase were also detected in the phloretin-treated cells. Finally, phloretin markedly increased caspase-3 activity as well as JNK and p38 mitogen-activated protein kinase signaling. Our findings suggest that the phloretin-induced apoptosis of breast tumor cells contributes to the chemopreventive potential of phloretin against breast cancer.

An ADPE Protest Primer: Lessons Learned from GSBCA Protest Decisions

DTIC Science & Technology

1991-06-01

reverse if necessary and identify, by block number) The General services Administration’s Board of Contract Appeals (GSBCA) is a significant venue for...David R. Whipple,i D ep a rtm en t of A d i i t a i eS ci ce iim ABSTRACT The General Services Administration’s Board of Contract Appeals (GSBCA) is a...Administration Board of Contract Appeals (GSBCA) ADPE protest decisions. In effect this study will serve as a primer to familiarize new Federal ADPE

Study of linear optical parameters of sodium sulphide nano-particles added ADP crystals

NASA Astrophysics Data System (ADS)

Kochuparampil, A. P.; Joshi, J. H.; Dixit, K. P.; Jethva, H. O.; Joshi, M. J.

2017-05-01

Ammonium Dihydrogen Phosphate (ADP) is one of the nonlinear optical crystals. It is having various applications like optical mixing, electro-optical modulator, harmonic generators, etc. Chalcogenide compounds are poorly soluble in water and difficult to add in the water soluble ADP crystals. The solubility of Chalcogenide compounds can be increased by synthesizing the nano-structured samples with suitable capping agent. In the present study sodium sulphide was added in to ADP to modify its linear optical parameters. Sodium sulphide nano particles were synthesized by co-precipitation technique using Ethylene diamine as capping agent followed by microwave irradiation. The powder XRD confirmed the nano-structured nature of sodium sulphide nano particles. The solubility of nanoparticles of sodium sulphide increased significantly in water compared to the bulk. Pure and Na2S added ADP crystals were grown by slow solvent evaporation method at room temperature. The presence of sodium in ADP was confirmed by AAS. The UV-Vis spectra were recorded for all crystals. Various optical parameters like, transmittance, energy band gap, extinction coefficient, refractive index, optical conductivity, etc. were evaluated. The electronic polarizibility of pure and doped crystals calculated from energy band gap. The effect of doping concentration was found on various parameters.

In the absence of phosphate shuttling, exercise reveals the in vivo importance of creatine-independent mitochondrial ADP transport.

PubMed

Miotto, Paula M; Holloway, Graham P

2016-09-15

The transport of cytosolic adenosine diphosphate (ADP) into the mitochondria is a major control point in metabolic homeostasis, as ADP concentrations directly affect glycolytic flux and oxidative phosphorylation rates within mitochondria. A large contributor to the efficiency of this process is thought to involve phosphocreatine (PCr)/Creatine (Cr) shuttling through mitochondrial creatine kinase (Mi-CK), whereas the biological importance of alterations in Cr-independent ADP transport during exercise remains unknown. Therefore, we utilized an Mi-CK knockout (KO) model to determine whether in vivo Cr-independent mechanisms are biologically important for sustaining energy homeostasis during exercise. Ablating Mi-CK did not alter exercise tolerance, as the time to volitional fatigue was similar between wild-type (WT) and KO mice at various exercise intensities. In addition, skeletal muscle metabolic profiles after exercise, including glycogen, PCr/Cr ratios, free ADP/adenosine monophosphate (AMP), and lactate, were similar between genotypes. While these data suggest that the absence of PCr/Cr shuttling is not detrimental to maintaining energy homeostasis during exercise, KO mice displayed a dramatic increase in Cr-independent mitochondrial ADP sensitivity after exercise. Specifically, whereas mitochondrial ADP sensitivity decreased with exercise in WT mice, in stark contrast, exercise increased mitochondrial Cr-independent ADP sensitivity in KO mice. As a result, the apparent ADP Km was 50% lower in KO mice after exercise, suggesting that in vivo activation of voltage-dependent anion channel (VDAC)/adenine nucleotide translocase (ANT) can support mitochondrial ADP transport. Altogether, we provide insight that Cr-independent ADP transport mechanisms are biologically important for regulating ADP sensitivity during exercise, while highlighting complex regulation and the plasticity of the VDAC/ANT axis to support adenosine triphosphate demand. © 2016 The Author

Overexpression of the ADP (E3-11.6K) protein increases cell lysis and spread of adenovirus.

PubMed

Doronin, Konstantin; Toth, Karoly; Kuppuswamy, Mohan; Krajcsi, Peter; Tollefson, Ann E; Wold, William S M

2003-01-20

Adenoviruses replicate in the nucleus and induce lytic cell death. We have shown previously that efficient cell lysis and release of adenovirus from infected cells requires an 11.6-kDa protein named Adenovirus Death Protein (ADP). The adp gene is located in the early E3 transcription unit, but the gene is expressed primarily at very late stages of infection. The putative function of ADP was discerned previously from the use of virus mutants that lack functional ADP. Here we describe two adenovirus mutants, named VRX-006 and VRX-007, that overexpress ADP. VRX-006 lacks all other genes in the E3 region, and VRX-007 lacks all other E3 genes except 12.5K. VRX-006 and VRX-007 display the phenotype predicted by the proposed function for ADP: they produce early cytopathic effect, early cell lysis, large plaques, and increased cell-to-cell spread. They grow as well in cultured cells as does adenovirus type 5. These results are consistent with the conclusion that ADP functions in adenovirus infections to promote virus release from cells at the culmination of infection.

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

PubMed

Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

2012-12-01

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio

PubMed Central

Tantama, Mathew; Martínez-François, Juan Ramón; Mongeon, Rebecca; Yellen, Gary

2013-01-01

The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically-encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research. PMID:24096541

Photoaffinity labeling of the TF1-ATPase from the thermophilic bacterium PS3 with 3'-O-(4-benzoyl)benzoyl ADP.

PubMed

Bar-Zvi, D; Yoshida, M; Shavit, N

1985-05-31

3'-O-(4-Benzoyl)benzoyl ADP (BzADP) was used as a photoaffinity label for covalent binding of adenine nucleotide analogs to the nucleotide binding site(s) of the thermophilic bacterium PS3 ATPase (TF1). As with the CF1-ATPase (Bar-Zvi, D. and Shavit, N. (1984) Biochim. Biophys. Acta 765, 340-356) noncovalently bound BzADP is a reversible inhibitor of the TF1-ATPase. BzADP changes the kinetics of ATP hydrolysis from noncooperative to cooperative in the same way as ADP does, but, in contrast to the effect on the CF1-ATPase, it has no effect on the Vmax. In the absence of Mg2+ 1 mol BzADP binds noncovalently to TF1, while with Mg2+ 3 mol are bound. Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF1 and correlates with the inactivation of the ATPase. Complete inactivation of the TF1-ATPase occurs after covalent binding of 2 mol BzADP/mol TF1. Photoinactivation of TF1 by BzADP is prevented if excess of either ADP or ATP is present during irradiation. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the Bz[3H]ADP-labeled TF1-ATPase shows that all the radioactivity is incorporated into the beta subunit.

Practical Experience of Discharge Measurement in Flood Conditions with ADP

NASA Astrophysics Data System (ADS)

Vidmar, A.; Brilly, M.; Rusjan, S.

2009-04-01

Accurate discharge estimation is important for an efficient river basin management and especially for flood forecasting. The traditional way of estimating the discharge in hydrological practice is to measure the water stage and to convert the recorded water stage values into discharge by using the single-valued rating curve .Relationship between the stage and discharge values of the rating curve for the extreme events are usually extrapolated by using different mathematical methods and are not directly measured. Our practice shows that by using the Accoustic Doppler Profiler (ADP) instrument we can record the actual relation between the water stage and the flow velocity at the occurrence of flood waves very successfully. Measurement in flood conditions it is not easy task, because of high water surface velocity and large amounts of sediments in the water and floating objects on the surface like branches, bushes, trees, piles and others which can also easily damage ADP instrument. We made several measurements in such extreme events on the Sava River down to the nuclear power plant Kr\\vsko where we have install fixed cable way. During the several measurement with traditional "moving-boat" measurement technique a mowing bed phenomenon was clearly seen. Measuring flow accurately using ADP that uses the "moving-boat" technique, the system needs a reference against which to relate water velocities to. This reference is river bed and must not move. During flood events we detected difficulty finding a static bed surface to which to relate water velocities. This is caused by motion of the surface layer of bed material or also sediments suspended in the water near bed very densely. So these traditional »moving-boat« measurement techniques that we normally use completely fail. Using stationary measurement method to making individual velocity profile measurements, using an Acoustic Doppler Profiler (ADP), at certain time at fixed locations across the width of a stream gave

ADP binding to TF1 and its subunits induces ultraviolet spectral changes.

PubMed

Hisabori, T; Yoshida, M; Sakurai, H

1986-09-01

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.

Effects of n-acetylcysteine in a rat model of ischemia and reperfusion injury.

PubMed

Cuzzocrea, S; Mazzon, E; Costantino, G; Serraino, I; De Sarro, A; Caputi, A P

2000-08-18

Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of n-acetylcysteine (NAC), a free radical scavenger, in rats subjected to SAO shock. Treatment of rats with NAC (applied at 20 mg/kg, 5 min prior to reperfusion, followed by an infusion of 20 mg/kg/h) attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. NAC also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine in the plasma of the SAO-shocked rats after reperfusion. Immunohistochemical analysis for nitrotyrosine and for poly(ADP-ribose) synthetase (PARS) revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine and PARS were markedly reduced in tissue sections obtained from SAO-shocked rats which had received NAC. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localised in the vascular endothelial cells. Ileum tissue section obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. NAC treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats. In addition, in ex vivo studies in aortic rings from shocked rats, we found reduced contractions to noradrenaline and reduced responsiveness to a relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). NAC treatment improved contractile responsiveness to noradrenaline, enhanced the endothelium-dependent relaxations and significantly improved survival. Taken together, our results clearly demonstrate that NAC

Dynamics of NAD-metabolism: everything but constant.

PubMed

Opitz, Christiane A; Heiland, Ines

2015-12-01

NAD, as well as its phosphorylated form, NADP, are best known as electron carriers and co-substrates of various redox reactions. As such they participate in approximately one quarter of all reactions listed in the reaction database KEGG. In metabolic pathway analysis, the total amount of NAD is usually assumed to be constant. That means that changes in the redox state might be considered, but concentration changes of the NAD moiety are usually neglected. However, a growing number of NAD-consuming reactions have been identified, showing that this assumption does not hold true in general. NAD-consuming reactions are common characteristics of NAD(+)-dependent signalling pathways and include mono- and poly-ADP-ribosylation of proteins, NAD(+)-dependent deacetylation by sirtuins and the formation of messenger molecules such as cyclic ADP-ribose (cADPR) and nicotinic acid (NA)-ADP (NAADP). NAD-consuming reactions are thus involved in major signalling and gene regulation pathways such as DNA-repair or regulation of enzymes central in metabolism. All known NAD(+)-dependent signalling processes include the release of nicotinamide (Nam). Thus cellular NAD pools need to be constantly replenished, mostly by recycling Nam to NAD(+). This process is, among others, regulated by the circadian clock, causing complex dynamic changes in NAD concentration. As disturbances in NAD homoeostasis are associated with a large number of diseases ranging from cancer to diabetes, it is important to better understand the dynamics of NAD metabolism to develop efficient pharmacological invention strategies to target this pathway. © 2015 Authors; published by Portland Press Limited.

Mw Spectroscopy Coupled with Ultrafast UV Laser Vaporization: {RIBOSE} Found in the Gas Phase

NASA Astrophysics Data System (ADS)

Cocinero, Emilio J.; Ecija, Patricia; Basterretxea, Francisco J.; Fernandez, Jose A.; Castano, Fernando; Lesarri, Alberto; Grabow, Jens-Uwe

2012-06-01

Sugars are aldoses or ketoses with multiple hydroxy groups which have been elusive to spectroscopic studies. Here we report a rotational study of the aldopentose ribose. According to any standard textbook aldopentoses can exhibit either linear forms, cyclic five-membered (furanose) structures or six-membered (pyranose) rings, occurring either as α- or β- anomers depending on the orientation of the hydroxy group at C-1 (anomeric carbon). β-Furanose is predominant in ribonucleosides, RNA, ATP and other biochemically relevant derivatives, but is β-furanose the native form also of free ribose? Recent condensed-phase X-ray and older NMR studies delivered conflicting results. In order to solve this question we conducted a microwave study on D-ribose that, owing to ultrafast UV laser vaporization, has become the first C-5 sugar observed with rotational resolution. The spectrum revealed six conformations of free ribose, preferentially adopting β-pyranose chairs as well as higher-energy α-pyranose forms. The method also allowed for unambiguous distinction between different orientations of the hydroxy groups, which stabilize the structures by cooperative hydrogen-bond networks. No evidence was observed of the α-/β-furanoses or linear forms found in the biochemical derivatives. i) D. Šišak, L. B. McCusker, G. Zandomeneghi, B. H. Meier, D. Bläser, R. Boese, W. B. Schweizer, R. Gylmour and J. D. Dunitz Angew. Chem. Int. Ed. 49, 4503, 2010. ii) W. Saenger Angew. Chem. Int. Ed. 49, 6487, 2010. i) M. Rudrum, and D. F. Shaw, J. Chem. Soc. 52, 1965. ii) R. U. Lemieux and J. D. Stevens Can. J. Chem. 44, 249, 1966. iii) E. Breitmaier and U. Hollstein Org. Magn. Reson. 8, 573, 1976. E. J. Cocinero, A. Lesarri, P. Écija, F. J. Basterretxea, J. U. Grabow, J. A. Fernández and F. Castaño Angew. Chem. Int. Ed. in press: DOI: 10.1002/anie.201107973, 2012.

Biosynthesis of 2-deoxysugars using whole-cell catalyst expressing 2-deoxy-D-ribose 5-phosphate aldolase.

PubMed

Li, Jitao; Yang, Jiangang; Men, Yan; Zeng, Yan; Zhu, Yueming; Dong, Caixia; Sun, Yuanxia; Ma, Yanhe

2015-10-01

2-Deoxy-D-ribose 5-phosphate aldolase (DERA) accepts a wide variety of aldehydes and is used in de novo synthesis of 2-deoxysugars, which have important applications in drug manufacturing. However, DERA has low preference for non-phosphorylated substrates. In this study, DERA from Klebsiella pneumoniae (KDERA) was mutated to increase its enzyme activity and substrate tolerance towards non-phosphorylated polyhydroxy aldehyde. Mutant KDERA(K12) (S238D/F200I/ΔY259) showed a 3.15-fold improvement in enzyme activity and a 1.54-fold increase in substrate tolerance towards D-glyceraldehyde compared with the wild type. Furthermore, a whole-cell transformation strategy using resting cells of the BL21(pKDERA12) strain, containing the expressed plasmid pKDERA12, resulted in increase in 2-deoxy-D-ribose yield from 0.41 mol/mol D-glyceraldehyde to 0.81 mol/mol D-glyceraldehyde and higher substrate tolerance from 0.5 to 3 M compared to in vitro assays. With further optimization of the transformation process, the BL21(pKDERA12) strain produced 2.14 M (287.06 g/L) 2-deoxy-D-robose (DR), with a yield of 0.71 mol/mol D-glyceraldehyde and average productivity of 0.13 mol/L·h (17.94 g/L·h). These results demonstrate the potential for large-scale production of 2-deoxy-D-ribose using the BL21(pKDERA12) strain. Furthermore, the BL21(pKDERA12) strain also exhibited the ability to efficiently produce 2-deoxy-D-altrose from D-erythrose, as well as 2-deoxy-L-xylose and 2-deoxy-L-ribose from L-glyceraldehyde.

Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

PubMed Central

Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

2015-01-01

Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. PMID:26713879

Characteristic antioxidant activity and comprehensive flavor compound profile of scallop (Chlamys farreri) mantle hydrolysates-ribose Maillard reaction products.

PubMed

Han, Jia-Run; Yan, Jia-Nan; Sun, Shi-Guang; Tang, Yue; Shang, Wen-Hui; Li, Ao-Ting; Guo, Xiao-Kun; Du, Yi-Nan; Wu, Hai-Tao; Zhu, Bei-Wei; Xiong, Youling L

2018-09-30

The objective of the present study was to improve the utilization of scallop (Chlamys farreri) byproducts by using Maillard reaction. Scallop mantle hydrolysates (SMHs) were prepared using neutrase then reacted with ribose. Thirty-four peptides were identified from SMHs by UPLC-Q-TOF-MS, and the abundance of Asp and Lys suggested the strong Maillard reactivity. The formation of Schiff's base as well as modification of amide I, II and III bands in Maillard reaction products (MRPs) was confirmed by ultraviolet-visible, fluorescence, and Fourier transform infrared spectroscopy. Thirty volatile compounds were produced by the reaction of SMHs with ribose. Moreover, MRPs with enhanced radical scavenging and anti-linoleic acid peroxidation activities over SMHs promoted the survival and reduced the DNA damage of HepG2 cells treated with hydrogen peroxide. These results suggest that SMHs-ribose MRPs can be potentially used as food antioxidant for suppressing of lipid oxidation or protecting of cell from oxidative damage. Copyright © 2018 Elsevier Ltd. All rights reserved.

Inflammatory and age-related pathologies in mice with ectopic expression of human PARP-1.

PubMed

Mangerich, Aswin; Herbach, Nadja; Hanf, Benjamin; Fischbach, Arthur; Popp, Oliver; Moreno-Villanueva, María; Bruns, Oliver T; Bürkle, Alexander

2010-06-01

Poly(ADP-ribose) polymerase-1 (PARP-1) is a sensor for DNA strand breaks and some unusual DNA structures and catalyzes poly(ADP-ribosyl)ation of nuclear proteins with NAD(+) serving as substrate. PARP-1 is involved in the regulation of genomic integrity, transcription, inflammation, and cell death. Due to its versatile role, PARP-1 is discussed both as a longevity factor and as an aging-promoting factor. Recently, we generated a mouse model with ectopic integration of full-length hPARP-1 [Mangerich, A., Scherthan, H., Diefenbach, J., Kloz, U., van der Hoeven, F., Beneke, S. and Bürkle, A., 2009. A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1. Transgenic Res. 18, 261-279]. Here, we show that hPARP-1 mice exhibit impaired survival rates accompanied by reduced hair growth and premature development of several inflammation and age-associated pathologies, such as adiposity, kyphosis, nephropathy, dermatitis, pneumonitis, cardiomyopathy, hepatitis, and anemia. Moreover, mutant male mice showed impaired glucose tolerance, yet without developing manifest diabetes. Overall tumor burden was comparable in wild-type and hPARP-1 mice, but tumor spectrum was shifted in mutant mice, showing lower incidence of sarcomas, but increased incidence of carcinomas. Furthermore, DNA repair was delayed in splenocytes of hPARP-1 mice, and gene expression of pro-inflammatory cytokines was dysregulated. Our results suggest that in hPARP-1 mice impaired DNA repair, accompanied by a continuous low-level increase in pro-inflammatory stimuli, causes development of chronic diseases leading to impaired survival. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

PTPL1 and PKCδ contribute to proapoptotic signalling in prostate cancer cells.

PubMed

Castilla, C; Chinchón, D; Medina, R; Torrubia, F J; Japón, M A; Sáez, C

2013-04-04

PTPL1 is a non-receptor protein tyrosine phosphatase involved in apoptosis regulation, although controversial findings have been reported in different cancer types. We report here a proapoptotic role for PTPL1 in PC3 and LNCaP prostate cancer cells, as its absence induces apoptosis resistance upon treatment with different drugs. In PC3 cells, PTPL1 silencing by small interfering RNA influences the expression levels of Bcl-xL and Mcl-1(S) proteins as well as final events in the apoptotic process such as activation of caspases and caspase-mediated cleavage of proteins like Mcl-1 or poly (ADP-ribose) polymerase. We have identified PKCδ as an intermediary of PTPL1-mediated apoptotic signalling and that phosphorylation status of NF-κB and IκBα is influenced by PTPL1 and PKCδ. Furthermore, the loss of PTPL1 and PKCδ expression in poorly differentiated, more aggressive human prostate cancers also indicate that their absence could be related to apoptosis resistance and tumour progression.

Oleanolic acid and its synthetic derivatives for the prevention and therapy of cancer: Preclinical and clinical evidence

PubMed Central

Shanmugam, Muthu K.; Dai, Xiaoyun; Kumar, Alan Prem; Tan, Benny KH; Sethi, Gautam; Bishayee, Anupam

2014-01-01

Oleanolic acid (OA, 3β-hydroxyolean-12-en-28-oic acid) is a ubiquitous pentacyclic multifunctional triterpenoid, widely found in several dietary and medicinal plants. Natural and synthetic OA derivatives can modulate multiple signaling pathways including nuclear factor-κB, AKT, signal transducer and activator of transcription 3, mammalian target of rapamycin, caspases, intercellular adhesion molecule 1, vascular endothelial growth factor, and poly (ADP-ribose) polymerase in a variety of tumor cells. Importantly, synthetic derivative of OA, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), and its C-28 methyl ester (CDDO-Me) and C28 imidazole (CDDO-Im) have demonstrated potent antiangiogenic and antitumor activities in rodent cancer models. These agents are presently under evaluation in phase I studies in cancer patients. This review summarizes the diverse molecular targets of OA and its derivatives and also provides clear evidence on their promising potential in preclinical and clinical situations. PMID:24486850

Quantitative Analysis of NAD Synthesis-Breakdown Fluxes.

PubMed

Liu, Ling; Su, Xiaoyang; Quinn, William J; Hui, Sheng; Krukenberg, Kristin; Frederick, David W; Redpath, Philip; Zhan, Le; Chellappa, Karthikeyani; White, Eileen; Migaud, Marie; Mitchison, Timothy J; Baur, Joseph A; Rabinowitz, Joshua D

2018-05-01

The redox cofactor nicotinamide adenine dinucleotide (NAD) plays a central role in metabolism and is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. NAD concentration falls during aging, which has triggered intense interest in strategies to boost NAD levels. A limitation in understanding NAD metabolism has been reliance on concentration measurements. Here, we present isotope-tracer methods for NAD flux quantitation. In cell lines, NAD was made from nicotinamide and consumed largely by PARPs and sirtuins. In vivo, NAD was made from tryptophan selectively in the liver, which then excreted nicotinamide. NAD fluxes varied widely across tissues, with high flux in the small intestine and spleen and low flux in the skeletal muscle. Intravenous administration of nicotinamide riboside or mononucleotide delivered intact molecules to multiple tissues, but the same agents given orally were metabolized to nicotinamide in the liver. Thus, flux analysis can reveal tissue-specific NAD metabolism. Copyright © 2018 Elsevier Inc. All rights reserved.

Enriched Environment Increases PCNA and PARP1 Levels in Octopus vulgaris Central Nervous System: First Evidence of Adult Neurogenesis in Lophotrochozoa.

PubMed

Bertapelle, Carla; Polese, Gianluca; Di Cosmo, Anna

2017-06-01

Organisms showing a complex and centralized nervous system, such as teleosts, amphibians, reptiles, birds and mammals, and among invertebrates, crustaceans and insects, can adjust their behavior according to the environmental challenges. Proliferation, differentiation, migration, and axonal and dendritic development of newborn neurons take place in brain areas where structural plasticity, involved in learning, memory, and sensory stimuli integration, occurs. Octopus vulgaris has a complex and centralized nervous system, located between the eyes, with a hierarchical organization. It is considered the most "intelligent" invertebrate for its advanced cognitive capabilities, as learning and memory, and its sophisticated behaviors. The experimental data obtained by immunohistochemistry and western blot assay using proliferating cell nuclear antigen and poli (ADP-ribose) polymerase 1 as marker of cell proliferation and synaptogenesis, respectively, reviled cell proliferation in areas of brain involved in learning, memory, and sensory stimuli integration. Furthermore, we showed how enriched environmental conditions affect adult neurogenesis. © 2017 Wiley Periodicals, Inc.

[Free radicals and hepatic ischemia-reperfusion].

PubMed

Szijártó, Attila

2015-11-22

The critical importance of the ischemic-reperfusive injury is well documented with regards to numerous organs and clinical conditions. Oxygen free radicals play a central role in the mediation of the injury, which dominantly influences the prevalence of postoperative complications, (long term) organ damage, and the potential manifestation of systemic reactions. The both anatomically and pathophysiologically unique ischemic-reperfusive injury of the liver, which is expressively vulnerable to free radicals, is of utmost importance in liver surgery. Several techniques (adaptive maneuvers, chemical agents) are known to ameliorate the reperfusive injury. Based on the prior research of the workgroup of the author, the aim of the current article is to overview the set of measures capable of attenuating ischemic-reperfusive injury (ischemic preconditioning, -perconditioning, administration of adenosine, -inosine, -levosimendan, and -poly-ADP-ribose-polymerase inhibitor), with special attention to the ischemic-reperfusive injury of the liver, as well as the special pathophysiological role of free radicals in mediating hepatic damage.

Licochalcone C induces apoptosis via B-cell lymphoma 2 family proteins in T24 cells.

PubMed

Wang, Penglong; Yuan, Xuan; Wang, Yan; Zhao, Hong; Sun, Xiling; Zheng, Qiusheng

2015-11-01

The current study investigated the mechanisms by which licochalcone C induces apoptosis of T24 human malignant bladder cancer cells. Cell viability was evaluated using an MTT assay. Apoptosis was investigated using a morphological assay, flow cytometry and a caspase‑3 activity assay. Alterations in the gene expression levels of Bcl‑2 family members were measured by semi‑quantitative reverse transcription‑polymerase chain reaction assays. The protein levels of pro‑caspase‑3 and cleaved poly(ADP ribose) polymerase were measured using western blotting. The results indicated that licochalcone C induced T24 cell apoptosis in a concentration‑dependent manner. Licochalcone C treatment reduced the levels of the anti‑apoptotic mRNAs (Bcl‑2, Bcl‑w and Bcl‑XL) and increased expression of the pro‑apoptotic mRNAs (Bax and Bim). The Bcl‑2 family inhibitor (ABT‑737) reduced apoptosis induced by licochalcone C in T24 cells. The current study demonstrated that licochalcone C may be a potential adjuvant therapeutic agent for bladder cancer.

Anticancer effects on human pancreatic cancer cells of triterpenoids, polysaccharides and 1,3-β-D-glucan derived from the fruiting body of Antrodia camphorata.

PubMed

Lee, Chu-I; Wu, Chih-Chung; Hsieh, Shu-Ling; Lee, Chun-Lin; Chang, Yueh-Ping; Chang, Chih-Chuan; Wang, Yi-Zhen; Wang, Jyh-Jye

2014-12-01

Antrodia camphorata is a fungus native to Taiwan, and it is considered a precious medicinal agent. We analyzed triterpenoids, polysaccharides and 1,3-β-D-glucan, three major effective components in A. camphorata extracts (ACE). ACE exhibited a selective cytotoxic effect on BxPC-3 human pancreatic cancer cells. ACE markedly inhibited the migration ability of BxPC-3 cells. Treatment of BxPC-3 cells with ACE resulted in the increase of cells in the sub-G1 phase and G2/M phase arrest. Apoptosis was confirmed by validating phosphatidylserine externalization, the observation of characteristic chromatin condensation, and nuclear DNA fragmentation. ACE induced apoptosis in BxPC-3 cells through a mitochondria-dependent pathway by triggering an appropriate balance of bax/bcl-2, cytochrome c release, activation of caspase-9 and -3, and poly(ADP-ribose) polymerase cleavage. ACE shows great therapeutic potential due to its cytotoxic effects against BxPC-3 cells which include inhibiting cell migration and inducing mitochondria-mediated apoptosis.

A Selenium-Modified Ginseng Polysaccharide Promotes the Apoptosis in Human Promyelocytic Leukemia (HL-60) Cells via a Mitochondrial-Mediated Pathway.

PubMed

Liao, Kainan; Bian, Zedong; Xie, Dongke; Peng, Qiang

2017-05-01

A ginseng polysaccharide was extracted, purified, and modified by nitric acid-selenious acid (HNO 3 -H 2 SeO 3 ) method to yield one selenylation-modified polysaccharide (sGP). We reported for the first time the anticancer potential of sGP on the human promyelocytic leukemia HL-60 cell line and evaluated its relevant underlying mechanism. Our results showed that sGP markedly inhibited the growth of HL-60 cells via induction of apoptosis. The event of apoptosis was accompanied by the formation of apoptotic bodies; the release of cytochrome c; loss of mitochondrial membrane potential; and activation of caspase-9, caspase-3, and cleavage of poly ADP ribose polymerase (PARP) in HL-60 cells. In addition, western blot analysis showed that sGP inhibited antiapoptotic Bcl-2 protein expression and increased proapoptotic Bax protein expression in cells under identical conditions. Together, our study suggests that sGP induces apoptosis of HL-60 cells through the mitochondrial-dependent pathway.

Beyond Breast and Ovarian Cancers: PARP Inhibitors for BRCA Mutation-Associated and BRCA-Like Solid Tumors

PubMed Central

O’Sullivan, Ciara C.; Moon, Dominic H.; Kohn, Elise C.; Lee, Jung-Min

2014-01-01

Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown clinical activity in patients with germline BRCA1/2 mutation (gBRCAm)-associated breast and ovarian cancers. Accumulating evidence suggests that PARPi may have a wider application in the treatment of cancers defective in DNA damage repair pathways, such as prostate, lung, endometrial, and pancreatic cancers. Several PARPi are currently in phase I/II clinical investigation, as single-agents and/or combination therapy in these solid tumors. Understanding more about the molecular abnormalities involved in BRCA-like phenotype in solid tumors beyond breast and ovarian cancers, exploring novel therapeutic trial strategies and drug combinations, and defining potential predictive biomarkers are critical to expanding the scope of PARPi therapy. This will improve clinical outcome in advanced solid tumors. Here, we briefly review the preclinical data and clinical development of PARPi, and discuss its future development in solid tumors beyond gBRCAm-associated breast and ovarian cancers. PMID:24616882

A potential oral anticancer drug candidate, Moringa oleifera leaf extract, induces the apoptosis of human hepatocellular carcinoma cells

PubMed Central

JUNG, IL LAE; LEE, JU HYE; KANG, SE CHAN

2015-01-01

It has previously been reported that cold water-extracts of Moringa oleifera leaf have anticancer activity against various human cancer cell lines, including non-small cell lung cancer. In the present study, the anticancer activity of M. oleifera leaf extracts was investigated in human hepatocellular carcinoma HepG2 cells. By the analysis of apoptotic signals, including the induction of caspase or poly(ADP-ribose) polymerase cleavage, and the Annexin V and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, it was demonstrated that M. oleifera leaf extracts induce the apoptosis of HepG2 cells. In the hollow fiber assay, oral administration of the leaf extracts significantly reduced (44–52%) the proliferation of the HepG2 cells and A549 non-small cell lung cancer cells. These results support the potential of soluble extracts of M. oleifera leaf as orally administered therapeutics for the treatment of human liver and lung cancers. PMID:26622717

Theophylline prevents NAD{sup +} depletion via PARP-1 inhibition in human pulmonary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moonen, Harald J.J.; Geraets, Liesbeth; Vaarhorst, Anika

2005-12-30

Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD{sup +}, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD{sup +} pool, and of NAD{sup +}-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD{sup +} levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzymemore » inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.« less

The methylene chloride fraction of Trichosanthis Fructus induces apoptosis in U937 cells through the mitochondrial pathway.

PubMed

Lee, Eun-Ok; Lee, Ju-Ryoung; Kim, Kwan-Hyun; Baek, Nam-In; Lee, Soo-Jin; Lee, Bog-Hieu; Cho, Kyung-Dong; Ahn, Kyoo-Seok; Kim, Sung-Hoon

2006-01-01

Trichosanthis kirilowii MAXIM has been used as a folk remedy to treat diabetes, leukemia, and breast cancer. In the present study, the apoptotic mechanism of the methylene chloride fraction of Trichosanthis Fructus (MCTF) was investigated in human leukemic U937 cells. MCTF exhibited antiproliferative effectsagainst U937 cells (IC50=ca. 8 microg/ml). Apoptotic bodies were observed in MCTF-treated U937 cells in the TUNEL assay. We also confirmed that MCTF significantly increases annexin V(+)/propidium iodide-cells using FACS analysis. MCTF treatment activated caspase-8, -9 and -3, and led to cleaved poly (ADP-ribose) polymerase and release of cytochrome c into cytosol in a concentration-dependent manner, while MCTF did not affect Bax or Bcl-2 protein levels as shown by Western blot analysis. Taken together, these results indicate that MCTF can induce apoptosis in U937 cells chiefly via a mitochondrial-mediated pathway and suggest that Trichosanthis Fructus can be used in cancer treatment as a chemopreventive agent.

Targeted PET imaging strategy to differentiate malignant from inflamed lymph nodes in diffuse large B-cell lymphoma

PubMed Central

Salloum, Darin; Carney, Brandon; Brand, Christian; Kossatz, Susanne; Sadique, Ahmad; Lewis, Jason S.; Weber, Wolfgang A.; Wendel, Hans-Guido; Reiner, Thomas

2017-01-01

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults. DLBCL exhibits highly aggressive and systemic progression into multiple tissues in patients, particularly in lymph nodes. Whole-body 18F-fluodeoxyglucose positron emission tomography ([18F]FDG-PET) imaging has an essential role in diagnosing DLBCL in the clinic; however, [18F]FDG-PET often faces difficulty in differentiating malignant tissues from certain nonmalignant tissues with high glucose uptake. We have developed a PET imaging strategy for DLBCL that targets poly[ADP ribose] polymerase 1 (PARP1), the expression of which has been found to be much higher in DLBCL than in healthy tissues. In a syngeneic DLBCL mouse model, this PARP1-targeted PET imaging approach allowed us to discriminate between malignant and inflamed lymph nodes, whereas [18F]FDG-PET failed to do so. Our PARP1-targeted PET imaging approach may be an attractive addition to the current PET imaging strategy to differentiate inflammation from malignancy in DLBCL. PMID:28827325

Ethanol Extract of Dianthus chinensis L. Induces Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells In Vitro

PubMed Central

Nho, Kyoung Jin; Chun, Jin Mi; Kim, Ho Kyoung

2012-01-01

Dianthus chinensis L. is used to treat various diseases including cancer; however, the molecular mechanism by which the ethanol extract of Dianthus chinensis L. (EDCL) induces apoptosis is unknown. In this study, the apoptotic effects of EDCL were investigated in human HepG2 hepatocellular carcinoma cells. Treatment with EDCL significantly inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. This induction was associated with chromatin condensation, activation of caspases, and cleavage of poly (ADP-ribose) polymerase protein. However, apoptosis induced by EDCL was attenuated by caspase inhibitor, indicating an important role for caspases in EDCL responses. Furthermore, EDCL did not alter the expression of bax in HepG2 cells but did selectively downregulate the expression of bcl-2 and bcl-xl, resulting in an increase in the ratio of bax:bcl-2 and bax:bcl-xl. These results support a mechanism whereby EDCL induces apoptosis through the mitochondrial pathway and caspase activation in HepG2 cells. PMID:22645629

Dihydromyricetin induces mitochondria-mediated apoptosis in HepG2 cells through down-regulation of the Akt/Bad pathway.

PubMed

Zhang, Zhuangwei; Zhang, Huiqin; Chen, Shiyong; Xu, Yan; Yao, Anjun; Liao, Qi; Han, Liyuan; Zou, Zuquan; Zhang, Xiaohong

2017-02-01

The plant flavonol dihydromyricetin (DHM) was reported to induce apoptosis in human hepatocarcinoma HepG2 cells. This study was undertaken to elucidate the underlying molecular mechanism of action of DHM. In the study, DHM down-regulated Akt expression and its phosphorylation at Ser473, up-regulated the levels of mitochondrial proapoptotic proteins Bax and Bad, and inhibited the phosphorylation of Bad at Ser136 and Ser112. It also inhibited the expression of the antiapoptotic protein Bcl-2 and enhanced the cleavage and activation of caspase-3 as well as the degradation of its downstream target poly(ADP-ribose) polymerase. Our results for the first time suggest that DHM-induced apoptosis in HepG2 cells may come about by the inhibition of the Akt/Bad signaling pathway and stimulation of the mitochondrial apoptotic pathway. Dihydromyricetin may be a promising therapeutic medication for hepatocellular carcinoma. Copyright © 2017 Elsevier Inc. All rights reserved.

Triple-negative breast cancer--current status and future directions.

PubMed

Gluz, O; Liedtke, C; Gottschalk, N; Pusztai, L; Nitz, U; Harbeck, N

2009-12-01

Triple-negative breast cancer (TNBC) is defined by a lack of expression of both estrogen and progesterone receptor as well as human epidermal growth factor receptor 2. It is characterized by distinct molecular, histological and clinical features including a particularly unfavorable prognosis despite increased sensitivity to standard cytotoxic chemotherapy regimens. TNBC is highly though not completely concordant with various definitions of basal-like breast cancer (BLBC) defined by high-throughput gene expression analyses. The lack in complete concordance may in part be explained by both BLBC and TNBC comprising entities that in themselves are heterogeneous. Numerous efforts are currently being undertaken to improve prognosis for patients with TNBC. They comprise both optimization of choice and scheduling of common cytotoxic agents (i.e. addition of platinum salts or dose intensification strategies) and introduction of novel agents (i.e. poly-ADP-ribose-polymerase-1 inhibitors, agents targeting the epidermal growth factor receptor, multityrosine kinase inhibitors or antiangiogenic agents).

Development and validation of a high-performance liquid chromatography method for the quantification of talazoparib in rat plasma: Application to plasma protein binding studies.

PubMed

Hidau, Mahendra Kumar; Kolluru, Srikanth; Palakurthi, Srinath

2018-02-01

A sensitive and selective RP-HPLC method has been developed and validated for the quantification of a highly potent poly ADP ribose polymerase inhibitor talazoparib (TZP) in rat plasma. Chromatographic separation was performed with isocratic elution method. Absorbance for TZP was measured with a UV detector (SPD-20A UV-vis) at a λ max of 227 nm. Protein precipitation was used to extract the drug from plasma samples using methanol-acetonitrile (65:35) as the precipitating solvent. The method proved to be sensitive and reproducible over a 100-2000 ng/mL linearity range with a lower limit of quantification (LLQC) of 100 ng/mL. TZP recovery was found to be >85%. Following analytical method development and validation, it was successfully employed to determine the plasma protein binding of TZP. TZP has a high level of protein binding in rat plasma (95.76 ± 0.38%) as determined by dialysis method. Copyright © 2017 John Wiley & Sons, Ltd.

Induction of apoptosis in HT-29 colon cancer cells by phloretin.

PubMed

Park, So Young; Kim, Eun Ji; Shin, Hyun-Kyung; Kwon, Dae Young; Kim, Myung Sunny; Surh, Young-Joon; Park, Jung Han Yoon

2007-12-01

Phloretin, which is present in apples and pears, has been found to inhibit the growth of several cancer cells and induce apoptosis of B16 melanoma and HL60 human leukemia cells. The present study examined whether and how phloretin induces apoptosis of HT-29 human colon cancer cells. Phloretin (0-100 micromol/L) substantially decreased viable cell number and induced apoptosis of HT-29 cells in a dose-dependent manner. Western blot analysis of total cell lysates revealed that phloretin increased the protein levels of Bax but had no effect on Bcl-2. In addition, phloretin induced cleavage of caspase-8, -9, -7, and -3 and poly(ADP-ribose) polymerase. Furthermore, phloretin increased the levels of cytochrome c and Smac/Diablo in the cytosol. The present results indicate that phloretin inhibits HT-29 cell growth by inducing apoptosis, which may be mediated through changes in mitochondrial membrane permeability and activation of the caspase pathways.

NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells.

PubMed

Ratajczak, Joanna; Joffraud, Magali; Trammell, Samuel A J; Ras, Rosa; Canela, Núria; Boutant, Marie; Kulkarni, Sameer S; Rodrigues, Marcelo; Redpath, Philip; Migaud, Marie E; Auwerx, Johan; Yanes, Oscar; Brenner, Charles; Cantó, Carles

2016-10-11

NAD + is a vital redox cofactor and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Supplementation with NAD + precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we show that nicotinamide riboside kinase 1 (NRK1) is necessary and rate-limiting for the use of exogenous NR and NMN for NAD + synthesis. Using genetic gain- and loss-of-function models, we further demonstrate that the role of NRK1 in driving NAD + synthesis from other NAD + precursors, such as nicotinamide or nicotinic acid, is dispensable. Using stable isotope-labelled compounds, we confirm NMN is metabolized extracellularly to NR that is then taken up by the cell and converted into NAD + . Our results indicate that mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD + synthesis, explaining the overlapping metabolic effects observed with the two compounds.

NAD+-Dependent Activation of Sirt1 Corrects the Phenotype in a Mouse Model of Mitochondrial Disease

PubMed Central

Cerutti, Raffaele; Pirinen, Eija; Lamperti, Costanza; Marchet, Silvia; Sauve, Anthony A.; Li, Wei; Leoni, Valerio; Schon, Eric A.; Dantzer, Françoise; Auwerx, Johan; Viscomi, Carlo; Zeviani, Massimo

2014-01-01

Summary Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD+-dependent protein deacetylase. As NAD+ boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD+ play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD+ precursor, or reduction of NAD+ consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans. PMID:24814483

The Vitamin Nicotinamide: Translating Nutrition into Clinical Care

PubMed Central

Maiese, Kenneth; Chong, Zhao Zhong; Hou, Jinling; Shang, Yan Chen

2009-01-01

Nicotinamide, the amide form of vitamin B3 (niacin), is changed to its mononucleotide compound with the enzyme nicotinic acide/nicotinamide adenylyl-transferase, and participates in the cellular energy metabolism that directly impacts normal physiology. However, nicotinamide also influences oxidative stress and modulates multiple pathways tied to both cellular survival and death. During disorders that include immune system dysfunction, diabetes, and aging-related diseases, nicotinamide is a robust cytoprotectant that blocks cellular inflammatory cell activation, early apoptotic phosphatidylserine exposure, and late nuclear DNA degradation. Nicotinamide relies upon unique cellular pathways that involve forkhead transcription factors, sirtuins, protein kinase B (Akt), Bad, caspases, and poly (ADP-ribose) polymerase that may offer a fine line with determining cellular longevity, cell survival, and unwanted cancer progression. If one is cognizant of the these considerations, it becomes evident that nicotinamide holds great potential for multiple disease entities, but the development of new therapeutic strategies rests heavily upon the elucidation of the novel cellular pathways that nicotinamide closely governs. PMID:19783937

NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells

PubMed Central

Ratajczak, Joanna; Joffraud, Magali; Trammell, Samuel A. J.; Ras, Rosa; Canela, Núria; Boutant, Marie; Kulkarni, Sameer S.; Rodrigues, Marcelo; Redpath, Philip; Migaud, Marie E.; Auwerx, Johan; Yanes, Oscar; Brenner, Charles; Cantó, Carles

2016-01-01

NAD+ is a vital redox cofactor and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Supplementation with NAD+ precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we show that nicotinamide riboside kinase 1 (NRK1) is necessary and rate-limiting for the use of exogenous NR and NMN for NAD+ synthesis. Using genetic gain- and loss-of-function models, we further demonstrate that the role of NRK1 in driving NAD+ synthesis from other NAD+ precursors, such as nicotinamide or nicotinic acid, is dispensable. Using stable isotope-labelled compounds, we confirm NMN is metabolized extracellularly to NR that is then taken up by the cell and converted into NAD+. Our results indicate that mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD+ synthesis, explaining the overlapping metabolic effects observed with the two compounds. PMID:27725675

Persimmon Leaves (Diospyros kaki) Extract Protects Optic Nerve Crush-Induced Retinal Degeneration

PubMed Central

Ryul Ahn, Hong; Kim, Kyung-A; Kang, Suk Woo; Lee, Joo Young; Kim, Tae-Jin; Jung, Sang Hoon

2017-01-01

Retinal ganglion cell (RGC) death is part of many retinal diseases. Here, we report that the ethanol extract of Diospyros kaki (EEDK) exhibits protective properties against retinal degeneration, both in vitro and in vivo. Upon exposure to cytotoxic compounds, RGC-5 cells showed approximately 40% cell viability versus the control, while pre-treatment with EEDK markedly increased cell viability in a concentration-dependent manner. Further studies revealed that cell survival induced by EEDK was associated with decreased levels of apoptotic proteins, such as poly (ADP-ribose) polymerase, p53, and cleaved caspase-3. In addition to apoptotic pathways, we demonstrated that expression levels of antioxidant-associated proteins, such as superoxide dismutase-1, glutathione S-transferase, and glutathione peroxidase-1, were positively modulated by EEDK. In a partial optic nerve crush mouse model, EEDK had similar ameliorating effects on retinal degeneration resulting from mechanical damages. Therefore, our results suggest that EEDK may have therapeutic potential against retinal degenerative disorders, such as glaucoma. PMID:28425487

Chemical Characterization and Antitumor Activities of Polysaccharide Extracted from Ganoderma lucidum

PubMed Central

Liang, Zengenni; Yi, Youjin; Guo, Yutong; Wang, Rencai; Hu, Qiulong; Xiong, Xingyao

2014-01-01

Ganoderma lucidum polysaccharide (GLP) is a biologically active substance reported to possess anti-tumor ability. Nonetheless, the mechanisms of GLP-stimulated apoptosis are still unclear. This study aims to determine the inhibitory and apoptosis-inducing effects of GLP on HCT-116 cells. We found that GLP reduced cell viability on HCT-116 cells in a time- and dose-dependent manner, which in turn, induced cell apoptosis. The observed apoptosis was characterized by morphological changes, DNA fragmentation, mitochondrial membrane potential decrease, S phase population increase, and caspase-3 and -9 activation. Furthermore, inhibition of c-Jun N-terminal kinase (JNK) by SP600125 led to a dramatic decrease of the GLP-induced apoptosis. Western blot analysis unveiled that GLP up-regulated the expression of Bax/Bcl-2, caspase-3 and poly (ADP-ribose) polymerase (PARP). These results demonstrate that apoptosis stimulated by GLP in human colorectal cancer cells is associated with activation of mitochondrial and mitogen-activated protein kinase (MAPK) pathways. PMID:24857920

Effects of etoposide alone and in combination with piroxicam on canine osteosarcoma cell lines.

PubMed

Ong, S M; Saeki, K; Tanaka, Y; Nishimura, R; Nakagawa, T

2016-12-01

Osteosarcoma (OSA) is the most common primary bone tumour in dogs. The poor survival rate in dogs with OSA highlights the need for new therapeutic approaches. This study evaluated the cytotoxic effects of etoposide, alone and in combination with piroxicam, on canine OSA cell cultures. Etoposide alone significantly suppressed cell growth and viability, whereas etoposide in combination with piroxicam exhibited concentration dependent cytotoxicity. The anti-proliferative effect was a result of inactivity of the Cdc2-cyclin B1 complex, which correlated with an increase in the G 2 /M fraction. This subsequently activated the apoptosis cascade, as indicated by elevated apoptosis levels and up-regulation of poly (ADP-ribose) polymerase proteolytic cleavage. Down-regulation of survivin expression induced by the combination treatment may have contributed to the enhanced cytotoxicity. The results of this study suggest that further investigation of etoposide and piroxicam as a therapeutic combination for canine OSA is warranted. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Latest international guidelines for screening, prevention and treatment of familial breast cancer - implications for the relevant practice in Hungary].

PubMed

Romics, László; Kocsis, Judit; Ormándi, Katalin; Molnár, Béla Ákos

2016-07-01

Screening, prevention and treatment of familial breast cancer require a multidisciplinary approach. New guidelines were published in the United Kingdom for the management of familial breast cancer. The authors summarise these new guidelines and analyse the relevant practice in Hungary. Relevant guidelines of the National Institute for Health and Care Excellence and Familial Breast Cancer Report (NHS Scotland) are described. New guidelines will increase the number of genetic tests as well as genetic counselling. An increase in the number of breast magnetic resonance imaging is expected, too. Chemoprevention can be offered for individuals with medium risk and above. Promising trials are underway with platinum based chemotherapy and polyADP-ribose polimerase inhibitors for the systemic treatment of familial breast cancer. The increase in the number of genetic tests, counselling, and breast magnetic resonance imaging may have a significant impact on health care budget. These guidelines will change some aspects of the current management of familial breast cancer. Orv. Hetil., 2016, 157(28), 1117-1125.

Bupivacaine induces apoptosis via ROS in the Schwann cell line.

PubMed

Park, C J; Park, S A; Yoon, T G; Lee, S J; Yum, K W; Kim, H J

2005-09-01

Local anesthetics have been generally accepted as being safe. However, recent clinical trials and basic studies have provided strong evidence for the neurotoxicity of local anesthetics, especially through apoptosis. We hypothesized that local anesthetics cause neural complications through Schwann cell apoptosis. Among local anesthetics tested on the Schwann cell line, RT4-D6P2T, bupivacaine significantly induced cell death, measured by the methyl tetrazolium (MTT) assay, in a dose- (LD50 = 476 microM) and time-dependent manner. The bupivacaine-induced generation of reactive oxygen species (ROS), which was initiated within 5 hrs and preceded the activation of caspase-3 and poly ADP-ribose polymerase (PARP) degradation, was suggested to trigger apoptosis, exhibited by Hoechst 33258 nuclear staining and DNA fragmentation. Furthermore, concomitant block of ROS by anti-oxidants significantly inhibited bupivacaine-induced apoptosis. Among the local anesthetics for peripheral neural blocks, bupivacaine induced apoptosis in the Schwann cell line, which may be associated with ROS production.

Nitrosative stress mediated misfolded protein aggregation mitigated by Na-D-β-hydroxybutyrate intervention.

PubMed

Kabiraj, Parijat; Pal, Rituraj; Varela-Ramirez, Armando; Miranda, Manuel; Narayan, Mahesh

2012-09-28

Mitochondrial dysfunction, leading to elevated levels of reactive oxygen species, is associated with the pathogenesis of neurodegenerative disorders. Rotenone, a mitochondrial stressor induces caspase-9 and caspase-3 activation leading proteolytic cleavage of substrate nuclear poly(ADP-ribose) polymerase (PARP). PARP cleavage is directly related to apoptotic cell death. In this study, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1, as a rotenone-induced Parkinsonia-onset biomarker. We report that the innate ketone body, Na-D-β-hydroxybutyrate (NaβHB) reduces markedly the incidence of synphilin-1 aggregation. Furthermore, our data reveal that the metabolic byproduct also prevents rotenone-induced caspase-activated apoptotic cell death in dopaminergic SH-SY5Y cells. Together, these results suggest that NaβHB is neuroprotective; it attenuates effects originating from mitochondrial insult and can serve as a scaffold for the design and development of sporadic neuropathies. Copyright © 2012 Elsevier Inc. All rights reserved.

NADP/sup +/ enhances cholera and pertussis toxin-catalyzed ADP-ribosylation of membrane proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawai, Y.; Whitsel, C.; Arinze, I.J.

1986-05-01

Cholera or pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation is frequently used to estimate the concentration of the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins which modulate the activity of adenylate cyclase. With this assay, however, the degradation of the substrate, NAD/sup +/, by endogenous enzymes such as NAD/sup +/-glycohydrolase (NADase) present in the test membranes can influence the results. In this study the authors show that both cholera and pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation of liver membrane proteins is markedly enhanced by NADP/sup +/. The effect is concentration dependent; with 20 ..mu..M (/sup 32/P)NAD/sup +/ as substrate maximal enhancement is obtainedmore » at 0.5-1.0 mM NADP/sup +/. The enhancement of (/sup 32/P)ADP-ribosylation by NADP/sup +/ was much greater than that by other known effectors such as Mg/sup 2 +/, phosphate or isoniazid. The effect of NADP/sup +/ on ADP-ribosylation may occur by inhibition of the degradation of NAD/sup +/ probably by acting as an alternate substrate for NADase. Among inhibitors tested (NADP/sup +/, isoniazid, imidazole, nicotinamide, L-Arg-methyl-ester and HgCl/sub 2/) to suppress NADase activity, NADP/sup +/ was the most effective and, 10 mM, inhibited activity of the enzyme by about 90%. In membranes which contain substantial activities of NADase the inclusion of NADP/sup +/ in the assay is necessary to obtain maximal ADP-ribosylation.« less

Aero-Propulsion Technology (APT) Task V Low Noise ADP Engine Definition Study

NASA Technical Reports Server (NTRS)

Holcombe, V.

2003-01-01

A study was conducted to identify and evaluate noise reduction technologies for advanced ducted prop propulsion systems that would allow increased capacity operation and result in an economically competitive commercial transport. The study investigated the aero/acoustic/structural advancements in fan and nacelle technology required to match or exceed the fuel burned and economic benefits of a constrained diameter large Advanced Ducted Propeller (ADP) compared to an unconstrained ADP propulsion system with a noise goal of 5 to 10 EPNDB reduction relative to FAR 36 Stage 3 at each of the three measuring stations namely, takeoff (cutback), approach and sideline. A second generation ADP was selected to operate within the maximum nacelle diameter constrain of 160 deg to allow installation under the wing. The impact of fan and nacelle technologies of the second generation ADP on fuel burn and direct operating costs for a typical 3000 nm mission was evaluated through use of a large, twin engine commercial airplane simulation model. The major emphasis of this study focused on fan blade aero/acoustic and structural technology evaluations and advanced nacelle designs. Results of this study have identified the testing required to verify the interactive performance of these components, along with noise characteristics, by wind tunnel testing utilizing and advanced interaction rig.

Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin.

PubMed

Popoff, M R; Boquet, P

1988-05-16

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.

Improved triacylglycerol production in Acinetobacter baylyi ADP1 by metabolic engineering.

PubMed

Santala, Suvi; Efimova, Elena; Kivinen, Virpi; Larjo, Antti; Aho, Tommi; Karp, Matti; Santala, Ville

2011-05-18

Triacylglycerols are used in various purposes including food applications, cosmetics, oleochemicals and biofuels. Currently the main sources for triacylglycerol are vegetable oils, and microbial triacylglycerol has been suggested as an alternative for these. Due to the low production rates and yields of microbial processes, the role of metabolic engineering has become more significant. As a robust model organism for genetic and metabolic studies, and for the natural capability to produce triacylglycerol, Acinetobacter baylyi ADP1 serves as an excellent organism for modelling the effects of metabolic engineering for energy molecule biosynthesis. Beneficial gene deletions regarding triacylglycerol production were screened by computational means exploiting the metabolic model of ADP1. Four deletions, acr1, poxB, dgkA, and a triacylglycerol lipase were chosen to be studied experimentally both separately and concurrently by constructing a knock-out strain (MT) with three of the deletions. Improvements in triacylglycerol production were observed: the strain MT produced 5.6 fold more triacylglycerol (mg/g cell dry weight) compared to the wild type strain, and the proportion of triacylglycerol in total lipids was increased by 8-fold. In silico predictions of beneficial gene deletions were verified experimentally. The chosen single and multiple gene deletions affected beneficially the natural triacylglycerol metabolism of A. baylyi ADP1. This study demonstrates the importance of single gene deletions in triacylglycerol metabolism, and proposes Acinetobacter sp. ADP1 as a model system for bioenergetic studies regarding metabolic engineering.

Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae

PubMed Central

Matsumoto, Yasuko; Nakano, Tsuyoshi; Yamamoto, Masafumi; Matsushima-Hibiya, Yuko; Odagiri, Ken-Ichi; Yata, Osamu; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

2008-01-01

Cabbage butterflies, Pieris rapae and Pieris brassicae, contain strong cytotoxic proteins, designated as pierisin-1 and -2, against cancer cell lines. These proteins exhibit DNA ADP-ribosylating activity. To determine the distribution of substances with cytotoxicity and DNA ADP-ribosylating activity among other species, crude extracts from 20 species of the family Pieridae were examined for cytotoxicity in HeLa cells and DNA ADP-ribosylating activity. Both activities were detected in extracts from 13 species: subtribes Pierina (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Pieris brassicae, Pontia daplidice, and Talbotia naganum), Aporiina (Aporia gigantea, Aporia crataegi, Aporia hippia, and Delias pasithoe), and Appiadina (Appias nero and Appias paulina). All of these extracts contained substances recognized by anti-pierisin-1 antibodies, with a molecular mass of ≈100 kDa established earlier for pierisin-1. Moreover, sequences containing NAD-binding sites, conserved in ADP-ribosyltransferases, were amplified from genomic DNA from 13 species of butterflies with cytotoxicity and DNA ADP-ribosylating activity by PCR. Extracts from seven species, Appias lyncida, Leptosia nina, Anthocharis scolymus, Eurema hecabe, Catopsilia pomona, Catopsilia scylla, and Colias erate, showed neither cytotoxicity nor DNA ADP-ribosylating activity, and did not contain substances recognized by anti-pierisin-1 antibodies. Sequences containing NAD-binding sites were not amplified from genomic DNA from these seven species. Thus, pierisin-like proteins, showing cytotoxicity and DNA ADP-ribosylating activity, are suggested to be present in the extracts from butterflies not only among the subtribe Pierina, but also among the subtribes Aporiina and Appiadina. These findings offer insight to understanding the nature of DNA ADP-ribosylating activity in the butterfly. PMID:18256183

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration

PubMed Central

Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

2014-01-01

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg2+ concentrations must be considered as well. Here we developed in vivo/in vitro techniques using 31P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg2+ concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg2+ in the mitochondrial matrix, where [Mg2+] is tenfold higher. In contrast, owing to a much higher affinity for Mg2+, ATP is mostly complexed by Mg2+ in both compartments. Mg2+ starvation used to alter cytosolic and mitochondrial [Mg2+] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg2+ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis. PMID:25313036

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

PubMed

Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

2014-10-28

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

7 CFR 277.18 - Establishment of an Automated Data Processing (ADP) and Information Retrieval System.

Code of Federal Regulations, 2012 CFR

2012-01-01

...) and Information Retrieval System. 277.18 Section 277.18 Agriculture Regulations of the Department of... Data Processing (ADP) and Information Retrieval System. (a) Scope and application. This section... costs of planning, design, development or installation of ADP and information retrieval systems if the...

7 CFR 277.18 - Establishment of an Automated Data Processing (ADP) and Information Retrieval System.

Code of Federal Regulations, 2014 CFR

2014-01-01

...) and Information Retrieval System. 277.18 Section 277.18 Agriculture Regulations of the Department of... Data Processing (ADP) and Information Retrieval System. (a) Scope and application. This section... costs of planning, design, development or installation of ADP and information retrieval systems if the...

7 CFR 277.18 - Establishment of an Automated Data Processing (ADP) and Information Retrieval System.

Code of Federal Regulations, 2011 CFR

2011-01-01

...) and Information Retrieval System. 277.18 Section 277.18 Agriculture Regulations of the Department of... Data Processing (ADP) and Information Retrieval System. (a) Scope and application. This section... costs of planning, design, development or installation of ADP and information retrieval systems if the...

7 CFR 277.18 - Establishment of an Automated Data Processing (ADP) and Information Retrieval System.

Code of Federal Regulations, 2013 CFR

2013-01-01

...) and Information Retrieval System. 277.18 Section 277.18 Agriculture Regulations of the Department of... Data Processing (ADP) and Information Retrieval System. (a) Scope and application. This section... costs of planning, design, development or installation of ADP and information retrieval systems if the...

The ribokinases of Arabidopsis thaliana and Saccharomyces cerevisiae are required for ribose recycling from nucleotide catabolism, which in plants is not essential to survive prolonged dark stress.

PubMed

Schroeder, Rebekka Y; Zhu, Anting; Eubel, Holger; Dahncke, Kathleen; Witte, Claus-Peter

2018-01-01

Nucleotide catabolism in Arabidopsis thaliana and Saccharomyces cerevisiae leads to the release of ribose, which requires phosphorylation to ribose-5-phosphate mediated by ribokinase (RBSK). We aimed to characterize RBSK in plants and yeast, to quantify the contribution of plant nucleotide catabolism to the ribose pool, and to investigate whether ribose carbon contributes to dark stress survival of plants. We performed a phylogenetic analysis and determined the kinetic constants of plant-expressed Arabidopsis and yeast RBSKs. Using mass spectrometry, several metabolites were quantified in AtRBSK mutants and double mutants with genes of nucleoside catabolism. Additionally, the dark stress performance of several nucleotide metabolism mutants and rbsk was compared. The plant PfkB family of sugar kinases forms nine major clades likely representing distinct biochemical functions, one of them RBSK. Nucleotide catabolism is the dominant ribose source in plant metabolism and is highly induced by dark stress. However, rbsk cannot be discerned from the wild type in dark stress. Interestingly, the accumulation of guanosine in a guanosine deaminase mutant strongly enhances dark stress symptoms. Although nucleotide catabolism contributes to carbon mobilization upon darkness and is the dominant source of ribose, the contribution appears to be of minor importance for dark stress survival. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Molecular structure of human KATP in complex with ATP and ADP

PubMed Central

Lee, Kenneth Pak Kin

2017-01-01

In many excitable cells, KATP channels respond to intracellular adenosine nucleotides: ATP inhibits while ADP activates. We present two structures of the human pancreatic KATP channel, containing the ABC transporter SUR1 and the inward-rectifier K+ channel Kir6.2, in the presence of Mg2+ and nucleotides. These structures, referred to as quatrefoil and propeller forms, were determined by single-particle cryo-EM at 3.9 Å and 5.6 Å, respectively. In both forms, ATP occupies the inhibitory site in Kir6.2. The nucleotide-binding domains of SUR1 are dimerized with Mg2+-ATP in the degenerate site and Mg2+-ADP in the consensus site. A lasso extension forms an interface between SUR1 and Kir6.2 adjacent to the ATP site in the propeller form and is disrupted in the quatrefoil form. These structures support the role of SUR1 as an ADP sensor and highlight the lasso extension as a key regulatory element in ADP’s ability to override ATP inhibition. PMID:29286281

Adrenaline potentiates PI 3-kinase in platelets stimulated with thrombin and SFRLLN: role of secreted ADP.

PubMed

Selheim, F; Frøyset, A K; Strand, I; Vassbotn, F S; Holmsen, H

2000-11-17

Adrenaline significantly potentiated late thrombin- and SFRLLN-induced PtdIns(3,4)P(2) production. Furthermore, the potentiating effect of adrenaline on thrombin-induced PtdIns(3, 4)P(2) production was independent on secreted ADP, whereas, the effect of adrenaline on SFRLLN-induced PtdIns(3,4)P(2) production was completely dependent of secreted ADP. However, the ADP-dependent accumulation of PtdIns(3,4)P(2) was not required for irreversible platelet aggregation induced by SFRLLN in the presence of adrenaline. It is concluded that adrenaline can replace secreted ADP to potentiate PtdIns(3,4)P(2) production in thrombin-stimulated but not in SFRLLN-stimulated platelets, thus demonstrating a qualitative difference between platelet stimulation by thrombin and the thrombin receptor activating peptide SFRLLN.

PAR-1 and PAR-2 Expression Is Enhanced in Inflamed Odontoblast Cells.

PubMed

Alvarez, M M P; Moura, G E; Machado, M F M; Viana, G M; de Souza Costa, C A; Tjäderhane, L; Nader, H B; Tersariol, I L S; Nascimento, F D

2017-12-01

Protease-activated receptors (PARs) are G protein-coupled receptors, which are activated by proteolytical cleavage of the amino-terminus and act as sensors for extracellular proteases. We hypothesized that PAR-1 and PAR-2 can be modulated by inflammatory stimulus in human dental pulp cells. PAR-1 and PAR-2 gene expression in human pulp tissue and MDPC-23 cells were analyzed by quantitative polymerase chain reaction. Monoclonal PAR-1 and PAR-2 antibodies were used to investigate the cellular expression of these receptors using Western blot, flow cytometry, and confocal microscopy in MDPC-23 cells. Immunofluorescence assays of human intact and carious teeth were performed to assess the presence of PAR-1 and PAR-2 in the dentin-pulp complex. The results show for the first time that human odontoblasts and MDPC-23 cells constitutively express PAR-1 and PAR-2. PAR-2 activation increased significantly the messenger RNA expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MMP-14 in MDPC-23 cells ( P < 0.05), while the expression of these enzymes decreased significantly in the PAR-1 agonist group ( P < 0.05). The high-performance liquid chromatography and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis showed the presence of MMP-13 activity cleaving PAR-1 at specific, noncanonical site TLDPRS 42 ↓F 43 LL in human dental pulp tissues. Also, we detected a presence of a trypsin-like activity cleaving PAR-2 at canonical site SKGR 20 ↓S 21 LIGRL in pulp tissues. Confocal microscopy analysis of human dentin-pulp complex showed intense positive staining of PAR-1 and PAR-2 in the odontoblast processes in dentinal tubules of carious teeth compared to intact ones. The present results support the hypothesis of activation of the upregulated PAR-1 and PAR-2 by endogenous proteases abundant during the inflammatory response in dentin-pulp complex.

Intratumoral delivery of docetaxel enhances antitumor activity of Ad-p53 in murine head and neck cancer xenograft model.

PubMed

Yoo, George H; Subramanian, Geetha; Ezzat, Waleed H; Tulunay, Ozlem E; Tran, Vivian R; Lonardo, Fulvio; Ensley, John F; Kim, Harold; Won, Joshua; Stevens, Timothy; Zumstein, Louis A; Lin, Ho-Sheng

2010-01-01

The aim of this study is to determine the ability of intratumorally delivered docetaxel to enhance the antitumor activity of adenovirus-mediated delivery of p53 (Ad-p53) in murine head and neck cancer xenograft model. A xenograft head and neck squamous cell carcinoma mouse model was used. Mice were randomized into 4 groups of 6 mice receiving 6 weeks of biweekly intratumoral injection of (a) diluent, (b) Ad-p53 (1 x 10(10) viral particles per injection), (c) docetaxel (1 mg/kg per injection), and (d) combination of Ad-p53 (1 x 10(10) viral particles per injection) and docetaxel (1 mg/kg per injection). Tumor size, weight, toxicity, and overall and disease-free survival rates were determined. Intratumoral treatments with either docetaxel alone or Ad-p53 alone resulted in statistically significant antitumor activity and improved survival compared with control group. Furthermore, combined delivery of Ad-p53 and docetaxel resulted in a statistically significant reduction in tumor weight when compared to treatment with either Ad-p53 or docetaxel alone. Intratumoral delivery of docetaxel enhanced the antitumor effect of Ad-p53 in murine head and neck cancer xenograft model. The result of this preclinical in vivo study is promising and supports further clinical testing to evaluate efficacy of combined intratumoral docetaxel and Ad-p53 in treatment of head and neck cancer. Copyright (c) 2010 Elsevier Inc. All rights reserved.

7 CFR 272.10 - ADP/CIS Model Plan.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 4 2010-01-01 2010-01-01 false ADP/CIS Model Plan. 272.10 Section 272.10 Agriculture Regulations of the Department of Agriculture (Continued) FOOD AND NUTRITION SERVICE, DEPARTMENT OF AGRICULTURE... benefit computation (including but not limited to all household members' names, addresses, dates of birth...

Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis

PubMed Central

Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C Arnold

2015-01-01

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced lung fibrosis is diminished in both PAR-1 and PAR-2 deficient mice. We thus have been suggested that combined inactivation of PAR-1 and PAR-2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR-1 and PAR-2 agonists in the absence or presence of specific PAR-1 or PAR-2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and PAR-2 deficient mice with or without a specific PAR-1 antagonist (P1pal-12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR-1 and/or PAR-2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR-1 and PAR-2 did not show any additive effects on these pro-fibrotic responses. Strikingly, PAR-2 deficiency as well as pharmacological PAR-1 inhibition reduced bleomycin-induced pulmonary fibrosis to a similar extent. PAR-1 inhibition in PAR-2 deficient mice did not further diminish bleomycin-induced pulmonary fibrosis. Finally, we show that the PAR-1-dependent pro-fibrotic responses are inhibited by the PAR-2 specific antagonist. Targeting PAR-1 and PAR-2 simultaneously is not superior to targeting either receptor alone in bleomycin-induced pulmonary fibrosis. We postulate that the pro-fibrotic effects of PAR-1 require the presence of PAR-2. PMID:25689283

Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis.

PubMed

Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C Arnold

2015-06-01

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced lung fibrosis is diminished in both PAR-1 and PAR-2 deficient mice. We thus have been suggested that combined inactivation of PAR-1 and PAR-2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR-1 and PAR-2 agonists in the absence or presence of specific PAR-1 or PAR-2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and PAR-2 deficient mice with or without a specific PAR-1 antagonist (P1pal-12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR-1 and/or PAR-2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR-1 and PAR-2 did not show any additive effects on these pro-fibrotic responses. Strikingly, PAR-2 deficiency as well as pharmacological PAR-1 inhibition reduced bleomycin-induced pulmonary fibrosis to a similar extent. PAR-1 inhibition in PAR-2 deficient mice did not further diminish bleomycin-induced pulmonary fibrosis. Finally, we show that the PAR-1-dependent pro-fibrotic responses are inhibited by the PAR-2 specific antagonist. Targeting PAR-1 and PAR-2 simultaneously is not superior to targeting either receptor alone in bleomycin-induced pulmonary fibrosis. We postulate that the pro-fibrotic effects of PAR-1 require the presence of PAR-2. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Caffeic acid phenethyl ester suppresses melanoma tumor growth by inhibiting PI3K/AKT/XIAP pathway.

PubMed

Pramanik, Kartick C; Kudugunti, Shashi K; Fofaria, Neel M; Moridani, Majid Y; Srivastava, Sanjay K

2013-09-01

Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE

Caffeic acid phenethyl ester suppresses melanoma tumor growth by inhibiting PI3K/AKT/XIAP pathway

PubMed Central

Srivastava, Sanjay K.

2013-01-01

Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE

Three-Dimensional Structures Reveal Multiple ADP/ATP Binding Modes

DOE Office of Scientific and Technical Information (OSTI.GOV)

C Simmons; C Magee; D Smith

The creation of synthetic enzymes with predefined functions represents a major challenge in future synthetic biology applications. Here, we describe six structures of de novo proteins that have been determined using protein crystallography to address how simple enzymes perform catalysis. Three structures are of a protein, DX, selected for its stability and ability to tightly bind ATP. Despite the addition of ATP to the crystallization conditions, the presence of a bound but distorted ATP was found only under excess ATP conditions, with ADP being present under equimolar conditions or when crystallized for a prolonged period of time. A bound ADPmore » cofactor was evident when Asp was substituted for Val at residue 65, but ATP in a linear configuration is present when Phe was substituted for Tyr at residue 43. These new structures complement previously determined structures of DX and the protein with the Phe 43 to Tyr substitution [Simmons, C. R., et al. (2009) ACS Chem. Biol. 4, 649-658] and together demonstrate the multiple ADP/ATP binding modes from which a model emerges in which the DX protein binds ATP in a configuration that represents a transitional state for the catalysis of ATP to ADP through a slow, metal-free reaction capable of multiple turnovers. This unusual observation suggests that design-free methods can be used to generate novel protein scaffolds that are tailor-made for catalysis.« less

Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+.

PubMed

Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D

2005-03-01

Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

NASA Technical Reports Server (NTRS)

Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D.

2005-01-01

Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

Pleiotropic regulatory genes bldA, adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin.

PubMed

Makitrynskyy, Roman; Ostash, Bohdan; Tsypik, Olga; Rebets, Yuriy; Doud, Emma; Meredith, Timothy; Luzhetskyy, Andriy; Bechthold, Andreas; Walker, Suzanne; Fedorenko, Victor

2013-10-23

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production-bldA, adpA and absB-exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA(Leu)UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs-that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.

Targeting Homology-Directed Recombinational Repair (HDR) of Chromosomal Breaks to Sensitize Prostate Cancer Cells to Poly (ADP-Ribose) Polymerase (PARP) Inhibition

DTIC Science & Technology

2013-08-01

The views, opinions and/or findings contained in this report are those of the author( s ) and should not be construed as an official Department of...on p53. To assess whether BRCA1 nuclear export following IR in prostate cancer cells is also p53 dependent, we next performed the above experiments...Task 1B. Previous reports suggest that IR-induced BRCA1 export is also dependent on CRM1. To test this hypothesis, we proposed that the CRM1

Targeting Homology-Directed Recombinational Repair (HDR) of Chromosomal Breaks to Sensitize Prostate Cancer Cells to Poly (ADP-Ribose) Polymerase (PARP) Inhibition

DTIC Science & Technology

2012-08-01

Investigator 15 UAB X1219: Molecular determinants of cellular susceptibility to PARP inhibition in an ex- vivo model of human cholangiocarcinoma Role...cellular susceptibility to PARP inhibition in an ex-vivo model of human cholangiocarcinoma Role: Co-Prinicipal Investigator Career Development

Class I ADP-Ribosylation Factors Are Involved in Enterovirus 71 Replication

PubMed Central

Wang, Jianmin; Du, Jiang; Jin, Qi

2014-01-01

Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies. PMID:24911624

Molecular Basis of ADP Inhibition of Vacuolar (V)-type ATPase/Synthase*

PubMed Central

Kishikawa, Jun-ichi; Nakanishi, Atsuko; Furuike, Shou; Tamakoshi, Masatada; Yokoyama, Ken

2014-01-01

Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V0V1) as a result of ADP inhibition occurs as part of the normal mechanism of V0V1 of Thermus thermophilus but not V0V1 of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V1 consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal domains of the catalytic A subunit from E. hirae V1 is central to increasing binding affinity of the chimeric V1 for phosphate, resulting in reduction of the ADP inhibition. These findings together with a comparison of the crystal structures of T. thermophilus V1 with E. hirae V1 strongly suggest that the A subunit adopts a conformation in T. thermophilus V1 different from that in E. hirae V1. This key difference results in ADP inhibition of T. thermophilus V1 by abolishing the binding affinity for phosphate during ATP hydrolysis. PMID:24247239

The 1994 NASA/USRA/ADP Design Projects

NASA Technical Reports Server (NTRS)

Cruse, Thomas; Richardson, Joseph; Tryon, Robert

1994-01-01

The NASA/USRA/ADP Design Projects from Vanderbilt University, Department of Mechanical Engineering (1994) are enclosed in this final report. Design projects include: (1) Protein Crystal Growth, both facilities and methodology; (2) ACES Deployable Space Boom; (3) Hybrid Launch System designs for both manned and unmanned systems; (4) LH2 Fuel Tank design (SSTO); (5) SSTO design; and (6) Pressure Tank Feed System design.

Design, Synthesis and Evaluation of Ribose-modified Anilinopyrimidine Derivatives as EGFR Tyrosine Kinase Inhibitors

NASA Astrophysics Data System (ADS)

Hu, Xiuqin; Wang, Disha; Tong, Yi; Tong, Linjiang; Wang, Xia; Zhu, Lili; Xie, Hua; Li, Shiliang; Yang, You; Xu, Yufang

2017-11-01

The synthesis of a series of ribose-modified anilinopyrimidine derivatives was efficiently achieved by utilizing DBU or tBuOLi-promoted coupling of ribosyl alcohols with 2,4,5-trichloropyrimidine as key step. Preliminary biological evaluation of this type of compounds as new EGFR tyrosine kinase inhibitors for combating EGFR L858R/T790M mutant associated with drug resistance in the treatment of non-small cell lung cancer revealed that 3-N-acryloyl-5-O-anilinopyrimidine ribose derivative 1a possessed potent and specific inhibitory activity against EGFR L858R/T790M over WT EGFR. Based upon molecular docking studies of the binding mode between compound 1a and EGFR, the distance between the Michael receptor and the pyrimidine scaffold is considered as an important factor for the inhibitory potency and future design of selective EGFR tyrosine kinase inhibitors against EGFR L858R/T790M mutants.

Preliminary crystallographic analysis of ADP-glucose pyrophosphorylase from Agrobacterium tumefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cupp-Vickery, Jill R., E-mail: jvickery@uci.edu; Igarashi, Robert Y.; Meyer, Christopher R.

2005-03-01

Crystallization and X-ray diffraction methods for native A. tumefaciens ADP-glucose pyrophosphorylase and its selenomethionyl derivative are described. Two crystal forms are identified, both of which diffract to 2 Å.

External validation of the emergency department assessment of chest pain score accelerated diagnostic pathway (EDACS-ADP).

PubMed

Flaws, Dylan; Than, Martin; Scheuermeyer, Frank Xavier; Christenson, James; Boychuk, Barbara; Greenslade, Jaimi H; Aldous, Sally; Hammett, Christopher J; Parsonage, William A; Deely, Joanne M; Pickering, John W; Cullen, Louise

2016-09-01

The emergency department assessment of chest pain score accelerated diagnostic pathway (EDACS-ADP) facilitates low-risk ED chest pain patients early to outpatient investigation. We aimed to validate this rule in a North American population. We performed a retrospective validation of the EDACS-ADP using 763 chest pain patients who presented to St Paul's Hospital, Vancouver, Canada, between June 2000 and January 2003. Patients were classified as low risk if they had an EDACS <16, no new ischaemia on ECG and non-elevated serial 0-hour and 2-hour cardiac troponin concentrations. The primary outcome was the number of patients who had a predetermined major adverse cardiac event (MACE) at 30 days after presentation. Of the 763 patients, 317 (41.6%) were classified as low risk by the EDACS-ADP. The sensitivity, specificity, negative predictive value and positive predictive value of the EDACS-ADP for 30-day MACE were 100% (95% CI 94.2% to 100%), 46.4% (95% CI 42.6% to 50.2%), 100% (95% CI 98.5% to 100.0%) and 17.5% (95% CI 14.1% to 21.3%), respectively. This study validated the EDACS-ADP in a novel context and supports its safe use in a North American population. It confirms that EDACS-ADP can facilitate progression to early outpatient investigation in up to 40% of ED chest pain patients within 2 hours. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Bacillus cereus Certhrax ADP-ribosylates Vinculin to Disrupt Focal Adhesion Complexes and Cell Adhesion*

PubMed Central

Simon, Nathan C.; Barbieri, Joseph T.

2014-01-01

Bacillus cereus is often associated with mild to moderate gastroenteritis; however, some recent isolates cause inhalational anthrax-like diseases and death. These potential emerging human pathogens express multiple virulence factors. B. cereus strain G9241 expresses anthrax toxin, several polysaccharide capsules, and the novel ADP-ribosyltransferase, Certhrax. In this study, we show that Certhrax ADP-ribosylates Arg-433 of vinculin, a protein that coordinates actin cytoskeleton and extracellular matrix interactions. ADP-ribosylation of vinculin disrupted focal adhesion complexes and redistributed vinculin to the cytoplasm. Exogenous vinculin rescued these phenotypes. This provides a mechanism for strain G9241 to breach host barrier defenses and promote bacterial growth and spread. Certhrax is the first bacterial toxin to add a post-translational modification to vinculin to disrupt the actin cytoskeleton. PMID:24573681

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

PubMed Central

Hu, Longhua; Vecchiarelli, Anthony G.; Mizuuchi, Kiyoshi; Neuman, Keir C.; Liu, Jian

2015-01-01

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA–nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos—an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria. PMID:26647183

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system.

PubMed

Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi; Neuman, Keir C; Liu, Jian

2015-12-22

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA-nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos-an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

NASA Astrophysics Data System (ADS)

Hu, Longhua; Vecchiarelli, Anthony G.; Mizuuchi, Kiyoshi; Neuman, Keir C.; Liu, Jian

The segregation of DNA prior to cell division is essential for faithful genetic inheritance. In many bacteria, segregation of the low-copy-number plasmids involves an active partition system composed of ParA ATPase and its stimulator protein ParB. Recent experiments suggest that ParA/ParB system motility is driven by a diffusion-ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. We develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA-nucleoid affinity to the motion of the ParB bound cargo. Paradoxically, the resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work sheds light on a new emergent phenomenon in which non-motor proteins work collectively via mechanochemical coupling to propel cargos -- an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.

Deciphering the kinetic mechanisms controlling selected plant ADP-glucose pyrophosphorylases.

PubMed

Boehlein, Susan K; Shaw, Janine R; Hwang, Seon K; Stewart, Jon D; Curtis Hannah, L

2013-07-15

ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes. With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last. 3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms. By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes. Redox state does not affect kcat of the two potato isoforms. Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex. This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme. Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis. Copyright © 2013 Elsevier Inc. All rights reserved.

Third-order nonlinear optical properties of ADP crystal

NASA Astrophysics Data System (ADS)

Wang, Mengxia; Wang, Zhengping; Chai, Xiangxu; Sun, Yuxiang; Sui, Tingting; Sun, Xun; Xu, Xinguang

2018-05-01

By using the Z-scan method, we investigated the third-order nonlinear optical (NLO) properties of ADP crystal at different wavelengths (355, 532, and 1064 nm) and different orientations ([001], [100], [110], I and II). The experimental data were fitted by NLO theory, to give out the two photon absorption (TPA) coefficient β 2 and the nonlinear refractive index n 2. When the light source changed from a 40 ps, 1064 nm fundamental laser to a 30 ps, 355 nm third-harmonic-generation (THG) laser, the β 2 value increased about 5 times (0.2 × 10‑2 → 1 × 10‑2 cm GW‑1), and the n 2 value increased about 1.5 times (1.5 × 10‑16 → 2.2 × 10‑16 cm2 W‑1). Among all of the orientations, the [110] sample exhibits the smallest β 2, and the second smallest n 2. It indicates that this orientation and its surroundings will be the preferred directions for high-power laser applications of ADP crystal.

Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fendrick, J.L.; Iglewski, W.J.

1989-01-01

Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing ({sup 32}P)orthophosphate and 105 (vol/vol) fetal bovine serum. A {sup 32}P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The {sup 32}P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The {sup 32}P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as ({sup 32}P)AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of trypticmore » peptides of the {sup 32}P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and ({sup 32}P)NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When {sup 32}P-labeled cultures were incubated in medium containing unlabeled phosphate, the {sup 32}P label was lost from the EF-2 within 30 min.« less

Ab initio treatment of ion-induced charge transfer dynamics of isolated 2-deoxy-D-ribose.

PubMed

Bacchus-Montabonel, Marie-Christine

2014-08-21

Modeling-induced radiation damage in biological systems, in particular, in DNA building blocks, is of major concern in cancer therapy studies. Ion-induced charge-transfer dynamics may indeed be involved in proton and hadrontherapy treatments. We have thus performed a theoretical approach of the charge-transfer dynamics in collision of C(4+) ions and protons with isolated 2-deoxy-D-ribose in a wide collision energy range by means of ab initio quantum chemistry molecular methods. The comparison of both projectile ions has been performed with regard to previous theoretical and experimental results. The charge transfer appears markedly less efficient with the 2-deoxy-D-ribose target than that with pyrimidine nucleobases, which would induce an enhancement of the fragmentation process in agreement with experimental measurements. The mechanism has been analyzed with regard to inner orbital excitations, and qualitative tendencies have been pointed out for studies on DNA buiding block damage.

Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue.

PubMed

Rovira, Alexander R; Fin, Andrea; Tor, Yitzhak

2017-11-08

The synthesis, photophysics, and biochemical utility of a fluorescent NAD + analogue based on an isothiazolo[4,3-d]pyrimidine core (N tz AD + ) are described. Enzymatic reactions, photophysically monitored in real time, show N tz AD + and N tz ADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as N tz AD + is converted to N tz ADH, reflecting a complementary photophysical behavior to that of the native NAD + /NADH. N tz AD + and N tz ADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding tz ADP-ribose. N tz AD + also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer tz ADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD + .

Uridylylation of Herbaspirillum seropedicae GlnB and GlnK proteins is differentially affected by ATP, ADP and 2-oxoglutarate in vitro.

PubMed

Bonatto, Ana C; Souza, Emanuel M; Oliveira, Marco A S; Monteiro, Rose A; Chubatsu, Leda S; Huergo, Luciano F; Pedrosa, Fábio O

2012-08-01

PII are signal-transducing proteins that integrate metabolic signals and transmit this information to a large number of proteins. In proteobacteria, PII are modified by GlnD (uridylyltransferase/uridylyl-removing enzyme) in response to the nitrogen status. The uridylylation/deuridylylation cycle of PII is also regulated by carbon and energy signals such as ATP, ADP and 2-oxoglutarate (2-OG). These molecules bind to PII proteins and alter their tridimensional structure/conformation and activity. In this work, we determined the effects of ATP, ADP and 2-OG levels on the in vitro uridylylation of Herbaspirillum seropedicae PII proteins, GlnB and GlnK. Both proteins were uridylylated by GlnD in the presence of ATP or ADP, although the uridylylation levels were higher in the presence of ATP and under high 2-OG levels. Under excess of 2-OG, the GlnB uridylylation level was higher in the presence of ATP than with ADP, while GlnK uridylylation was similar with ATP or ADP. Moreover, in the presence of ADP/ATP molar ratios varying from 10/1 to 1/10, GlnB uridylylation level decreased as ADP concentration increased, whereas GlnK uridylylation remained constant. The results suggest that uridylylation of both GlnB and GlnK responds to 2-OG levels, but only GlnB responds effectively to variation on ADP/ATP ratio.

Growth, structural, spectroscopic, thermal, dielectric and optical study of cobalt sulphide-doped ADP crystals

NASA Astrophysics Data System (ADS)

Kochuparampil, A. P.; Joshi, J. H.; Joshi, M. J.

2017-09-01

As ammonium dihydrogen phosphate (ADP) is a popular nonlinear optical crystal, to engineer its linear and nonlinear optical properties, the chalcogenide compound cobalt sulphide (CoS) was doped and the crystals were grown by the slow solvent evaporation method. To increase the solubility of CoS in water, its nanoparticles were synthesized by wet chemical technique using ethylene diamine as the capping agent followed by microwave irradiation. The nanoparticle sample exhibited finite solubility in water and was used to dope in ADP crystals. The powder XRD patterns showed the single phase nature of the doped crystals. The FTIR spectra confirmed the presence of various functional groups and EDAX gave the estimation of Co and S elements. The EPR spectroscopy also confirmed the presence of cobalt in the doped samples. TGA indicated slightly less thermal stability of the doped crystals compared to the pure ADP. The dielectric study was carried out at room temperature in the frequency range from 100Hz to 1MHz. Also, various linear optical parameters were evaluated for pure and doped crystals using UV-Vis spectroscopy. The second harmonic generation (SHG) efficiency of Nd:YAG laser was evaluated by the Kurtz and Parry method for the doped samples, it was found to be slightly lesser than that of the pure ADP crystals.

Nuclear CD38 in retinoic acid-induced HL-60 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yalcintepe, Leman; Albeniz, Isil; Adin-Cinar, Suzan

2005-02-01

The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD{sup +} and hydrolysis of either NAD{sup +} or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD{sup +} glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. Withmore » Western blotting, we detected in the nuclear protein fraction from RA-treated cells a {approx}43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the {approx}43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.« less

Dynein-ADP as a force-generating intermediate revealed by a rapid reactivation of flagellar axoneme.

PubMed Central

Tani, T; Kamimura, S

1999-01-01

Fragmented flagellar axonemes of sand dollar spermatozoa were reactivated by rapid photolysis of caged ATP. After a time lag of 10 ms, axonemes treated with protease started sliding disintegration. Axonemes without protease digestion started nanometer-scale high-frequency oscillation after a similar time lag. Force development in the sliding disintegration was measured with a flexible glass needle and its time course was corresponded well to that of the dynein-ADP intermediate production estimated using kinetic rates previously reported. However, with a high concentration ( approximately 80 microM) of vanadate, which binds to the dynein-ADP intermediate and forms a stable complex of dynein-ADP-vanadate, the time course of force development in sliding disintegration was not affected at all. In the case of high frequency oscillation, the time lag to start the oscillation, the initial amplitude, and the initial frequency were not affected by vanadate, though the oscillation once started was damped more quickly at higher concentrations of vanadate. These results suggest that during the initial turnover of ATP hydrolysis, force generation of dynein is not blocked by vanadate. A vanadate-insensitive dynein-ADP is postulated as a force-generating intermediate. PMID:10465762

Water-mediated protein-fluorophore interactions modulate the affinity of an ABC-ATPase/TNP-ADP complex.

PubMed

Oswald, Christine; Jenewein, Stefan; Smits, Sander H J; Holland, I Barry; Schmitt, Lutz

2008-04-01

TNP-modified nucleotides have been used extensively to study protein-nucleotide interactions. In the case of ABC-ATPases, application of these powerful tools has been greatly restricted due to the significantly higher affinity of the TNP-nucleotide for the corresponding ABC-ATPase in comparison to the non-modified nucleotides. To understand the molecular changes occurring upon binding of the TNP-nucleotide to an ABC-ATPase, we have determined the crystal structure of the TNP-ADP/HlyB-NBD complex at 1.6A resolution. Despite the higher affinity of TNP-ADP, no direct fluorophore-protein interactions were observed. Unexpectedly, only water-mediated interactions were detected between the TNP moiety and Tyr(477), that is engaged in pi-pi stacking with the adenine ring, as well as with two serine residues (Ser(504) and Ser(509)) of the Walker A motif. Interestingly, the side chains of these two serine residues adopt novel conformations that are not observed in the corresponding ADP structure. However, in the crystal structure of the S504A mutant, which binds TNP-ADP with similar affinity to the wild type enzyme, a novel TNP-water interaction compensates for the missing serine side chain. Since this water molecule is not present in the wild type enzyme, these results suggest that only water-mediated interactions provide a structural explanation for the increased affinity of TNP-nucleotides towards ABC-ATPases. However, our results also imply that in silico approaches such as docking or modeling cannot directly be applied to generate 'affinity-adopted' ADP- or ATP-analogs for ABC-ATPases.