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Sample records for poly-adp ribose polymerase

  1. Targeting poly(ADP-ribose) polymerase activity for cancer therapy

    PubMed Central

    Mégnin-Chanet, Frédérique; Bollet, Marc A.

    2010-01-01

    Poly(ADP-ribosyl)ation is a ubiquitous protein modification found in mammalian cells that modulates many cellular responses, including DNA repair. The poly(ADP-ribose) polymerase (PARP) family catalyze the formation and addition onto proteins of negatively charged ADP-ribose polymers synthesized from NAD+. The absence of PARP-1 and PARP-2, both of which are activated by DNA damage, results in hypersensitivity to ionizing radiation and alkylating agents. PARP inhibitors that compete with NAD+ at the enzyme’s activity site are effective chemo- and radiopotentiation agents and, in BRCA-deficient tumors, can be used as single-agent therapies acting through the principle of synthetic lethality. Through extensive drug-development programs, third-generation inhibitors have now entered clinical trials and are showing great promise. However, both PARP-1 and PARP-2 are not only involved in DNA repair but also in transcription regulation, chromatin modification, and cellular homeostasis. The impact on these processes of PARP inhibition on long-term therapeutic responses needs to be investigated. PMID:20725763

  2. Metabolic roles of poly(ADP-ribose) polymerases.

    PubMed

    Vida, András; Márton, Judit; Mikó, Edit; Bai, Péter

    2017-03-01

    Poly(ADP-ribosyl)ation (PARylation) is an evolutionarily conserved reaction that had been associated with numerous cellular processes such as DNA repair, protein turnover, inflammatory regulation, aging or metabolic regulation. The metabolic regulatory tasks of poly(ADP-ribose) polymerases (PARPs) are complex, it is based on the regulation of metabolic transcription factors (e.g. SIRT1, nuclear receptors, SREBPs) and certain cellular energy sensors. PARP over-activation can cause damage to mitochondrial terminal oxidation, while the inhibition of PARP-1 or PARP-2 can induce mitochondrial oxidation by enhancing the mitotropic tone of gene transcription and signal transduction. These PARP-mediated processes impact on higher order metabolic regulation that modulates lipid metabolism, circadian oscillations and insulin secretion and signaling. PARP-1, PARP-2 and PARP-7 are related to metabolic diseases such as diabetes, alcoholic and non-alcoholic fatty liver disease (AFLD, NAFLD), or on a broader perspective to Warburg metabolism in cancer or the metabolic diseases accompanying aging.

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

    PubMed

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

    2013-12-01

    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 aging). Finally, we give an overview of the possibilities of pharmacological intervention to modulate PARP and SIRT enzymes either directly, or through modulating NAD(+) homeostasis.

  4. Pharmacological inhibition of poly(ADP-ribose) polymerase inhibits angiogenesis

    SciTech Connect

    Rajesh, Mohanraj; Mukhopadhyay, Partha; Batkai, Sandor; Godlewski, Grzegorz; Hasko, Gyoergy; Liaudet, Lucas; Pacher, Pal . E-mail: pacher@mail.nih.gov

    2006-11-17

    Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which plays an important role in regulating cell death and cellular responses to DNA repair. Pharmacological inhibitors of PARP are being considered as treatment for cancer both in monotherapy as well as in combination with chemotherapeutic agents and radiation, and were also reported to be protective against untoward effects exerted by certain anticancer drugs. Here we show that pharmacological inhibition of PARP with 3-aminobenzamide or PJ-34 dose-dependently reduces VEGF-induced proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro. These results suggest that treatment with PARP inhibitors may exert additional benefits in various cancers and retinopathies by decreasing angiogenesis.

  5. 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

  6. Poly(ADP-Ribose) Polymerases in Aging - Friend or Foe?

    PubMed

    Vida, András; Abdul-Rahman, Omar; Mikó, Edit; Brunyánszki, Attila; Bai, Peter

    2016-01-01

    Poly(ADP-ribose) polymerases were originally described as DNA repair enzymes. PARP-1, PARP-2 and PARP-3 can be activated by DNA damage and the resulting activation of these enzymes that facilitate DNA repair, seems to be a prerequisite of successful aging. PARP activation helps to maintain genomic integrity through supporting DNA repair systems; however, in parallel these enzymes limit metabolic fitness and make the organism more prone for metabolic diseases. In addition, several other pathways (e.g., proteostasis, nutrient sensing, stem cell proliferation or cellular communication) all contributing to aging, were shown to be PARP mediated. In this review we aim to summarize our current knowledge on the role of PARPs in aging.

  7. Proteomic investigation of phosphorylation sites in poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase.

    PubMed

    Gagné, Jean-Philippe; Moreel, Xavier; Gagné, Pierre; Labelle, Yves; Droit, Arnaud; Chevalier-Paré, Mélissa; Bourassa, Sylvie; McDonald, Darin; Hendzel, Michael J; Prigent, Claude; Poirier, Guy G

    2009-02-01

    Phosphorylation is a very common post-translational modification event known to modulate a wide range of biological responses. Beyond the regulation of protein activity, the interrelation of phosphorylation with other post-translational mechanisms is responsible for the control of diverse signaling pathways. Several observations suggest that phosphorylation of poly(ADP-ribose) polymerase-1 (PARP-1) regulates its activity. There is also accumulating evidence to suggest the establishment of phosphorylation-dependent assembly of PARP-1-associated multiprotein complexes. Although it is relatively straightforward to demonstrate phosphorylation of a defined target, identification of the actual amino acids involved still represents a technical challenge for many laboratories. With the use of a combination of bioinformatics-based predictions tools for generic and kinase-specific phosphorylation sites, in vitro phosphorylation assays and mass spectrometry analysis, we investigated the phosphorylation profile of PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), two major enzymes responsible for poly(ADP-ribose) turnover. Mass spectrometry analysis revealed the phosphorylation of several serine/threonine residues within important regulatory domains and motifs of both enzymes. With the use of in vivo microirradiation-induced DNA damage, we show that altered phosphorylation at specific sites can modify the dynamics of assembly and disassembly of PARP-1 at sites of DNA damage. By documenting and annotating a collection of known and newly identified phosphorylation sites, this targeted proteomics study significantly advances our understanding of the roles of phosphorylation in the regulation of PARP-1 and PARG.

  8. Poly(ADP-ribose)polymerase inhibition decreases angiogenesis

    SciTech Connect

    Rajesh, Mohanraj; Mukhopadhyay, Partha; Godlewski, Grzegorz; Batkai, Sandor; Hasko, Gyoergy; Liaudet, Lucas; Pacher, Pal . E-mail: pacher@mail.nih.gov

    2006-12-01

    Inhibitors of poly(ADP-ribose)polymerase (PARP), a nuclear enzyme involved in regulating cell death and cellular responses to DNA repair, show considerable promise in the treatment of cancer both in monotherapy as well as in combination with chemotherapeutic agents and radiation. We have recently demonstrated that PARP inhibition with 3-aminobenzamide or PJ-34 reduced vascular endothelial growth factor (VEGF)-induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Here, we show dose-dependent reduction of VEGF- and basic fibroblast growth factor (bFGF)-induced proliferation, migration, and tube formation of HUVECs in vitro by two potent PARP inhibitors 5-aminoisoquinolinone-hydrochloride (5-AIQ) and 1,5-isoquinolinediol (IQD). Moreover, PARP inhibitors prevented the sprouting of rat aortic ring explants in an ex vivo assay of angiogenesis. These results establish the novel concept that PARP inhibitors have antiangiogenic effects, which may have tremendous clinical implications for the treatment of various cancers, tumor metastases, and certain retinopathies.

  9. Inhibiting poly(ADP-ribose) polymerase: a potential therapy against oligodendrocyte death

    PubMed Central

    Veto, Sara; Acs, Peter; Bauer, Jan; Lassmann, Hans; Berente, Zoltan; Setalo, Gyorgy; Borgulya, Gabor; Sumegi, Balazs; Komoly, Samuel; Gallyas, Ferenc; Illes, Zsolt

    2010-01-01

    Oligodendrocyte loss and demyelination are major pathological hallmarks of multiple sclerosis. In pattern III lesions, inflammation is minor in the early stages, and oligodendrocyte apoptosis prevails, which appears to be mediated at least in part through mitochondrial injury. Here, we demonstrate poly(ADP-ribose) polymerase activation and apoptosis inducing factor nuclear translocation within apoptotic oligodendrocytes in such multiple sclerosis lesions. The same morphological and molecular pathology was observed in an experimental model of primary demyelination, induced by the mitochondrial toxin cuprizone. Inhibition of poly(ADP-ribose) polymerase in this model attenuated oligodendrocyte depletion and decreased demyelination. Poly(ADP-ribose) polymerase inhibition suppressed c-Jun N-terminal kinase and p38 mitogen-activated protein kinase phosphorylation, increased the activation of the cytoprotective phosphatidylinositol-3 kinase-Akt pathway and prevented caspase-independent apoptosis inducing factor-mediated apoptosis. Our data indicate that poly(ADP-ribose) polymerase activation plays a crucial role in the pathogenesis of pattern III multiple sclerosis lesions. Since poly(ADP-ribose) polymerase inhibition was also effective in the inflammatory model of multiple sclerosis, it may target all subtypes of multiple sclerosis, either by preventing oligodendrocyte death or attenuating inflammation. PMID:20157013

  10. Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

    PubMed

    De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

    2013-09-01

    Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity.

  11. Regulation of NFAT by poly(ADP-ribose) polymerase activity in T cells.

    PubMed

    Valdor, Rut; Schreiber, Valérie; Saenz, Luis; Martínez, Teresa; Muñoz-Suano, Alba; Dominguez-Villar, Margarita; Ramírez, Pablo; Parrilla, Pascual; Aguado, Enrique; García-Cózar, Francisco; Yélamos, José

    2008-04-01

    The nuclear factor of activated T cells (NFAT) family of transcription factors is pivotal for T lymphocyte functionality. All relevant NFAT activation events upon T cells stimulation such as nuclear translocation, DNA binding, and transcriptional activity have been shown to be dictated by its phosphorylation state. Here, we provide evidence for a novel post-translational modification that regulates NFAT. Indeed, NFATc1 and NFATc2 are poly(ADP-ribosyl)ated by poly-ADP-ribose polymerase-1 (PARP-1). Moreover, we have also found a physical interaction between PARP-1 and both NFATc1 and NFATc2. Interestingly, PARP is activated during T cell stimulation in the absence of DNA damage, leading to ADP-ribose polymers formation and transfer to nuclear acceptor proteins. Our data suggest that poly(ADP-ribosyl)ation modulates the activation of NFAT in T cells, as PARP inhibition causes an increase in NFAT-dependent transactivation and a delay in NFAT nuclear export. Poly(ADP-ribosyl)ation will expedited NFAT export from the nucleus directly or by priming/facilitating NFAT phosphorylation. Altogether, these data point to PARP-1 and poly(ADP-ribosyl)ation as a novel regulatory mechanism of NFAT at nuclear level, suggesting a potential use of PARP as a new therapeutic target in the modulation of NFAT.

  12. Inhibition of Poly(ADP-Ribose) Polymerase by Nucleic Acid Metabolite 7-Methylguanine

    PubMed Central

    Nilov, D. K.; Tararov, V. I.; Kulikov, A. V.; Zakharenko, A. L.; Gushchina, I. V.; Mikhailov, S. N.; Lavrik, O. I.; Švedas, V. K.

    2016-01-01

    The ability of 7-methylguanine, a nucleic acid metabolite, to inhibit poly(ADP-ribose)polymerase-1 (PARP-1) and poly(ADP-ribose)polymerase-2 (PARP-2) has been identified in silico and studied experimentally. The amino group at position 2 and the methyl group at position 7 were shown to be important substituents for the efficient binding of purine derivatives to PARPs. The activity of both tested enzymes, PARP-1 and PARP-2, was suppressed by 7-methylguanine with IC50 values of 150 and 50 μM, respectively. At the PARP inhibitory concentration, 7-methylguanine itself was not cytotoxic, but it was able to accelerate apoptotic death of BRCA1-deficient breast cancer cells induced by cisplatin and doxorubicin, the widely used DNA-damaging chemotherapeutic agents. 7-Methylguanine possesses attractive predictable pharmacokinetics and an adverse-effect profile and may be considered as a new additive to chemotherapeutic treatment. PMID:27437145

  13. Poly(ADP-ribose) polymerase 1 at the crossroad of metabolic stress and inflammation in aging

    PubMed Central

    Altmeyer, Matthias; Hottiger, Michael O.

    2009-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein, which functions as molecular stress sensor. Reactive oxygen species, responsible for the most plausible and currently acceptable global mechanism to explain the aging process, strongly activate the enzymatic activity of PARP1 and the formation of poly(ADP-ribose) (PAR) from NAD+. Consumption of NAD+ links PARP1 to energy metabolism and to a large number of NAD+-dependent enzymes, such as the sirtuins. As transcriptional cofactor for NF-κB-dependent gene expression, PARP1 is also connected to the immune response, which is implicated in almost all age-related or associated diseases. Accordingly, numerous experimental studies have demonstrated the beneficial effects of PARP inhibition for several age-related diseases. This review summarizes recent findings on PARP1 and puts them in the context of metabolic stress and inflammation in aging. PMID:20157531

  14. Does inhibition of poly(ADP-ribose) polymerase prevent energy overconsumption under microgravity?

    NASA Astrophysics Data System (ADS)

    Dobrota, C.; Piso, M. I.; Keul, A.

    When plants are exposed to a stress signal they expend a lot of energy and exhibit enhanced respiration rates This is partially due to a breakdown in the NAD pool caused by the enhanced activity PARP which uses NAD as a substrate to synthesize polymers of ADP-ribose Stress-induced depletion of NAD results in a similar depletion of energy since ATP molecules are required to resynthesize the depleted NAD It seems that plants with lowered poly ADP ribosyl ation activity appear tolerant to multiple stresses Inhibiting PARP activity prevents energy overconsumption under stress allowing normal mitochondrial respiration We intend to study if the microgravity is perceived by plants as a stress factor and if experimental inhibition of poly ADP-ribose polymerase may improve the energetic level of the cells References DeBlock M Verduyn C De Brouwer D and Cornelissen M 2005 Poly ADP-ribose polymerase in plants affects energy homeostasis cell death and stress tolerance The Plant Journal 41 95--106 Huang S Greenway H Colmerm T D and Millar A H 2005 Protein synthesis by rice coleoptiles during prolonged anoxia Implications for glycolysis growth and energy utilization Annals of Botany 96 703--715 Mittler R Vanderauwera S Gollery M and Van Breusegem F 2005 Reactive oxygen gene network of plants Trends in Plant Science 9 10 490-498

  15. PARP1 Is a TRF2-associated Poly(ADP-Ribose)Polymerase and Protects Eroded Telomeres

    SciTech Connect

    Liu, Yie; Wu, Jun; Schreiber, Valerie; Dunlap, John; Dantzer, Francoise; Wang, Yisong

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.

  16. Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro

    PubMed Central

    Talhaoui, Ibtissam; Lebedeva, Natalia A.; Zarkovic, Gabriella; Saint-Pierre, Christine; Kutuzov, Mikhail M.; Sukhanova, Maria V.; Matkarimov, Bakhyt T.; Gasparutto, Didier; Saparbaev, Murat K.; Lavrik, Olga I.; Ishchenko, Alexander A.

    2016-01-01

    Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3′-cordycepin, 5′- and 3′-phosphate and also to 5′-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5′-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2′-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2′,1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1′ of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs. PMID:27471034

  17. Poly(ADP-ribose) polymerase-1 polymorphisms, expression and activity in selected human tumour cell lines

    PubMed Central

    Zaremba, T; Ketzer, P; Cole, M; Coulthard, S; Plummer, E R; Curtin, N J

    2009-01-01

    Background: Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA-binding enzyme activated by DNA breaks and involved in DNA repair and other cellular processes. Poly(ADP-ribose) polymerase activity can be higher in cancer than in adjacent normal tissue, but cancer predisposition is reported to be greater in individuals with a single-nucleotide polymorphism (SNP) V762A (T2444C) in the catalytic domain that reduces PARP-1 activity. Methods: To resolve these divergent observations, we determined PARP-1 polymorphisms, PARP-1 protein expression and activity in a panel of 19 solid and haematological, adult and paediatric human cancer cell lines. Results: There was a wide variation in PARP activity in the cell line panel (coefficient of variation, CV=103%), with the lowest and the highest activity being 2460 pmol PAR/106 (HS-5 cells) and 85 750 pmol PAR/106 (NGP cells). Lower variation (CV=32%) was observed in PARP-1 protein expression with the lowest expression being 2.0 ng μg−1 (HS-5 cells) and the highest being 7.1 ng μg−1 (ML-1 cells). The mean activity in the cancer cells was 45-fold higher than the mean activity in normal human lymphocytes and the PARP-1 protein levels were 23-fold higher. Conclusions: Surprisingly, there was no significant correlation between PARP activity and PARP-1 protein level or the investigated polymorphisms, T2444C and CA. PMID:19568233

  18. Alternative mechanisms of inhibiting activity of poly (ADP-ribose) polymerase-1.

    PubMed

    Sriram, Chandra Shaker; Jangra, Ashok; Bezbaruah, Babul Kumar; V, Athira K; Sykam, Shivaji

    2016-01-01

    Poly ADP-ribose polymerase (PARP-1), a DNA nick-sensor enzyme, is an abundant nuclear protein. Upon sensing DNA breaks, PARP-1 gets activated and cleaves NAD into nicotinamide and ADP-ribose and polymerizes the latter onto nuclear acceptor proteins including histones, transcription factors, and PARP-1 itself. Poly(ADP-ribosylation) mainly contributes to DNA repairing mechanism. However, oxidative stress-induced over-activation of PARP-1 consumes excess of NAD and consequently ATP, culminating into cell necrosis. This cellular suicide pathway has been implicated in several conditions such as stroke, myocardial ischemia, diabetes. Thus, it can be a rationale approach to inhibit the activity of PARP-1 for reducing detrimental effects associated with oxidative stress-induced over-activation of PARP-1. Several preclinical as well as clinical studies of PARP-1 inhibitors have been used in conditions such as cancer, stroke and traumatic brain injury. Conventionally, there are many studies which employed the concept of direct inhibition of PARP-1 by competing with NAD. Here, in the present review, we highlight several prospective alternative approaches for the inhibition of PARP-1 activity.

  19. Poly(ADP-ribose) polymerase 1 is a novel target to promote axonal regeneration

    PubMed Central

    Brochier, Camille; Jones, James I.; Willis, Dianna E.; Langley, Brett

    2015-01-01

    Therapeutic options for the restoration of neurological functions after acute axonal injury are severely limited. In addition to limiting neuronal loss, effective treatments face the challenge of restoring axonal growth within an injury environment where inhibitory molecules from damaged myelin and activated astrocytes act as molecular and physical barriers. Overcoming these barriers to permit axon growth is critical for the development of any repair strategy in the central nervous system. Here, we identify poly(ADP-ribose) polymerase 1 (PARP1) as a previously unidentified and critical mediator of multiple growth-inhibitory signals. We show that exposure of neurons to growth-limiting molecules—such as myelin-derived Nogo and myelin-associated glycoprotein—or reactive astrocyte-produced chondroitin sulfate proteoglycans activates PARP1, resulting in the accumulation of poly(ADP-ribose) in the cell body and axon and limited axonal growth. Accordingly, we find that pharmacological inhibition or genetic loss of PARP1 markedly facilitates axon regeneration over nonpermissive substrates. Together, our findings provide critical insights into the molecular mechanisms of axon growth inhibition and identify PARP1 as an effective target to promote axon regeneration. PMID:26598704

  20. Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities

    PubMed Central

    Khadka, Prabhat; Hsu, Joseph K.; Veith, Sebastian; Tadokoro, Takashi; Shamanna, Raghavendra A.; Mangerich, Aswin; Croteau, Deborah L.

    2015-01-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response. PMID:26391948

  1. Global analysis of transcriptional regulation by poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase in MCF-7 human breast cancer cells.

    PubMed

    Frizzell, Kristine M; Gamble, Matthew J; Berrocal, Jhoanna G; Zhang, Tong; Krishnakumar, Raga; Cen, Yana; Sauve, Anthony A; Kraus, W Lee

    2009-12-04

    Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs to stably knock down PARP-1 or PARG in MCF-7 cells followed by expression microarray analyses. Correlation analyses showed that the majority of genes affected by the knockdown of one factor were similarly affected by the knockdown of the other factor. The most robustly regulated common genes were enriched for stress-response and metabolic functions. In chromatin immunoprecipitation assays, PARP-1 and PARG localized to the promoters of positively and negatively regulated target genes. The levels of chromatin-bound PARG at a given promoter generally correlated with the levels of PARP-1 across the subset of promoters tested. For about half of the genes tested, the binding of PARP-1 at the promoter was dependent on the binding of PARG. Experiments using stable re-expression of short hairpin RNA-resistant catalytic mutants showed that PARP-1 and PARG enzymatic activities are required for some, but not all, target genes. Collectively, our results indicate that PARP-1 and PARG, nuclear enzymes with opposing enzymatic activities, localize to target promoters and act in a similar, rather than antagonistic, manner to regulate gene expression.

  2. Tankyrase Polymerization Is Controlled by Its Sterile Alpha Motif and Poly(ADP-Ribose) Polymerase Domains

    PubMed Central

    De Rycker, Manu; Price, Carolyn M.

    2004-01-01

    Tankyrases are novel poly(ADP-ribose) polymerases that have SAM and ankyrin protein-interaction domains. They are found at telomeres, centrosomes, nuclear pores, and Golgi vesicles and have been shown to participate in telomere length regulation. Their other function(s) are unknown, and it has been difficult to envision a common role at such diverse cellular locations. We have shown that tankyrase 1 polymerizes through its sterile alpha motif (SAM) domain to assemble large protein complexes. In vitro polymerization is reversible and still allows interaction with ankyrin-domain binding proteins. Polymerization can also occur in vivo, with SAM-dependent association of overexpressed tankyrase leading to formation of large tankyrase-containing vesicles, disruption of Golgi structure, and inhibition of apical secretion. Finally, tankyrase polymers are dissociated efficiently by poly(ADP-ribosy)lation. This disassembly is prevented by mutation of the PARP domain. Our findings indicate that tankyrase 1 has the unique capacity to promote both assembly and disassembly of large protein complexes. Thus, tankyrases appear to be master scaffolding proteins that regulate the formation of dynamic protein networks at different cellular locations. This implies a common scaffolding function for tankyrases at each location, with specific tankyrase interaction partners conferring location-specific roles to each network, e.g., telomere compaction or regulation of vesicle trafficking. PMID:15509784

  3. Poly(ADP-ribose)polymerase-1 (PARP1) controls adipogenic gene expression and adipocyte function.

    PubMed

    Erener, Süheda; Hesse, Mareike; Kostadinova, Radina; Hottiger, Michael O

    2012-01-01

    Poly(ADP-ribose)polymerase-1 (PARP1) is a chromatin-associated enzyme that was described to affect chromatin compaction. Previous reports suggested a dynamic modulation of the chromatin landscape during adipocyte differentiation. We thus hypothesized that PARP1 plays an important transcriptional role in adipogenesis and metabolism and therefore used adipocyte development and function as a model to elucidate the molecular action of PARP1 in obesity-related diseases. Our results show that PARP1-dependent ADP-ribose polymer (PAR) formation increases during adipocyte development and, at late time points of adipogenesis, is involved in the sustained expression of PPARγ2 and of PPARγ2 target genes. During adipogenesis, PARP1 was recruited to PPARγ2 target genes such as CD36 or aP2 in a PAR-dependent manner. Our results also reveal a PAR-dependent decrease in repressory histone marks (e.g. H3K9me3) and an increase in stimulatory marks (e.g. H3K4me3) at the PPARγ2 promoter, suggesting that PARP1 may exert its regulatory function during adipogenesis by altering histone marks. Interestingly, activation of PARP1 enzymatic activity was prevented with a topoisomerase II inhibitor. These data hint at topoisomerase II-dependent, transient, site-specific double-strand DNA breaks as the cause for poly(ADP)-ribose formation, adipogenic gene expression, and adipocyte function. Together, our study identifies PARP1 as a critical regulator of PPARγ2-dependent gene expression with implications in adipocyte function and obesity-related disease models.

  4. Curcumin enhances poly(ADP-ribose) polymerase inhibitor sensitivity to chemotherapy in breast cancer cells.

    PubMed

    Choi, Young Eun; Park, Eunmi

    2015-12-01

    Poly(ADP-ribose) polymerase (PARP) inhibitor has shown promising responses in homologous recombination (HR) repair-deficient cancer cells. More specifically, targeting HR pathway in combination with PARP inhibitor has been an effective chemotherapy strategy by so far. Curcumin has been recognized as anticancer agents for several types of cancers. Here, we demonstrate that curcumin inhibits a critical step in HR pathway, Rad51 foci formation, and accumulates γ-H2AX levels in MDA-MB-231 breast cancer cells. Curcumin also directly reduces HR and induces cell death with cotreatment of PARP inhibitor in MDA-MB-231 breast cancer cells. Moreover, curcumin, when combined with ABT-888, could effectively delayed breast tumor formation in vivo. Our study indicates that cotreatment of curcumin and PARP inhibitor might be useful for the combination chemotherapy for aggressive breast cancer treatment as a natural bioactive compound.

  5. Regulation of microglial expression of integrins by poly(ADP-ribose) polymerase-1.

    PubMed

    Ullrich, O; Diestel, A; Eyüpoglu, I Y; Nitsch, R

    2001-12-01

    Excitotoxic brain lesions initially result in the primary destruction of brain parenchyma, after which microglial cells migrate towards the sites of injury. At these sites, the cells produce large quantities of oxygen radicals and cause secondary damage that accounts for most of the loss of brain function. Here we show that this microglial migration is strongly controlled in living brain tissue by expression of the integrin CD11a, regulated by the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) through the formation of a nuclear PARP-NF-kappaB-protein complex. Downregulation of PARP or CD11a by transfection with antisense DNA abrogated microglial migration almost completely and prevented neurons from secondary damage.

  6. Poly(ADP-ribose) polymerase-13 and RNA regulation in immunity and cancer.

    PubMed

    Todorova, Tanya; Bock, Florian J; Chang, Paul

    2015-06-01

    Post-transcriptional 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 affect 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 prosurvival cytokine receptor tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 4 (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.

  7. Association of sperm morphology and the sperm deformity index (SDI) with poly (ADP-ribose) polymerase (PARP) cleavage inhibition.

    PubMed

    Aziz, Nabil; Sharma, Rakesh K; Mahfouz, Reda; Jha, Rajesh; Agarwal, Ashok

    2011-06-30

    Apoptosis was induced in immature and mature sperm in the presence or absence of poly (ADP-ribose) polymerase (PARP) inhibitor. The association of cleaved (cPARP) with sperm morphology was examined using sperm deformity index (SDI) score. The SDI scores are associated with PARP cleavage as an early marker of apoptosis.

  8. Purification and properties of poly(ADP-ribose)polymerase from Crithidia fasciculata. Automodification and poly(ADP-ribosyl)ation of DNA topoisomerase I.

    PubMed

    Podestá, Dolores; García-Herreros, María I; Cannata, Joaquín J B; Stoppani, Andrés O M; Fernández Villamil, Silvia H

    2004-06-01

    Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.

  9. Transcriptional regulation by Poly(ADP-ribose) polymerase-1 during T cell activation

    PubMed Central

    Saenz, Luis; Lozano, Juan J; Valdor, Rut; Baroja-Mazo, Alberto; Ramirez, Pablo; Parrilla, Pascual; Aparicio, Pedro; Sumoy, Lauro; Yélamos, José

    2008-01-01

    Background Accumulating evidence suggests an important role for the enzyme poly(ADP-ribose) polymerase-1 (PARP-1) as an integral part of the gene expression regulatory machinery during development and in response to specific cellular signals. PARP-1 might modulate gene expression through its catalytic activity leading to poly(ADP-ribosyl)ation of nuclear proteins or by its physical association with relevant proteins. Recently, we have shown that PARP-1 is activated during T cell activation. However, the proposed role of PARP-1 in reprogramming T cell gene expression upon activation remains largely unexplored. Results In the present study we use oligonucleotide microarray analysis to gain more insight into the role played by PARP-1 during the gene expression reprogramming that takes place in T cells upon activation with anti-CD3 stimulation alone, or in combination with anti-CD28 co-stimulation. We have identified several groups of genes with expression modulated by PARP-1. The expression of 129 early-response genes to anti-CD3 seems to be regulated by PARP-1 either in a positive (45 genes) or in a negative manner (84 genes). Likewise, in the presence of co-stimulation (anti-CD3 + anti-CD28 stimulation), the expression of 203 genes is also regulated by PARP-1 either up (173 genes) or down (30 genes). Interestingly, PARP-1 deficiency significantly alters expression of genes associated with the immune response such as chemokines and genes involved in the Th1/Th2 balance. Conclusion This study provides new insights into changes in gene expression mediated by PARP-1 upon T cell activation. Pathway analysis of PARP-1 as a nuclear signalling molecule in T cells would be of relevance for the future development of new therapeutic approaches targeting PARP-1 in the acquired immune response. PMID:18412984

  10. Poly (ADP-Ribose) Polymerase Mediates Diabetes-Induced Retinal Neuropathy

    PubMed Central

    Mohammad, Ghulam; Siddiquei, Mohammad Mairaj

    2013-01-01

    Retinal neuropathy is an early event in the development of diabetic retinopathy. One of the potential enzymes that are activated by oxidative stress in the diabetic retina is poly (ADP-ribose) polymerase (PARP). We investigated the effect of the PARP inhibitor 1,5-isoquinolinediol on the expression of the neurodegeneration mediators and markers in the retinas of diabetic rats. After two weeks of streptozotocin-induced diabetes, rats were treated with 1,5-isoquinolinediol (3 mg/kg/day). After 4 weeks of diabetes, the retinas were harvested and the levels of reactive oxygen species (ROS) were determined fluorometrically and the expressions of PARP, phosporylated-ERK1/2, BDNF, synaptophysin, glutamine synthetase (GS), and caspase-3 were determined by Western blot analysis. Retinal levels of ROS, PARP-1/2, phosphorylated ERK1/2, and cleaved caspase-3 were significantly increased, whereas the expressions of BDNF synaptophysin and GS were significantly decreased in the retinas of diabetic rats, compared to nondiabetic rats. Administration of 1,5-isoquinolinediol did not affect the metabolic status of the diabetic rats, but it significantly attenuated diabetes-induced upregulation of PARP, ROS, ERK1/2 phosphorylation, and cleaved caspase-3 and downregulation of BDNF, synaptophysin, and GS. These findings suggest a beneficial effect of the PARP inhibitor in increasing neurotrophic support and ameliorating early retinal neuropathy induced by diabetes. PMID:24347828

  11. Poly (ADP-ribose) Polymerase 1 Protein Expression in Normal and Neoplastic Prostatic Tissue

    PubMed Central

    Salemi, M.; Galia, A.; Fraggetta, F.; La Corte, C.; Pepe, P.; La Vignera, S.; Improta, G.; Bosco, P.; Calogero, A.E.

    2013-01-01

    A genetic background has been implicated in the development of prostate cancer. Protein microarrays have enabled the identification of proteins, some of which associated with apoptosis, that may play a role in the development of such a tumor. Inhibition of apoptosis is a co-factor that contributes to the onset and progression of prostate cancer, though the molecular mechanisms are not entirely understood. Poly (ADP-ribose) polymerase 1 (PARP-1) gene is required for translocation of the apoptosis-inducing factor (AIF) from the mitochondria to the nucleus. Hence, it is involved in programmed cell death. Different PARP-1 gene expression has been observed in various tumors such as glioblastoma, lung, ovarian, endometrial, and skin cancers. We evaluated the expression of PARP-1 protein in prostatic cancer and normal prostate tissues by immunohistochemistry in 40 men with prostate cancer and in 37 normal men. Positive nuclear PARP-1 staining was found in all samples (normal prostate and prostate cancer tissues). No cytoplasmic staining was observed in any sample. PARP-1-positive cells resulted significantly higher in patients with prostate carcinoma compared with controls (P<0.001). PARP-1 over-expression in prostate cancer tissue compared with normal prostate suggests a greater activity of PARP-1 in these tumors. These findings suggest that PARP-1 expression in prostate cancer is an attempt to trigger apoptosis in this type of tumor similarly to what reported in other cancers. PMID:23807292

  12. 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.

  13. 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.

  14. Poly(ADP-Ribose)Polymerase Activity Controls Plant Growth by Promoting Leaf Cell Number

    PubMed Central

    Schulz, Philipp; Jansseune, Karel; Degenkolbe, Thomas; Méret, Michaël; Claeys, Hannes; Skirycz, Aleksandra; Teige, Markus; Willmitzer, Lothar; Hannah, Matthew A.

    2014-01-01

    A changing global environment, rising population and increasing demand for biofuels are challenging agriculture and creating a need for technologies to increase biomass production. Here we demonstrate that the inhibition of poly (ADP-ribose) polymerase activity is a promising technology to achieve this under non-stress conditions. Furthermore, we investigate the basis of this growth enhancement via leaf series and kinematic cell analysis as well as single leaf transcriptomics and plant metabolomics under non-stress conditions. These data indicate a regulatory function of PARP within cell growth and potentially development. PARP inhibition enhances growth of Arabidopsis thaliana by enhancing the cell number. Time course single leaf transcriptomics shows that PARP inhibition regulates a small subset of genes which are related to growth promotion, cell cycle and the control of metabolism. This is supported by metabolite analysis showing overall changes in primary and particularly secondary metabolism. Taken together the results indicate a versatile function of PARP beyond its previously reported roles in controlling plant stress tolerance and thus can be a useful target for enhancing biomass production. PMID:24587323

  15. Inhibition of poly(ADP-ribose) polymerase attenuates ischemic renal injury in rats.

    PubMed

    Martin, D R; Lewington, A J; Hammerman, M R; Padanilam, B J

    2000-11-01

    The enzyme, poly(ADP-ribose) polymerase (PARP), effects repair of DNA after ischemia-reperfusion (I/R) injury to cells in nerve and muscle tissue. However, its activation in severely damaged cells can lead to ATP depletion and death. We show that PARP expression is enhanced in damaged renal proximal tubules beginning at 6-12 h after I/R injury. Intraperitoneal administration of PARP inhibitors, benzamide or 3-amino benzamide, after I/R injury accelerates the recovery of normal renal function, as assessed by monitoring the levels of plasma creatinine and blood urea nitrogen during 6 days postischemia. PARP inhibition leads to increased cell proliferation at 1 day postinjury as assessed by proliferating cell nuclear antigen and improves the histopathological appearance of kidneys examined at 7 days postinjury. Furthermore, inhibition of PARP increases levels of ATP measured at 24 h postischemia compared with those in vehicle-treated animals. Our data indicate that PARP activation is a part of the cascade of molecular events that occurs after I/R injury in the kidney. Although caution is advised, transient inhibition of PARP postischemia may constitute a novel therapy for acute renal failure.

  16. 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

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

    PubMed Central

    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.

    2014-01-01

    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. PMID:24987120

  18. Poly (ADP) ribose polymerase inhibition: A potential treatment of malignant peripheral nerve sheath tumor.

    PubMed

    Kivlin, Christine M; Watson, Kelsey L; Al Sannaa, Ghadah A; Belousov, Roman; Ingram, Davis R; Huang, Kai-Lieh; May, Caitlin D; Bolshakov, Svetlana; Landers, Sharon M; Kalam, Azad Abul; Slopis, John M; McCutcheon, Ian E; Pollock, Raphael E; Lev, Dina; Lazar, Alexander J; Torres, Keila E

    2016-01-01

    Poly (ADP) ribose polymerase (PARP) inhibitors, first evaluated nearly a decade ago, are primarily used in malignancies with known defects in DNA repair genes, such as alterations in breast cancer, early onset 1/2 (BRCA1/2). While no specific mutations in BRCA1/2 have been reported in malignant peripheral nerve sheath tumors (MPNSTs), MPNST cells could be effectively targeted with a PARP inhibitor to drive cells to synthetic lethality due to their complex karyotype and high level of inherent genomic instability. In this study, we assessed the expression levels of PARP1 and PARP2 in MPNST patient tumor samples and correlated these findings with overall survival. We also determined the level of PARP activity in MPNST cell lines. In addition, we evaluated the efficacy of the PARP inhibitor AZD2281 (Olaparib) in MPNST cell lines. We observed decreased MPNST cell proliferation and enhanced apoptosis in vitro at doses similar to, or less than, the doses used in cell lines with established defective DNA repair genes. Furthermore, AZD2281 significantly reduced local growth of MPNST xenografts, decreased the development of macroscopic lung metastases, and increased survival of mice with metastatic disease. Our results suggest that AZD2281 could be an effective therapeutic option in MPNST and should be further investigated for its potential clinical use in this malignancy.

  19. Poly(ADP-ribose)polymerase activity controls plant growth by promoting leaf cell number.

    PubMed

    Schulz, Philipp; Jansseune, Karel; Degenkolbe, Thomas; Méret, Michaël; Claeys, Hannes; Skirycz, Aleksandra; Teige, Markus; Willmitzer, Lothar; Hannah, Matthew A

    2014-01-01

    A changing global environment, rising population and increasing demand for biofuels are challenging agriculture and creating a need for technologies to increase biomass production. Here we demonstrate that the inhibition of poly (ADP-ribose) polymerase activity is a promising technology to achieve this under non-stress conditions. Furthermore, we investigate the basis of this growth enhancement via leaf series and kinematic cell analysis as well as single leaf transcriptomics and plant metabolomics under non-stress conditions. These data indicate a regulatory function of PARP within cell growth and potentially development. PARP inhibition enhances growth of Arabidopsis thaliana by enhancing the cell number. Time course single leaf transcriptomics shows that PARP inhibition regulates a small subset of genes which are related to growth promotion, cell cycle and the control of metabolism. This is supported by metabolite analysis showing overall changes in primary and particularly secondary metabolism. Taken together the results indicate a versatile function of PARP beyond its previously reported roles in controlling plant stress tolerance and thus can be a useful target for enhancing biomass production.

  20. Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells

    PubMed Central

    Du, Xueliang; Matsumura, Takeshi; Edelstein, Diane; Rossetti, Luciano; Zsengellér, Zsuzsanna; Szabó, Csaba; Brownlee, Michael

    2003-01-01

    In this report, we show that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron transport chain activates the three major pathways of hyperglycemic damage found in aortic endothelial cells by inhibiting GAPDH activity. In bovine aortic endothelial cells, GAPDH antisense oligonucleotides activated each of the pathways of hyperglycemic vascular damage in cells cultured in 5 mM glucose to the same extent as that induced by culturing cells in 30 mM glucose. Hyperglycemia-induced GAPDH inhibition was found to be a consequence of poly(ADP-ribosyl)ation of GAPDH by poly(ADP-ribose) polymerase (PARP), which was activated by DNA strand breaks produced by mitochondrial superoxide overproduction. Both the hyperglycemia-induced decrease in activity of GAPDH and its poly(ADP-ribosyl)ation were prevented by overexpression of either uncoupling protein–1 (UCP-1) or manganese superoxide dismutase (MnSOD), which decrease hyperglycemia-induced superoxide. Overexpression of UCP-1 or MnSOD also prevented hyperglycemia-induced DNA strand breaks and activation of PARP. Hyperglycemia-induced activation of each of the pathways of vascular damage was abolished by blocking PARP activity with the competitive PARP inhibitors PJ34 or INO-1001. Elevated glucose increased poly(ADP-ribosyl)ation of GAPDH in WT aortae, but not in the aortae from PARP-1–deficient mice. Thus, inhibition of PARP blocks hyperglycemia-induced activation of multiple pathways of vascular damage. PMID:14523042

  1. Poly(ADP-ribose) polymerase is hyperactivated in homologous recombination-defective cells.

    PubMed

    Gottipati, Ponnari; Vischioni, Barbara; Schultz, Niklas; Solomons, Joyce; Bryant, Helen E; Djureinovic, Tatjana; Issaeva, Natalia; Sleeth, Kate; Sharma, Ricky A; Helleday, Thomas

    2010-07-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gammaH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers.

  2. Poly(ADP-ribose) polymerase activity in mononuclear leukocytes of 13 mammalian species correlates with species-specific life span.

    PubMed Central

    Grube, K; Bürkle, A

    1992-01-01

    Poly(ADP-ribosyl)ation is a eukaryotic posttranslational modification of proteins that is strongly induced by the presence of DNA strand breaks and plays a role in DNA repair and the recovery of cells from DNA damage. We compared poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) activities in Percoll gradient-purified, permeabilized mononuclear leukocytes from mammalian species of different maximal life span. Saturating concentrations of a double-stranded octameric oligonucleotide were applied to provide a direct and maximal stimulation of PARP. Our results on 132 individuals from 13 different species yield a strong positive correlation between PARP activity and life span (r = 0.84; P << 0.001), with human cells displaying approximately 5 times the activity of rat cells. Intraspecies comparisons with both rat and human cells from donors of all age groups revealed some decline of PARP activity with advancing age, but it was only weakly correlated. No significant polymer degradation was detectable under our assay conditions, ruling out any interference by poly(ADP-ribose) glycohydrolase activity. By Western blot analysis of mononuclear leukocytes from 11 species, using a crossreactive antiserum directed against the extremely well-conserved NAD-binding domain, no correlation between the amount of PARP protein and the species' life spans was found, suggesting a greater specific enzyme activity in longer-lived species. We propose that a higher poly(ADP-ribosyl)ation capacity in cells from long-lived species might contribute to the efficient maintenance of genome integrity and stability over their longer life span. Images PMID:1465394

  3. The genes pme-1 and pme-2 encode two poly(ADP-ribose) polymerases in Caenorhabditis elegans.

    PubMed

    Gagnon, Steve N; Hengartner, Michael O; Desnoyers, Serge

    2002-11-15

    Poly(ADP-ribose) polymerases (PARPs) are an expanding, well-conserved family of enzymes found in many metazoan species, including plants. The enzyme catalyses poly(ADP-ribosyl)ation, a post-translational modification that is important in DNA repair and programmed cell death. In the present study, we report the finding of an endogenous source of poly(ADP-ribosyl)ation in total extracts of the nematode Caenorhabditis elegans. Two cDNAs encoding highly similar proteins to human PARP-1 (huPARP-1) and huPARP-2 are described, and we propose to name the corresponding enzymes poly(ADP-ribose) metabolism enzyme 1 (PME-1) and PME-2 respectively. PME-1 (108 kDa) shares 31% identity with huPARP-1 and has an overall structure similar to other PARP-1 subfamily members. It contains sequences having considerable similarity to zinc-finger motifs I and II, as well as with the catalytic domain of huPARP-1. PME-2 (61 kDa) has structural similarities with the catalytic domain of PARPs in general and shares 24% identity with huPARP-2. Recombinant PME-1 and PME-2 display PARP activity, which may partially account for the similar activity found in the worm. A partial duplication of the pme-1 gene with pseudogene-like features was found in the nematode genome. Messenger RNA for pme-1 are 5'-tagged with splice leader 1, whereas those for pme - 2 are tagged with splice leader 2, suggesting an operon-like expression for pme - 2. The expression pattern of pme-1 and pme-2 is also developmentally regulated. Together, these results show that PARP-1 and -2 are conserved in evolution and must have important functions in multicellular organisms. We propose using C. elegans as a model to understand better the functions of these enzymes.

  4. Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

    PubMed

    Pham, Phuong Anh; Wahl, Vanessa; Tohge, Takayuki; de Souza, Laise Rosado; Zhang, Youjun; Do, Phuc Thi; Olas, Justyna J; Stitt, Mark; Araújo, Wagner L; Fernie, Alisdair R

    2015-11-01

    The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds.

  5. Prevention of tumorigenesis of oncogene-transformed rat fibroblasts with DNA site inhibitors of poly(ADP ribose) polymerase

    SciTech Connect

    Tseng, A. Jr.; Lee, W.M.F.; Kirsten, E.; Hakam, A.; McLick, J.; Buki, K.; Kun, E.

    1987-02-01

    The EJ-ras gene was placed under the transcriptional control of the steroid-inducible mouse mammary tumor virus promoter/enhancer and introduced into Rat-1 fibroblasts, yielding the 14C cell line. When these cells were exposed to dexamethasone in vitro, EJ-ras mRNA was induced 15- to 20-fold, the cells grew in agar, and, after injection of cells into syngenic Fischer 344 rats, they produced lethal fibrosarcomas. Inhibitors of poly(ADP ribose) polymerase, which prevent the activation of the purified enzyme by a synthrtic octadeoxyribonucleotide duplex, inhibited both in vivo tumorigenicity and in vitro growth in soft agar. The enzyme inhibitor 1,2-benzopyrone, which was studied in detail, and other polymerase inhibitors had no effect on EJ-ras mRNA or p21 protein expression. Poly(ADP ribose) polymerase was inhibited by the drug in both untreated and dexamethasone-treated cells both in vitro and in vivo to the same extent, but biological consequences of enzyme inhibition were manifest only when the cells were in the transformed tumorigenic state.

  6. Poly(ADP-ribose) polymerase (PARP-1) is not involved in DNA double-strand break recovery

    PubMed Central

    Noël, Georges; Giocanti, Nicole; Fernet, Marie; Mégnin-Chanet, Frédérique; Favaudon, Vincent

    2003-01-01

    Background The cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose) polymerase (PARP-1) in DNA double-strand break repair. Results PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose) synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2. Conclusions The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose) synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks. PMID:12866953

  7. NGF promotes long-term memory formation by activating poly(ADP-ribose)polymerase-1.

    PubMed

    Wang, Shao-Hui; Liao, Xiao-Mei; Liu, Dan; Hu, Juan; Yin, Yang-Yang; Wang, Jian-Zhi; Zhu, Ling-Qiang

    2012-11-01

    Nerve growth factor (NGF) is a critical secreted protein that plays an important role in development, survival, and function of the mammalian nervous system. Previously reports suggest that endogenous NGF is essential for the hippocampal plasticity/memory and NGF deprivation induces the impairment of hippocampus-related memory and synaptic plasticity. However, whether exogenous supplement of NGF could promote the hippocampus-dependent synaptic plasticity/memory and the possible underlying mechanisms are not clear. In this study we found that NGF administration facilitates the hippocampus-dependent long-term memory and synaptic plasticity by increasing the activity of PARP-1, a polymerase mediating the PolyADP-ribosylation and important for the memory formation. Co-application of 3-Aminobenzamide (3-AB), a specific inhibitor of PARP-1, distinctly blocked the boosting effect of NGF on memory and synaptic plasticity, and the activation of downstream PKA-CREB signal pathway. Our data provide the first evidence that NGF supplement facilitates synaptic plasticity and the memory ability through PARP-1-mediated protein polyADP-ribosylation and activation of PKA-CREB pathway.

  8. Poly(ADP-ribose)polymerase (PARP) inhibition and anticancer activity of simmiparib, a new inhibitor undergoing clinical trials.

    PubMed

    Yuan, Bo; Ye, Na; Song, Shan-Shan; Wang, Yu-Ting; Song, Zilan; Chen, Hua-Dong; Chen, Chuan-Huizi; Huan, Xia-Juan; Wang, Ying-Qing; Su, Yi; Shen, Yan-Yan; Sun, Yi-Ming; Yang, Xin-Ying; Chen, Yi; Guo, Shi-Yan; Gan, Yong; Gao, Zhi-Wei; Chen, Xiao-Yan; Ding, Jian; He, Jin-Xue; Zhang, Ao; Miao, Ze-Hong

    2017-02-01

    Poly(ADP-ribose)polymerase (PARP)1/2 inhibitors have been proved to be clinically effective anticancer drugs. Here we report a new PARP1/2 inhibitor, simmiparib, displaying apparently improved preclinical anticancer activities relative to the first approved inhibitor olaparib. Simmiparib inhibited PARP1/2 approximately 2-fold more potently than olaparib, with more than 90-fold selectivity over the other tested PARP family members. Simmiparib and olaparib caused similar cellular PARP1-DNA trapping. Simmiparib selectively induced the accumulation of DNA double-strand breaks, G2/M arrest and apoptosis in homologous recombination repair (HR)-deficient cells. Consistently, simmiparib showed 26- to 235-fold selectivity in its antiproliferative activity against HR-deficient cells over the corresponding isogenic HR-proficient cells. Notably, its antiproliferative activity was 43.8-fold more potent than that of olaparib in 11 HR-deficient cancer cell lines. Simmiparib also potentiated the proliferative inhibition of several conventional anticancer drugs. Simmiparib reduced the poly(ADP-ribose) formation in HR-deficient cancer cells and xenografts. When orally administered to nude mice bearing xenografts, simmiparib revealed excellent pharmacokinetic properties. Simmiparib caused approximately 10-fold greater growth inhibition than olaparib against HR-deficient human cancer cell- or tissue-derived xenografts in nude mice. Collectively, these findings support the undergoing clinical trials of simmiparib.

  9. Activation of Poly(ADP-Ribose) Polymerase by Myocardial Ischemia and Coronary Reperfusion in Human Circulating Leukocytes

    PubMed Central

    Tóth-Zsámboki, Emese; Horváth, Eszter; Vargova, Katarina; Pankotai, Eszter; Murthy, Kanneganti; Zsengellér, Zsuzsanna; Bárány, Tamás; Pék, Tamás; Fekete, Katalin; Kiss, Róbert Gábor; Préda, István; Lacza, Zsombor; Gerö, Domokos; Szabó, Csaba

    2006-01-01

    Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 24 and 96 h later, we determined serum hydrogen peroxide concentrations, plasma levels of the oxidative DNA adduct 8-hydroxy-2′-deoxyguanosine (8OHdG), tyrosine nitration, PARP activation, and translocation of apoptosis-inducing factor (AIF) in circulating leukocytes. Plasma 8OHdG levels and leukocyte tyrosine nitration were rapidly increased by PCI. Similarly, poly(ADP-ribose) content of the leukocytes increased in cells isolated just after PCI, indicating immediate PARP activation triggered by reperfusion of the myocardium. In contrast, serum hydrogen peroxide concentrations and the translocation of AIF gradually increased over time and were most pronounced at 96 h. Reperfusion-related oxidative/nitrosative stress triggers DNA damage, which leads to PARP activation in circulating leukocytes. Translocation of AIF and lipid peroxidation occurs at a later stage. These results represent the first direct demonstration of PARP activation in human myocardial infarction. Future work is required to test whether pharmacological inhibition of PARP may offer myocardial protection during primary PCI. PMID:17225870

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

    SciTech Connect

    M Langelier; J Planck; S Roy; J Pascal

    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 DNA 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.

  11. Postnatal Age Influences Hypoglycemia-induced Poly(ADP-ribose) Polymerase-1 Activation in the Brain Regions of Rats

    PubMed Central

    Rao, Raghavendra; Sperr, Dustin; Ennis, Kathleen; Tran, Phu

    2009-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) overactivation plays a significant role in hypoglycemia-induced brain injury in adult rats. To determine the influence of postnatal age on PARP-1 activation, developing and adult male rats were subjected to acute hypoglycemia of equivalent severity and duration. The expression of PARP-1 and its downstream effectors, apoptosis inducing factor (Aifm1), caspase 3 (Casp3), NF-κB (Nfkb1) and bcl-2 (Bcl2), and cellular poly(ADP-ribose) (PAR) polymer expression was assessed in the cerebral cortex, hippocampus, striatum and hypothalamus at 0 h and 24 h post-hypoglycemia. Compared with the control group, PARP-1 expression increased in the cerebral cortex of adult rats 24 h post-hypoglycemia, but not at 0 h, and was accompanied by increased number of PAR-positive cells. The expression was not altered in other brain regions. Aifm1, Nfkb1, Casp3, and Bcl2 expression also increased in the cerebral cortex of adult rats 24 h post-hypoglycemia. Conversely, hypoglycemia did not alter PARP-1 expression and its downstream effectors in any brain region in developing rats. These data parallel the previously demonstrated pattern of hypoglycemia-induced brain injury and suggest that PARP-1 overactivation may determine age- and region-specific vulnerability during hypoglycemia. PMID:19687776

  12. Activation of poly(ADP-ribose) polymerase by myocardial ischemia and coronary reperfusion in human circulating leukocytes.

    PubMed

    Tóth-Zsámboki, Emese; Horváth, Eszter; Vargova, Katarina; Pankotai, Eszter; Murthy, Kanneganti; Zsengellér, Zsuzsanna; Bárány, Tamás; Pék, Tamás; Fekete, Katalin; Kiss, Róbert Gábor; Préda, István; Lacza, Zsombor; Gerö, Domokos; Szabó, Csaba

    2006-01-01

    Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 24 and 96 h later, we determined serum hydrogen peroxide concentrations, plasma levels of the oxidative DNA adduct 8-hydroxy-2'-deoxyguanosine (8OHdG), tyrosine nitration, PARP activation, and translocation of apoptosis-inducing factor (AIF) in circulating leukocytes. Plasma 8OHdG levels and leukocyte tyrosine nitration were rapidly increased by PCI. Similarly, poly(ADP-ribose) content of the leukocytes increased in cells isolated just after PCI, indicating immediate PARP activation triggered by reperfusion of the myocardium. In contrast, serum hydrogen peroxide concentrations and the translocation of AIF gradually increased over time and were most pronounced at 96 h. Reperfusion-related oxidative/nitrosative stress triggers DNA damage, which leads to PARP activation in circulating leukocytes. Translocation of AIF and lipid peroxidation occurs at a later stage. These results represent the first direct demonstration of PARP activation in human myocardial infarction. Future work is required to test whether pharmacological inhibition of PARP may offer myocardial protection during primary PCI.

  13. Mass spectrometry-based functional proteomics of poly(ADP-ribose) polymerase-1.

    PubMed

    Pic, Emilie; Gagné, Jean-Philippe; Poirier, Guy G

    2011-12-01

    PARP-1 is an abundant nuclear protein that plays an essential role in the regulation of many genome integrity and chromatin-based processes, such as DNA repair, replication or transcriptional regulation. PARP-1 modulates the function of chromatin and nuclear proteins through several poly(ADP-ribose) (pADPr)-dependent pathways. Aside from the clearly established role of PARP-1 in the maintenance of genome stability, PARP-1 also emerged as an important regulator that links chromatin functions with extranuclear compartments. pADPr signaling has notably been found to be responsible for PARP-1-mediated mitochondrial dysfunction and cell death. Defining the mechanisms that govern the intrinsic functions of PARP-1 is fundamental to the understanding of signaling networks regulated by pADPr. The emergence of mass spectrometry-based proteomics and its broad applications in the study of biological systems represents an outstanding opportunity to widen our knowledge of the functional spectrum of PARP-1. In this article, we summarize various PARP-1 targeted proteomics studies and proteome-wide analyses that shed light on its protein interaction partners, expression levels and post-translational modifications.

  14. 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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27904474

  16. An Update on Poly(ADP-ribose)polymerase-1 (PARP-1) Inhibitors: Opportunities and Challenges in Cancer Therapy.

    PubMed

    Wang, Ying-Qing; Wang, Ping-Yuan; Wang, Yu-Ting; Yang, Guang-Fu; Zhang, Ao; Miao, Ze-Hong

    2016-11-10

    Poly(ADP-ribose)polymerase-1 (PARP-1) is a critical DNA repair enzyme in the base excision repair pathway. Inhibitors of this enzyme comprise a new type of anticancer drug that selectively kills cancer cells by targeting homologous recombination repair defects. Since 2010, important advances have been achieved in PARP-1 inhibitors. Specifically, the approval of olaparib in 2014 for the treatment of ovarian cancer with BRCA mutations validated PARP-1 as an anticancer target and established its clinical importance in cancer therapy. Here, we provide an update on PARP-1 inhibitors, focusing on breakthroughs in their clinical applications and investigations into relevant mechanisms of action, biomarkers, and drug resistance. We also provide an update on the design strategies and the structural types of PARP-1 inhibitors. Opportunities and challenges in PARP-1 inhibitors for cancer therapy will be discussed based on the above advances.

  17. A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1

    PubMed Central

    Wang, Yingfei; An, Ran; Umanah, George K.; Park, Hyejin; Nambiar, Kalyani; Eacker, Stephen M.; Kim, BongWoo; Bao, Lei; Harraz, Maged M.; Chang, Calvin; Chen, Rong; Wang, Jennifer E.; Kam, Tae-In; Jeong, Jun Seop; Xie, Zhi; Neifert, Stewart; Qian, Jiang; Andrabi, Shaida A.; Blackshaw, Seth; Zhu, Heng; Song, Hongjun; Ming, Guo-li; Dawson, Valina L.; Dawson, Ted M.

    2016-01-01

    Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1–dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1–dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF's nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation. PMID:27846469

  18. Inhibitor and NAD+ binding to poly(ADP-ribose) polymerase as derived from crystal structures and homology modeling.

    PubMed

    Ruf, A; de Murcia, G; Schulz, G E

    1998-03-17

    Inhibitors of poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) are of clinical interest because they have potential for improving radiation therapy and chemotherapy of cancer. The refined binding structures of four such inhibitors are reported together with the refined structure of the unligated catalytic fragment of the enzyme. Following their design, all inhibitors bind at the position of the nicotinamide moiety of the substrate NAD+. The observed binding mode suggests inhibitor improvements that avoid other NAD(+)-binding enzymes. Because the binding pocket of NAD+ has been strongly conserved during evolution, the homology with ADP-ribosylating bacterial toxins could be used to extend the bound nicotinamide, which is marked by the inhibitors, to the full NAD+ molecule.

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

    DTIC Science & Technology

    2004-08-01

    The proteins were transferred to a nitrocellulose membrane and PARP was detected using anti-human PARP monoclonal antibody. Since PARP is a basic...to check if this modification is due to poly(ADP-ribosyl)ation of the protein , the membrane was stripped off and re-probed with anti-PAR polyclonal...detect any poly(ADP- ribosyl)ated proteins corresponding to the molecular weight of PARP (116 kDa) (Figure 18 ), we initiated experiment to test possible

  20. Poly(ADP-ribose) polymerase 1 regulates both the exonuclease and helicase activities of the Werner syndrome protein.

    PubMed

    von Kobbe, Cayetano; Harrigan, Jeanine A; Schreiber, Valérie; Stiegler, Patrick; Piotrowski, Jason; Dawut, Lale; Bohr, Vilhelm A

    2004-01-01

    Werner syndrome (WS) is a genetic premature aging disorder in which patients appear much older than their chronological age. The gene mutated in WS encodes a nuclear protein (WRN) which possesses 3'-5' exonuclease and ATPase-dependent 3'-5' helicase activities. The genomic instability associated with WS cells and the biochemical characteristics of WRN suggest that WRN plays a role in DNA metabolic pathways such as transcription, replication, recombination and repair. Recently we have identified poly(ADP-ribose) polymerase-1 (PARP-1) as a new WRN interacting protein. In this paper, we further mapped the interacting domains. We found that PARP-1 bound to the N-terminus of WRN and to the C-terminus containing the RecQ-conserved (RQC) domain. WRN bound to the N-terminus of PARP-1 containing DNA binding and BRCA1 C-terminal (BRCT) domains. We show that unmodified PARP-1 inhibited both WRN exonuclease and helicase activities, and to our knowledge is the only known WRN protein partner that inactivates both of the WRN's catalytic activities suggesting a biologically significant regulation. Moreover, this dual inhibition seems to be specific for PARP-1, as PARP-2 did not affect WRN helicase activity and only slightly inhibited WRN exonuclease activity. The differential effect of PARP-1 and PARP-2 on WRN catalytic activity was not due to differences in affinity for WRN or the DNA substrate. Finally, we demonstrate that the inhibition of WRN by PARP-1 was influenced by the poly(ADP-ribosyl)ation state of PARP-1. The biological relevance of the specific modulation of WRN catalytic activities by PARP-1 are discussed in the context of pathways in which these proteins may function together, namely in the repair of DNA strand breaks.

  1. Poly(ADP-ribose) Polymerase 1 Represses Liver X Receptor-mediated ABCA1 Expression and Cholesterol Efflux in Macrophages.

    PubMed

    Shrestha, Elina; Hussein, Maryem A; Savas, Jeffery N; Ouimet, Mireille; Barrett, Tessa J; Leone, Sarah; Yates, John R; Moore, Kathryn J; Fisher, Edward A; Garabedian, Michael J

    2016-05-20

    Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport through up-regulation of ATP-binding cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRα complexes and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRα and LXRβ. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow-derived macrophages but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor 3-aminobenzamide enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI. These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis.

  2. Review of Poly (ADP-ribose) Polymerase (PARP) Mechanisms of Action and Rationale for Targeting in Cancer and Other Diseases

    PubMed Central

    Morales, Julio C.; Li, Longshan; Fattah, Farjana J.; Dong, Ying; Bey, Erik A.; Patel, Malina; Gao, Jinming; Boothman, David A.

    2016-01-01

    Poly (ADP-ribose) polymerases (PARPs) are a family of related enzymes that share the ability to catalyze the transfer of ADP-ribose to target proteins. PARPs play an important role in various cellular processes, including modulation of chromatin structure, transcription, replication, recombination, and DNA repair. The role of PARP proteins in DNA repair is of particular interest, in view of the finding that certain tumors defective in homologous recombination mechanisms, may rely on PARP-mediated DNA repair for survival, and are sensitive to its inhibition. PARP inhibitors may also increase tumor sensitivity to DNA-damaging agents. Clinical trials of PARP inhibitors are investigating the utility of these approaches in cancer. The hyperactivation of PARP has also been shown to result in a specific programmed cell death pathway involving NAD+/ATP depletion, mu-calpain activation, loss of mitochondrial membrane potential, and the release of apoptosis inducing factor. Hyperactivation of the PARP pathway may be exploited to selectively kill cancer cells. Other PARP forms, including tankyrase 1 (PARP 5a), which plays an important role in enhancing telomere elongation by telomerase, have been found to be potential targets in cancer therapy. The PARP pathway and its inhibition thus offers a number of opportunities for therapeutic intervention in both cancer and other disease states. PMID:24579667

  3. Association of poly(ADP-ribose) polymerase with nuclear subfractions catalyzed with sodium tetrathionate and hydrogene peroxide crosslinks.

    PubMed

    Desnoyers, S; Kirkland, J B; Poirier, G G

    1996-06-21

    Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which catalyzes the transfer of ADP-ribose units from NAD+ to a variety of nuclear proteins under the stimulation of DNA strand break. To examine its role in DNA repair, we have been studying the interaction of PARP with other nuclear proteins using disulfide cross-linking, initiated by sodium tetrathionate (NaTT). Chinese Hamster Ovary (CHO) cells were extracted sequentially with Nonidet P40 (detergent), nucleases (DNase+RNase), and high salt (1.6 M NaCl) with and without the addition of a sulfhydryl reducing agent. The residual structures are referred to as the nuclear matrix, and are implicated in the organization of DNA repair and replication. Treatment of the cells with NaTT causes the crosslinking of PARP to the nuclear matrix. Activating PARP by pretreating the cells with H2O2 did not increase the cross-linking of PARP with the nuclear matrix, suggesting a lack of additional interaction of the enzyme with the nuclear matrix during DNA repair. Both NaTT and H2O2 induced crosslinks of PARP that were extractable with high salt. To shorten the procedure, these crosslinks were extracted from cells without nucleases and high salt treatment, using phosphate buffer. Using western blotting, these crosslinks appeared as a smear of high molecular weight species including a possible dimer of PARP at 230 kDa, which return to 116 kDa following reduction with beta-mercaptoethanol.

  4. Poly(ADP-ribose) polymerase inhibitors suppress UV-induced human immunodeficiency virus type 1 gene expression at the posttranscriptional level.

    PubMed Central

    Yamagoe, S; Kohda, T; Oishi, M

    1991-01-01

    Gene expression of human immunodeficiency virus type 1 (HIV-1) is induced not only by trans activation mediated through a gene product (tat) encoded by the virus but also by treatment of virus-carrying cells with DNA-damaging agents such as UV light. Employing an artificially constructed DNA in which the chloramphenicol acetyltransferase gene was placed under the control of the HIV-1 long terminal repeat, we analyzed the induction process in HeLa cells and found that inhibitors of poly(ADP-ribose) polymerase suppressed UV-induced HIV-1 gene expression but not tat-mediated expression. We also found that suppression occurs at the posttranscriptional level. These results indicate that HIV-1 gene expression is activated by at least two different mechanisms, one of which involves poly-ADP ribosylation. A possible new role of poly-ADP ribosylation in the regulation of specific gene expression is also discussed. Images PMID:1828533

  5. Acyl-CoA-binding domain containing 3 modulates NAD+ metabolism through activating poly(ADP-ribose) polymerase 1.

    PubMed

    Chen, Yong; Bang, Sookhee; Park, Soohyun; Shi, Hanyuan; Kim, Sangwon F

    2015-07-15

    NAD(+) plays essential roles in cellular energy homoeostasis and redox state, functioning as a cofactor along the glycolysis and citric acid cycle pathways. Recent discoveries indicated that, through the NAD(+)-consuming enzymes, this molecule may also be involved in many other cellular and biological outcomes such as chromatin remodelling, gene transcription, genomic integrity, cell division, calcium signalling, circadian clock and pluripotency. Poly(ADP-ribose) polymerase 1 (PARP1) is such an enzyme and dysfunctional PARP1 has been linked with the onset and development of various human diseases, including cancer, aging, traumatic brain injury, atherosclerosis, diabetes and inflammation. In the present study, we showed that overexpressed acyl-CoA-binding domain containing 3 (ACBD3), a Golgi-bound protein, significantly reduced cellular NAD(+) content via enhancing PARP1's polymerase activity and enhancing auto-modification of the enzyme in a DNA damage-independent manner. We identified that extracellular signal-regulated kinase (ERK)1/2 as well as de novo fatty acid biosynthesis pathways are involved in ACBD3-mediated activation of PARP1. Importantly, oxidative stress-induced PARP1 activation is greatly attenuated by knocking down the ACBD3 gene. Taken together, these findings suggest that ACBD3 has prominent impacts on cellular NAD(+) metabolism via regulating PARP1 activation-dependent auto-modification and thus cell metabolism and function.

  6. Glutaminase and poly(ADP-ribose) polymerase inhibitors suppress pyrimidine synthesis and VHL-deficient renal cancers.

    PubMed

    Okazaki, Arimichi; Gameiro, Paulo A; Christodoulou, Danos; Laviollette, Laura; Schneider, Meike; Chaves, Frances; Stemmer-Rachamimov, Anat; Yazinski, Stephanie A; Lee, Richard; Stephanopoulos, Gregory; Zou, Lee; Iliopoulos, Othon

    2017-03-27

    Many cancer-associated mutations that deregulate cellular metabolic responses to hypoxia also reprogram carbon metabolism to promote utilization of glutamine. In renal cell carcinoma (RCC), cells deficient in the von Hippel-Lindau (VHL) tumor suppressor gene use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate (αKG). Glutamine can also generate aspartate, the carbon source for pyrimidine biosynthesis, and glutathione for redox balance. Here we have shown that VHL-/- RCC cells rely on RC-derived aspartate to maintain de novo pyrimidine biosynthesis. Glutaminase 1 (GLS1) inhibitors depleted pyrimidines and increased ROS in VHL-/- cells but not in VHL+/+ cells, which utilized glucose oxidation for glutamate and aspartate production. GLS1 inhibitor-induced nucleoside depletion and ROS enhancement led to DNA replication stress and activation of an intra-S phase checkpoint, and suppressed the growth of VHL-/- RCC cells. These effects were rescued by administration of glutamate, αKG, or nucleobases with N-acetylcysteine. Further, we observed that the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib synergizes with GLS1 inhibitors to suppress the growth of VHL-/- cells in vitro and in vivo. This work describes a mechanism that explains the sensitivity of RCC tumor growth to GLS1 inhibitors and supports the development of therapeutic strategies for targeting VHL-deficient RCC.

  7. Targeting Human Poly(ADP-Ribose) Polymerase-1 with Natural Medicines and Its Potential Applications in Ovarian Cancer Therapeutics.

    PubMed

    Song, Min; Li, Jun-Lan; Li, Xiao-Ping; Kan, Shi-Feng

    2015-09-07

    Targeting poly(ADP-ribose) polymerase-1 (PARP-1) has been established as an efficient therapeutics for advanced ovarian cancer. In this study, we describe an integrated procedure that combines virtual computer screening and an experimental enzyme assay to discover novel potent PARP-1 inhibitors from more than 130000 commercially available natural products. The protocol employed a stepwise strategy to fast exclude typical PARP-1 non-binders and then performing rigorous prediction to identify promising candidates with high potency against PARP-1. Consequently, eight natural products were hit and tested to determine their inhibitory activities against the PARP-1 catalytic domain. From these, four compounds, i.e., puerarin, phloretin, chlorogenic acid, and biochanin A, were found to have high or moderate potencies with inhibitory IC50 values of 6, 470, 25, and 86 nM, respectively. The values are comparable to that (IC50  = 1.94 nM) of the FDA-approved agent olaparib. Structural and energetic analyses of the modeled structures of the PARP-1 catalytic domain complexed with the newly identified inhibitors revealed a common binding mode in the complexes: the active site of PARP-1 is composed of a thin polar helix and a flat non-polar pocket; the inhibitors can form a number of hydrogen bonds and electrostatic forces with the helix, while tightly packing against the pocket to define chemical interactions.

  8. Effect of silicon dioxide on expression of poly (ADP-ribose) polymerase mRNA and protein.

    PubMed

    Gao, Ai; Song, Shanshan; Wang, Danlin; Peng, Wei; Tian, Lin

    2009-07-01

    Silicon dioxide induces acute injury and chronic pulmonary fibrosis. International Agency for Research on Cancer (IARC) listed it as a human carcinogen in 1996. However, the molecular mechanisms to induce cancer are not understood yet. The content of poly (ADP-ribose) polymerases (PARP) mRNA and protein in Hela cells treated with concentrations of silicon dioxide up to 400microg/ml was determined by real-time fluorogenetic quantitative PCR (RQ-PCR) and immunofluorescence assay, respectively. MTT assay was used to determine cell viability. The results showed that viability at 400microg/ml silica was significantly decreased but not at lower concentrations. The protein content of gamma-H2AX in silica-treated group was significantly higher than the controls. The PARP mRNA and protein levels were significantly reduced with a dose response manner from the lowest silicon dioxide level. Our findings suggested that silicon dioxide increased the expression of gamma-H2AX and inhibited the expression of PARP mRNA and protein in Hela cells.

  9. Inhibition of Poly(ADP-Ribose) Polymerase Enhances Radiochemosensitivity in Cancers Proficient in DNA Double-Strand Break Repair.

    PubMed

    Shunkwiler, Lauren; Ferris, Gina; Kunos, Charles

    2013-02-08

    Pharmacologic inhibitors of poly(ADP-ribose) polymerase (PARP) putatively enhance radiation toxicity in cancer cells. Although there is considerable information on the molecular interactions of PARP and BRCA1- and BRCA2-deficient cancers, very little is known of the PARP inhibition effect upon cancers proficient in DNA double-strand break repair after ionizing radiation or after stalled replication forks. In this work, we investigate whether PARP inhibition by ABT-888 (veliparib) augments death-provoking effects of ionizing radiation, or of the topoisomerase I poison topotecan, within uterine cervix cancers cells harboring an unfettered, overactive ribonucleotide reductase facilitating DNA double-strand break repair and contrast these findings with ovarian cancer cells whose regulation of ribonucleotide reductase is relatively intact. Cell lethality of a radiation-ABT-888 combination is radiation and drug dose dependent. Data particularly highlight an enhanced topotecan-ABT-888 cytotoxicity, and corresponds to an increased number of unrepaired DNA double-strand breaks. Overall, our findings support enhanced radiochemotherapy toxicity in cancers proficient in DNA double-strand break repair when PARP is inhibited by ABT-888.

  10. Niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase inhibitor, radiosensitizes human lung and breast cancer cells

    PubMed Central

    Bridges, Kathleen A.; Toniatti, Carlo; Buser, Carolyn A.; Liu, Huifeng; Buchholz, Thomas A.; Meyn, Raymond E.

    2014-01-01

    The aim of this study was to assess niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast and prostate cancers were tested for radiosensitization by niraparib using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of niraparib to alter the repair of radiation-induced DNA double strand breaks (DSBs) was determined using detection of γ-H2AX foci and RAD51 foci. Clonogenic survival analyses indicated that micromolar concentrations of niraparib radiosensitized tumor cell lines derived from lung, breast, and prostate cancers independently of their p53 status but not cell lines derived from normal tissues. Niraparib also sensitized tumor cells to H2O2 and converted H2O2-induced single strand breaks (SSBs) into DSBs during DNA replication. These results indicate that human tumor cells are significantly radiosensitized by the potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The mechanism of this effect appears to involve a conversion of sublethal SSBs into lethal DSBs during DNA replication due to the inhibition of base excision repair by the drug. Taken together, our findings strongly support the clinical evaluation of niraparib in combination with radiation. PMID:24970803

  11. 3-aminobenzamide, one of poly(ADP-ribose)polymerase-1 inhibitors, rescuesapoptosisin rat models of spinal cord injury.

    PubMed

    Meng, Xianqing; Song, Wenqi; Deng, Bin; Xing, Ziling; Zhang, Weihong

    2015-01-01

    Poly(ADP-ribose)polymerase-1 (PARP-1) is anubiquitous, DNA repair-associated enzyme, which participates in gene expression, cell death, central nerve system (CNS) disorders and oxidative stress. According to the previous studies, PARP-1 over-activation may lead to over-consumption of ATP and even cell apoptosis. Spinal cord injury (SCI) is an inducement towards PARP-1 over-activation due to its massive damage to DNA. 3-aminobenzamide (3-AB) is a kind of PARP-1 inhibitors. The relationship among PARP-1, 3-AB, SCI and apoptosis has not been fully understood. Hence, in the present study, we focused on the effects of 3-AB on cell apoptosis after SCI. Accordingly, SCI model was constructed artificially, and 3-AB was injected intrathecally into the Sprague-Dawley (SD) rats. The results demonstrated an increase in cell apoptosis after SCI. Furthermore, PARP-1 was over-activated after SCI but inhibited by 3-AB injection. In addition, apoptosis-inducing factor (AIF) was inhibited but B-cell lymphoma-2 (Bcl-2) was up-regulated by 3-AB. Interestingly, caspase-3 was not significantly altered with or without 3-AB. In conclusion, our experiments showed that 3-AB, as a PARP-1 inhibitor, could inhibit cell apoptosis after SCI in caspase-independent way, which could provide a better therapeutic target for the treatment of SCI.

  12. 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.

  13. Poly(ADP-ribose)polymerase 1 stimulates the AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1.

    PubMed

    Lebedeva, Natalia A; Anarbaev, Rashid O; Sukhanova, Maria; Vasil'eva, Inna A; Rechkunova, Nadejda I; Lavrik, Olga I

    2015-06-15

    The influence of poly(ADP-ribose)polymerase 1 (PARP1) on the apurinic/apyrimidinic (AP)-site cleavage activity of tyrosyl-DNA phosphodiesterase 1 (TDP1) and interaction of PARP1 and TDP1 were studied. The efficiency of single or clustered AP-site hydrolysis catalysed by TDP1 was estimated. It was shown that the efficiency of AP-site cleavage increases in the presence of an additional AP-site in the opposite DNA strand depending on its position. PARP1 stimulates TDP1; the stimulation effect was abolished in the presence of NAD(+). The interaction of these two proteins was characterized quantitatively by measuring the dissociation constant for the TDP1-PARP1 complex using fluorescently-labelled proteins. The distance between the N-termini of the proteins within the complex was estimated using FRET. The data obtained suggest that PARP1 and TDP1 bind in an antiparallel orientation; the N-terminus of the former protein interacts with the C-terminal domain of the latter. The functional significance of PARP1 and TDP1 interaction in the process of DNA repair was demonstrated for the first time.

  14. The rise and fall of poly(ADP-ribose): An enzymatic perspective.

    PubMed

    Pascal, John M; Ellenberger, Tom

    2015-08-01

    Human cells respond to DNA damage with an acute and transient burst in production of poly(ADP-ribose), a posttranslational modification that expedites damage repair and plays a pivotal role in cell fate decisions. Poly(ADP-ribose) polymerases (PARPs) and glycohydrolase (PARG) are the key set of enzymes that orchestrate the rise and fall in cellular levels of poly(ADP-ribose). In this perspective, we focus on recent structural and mechanistic insights into the enzymes involved in poly(ADP-ribose) production and turnover, and we highlight important questions that remain to be answered.

  15. The Rise and Fall of Poly (ADP-ribose). An Enzymatic Perspective

    PubMed Central

    Pascal, John M.; Ellenberger, Tom

    2015-01-01

    Human cells respond to DNA damage with an acute and transient burst in production of poly(ADP-ribose), a posttranslational modification that expedites damage repair and plays a pivotal role in cell fate decisions. Poly(ADP-ribose) polymerases (PARPs) and glycohydrolase (PARG) are the key set of enzymes that orchestrate the rise and fall in cellular levels of poly(ADP-ribose). In this perspective, we focus on recent structural and mechanistic insights into the enzymes involved in poly(ADP-ribose) production and turnover, and we highlight important questions that remain to be answered. PMID:25963443

  16. Neurological and histological consequences induced by in vivo cerebral oxidative stress: evidence for beneficial effects of SRT1720, a sirtuin 1 activator, and sirtuin 1-mediated neuroprotective effects of poly(ADP-ribose) polymerase inhibition.

    PubMed

    Gueguen, Cindy; Palmier, Bruno; Plotkine, Michel; Marchand-Leroux, Catherine; Besson, Valérie C

    2014-01-01

    Poly(ADP-ribose)polymerase and sirtuin 1 are both NAD(+)-dependent enzymes. In vitro oxidative stress activates poly(ADP-ribose)polymerase, decreases NAD(+) level, sirtuin 1 activity and finally leads to cell death. Poly(ADP-ribose)polymerase hyperactivation contributes to cell death. In addition, poly(ADP-ribose)polymerase inhibition restores NAD(+) level and sirtuin 1 activity in vitro. In vitro sirtuin 1 induction protects neurons from cell loss induced by oxidative stress. In this context, the role of sirtuin 1 and its involvement in beneficial effects of poly(ADP-ribose)polymerase inhibition were evaluated in vivo in a model of cerebral oxidative stress induced by intrastriatal infusion of malonate in rat. Malonate promoted a NAD(+) decrease that was not prevented by 3-aminobenzamide, a poly(ADP-ribose)polymerase inhibitor, at 4 and 24 hours. However, 3-aminobenzamide increased nuclear SIRT1 activity/expression ratio after oxidative stress. Malonate induced a neurological deficit associated with a striatal lesion. Both were reduced by 3-aminobenzamide and SRT1720, a sirtuin 1 activator, showing beneficial effects of poly(ADP-ribose)polymerase inhibition and sirtuin 1 activation on oxidative stress consequences. EX527, a sirtuin 1 inhibitor, given alone, modified neither the score nor the lesion, suggesting that endogenous sirtuin 1 was not activated during cerebral oxidative stress. However, its association with 3-aminobenzamide suppressed the neurological improvement and the lesion reduction induced by 3-aminobenzamide. The association of 3-aminobenzamide with SRT1720, the sirtuin 1 activator, did not lead to a better protection than 3-aminobenzamide alone. The present data represent the first demonstration that the sirtuin 1 activator SRT1720 is neuroprotective during in vivo cerebral oxidative stress. Furthermore sirtuin 1 activation is involved in the beneficial effects of poly(ADP-ribose)polymerase inhibition after in vivo cerebral oxidative stress.

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

    SciTech Connect

    Gebhard, Catherine; Staehli, Barbara E.; Shi, Yi; Camici, Giovanni G.; Akhmedov, Alexander; Hoegger, Lisa; Lohmann, Christine; Matter, Christian M.; Hassa, Paul O.; Hottiger, Michael O.; Malinski, Tadeusz; Luescher, Thomas F.; and others

    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 were 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.

  18. Apurinic/apyrimidinic (AP) site recognition by the 5'-dRP/AP lyase in poly(ADP-ribose) polymerase-1 (PARP-1).

    PubMed

    Khodyreva, S N; Prasad, R; Ilina, E S; Sukhanova, M V; Kutuzov, M M; Liu, Y; Hou, E W; Wilson, S H; Lavrik, O I

    2010-12-21

    The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction of the PARP-1/AP site DNA complex resulted in covalent cross-linking of PARP-1 to DNA; the identity of cross-linked PARP-1 was confirmed by mass spectrometry. Using purified human PARP-1, the specificity of PARP-1 binding to AP site-containing DNA was confirmed in competition binding experiments. PARP-1 was only weakly activated to conduct poly(ADP-ribose) synthesis upon binding to AP site-containing DNA, but was strongly activated for poly(ADP-ribose) synthesis upon strand incision by AP endonuclease 1 (APE1). By virtue of its binding to AP sites, PARP-1 could be poised for its role in base excision repair, pending DNA strand incision by APE1 or the 5'-dRP/AP lyase activity in PARP-1.

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

    DTIC Science & Technology

    1985-12-15

    mechanism is operative from NOD either directly ADY-ribosy- latintr "acceptor" proteins (%.g., hiatone), a reaction presumably catalyzed by the...24 Figures 1through 5. .. ............................. .. .. .. .. .........25-29’ 2. Mechanisms of’Poly(ADP-Ribose) Poiymerase Catalysis...Figures 1 through 3 .. ...................... ...........................46-48 3. Mechanism of inactivation of Poly(ADP-Ribose) Polymerase of Rat Liver

  20. Neuropilin-1 modulates vascular endothelial growth factor-induced poly(ADP-ribose)-polymerase leading to reduced cerebrovascular apoptosis.

    PubMed

    Mey, Lilli; Hörmann, Mareike; Schleicher, Nadine; Reuter, Peter; Dönges, Simone; Kinscherf, Ralf; Gassmann, Max; Gerriets, Tibo; Al-Fakhri, Nadia

    2013-11-01

    Cerebral ischemia is encompassed by cerebrovascular apoptosis, yet the mechanisms behind apoptosis regulation are not fully understood. We previously demonstrated inhibition of endothelial apoptosis by vascular endothelial growth factor (VEGF) through upregulation of poly(ADP-ribose)-polymerase (PARP) expression. However, PARP overactivation through oxidative stress can lead to necrosis. This study tested the hypothesis that neuropilin-1 (NP-1), an alternative VEGF receptor, regulates the response to cerebral ischemia by modulating PARP expression and, in turn, apoptosis inhibition by VEGF. In endothelial cell culture, NP-1 colocalized with VEGF receptor-2 (VEGFR-2) and acted as its coreceptor. This significantly enhanced VEGF-induced PARP mRNA and protein expression demonstrated by receptor-specific inhibitors and VEGF-A isoforms. NP-1 augmented the inhibitory effect of VEGF/VEGFR-2 interaction on apoptosis induced by adhesion inhibition through the αV-integrin inhibitor cRGDfV. NP-1/VEGFR-2 signal transduction involved JNK and Akt. In rat models of permanent and temporary middle cerebral artery occlusion, the ischemic cerebral hemispheres displayed endothelial and neuronal apoptosis next to increased endothelial NP-1 and VEGFR-2 expression compared to non-ischemic cerebral hemispheres, sham-operated or untreated controls. Increased vascular superoxide dismutase-1 and catalase expression as well as decreased glycogen reserves indicated oxidative stress in the ischemic brain. Of note, protein levels of intact PARP remained stable despite pro-apoptotic conditions through increased PARP mRNA production during cerebral ischemia. In conclusion, NP-1 is upregulated in conditions of imminent cerebrovascular apoptosis to reinforce apoptosis inhibition and modulate VEGF-dependent PARP expression and activation. We propose that NP-1 is a key modulator of VEGF maintaining cerebrovascular integrity during ischemia. Modulating the function of NP-1 to target PARP could help to

  1. Poly (ADP-ribose) polymerase-1 inhibitor, 3-aminobenzamide pretreatment ameliorates lipopolysaccharide-induced neurobehavioral and neurochemical anomalies in mice.

    PubMed

    Sriram, Chandra Shaker; Jangra, Ashok; Gurjar, Satendra Singh; Hussain, Md Iftikar; Borah, Probodh; Lahkar, Mangala; Mohan, Pritam; Bezbaruah, Babul Kumar

    2015-06-01

    Poly (ADP-ribose) polymerase-1 (PARP-1) functions at the center of cellular stress and sways the immune system at several key points, thus modulates inflammatory diseases. The antiinflammatory properties of PARP-1 inhibitors have been demonstrated ameliorating effect in various neuroinflammatory disorders. It has been reported that there is a close relationship between the inflammatory processes and major depressive disorder. In the present study, we have elucidated the role of oxidative-nitrosative stress-PARP-1 pathway in lipopolysaccharide (LPS)-induced neurobehavioral and neurochemical alterations in mice. 3-Aminobenzamide (10 and 30mg/kg) and imipramine (10 and 30mg/kg) were administered once daily for 14days. Mice were challenged with LPS (1mg/kg, i.p.) 30min after drug administration on the 14th day. The mRNA expression level of PARP-1 (12h after LPS injection) in the hippocampus was measured through quantitative real-time PCR. All the behavioral and biochemical parameters were assessed at 24h after LPS injection. The expression level of PARP-1mRNA was found significantly up-regulated in the hippocampus at 12h after LPS administration. Results showed that the LPS-challenged mice exhibited an increase in immobility time seen in forced swimming test and tail suspension test. LPS increased the levels of proinflammatory cytokines and oxido-nitrosative stress parameters in the hippocampus. However, pretreatment with 3-aminobenzamide (30mg/kg) significantly reversed the LPS-induced alterations in behavioral parameters, proinflammatory cytokines, oxidative-nitrosative stress and PARP-1 mRNA levels. Imipramine failed to prevent the up-regulation of PARP-1 induced by LPS administration. Our results emphasized that oxidative-nitrosative stress-PARP-1 cascade can play a key role in LPS-induced neurobehavioral and neurochemical anomalies.

  2. Radiosensitization effect of poly(ADP-ribose) polymerase inhibition in cells exposed to low and high liner energy transfer radiation.

    PubMed

    Hirai, Takahisa; Shirai, Hidenori; Fujimori, Hiroaki; Okayasu, Ryuichi; Sasai, Keisuke; Masutani, Mitsuko

    2012-06-01

    Poly(ADP-ribose) polymerase (PARP)-1 promotes base excision repair and DNA strand break repair. Inhibitors of PARP enhance the cytotoxic effects of γ-irradiation and X-irradiation. We investigated the impact of PARP inhibition on the responses to γ-irradiation (low liner energy transfer [LET] radiation) and carbon-ion irradiation (high LET radiation) in the human pancreatic cancer cell line MIA PaCa-2. Cell survival was assessed by colony formation assay after combination treatment with the PARP inhibitor AZD2281 and single fraction γ-irradiation and carbon-ion irradiation (13 and 70 keV/μm [LET 13 and LET 70]). The DNA damage response (DDR) was assessed by pulse field gel electrophoresis, western blotting and flow cytometry. Treatment with a PARP inhibitor enhanced the cytotoxic effect of γ-irradiation and LET 13 and LET 70 carbon-ion irradiation. Moreover, the radiosensitization effect was greater for LET 70 than for LET 13 irradiation. Prolonged and increased levels of γ-H2AX were observed both after γ-irradiation and carbon-ion irradiation in the presence of the PARP inhibitor. Enhanced level of phosphorylated-p53 (Ser-15) was observed after γ-irradiation but not after carbon-ion irradiation. PARP inhibitor treatment induced S phase arrest and enhanced subsequent G2/M arrest both after γ-irradiation and carbon-ion irradiation. These results suggest that the induction of S phase arrest through an enhanced DDR and a local delay in DNA double strand break processing by PARP inhibition caused sensitization to γ-irradiation and carbon-ion irradiation. Taken together, PARP inhibitors might be applicable to a wide therapeutic range of LET radiation through their effects on the DDR.

  3. Poly(ADP-ribose) polymerase 1 contributes to oxidative stress through downregulation of sirtuin 3 during cisplatin nephrotoxicity

    PubMed Central

    Yoon, Sang Pil

    2016-01-01

    Enhanced oxidative stress is a hallmark of cisplatin nephrotoxicity, and inhibition of poly(ADP-ribose) polymerase 1 (PARP1) attenuates oxidative stress during cisplatin nephrotoxicity; however, the precise mechanisms behind its action remain elusive. Here, using an in vitro model of cisplatin-induced injury to human kidney proximal tubular cells, we demonstrated that the protective effect of PARP1 inhibition on oxidative stress is associated with sirtuin 3 (SIRT3) activation. Exposure to 400 µM cisplatin for 8 hours in cells decreased activity and expression of manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase (GPX), and SIRT3, while it increased their lysine acetylation. However, treatment with 1 µM PJ34 hydrochloride, a potent PARP1 inhibitor, restored activity and/or expression in those antioxidant enzymes, decreased lysine acetylation of those enzymes, and improved SIRT3 expression and activity in the cisplatin-injured cells. Using transfection with SIRT3 double nickase plasmids, SIRT3-deficient cells given cisplatin did not show the ameliorable effect of PARP1 inhibition on lysine acetylation and activity of antioxidant enzymes, including MnSOD, catalase and GPX. Furthermore, SIRT3 deficiency in cisplatin-injured cells prevented PARP1 inhibition-induced increase in forkhead box O3a transcriptional activity, and upregulation of MnSOD and catalase. Finally, loss of SIRT3 in cisplatin-exposed cells removed the protective effect of PARP1 inhibition against oxidative stress, represented by the concentration of lipid hydroperoxide and 8-hydroxy-2'-deoxyguanosine; and necrotic cell death represented by a percentage of propidium iodide–positively stained cells. Taken together, these results indicate that PARP1 inhibition protects kidney proximal tubular cells against oxidative stress through SIRT3 activation during cisplatin nephrotoxicity. PMID:27722009

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

    SciTech Connect

    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 of 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.

  5. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    PubMed Central

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  6. Synthesis and biological evaluation of substituted 2-phenyl-2H-indazole-7-carboxamides as potent poly(ADP-ribose) polymerase (PARP) inhibitors.

    PubMed

    Scarpelli, Rita; Boueres, Julia K; Cerretani, Mauro; Ferrigno, Federica; Ontoria, Jesus M; Rowley, Michael; Schultz-Fademrecht, Carsten; Toniatti, Carlo; Jones, Philip

    2010-01-15

    A potent series of substituted 2-phenyl-2H-indazole-7-carboxamides were synthesized and evaluated as inhibitors of poly (ADP-ribose) polymerase (PARP). This extensive SAR exploration culminated with the identification of substituted 5-fluoro-2-phenyl-2H-indazole-7-carboxamide analog 48 which displayed excellent PARP enzyme inhibition with IC(50)=4nM, inhibited proliferation of cancer cell lines deficient in BRCA-1 with CC(50)=42nM and showed encouraging pharmacokinetic properties in rats compared to the lead 6.

  7. 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.

  8. Inhibition of poly(ADP-ribose) polymerase-1 or poly(ADP-ribose) glycohydrolase individually, but not in combination, leads to improved chemotherapeutic efficacy in HeLa cells

    PubMed Central

    FENG, XIAOXING; KOH, DAVID W.

    2013-01-01

    The genome-protecting role of poly(ADP-ribose) (PAR) has identified PAR polymerase-1 (PARP-1) and PAR glycohydrolase (PARG), two enzymes responsible for the synthesis and hydrolysis of PAR, as chemotherapeutic targets. Each has been previously individually evaluated in chemotherapy, but the effects of combination PARP-1 and PARG inhibition in cancer cells are not known. Here we determined the effects of the inhibition of PARP-1 and the absence or RNAi knockdown of PARG on PAR synthesis, cell death after chemotherapy and long-term viability. Using three experimental/clinical PARP-1 inhibitors in PARG-null cells, we show decreased levels of PAR and increased short-term and long-term viability with each inhibitor, with the exception of DPQ. Treatment with the experimental chemotherapeutic agent, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), led to increased cell death in PARG-null cells, but decreased cell death when pretreated with each PARP-1 inhibitor. Similar results were observed in MNNG-treated HeLa cells, where RNAi knockdown of PARG or pretreatment with ABT-888 led to increased HeLa cell death, whereas combination PARG RNAi knockdown + ABT-888 failed to produce increased cell death. The results demonstrate the ability of the PARP-1 inhibitors to decrease PAR levels, maintain viability and decrease PAR-mediated cell death after chemotherapeutic treatment in the absence of PARG. Further, the results demonstrate that the combination of PARP-1 and PARG inhibition in chemotherapy does not produce increased HeLa cell death. Thus, the results indicate that inhibiting both PARP-1 and PARG, which both are chemotherapeutic targets that increase cancer cell death, does not lead to synergistic cell death in HeLa cells. Therefore, strategies that target PAR metabolism for the improved treatment of cancer may be required to target PARP-1 and PARG individually in order to optimize cancer cell death. PMID:23254695

  9. Stable and Reusable Electrochemical Biosensor for Poly(ADP-ribose) Polymerase and Its Inhibitor Based on Enzyme-Initiated Auto-PARylation.

    PubMed

    Xu, Yuanyuan; Liu, Li; Wang, Zhaoyin; Dai, Zhihui

    2016-07-27

    A stable and reusable electrochemical biosensor for the label-free detection of poly(ADP-ribose) polymerase (PARP) is designed in this work. C-kit-1, a thiol-modified G-quadruplex oligonucleotide, is first self-assembled on a gold electrode surface. The G-quadruplex structure of c-kit-1 can specifically tether and activate PARP, resulting in the generation of negatively charged poly(ADP-ribose) polymer (PAR). On the basis of electrostatic attraction, PAR facilitates the surface accumulation of positively charged electrochemical signal molecules. Through the characterization of electrochemical signal molecules, the label-free quantification of PARP is simply implemented. On the basis of the proposed method, selective quantification of PARP can be achieved over the linear range from 0.01 to 1 U with a calculated detection limit of 0.003U. Further studies also demonstrate the applicability of the proposed method to biosamples revealing the broad potential in practical applications. Furthermore, inhibitor of PARP has also been detected with this biosensor. Meanwhile, benefited from self-assembly on solid surface, this biosensor possesses two important features, i.e., reusability and stability, which are desirable in related biosensors.

  10. Regulation of kinase cascade activation and heat shock protein expression by poly(ADP-ribose) polymerase inhibition in doxorubicin-induced heart failure.

    PubMed

    Bartha, Eva; Solti, Izabella; Szabo, Aliz; Olah, Gabor; Magyar, Klara; Szabados, Eszter; Kalai, Tamas; Hideg, Kalman; Toth, Kalman; Gero, Domokos; Szabo, Csaba; Sumegi, Balazs; Halmosi, Robert

    2011-10-01

    Cardiomyopathy is one of the most severe side effects of the chemotherapeutic agent doxorubicin (DOX). The formation of reactive oxygen species plays a critical role in the development of cardiomyopathies, and the pathophysiological cascade activates nuclear enzyme poly(ADP-ribose) polymerase (PARP), and kinase pathways. We characterized the effects of the PARP-inhibitor and kinase-modulator compound L-2286 in DOX-induced cardiac injury models. We studied the effect of the established superoxide dismutase-mimic Tempol and compared the effects of this agent with those of the PARP inhibitor. In the rat H9C2 cardiomyocytes, in which DOX-induced poly(ADP-ribosyl)ation, L-2286 protected them from the DOX-induced injury in a concentration-dependent manner. In the in vivo studies, mice were pretreated (for 1 week) with L-2286 or Tempol before the DOX treatment. Both the agents improved the activation of cytoprotective kinases, Akt, phospho-specific protein kinase C ϵ, ζ/λ and suppressed the activity of cell death promoting kinases glycogen synthase kinase-3β, JNK, and p38 mitogen-activated protein kinase, but the effect of PARP inhibitor was more pronounced and improved the survival as well. L-2286 activated the phosphorylation of proapoptotic transcription factor FKHR1 and promoted the expression of Hsp72 and Hsp90. These data suggest that the mode of the cytoprotective action of the PARP inhibitor may include the modulation of kinase pathways and heat shock protein expression.

  11. The Sound of Silence: RNAi in Poly (ADP-Ribose) Research

    PubMed Central

    Blenn, Christian; Wyrsch, Philippe; Althaus, Felix R.

    2012-01-01

    Poly(ADP-ribosyl)-ation is a nonprotein posttranslational modification of proteins and plays an integral part in cell physiology and pathology. The metabolism of poly(ADP-ribose) (PAR) is regulated by its synthesis by poly(ADP-ribose) polymerases (PARPs) and on the catabolic side by poly(ADP-ribose) glycohydrolase (PARG). PARPs convert NAD+ molecules into PAR chains that interact covalently or noncovalently with target proteins and thereby modify their structure and functions. PAR synthesis is activated when PARP1 and PARP2 bind to DNA breaks and these two enzymes account for almost all PAR formation after genotoxic stress. PARG cleaves PAR molecules into free PAR and finally ADP-ribose (ADPR) moieties, both acting as messengers in cellular stress signaling. In this review, we discuss the potential of RNAi to manipulate the levels of PARPs and PARG, and consequently those of PAR and ADPR, and compare the results with those obtained after genetic or chemical disruption. PMID:24705085

  12. Synthesis and SAR of novel, potent and orally bioavailable benzimidazole inhibitors of poly(ADP-ribose) polymerase (PARP) with a quaternary methylene-amino substituent.

    PubMed

    Zhu, Gui-Dong; Gandhi, Viraj B; Gong, Jianchun; Thomas, Sheela; Luo, Yan; Liu, Xuesong; Shi, Yan; Klinghofer, Vered; Johnson, Eric F; Frost, David; Donawho, Cherrie; Jarvis, Ken; Bouska, Jennifer; Marsh, Kennan C; Rosenberg, Saul H; Giranda, Vincent L; Penning, Thomas D

    2008-07-15

    Poly(ADP-ribose) polymerases (PARPs) play significant roles in various cellular functions including DNA repair and control of RNA transcription. PARP inhibitors have been demonstrated to potentiate the effect of cytotoxic agents or radiation in a number of animal tumor models. Utilizing a benzimidazole carboxamide scaffold in which the amide forms a key intramolecular hydrogen bond for optimal interaction with the enzyme, we have identified a novel series of PARP inhibitors containing a quaternary methylene-amino substituent at the C-2 position of the benzimidazole. Geminal dimethyl analogs at the methylene-amino substituent were typically more potent than mono-methyl derivatives in both intrinsic and cellular assays. Smaller cycloalkanes such as cyclopropyl or cyclobutyl were tolerated at the quaternary carbon while larger rings were detrimental to potency. In vivo efficacy data in a B16F10 murine flank melanoma model in combination with temozolomide (TMZ) are described for two optimized analogs.

  13. Design and synthesis of N-substituted indazole-3-carboxamides as poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors(†).

    PubMed

    Patel, Maulik R; Pandya, Kashyap G; Lau-Cam, Cesar A; Singh, Satyakam; Pino, Maria A; Billack, Blase; Degenhardt, Kurt; Talele, Tanaji T

    2012-04-01

    A group of novel N-1-substituted indazole-3-carboxamide derivatives were synthesized and evaluated as inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1). A structure-based design strategy was applied to a weakly active unsubstituted 1H-indazole-3-carboxamide 2, by introducing a three carbon linker between 1H-indazole-3-carboxamide and different heterocycles, and led to compounds 4 [1-(3-(piperidine-1-yl)propyl)-1H-indazole-3-carboxamide, IC(50) =36μm] and 5 [1-(3-(2,3-dioxoindolin-1-yl)propyl)-1H-indazole-3-carboxamide, IC(50) = 6.8μm]. Compound 5 was evaluated in rats for its protective action against diabetes induced by a treatment with streptozotocin, a known diabetogenic agent. In addition to preserving the ability of the pancreas to secrete insulin, compound 5 was also able to attenuate the ensuing hyperglycemic response to a significant extent.

  14. Zinc carnosine protects against hydrogen peroxide-induced DNA damage in WIL2-NS lymphoblastoid cell line independent of poly (ADP-Ribose) polymerase expression.

    PubMed

    Ooi, Theng Choon; Mohammad, Nur Hafiza; Sharif, Razinah

    2014-12-01

    The aim of this study is to investigate the ability of zinc carnosine to protect the human lymphoblastoid (WIL2-NS) cell line from hydrogen peroxide-induced DNA damage. Cells were cultured with medium containing zinc carnosine at the concentrations of 0.4, 4, 16 and 32 μM for 9 days prior to treatment with 30 μM of hydrogen peroxide (30 min). Zinc carnosine at the concentration 16 μM was optimal in protecting cells from hydrogen peroxide-induced cytotoxicity and gave the lowest percentage of apoptotic and necrotic cells. Results showed that zinc carnosine was able to induce glutathione production and protect cells from hydrogen peroxide-induced oxidative stress at all concentration and the highest protection was observed at 32-μM zinc carnosine culture. Cytokinesis-block micronucleus cytome assay showed that cells cultured with 4-32 μM of zinc carnosine showed significant reduction in micronuclei formation, nucleoplasmic bridges and nuclear bud frequencies (p < 0.05), suggesting that these concentrations maybe optimal in protecting cells from hydrogen peroxide-induced DNA damage. However, after being challenged with hydrogen peroxide, no increase in poly(ADP-ribose) polymerase expression was observed. Thus, results from this study demonstrate that zinc carnosines possess antioxidant properties and are able to reduce hydrogen peroxide-induced DNA damage in vitro independent of poly(ADP-ribose) polymerase. Further studies are warranted to understand the mechanism of protection of zinc carnosine against hydrogen peroxide-induced damage.

  15. The Angiotensin-converting enzyme inhibitor captopril inhibits poly(adp-ribose) polymerase activation and exerts beneficial effects in an ovine model of burn and smoke injury.

    PubMed

    Asmussen, Sven; Bartha, Eva; Olah, Gabor; Sbrana, Elena; Rehberg, Sebastian W; Yamamoto, Yusuke; Enkhbaatar, Perenlei; Hawkins, Hal K; Ito, Hiroshi; Cox, Robert A; Traber, Lillian D; Traber, Daniel L; Szabo, Csaba

    2011-10-01

    We investigated the effect of the angiotensin-converting enzyme (ACE) inhibitor captopril in a clinically relevant ovine model of smoke and burn injury, with special reference to oxidative stress and activation of poly(ADP-ribose) polymerase, in the lung and in circulating leukocytes. Female, adult sheep (28-40 kg) were divided into three groups. After tracheostomy and under deep anesthesia, both vehicle-control-treated (n = 5) and captopril-treated (20 mg/kg per day, i.v., starting 0.5 h before the injury) (n = 5) groups were subjected to 2 × 20%, third-degree burn injury and were insufflated with 48 breaths of cotton smoke. A sham group not receiving burn/smoke was also studied (n = 5). Animals were mechanically ventilated and fluid resuscitated for 24 h in the awake state. Burn and smoke injury resulted in an upregulation of ACE in the lung, evidenced by immunohistochemical determination and Western blotting. Burn and smoke injury resulted in pulmonary dysfunction, as well as systemic hemodynamic alterations. Captopril treatment of burn and smoke animals improved PaO2/FiO2 ratio and pulmonary shunt fraction and reduced the degree of lung edema. There was a marked increase in PAR levels in circulating leukocytes after burn/smoke injury, which was significantly decreased by captopril. The pulmonary level of ACE and the elevated pulmonary levels of transforming growth factor β in response to burn and smoke injury were significantly decreased by captopril treatment. Our results suggest that the ACE inhibitor captopril exerts beneficial effects on the pulmonary function in burn/smoke injury. The effects of the ACE inhibitor may be related to the prevention of reactive oxygen species-induced poly(ADP-ribose)polymerase overactivation. Angiotensin-converting enzyme inhibition may also exert additional beneficial effects by inhibiting the expression of the profibrotic mediator transforming growth factor β.

  16. Reduced estradiol-induced vasodilation and poly-(ADP-ribose) polymerase (PARP) activity in the aortas of rats with experimental polycystic ovary syndrome (PCOS).

    PubMed

    Masszi, Gabriella; Horvath, Eszter Maria; Tarszabo, Robert; Benko, Rita; Novak, Agnes; Buday, Anna; Tokes, Anna-Maria; Nadasy, Gyorgy L; Hamar, Peter; Benyó, Zoltán; Varbiro, Szabolcs

    2013-01-01

    Polycystic ovary syndrome (PCOS) is a complex endocrine disorder characterized by hyperandrogenism and insulin resistance, both of which have been connected to atherosclerosis. Indeed, an increased risk of clinical manifestations of arterial vascular diseases has been described in PCOS. On the other hand endothelial dysfunction can be detected early on, before atherosclerosis develops. Thus we assumed that vascular dysfunction is also related directly to the hormonal imbalance rather than to its metabolic consequences. To detect early functional changes, we applied a novel rodent model of PCOS: rats were either sham operated or hyperandrogenism was achieved by implanting subcutaneous pellets of dihydrotestosterone (DHT). After ten weeks, myograph measurements were performed on isolated aortic rings. Previously we described an increased contractility to norepinephrine (NE). Here we found a reduced immediate relaxation to estradiol treatment in pre-contracted aortic rings from hyperandrogenic rats. Although the administration of vitamin D3 along with DHT reduced responsiveness to NE, it did not restore relaxation to estradiol. Poly-(ADP-ribose) polymerase (PARP) activity was assessed by poly-ADP-ribose immunostaining. Increased PAR staining in ovaries and circulating leukocytes from DHT rats showed enhanced DNA damage, which was reduced by concomitant vitamin D3 treatment. Surprisingly, PAR staining was reduced in both the endothelium and vascular smooth muscle cells of the aorta rings from hyperandrogenic rats. Thus in the early phase of PCOS, vascular tone is already shifted towards vasoconstriction, characterized by reduced vasorelaxation and vascular dysfunction is concomitant with altered PARP activity. Based on our findings, PARP inhibitors might have a future perspective in restoring metabolic disorders in PCOS.

  17. Overview on poly(ADP-ribose) immuno-biomedicine and future prospects

    PubMed Central

    KANAI, Yoshiyuki

    2016-01-01

    Poly(ADP-ribose), identified in 1966 independently by three groups Strassbourg, Kyoto and Tokyo, is synthesized by poly(ADP-ribose) polymerases (PARP) from NAD+ as a substrate in the presence of Mg2+. The structure was unique in that it has ribose-ribose linkage. In the early-1970s, however, its function in vivo/in vitro was still controversial and the antibody against it was desired to help clear its significance. Thereupon, the author tried to produce antibody against poly(ADP-ribose) in rabbits and succeeded in it for the first time in the world. Eventually, this success has led to the following two groundbreaking papers in Nature: “Naturally-occurring antibody against poly(ADP-ribose) in patients with autoimmune disease SLE”, and “Induction of anti-poly(ADP-ribose) antibody by immunization with synthetic double-stranded RNA, poly(A)·poly(U)”. On the way to the publication of the first paper, a reviewer gave me a friendly comment that there is “heteroclitic” fashion as a mechanism of the production of natural antibody. This comment was really a God-send for me, and became a train of power for publication of another paper, as described above. Accordingly, I thought this, I would say, episode is worth describing herein. Because of its importance in biomedical phenomena, a certain number of articles related to “heteroclitic” have become to be introduced in this review, although they were not always directly related to immuno-biological works on poly(ADP-ribose). Also, I tried to speculate on the future prospects of poly(ADP-ribose), product of PARP, as an immuno-regulatory molecule, including either induced or naturally-occurring antibodies, in view of “heteroclitic”. PMID:27477457

  18. Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    PubMed Central

    Walko, Thomas D; Di Caro, Valentina; Piganelli, Jon; Billiar, Timothy R; Clark, Robert SB; Aneja, Rajesh K

    2014-01-01

    Pathophysiological conditions that lead to the release of the prototypic damage-associated molecular pattern molecule high mobility group box 1 (HMGB1) also result in activation of poly(ADP-ribose) polymerase 1 (PARP1; now known as ADP-ribosyl transferase 1 [ARTD1]). Persistent activation of PARP1 promotes energy failure and cell death. The role of poly(ADP-ribosyl)ation in HMGB1 release has been explored previously; however, PARP1 is a versatile enzyme and performs several other functions including cross-talk with another nicotinamide adenine dinucleotide- (NAD+) dependent member of the Class III histone deacetylases (HDACs), sirtuin-1 (SIRT1). Previously, it has been shown that the hyperacetylation of HMGB1 is a seminal event prior to its secretion, a process that also is dependent on HDACs. Therefore, in this study, we seek to determine if PARP1 inhibition alters LPS-mediated HMGB1 hyperacetylation and subsequent secretion due to its effect on SIRT1. We demonstrate in an in vitro model that LPS treatment leads to hyperacetylated HMGB1 with concomitant reduction in nuclear HDAC activity. Treatment with PARP1 inhibitors mitigates the LPS-mediated reduction in nuclear HDAC activity and decreases HMGB1 acetylation. By utilizing an NAD+-based mechanism, PARP1 inhibition increases the activity of SIRT1. Consequently, there is an increased nuclear retention and decreased extracellular secretion of HMGB1. We also demonstrate that PARP1 physically interacts with SIRT1. Further confirmation of this data was obtained in a murine model of sepsis, that is, administration of PJ-34, a specific PARP1 inhibitor, led to decreased serum HMGB1 concentrations in mice subjected to cecal ligation and puncture (CLP) as compared with untreated mice. In conclusion, our study provides new insights in understanding the molecular mechanisms of HMGB1 secretion in sepsis. PMID:25517228

  19. Poly(ADP-Ribose) Polymerase-1 (PARP-1) Inhibitors Reduce Reactive Gliosis and Improve Angiostatin Levels in Retina of Diabetic Rats.

    PubMed

    Guzyk, Mykhailo M; Tykhomyrov, Artem A; Nedzvetsky, Victor S; Prischepa, Irina V; Grinenko, Tatiana V; Yanitska, Lesya V; Kuchmerovska, Tamara M

    2016-10-01

    Diabetic retinopathy (DR) is a multifactorial disease characterized by reactive gliosis and disbalance of angiogenesis regulators, contributing to endothelial dysfunction and microvascular complications. This study was organized to elucidate whether poly(ADP-ribose) polymerase-1 (PARP-1) inhibition could attenuate diabetes-induced damage to macroglia and correct angiogenic disbalance in diabetic rat retina. After 8 weeks of streptozotocin (STZ)-induced diabetes, Wistar male rats were treated with PARP-1 inhibitors, nicotinamide (NAm) or 3-aminobenzamide (3-AB) (100 and 30 mg/kg/daily i.p., respectively), for 14 days. After the 10-weeks experiment period, retinas were undergone an immunohistochemical staining for glial fibrillary acidic protein (GFAP), while western blots were performed to evaluate effects of PAPR-1 inhibitors on the levels of PARP-1, poly(ADP-ribosyl)ated proteins (PARs), GFAP, and angiostatin isoforms. Diabetes induced significant up-regulation and activation of retinal PARP-1, reactive gliosis development, and GFAP overexpression compared to non-diabetic control. Moreover, extensive fragmentation of both PARP-1 and GFAP (hallmarks of apoptosis and macroglia reactivation, respectively) in diabetic retina was also observed. Levels of angiostatin isoforms were dramatically decreased in diabetic retina, sustaining aberrant pro-angiogenic condition. Both NAm and 3-AB markedly attenuated damage to macroglia, evidenced by down-regulation of PARP-1, PARs and total GFAP compared to diabetic non-treated group. PARP-1-inhibitory therapy prevented formation of PARP-1 and GFAP cleavage-derived products. In retinas of anti-PARP-treated diabetic animals, partial restoration of angiostatin's levels was shown. Therefore, PARP-1 inhibitors counteract diabetes-induced injuries and manifest retinoprotective effects, including attenuation of reactive gliosis and improvement of angiogenic status, thus, such agents could be considered as promising candidates for DR

  20. Increased transcript level of poly(ADP-ribose) polymerase (PARP-1) in human tricuspid compared with bicuspid aortic valves correlates with the stenosis severity

    SciTech Connect

    Nagy, Edit; Caidahl, Kenneth; Franco-Cereceda, Anders; Baeck, Magnus

    2012-04-13

    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, 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 valves

  1. 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.

  2. 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

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

    SciTech Connect

    Aoyagi-Scharber, Mika; Gardberg, Anna S.; Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A.

    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. 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.

  4. Ca2+-dependent generation of mitochondrial reactive oxygen species serves as a signal for poly(ADP-ribose) polymerase-1 activation during glutamate excitotoxicity

    PubMed Central

    Duan, Yuntao; Gross, Robert A; Sheu, Shey-Shing

    2007-01-01

    Mitochondrial Ca2+ uptake and poly(ADP-ribose) polymerase-1 (PARP-1) activation are both required for glutamate-induced excitotoxic neuronal death. Since activation of the glutamate receptors can induce increased levels of reactive oxygen species (ROS), we investigated the relationship of mitochondrial Ca2+ uptake and ROS generation, and the possibility that ROS increase is a required signal for PARP-1 activation in cultured striatal neurons. Based on the spatial profile of NMDA-induced ROS generation, we found that only mitochondria showed a significant ROS increase within 30 min after NMDA receptor activation. This ROS increase was inhibited by the mitochondrial complex inhibitors rotenone and oligomycin, but not by the cytosolic phospholipase A2 or xanthine oxidase inhibitors. Mitochondrial ROS generation was also inhibited by both removal of Ca2+ from extracellular medium and blockage of mitochondrial Ca2+ uptake by either a mitochondrial uncoupler or a Ca2+ uniporter inhibitor. Furthermore, both DNA damage and PARP-1 activation induced by NMDA treatment was inhibited by blocking mitochondrial Ca2+ uptake or by antioxidants. Our results demonstrate that ROS production during the early stage of acute excitotoxicity derives primarily from mitochondria and is Ca2+-dependent. More importantly, the increase of mitochondrial ROS serves as a signal for PARP-1 activation, suggesting that concomitant mitochondrial Ca2+ uptake and PARP-1 activation constitute a unified mechanism for excitotoxic neuronal death. PMID:17947304

  5. Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo.

    PubMed

    Liu, Yie; Snow, Bryan E; Kickhoefer, Valerie A; Erdmann, Natalie; Zhou, Wen; Wakeham, Andrew; Gomez, Marla; Rome, Leonard H; Harrington, Lea

    2004-06-01

    Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with exogenously expressed telomerase-associated protein 1 (TEP1) in human cells. To study the possible role of VPARP in telomerase and vault complexes in vivo, mVparp-deficient mice were generated. Mice deficient in mVparp were viable and fertile for up to five generations, with no apparent changes in telomerase activity or telomere length. Vaults purified from mVparp-deficient mouse liver appeared intact, and no defect in association with other vault components was observed. Mice deficient in mTep1, whose disruption alone does not affect telomere function but does affect the stability of vault RNA, showed no additional telomerase or telomere-related phenotypes when the mTep1 deficiency was combined with an mVparp deficiency. These data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo.

  6. Multi-targeted organometallic ruthenium(II)-arene anticancer complexes bearing inhibitors of poly(ADP-ribose) polymerase-1: A strategy to improve cytotoxicity.

    PubMed

    Wang, Zhigang; Qian, Hui; Yiu, Shek-Man; Sun, Jianwei; Zhu, Guangyu

    2014-02-01

    Small-molecule inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) have currently drawn much attention as promising chemotherapeutic drug candidates, and there is a need to develop more potent PARP inhibitors with improved bioavailability. Here we report a strategy to improve the cytotoxicity of PARP inhibitors by conjugation with organometallic ruthenium(II)-arene compounds. We also report a systematic study to reveal the mechanism of action of these ruthenium-PARP inhibitor conjugates. The complexes have been synthesized and characterized spectroscopically. The improved antiproliferative activity from the as-prepared complexes in four human cancer cell lines has indicated their potential for further development as antitumor drugs. Cellular uptake study reveals that the most active complex 3 easily entered into cells. Target validation assays show that the complexes inhibited PARP-1 slightly better than the original PARP inhibitors, that complex 3 strongly bound to DNA and inhibited transcription, and that this complex arrested the cell cycle at the G0/G1 stage. This type of information could shed light on the design of the next generation of more active ruthenium-PARP inhibitor conjugates.

  7. Potentiation of anti-cancer agent cytotoxicity by the potent poly(ADP-ribose) polymerase inhibitors NU1025 and NU1064.

    PubMed Central

    Bowman, K. J.; White, A.; Golding, B. T.; Griffin, R. J.; Curtin, N. J.

    1998-01-01

    The ability of the potent poly(ADP-ribose) polymerase (PARP) inhibitor, NU1025 (8-hydroxy-2-methyl-quinazolin-4-[3H]one) to potentiate the cytotoxicity of a panel of mechanistically diverse anti-cancer agents was evaluated in L1210 cells. NU1025 enhanced the cytotoxicity of the DNA-methylating agent MTIC, gamma-irradiation and bleomycin 3.5-, 1.4- and 2-fold respectively. The cytotoxicities of the thymidylate synthase inhibitor, nolatrexed, and the cytotoxic nucleoside, gemcitabine, were not increased. Potentiation of MTIC cytotoxicity by a delayed exposure to NU1025 was equally effective as by a simultaneous exposure to NU1025, indicating that the effects of NU1025 were mediated by an inhibition of the cellular recovery. The recovery from potentially lethal gamma-irradiation damage cytotoxicity in plateau-phase cells was also inhibited by NU1025. Investigation of DNA strand breakage and repair in gamma-irradiated cells by alkaline elution demonstrated that NU1025 caused a marked retardation of DNA repair. A structurally different PARP inhibitor, NU1064 (2-methylbenzimidazole-4-carboxamide), also potentiated the cytotoxicity of MTIC, to a similar extent to NU1025. NU1064 potentiated a sublethal concentration of a DNA methylating agent in a concentration-dependent manner. Collectively, these data suggest that the most suitable cytotoxic agents for use in combination with PARP inhibitors are methylating agents, bleomycin and ionizing radiation, but not anti-metabolites. PMID:9823965

  8. Induction of Poly(ADP-ribose) Polymerase in Mouse Bone Marrow Stromal Cells Exposed to 900 MHz Radiofrequency Fields: Preliminary Observations

    PubMed Central

    He, Qina; Sun, Yulong; Zong, Lin; Tong, Jian; Cao, Yi

    2016-01-01

    Background. Several investigators have reported increased levels of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme which plays an important role in the repair of damaged DNA, in cells exposed to extremely low dose ionizing radiation which does not cause measurable DNA damage. Objective. To examine whether exposure of the cells to nonionizing radiofrequency fields (RF) is capable of increasing messenger RNA of PARP-1 and its protein levels in mouse bone marrow stromal cells (BMSCs). Methods. BMSCs were exposed to 900 MHz RF at 120 μW/cm2 power intensity for 3 hours/day for 5 days. PARP-1 mRNA and its protein levels were examined at 0, 0.5, 1, 2, 4, 6, 8, and 10 hours after exposure using RT-PCR and Western blot analyses. Sham-exposed (SH) cells and those exposed to ionizing radiation were used as unexposed and positive control cells. Results. BMSCs exposed to RF showed significantly increased expression of PARP-1 mRNA and its protein levels after exposure to RF while such changes were not observed in SH-exposed cells. Conclusion. Nonionizing RF exposure is capable of inducing PARP-1. PMID:27190989

  9. Regulation of mitochondrial poly(ADP-Ribose) polymerase activation by the β-adrenoceptor/cAMP/protein kinase A axis during oxidative stress.

    PubMed

    Brunyanszki, Attila; Olah, Gabor; Coletta, Ciro; Szczesny, Bartosz; Szabo, Csaba

    2014-10-01

    We investigated the regulation of mitochondrial poly(ADP-ribose) polymerase 1 (PARP1) by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) system during oxidative stress in U937 monocytes. Oxidative stress induced an early (10 minutes) mitochondrial DNA damage, and concomitant activation of PARP1 in the mitochondria. These early events were followed by a progressive mitochondrial oxidant production and nuclear PARP1 activation (by 6 hours). These processes led to a functional impairment of mitochondria, culminating in cell death of mixed (necrotic/apoptotic) type. β-Adrenoceptor blockade with propranolol or inhibition of its downstream cAMP/PKA signaling attenuated, while β-adrenoceptor agonists and cAMP/PKA activators enhanced, the oxidant-mediated PARP1 activation. In the presence of cAMP, recombinant PKA directly phosphorylated recombinant PARP1 on serines 465 (in the automodification domain) and 782 and 785 (both in the catalytic domain). Inhibition of the β-adrenergic receptor/cAMP/PKA axis protected against the oxidant-mediated cell injury. Propranolol also suppressed PARP1 activation in peripheral blood leukocytes during bacterial lipopolysaccharide (LPS)-induced systemic inflammation in mice. We conclude that the activation of mitochondrial PARP1 is an early, active participant in oxidant-induced cell death, which is under the control of β-adrenoceptor/cAMP/PKA axis through the regulation of PARP1 activity by PARP1 phosphorylation.

  10. The 3′–5′ DNA Exonuclease TREX1 Directly Interacts with Poly(ADP-ribose) Polymerase-1 (PARP1) during the DNA Damage Response*

    PubMed Central

    Miyazaki, Takuya; Kim, Yong-Soo; Yoon, Jeongheon; Wang, Hongsheng; Suzuki, Teruhiko; Morse, Herbert C.

    2014-01-01

    The main function of the 3′–5′ DNA exonuclease TREX1 is to digest cytosolic single-stranded DNA to prevent activation of cell-intrinsic responses to immunostimulatory DNA. TREX1 translocates to the nucleus following DNA damage with its nuclear activities being less well defined. Although mutations in human TREX1 have been linked to autoimmune/inflammatory diseases, the mechanisms contributing to the pathogenesis of these diseases remain incompletely understood. Here, using mass spectrometry and co-immunoprecipitation assays and in vivo overexpression models, we show that TREX1 interacts with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear enzyme involved in the DNA damage response. Two zinc finger domains at the amino terminus of PARP1 were required for the interaction with TREX1 that occurs after nuclear translocation of TREX1 in response to DNA damage. Functional studies suggested that TREX1 may contribute to stabilization of PARP1 levels in the DNA damage response and its activity. These results provide new insights into the mechanisms of single-stranded DNA repair following DNA damage and alterations induced by gene mutations. PMID:25278026

  11. The Poly (ADP-Ribose) Polymerase Inhibitor Veliparib and Radiation Cause Significant Cell Line Dependent Metabolic Changes in Breast Cancer Cells

    PubMed Central

    Bhute, Vijesh J.; Ma, Yan; Bao, Xiaoping; Palecek, Sean P.

    2016-01-01

    Breast tumors are characterized into subtypes based on their surface marker expression, which affects their prognosis and treatment. Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in clinical trials, both as single agents and in combination with other chemotherapeutics, in several subtypes of breast cancer patients. Here, we used NMR-based metabolomics to probe cell line-specific effects of the PARP inhibitor Veliparib and radiation on metabolism in three breast cancer cell lines. Our data reveal several cell line-independent metabolic changes upon PARP inhibition. Pathway enrichment and topology analysis identified that nitrogen metabolism, glycine, serine and threonine metabolism, aminoacyl-tRNA biosynthesis and taurine and hypotaurine metabolism were enriched after PARP inhibition in all three breast cancer cell lines. Many metabolic changes due to radiation and PARP inhibition were cell line-dependent, highlighting the need to understand how these treatments affect cancer cell response via changes in metabolism. Finally, both PARP inhibition and radiation induced a similar metabolic responses in BRCA-mutant HCC1937 cells, but not in MCF7 and MDAMB231 cells, suggesting that radiation and PARP inhibition share similar interactions with metabolic pathways in BRCA mutant cells. Our study emphasizes the importance of differences in metabolic responses to cancer treatments in different subtypes of cancers. PMID:27811964

  12. Hydroxyurea-induced replication stress causes poly(ADP-ribose) polymerase-2 accumulation and changes its intranuclear location in root meristems of Vicia faba.

    PubMed

    Rybaczek, Dorota

    2016-07-01

    Replication stress induced by 24 and 48h exposure to 2.5mM hydroxyurea (HU) increased the activity of poly(ADP-ribose) polymerase-2 (PARP-2; EC 2.4.2.30) in root meristem cells of Vicia faba. An increase in the number of PARP-2 foci was accompanied by their delocalization from peripheral areas to the interior of the nucleus. Our results indicate that the increase in PARP-2 was connected with an increase in S139-phosphorylated H2AX histones. The findings suggest the possible role of PARP-2 in replication stress. We also confirm that the intranuclear location of PARP-2 depends on the duration of HU-induced replication stress, confirming the role of PARP-2 as an indicator of stress intensity. Finally, we conclude that the more intense the HU-mediated replication stress, the greater the probability of PARP-2 activation or H2AXS139 phosphorylation, but also the greater the chance of increasing the efficiency of repair processes and a return to normal cell cycle progression.

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

    PubMed Central

    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. PMID:26366077

  14. Effect of acute poly(ADP-ribose) polymerase inhibition by 3-AB on blood-brain barrier permeability and edema formation after focal traumatic brain injury in rats.

    PubMed

    Lescot, Thomas; Fulla-Oller, Laurence; Palmier, Bruno; Po, Christelle; Beziaud, Tiphaine; Puybasset, Louis; Plotkine, Michel; Gillet, Brigitte; Meric, Philippe; Marchand-Leroux, Catherine

    2010-06-01

    Recent evidence supports a crucial role for matrix metalloproteinase-9 (MMP-9) in blood-brain barrier (BBB) disruption and vasogenic edema formation after traumatic brain injury (TBI). Although the exact causes of MMP-9 upregulation after TBI are not fully understood, several arguments suggest a contribution of the enzyme poly(ADP-ribose)polymerase (PARP) in the neuroinflammatory response leading to MMP-9 activation. The objectives of this study were to evaluate the effect of PARP inhibition by 3-aminobenzamide (3-AB) (1) on MMP-9 upregulation and BBB integrity, (2) on edema formation as assessed by magnetic resonance imaging (MRI), (3) on neuron survival as assessed by (1)H magnetic resonance spectroscopy ((1)H-MRS), and (4) on neurological deficits at the acute phase of TBI. Western blots and zymograms showed blunting of MMP-9 upregulation 6 h after TBI. BBB permeability was decreased at the same time point in 3-AB-treated rats compared to vehicle-treated rats. Cerebral MRI showed less "free" water in 3-AB-treated than in vehicle-treated rats 6 h after TBI. MRI findings 24 h after TBI indicated predominant cytotoxic edema, and at this time point no significant differences were found between 3-AB- and vehicle-treated rats with regard to MMP-9 upregulation, BBB permeability, or MRI changes. At both 6 and 24 h, neurological function was better in the 3-AB-treated than in the vehicle-treated rats. These data suggest that PARP inhibition by 3-AB protected the BBB against hyperpermeability induced by MMP-9 upregulation, thereby decreasing vasogenic edema formation 6 h after TBI. Furthermore, our data confirm the neuroprotective effect of 3-AB at the very acute phase of TBI.

  15. Poly (ADP-ribose) polymerase (PARP) is essential for sulfur mustard-induced DNA damage repair, but has no role in DNA ligase activation.

    PubMed

    Bhat, K Ramachandra; Benton, Betty J; Ray, Radharaman

    2006-01-01

    Concurrent activation of poly (ADP-ribose) polymerase (PARP) and DNA ligase was observed in cultured human epidermal keratinocytes (HEK) exposed to the DNA alkylating compound sulfur mustard (SM), suggesting that DNA ligase activation could be due to its modification by PARP. Using HEK, intracellular 3H-labeled NAD+ (3H-adenine) was metabolically generated and then these cells were exposed to SM (1 mM). DNA ligase I isolated from these cells was not 3H-labeled, indicating that DNA ligase I is not a substrate for (ADP-ribosyl)ation by PARP. In HEK, when PARP was inhibited by 3-amino benzamide (3-AB, 2 mM), SM-activated DNA ligase had a half-life that was four-fold higher than that observed in the absence of 3-AB. These results suggest that DNA repair requires PARP, and that DNA ligase remains activated until DNA damage repair is complete. The results show that in SM-exposed HEK, DNA ligase I is activated by phosphorylation catalysed by DNA-dependent protein kinase (DNA-PK). Therefore, the role of PARP in DNA repair is other than that of DNA ligase I activation. By using the DNA ligase I phosphorylation assay and decreasing PARP chemically as well as by PARP anti-sense mRNA expression in the cells, it was confirmed that PARP does not modify DNA ligase I. In conclusion, it is proposed that PARP is essential for efficient DNA repair; however, PARP participates in DNA repair by altering the chromosomal structure to make the DNA damage site(s) accessible to the repair enzymes.

  16. Exploiting the Achilles heel of cancer: the therapeutic potential of poly(ADP-ribose) polymerase inhibitors in BRCA2-defective cancer.

    PubMed

    Kyle, S; Thomas, H D; Mitchell, J; Curtin, N J

    2008-10-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) facilitates DNA single-strand break-base excision repair to maintain genomic stability. Inhibition or loss of PARP activity leads to a recombinogenic phenotype characterized by increased sister chromatid exchange. Deficiency in homologous recombination (HR) owing to loss of BRCA1 or BRCA2 is associated with hereditary cancers of the breast, ovary, pancreas and prostate. We investigated the therapeutic potential of PARP inhibitors in HR and BRCA2-defective cells. We exposed cells defective in the HR component XRCC3 (irs1SF) and BRCA2 (V-C8) and their parental (AA8, V79) or deficiency corrected (CXR3, V-C8+B2) cells to the PARP inhibitors NU1025 and AG14361. Mice bearing BRCA2-deficient and BRCA2-proficient tumours were treated with AG14361. All HR-defective cells were hypersensitive to normally non-cytotoxic concentrations of PARP inhibitors. Cells lacking BRCA2 were 20 times more sensitive to PARP inhibitor-induced cytotoxicity. Three out of five BRCA2-defective xenografts responded to the potent PARP inhibitor, AG14361, and one tumour regressed completely, compared with non-responses in the BRCA2-proficient tumours treated with AG14361 or any mice treated with vehicle control. Untreated PARP-1(-/-) mouse embryo fibroblasts (MEFs) accumulated more DNA double-strand breaks than did PARP-1(+/+) MEFs. We believe the underlying cytotoxic mechanism is due to PARP inhibitor-mediated suppression of repair of DNA single-strand breaks, which are converted to DNA double-strand breaks at replication. These replication-associated double-strand breaks, which are normally repaired by HR, become cytotoxic in cells defective in HR. Using a DNA repair inhibitor alone to selectively kill a tumour represents an exciting new concept in cancer therapy.

  17. Oxidative DNA damage and augmentation of poly(ADP-ribose) polymerase/nuclear factor-kappa B signaling in patients with type 2 diabetes and microangiopathy.

    PubMed

    Adaikalakoteswari, Antonysunil; Rema, Mohan; Mohan, Viswanathan; Balasubramanyam, Muthuswamy

    2007-01-01

    Although oxidative stress and the subsequent DNA damage is one of the obligatory signals for poly(ADP-ribose) polymerase (PARP) activation and nuclear factor-kappa B (NFkappaB) alterations, these molecular aspects have not been collectively examined in epidemiological and clinical settings. Therefore, this study attempts to assess the oxidative DNA damage and its downstream effector signals in peripheral blood lymphocytes from Type 2 diabetes subjects without and with microangiopathy along with age-matched non-diabetic subjects. The basal DNA damage, lipid peroxidation and protein carbonyl content were significantly (p<0.05) higher in patients with and without microangiopathy compared to control subjects. Formamido Pyrimidine Glycosylase (FPG)-sensitive DNA strand breaks which represents reliable indicator of oxidative DNA damage were also significantly (p<0.001) higher in diabetic patients with (19.41+/-2.5) and without microangiopathy (16.53+/-2.0) compared to control subjects (1.38+/-0.85). Oxidative DNA damage was significantly correlated to poor glycemic control. PARP mRNA expression and PARP activity were significantly (p<0.05) increased in cells from diabetic patients with (0.31+/-0.03 densitometry units; 0.22+/-0.02PARPunits/mgprotein, respectively) and without (0.35+/-0.02; 0.42+/-0.05) microangiopathy compared to control (0.19+/-0.02; 0.11+/-0.02) subjects. Diabetic subjects with and without microangiopathy exhibited a significantly (p<0.05) higher (80%) NFkappaB binding activity compared to control subjects. In diabetic patients, FPG-sensitive DNA strand breaks correlated positively with PARP gene expression, PARP activity and NFkappaB binding activity. This study provides a comprehensive molecular evidence for increased oxidative stress and genomic instability in Type 2 diabetic subjects even prior to vascular pathology and hence reveals a window of opportunity for early therapeutic intervention.

  18. Effect of poly(ADP-ribose)polymerase and DNA topoisomerase I inhibitors on the p53/p63-dependent survival of carcinoma cells.

    PubMed

    Montariello, Daniela; Troiano, Annaelena; Di Girolamo, Daniela; Beneke, Sascha; Calabrò, Viola; Quesada, Piera

    2015-04-01

    Depending on their genetic background (p53(wt) versus p53(null)), carcinoma cells are more or less sensitive to drug-induced cell cycle arrest and/or apoptosis. Among the members of the p53 family, p63 is characterized by two N-terminal isoforms, TAp63 and ΔNp63. TAp63 isoform has p53-like functions, while ΔNp63 acts as a dominant negative inhibitor of p53. We have previously published that TAp63 is involved in poly(ADP-ribose)polymerase-1 (PARP-1) signaling of DNA damage deriving from DNA topoisomerase I (TOP I) inhibition in carcinoma cells. In the present study, we treated MCF7 breast carcinoma cells (p53(+)/ΔNp63(-)) or SCC022 (p53(-)/ΔNp63(+)) squamous carcinoma cells with the TOP I inhibitor topotecan (TPT) and the PJ34 PARP inhibitor, to compare their effects in the two different cell contexts. In MCF7 cells, we found that PJ34 addition reverts TPT-dependent PARP-1 auto-modification and triggers caspase-dependent PARP-1 proteolysis. Moreover, TPT as single agent stimulates p53(ser15) phosphorylation, p53 PARylation and occupancy of the p21WAF promoter by p53 resulting in an increase of p21WAF expression. Interestingly, PJ34 in combination with TPT enhances p53 occupancy at the BAX promoter and is associated with increased BAX protein level. In SCC022 cells, instead, TPT+PJ34 combined treatment reduces the level of the anti-apoptotic ΔNp63α protein without inducing apoptosis. Remarkably, in such cells, either exogenous p53 or TAp63 can rescue the apoptotic program in response to the treatment. All together our results suggest that in cancer cells PARP inhibitor(s) can operate in the choice between growth arrest and apoptosis by modulating p53 family-dependent signal.

  19. p63 involvement in poly(ADP-ribose) polymerase 1 signaling of topoisomerase I-dependent DNA damage in carcinoma cells.

    PubMed

    Montariello, Daniela; Troiano, Annaelena; Malanga, Maria; Calabrò, Viola; Quesada, Piera

    2013-04-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) inhibitors are thought as breakthrough for cancer treatment in solid tumors such as breast cancer through their effects on PARP's enzymatic activity. Our previous findings showed that the hydrophilic PARP inhibitor PJ34 enhances the sensitivity of p53 proficient MCF7 breast carcinoma cells to topotecan, a DNA Topoisomerase I (TOP 1) inhibitor. In the present study, we combine the classical TOP 1 poison camptothecin or its water-soluble derivative topotecan with PJ34 to investigate the potentiation of chemotherapeutic efficiency in MCF7 (p53(WT)), MDA-MB231 (p53(mut)) breast carcinoma cells and SCC022 (p53(null)) squamous carcinoma cells. We show that, following TPT-PJ34 combined treatment, MCF7 cells exhibit apoptotic death while MDA-MB231 and SCC022 cells are more resistant to these agents. Specifically, in MCF7, (i) PJ34 in combination with TPT causes a G2/M cell cycle arrest followed by massive apoptosis; (ii) PJ34 addition reverts TPT-dependent PARP-1 automodification and triggers caspase-dependent PARP-1 proteolysis; (iii) TPT, used as a single agent, stimulates p53 expression while in combination with PJ34 increases p53, TAp63α and TAp63γ protein levels with a concomitant reduction of MDM2 protein. The identification of p63 proteins as new players involved in the cancer cell response to TPT-PJ34 is relevant for a better understanding of the PARP1-dependent signaling of DNA damage. Furthermore, our data indicate that, in response to TPT-PJ34 combined chemotherapy, a functional cooperation between p53 and TAp63 proteins may occur and be essential to trigger apoptotic cell death.

  20. 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.

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

    SciTech Connect

    Mitchell, Jody; Smith, Graeme; Curtin, Nicola J.

    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 kinetics 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.

  2. 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.

  3. Effects of poly (ADP-ribose) polymerase inhibitor 3-aminobenzamide on blood-brain barrier and dopaminergic neurons of rats with lipopolysaccharide-induced Parkinson's disease.

    PubMed

    Wu, Xiao-li; Wang, Ping; Liu, Yun-hui; Xue, Yi-xue

    2014-05-01

    Neuro-inflammation and dysfunction of blood-brain barrier play an important role in the occurrence, development, and neuronal degeneration of Parkinson's disease (PD). Studies have demonstrated that a variety of cytokines such as TNF-α and IL-1β destroy the structure and function of blood-brain barrier. The damage to blood-brain barrier results in death of dopaminergic neurons, while protection of blood-brain barrier slows down the progression of PD. Also, it has been shown that activation of poly (ADP-ribose) polymerase (PARP) plays an important role in causing damage to blood-brain barrier. In addition, the PARP inhibitor 3-AB has been shown to protect blood-brain barrier from damage and has neuroprotective effects. In this study, using a lipopolysaccharide (LPS)-induced PD rat model, we investigated whether 3-AB protects blood-brain barrier and dopaminergic neurons from functional damage. LPS significantly increased Evans blue content in the substantia nigra which peaked at 12 h, while administration of 3-AB significantly inhibited the LPS-induced increase in Evans blue content and also significantly increased the expression of the tight junction-associated proteins claudin-5, occludin and ZO-1. 3-AB also increased the number of tyrosine hydroxylase positive cells and reduced the IL-1β and TNF-α content significantly. According to western blot analysis, 3-AB significantly reduced the p-ERK1/2 expression, while the expression of p-p38MAPK increased. These results suggest that 3-AB protects the blood-brain barrier from functional damage in an LPS-induced PD rat model and dopaminergic neurons are protected from degeneration by upregulation of tight junction-associated proteins. These protective effects of 3-AB may be related to modulation of the ERK1/2 pathway.

  4. The efficiency of Poly(ADP-Ribose) Polymerase (PARP) cleavage on detection of apoptosis in an experimental model of testicular torsion.

    PubMed

    Aslan Koşar, Pınar; Tuncer, Hamdi; Cihangir Uğuz, Abdülhadi; Espino Palma, Javier; Darıcı, Hakan; Onaran, İbrahim; Çiğ, Bilal; Koşar, Alim; Rodriguez Moratinos, Ana Beatriz

    2015-10-01

    The aim of this study was to evaluate the histopathological and apoptotic changes occurring in the rat ipsilateral and contralateral testes, after experimental spermatic cord torsion, and to explore and the role of poly(ADP-ribose) polymerase (PARP) cleavage in testicular torsion-detorsion injury. A total of 37 Wistar albino rats were subjected to 720° unilateral spermatic cord torsion for 1, 2 and 4 h, followed by 4-h reperfusion, or else to a sham operation (control group). Histology of the testicle was evaluated using haematoxylin-eosin (H&E) staining and Johnsen's scoring system. Germ cell apoptosis was evaluated via active caspase-3 immunostaining, and PARP expression levels were evaluated via Western blotting. The mean Johnsen's tubular biopsy scores (JTBS) of the ipsilateral testicles were lower for all torsion groups than for the controls (P < 0.05), but the JTBS of the contralateral testicles were only lower in the 4-h torsion group (P < 0.05). The mean apoptosis score (AS) of the ipsilateral and contralateral testicles was significantly higher in the torsion groups than in the sham group. AS increased correlatively with torsion time, in both testicles. The effect of testicular torsion on PARP cleavage was time dependent, with the highest effect observed after 4 h of testicular torsion (P < 0.05). Testicular torsion caused time-dependent histological changes, apoptosis and increases in PARP cleavage. Our results suggest that testicular torsion-detorsion injury caused cell damage and germ cell apoptosis that apparently involved cleavage of PARP. Increased PARP cleavage could, in turn, lead to enhanced apoptosis.

  5. Influence of 3-aminobenzamide, an inhibitor of poly(ADP-ribose)polymerase, in the evaluation of the genotoxicity of doxorubicin, cyclophosphamide and zidovudine in female mice.

    PubMed

    Yadav, L; Khan, S; Shekh, K; Jena, G B

    2014-08-01

    Testing new chemical entities for genotoxicity is an integral part of the preclinical drug-development process. Lowering the detection limit and enhancing the sensitivity of genotoxicity assays is required, as the standard test-battery fails to detect some carcinogens (non-genotoxic) and weak genotoxins. One of the mechanisms that affect the detection of weak genotoxins is related with the DNA-repair efficiency of the cell system used. In the present study, 3-aminobenzamide (3-AB, 30 mg/kg body-weight), a poly(ADP-ribose)polymerase inhibitor, was used to evaluate the DNA-damaging potential of zidovudine (AZT, 400 mg/kg bw), doxorubicin (DOX, 5 mg/kg bw) and cyclophosphamide (CP, 50 mg/kg bw, as a positive control) and sucrose (SUC, 3 g/kg bw, as a negative control) in Swiss female mice. The endpoints considered included micronucleus formation, DNA breakage (in peripheral blood lymphocytes, bone marrow and liver; comet assay) and chromosome aberrations, as well as immunohistochemistry of PARP-1 and phosphorylated histone H2AX (γ-H2AX). The results clearly indicate that the genotoxicity of zidovudine (AZT), doxorubicin (DOX) and cyclophosphamide (CP) was significantly increased in the combination treatments (3-AB+AZT, 3-AB+DOX, 3-AB+CP) as compared with the respective controls (treatment with AZT, DOX and CP alone). There was no increase in the genotoxicity per se after treatment with SUC, 3-AB or 3-AB+SUC, compared with the control (saline). Correlation analysis suggests that all genotoxicity parameters are well correlated with each other. The results clearly show that the genotoxicity of weak genotoxins can be enhanced and detected in the presence of 3-AB in mice. Thus, this approach can be used in the pre-clinical genotoxicity screening of weak genotoxins.

  6. 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.

  7. Association of poly(ADP-ribose) polymerase with the nuclear matrix: the role of intermolecular disulfide bond formation, RNA retention, and cell type.

    PubMed

    Kaufmann, S H; Brunet, G; Talbot, B; Lamarr, D; Dumas, C; Shaper, J H; Poirier, G

    1991-02-01

    The recovery of the enzyme poly(ADP-ribose) polymerase (pADPRp) in the nuclease- and 1.6 M NaCl-resistant nuclear subfraction prepared from a number of different sources was assessed by Western blotting. When rat liver nuclei were treated with DNase I and RNase A followed by 1.6 M NaCl, approximately 10% of the nuclear pADPRp was recovered in the sedimentable fraction. The proportion of pADPRp recovered with the residual fraction decreased to less than 5% of the total nuclear polymerase when nuclei were prepared in the presence of the sulfhydryl blocking reagent iodoacetamide and increased to approximately 50% of the total nuclear pADPRp when nuclei were treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to fractionation. To determine whether this effect of disulfide bond formation was unique to rat liver nuclei, nuclear matrix/cytoskeleton structures were prepared in situ by sequentially treating monolayers of tissue culture cells with Nonidet-P40, DNase I and RNase A, and 1.6 M NaCl (S.H. Kaufmann and J.H. Shaper (1991) Exp. Cell Res. 192, 511-523). When nuclear monolayers were prepared from HTC rat hepatoma cells, CaLu-1 human lung carcinoma cells, and CHO hamster ovary cells in the absence of NaTT, pADPRp was undetectable in the nuclease- and 1.6 M NaCl-resistant fraction. In contrast, when nuclear monolayers were isolated in the presence of NaTT, from 5% (CaLu-1) to 26% (HTC cells) of the total nuclear pADPRp was recovered with the nuclease- and salt-resistant fraction. Examination of these residual structures by SDS-polyacrylamide gel electrophoresis under nonreducing conditions suggested that pADPRp was present as a component of disulfide cross-linked complexes. Further analysis by immunofluorescence revealed that the pADPRp was diffusely distributed throughout the CaLu-1 or CHO nuclear matrix. In addition, when matrices were prepared in the absence of RNase A, pADPRp was also observed in the residual nucleoli. These

  8. Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion.

    PubMed

    Szabó, C; Dawson, V L

    1998-07-01

    Oxidative and nitrosative stress can trigger DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) synthetase (PARS). This enzyme has also been termed poly(ADP-ribose) polymerase (PARP) or poly(ADP-ribose) transferase (pADPRT). Rapid activation of the enzyme depletes the intracellular concentration of its substrate, nicotinamide adenine dinucleotide, thus slowing the rate of glycolysis, electron transport and subsequently ATP formation. This process can result in cell dysfunction and cell death. In this article, Csaba Szabó and Valina Dawson overview the impact of pharmacological inhibition or genetic inactivation of PARS on the course of oxidant-induced cell death in vitro, and in inflammation and reperfusion injury in vivo. A major trigger for DNA damage in pathophysiological conditions is peroxynitrite, a cytotoxic oxidant formed by the reaction between the free radicals nitric oxide and superoxide. The pharmacological inhibition of poly(ADP-ribose) synthetase is a novel approach for the experimental therapy of various forms of inflammation and shock, stroke, myocardial and intestinal ischaemia-reperfusion, and diabetes mellitus.

  9. Effects of poly (ADP-ribose) polymerase-1 (PARP-1) inhibition on sulfur mustard-induced cutaneous injuries in vitro and in vivo

    PubMed Central

    Liu, Feng; Jiang, Ning; Xiao, Zhi-yong; Cheng, Jun-ping; Mei, Yi-zhou; Zheng, Pan; Wang, Li; Zhang, Xiao-rui; Zhou, Xin-bo

    2016-01-01

    Early studies with first-generation poly (ADP-ribose) polymerase (PARP) inhibitors have already indicated some therapeutic potential for sulfur mustard (SM) injuries. The available novel and more potential PARP inhibitors, which are undergoing clinical trials as drugs for cancer treatment, bring it back to the centre of interest. However, the role of PARP-1 in SM-induced injury is not fully understood. In this study, we selected a high potent specific PARP inhibitor ABT-888 as an example to investigate the effect of PARP inhibitor in SM injury. The results showed that in both the mouse ear vesicant model (MEVM) and HaCaT cell model, PARP inhibitor ABT-888 can reduce cell damage induced by severe SM injury. ABT-888 significantly reduced SM induced edema and epidermal necrosis in MEVM. In the HaCaT cell model, ABT-888 can reduce SM-induced NAD+/ATP depletion and apoptosis/necrosis. Then, we studied the mechanism of PARP-1 in SM injury by knockdown of PARP-1 in HaCaT cells. Knockdown of PARP-1 protected cell viability and downregulated the apoptosis checkpoints, including p-JNK, p-p53, Caspase 9, Caspase 8, c-PARP and Caspase 3 following SM-induced injury. Furthermore, the activation of AKT can inhibit autophagy via the regulation of mTOR. Our results showed that SM exposure could significantly inhibit the activation of Akt/mTOR pathway. Knockdown of PARP-1 reversed the SM-induced suppression of the Akt/mTOR pathway. In summary, the results of our study indicated that the protective effects of downregulation of PARP-1 in SM injury may be due to the regulation of apoptosis, necrosis, energy crisis and autophagy. However, it should be noticed that PARP inhibitor ABT-888 further enhanced the phosphorylation of H2AX (S139) after SM exposure, which indicated that we should be very careful in the application of PARP inhibitors in SM injury treatment because of the enhancement of DNA damage. PMID:27077006

  10. No Silver Bullet – Canonical Poly(ADP-Ribose) Polymerases (PARPs) Are No Universal Factors of Abiotic and Biotic Stress Resistance of Arabidopsis thaliana

    PubMed Central

    Rissel, Dagmar; Heym, Peter P.; Thor, Kathrin; Brandt, Wolfgang; Wessjohann, Ludger A.; Peiter, Edgar

    2017-01-01

    Abiotic and biotic stress can have a detrimental impact on plant growth and productivity. Hence, there is a substantial demand for key factors of stress responses to improve yield stability of crops. Members of the poly(ADP-ribose)polymerase (PARP) protein family, which post-translationally modify (PARylate) nuclear proteins, have been suggested as such universal determinants of plant stress responses. A role under abiotic stress has been inferred from studies in which a genetic or, more commonly, pharmacological inhibition of PARP activity improved the performance of stressed plants. To further elucidate the role of PARP proteins under stress, T-DNA knockout mutants for the three Arabidopsis thaliana PARP genes were subjected to drought, osmotic, salt, and oxidative stress. To exclude a functional redundancy, which was indicated by a transcriptional upregulation of the remaining parp genes, a parp triple mutant was generated. Surprisingly, parp mutant plants did not differ from wild type plants in any of these stress experiments, independent from the number of PARP genes mutated. The parp triple mutant was also analyzed for callose formation in response to the pathogenassociated molecular pattern flg22. Unexpectedly, callose formation was unaltered in the mutant, albeit pharmacological PARP inhibition robustly blocked this immune response, confirming previous reports. Evidently, pharmacological inhibition appears to be more robust than the abolition of all PARP genes, indicating the presence of so-far undescribed proteins with PARP activity. This was supported by the finding that protein PARylation was not absent, but even increased in the parp triple mutant. Candidates for novel PARP-inhibitor targets may be found in the SRO protein family. These proteins harbor a catalytic PARP-like domain and are centrally involved in stress responses. Molecular modeling analyses, employing animal PARPs as templates, indeed indicated a capability of the SRO proteins RCD1 and

  11. 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.

  12. 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

    SciTech Connect

    Wnek, Shawn M.; Kuhlman, Christopher L.; Camarillo, Jeannie M.; Medeiros, Matthew K.; Liu, Ke J.; Lau, Serrine S.; Gandolfi, A.J.

    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-strand 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 MMA

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

    SciTech Connect

    Zhu, Yan; Li, Guodong; Dong, Yafeng; Zhou, Helen H.; Kong, Bo; Aleksunes, Lauren M.; Richardson, Jason R.; Li, Fei; Guo, Grace L.

    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 mice 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.

  14. Poly(ADP-ribose) protects vascular smooth muscle cells from oxidative DNA damage

    PubMed Central

    Zhang, Chao; Luo, Tao; Cui, Shijun; Gu, Yongquan; Bian, Chunjing; Chen, Yibin; Yu, Xiaochun; Wang, Zhonggao

    2015-01-01

    Vascular smooth muscle cells (VSMCs) undergo death during atherosclerosis, a widespread cardiovascular disease. Recent studies suggest that oxidative damage occurs in VSMCs and induces atherosclerosis. Here, we analyzed oxidative damage repair in VSMCs and found that VSMCs are hypersensitive to oxidative damage. Further analysis showed that oxidative damage repair in VSMCs is suppressed by a low level of poly (ADP-ribosyl)ation (PARylation), a key post-translational modification in oxidative damage repair. The low level of PARylation is not caused by the lack of PARP-1, the major poly(ADP-ribose) polymerase activated by oxidative damage. Instead, the expression of poly(ADP-ribose) glycohydrolase, PARG, the enzyme hydrolyzing poly(ADP-ribose), is significantly higher in VSMCs than that in the control cells. Using PARG inhibitor to suppress PARG activity facilitates oxidative damage-induced PARylation as well as DNA damage repair. Thus, our study demonstrates a novel molecular mechanism for oxidative damage-induced VSMCs death. This study also identifies the use of PARG inhibitors as a potential treatment for atherosclerosis. [BMB Reports 2015; 48(6): 354-359] PMID:25748172

  15. Poly(ADP-ribose) metabolism in brain and its role in ischemia pathology.

    PubMed

    Strosznajder, Robert Piotr; Czubowicz, Kinga; Jesko, Henryk; Strosznajder, Joanna Benigna

    2010-06-01

    The biological roles of poly(ADP-ribose) polymers (PAR) and poly(ADP-ribosyl)ation of proteins in the central nervous system are diverse. The homeostasis of PAR orchestrated by poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) is crucial for cell physiology and pathology. Both enzymes are ubiquitously distributed in neurons and glia; however, they are segregated at the subcellular level. PARP-1 serves as a "nick sensor" for single- or double-stranded breaks in DNA and is involved in long and short patch base-excision repair, while PARG breaks down PAR. The stimulation of PARP-1 and PAR formation can activate proinflammatory transcription factors, including nuclear factor kappa B. However, hyperactivation of PARP-1 can result in depletion of NAD/ATP, and in PAR-dependent mitochondrial pore formation leading to release of apoptosis inducing factor and cell death. The role of PAR as a death signaling molecule in brain ischemia-reperfusion and inflammation as well as the effect of gender and aging is presented in this review. Modulating the PAR level through pharmacological or genetic intervention on PARP-1/PARG activity and gene expression should be a valuable way for neuroprotective strategy.

  16. 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.

  17. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt.

    PubMed

    Lafon-Hughes, Laura; Vilchez Larrea, Salomé C; Kun, Alejandra; Fernández Villamil, Silvia H

    2014-01-01

    Poly-ADP-ribose (PAR) is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs) and degraded by poly-ADP-ribose-glycohydrolase (PARG). Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair). Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt). In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO). PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications.

  18. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

    PubMed Central

    Vilchez Larrea, Salomé C.; Kun, Alejandra

    2014-01-01

    Poly-ADP-ribose (PAR) is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs) and degraded by poly-ADP-ribose-glycohydrolase (PARG). Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair). Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt). In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO). PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications. PMID:25332845

  19. Noncovalent protein interaction with poly(ADP-ribose).

    PubMed

    Malanga, Maria; Althaus, Felix R

    2011-01-01

    Compared to most common posttranslational modifications of proteins, a peculiarity of poly(ADP-ribosyl)ation is the molecular heterogeneity and complexity of the reaction product, poly(ADP-ribose) (PAR). In fact, protein-bound PAR consists of variously sized (2-200 ADP-ribose residues) linear or branched molecules, negatively charged at physiological pH. It is now clear that PAR not only affects the function of the polypeptide to which it is covalently bound, but it can also influence the activity of other proteins by engaging specific noncovalent interactions. In the last 10 years, the family of PAR-binding proteins has been rapidly growing and functional studies have expanded the regulatory potential of noncovalent -protein targeting by PAR far beyond initial assumptions.In this chapter, methods are described for: (1) PAR synthesis and analysis; (2) detecting PAR-binding proteins in protein mixtures; (3) defining affinity and specificity of PAR binding to individual proteins or protein fragments; and (4) identifying PAR molecules selectively involved in the interaction.

  20. The alpha-glycosidic bonds of poly(ADP-ribose) are acid-labile.

    PubMed

    Panzeter, P L; Zweifel, B; Althaus, F R

    1992-04-15

    The poly(ADP-ribosyl)ation system of higher eukaryotes produces multiple ADP-ribose polymers of distinct sizes which exhibit different binding affinities for histones. Although precipitation with trichloroacetic acid (TCA) is the standard procedure for isolation of poly(ADP-ribose) from biological material, we show here that poly(ADP-ribose) is not stable under acidic conditions. Storage of poly(ADP-ribose) as TCA pellets results in acid hydrolysis of polymers, the extent of which is dependent on storage time and temperature. The alpha-glycosidic, inter-residue bonds are the preferred sites of attack, thus reducing polymer sizes by integral numbers of ADP-ribose to yield artefactually more and smaller polymers than originally present. Therefore, poly(ADP-ribosyl)ation studies involving TCA precipitation, histone extraction with acids, or acidic incubations of ADP-ribose polymers must account for the impact of acids on resulting polymer populations.

  1. Fermented wheat germ extract inhibits glycolysis/pentose cycle enzymes and induces apoptosis through poly(ADP-ribose) polymerase activation in Jurkat T-cell leukemia tumor cells.

    PubMed

    Comin-Anduix, Begona; Boros, Laszlo G; Marin, Silvia; Boren, Joan; Callol-Massot, Carles; Centelles, Josep J; Torres, Josep L; Agell, Neus; Bassilian, Sara; Cascante, Marta

    2002-11-29

    The fermented extract of wheat germ, trade name Avemar, is a complex mixture of biologically active molecules with potent anti-metastatic activities in various human malignancies. Here we report the effect of Avemar on Jurkat leukemia cell viability, proliferation, cell cycle distribution, apoptosis, and the activity of key glycolytic/pentose cycle enzymes that control carbon flow for nucleic acid synthesis. The cytotoxic IC(50) concentration of Avemar for Jurkat tumor cells is 0.2 mg/ml, and increasing doses of the crude powder inhibit Jurkat cell proliferation in a dose-dependent fashion. At concentrations higher than 0.2 mg/ml, Avemar inhibits cell growth by more than 50% (72 h of incubation), which is preceded by the appearance of a sub-G(1) peak on flow histograms at 48 h. Laser scanning cytometry of propidium iodide- and annexin V-stained cells indicated that the growth-inhibiting effect of Avemar was consistent with a strong induction of apoptosis. Inhibition by benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone of apoptosis but increased proteolysis of poly(ADP-ribose) indicate caspases mediate the cellular effects of Avemar. Activities of glucose-6-phosphate dehydrogenase and transketolase were inhibited in a dose-dependent fashion, which correlated with decreased (13)C incorporation and pentose cycle substrate flow into RNA ribose. This decrease in pentose cycle enzyme activities and carbon flow toward nucleic acid precursor synthesis provide the mechanistic understanding of the cell growth-controlling and apoptosis-inducing effects of fermented wheat germ. Avemar exhibits about a 50-fold higher IC(50) (10.02 mg/ml) for peripheral blood lymphocytes to induce a biological response, which provides the broad therapeutic window for this supplemental cancer treatment modality with no toxic effects.

  2. Optimization of Phenyl-Substituted Benzimidazole Carboxamide Poly(ADP-Ribose) Polymerase Inhibitors: Identification of (S)-2-(2-Fluoro-4-(pyrrolidin-2-yl)phenyl)-1H-benzimidazole-4-carboxamide (A-966492), a Highly Potent and Efficacious Inhibitor

    SciTech Connect

    Penning, Thomas D.; Zhu, Gui-Dong; Gong, Jianchun; Thomas, Sheela; Gandhi, Viraj B.; Liu, Xuesong; Shi, Yan; Klinghofer, Vered; Johnson, Eric F.; Park, Chang H.; Fry, Elizabeth H.; Donawho, Cherrie K.; Frost, David J.; Buchanan, Fritz G.; Bukofzer, Gail T.; Rodriguez, Luis E.; Bontcheva-Diaz, Velitchka; Bouska, Jennifer J.; Osterling, Donald J.; Olson, Amanda M.; Marsh, Kennan C.; Luo, Yan; Giranda, Vincent L.

    2010-06-21

    We have developed a series of phenylpyrrolidine- and phenylpiperidine-substituted benzimidazole carboxamide poly(ADP-ribose) polymerase (PARP) inhibitors with excellent PARP enzyme potency as well as single-digit nanomolar cellular potency. These efforts led to the identification of (S)-2-(2-fluoro-4-(pyrrolidin-2-yl)phenyl)-1H-benzimidazole-4-carboxamide (22b, A-966492). Compound 22b displayed excellent potency against the PARP-1 enzyme with a K{sub i} of 1 nM and an EC{sub 50} of 1 nM in a whole cell assay. In addition, 22b is orally bioavailable across multiple species, crosses the blood-brain barrier, and appears to distribute into tumor tissue. It also demonstrated good in vivo efficacy in a B16F10 subcutaneous murine melanoma model in combination with temozolomide and in an MX-1 breast cancer xenograft model both as a single agent and in combination with carboplatin.

  3. Drosophila Poly(ADP-Ribose) Glycohydrolase Mediates Chromatin Structure and SIR2-Dependent Silencing

    PubMed Central

    Tulin, Alexei; Naumova, Natalia M.; Menon, Ammini K.; Spradling, Allan C.

    2006-01-01

    Protein ADP ribosylation catalyzed by cellular poly(ADP-ribose) polymerases (PARPs) and tankyrases modulates chromatin structure, telomere elongation, DNA repair, and the transcription of genes involved in stress resistance, hormone responses, and immunity. Using Drosophila genetic tools, we characterize the expression and function of poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme responsible for degrading protein-bound ADP-ribose moieties. Strongly increasing or decreasing PARG levels mimics the effects of Parp mutation, supporting PARG's postulated roles in vivo both in removing ADP-ribose adducts and in facilitating multiple activity cycles by individual PARP molecules. PARP is largely absent from euchromatin in PARG mutants, but accumulates in large nuclear bodies that may be involved in protein recycling. Reducing the level of either PARG or the silencing protein SIR2 weakens copia transcriptional repression. In the absence of PARG, SIR2 is mislocalized and hypermodified. We propose that PARP and PARG promote chromatin silencing at least in part by regulating the localization and function of SIR2 and possibly other nuclear proteins. PMID:16219773

  4. An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells.

    PubMed

    James, Dominic I; Durant, Stephen; Eckersley, Kay; Fairweather, Emma; Griffiths, Louise A; Hamilton, Nicola; Kelly, Paul; O'Connor, Mark; Shea, Kerry; Waddell, Ian D; Ogilvie, Donald J

    2016-01-01

    After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

  5. The role of poly(ADP-ribose) in the DNA damage signaling network.

    PubMed

    Malanga, Maria; Althaus, Felix R

    2005-06-01

    DNA damage signaling is crucial for the maintenance of genome integrity. In higher eukaryotes a NAD+-dependent signal transduction mechanism has evolved to protect cells against the genome destabilizing effects of DNA strand breaks. The mechanism involves 2 nuclear enzymes that sense DNA strand breaks, poly(ADP-ribose) polymerase-1 and -2 (PARP-1 and PARP-2). When activated by DNA breaks, these PARPs use NAD+ to catalyze their automodification with negatively charged, long and branched ADP-ribose polymers. Through recruitment of specific proteins at the site of damage and regulation of their activities, these polymers may either directly participate in the repair process or coordinate repair through chromatin unfolding, cell cycle progression, and cell survival-cell death pathways. A number of proteins, including histones, DNA topoisomerases, DNA methyltransferase-1 as well as DNA damage repair and checkpoint proteins (p23, p21, DNA-PK, NF-kB, XRCC1, and others) can be targeted in this manner; the interaction involves a specific poly(ADP-ribose)-binding sequence motif of 20-26 amino acids in the target domains.

  6. Poly (ADP-ribose) glycohydrolase regulates retinoic acid receptor-mediated gene expression.

    PubMed

    Le May, Nicolas; Iltis, Izarn; Amé, Jean-Christophe; Zhovmer, Alexander; Biard, Denis; Egly, Jean-Marc; Schreiber, Valérie; Coin, Frédéric

    2012-12-14

    Poly-(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers synthesized by poly-(ADP-ribose) polymerases. Here, transcriptome profiling and differentiation assay revealed a requirement of PARG for retinoic acid receptor (RAR)-mediated transcription. Mechanistically, PARG accumulates early at promoters of RAR-responsive genes upon retinoic acid treatment to promote the formation of an appropriate chromatin environment suitable for transcription. Silencing of PARG or knockout of its enzymatic activity maintains the H3K9me2 mark at the promoter of the RAR-dependent genes, leading to the absence of preinitiation complex formation. In the absence of PARG, we found that the H3K9 demethylase KDM4D/JMJD2D became PARsylated. Mutation of two glutamic acids located in the Jumonji N domain of KDM4D inhibited PARsylation. PARG becomes dispensable for ligand-dependent transcription when either a PARP inhibitor or a non-PARsylable KDM4D/JMJD2D mutant is used. Our results define PARG as a coactivator regulating chromatin remodeling during RA-dependent gene expression.

  7. Thrombin or Ca(++)-ionophore-mediated fall in endothelial ATP levels independent of poly(ADP-Ribose) polymerase activity and NAD levels--comparison with the effects of hydrogen peroxide.

    PubMed

    Halldórsson, Haraldur; Thors, Brynhildur; Thorgeirsson, Gudmundur

    2015-01-01

    To test the hypothesis that a fall in cellular ATP following stimulation of endothelial cells with thrombin is secondary to a decrease in NAD levels caused by poly(ADP-Ribose)polymerase (PARP), we measured the levels of NAD and ATP in endothelial cells after treatment with thrombin, the Ca(++)-ionophore A23187, or hydrogen peroxide (H2O2), and compared the effects of inhibitors of PARP, NAD synthesis, and ADP-ribose breakdown on these responses. Neither thrombin nor A23187 caused a reduction in endothelial NAD levels and A23187 affected ATP levels independently of NAD levels or PARP activity. H2O2 induced lowering of NAD caused modest lowering of ATP but marked additional ATP-lowering, independent of PARP and NAD, was also demonstrated. We conclude that in endothelial cells ATP levels are largely independent of NAD and PARP, which do not play a role in thrombin or Ca(++)-ionophore-mediated lowering of ATP. H2O2 caused ATP lowering through a similar mechanism as thrombin and A23187 but, additionally, caused a further ATP lowering through its intense stimulation of PARP and marked lowering of NAD.

  8. Resveratrol inhibits inflammatory signaling implicated in ionizing radiation-induced premature ovarian failure through antagonistic crosstalk between silencing information regulator 1 (SIRT1) and poly(ADP-ribose) polymerase 1 (PARP-1).

    PubMed

    Said, Riham Soliman; El-Demerdash, Ebtehal; Nada, Ahmed Shafik; Kamal, Mohamed M

    2016-03-01

    This study hypothesized that resveratrol, a silencing information regulator 1 (SIRT1) activator, would counteract the inflammatory signaling associated with radiotherapy-induced premature ovarian failure (POF). Immature female Sprague-Dawley rats were subjected to a single dose of γ-radiation to induce POF and treated with resveratrol (25mg/kg) once daily for two weeks before and three days post irradiation. Resveratrol preserves the entire ovarian follicle pool manifested by increasing serum anti-Müllerian hormone (AMH) levels. Radiation triggered inflammatory process in the ovary through enhanced NF-κB and poly(ADP-ribose) polymerase (PARP)-1 expression which convinced the expression of inflammatory markers including IL-6, IL-8, and visfatin mRNA levels, as well as inducible nitric oxide synthase and cyclooxygenase-2 protein expression with a concomitant reduction in IL-10 mRNA levels. Resveratrol significantly counteracted the effect of radiation and upregulated the gene expression of peroxisome proliferator-activated receptor γ (PPAR-γ) and SIRT1. Resveratrol-activated SIRT1 expression was associated with inhibition of PARP-1 and NF-κB expression-mediated inflammatory cytokines. Our findings suggest that resveratrol restored ovarian function through increasing AMH levels, and diminishing ovarian inflammation, predominantly via upregulation of PPAR-γ and SIRT1 expression leading to inhibition of NF-κB provoked inflammatory cytokines.

  9. 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

  10. Chromosomal damage and micronucleus induction by MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor: Evidence for a non-DNA-reactive mode of action.

    PubMed

    Yamamura, Eiji; Muto, Shigeharu; Yamada, Katsuya; Sato, Yuko; Iwase, Yumiko; Uno, Yoshifumi

    2015-04-01

    MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that competes with the binding of the PARP substrate nicotinamide adenine dinucleotide (NAD), is being developed as a neuroprotective agent against acute ischemic stroke. MP-124 increased structural chromosomal aberration in CHL/IU cells, but showed negative results in the bacterial reverse mutation test, and the rat bone marrow micronucleus (MN) and the rat liver unscheduled DNA synthesis tests after the intravenous bolus injection. Thus, MP-124 did not appear to be direct-acting mutagen. Since, PARP-1 is a key enzyme in DNA repair, the effect of continuous PARP-1 inhibition by MP-124 was further examined in the rat MN test under 24-h intravenous infusion, and an increase in micronucleated immature erythrocytes (MNIE) was observed. The increase was clearly reduced by co-treatment with nicotinic acid, which resulted in increased intracellular NAD levels. This is consistent with the established activity of MP-124 as a competitive inhibitor of PARP and provides strong evidence that the DNA-damaging effect that leads to the increase in MNIE is a secondary effect of PARP-1 inhibition. This mechanism is expected to result in a threshold for the induction of MNIE by MP-124, and allows for the establishment of a safe margin of exposure for the therapeutic use of MP-124.

  11. Phase I, Dose-Escalation, 2-Part Trial of Poly(ADP-Ribose) Polymerase Inhibitor Talazoparib in Patients with Advanced Germline BRCA1/2 Mutations and Selected Sporadic Cancers.

    PubMed

    de Bono, Johann; Ramanathan, Ramesh K; Mina, Lida; Chugh, Rashmi; Glaspy, John; Rafii, Saeed; Kaye, Stan; Sachdev, Jasgit; Heymach, John; Smith, David C; Henshaw, Joshua W; Herriott, Ashleigh; Patterson, Miranda; Curtin, Nicola J; Byers, Lauren Averett; Wainberg, Zev A

    2017-02-27

    Talazoparib inhibits poly(ADP-ribose) polymerase (PARP) catalytic activity, trapping PARP1 on damaged DNA and causing cell death in BRCA1/2-mutated cells. We evaluated talazoparib therapy in this 2-part, phase I, first-in-human trial. Antitumor activity, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of once-daily talazoparib were determined in an open-label, multicenter, dose-escalation study (NCT01286987). The MTD was 1.0 mg/day, with an elimination half-life of 50 hours. Treatment-related adverse events included fatigue (26/71 patients; 37%) and anemia (25/71 patients; 35%). Grade 3 to 4 adverse events included anemia (17/71 patients; 24%) and thrombocytopenia (13/71 patients; 18%). Sustained PARP inhibition was observed at doses ≥0.60 mg/day. At 1.0 mg/day, confirmed responses were observed in 7/14 (50%) and 5/12 (42%) patients with BRCA mutation-associated breast and ovarian cancers, respectively, and in patients with pancreatic and small cell lung cancer. Talazoparib demonstrated single-agent antitumor activity and was well tolerated in patients at the recommended dose of 1.0 mg/day.

  12. Poly(ADP-ribose): Structure, Physicochemical Properties and Quantification In Vivo, with Special Reference to Poly(ADP-ribose) Binding Protein Modules.

    PubMed

    Miwa, Masanao; Ida, Chieri; Yamashita, Sachiko; Tanaka, Masakazu; Fujisawa, Junichi

    2016-01-01

    PolyADP-ribosylation is a unique posttranslational modification of proteins, involved in various cellular functions including stability of chromatin. PolyADP-ribosylation modifies acceptor proteins with a large negatively charged poly(ADP-ribose) (PAR) to greatly change the structure and function of the acceptor proteins. In addition various specific motifs of proteins were recently found to interact non-covalently with PAR thereby changing the spaciotemporal activity of protein-protein interaction in cells. However, the structure of PAR to which specific protein motifs should bind is not fully characterized. The present work will review the structure, physicochemical properties and quantification of PAR in vivo, with special reference to PAR binding protein modules.

  13. Poly(ADP) ribose polymerase-1 ablation alters eicosanoid and docosanoid signaling and metabolism in a murine model of contact hypersensitivity.

    PubMed

    Kiss, Borbála; Szántó, Magdolna; Szklenár, Mónika; Brunyánszki, Attila; Marosvölgyi, Tamás; Sárosi, Eszter; Remenyik, Éva; Gergely, Pál; Virág, László; Decsi, Tamás; Rühl, Ralph; Bai, Peter

    2015-04-01

    Poly(ADP‑ribose) polymerase (PARP)‑1 is a pro‑inflammatory protein. The inhibition of PARP‑1 reduces the activity of numerous pro‑inflammatory transcription factors, which results in the reduced production of pro‑inflammatory cytokines, chemokines, matrix metalloproteinases and inducible nitric oxide synthase, culminating in reduced inflammation of the skin and other organs. The aim of the present study was to investigate the effects of the deletion of PARP‑1 expression on polyunsaturated fatty acids (PUFA), and PUFA metabolite composition, in mice under control conditions or undergoing an oxazolone (OXA)‑induced contact hypersensitivity reaction (CHS). CHS was elicited using OXA in both the PARP‑1+/+ and PARP‑1/ mice, and the concentration of PUFAs and PUFA metabolites in the diseased skin were assessed using lipidomics experiments. The levels of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were shown to be increased in the PARP‑1/ mice, as compared with the control, unsensitized PARP‑1+/+ mice. In addition, higher expression levels of fatty acid binding protein 7 (FABP7) were detected in the PARP‑1/ mice. FABP7 is considered to be a specific carrier of DHA and EPA. Furthermore, the levels of the metabolites of DHA and EPA (considered mainly as anti‑inflammatory or pro‑resolving factors) were higher, as compared with the metabolites of arachidonic acid (considered mainly pro‑inflammatory), both in the unsensitized control and OXA‑sensitized PARP‑1/ mice. The results of the present study suggest that the genetic deletion of PARP‑1 may affect the PUFA‑homeostasis of the skin, resulting in an anti‑inflammatory milieu, including increased DHA and EPA levels, and DHA and EPA metabolite levels. This may be an important component of the anti‑inflammatory action of PARP‑1 inhibition.

  14. Common and unique genetic interactions of the poly(ADP-ribose) polymerases PARP1 and PARP2 with DNA double-strand break repair pathways.

    PubMed

    Ghosh, Rajib; Roy, Sanchita; Kamyab, Johan; Dantzer, Francoise; Franco, Sonia

    2016-09-01

    In mammalian cells, chromatin poly(ADP-ribos)ylation (PARylation) at sites of DNA Double-Strand Breaks (DSBs) is mediated by two highly related enzymes, PARP1 and PARP2. However, enzyme-specific genetic interactions with other DSB repair factors remain largely undefined. In this context, it was previously shown that mice lacking PARP1 and H2AX, a histone variant that promotes DSB repair throughout the cell cycle, or the core nonhomologous end-joining (NHEJ) factor Ku80 are not viable, while mice lacking PARP1 and the noncore NHEJ factor DNA-PKcs are severely growth retarded and markedly lymphoma-prone. Here, we have examined the requirement for PARP2 in these backgrounds. We find that, like PARP1, PARP2 is essential for viability in mice lacking H2AX. Moreover, treatment of H2AX-deficient primary fibroblasts or B lymphocytes with PARP inhibitors leads to activation of the G2/M checkpoint and accumulation of chromatid-type breaks in a lineage- and gene-dose dependent manner. In marked contrast to PARP1, loss of PARP2 does not result in additional phenotypes in growth, development or tumorigenesis in mice lacking either Ku80 or DNA-PKcs. Altogether these findings highlight specific nonoverlapping functions of PARP1 and PARP2 at H2AX-deficient chromatin during replicative phases of the cell cycle and uncover a unique requirement for PARP1 in NHEJ-deficient cells.

  15. Poly(ADP-ribose) binds to the splicing factor ASF/SF2 and regulates its phosphorylation by DNA topoisomerase I.

    PubMed

    Malanga, Maria; Czubaty, Alicja; Girstun, Agnieszka; Staron, Krzysztof; Althaus, Felix R

    2008-07-18

    Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SR-protein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression.

  16. Investigation of the action of poly(ADP-ribose)-synthesising enzymes on NAD+ analogues

    PubMed Central

    Wallrodt, Sarah; Simpson, Edward L

    2017-01-01

    ADP-ribosyl transferases with diphtheria toxin homology (ARTDs) catalyse the covalent addition of ADP-ribose onto different acceptors forming mono- or poly(ADP-ribos)ylated proteins. Out of the 18 members identified, only four are known to synthesise the complex poly(ADP-ribose) biopolymer. The investigation of this posttranslational modification is important due to its involvement in cancer and other diseases. Lately, metabolic labelling approaches comprising different reporter-modified NAD+ building blocks have stimulated and enriched proteomic studies and imaging applications of ADP-ribosylation processes. Herein, we compare the substrate scope and applicability of different NAD+ analogues for the investigation of the polymer-synthesising enzymes ARTD1, ARTD2, ARTD5 and ARTD6. By varying the site and size of the NAD+ modification, suitable probes were identified for each enzyme. This report provides guidelines for choosing analogues for studying poly(ADP-ribose)-synthesising enzymes. PMID:28382184

  17. Advanced oxidation protein products induce intestine epithelial cell death through a redox-dependent, c-jun N-terminal kinase and poly (ADP-ribose) polymerase-1-mediated pathway.

    PubMed

    Xie, F; Sun, S; Xu, A; Zheng, S; Xue, M; Wu, P; Zeng, J H; Bai, L

    2014-01-16

    Advanced oxidation protein products (AOPPs), a novel protein marker of oxidative damage, have been confirmed to accumulate in patients with inflammatory bowel disease (IBD), as well as those with diabetes and chronic kidney disease. However, the role of AOPPs in the intestinal epithelium remains unclear. This study was designed to investigate whether AOPPs have an effect on intestinal epithelial cell (IEC) death and intestinal injury. Immortalized rat intestinal epithelial (IEC-6) cells and normal Sprague Dawley rats were treated with AOPP-albumin prepared by incubation of rat serum albumin (RSA) with hypochlorous acid. Epithelial cell death, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit activity, reactive oxygen species (ROS) generation, apoptosis-related protein expression, and c-jun N-terminal kinase (JNK) phosphorylation were detected both in vivo and in vitro. In addition, we measured AOPPs deposition and IEC death in 23 subjects with Crohn's disease (CD). Extracellular AOPP-RSA accumulation induced apoptosis in IEC-6 cultures. The triggering effect of AOPPs was mainly mediated by a redox-dependent pathway, including NADPH oxidase-derived ROS generation, JNK phosphorylation, and poly (ADP-ribose) polymerase-1 (PARP-1) activation. Chronic AOPP-RSA administration to normal rats resulted in AOPPs deposition in the villous epithelial cells and in inflammatory cells in the lamina propria. These changes were companied with IEC death, inflammatory cellular infiltration, and intestinal injury. Both cell death and intestinal injury were ameliorated by chronic treatment with apocynin. Furthermore, AOPPs deposition was also observed in IECs and inflammatory cells in the lamina propria of patients with CD. The high immunoreactive score of AOPPs showed increased apoptosis. Our results demonstrate that AOPPs trigger IEC death and intestinal tissue injury via a redox-mediated pathway. These data suggest that AOPPs may represent a novel pathogenic factor

  18. Poly(ADP-Ribose)Polymerase 1 (PARP-1) Activation and Ca(2+) Permeable α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid (AMPA) Channels in Post-Ischemic Brain Damage: New Therapeutic Opportunities?

    PubMed

    Gerace, Elisabetta; Pellegrini-Giampietro, Domenico E; Moroni, Flavio; Mannaioni, Guido

    2015-01-01

    A significant number of laboratories observed that poly (ADP-ribose) polymerase (PARP) inhibitors, administered a few hours after ischemic or traumatic brain injury, may drastically reduce the subsequent neurological damage. It has also been shown that PARP inhibitors, administered for 24 hours to rats with permanent middle cerebral artery occlusion (MCAO), may reduce the number of dying neurons for a long period after surgery, thus suggesting that these agents could reduce the delayed brain damage and the neurological and cognitive impairment (dementia) frequently observed a few months after a stroke. In organotypic hippocampal slices exposed to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG), an alkylating agent able to activate PARP, a selective and delayed degeneration of the CA1 pyramidal cells which was anatomically similar to that observed after a short period of oxygen and glucose deprivation (OGD) has been described. Biochemical and electrophysiological approaches showed that MNNG exposure caused an increased expression and function of the calcium permeable α-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) channels in the CA1 but not in the CA3 hippocampal region. PARP inhibitors prevented this increase and reduced CA1 cell death. The AMPA receptor antagonist 2,3-dihydroxy-6- nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione or the selective Ca(2+) permeable AMPA channel blocker 1-Naphthyl acetyl spermine (NASPM), also reduced the MNNG-induced CA1 pyramidal cell death. Since activation of PARP-1 facilitate the expression of Ca(2+) permeable channels and the subsequent delayed cell death, PARP inhibitors administered a few hours after a stroke may not only reduce the early post-ischemic brain damage but also the late neuronal death frequently occurring after severe stroke.

  19. ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase.

    PubMed Central

    Vaziri, H; West, M D; Allsopp, R C; Davison, T S; Wu, Y S; Arrowsmith, C H; Poirier, G G; Benchimol, S

    1997-01-01

    Telomere loss has been proposed as a mechanism for counting cell divisions during aging in normal somatic cells. How such a mitotic clock initiates the intracellular signalling events that culminate in G1 cell cycle arrest and senescence to restrict the lifespan of normal human cells is not known. We investigated the possibility that critically short telomere length activates a DNA damage response pathway involving p53 and p21(WAF1) in aging cells. We show that the DNA binding and transcriptional activity of p53 protein increases with cell age in the absence of any marked increase in the level of p53 protein, and that p21(WAF1) promoter activity in senescent cells is dependent on both p53 and the transcriptional co-activator p300. Moreover, we detected increased specific activity of p53 protein in AT fibroblasts, which exhibit accelerated telomere loss and undergo premature senescence, compared with normal fibroblasts. We investigated the possibility that poly(ADP-ribose) polymerase is involved in the post-translational activation of p53 protein in aging cells. We show that p53 protein can associate with PARP and inhibition of PARP activity leads to abrogation of p21 and mdm2 expression in response to DNA damage. Moreover, inhibition of PARP activity leads to extension of cellular lifespan. In contrast, hyperoxia, an activator of PARP, is associated with accelerated telomere loss, activation of p53 and premature senescence. We propose that p53 is post-translationally activated not only in response to DNA damage but also in response to the critical shortening of telomeres that occurs during cellular aging. PMID:9312059

  20. The expanding role of poly(ADP-ribose) metabolism: current challenges and new perspectives.

    PubMed

    Gagné, Jean-Philippe; Hendzel, Michael J; Droit, Arnaud; Poirier, Guy G

    2006-04-01

    Recent discoveries have resulted in significant breakthroughs in the understanding of PARPs and PARG functions within a broad range of cellular processes. The novel and sometimes unexpected pathways that are regulated by poly(ADP-ribosylation) bring new questions and hypotheses, some of them being contentious. In this review, we highlight current areas of investigation such as the clinical potential of PARP and PARG inhibitors and the important mitotic regulatory functions of poly(ADP-ribose) in cell-cycle progression, a recent discovery that has broadened our knowledge regarding poly(ADP-ribose) functions. A special emphasis is placed on recent advances in relation to PARG that are stimulating new directions in future research. Noticeably, the existence of various PARG isoforms characterized by distinct cellular localizations and nucleocytoplasmic shuttling properties challenges our current comprehension of pADPr metabolism. Observations and suppositions towards functionally important regulatory elements in the N-terminal portion of PARG are also discussed.

  1. An affinity matrix for the purification of poly(ADP-ribose) glycohydrolase.

    PubMed Central

    Thomassin, H; Jacobson, M K; Guay, J; Verreault, A; Aboul-ela, N; Menard, L; Poirier, G G

    1990-01-01

    The preparation of quantities of poly(ADP-ribose) glycohydrolase sufficient for detailed structural and enzymatic characterizations has been difficult due to the very low tissue content of the enzyme and its lability in late stages of purification. To date, the only purification of this enzyme to apparent homogeneity has involved a procedure requiring 6 column chromatographic steps. Described here is the preparation of an affinity matrix which consists of ADP-ribose polymers bound to dihydroxyboronyl sepharose. An application is described for the purification of poly(ADP-ribose) glycohydrolase from calf thymus in which a single rapid affinity step was used to replace 3 column chromatographic steps yielding enzyme of greater than 90% purity with a 3 fold increase in yield. This matrix should also prove useful for other studies of ADP-ribose polymer metabolism and related clinical conditions. Images PMID:2395636

  2. On PAR with PARP: cellular stress signaling through poly(ADP-ribose) and PARP-1

    PubMed Central

    Luo, Xin; Kraus, W. Lee

    2012-01-01

    Cellular stress responses are mediated through a series of regulatory processes that occur at the genomic, transcriptional, post-transcriptional, translational, and post-translational levels. These responses require a complex network of sensors and effectors from multiple signaling pathways, including the abundant and ubiquitous nuclear enzyme poly(ADP-ribose) (PAR) polymerase-1 (PARP-1). PARP-1 functions at the center of cellular stress responses, where it processes diverse signals and, in response, directs cells to specific fates (e.g., DNA repair vs. cell death) based on the type and strength of the stress stimulus. Many of PARP-1's functions in stress response pathways are mediated by its regulated synthesis of PAR, a negatively charged polymer, using NAD+ as a donor of ADP-ribose units. Thus, PARP-1's functions are intimately tied to nuclear NAD+ metabolism and the broader metabolic profile of the cell. Recent studies in cell and animal models have highlighted the roles of PARP-1 and PAR in the response to a wide variety of extrinsic and intrinsic stress signals, including those initiated by oxidative, nitrosative, genotoxic, oncogenic, thermal, inflammatory, and metabolic stresses. These responses underlie pathological conditions, including cancer, inflammation-related diseases, and metabolic dysregulation. The development of PARP inhibitors is being pursued as a therapeutic approach to these conditions. In this review, we highlight the newest findings about PARP-1's role in stress responses in the context of the historical data. PMID:22391446

  3. Pyridine nucleotide cycling and control of intracellular redox state in relation to poly (ADP-ribose) polymerase activity and nuclear localization of glutathione during exponential growth of Arabidopsis cells in culture.

    PubMed

    Pellny, Till K; Locato, Vittoria; Vivancos, Pedro Diaz; Markovic, Jelena; De Gara, Laura; Pallardó, Federico V; Foyer, Christine H

    2009-05-01

    Pyridine nucleotides, ascorbate and glutathione are major redox metabolites in plant cells, with specific roles in cellular redox homeostasis and the regulation of the cell cycle. However, the regulation of these metabolite pools during exponential growth and their precise functions in the cell cycle remain to be characterized. The present analysis of the abundance of ascorbate, glutathione, and pyridine nucleotides during exponential growth of Arabidopsis cells in culture provides evidence for the differential regulation of each of these redox pools. Ascorbate was most abundant early in the growth cycle, but glutathione was low at this point. The cellular ascorbate to dehydroascorbate and reduced glutathione (GSH) to glutathione disulphide ratios were high and constant but the pyridine nucleotide pools were largely oxidized over the period of exponential growth and only became more reduced once growth had ceased. The glutathione pool increased in parallel with poly (ADP-ribose) polymerase (PARP) activities and with increases in the abundance of PARP1 and PARP2 mRNAs at a time of high cell cycle activity as indicated by transcriptome information. Marked changes in the intracellular partitioning of GSH between the cytoplasm and nucleus were observed. Extension of the exponential growth phase by dilution or changing the media led to increases in the glutathione and nicotinamide adenine dinucleotide, oxidized form (NAD)-plus-nicotinamide adenine dinucleotide, reduced form (NADH) pools and to higher NAD/NADH ratios but the nicotinamide adenine dinucleotide phosphate, oxidized form (NADP)-plus-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) pool sizes, and NAPD/NADPH ratios were much less affected. The ascorbate, glutathione, and pyridine nucleotide pools and PARP activity decreased before the exponential growth phase ended. We conclude that there are marked changes in intracellular redox state during the growth cycle but that redox homeostasis is

  4. Drug repurposing screen identifies lestaurtinib amplifies the ability of the poly (ADP-ribose) polymerase 1 inhibitor AG14361 to kill breast cancer associated gene-1 mutant and wild type breast cancer cells

    PubMed Central

    2014-01-01

    Introduction Breast cancer is a devastating disease that results in approximately 40,000 deaths each year in the USA. Current drug screening and chemopreventatitive methods are suboptimal, due in part to the poor specificity of compounds for cancer cells. Poly (ADP-ribose) polymerase 1 (PARP1) inhibitor (PARPi)-mediated therapy is a promising approach for familial breast cancers caused by mutations of breast cancer-associated gene-1 and -2 (BRCA1/2), yet drug resistance frequently occurs during the treatment. Moreover, PARPis exhibit very little effect on cancers that are proficient for DNA repair and clinical efficacy for PARPis as single-agent therapies has yet to be illustrated. Methods Using a quantitative high-throughput screening approach, we screened a library containing 2,816 drugs, most of which are approved for human or animal use by the Food and Drug Administration (FDA) or other countries, to identify compounds that sensitize breast cancer cells to PARPi. After initial screening, we performed further cellular and molecular analysis on lestaurtinib, which is an orally bioavailable multikinase inhibitor and has been used in clinical trials for myeloproliferative disorders and acute myelogenous leukemia. Results Our study indicated that lestaurtinib is highly potent against breast cancers as a mono-treatment agent. It also strongly enhanced the activity of the potent PARPi AG14361 on breast cancer cell growth both in vitro and in vivo conditions. The inhibition of cancer growth is measured by increased apoptosis and reduced cell proliferation. Consistent with this, the treatment results in activation of caspase 3/7, and accumulation of cells in the G2 phase of the cell cycle, irrespective of their BRCA1 status. Finally, we demonstrated that AG14361 inhibits NF-κB signaling, which is further enhanced by lestaurtinib treatment. Conclusions Lestaurtinib amplifies the ability of the PARP1 inhibitor AG14361 to kill BRCA1 mutant and wild-type breast cancer

  5. Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities

    PubMed Central

    Barkauskaite, Eva; Brassington, Amy; Tan, Edwin S.; Warwicker, Jim; Dunstan, Mark S.; Banos, Benito; Lafite, Pierre; Ahel, Marijan; Mitchison, Timothy J.; Ahel, Ivan; Leys, David

    2013-01-01

    Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios. PMID:23917065

  6. Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

    PubMed Central

    Vilchez Larrea, Salomé C.; Schlesinger, Mariana; Kevorkian, María L.; Flawiá, Mirtha M.; Alonso, Guillermo D.; Fernández Villamil, Silvia H.

    2013-01-01

    Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease. PMID:23776710

  7. Host cell poly(ADP-ribose) glycohydrolase is crucial for Trypanosoma cruzi infection cycle.

    PubMed

    Vilchez Larrea, Salomé C; Schlesinger, Mariana; Kevorkian, María L; Flawiá, Mirtha M; Alonso, Guillermo D; Fernández Villamil, Silvia H

    2013-01-01

    Trypanosoma cruzi, etiological agent of Chagas' disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas' disease.

  8. Poly(ADP-Ribose) Glycohydrolase (PARG) Silencing Suppresses Benzo(a)pyrene Induced Cell Transformation

    PubMed Central

    Huang, Peiwu; Zhuang, Zhixiong; Liu, Jianjun; Gao, Wei; Liu, Yinpin; Huang, Haiyan

    2016-01-01

    Benzo(a)pyrene (BaP) is a ubiquitously distributed environmental pollutant and known carcinogen, which can induce malignant transformation in rodent and human cells. Poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme that catalyzes the degradation of poly(ADP-ribose) (PAR), has been known to play an important role in regulating DNA damage repair and maintaining genomic stability. Although PARG has been shown to be a downstream effector of BaP, the role of PARG in BaP induced carcinogenesis remains unclear. In this study, we used the PARG-deficient human bronchial epithelial cell line (shPARG) as a model to examine how PARG contributed to the carcinogenesis induced by chronic BaP exposure under various concentrations (0, 10, 20 and 40 μM). Our results showed that PARG silencing dramatically reduced DNA damages, chromosome abnormalities, and micronuclei formations in the PARG-deficient human bronchial epithelial cells compared to the control cells (16HBE cells). Meanwhile, the wound healing assay showed that PARG silencing significantly inhibited BaP-induced cell migration. Furthermore, silencing of PARG significantly reduced the volume and weight of tumors in Balb/c nude mice injected with BaP induced transformed human bronchial epithelial cells. This was the first study that reported evidences to support an oncogenic role of PARG in BaP induced carcinogenesis, which provided a new perspective for our understanding in BaP exposure induced cancer. PMID:27003318

  9. Two small enzyme isoforms mediate mammalian mitochondrial poly(ADP-ribose) glycohydrolase (PARG) activity

    SciTech Connect

    Meyer, Ralph G. . E-mail: meyerg@vet.upenn.edu; Meyer-Ficca, Mirella L.; Whatcott, Clifford J.; Jacobson, Elaine L.; Jacobson, Myron K.

    2007-08-01

    Poly(ADP-ribose)glycohydrolase (PARG) is the major enzyme capable of rapidly hydrolyzing poly(ADP-ribose) (PAR) formed by the diverse members of the PARP enzyme family. This study presents an alternative splice mechanism by which two novel PARG protein isoforms of 60 kDa and 55 kDa are expressed from the human PARG gene, termed hPARG60 and hPARG55, respectively. Homologous forms were found in the mouse (mPARG63 and mPARG58) supporting the hypothesis that expression of small PARG isoforms is conserved among mammals. A PARG protein of {approx} 60 kDa has been described for decades but with its genetic basis unknown, it was hypothesized to be a product of posttranslational cleavage of larger PARG isoforms. While this is not excluded entirely, isolation and expression of cDNA clones from different sources of RNA indicate that alternative splicing leads to expression of a catalytically active hPARG60 in multiple cell compartments. A second enzyme, hPARG55, that can be expressed through alternative translation initiation from hPARG60 transcripts is strictly targeted to the mitochondria. Functional studies of a mitochondrial targeting signal (MTS) in PARG exon IV suggest that hPARG60 may be capable of shuttling between nucleus and mitochondria, which would be in line with a proposed function of PAR in genotoxic stress-dependent, nuclear-mitochondrial crosstalk.

  10. Readers of poly(ADP-ribose): designed to be fit for purpose

    PubMed Central

    Teloni, Federico; Altmeyer, Matthias

    2016-01-01

    Post-translational modifications (PTMs) regulate many aspects of protein function and are indispensable for the spatio-temporal regulation of cellular processes. The proteome-wide identification of PTM targets has made significant progress in recent years, as has the characterization of their writers, readers, modifiers and erasers. One of the most elusive PTMs is poly(ADP-ribosyl)ation (PARylation), a nucleic acid-like PTM involved in chromatin dynamics, genome stability maintenance, transcription, cell metabolism and development. In this article, we provide an overview on our current understanding of the writers of this modification and their targets, as well as the enzymes that degrade and thereby modify and erase poly(ADP-ribose) (PAR). Since many cellular functions of PARylation are exerted through dynamic interactions of PAR-binding proteins with PAR, we discuss the readers of this modification and provide a synthesis of recent findings, which suggest that multiple structurally highly diverse reader modules, ranging from completely folded PAR-binding domains to intrinsically disordered sequence stretches, evolved as PAR effectors to carry out specific cellular functions. PMID:26673700

  11. Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death.

    PubMed

    Blenn, Christian; Althaus, Felix R; Malanga, Maria

    2006-06-15

    PAR [poly(ADP-ribose)] is a structural and regulatory component of multiprotein complexes in eukaryotic cells. PAR catabolism is accelerated under genotoxic stress conditions and this is largely attributable to the activity of a PARG (PAR glycohydrolase). To overcome the early embryonic lethality of parg-knockout mice and gain more insights into the biological functions of PARG, we used an RNA interference approach. We found that as little as 10% of PARG protein is sufficient to ensure basic cellular functions: PARG-silenced murine and human cells proliferated normally through several subculturing rounds and they were able to repair DNA damage induced by sublethal doses of H2O2. However, cell survival following treatment with higher concentrations of H2O2 (0.05-1 mM) was increased. In fact, PARG-silenced cells were more resistant than their wild-type counterparts to oxidant-induced apoptosis while exhibiting delayed PAR degradation and transient accumulation of ADP-ribose polymers longer than 15-mers at early stages of drug treatment. No difference was observed in response to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, suggesting a specific involvement of PARG in the cellular response to oxidative DNA damage.

  12. Structures of the human poly (ADP-ribose) glycohydrolase catalytic domain confirm catalytic mechanism and explain inhibition by ADP-HPD derivatives.

    PubMed

    Tucker, Julie A; Bennett, Neil; Brassington, Claire; Durant, Stephen T; Hassall, Giles; Holdgate, Geoff; McAlister, Mark; Nissink, J Willem M; Truman, Caroline; Watson, Martin

    2012-01-01

    Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5'-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.

  13. A specific isoform of poly(ADP-ribose) glycohydrolase is targeted to the mitochondrial matrix by a N-terminal mitochondrial targeting sequence

    SciTech Connect

    Whatcott, Clifford J.; Meyer-Ficca, Mirella L.; Meyer, Ralph G.; Jacobson, Myron K.

    2009-12-10

    Poly(ADP-ribose) polymerases (PARPs) convert NAD to polymers of ADP-ribose that are converted to free ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG). The activation of the nuclear enzyme PARP-1 following genotoxic stress has been linked to release of apoptosis inducing factor from the mitochondria, but the mechanisms by which signals are transmitted between nuclear and mitochondrial compartments are not well understood. The study reported here has examined the relationship between PARG and mitochondria in HeLa cells. Endogenous PARG associated with the mitochondrial fraction migrated in the range of 60 kDa. Transient transfection of cells with PARG expression constructs with amino acids encoded by exon 4 at the N-terminus was targeted to the mitochondria as demonstrated by subcellular fractionation and immunofluorescence microscopy of whole cells. Deletion and missense mutants allowed identification of a canonical N-terminal mitochondrial targeting sequence consisting of the first 16 amino acids encoded by PARG exon 4. Sub-mitochondrial localization experiments indicate that this mitochondrial PARG isoform is targeted to the mitochondrial matrix. The identification of a PARG isoform as a component of the mitochondrial matrix raises several interesting possibilities concerning mechanisms of nuclear-mitochondrial cross talk involved in regulation of cell death pathways.

  14. PKCα and HMGB1 antagonistically control hydrogen peroxide-induced poly-ADP-ribose formation

    PubMed Central

    Andersson, Anneli; Bluwstein, Andrej; Kumar, Nitin; Teloni, Federico; Traenkle, Jens; Baudis, Michael; Altmeyer, Matthias; Hottiger, Michael O.

    2016-01-01

    Harmful oxidation of proteins, lipids and nucleic acids is observed when reactive oxygen species (ROS) are produced excessively and/or the antioxidant capacity is reduced, causing ‘oxidative stress’. Nuclear poly-ADP-ribose (PAR) formation is thought to be induced in response to oxidative DNA damage and to promote cell death under sustained oxidative stress conditions. However, what exactly triggers PAR induction in response to oxidative stress is incompletely understood. Using reverse phase protein array (RPPA) and in-depth analysis of key stress signaling components, we observed that PAR formation induced by H2O2 was mediated by the PLC/IP3R/Ca2+/PKCα signaling axis. Mechanistically, H2O2-induced PAR formation correlated with Ca2+-dependent DNA damage, which, however, was PKCα-independent. In contrast, PAR formation was completely lost upon knockdown of PKCα, suggesting that DNA damage alone was not sufficient for inducing PAR formation, but required a PKCα-dependent process. Intriguingly, the loss of PAR formation observed upon PKCα depletion was overcome when the chromatin structure-modifying protein HMGB1 was co-depleted with PKCα, suggesting that activation and nuclear translocation of PKCα releases the inhibitory effect of HMGB1 on PAR formation. Together, these results identify PKCα and HMGB1 as important co-regulators involved in H2O2-induced PAR formation, a finding that may have important relevance for oxidative stress-associated pathophysiological conditions. PMID:27198223

  15. The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response

    PubMed Central

    Zhang, Feng; Shi, Jiazhong; Chen, Shih-Hsun; Bian, Chunjing; Yu, Xiaochun

    2015-01-01

    Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response. PMID:26400172

  16. The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage

    PubMed Central

    Sellou, Hafida; Lebeaupin, Théo; Chapuis, Catherine; Smith, Rebecca; Hegele, Anna; Singh, Hari R.; Kozlowski, Marek; Bultmann, Sebastian; Ladurner, Andreas G.; Timinszky, Gyula; Huet, Sébastien

    2016-01-01

    Chromatin relaxation is one of the earliest cellular responses to DNA damage. However, what determines these structural changes, including their ATP requirement, is not well understood. Using live-cell imaging and laser microirradiation to induce DNA lesions, we show that the local chromatin relaxation at DNA damage sites is regulated by PARP1 enzymatic activity. We also report that H1 is mobilized at DNA damage sites, but, since this mobilization is largely independent of poly(ADP-ribosyl)ation, it cannot solely explain the chromatin relaxation. Finally, we demonstrate the involvement of Alc1, a poly(ADP-ribose)- and ATP-dependent remodeler, in the chromatin-relaxation process. Deletion of Alc1 impairs chromatin relaxation after DNA damage, while its overexpression strongly enhances relaxation. Altogether our results identify Alc1 as an important player in the fast kinetics of the NAD+- and ATP-dependent chromatin relaxation upon DNA damage in vivo. PMID:27733626

  17. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.

    PubMed Central

    D'Amours, D; Desnoyers, S; D'Silva, I; Poirier, G G

    1999-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism. PMID:10455009

  18. Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage

    PubMed Central

    Kang, Ho Chul; Lee, Yun-Il; Shin, Joo-Ho; Andrabi, Shaida A.; Chi, Zhikai; Gagné, Jean-Philippe; Lee, Yunjong; Ko, Han Seok; Lee, Byoung Dae; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2011-01-01

    Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna’s E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna’s PAR binding and Iduna’s E3 ligase activity. Iduna’s E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna’s PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair. PMID:21825151

  19. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint

    SciTech Connect

    Nakadate, Yusuke; Kodera, Yasuo; Kitamura, Yuka; Tachibana, Taro; Tamura, Tomohide; Koizumi, Fumiaki

    2013-11-29

    Highlights: •Radiosensitization by PARG silencing was observed in multiple lung cancer cells. •PAR accumulation was enhanced by PARG silencing after DNA damage. •Radiation-induced G2/M arrest and checkpoint activation were impaired by PARG siRNA. -- Abstract: Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy.

  20. Poly(ADP-ribose) synthesis following DNA damage in cells heterozygous or homozygous for the xeroderma pigmentosum genotype

    SciTech Connect

    McCurry, L.S.; Jacobson, M.K.

    1981-01-25

    Treatment of normal human cells with DNA-damaging agents such as uv light or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) stimulates the conversion of NAD to the chromosomal polymer poly(ADP-ribose) which in turn results in a rapid depletion of the cellular NAD pool. The effect of uv light or MNNG on the NAD pools of seven cell lines of human fibroblasts either homozygous or heterozygous for the xeroderma pigmentosum genotype has been studied. Xeroderma pigmentosum cells of genetic complementation groups A, C, and D are deficient in the excision repair of DNA damage caused by uv light. Following uv treatment, the NAD content of these cells was unchanged or only slightly reduced. All of the cell lines are able to excise DNA damage caused by MNNG and all of the cell lines had a greatly reduced content of NAD following MNNG treatment. The results demonstrate a close relationship between the conversion of NAD to poly(ADP-ribose) and DNA excision repair in human cells.

  1. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

    SciTech Connect

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

    2013-08-30

    Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.

  2. Role of free radicals and poly(ADP-ribose) synthetase in intestinal tight junction permeability.

    PubMed Central

    Cuzzocrea, S.; Mazzon, E.; De Sarro, A.; Caputi, A. P.

    2000-01-01

    BACKGROUND: Small intestine permeability is frequently altered in inflammatory bowel disease and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJ) and adherence (1,2). The role of hydrogen peroxide (H2O2), and nitric oxide (NO) and PARS in the permeability and structure of small intestine TJ is not clearly understood. MATERIALS AND METHODS: In vitro study, MDCK (Madin-Darby Canine Kidney) cells were exposed to H2O2 (100 microM for 2h), or zymosan (200 microl of stock solution 1 mg/ml for 4h), in the presence or absence of a treatment with poly(ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB: 3 mM) or with n-acetylcysteine (NAC 10 mM). In vivo study, wild-type mice (WT) and mice lacking (KO) of the inducible (or type 2) nitric oxide synthase (iNOS) were treated with zymosan (500 mg/kg, suspended in saline solution, i.p.). In addition INOSWT mice were treated with 3-AB (10 mg/kg, i.p.) or with NAC (40 mg/kg, i.p.) 1 hour and 6 h after zymosan administration. RESULTS: Exposure of MDCK cells to hydrogen peroxide caused a significant impairment in mitochondrial respiration that was associated with a reduction of cells adherence as well as derangement of the junctional proteins. A significant increase of nitrate and nitrite levels, stable metabolites of nitric oxide (NO), were found in MDCK supernatant after zymosan incubation. NO production was associated with a significant reduction of cell adherence and impairment of occludin protein. Pre-treatment of the cells with 3-AB or with NAC caused a significant prevention of H2O2-mediated occludin junctional damage as well as reduced the NO-induced occludin damage. In addition, H2O2 and NO are able to induce a significant derangement of beta-catenin and Zonula Ocludence-1 (ZO-1). We found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the terminal ileal TJs in zymosan-treated iNOSWT mice compared with

  3. Immunoaffinity fractionation of the poly(ADP-ribosyl)ated domains of chromatin.

    PubMed Central

    Malik, N; Miwa, M; Sugimura, T; Thraves, P; Smulson, M

    1983-01-01

    Antibody to poly(ADP-ribose) has been covalently coupled to Sepharose and utilized to isolate selectively oligonucleosomes undergoing the poly(ADP-ribosyl)ation reaction from the bulk of chromatin. Approximately 12% of the unfractionated oligonucleosomes were bound to the immunoaffinity column and these represented essentially 100% of the original poly(ADP-ribosyl)ated nucleosomal species in the unfractionated chromatin. Poly(ADP-ribosyl)ated chromatin was not bound by preimmune IgG columns. KSCN eluted the modified nucleosomes in the form of nucleoprotein complexes. The eluted chromatin components were shown to contain poly(ADP-ribosyl)ated histones as well as automodified poly(ADP-ribose) polymerase. By using [3H]lysine- and [3H]arginine-labeled chromatin, it was shown that the poly-(ADP-ribosyl)ated histones, attached to stretches of oligonucleosomes bound to the column, had a 6-fold enrichment of the modification compared to histones of the unfractionated chromatin. This indicated that non-poly(ADP-ribosyl)ated nucleosomes, connected and proximal to the modified regions, were copurified by this procedure. This allowed characterization of the oligonucleosomal DNA around poly(ADP-ribosyl)ated chromatin domains to be compared with the unbound bulk chromatin. The data indicated that immunofractionated poly(ADP-ribosyl)ated oligonucleosomal DNA contained significant amounts of internal single-strand breaks compared with bulk chromatin. The bound nucleo-protein complexes were found to be enzymatically active for poly(ADP-ribose) polymerase after elution from the antibody column. In contrast, the unbound nucleosomes, representing 90% of the unfractionated chromatin, were totally inactive in the poly(ADP-ribosyl)ation reaction. Images PMID:6573670

  4. Poly (ADP-Ribose) synthetase. Separation and identification of three proteolytic fragments as the substrate-binding domain, the DNA-binding domain, and the automodification domain.

    PubMed

    Kameshita, I; Matsuda, Z; Taniguchi, T; Shizuta, Y

    1984-04-25

    Poly(ADP-ribose) synthetase of Mr = 120,000 is cleaved by limited proteolysis with alpha-chymotrypsin into two fragments of Mr = 54,000 (54K) and Mr = 66,000 (66K). When the native enzyme is modified with 3-(bromoacetyl)pyridine, both portions of the enzyme are alkylated; however, alkylation of the 54K portions of the enzyme is protected by the addition of the substrate, NAD, or its analog, nicotinamide, suggesting that the substrate-binding site is localized in the 54K fragment. When the enzyme previously automodified with a low concentration of [adenine-U-14C] NAD is digested with alpha-chymotrypsin, the radioactivity is detected exclusively in the 66K fragment. The 66K fragment thus labeled is further cleaved with papain into two fragments of Mr = 46,000 and Mr = 22,000. With these two fragments, the label is detected only in the 22K fragment, but not in the 46K fragment. The 46K fragment binds to a DNA-cellulose column with the same affinity as that of the native enzyme, while the 22K fragment and the 54K fragment have little affinity for the DNA ligand. These results indicate that poly (ADP-ribose) synthetase contains three separable domains, the first possessing the site for binding of the substrate, NAD, the second containing the site for binding of DNA, and the third acting as the site(s) for accepting poly(ADP-ribose).

  5. Molecular biology basis for the response of poly(ADP-rib) polymerase and NAD metabolism to dna damage caused by mustard alkylating agents. Final report, 30 April 1990-30 July 1994

    SciTech Connect

    Smulson, M.E.

    1994-08-30

    During the course of this contract, we have performed a variety of experiments whose intent has been to provide a strategy to modulate the nuclear enzyme poly(ADP-ribose) polymerase (PADPRP) in cultured keratinocytes. During this study, human keratinocyte lines were stably transfected with the cDNA for human PADPRP in the antisense orientation under an inducible promoter. Induction of this antisense RNA by dexamethasone in cultured cells selectively lowered levels of PADPRP in RNA, protein, and enzyme activity. Induction of antisense RNA led to a reduction in the levels of PADPRP in individual cell nuclei, as well as the loss of the ability of cells to synthesize and modify proteins by poly(ADP-ribose) polymer in response to an alkylating agent. When keratinocyte clones containing the antisense construct or empty vector alone were grafted onto nude mice they formed histologically normal human skin. The PADPRP antisense construct was also inducible in vivo by the topical application of dexamethasone to the reconstituted epidermis. In addition, poly(ADP-ribose) polymer could be induced and detected in vivo following the topical application of a sulfur mustard to the grafted transfected skin layers. Accordingly, a model system has been developed in which the levels of PADPRP can be selectively manipulated in human keratinocytes in cell culture, and potentially in reconstituted epidermis as well.

  6. Peroxynitrite-induced thymocyte apoptosis: the role of caspases and poly (ADP-ribose) synthetase (PARS) activation.

    PubMed Central

    Virág, L; Scott, G S; Cuzzocrea, S; Marmer, D; Salzman, A L; Szabó, C

    1998-01-01

    The mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cross-linking of T-cell receptor leads to stromal cell activation, expression of inducible nitric oxide synthase (iNOS) and, subsequently, to thymocyte apoptosis. Therefore we examined, whether NO directly or indirectly, through peroxynitrite formation, causes thymocyte apoptosis. Immuno-histochemical detection of nitrotyrosine revealed in vivo peroxynitrite formation in the thymi of naive mice. Nitrotyrosine, the footprint of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concentrations (10 microM) of peroxynitrite led to apoptosis, whereas higher concentrations (50 microM) resulted in intense cell death with the characteristics of necrosis. We also investigated the effect of poly (ADP-ribose) synthetase (PARS) inhibition on thymocyte apoptosis. Using the PARS inhibitor 3-aminobenzamide (3-AB), or thymocytes from PARS-deficient animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis in response to peroxynitrite were prevented by PARS inhibition. Therefore, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H2O2 treatment, while apoptosis induced by non-oxidative stimuli such as dexamethasone or anti-FAS antibody was unaffected by PARS inhibition. In conclusion, we propose that peroxynitrite-induced apoptosis may play a role in the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apoptosis may serve as an energy-conserving step, enabling the cell to complete the process of apoptosis

  7. 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.

  8. 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

  9. Protein poly(ADP-ribosyl)ation regulates arabidopsis immune gene expression and defense responses.

    PubMed

    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.

  10. The N-terminal Region of Chromodomain Helicase DNA-binding Protein 4 (CHD4) Is Essential for Activity and Contains a High Mobility Group (HMG) Box-like-domain That Can Bind Poly(ADP-ribose)*

    PubMed Central

    Silva, Ana P. G.; Ryan, Daniel P.; Galanty, Yaron; Low, Jason K. K.; Vandevenne, Marylene; Jackson, Stephen P.; Mackay, Joel P.

    2016-01-01

    Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA-damage responses through its N-terminal region in a poly(ADP-ribose) polymerase-dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this N-terminal region. The-fold consists of a four-α-helix bundle with structural similarity to the high mobility group box, a domain that is well known as a DNA binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4-NuRD acts to regulate gene expression and participates in the DNA-damage response. PMID:26565020

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

    DTIC Science & Technology

    1985-02-01

    portions (100 ug/mg nuclear protein) and incubation for 2 x 20 min. at 37*. In (A) adducts were hydrolyzed by 1 M NaOH by incubation at 55 ° for two hours...indicate NAD-glycohydrolase activities that are probably unrelated to the polymerase reaction. When polymer- I" . protein adducts are hydrolyzed by...cells, were dispensed in Falcon Petri dishes (60-100 mm diameter) precoated with collagen , previously sterilized by UV radiation. Collagen was isolated

  12. The pioneering spirit of Takashi Sugimura: his studies of the biochemistry of poly(ADP-ribosylation) and of cancer.

    PubMed

    Masutani, Mitsuko

    2012-03-01

    Takashi Sugimura has accomplished many scientific achievements in the field of biochemistry and in cancer research. Sugimura's group identified the novel polymer poly(ADP-ribose) in parallel to P. Mandel's and O. Hayaishi's groups and demonstrated the presence of the enzyme poly(ADP-ribose) polymerase (PARP). He also discovered the cognate catabolic enzyme, poly(ADP-ribose) glycohydrolase (PARG) and further elucidated the biology of poly(ADP-ribose). The astonishing discovery of pierisin, an apoptogenic peptide that ADP-ribosyaltes DNA, profoundly illuminates his scientific character and curiosity as well. Sugimura's work in cancer research shows an extraordinarily wide range, which includes the establishment of new methods in chemical carcinogenesis, the identification of various environmental mutagens/carcinogens and new tumour promoters. He also established the concept that cancer is a disease of DNA and contributed to the development of the concept of the multi-step model of carcinogenesis.

  13. Detection and quantification of poly-ADP-ribosylated cellular proteins of spleen and liver tissues of mice in vivo by slot and Western blot immunoprobing using polyclonal antibody against mouse ADP-ribose polymer.

    PubMed

    Sharan, R N; Devi, B Jaylata; Humtsoe, J O; Saikia, Jyoti R; Kma, L

    2005-10-01

    Poly-ADP-ribosylation (PAR) of cellular proteins has been shown to have decisive roles in diverse cellular functions including carcinogenesis. There are indications that metabolic level of poly-ADP-ribosylated cellular proteins might indicate carcinogenesis and, therefore, could be potentially used in cancer screening program. Keeping in mind the limitations of currently available assays of cellular PAR, a new assay is being reported that measures the metabolic level of poly-ADP-ribosylated cellular proteins. The ELISA based slot and Western blot immunoassay used polyclonal antibody against natural, heterogeneous ADP-ribose polymers. It could be successfully employed to qualitatively and quantitatively assay metabolic levels of poly-ADP-ribosylated proteins of spleen and liver tissues of normal mice or mice exposed to dimethylnitrosamine for up to 8 weeks; potentially PAR of cellular proteins could be assayed in any tissue or biopsy. Implications of the results in cancer screening program have been discussed.

  14. Solution structures of the two PBZ domains from human APLF and their interaction with poly(ADP-ribose)

    PubMed Central

    Eustermann, Sebastian; Brockmann, Christoph; Mehrotra, Pawan Vinod; Yang, Ji-Chun; Loakes, David; West, Stephen C.; Ahel, Ivan; Neuhaus, David

    2010-01-01

    Poly(ADP-ribosyl)ation represents an important post-translational modification in higher eukaryotes. Several DNA repair/checkpoint proteins possess specific PAR-Binding Zinc finger (PBZ) modules critical for function. Here, we present solution structures of the two PBZ modules of APLF (Aprataxin and PNK-like factor), revealing a novel type of zinc finger. By combining in vivo PAR-binding data with NMR interaction data using PAR fragments, we suggest a structural basis for PBZ-PAR recognition. PMID:20098424

  15. Molecular biological basis for the response of poly(ADP-rib) polymerase and NAD metabolism to DNA damage caused by mustard alkylating agents. Midterm report

    SciTech Connect

    Smulson, M.E.

    1996-07-01

    During the course of this contract, we have performed a variety of experiments to provide a strategy to modulate the nuclear enzyme poly(ADP-ribose) polymerase (PARP), in cultured keratinocytes. This enzyme modifies a variety of nuclear proteins utilizing NAD. DNA is required for the catalytic activity of the enzyme and the activity is dependent upon the presence of strand breaks in this DNA. It has been hypothesized that human skin exposed to mustards may develop blisters due to a generalized lowering of NAD in exposed skin cells. During the contract period, we have established a stably transfected human keratinocyte cell line which expresses antisense transcripts to PARP mRNA when these keratinocyte were grafted onto nude mice they formed histologically normal human skin. Accordingly, a model system has been developed in which the levels of PARP can be selectively manipulated in human keratinocytes in reconstituted epidermis as well. We also showed that PARP was proteolytically cleaved at the onset of spontaneous apoptosis following proteolytic conversion of CPP32b to its active form, termed `apopain`. Having characterized the events associated with apoptosis, we determined, during the last period, whether any or all of these features could be observed following exposure of keratinocytes to SM.

  16. Poly(ADP-ribosylation) and neoplastic transformation: effect of PARP inhibitors.

    PubMed

    Donà, Francesca; Chiodi, Ilaria; Belgiovine, Cristina; Raineri, Tatiana; Ricotti, Roberta; Mondello, Chiara; Scovassi, Anna Ivana

    2013-01-01

    Poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribosylation) play essential roles in several biological processes, among which neoplastic transformation and telomere maintenance. In this paper, we review the poly(ADP-ribosylation) process together with the highly appealing use of PARP inhibitors for the treatment of cancer. In addition, we report our results concerning poly(ADP-ribosylation) in a cellular model system for neoplastic transformation developed in our laboratory. Here we show that PARP-1 and PARP-2 expression increases during neoplastic transformation, together with the basal levels of poly(ADP-ribosylation). Furthermore, we demonstrate a greater effect of the PARP inhibitor 3-aminobenzamide (3AB) on cellular viability in neoplastically transformed cells compared to normal fibroblasts and we show that prolonged 3AB administration to tumorigenic cells causes a decrease in telomere length. Taken together, our data support an active involvement of poly(ADP-ribosylation) in neoplastic transformation and telomere length maintenance and confirm the relevant role of poly(ADP-ribosylation) inhibition for the treatment of cancer.

  17. Chromosomal protein poly(ADP-ribosyl)ation in pancreatic nucleosomes.

    PubMed

    Aubin, R J; Dam, V T; Miclette, J; Brousseau, Y; Poirier, G G

    1982-03-01

    When pancreatic chromatin fragments were prepared and resolved in the presence of 80 mM NaCl, endogenous poly(ADP-ribose) polymerase activity was found to be maximal in nucleosome periodicities of four to five units and did not respond to any further increases in nucleosomal architecture. Furthermore, in nucleosome complexities spanning 1 through 14 and over unit lengths, polyacrylamide gel electrophoresis on acid-urea and acid-urea-Triton gels has shown pancreatic histone H1 to be the only actively ADP-ribosylated histone species. The extent of ADP-ribosylation of histone H1 was also demonstrated to retard the protein's mobility in acid-urea, acid-urea-Triton, and lithium dodecyl sulfate polyacrylamide gels and to consist of at least 12 distinct ADP-ribosylated species extractable in all nucleosome complexities studied. Finally, extraction and subsequent electrophoresis of total chromosomal proteins in the presence of lithium dodecyl sulfate also evidenced heavy ADP-ribosylation at the level of nonhistone chromosomal proteins of the high mobility group comigrating in the core histone region, as well as in the topmost region of the gels where poly(ADP-ribose) polymerase was found to form a poly(ADP-ribosyl)ated aggregate.

  18. Extracellular poly(ADP-ribose) is a neurotrophic signal that upregulates glial cell line-derived neurotrophic factor (GDNF) levels in vitro and in vivo.

    PubMed

    Nakajima, Hidemitsu; Itakura, Masanori; Sato, Keishi; Nakamura, Sunao; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

    2017-03-04

    Synthesis of poly(ADP-ribose) (PAR) is catalyzed by PAR polymerase-1 (PARP-1) in neurons. PARP1 plays a role in various types of brain damage in neurodegenerative disorders. In neurons, overactivation of PARP-1 during oxidative stress induces robust PAR formation, which depletes nicotinamide adenine dinucleotide levels and leads to cell death. However, the role of the newly-formed PAR in neurodegenerative disorders remains elusive. We hypothesized that the effects of PAR could occur in the extracellular space after it is leaked from damaged neurons. Here we report that extracellular PAR (EC-PAR) functions as a neuroprotective molecule by inducing the synthesis of glial cell line-derived neurotrophic factor (GDNF) in astrocytes during neuronal cell death, both in vitro and in vivo. In primary rat astrocytes, exogenous treatment with EC-PAR produced GDNF but not other neurotrophic factors. The effect was concentration-dependent and did not affect cell viability in rat C6 astrocytoma cells. Topical injection of EC-PAR into rat striatum upregulated GDNF levels in activated astrocytes and improved pathogenic rotation behavior in a unilateral 6-hydroxydopamine model of Parkinson disease in rats. These findings indicate that EC-PAR acts as a neurotrophic enhancer by upregulating GDNF levels. This effect protects the remaining neurons following oxidative stress-induced brain damage, such as that seen with Parkinson disease.

  19. Poly(ADP-ribosyl)ation as a new posttranslational modification of YB-1.

    PubMed

    Alemasova, Elizaveta E; Pestryakov, Pavel E; Sukhanova, Maria V; Kretov, Dmitry A; Moor, Nina A; Curmi, Patrick A; Ovchinnikov, Lev P; Lavrik, Olga I

    2015-12-01

    Multifunctional Y-box binding protein 1 (YB-1) is actively studied as one of the components of cellular response to genotoxic stress. However, the precise role of YB-1 in the process of DNA repair is still obscure. In the present work we report for the first time new posttranslational modification of YB-1 - poly(ADP-ribosyl)ation, catalyzed by one of the main regulatory enzymes of DNA repair - poly(ADP-ribose)polymerase 1 (PARP1) in the presence of model DNA substrate carrying multiple DNA lesions. Therefore, poly(ADP-ribosyl)ation of YB-1 catalyzed with PARP1, can be stimulated by damaged DNA. The observed property of YB-1 underlines its ability to participate in the DNA repair by its involvement in the regulatory cascades of DNA repair.

  20. The Key Involvement of Poly (ADP-Ribosylation) in Defense against Toxic Agents in Molecular Biology Studies

    DTIC Science & Technology

    1989-11-15

    polymerase increased very early and remained high for up to 48 after which it decreased to pre-induced levels. Polymerase transcript levels did not change...the Ub- PADPRP junction. HUMAN POLY(ADP-RIBOSE) POLYMERASE IS FUNCTIONAL IN SCHr2OSACCHAROMYCES POMBE (MS IN PREP.) The full length cDNA for human...PADPRP has been introduced into the yeast Schizosaccharomyces pombe under the transcriptional control of the SV40 early promoter. A number of haploid

  1. The Key Involvement of Poly(ADP-Ribosylation) in Defense Against Toxic Agents in Molecular Biology Studies

    DTIC Science & Technology

    1991-12-17

    for the polymerase increased very early and remained high for up to 48 after which it decreased to pre-induced levels. Polymerase transcript levels...cleave the Ub-PADPRP Junction. HUMAN POLY(ADP-RIBOSE) POLYMERASE IS FUNCTIONAL IN SC.=OSACCHAROMYCES POMBE (MS IN PREP.) The full length cDNA for human...PADPRP has been introduced into the yeast Schizosaccharomyces pombe under the transcriptional control of the SV40 early promoter. A number of haploid

  2. Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

    PubMed Central

    Perina, Dragutin; Mikoč, Andreja; Ahel, Josip; Ćetković, Helena; Žaja, Roko; Ahel, Ivan

    2014-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation. Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment. PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome. Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as

  3. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    SciTech Connect

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-08-14

    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 did 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.

  4. Arabidopsis PARG1 is the key factor promoting cell survival among the enzymes regulating post-translational poly(ADP-ribosyl)ation

    PubMed Central

    Zhang, Hailei; Gu, Zongying; Wu, Qiao; Yang, Lifeng; Liu, Caifeng; Ma, Hong; Xia, Yiji; Ge, Xiaochun

    2015-01-01

    Poly(ADP-ribosyl)ation is a reversible post-translational modification of proteins, characterized by the addition of poly(ADP-ribose) (PAR) to proteins by poly(ADP-ribose) polymerase (PARP), and removal of PAR by poly(ADP-ribose) glycohydrolase (PARG). Three PARPs and two PARGs have been found in Arabidopsis, but their respective roles are not fully understood. In this study, the functions of each PARP and PARG in DNA repair were analyzed based on their mutant phenotypes under genotoxic stresses. Double or triple mutant analysis revealed that PARP1 and PARP2, but not PARP3, play a similar but not critical role in DNA repair in Arabidopsis seedlings. PARG1 and PARG2 play an essential and a minor role, respectively under the same conditions. Mutation of PARG1 results in increased DNA damage level and enhanced cell death in plants after bleomycin treatment. PARG1 expression is induced primarily in root and shoot meristems by bleomycin and induction of PARG1 is dependent on ATM and ATR kinases. PARG1 also antagonistically modulates the DNA repair process by preventing the over-induction of DNA repair genes. Our study determined the contribution of each PARP and PARG member in DNA repair and indicated that PARG1 plays a critical role in this process. PMID:26516022

  5. ZMYND8 Co-localizes with NuRD on Target Genes and Regulates Poly(ADP-Ribose)-Dependent Recruitment of GATAD2A/NuRD to Sites of DNA Damage.

    PubMed

    Spruijt, Cornelia G; Luijsterburg, Martijn S; Menafra, Roberta; Lindeboom, Rik G H; Jansen, Pascal W T C; Edupuganti, Raghu Ram; Baltissen, Marijke P; Wiegant, Wouter W; Voelker-Albert, Moritz C; Matarese, Filomena; Mensinga, Anneloes; Poser, Ina; Vos, Harmjan R; Stunnenberg, Hendrik G; van Attikum, Haico; Vermeulen, Michiel

    2016-10-11

    NuRD (nucleosome remodeling and histone deacetylase) is a versatile multi-protein complex with roles in transcription regulation and the DNA damage response. Here, we show that ZMYND8 bridges NuRD to a number of putative DNA-binding zinc finger proteins. The MYND domain of ZMYND8 directly interacts with PPPLΦ motifs in the NuRD subunit GATAD2A. Both GATAD2A and GATAD2B exclusively form homodimers and define mutually exclusive NuRD subcomplexes. ZMYND8 and NuRD share a large number of genome-wide binding sites, mostly active promoters and enhancers. Depletion of ZMYND8 does not affect NuRD occupancy genome-wide and only slightly affects expression of NuRD/ZMYND8 target genes. In contrast, the MYND domain in ZMYND8 facilitates the rapid, poly(ADP-ribose)-dependent recruitment of GATAD2A/NuRD to sites of DNA damage to promote repair by homologous recombination. Thus, these results show that a specific substoichiometric interaction with a NuRD subunit paralogue provides unique functionality to distinct NuRD subcomplexes.

  6. S100B impairs glycolysis via enhanced poly(ADP-ribosyl)ation of glyceraldehyde 3-phosphate dehydrogenase in rodent muscle cells.

    PubMed

    Hosokawa, Kaori; Hamada, Yoji; Fujiya, Atsushi; Murase, Masatoshi; Maekawa, Ryuya; Niwa, Yasuhiro; Izumoto, Takako; Seino, Yusuke; Tsunekawa, Shin; Arima, Hiroshi

    2017-02-07

    S100 calcium-binding protein B (S100B), a multifunctional macromolecule mainly expressed in nerve tissues and adipocytes, has been suggested to contribute to the pathogenesis of obesity. To clarify the role of S100B in insulin action and glucose metabolism in peripheral tissues, we investigated the effect of S100B on glycolysis in myoblast and myotube cells. Rat myoblast L6 cells were treated with recombinant mouse S100B to examine glucose consumption, lactate production, glycogen accumulation, glycolytic metabolites and enzyme activity, insulin signaling, and poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Glycolytic metabolites were investigated by enzyme assays or metabolome analysis, and insulin signaling was assessed by western blot analysis. Enzyme activity and poly(ADP-ribosyl)ation of GAPDH was evaluated by an enzyme assay and immunoprecipitation followed by dot blot with an anti-poly(ADP-ribose) antibody, respectively. S100B significantly decreased glucose consumption, glucose analog uptake, and lactate production in L6 cells, in either the presence or absence of insulin. In contrast, S100B had no effect on glycogen accumulation and insulin signaling. Metabolome analysis revealed that S100B increased the concentration of glycolytic intermediates upstream of GAPDH. S100B impaired GAPDH activity and increased poly(ADP-ribosyl)ated GAPDH proteins. The effects of S100B on glucose metabolism were mostly canceled by a poly(ADP-ribose) polymerase (PARP) inhibitor. Similar results were obtained in C2C12 myotube cells. We conclude that S100B as a humoral factor may impair glycolysis in muscle cells independently of insulin action, and the effect may be attributed to the inhibition of GAPDH activity from enhanced poly(ADP-ribosyl)ation of the enzyme.

  7. Mechanisms controlling the smooth muscle cell death in progeria via down-regulation of poly(ADP-ribose) polymerase 1.

    PubMed

    Zhang, Haoyue; Xiong, Zheng-Mei; Cao, Kan

    2014-06-03

    Hutchinson-Gilford progeria syndrome (HGPS) is a severe human premature aging disorder caused by a lamin A mutant named progerin. Death occurs at a mean age of 13 y from cardiovascular problems. Previous studies revealed loss of vascular smooth muscle cells (SMCs) in the media of large arteries in a patient with HGPS and two mouse models, suggesting a causal connection between the SMC loss and cardiovascular malfunction. However, the mechanisms of how progerin leads to massive SMC loss are unknown. In this study, using SMCs differentiated from HGPS induced pluripotent stem cells, we show that HGPS SMCs exhibit a profound proliferative defect, which is primarily caused by caspase-independent cell death. Importantly, progerin accumulation stimulates a powerful suppression of PARP1 and consequently triggers an activation of the error-prone nonhomologous end joining response. As a result, most HGPS SMCs exhibit prolonged mitosis and die of mitotic catastrophe. This study demonstrates a critical role of PARP1 in mediating SMC loss in patients with HGPS and elucidates a molecular pathway underlying the progressive SMC loss in progeria.

  8. Inhibition of DNA Binding by the Phosphorylation of Poly ADP-Ribose Polymerase Protein Catalyzed by Protein Kinase C

    DTIC Science & Technology

    1993-04-21

    drying and authentic Ser-P and Thr-P were added as standards followed by paper electrophoresis (27). The paper strips were ninhydrin -stained, dried and... tested with the specific oligopeptide substrate of PKC (26) and therefore the inhibition by DNA is due to a ADPRT-DNA interaction (Fig 3...correctness of this conclusion was tested also by "opposite" labeling, i.e. radioiodination of the major chymotryptic polypeptides of ADPRT (i.e. the 56 and 64

  9. CD38 Knockout Mice Show Significant Protection Against Ischemic Brain Damage Despite High Level Poly-ADP-Ribosylation.

    PubMed

    Long, Aaron; Park, Ji H; Klimova, Nina; Fowler, Carol; Loane, David J; Kristian, Tibor

    2017-01-01

    Several enzymes in cellular bioenergetics metabolism require NAD(+) as an essential cofactor for their activity. NAD(+) depletion following ischemic insult can result in cell death and has been associated with over-activation of poly-ADP-ribose polymerase PARP1 as well as an increase in NAD(+) consuming enzyme CD38. CD38 is an NAD(+) glycohydrolase that plays an important role in inflammatory responses. To determine the contribution of CD38 activity to the mechanisms of post-ischemic brain damage we subjected CD38 knockout (CD38KO) mice and wild-type (WT) mice to transient forebrain ischemia. The CD38KO mice showed a significant amelioration in both histological and neurologic outcome following ischemic insult. Decrease of hippocampal NAD(+) levels detected during reperfusion in WT mice was only transient in CD38KO animals, suggesting that CD38 contributes to post-ischemic NAD(+) catabolism. Surprisingly, pre-ischemic poly-ADP-ribose (PAR) levels were dramatically higher in CD38KO animals compared to WT animals and exhibited reduction post-ischemia in contrast to the increased levels in WT animals. The high PAR levels in CD38 mice were due to reduced expression levels of poly-ADP-ribose glycohydrolase (PARG). Thus, the absence of CD38 activity can not only directly affect inflammatory response, but also result in unpredicted alterations in the expression levels of enzymes participating in NAD(+) metabolism. Although the CD38KO mice showed significant protection against ischemic brain injury, the changes in enzyme activity related to NAD(+) metabolism makes the determination of the role of CD38 in mechanisms of ischemic brain damage more complex.

  10. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure*

    PubMed Central

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O.; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M. Cristina

    2016-01-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1−/− compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  11. Cell cycle-dependent intervention by benzamide of carcinogen-induced neoplastic transformation and in vitro poly(ADP-ribosyl)ation of nuclear proteins in human fibroblasts.

    PubMed Central

    Kun, E; Kirsten, E; Milo, G E; Kurian, P; Kumari, H L

    1983-01-01

    Human fibroblasts were subjected to nutritionally induced G1 block, followed by release and subsequent entry into S phase, and exposed to nontoxic concentrations of carcinogens in early S phase. Cell transformation occurred as determined by early morphologic cell alterations, anchorage-independent colony formation, cell invasiveness, and augmentation of Ab 376 human malignancy-specific cell-surface antigenic determinant. Methylazoxymethanol acetate was the most potent transforming agent at doses that were negative in toxicity tests. Benzamide (10 microM intracellular concentration), a specific inhibitor of poly(ADP-ribose) polymerase, prevented transformation in a cell cycle-specific manner, maximal prevention coinciding with early S phase, also characteristic of maximal susceptibility to transformation. Neither an interference of carcinogen deoxyguanosine nucleoside adduct formation nor a chemical reaction between benzamide and carcinogens was detected. Methylazoxymethanol acetate at transforming but nontoxic dose partially inhibited poly(ADP-ribosyl)ation to about the same extent as benzamide. However, simultaneous exposure of cells to both agents in early S phase, resulting in the prevention of transformation, augmented poly(ADP-ribosyl)ation above the controls. Enzymatic activities ran parallel with the formation of DNA-associating polymer-nonhistone protein adducts that are assumed to regulate the physiological function of chromatin at the structural level. Images PMID:6196785

  12. 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.

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

    SciTech Connect

    Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li; Yang, Wen-Ming

    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 of 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.

  14. Inhibition of Poly-ADP-Ribosylation Fails to Increase Axonal Regeneration or Improve Functional Recovery after Adult Mammalian CNS Injury

    PubMed Central

    Wang, Xingxing; Byrne, Alexandra B.

    2016-01-01

    Abstract After traumatic damage of the brain or spinal cord, many surviving neurons are disconnected, and recovery of function is limited by poor axon regeneration. Recent data have suggested that poly ADP-ribosylation plays a role in limiting axonal regrowth such that inhibition of poly (ADP-ribose) polymerase (PARP) may have therapeutic efficacy for neurological recovery after trauma. Here, we tested systemic administration of the PARP inhibitor, veliparib, and showed effective suppression of PARylation in the mouse CNS. After optic nerve crush injury or dorsal hemisection of the thoracic spinal cord in mice, treatment with veliparib at doses with pharmacodynamic action had no benefit for axonal regeneration or functional recovery. We considered whether PARP gene family specificity might play a role. In vitro mouse cerebral cortex axon regeneration experiments revealed that short hairpin RNA (shRNA)-mediated suppression of PARP1 promoted axonal regeneration, whereas suppression of other PARP isoforms either had no effect or decreased regeneration. Therefore, we examined recovery from neurological trauma in mice lacking PARP1. No increase of axonal regeneration was observed in Parp1–/– mice after optic nerve crush injury or dorsal hemisection of the thoracic spinal cord, and there was no improvement in motor function recovery. Thus, comprehensive in vivo analysis reveals no indication that clinical PARP inhibitors will on their own provide benefit for recovery from CNS trauma. PMID:28032120

  15. Poly(ADP-ribosyl)ation in carcinogenesis.

    PubMed

    Masutani, Mitsuko; Fujimori, Hiroaki

    2013-12-01

    Cancer develops through diverse genetic, epigenetic and other changes, so-called 'multi-step carcinogenesis', and each cancer harbors different alterations and properties. Here in this article we review how poly(ADP-ribosyl)ation is involved in multi-step and diverse pathways of carcinogenesis. Involvement of poly- and mono-ADP-ribosylation in carcinogenesis has been studied at molecular and cellular levels, and further by animal models and human genetic approaches. PolyADP-ribosylation acts in DNA damage repair response and maintenance mechanisms of genomic stability. Several DNA repair pathways, including base-excision repair and double strand break repair pathways, involve PARP and PARG functions. These care-taker functions of poly(ADP-ribosyl)ation suggest that polyADP-ribosyation may mainly act in a tumor suppressive manner because genomic instability caused by defective DNA repair response could serve as a driving force for tumor progression, leading to invasion, metastasis and relapse of cancer. On the other hand, the new concept of 'synthetic lethality by PARP inhibition' suggests the significance of PARP activities for survival of cancer cells that harbor defects in DNA repair. Accumulating evidence has revealed that some PARP family molecules are involved in various signaling cascades other than DNA repair, including epigenetic and transcriptional regulations, inflammation/immune response and epithelial-mesenchymal transition, suggesting that poly(ADP-ribosyl)ation both promotes and suppresses carcinogenic processes depending on the conditions. Expanding understanding of poly(ADP-ribosyl)ation suggests that strategies to achieve cancer prevention targeting poly(ADP-ribosyl)ation for genome protection against life-long exposure to environmental carcinogens and endogenous carcinogenic stimuli.

  16. 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

  17. Further evidence for poly-ADP-ribosylated histones as DNA suppressors

    SciTech Connect

    Yu, F.L.; Geronimo, I.H.; Bender, W.; Meginniss, K.E.

    1986-05-01

    For many years histones have been considered to be the gene suppressors in eukaryotic cells. Recently, the authors have found strong evidence indicating that poly-ADP-ribosylated histones, rather than histones, are the potent inhibitors of DNA-dependent RNA synthesis. They now report additional evidence for this concept: 1) using histone inhibitor isolated directly from nuclei, the authors are able to confirm their earlier findings that the inhibitor substances are sensitive to pronase, snake venom phosphodiesterase digestion and 0.1N KOH hydrolysis, and are resistant to DNase I and RNase A digestion, 2) the O.D. 260/O.D.280 ratio of the histone inhibitor is between pure protein and nuclei acid, suggesting the inhibitor substance is a nucleoprotein hybrid. This result directly supports the fact that the isolated histone inhibitor is radioactive poly (ADP-ribose) labeled, 3) commercial histones show big differences in inhibitor activity. The authors believe this reflects the variation in poly-ADP-ribosylation among commercial histones, and 4) 0.1N KOH hydrolysis eliminates the poly (ADP-ribose) radioactivity from the acceptor proteins as well as histone inhibitor activity. Yet, on gel, the inhibitor shows identical histone bands and stain intensity before and after hydrolysis, indicating the histones per se are qualitatively and quantitatively unaffected by alkaline treatment. This result strongly suggests that histones themselves are not capable of inhibiting DNA-dependent RNA synthesis.

  18. Poly (ADP-Ribose) Polymerase is Involved in the Repair of DNA Damage Due to Sulfur Mustard by a Mechanism Other Than DNA Ligase I Activation

    DTIC Science & Technology

    2004-11-16

    agents including sulfur mustard (SM). We observed concurrent activation of PARP and DNA ligase in SM-exposed human epidermal keratinocytes (HEK...Previous reports from other laboratories suggested that DNA ligase activation could be due to its modification by PARP. In humans, there are three distinct...DNA ligases, I, II and IV of which DNA ligase I participates in DNA replication and repair. By metabolically labeling HEK using 3H-adenosine

  19. The Effect of Poly(ADP-ribose) Polymerase-1 Gene 3'Untranslated Region Polymorphism in Colorectal Cancer Risk among Saudi Cohort.

    PubMed

    Alhadheq, Abdullah M; Purusottapatnam Shaik, Jilani; Alamri, Abdullah; Aljebreen, Abdulrahman M; Alharbi, Othman; Almadi, Majid A; Alhadeq, Faten; Azzam, Nahla A; Semlali, Abdelhabib; Alanazi, Mohammad; Bazzi, Mohammad D; Reddy Parine, Narasimha

    2016-01-01

    Background. DNA repair systems are essential for each cell to repair and maintain the genome integrity. Base excision repair pathway is one of the crucial pathways to maintain genome integrity and PARP-1 plays a key role in BER pathway. The purpose of this study is to evaluate the association between polymorphisms in PARP-1 3'untranslated region (3'UTR) SNP rs8679 and its expression in colorectal cancer. Methods. Genotyping and gene expression were performed using TaqMan assays. The effects of age, gender, and tumor location were evaluated in cases and controls regarding the genotyping results. Resulting data was analyzed using SPSS software. Results and Conclusions. Genotyping analysis for SNP rs8679 showed decreased susceptibility to colorectal cancer at heterozygous TC allele and at minor allele C. Further this protective association was also observed in younger age patients (≤57), in female patients, and also in patients with tumors located at colon and rectum. PARP-1 expression levels are significantly different in colorectal cancer compared to matched normal tissue. Our findings proved that the upregulation of PARP-1 is associated with tumor progression and poor prognosis in Saudi patients with colorectal cancer, suggesting that PARP-1 can be novel and valuable signatures for predicting the clinical outcome of patients with colorectal cancer.

  20. Poly (ADP-Ribose) Polymerase (PARP) is Essential for Sulfur Mustard-Induced DNA Damage Repair, But Has No Role in DNA Ligase Activation

    DTIC Science & Technology

    2006-01-01

    ligase activation could be due to its modification by PARP. Using HEK, intracellular "H-labeled NAD÷ (H-adenine) was metabolically generated and then... acetic acid methyl ester) (Bhat et al., 1998). These observations indicate a Stock HEK from adult skin of a single donor at passage need for a better...0.76 HEK were used in which NAD’ was metabolically 3H- Z-VAD-FMK (4 pm) 0.55CDO5 antibodly (2 pag ml )/(.( labeled at adenine (Malanga and Althaus

  1. 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

    Treatment % G FP p os iti ve Figure 7. Leptomycin B abolishes the HR deficit induced by HR. additionally, it may enhance HR. 4 Task 2. To transiently...Stanley, J , and Yang, ES*. PARP-1: Friend or foe of DNA damage and repair in tumorigenesis? Cancers 2013. 5(3), 943-958. *Corresponding author 7...University School of Medicine, Nashville, TN ABR Holman Research Scholar Nucletron Prostate HDR Training Course 2009 Chief Resident 2009-2010 2005

  2. The Treatment of BRCA1/2 Hereditary BRCA1/2 and Sporadic Breast Cancer with Poly(ADP-Ribose) Polymerase Inhibitors and Chemotherapy

    DTIC Science & Technology

    2009-09-01

    increased antineoplastic activity  and bone marrow protection. Cell Mol Biol. 2007 May 15;53(3):18‐26.    De Soto JA, Mandatory  HPV   vaccination ? , J. Fam...about MRSA? The Journal  29 Oct, 2007. pgs C1‐ 2.    De Soto JA, Should your daughter be  vaccinated  for  HPV ? The Journal Sept. 24,  2007. pgs C1‐2...The Journal, Oct 31, 2005, pgs C1‐2.        Other publications (including web pages)    De Soto JA,  HPV   Vaccine , http://en.wikipedia.org/wiki

  3. 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

    abolishes synthetic lethality between IR and PARP inhibition. - + 0 1 2 3 4 5 Control LMB 4Gy Radiation Treatment % G FP p os iti ve Figure 7...formation ability (Months 21-24) Progress on Task 2. We have had trouble generat ing stable cell lines expressing both the DRGF P repair substrate as...4. Knight- Krajewski , S, Welsh, CF, Liu, YQ, Lyons, L, Faysal, J, Yang, ES, and Burnstein, KL. Deregulation of the Rho GTPase, Rac1, suppresses

  4. Persistence of histone H2AX phosphorylation after meiotic chromosome synapsis and abnormal centromere cohesion in Poly (ADP-ribose) polymerase (Parp-1) null oocytes

    PubMed Central

    Yang, Feikun; Baumann, Claudia; De La Fuente, Rabindranath

    2009-01-01

    In spite of the impact of aneuploidy on human health little is known concerning the molecular mechanisms involved in the formation of structural or numerical chromosome abnormalities during meiosis. Here, we provide novel evidence indicating that lack of PARP-1 function during oogenesis predisposes the female gamete to genome instability. During prophase I of meiosis, a high proportion of Parp-1 (−/−) mouse oocytes exhibit a spectrum of meiotic defects including incomplete homologous chromosome synapsis or persistent histone H2AX phosphorylation in fully synapsed chromosomes at the late pachytene stage. Moreover, the X chromosome bivalent is also prone to exhibit persistent double strand DNA breaks (DSBs). In striking contrast, such defects were not detected in mutant pachytene spermatocytes. In fully-grown wild type oocytes at the germinal vesicle stage, PARP-1 protein associates with nuclear speckles and upon meiotic resumption, undergoes a striking re-localization towards spindle poles as well as pericentric heterochromatin domains at the metaphase II stage. Notably, a high proportion of in vivo matured Parp-1 (−/−) oocytes show lack of recruitment of the kinetochore-associated protein BUB3 to centromeric domains and fail to maintain metaphase II arrest. Defects in chromatin modifications in the form of persistent histone H2AX phosphorylation during prophase I of meiosis and deficient sister chromatid cohesion during metaphase II predispose mutant oocytes to premature anaphase II onset upon removal from the oviductal environment. Our results indicate that PARP-1 plays a critical role in the maintenance of chromosome stability at key stages of meiosis in the female germ line. Moreover, in the metaphase II stage oocyte PARP-1 is required for the regulation of centromere structure and function through a mechanism that involves the recruitment of BUB3 protein to centromeric domains. PMID:19463809

  5. The Effect of Poly(ADP-ribose) Polymerase-1 Gene 3′Untranslated Region Polymorphism in Colorectal Cancer Risk among Saudi Cohort

    PubMed Central

    Alhadheq, Abdullah M.; Purusottapatnam Shaik, Jilani; Alamri, Abdullah; Aljebreen, Abdulrahman M.; Alharbi, Othman; Almadi, Majid A.; Alhadeq, Faten; Azzam, Nahla A.; Alanazi, Mohammad; Bazzi, Mohammad D.

    2016-01-01

    Background. DNA repair systems are essential for each cell to repair and maintain the genome integrity. Base excision repair pathway is one of the crucial pathways to maintain genome integrity and PARP-1 plays a key role in BER pathway. The purpose of this study is to evaluate the association between polymorphisms in PARP-1 3′untranslated region (3′UTR) SNP rs8679 and its expression in colorectal cancer. Methods. Genotyping and gene expression were performed using TaqMan assays. The effects of age, gender, and tumor location were evaluated in cases and controls regarding the genotyping results. Resulting data was analyzed using SPSS software. Results and Conclusions. Genotyping analysis for SNP rs8679 showed decreased susceptibility to colorectal cancer at heterozygous TC allele and at minor allele C. Further this protective association was also observed in younger age patients (≤57), in female patients, and also in patients with tumors located at colon and rectum. PARP-1 expression levels are significantly different in colorectal cancer compared to matched normal tissue. Our findings proved that the upregulation of PARP-1 is associated with tumor progression and poor prognosis in Saudi patients with colorectal cancer, suggesting that PARP-1 can be novel and valuable signatures for predicting the clinical outcome of patients with colorectal cancer. PMID:27746584

  6. Poly(ADP-ribosyl)ation of a herpes simplex virus immediate early polypeptide

    SciTech Connect

    Preston, C.M.; Notarianni, E.L.

    1983-12-01

    In vitro poly(ADP-ribosyl)ation of the herpes simplex virus type 1 (HSV-1) immediate early polypeptide Vmw175 is reported. The phenomenon was most clearly observed by use of the temperature-sensitive mutant tsK, which overproduces Vmw175 at the nonpermissive temperature (NPT) and has a mutation in the coding sequences for this polypeptide. Nuclei prepared from cells which were infected with tsK at NPT and subsequently downshifted to the permissive temperature incorporated (/sup 32/P)NAD into Vmw175. This reaction did not occur when nuclei were prepared from cells constantly maintained at NPT, showing that only functional Vmw175 can be radiolabeled with (/sup 32/P)NAD. The identity of the acceptor protein was confirmed by demonstrating the expected electrophoretic mobility differences between the HSV-1 and HSV-2 counterparts of Vmw175. The use of suitable inhibitors demonstrated that the reaction represented mono- or poly(ADP-ribosyl)ation, and further analysis showed the presence of long poly(ADP-ribose) chains attached to Vmw175. Poly(ADP-ribosyl)ation may be important as a cause or result of the regulation of viral transcription by Vmw175. Radiolabeling of another virus-specified polypeptide (approximate molecular weight 38,000), thought to be a structural component of the input virus, is also reported.

  7. Excessive stimulation of poly(ADP-ribosyl)ation contributes to endothelial dysfunction in pre-eclampsia

    PubMed Central

    Crocker, Ian P; Kenny, Louise C; Thornton, Wayne A; Szabo, Csaba; Baker, Philip N

    2004-01-01

    Pre-eclampsia is a serious pregnancy disorder associated with widespread activation of the maternal vascular endothelium. Recent evidence implicates a role for oxidative stress in the aetiology of this condition. Reactive oxygen species, particularly superoxide anions, invokes endothelial cell activation through many pathways. Oxidant-induced cell injury triggers the activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP) leading to endothelial dysfunction in various pathophysiological conditions (reperfusion, shock, diabetes). We have studied whether the loss of endothelial function in pre-eclampsia is dependent on PARP activity. Endothelium-dependent responses of myometrial arteries were tested following exposure to either plasma from women with pre-eclampsia or normal pregnant women in the presence and absence of a novel potent inhibitor of PARP, PJ34. Additional effects of plasma and PJ34 inhibition were identified in microvascular endothelial cell cultures. In myometrial arteries, PARP inhibition blocked the attenuation of endothelium-dependent responses following exposure to plasma from women with pre-eclampsia. In endothelial cell cultures, plasma from pre-eclamptics induced measurable oxidative stress and a concomitant increase in PARP activity and reduction in cellular ATP. Again, these biochemical changes were reversed by PJ34. These results suggest that PARP activity plays a pathogenic role in the development of endothelial dysfunction in pre-eclampsia and promotes PARP inhibition as a potential therapy in this condition. PMID:15778700

  8. Poly-ADP ribosylation of PTEN by tankyrases promotes PTEN degradation and tumor growth

    PubMed Central

    Li, Nan; Zhang, Yajie; Han, Xin; Liang, Ke; Wang, Jiadong; Feng, Lin; Wang, Wenqi; Songyang, Zhou; Lin, Chunru; Yang, Liuqing; Yu, Yonghao

    2015-01-01

    PTEN [phosphatidylinositol (3,4,5)-trisphosphate phosphatase and tensin homolog deleted from chromosome 10], a phosphatase and critical tumor suppressor, is regulated by numerous post-translational modifications, including phosphorylation, ubiquitination, acetylation, and SUMOylation, which affect PTEN localization and protein stability. Here we report ADP-ribosylation as a new post-translational modification of PTEN. We identified PTEN as a novel substrate of tankyrases, which are members of the poly(ADP-ribose) polymerases (PARPs). We showed that tankyrases interact with and ribosylate PTEN, which promotes the recognition of PTEN by a PAR-binding E3 ubiquitin ligase, RNF146, leading to PTEN ubiquitination and degradation. Double knockdown of tankyrase1/2 stabilized PTEN, resulting in the subsequent down-regulation of AKT phosphorylation and thus suppressed cell proliferation and glycolysis in vitro and tumor growth in vivo. Furthermore, tankyrases were up-regulated and negatively correlated with PTEN expression in human colon carcinomas. Together, our study revealed a new regulation of PTEN and highlighted a role for tankyrases in the PTEN–AKT pathway that can be explored further for cancer treatment. PMID:25547115

  9. Poly(ADP-ribosyl)ation enhances H-RAS protein stability and causes abnormal cell cycle progression in human TK6 lymphoblastoid cells treated with hydroquinone.

    PubMed

    Liu, Linhua; Ling, Xiaoxuan; Tang, Huanwen; Chen, Jialong; Wen, Qiaosheng; Zou, Fei

    2015-08-05

    Hydroquinone (HQ), one of the most important benzene-derived metabolites, can induce aberrant cell cycle progression; however, the mechanism of this induction remains unclear. Poly(ADP-ribosyl)ation (PARylation), which is catalysed primarily by poly(ADP-ribose) polymerase-1 (PARP-1), participates in various biological processes, including cell cycle control. The results of the present study show an accumulation in G1 phase versus S phase of TK6 human lymphoblast cells treated with HQ for 48h compared with PBS-treated cells; after 72h of HQ treatment, the cells transitioned from G1 arrest to S phase arrest. We examined the expression of six genes related to the cell cycle or leukaemia to further explore the reason for this phenomenon. Among these genes, H-RAS was found to be associated with this phenomenon because its mRNA and protein expression decreased at 48h and increased at 72h. Experiments for PARP activity induction and inhibition revealed that the observed PARylation was positively associated with H-RAS expression. Moreover, in cells treated with HQ in conjunction with PARP-1 knockdown, expression of the H-RAS protein decreased and the number of cells in G1 phase increased. The degree of poly(ADP-ribosyl) modification of the H-RAS protein increased in cells treated with HQ for 72h, further supporting that changes in PARylation contributed to the rapid alteration of H-RAS protein expression, followed by abnormal progression of the cell cycle. Co-immunoprecipitation (co-IP) assays were employed to determine whether protein complexes were formed by PARP-1 and H-RAS proteins, and the direct interaction between these proteins indicated that PARylation regulated H-RAS expression. As detected by confocal microscopy, the H-RAS protein was found in the nucleus and cytoplasm. To our knowledge, this study is the first to reveal that H-RAS protein can be modified by PARylation.

  10. CTCF participates in DNA damage response via poly(ADP-ribosyl)ation

    PubMed Central

    Han, Deqiang; Chen, Qian; Shi, Jiazhong; Zhang, Feng; Yu, Xiaochun

    2017-01-01

    CCCTC-binding factor (CTCF) plays an essential role in regulating the structure of chromatin by binding DNA strands for defining the boundary between active and heterochromatic DNA. However, the role of CTCF in DNA damage response remains elusive. Here, we show that CTCF is quickly recruited to the sites of DNA damage. The fast recruitment is mediated by the zinc finger domain and poly (ADP-ribose) (PAR). Further analyses show that only three zinc finger motifs are essential for PAR recognition. Moreover, CTCF-deficient cells are hypersensitive to genotoxic stress such as ionizing radiation (IR). Taken together, these results suggest that CTCF participate in DNA damage response via poly(ADP-ribosylation). PMID:28262757

  11. The effect of poly(ADP-ribosyl)ation inhibition on the porcine cumulus-oocyte complex during in vitro maturation.

    PubMed

    Kim, Duk Hyoun; Lee, Hye Ran; Kim, Min Gyeong; Lee, Jun Sung; Jin, Su Jin; Lee, Hoon Taek

    2017-01-29

    Poly(ADP-ribosyl)ation (PARylation) plays important roles in DNA repair, apoptosis, transcriptional regulation, and cell death, and occurs via the activity of poly(ADP-ribose) polymerases (PARPs). Previous studies have shown that PARylation affects mouse and porcine pre-implantation development and participates in mechanisms of autophagy. However, there have not yet been reported the role of PARylation during in vitro maturation (IVM) of porcine oocytes. Thus, we investigated the effect of PARylation inhibition on this process; cumulus-oocyte complexes (COCs) were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during porcine IVM. Full cumulus expansion was significantly reduced (10.34 ± 1.23 [3-ABA] vs. 48.17 ± 2.03% [control]), but nuclear maturation rates were not changed in the 3-ABA treatment group. Especially, we observed that cumulus cells were little expanded after 22 h in 3-ABA treated COCs. The mRNA expression levels of oocyte maturation- and cumulus expansion-related genes were evaluated at 22 and 44 h. GDF9, BMP15, COX-2, and PTX3 expression were upregulated at 44 h, whereas the levels of HAS2 and TNFAIP6 were downregulated in the 3-ABA treated group. Furthermore, 3-ABA treatment significantly decreased the developmental rate (28.24 ± 1.06 vs. 40.24 ± 3.03%) and total cell number (41.12 ± 2.10 vs. 50.38 ± 2.27), but increased the total apoptotic index (6.44 ± 0.81 vs. 3.08 ± 0.51) in parthenogenetically activated embryos. In conclusion, these results showed that PARylation regulates cumulus expansion through the regulation of gene expression and affects developmental competence and quality in parthenogenetic embryos.

  12. Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.

    PubMed

    Mangerich, Aswin; Debiak, Malgorzata; Birtel, Matthias; Ponath, Viviane; Balszuweit, Frank; Lex, Kirsten; Martello, Rita; Burckhardt-Boer, Waltraud; Strobelt, Romano; Siegert, Markus; Thiermann, Horst; Steinritz, Dirk; Schmidt, Annette; Bürkle, Alexander

    2016-02-26

    Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of

  13. Suppression of dexamethasone-stimulated DNA synthesis in an oncogene construct containing rat cell line by a DNA site-oriented ligand of poly-ADP-ribose polymerase: 6-amino-1,2-benzopyrone

    SciTech Connect

    Kirsten, E.; Bauer, P.I.; Kun, E. San Francisco State Univ., CA )

    1991-03-01

    The cellular inhibitory effects of 6-amino-1,2-benzopyrone (6-ABP), a DNA site-specific ligand of adenosine diphosphoribosyl transferase (ADPRT), were determined in a dexamethasone-sensitive EJ-ras gene construct containing cell line (14C cells). Dexamethansone in vitro transforms these cells to a tumorigenic phenotype and also stimulates cell replication. AT a nontoxic concentration 6-ABP treatment of intact cells for 4 days inhibits the dexamethasone-stimulated increment of cellular DNA content, depresses replicative DNA synthesis as assayed by thymidine incorporation to the level of cells that were not exposed to dexamethasone, and in permeabilized cells reduces the dexamethasone-stimulated increase of deoxyribonucleotide incorporation into DNA to the level of untreated cells. In situ pulse labeling of cells pretreated with 6-ABP indicated an inhibition of DNA synthesis at a stage prior to the formation of the 10-kb intermediate species. Neither dexamethasone nor the drug influenced the cellular quantity of ADPRT molecules, tested immunochemically.

  14. Poly ADP ribosylation as a possible mechanism of microwave--biointeraction.

    PubMed

    Singh, N; Rudra, N; Bansal, P; Mathur, R; Behari, J; Nayar, U

    1994-07-01

    Electromagnetic fields (EMFs) affect the metabolism of the body including the nervous, endocrine, cardiovascular, hematological as well as the reproductive system. EMFs are environmental pollutants, thus posing a health hazard which can cause steric changes in the molecule located at the cell surface. Microwaves are known to cause chromosomal abberations and act as tumor promoters. The process involves a stream of signals from cell membrane to nucleus and other organelles. The present investigations aim to understand the mechanism of biological effects of microwaves (2.45 GHz). The effect was studied on poly ADP-ribosylation, which is a post translational modification of chromatin protein catalysed by the enzyme poly ADPR polymerase using NAD+ as the substrate. Poly ADP-ribosylation has been shown to be involved in several aspects of chromatin structure and function. Twenty-three days old rats weighing 42-48 gms were exposed at a microwave dose level of 1.0 mW/cm2. After exposure for sixty days the animals were sacrificed and an estimation of poly ADPR polymerase activity was undertaken in different organs of these animals. There was an increase of 20% in its activity in liver, 35% in testis, whereas brain showed a 53% decrease in diencephalon and 20% decrease in the cortex in the exposed animals as compared to their respective controls. There was no change in enzyme activity in spleen and kidney. This was accompanied by concomitant changes in NAD+ levels. The above results may be cited as important events in carcinogenesis and tumor promotion related to microwave exposure and the signal transduction mechanism involved. The goal is to shed light on complex ecogenetic interactions leading to cancer modulation of gene expression by epigenetic mechanism.

  15. Tankyrase-1 Ankyrin Repeats Form an Adaptable Binding Platform for Targets of ADP-Ribose Modification.

    PubMed

    Eisemann, Travis; McCauley, Michael; Langelier, Marie-France; Gupta, Kushol; Roy, Swati; Van Duyne, Gregory D; Pascal, John M

    2016-10-04

    The poly(ADP-ribose) polymerase enzyme Tankyrase-1 (TNKS) regulates multiple cellular processes and interacts with diverse proteins using five ankyrin repeat clusters (ARCs). There are limited structural insights into functional roles of the multiple ARCs of TNKS. Here we present the ARC1-3 crystal structure and employ small-angle X-ray scattering (SAXS) to investigate solution conformations of the complete ankyrin repeat domain. Mutagenesis and binding studies using the bivalent TNKS binding domain of Axin1 demonstrate that only certain ARC combinations function together. The physical basis for these restrictions is explained by both rigid and flexible ankyrin repeat elements determined in our structural analysis. SAXS analysis is consistent with a dynamic ensemble of TNKS ankyrin repeat conformations modulated by Axin1 interaction. TNKS ankyrin repeat domain is thus an adaptable binding platform with structural features that can explain selectivity toward diverse proteins, and has implications for TNKS positioning of bound targets for poly(ADP-ribose) modification.

  16. Inhibition of poly(adenosine diphosphate-ribose) polymerase by the active form of vitamin D

    PubMed Central

    MABLEY, JON G.; WALLACE, REBECCA; PACHER, PÁL; MURPHY, KANNEGANTI; SZABÓ, CSABA

    2008-01-01

    Vitamin D is well characterized for its role in mineral homeostasis and maintenance of normal skeletal architecture. Vitamin D has been demonstrated to exert anti-inflammatory effects in a variety of disease states including diabetes, arthritis and inflammatory bowel disease. In these diseases poly[adenosine diphosphate (ADP)-ribose] polymerase (PARP) inhibitors have also proved effective as anti-inflammatory agents. Here we present data demonstrating that the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D3, is a PARP inhibitor. UV irradiation-mediated PARP activation in human keratinocytes can be inhibited by treatment with vitamin D, 7-dehydrocholesterol or 1α,25-dihydroxyvitamin D3. Inhibition of cytochrome P450 reversed the PARP inhibitory action of vitamin D and 7-dehydrocholesterol, indicating that conversion to 1α,25-dihydroxyvitamin D3 mediates their PARP inhibitory action. Vitamin D may protect keratinocytes against over-activation of PARP resulting from exposure to sunlight. PARP inhibition may contribute to the pharmacological and anti-inflammatory effects of vitamin D. PMID:17487428

  17. Activation and modulation of cardiac poly-adenosine diphosphate ribose polymerase activity in a rat model of brain death.

    PubMed

    Brain, John G; Rostron, Anthony J; Dark, John H; Kirby, John A

    2008-05-15

    DNA damage during transplantation can activate poly-adenosine diphosphate ribose polymerase (PARP) resulting in the generation of polymers of adenosine diphosphate-ribose (PAR). Excessive linkage of PAR to nuclear proteins can induce cell death, thereby limiting the function of transplanted organs. This study uses a rat model of brain death to determine the profile of PARP activation and whether mechanisms that lead to cell death can be ameliorated by appropriate donor resuscitation. The expression of PAR-linked nuclear proteins within cardiac myocytes was greatly increased after the induction of donor brain death. Importantly, infusion of noradrenaline or vasopressin to normalize the chronic hypotension produced by brain death reduced the expression of PAR to a level below baseline. These data suggest that chronic hypotension after donor brain death has the potential to limit cardiac function through the activation of PARP; however, this early cause of graft damage can be mitigated by appropriate donor resuscitation.

  18. ADP-ribose polymer - a novel and general biomarker of human cancers of head & neck, breast, and cervix

    PubMed Central

    2010-01-01

    Background Poly-ADP-ribosylation, a reversible post-translational modification of primarily chromosomal proteins, is involved in various cellular and molecular processes including carcinogenesis. ADP-ribose polymer or poly-ADP-ribose adducts are enzymatically added onto or stripped off the target chromosomal proteins during this metabolic process. Due to this, the chromatin superstructure is reversibly altered, which significantly influences the pattern of gene expression. We hypothesize that a decrease in the concentration of total poly-ADP-ribose adducts of peripheral blood lymphocyte (PBL) proteins strongly correlates with the incidence of human cancer. Results Using a novel immunoprobe assay, we show a statistically significant (P ≤ 0.001) reduction (~ 42 to 49%) in the level of poly-ADP-ribose adducts of PBL proteins of patients with advanced cancers of head & neck (H & N) region (comprising fourteen distinct cancers at different sites), breast and cervix in comparison to healthy controls. Conclusions These findings imply potential utility of the poly-ADP-ribose adducts of PBL proteins as a novel and general biomarker of human cancers with potentials of significant clinical and epidemiological applications. PMID:21034502

  19. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

    PubMed Central

    Sharifi, Reza; Morra, Rosa; Denise Appel, C; Tallis, Michael; Chioza, Barry; Jankevicius, Gytis; Simpson, Michael A; Matic, Ivan; Ozkan, Ege; Golia, Barbara; Schellenberg, Matthew J; Weston, Ria; Williams, Jason G; Rossi, Marianna N; Galehdari, Hamid; Krahn, Juno; Wan, Alexander; Trembath, Richard C; Crosby, Andrew H; Ahel, Dragana; Hay, Ron; Ladurner, Andreas G; Timinszky, Gyula; Williams, R Scott; Ahel, Ivan

    2013-01-01

    Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans. PMID:23481255

  20. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    PubMed

    Neuvonen, Maarit; Ahola, Tero

    2009-01-09

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  1. In silico characterization of the family of PARP-like poly(ADP-ribosyl)transferases (pARTs)

    PubMed Central

    Otto, Helge; Reche, Pedro A; Bazan, Fernando; Dittmar, Katharina; Haag, Friedrich; Koch-Nolte, Friedrich

    2005-01-01

    Background ADP-ribosylation is an enzyme-catalyzed posttranslational protein modification in which mono(ADP-ribosyl)transferases (mARTs) and poly(ADP-ribosyl)transferases (pARTs) transfer the ADP-ribose moiety from NAD onto specific amino acid side chains and/or ADP-ribose units on target proteins. Results Using a combination of database search tools we identified the genes encoding recognizable pART domains in the public genome databases. In humans, the pART family encompasses 17 members. For 16 of these genes, an orthologue exists also in the mouse, rat, and pufferfish. Based on the degree of amino acid sequence similarity in the catalytic domain, conserved intron positions, and fused protein domains, pARTs can be divided into five major subgroups. All six members of groups 1 and 2 contain the H-Y-E trias of amino acid residues found also in the active sites of Diphtheria toxin and Pseudomonas exotoxin A, while the eleven members of groups 3 – 5 carry variations of this motif. The pART catalytic domain is found associated in Lego-like fashion with a variety of domains, including nucleic acid-binding, protein-protein interaction, and ubiquitylation domains. Some of these domain associations appear to be very ancient since they are observed also in insects, fungi, amoebae, and plants. The recently completed genome of the pufferfish T. nigroviridis contains recognizable orthologues for all pARTs except for pART7. The nearly completed albeit still fragmentary chicken genome contains recognizable orthologues for twelve pARTs. Simpler eucaryotes generally contain fewer pARTs: two in the fly D. melanogaster, three each in the mosquito A. gambiae, the nematode C. elegans, and the ascomycete microfungus G. zeae, six in the amoeba E. histolytica, nine in the slime mold D. discoideum, and ten in the cress plant A. thaliana. GenBank contains two pART homologues from the large double stranded DNA viruses Chilo iridescent virus and Bacteriophage Aeh1 and only a single entry

  2. Prognostic and clinicopathological value of poly (adenosine diphosphate-ribose) polymerase expression in breast cancer: A meta-analysis

    PubMed Central

    Qiao, Weiqiang; Pan, Linlin; Kou, Changgui; Li, Ke; Yang, Ming

    2017-01-01

    Background Previous studies have shown that the poly (adenosine diphosphate-ribose) polymerase (PARP) level is a promising indicator of breast cancer. However, its prognostic value remains controversial. The present meta-analysis evaluated the prognostic value of PARP expression in breast cancer. Materials and methods Eligible studies were retrieved from the PubMed, Web of Science, Embase, and Cochrane Library databases through July 20, 2016. Studies investigating PARP expression as well as reporting survival data in breast cancer were included. Two independent reviewers carried out all literature searches. The pooled relative risk (RR) and hazard ratio (HR) with 95% confidence interval (95% CI) were applied to assess the association between PARP expression and the clinicopathological features and survival outcome in breast cancer. Results A total of 3506 patients from eight eligible studies were included. We found that higher PARP expression indicated a worse clinical outcome in early stage breast cancer, with a HR of 3.08 (95% CI, 1.14–8.29, P = 0.03) for disease-free survival and a HR of 1.82 (95% CI, 1.20–2.76; P = 0.005) for overall survival. Moreover, increased PARP expression was significantly associated with higher nuclear grade (RR, 1.51; 95% CI, 1.12–2.04; P = 0.008) in breast cancer. A similar correlation was detected in triple-negative breast cancer (TNBC; RR, 1.81; 95% CI, 1.04–3.17; P = 0.04). Conclusions Our findings indicated that elevated PARP expression correlated with worse prognosis in early stage breast cancer. Furthermore, high PARP expression was associated with higher nuclear grade and TNBC. PMID:28212434

  3. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging

    PubMed Central

    Sukhanova, Maria V.; Abrakhi, Sanae; Joshi, Vandana; Pastre, David; Kutuzov, Mikhail M.; Anarbaev, Rashid O.; Curmi, Patrick A.; Hamon, Loic; Lavrik, Olga I.

    2016-01-01

    PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages. PMID:26673720

  4. Real-Time Cellular Imaging of Protein Poly(ADP-ribos)ylation.

    PubMed

    Buntz, Annette; Wallrodt, Sarah; Gwosch, Eva; Schmalz, Michael; Beneke, Sascha; Ferrando-May, Elisa; Marx, Andreas; Zumbusch, Andreas

    2016-09-05

    Poly(ADP-ribos)ylation (PARylation) is an important posttranslational protein modification, and is involved in major cellular processes such as gene regulation and DNA repair. Its dysregulation has been linked to several diseases, including cancer. Despite its importance, methods to observe PARylation dynamics within cells are rare. By following a chemical biology approach, we developed a fluorescent NAD(+) analogue that proved to be a competitive building block for protein PARylation in vitro and in cells. This allowed us to directly monitor the turnover of PAR in living cells at DNA damage sites after near-infrared (NIR) microirradiation. Additionally, covalent and noncovalent interactions of selected target proteins with PAR chains were visualized in cells by using FLIM-FRET microscopy. Our results open up new opportunities for the study of protein PARylation in real time and in live cells, and will thus contribute to a better understanding of its significance in a cellular context.

  5. Poly(ADP-ribosylation) regulates chromatin organization through histone H3 modification and DNA methylation of the first cell cycle of mouse embryos

    SciTech Connect

    Osada, Tomoharu; Rydén, Anna-Margareta; Masutani, Mitsuko

    2013-04-26

    Highlights: •Histone modification of the mouse pronuclei is regulated by poly(ADP-ribosylation). •Hypermethylation of the mouse female pronuclei is maintained by poly(ADP-ribosylation). •Parp1 is physically interacted with Suz12, which may function in the pronuclei. •Poly(ADP-ribosylation) affects ultrastructure of chromatin of the mouse pronucleus. -- Abstract: We examined the roles of poly(ADP-ribosylation) in chromatin remodeling during the first cell cycle of mouse embryos. Drug-based inhibition of poly(ADP-ribosylation) by a PARP inhibitor, PJ-34, revealed up-regulation of dimethylation of histone H3 at lysine 4 in male pronuclei and down-regulation of dimethylation of histone H3 at lysine 9 (H3K9) and lysine 27 (H3K27). Association of poly(ADP-ribosylation) with histone modification was suggested to be supported by the interaction of Suz12, a histone methyltransferase in the polycomb complex, with Parp1. PARP activity was suggested to be required for a proper localization and maintenance of Suz12 on chromosomes. Notably, DNA methylation level of female pronuclei in one-cell embryos was robustly decreased by PJ-34. Electron microscopic analysis showed a frequent appearance of unusual electron-dense areas within the female pronuclei, implying the disorganized and hypercondensed chromatin ultrastructure. These results show that poly(ADP-ribosylation) is important for the integrity of non-equivalent epigenetic dynamics of pronuclei during the first cell cycle of mouse embryos.

  6. 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

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

    PubMed

    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

    2016-12-01

    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.

  8. Reduced nicotinamide adenine dinucleotide fluorescence lifetime detected poly(adenosine-5'-diphosphate-ribose) polymerase-1-mediated cell death and therapeutic effect of pyruvate

    NASA Astrophysics Data System (ADS)

    Guo, Han-Wen; Wei, Yau-Huei; Wang, Hsing-Wen

    2011-06-01

    Noninvasive detection of cell death has the potential for definitive diagnosis and monitoring treatment outcomes in real time. Reduced nicotinamide adenine dinucleotide (NADH) fluorescence intensity has long been used as a noninvasive optical probe of metabolic states. NADH fluorescence lifetime has recently been studied for its potential as an alternative optical probe of cellular metabolic states and cell death. In this study, we investigated the potential using NADH fluorescence intensity and/or lifetime to detect poly(adenosine-5'-diphosphate-ribose) polymerase-1 (PARP-1)-mediated cell death in HeLa cells. We also examined if NADH signals respond to treatment by pyruvate. The mechanism of PARP-1-mediated cell death has been well studied that extensive PARP-1 activation leads to cytosolic nicotinamide adenine dinucleotide depletion resulting in glycolytic inhibition, mitochondrial failure, and death. Pyruvate could restore electron transport chain to prevent energy failure and death. Our results show that NADH fluorescence lifetime, not intensity, responded to PARP-1-mediated cell death and the rescue effect of pyruvate. This lifetime change of NADH fluorescence happened before the collapse of mitochondrial membrane potential and mitochondrial uncoupling. Together with our previous findings in staurosporine-induced cell death, we suggest that NADH fluorescence lifetime increase during cell death is mainly due to increased protein-protein interactions but not the intracellular NADH content.

  9. Ribose 2'-O methylation of the vesicular stomatitis virus mRNA cap precedes and facilitates subsequent guanine-N-7 methylation by the large polymerase protein.

    PubMed

    Rahmeh, Amal A; Li, Jianrong; Kranzusch, Philip J; Whelan, Sean P J

    2009-11-01

    During conventional mRNA cap formation, two separate methyltransferases sequentially modify the cap structure, first at the guanine-N-7 (G-N-7) position and subsequently at the ribose 2'-O position. For vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses, the two methylase activities share a binding site for the methyl donor S-adenosyl-l-methionine and are inhibited by individual amino acid substitutions within the C-terminal domain of the large (L) polymerase protein. This led to the suggestion that a single methylase domain functions for both 2'-O and G-N-7 methylations. Here we report a trans-methylation assay that recapitulates both ribose 2'-O and G-N-7 modifications by using purified recombinant L and in vitro-synthesized RNA. Using this assay, we demonstrate that VSV L typically modifies the 2'-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2'-O methylation of the substrate RNA. Amino acid substitutions in the C terminus of L that prevent all cap methylation in recombinant VSV (rVSV) partially retain the ability to G-N-7 methylate a pre-2'-O-methylated RNA, therefore uncoupling the effect of substitutions in the C terminus of the L protein on the two methylations. In addition, we show that the 2'-O and G-N-7 methylase activities act specifically on RNA substrates that contain the conserved elements of a VSV mRNA start at the 5' terminus. This study provides new mechanistic insights into the mRNA cap methylase activities of VSV L, demonstrates that 2'-O methylation precedes and facilitates subsequent G-N-7 methylation, and reveals an RNA sequence and length requirement for the two methylase activities. We propose a model of regulation of the activity of the C terminus of L protein in 2'-O and G-N-7 methylation of the cap structure.

  10. Co-targeting Deoxyribonucleic Acid–Dependent Protein Kinase and Poly(Adenosine Diphosphate-Ribose) Polymerase-1 Promotes Accelerated Senescence of Irradiated Cancer Cells

    SciTech Connect

    Azad, Arun; Bukczynska, Patricia; Jackson, Susan; Haput, Ygal; Cullinane, Carleen; McArthur, Grant A.; Solomon, Benjamin

    2014-02-01

    Purpose: 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. Methods and Materials: 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. Results: 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. Conclusion: 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.

  11. Characterization of polymers of adenosine diphosphate ribose generated in vitro and in vivo.

    PubMed

    Alvarez-Gonzalez, R; Jacobson, M K

    1987-06-02

    Methods have been developed and applied to determine the size and branching frequency of polymers of ADP-ribose synthesized in nucleotide-permeable cultured mouse cells and in intact cultured cells. Polymers were purified by affinity chromatography with a boronate resin and were fractionated according to size molecular sieve high-performance liquid chromatography. Fractions were enzymatically digested to nucleotides, which were separated by strong anion exchange high-performance liquid chromatography. From these data, average polymer size and branching frequency were calculated. A wide range of polymer sizes was observed. Polymers as large as 190 residues with at least five points of branching per molecule were generated in vitro. Polymers of up to 67 residues containing up to two points of branching per molecule were isolated from intact cells following treatment with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Cells treated with hyperthermia prior to DNA damage contained polymers of an average maximum size of 244 residues containing up to six points of branching per molecule. The detection of large polymers of ADP-ribose in intact cells suggests that alterations in chromatin organization effected by poly(ADP-ribosylation) may extend beyond the covalently modified proteins and very likely involve noncovalent interactions of poly(ADP-ribose) with other components of chromatin.

  12. Role of CTCF poly(ADP-Ribosyl)ation in the regulation of apoptosis in breast cancer cells

    PubMed Central

    Venkatraman, Bhooma; Klenova, Elena

    2015-01-01

    Introduction: CTCF is a candidate tumor suppressor gene encoding a multifunctional transcription factor. CTCF function is controlled by posttranslational modification and interaction with other proteins. Research findings suggested that CTCF function can be regulated by poly(ADP-ribosyl)ation (PARlation) and has specific anti-apoptotic function in breast cancer cells. The aim of this study is to assess the effect of CTCF-wild type (WT) and CTCF complete mutant, which is deficient of PARlation in regulating apoptosis in breast cancer cells. Materials and Methods: The effect of CTCF-WT and CTCF complete mutant was expressed in breast cancer cell-lines by DNA-mediated transfection technique monitored by enhanced green fluorescent protein fluorescence. Evaluation of apoptotic cell death was carried out with immunohistochemical staining using 4’-6’-diamino-2 phenylindole and scoring by fluorescent microscopy. Results: CTCF-WT supports survival of breast cancer cells and was observed that CTCF complete mutant interferes with the functions of the CTCF-WT and there was a considerable apoptotic cell death in the breast cancer cell lines such as MDA-MB-435, CAMA-1 and MCF-7. Conclusion: The study enlighten CTCF as a Biological Marker for breast cancer and the role of CTCF PARlation may be involved in breast carcinogenesis. PMID:25810575

  13. DNA polymerase β-dependent cell survival independent of XRCC1 expression

    PubMed Central

    Horton, Julie K.; Gassman, Natalie R.; Dunigan, Brittany B.; Stefanick, Donna F.; Wilson, Samuel H.

    2014-01-01

    Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1+/+ and Xrcc1−/− mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β- dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants. PMID:25541391

  14. The Treatment of BRCA1/2 Hereditary Breast Cancer and Sporadic Breast Cancer with Poly(ADP-ribose) PARP-1 Inhibitors and Chemotherapy

    DTIC Science & Technology

    2008-09-01

    American population d) D) Obesity, and breast cancer J. of Nursing and Bariatric Surgery . 2008 submitting. This paper uses in part mechanisms worked...National Med. Society. 2008 submitting D) Obesity, and breast cancer J. of Nursing and Bariatric Surgery . 2008 submitting Abstracts: A) De Soto JA...submitting De Soto JA. Obesity, breast cancer and bariatric surgery . J. of Nursing and Bariatric Surgery . 2008 submitting Davis JH, De Soto JA

  15. 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

  16. Activation of Telomerase by Ionizing Radiation: Differential Response to the Inhibition of DNA Double-Strand Break Repair by Abrogation of Poly(ADP-ribosyl)ation, by LY294002, or by Wortmannin

    SciTech Connect

    Neuhof, Dirk Zwicker, Felix; Kuepper, Jan-Heiner; Debus, Juergen; Weber, Klaus-Josef

    2007-11-01

    Purpose: Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. Methods and Materials: COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10 Gy. Phosphorylation status of PKB/Akt and of PKC{alpha}/{beta}{sub II} was assessed by western blotting. Results: No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. Conclusions: Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than {alpha}/{beta}{sub II} cannot be ruled out.

  17. The Key Involvement of Poly(ADP-Ribosyl)ation in Defense Against Toxic Agents: Molecular Biology Studies

    DTIC Science & Technology

    2008-02-19

    in all species of phloem sap-feeding insects , was reported to contain only 182 open reading frames (Nakabachi, el al, 2006). Even though this gene...including treatment with ethidium bromide (EtBr) (King, and Attardi, 1989) and silencing of the mt-DNA polymerase by RNAi (Khan, and Bennett, 2004

  18. In vivo exposure of swiss albino mice to chronic low dose of dimethylnitrosamine (DMN) lowers poly-ADP-ribosylation (PAR) of bone marrow cell and blood lymphocyte proteins.

    PubMed

    Kma, L; Sharan, R N

    2006-08-01

    Efforts to identify an easy and convenient biomarker of carcinogenesis with potentials of application in mass screening program continue. In a series of investigations on mice exposed to different carcinogens, poly-ADP-ribosylation (PAR) of cellular proteins of different tissues has been shown to be a potential biomarker of carcinogenesis. Because blood based biomarker of carcinogenesis offers significant advantage in its use in a cancer screening program, this investigation was undertaken to find correlations between initiation of carcinogenesis and PAR of bone marrow cell (BMC) and blood lymphocyte (BL) proteins in mice chronically exposed to low dose of dimethylnitrosamine (DMN) for up to four weeks in vivo. The exposure was either alone or in combination with 3-aminobenzamide (3-AB), an inhibitor of PAR. Total PAR of cellular proteins and of histone H1 protein were monitored by slot and Western blot immunoprobe assays, respectively. The PAR of total cellular proteins as well as of histone H1 was down-regulated in duration of exposure dependent manners. The results suggest that BMC and BL mirrored status of PAR in other tissues. This finding opens up the possibility of using PAR as a biomarker of carcinogenesis in a blood based test utilizing immunoprobe assay of cellular PAR.

  19. Structure and function of the ARH family of ADP-ribosyl-acceptor hydrolases.

    PubMed

    Mashimo, Masato; Kato, Jiro; Moss, Joel

    2014-11-01

    ADP-ribosylation is a post-translational protein modification, in which ADP-ribose is transferred from nicotinamide adenine dinucleotide (NAD(+)) to specific acceptors, thereby altering their activities. The ADP-ribose transfer reactions are divided into mono- and poly-(ADP-ribosyl)ation. Cellular ADP-ribosylation levels are tightly regulated by enzymes that transfer ADP-ribose to acceptor proteins (e.g., ADP-ribosyltransferases, poly-(ADP-ribose) polymerases (PARP)) and those that cleave the linkage between ADP-ribose and acceptor (e.g., ADP-ribosyl-acceptor hydrolases (ARH), poly-(ADP-ribose) glycohydrolases (PARG)), thereby constituting an ADP-ribosylation cycle. This review summarizes current findings related to the ARH family of proteins. This family comprises three members (ARH1-3) with similar size (39kDa) and amino acid sequence. ARH1 catalyzes the hydrolysis of the N-glycosidic bond of mono-(ADP-ribosyl)ated arginine. ARH3 hydrolyzes poly-(ADP-ribose) (PAR) and O-acetyl-ADP-ribose. The different substrate specificities of ARH1 and ARH3 contribute to their unique roles in the cell. Based on a phenotype analysis of ARH1(-/-) and ARH3(-/-) mice, ARH1 is involved in the action by bacterial toxins as well as in tumorigenesis. ARH3 participates in the degradation of PAR that is synthesized by PARP1 in response to oxidative stress-induced DNA damage; this hydrolytic reaction suppresses PAR-mediated cell death, a pathway termed parthanatos.

  20. Negative correlation between poly-ADP-ribosylation of spleen cell histone proteins and initial duration of dimethylnitrosamine exposure to mice in vivo measured by Western blot immunoprobe assay: a possible biomarker for cancer detection.

    PubMed

    Devi, Brahmacharimayum J; Schneeweiss, Frank H A; Sharan, Rajeshwar N

    2005-01-01

    Improved cancer detection involving suitable biomarkers with easy applicability is a challenge to our fight against cancer. Poly-ADP-ribosylation (PAR) of proteins is a likely candidate biomarker for this purpose because it meets the criterion well. This report is a step towards testing suitability of PAR as a biomarker for cancer detection. Swiss albino mice were exposed to hepatocarcinogen, dimethylnitrosamine (DMN), at a chronic dose, which is known to induce carcinogenesis in liver. PAR was monitored by a Western blot immunoprobe assay in spleen, a lymphoid organ, to find a correlation between PAR of spleen histone proteins and duration of DMN exposure. A negative, non-linear correlation was found for most histone proteins. The inhibition of PAR of histones was significant from 4 weeks onwards until the end of the observation. The inhibition was potentiated when 3-aminobenzamide was simultaneously administered. The results open up the possibility of PAR of cellular proteins being used as biomarker for cancer detection.

  1. p63 in Development and Maintenance of the Prostate Epithelium

    DTIC Science & Technology

    2010-03-01

    by performing genetic lineage tracing experiments. We plan to generate mice expressing inducible Cre recombinase (Cre-ERT2) under the control of...caspase 3 levels and a concomitant decrease in the expression total caspase 3. Accordingly, cleaved poly (ADP-ribose) polymerase (PARP) protein

  2. Laying a trap to kill cancer cells: PARP inhibitors and their mechanisms of action.

    PubMed

    Pommier, Yves; O'Connor, Mark J; de Bono, Johann

    2016-10-26

    Poly(ADP-ribose) polymerase (PARP) inhibitors are the first DNA damage response targeted agents approved for cancer therapy. Here, we focus on their molecular mechanism of action by PARP "trapping" and what this means for both clinical monotherapy and combination with chemotherapeutic agents.

  3. The development of PARP inhibitors in ovarian cancer: from bench to bedside

    PubMed Central

    Drew, Yvette

    2015-01-01

    The nuclear enzyme poly (ADP-ribose) polymerase (PARP) represents an important novel target in the treatment of ovarian cancer. This article charts over 50 years of research from the discovery of the first PARP enzyme in 1963, to the approval and licensing in 2015 of the first PARP inhibitor, olaparib (Lynparza), in the treatment of BRCA-mutated ovarian cancer. PMID:26669452

  4. Ca2+, Mg2+-dependent endonuclease and ADP-ribosylation.

    PubMed

    Yoshihara, K; Tanaka, Y; Kamiya, T

    1983-01-01

    The molecular mechanism of the inhibition of Ca2+, Mg2+-dependent endonuclease by ADP-ribosylation was studied by using purified bull seminal plasma Ca2+, Mg2+-dependent endonuclease, endonuclease-stimulating proteins, and poly-(ADP-ribose) polymerase. The activity of an essentially homogeneous preparation of the endonuclease was markedly suppressed by its preincubation with NAD+, poly-(ADP-ribose) polymerase, DNA, and Mg2+. These four components of the incubation mixture were all essential for the suppression of the activity. Analyses of the initial and the chased reaction product by Sephadex G-100 column chromatography and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed that Ca2+, Mg2+-dependent endonuclease was ADP-ribosylated during the incubation and its activity was markedly inhibited by the elongation of the ADP-ribose polymer covalently attached to the endonuclease. When the suppressed enzymes were mildly treated with an alkaline pH of 10.0, the activity was restored almost to the level of the unmodified control sample. These facts indicate that the linkage between the enzyme and poly(ADP-ribose) is hydrolyzed at this pH, and that the liberated polymer itself does not appreciably affect the endonuclease activity. These results also suggest that an electric repulsion between negative charges on DNA and poly(ADP-ribose) attached to Ca2+, Mg2+-dependent endonuclease is the basis for the observed suppression of the enzyme by ADP-ribosylation. Though histone H2B and H1 are shown to be as good endonuclease-stimulators (1) as they are good acceptors of ADP-ribose in poly(ADP-ribose) polymerase reaction (2), ADP-ribosylation of these two proteins did not affect their endonuclease-stimulating ability appreciably, at least under the conditions used.

  5. Identification, validation, and targeting of the mutant p53-PARP-MCM chromatin axis in triple negative breast cancer.

    PubMed

    Qiu, Wei-Gang; Polotskaia, Alla; Xiao, Gu; Di, Lia; Zhao, Yuhan; Hu, Wenwei; Philip, John; Hendrickson, Ronald C; Bargonetti, Jill

    2017-01-01

    Over 80% of triple negative breast cancers express mutant p53. Mutant p53 often gains oncogenic function suggesting that triple negative breast cancers may be driven by p53 protein type. To determine the chromatin targets of this gain-of-function mutant p53 we used inducible knockdown of endogenous gain-of-function mtp53 in MDA-MB-468 cells in conjunction with stable isotope labeling with amino acids in cell culture and subcellular fractionation. We sequenced over 70,000 total peptides for each corresponding reciprocal data set and were able to identify 3010 unique cytoplasmic fraction proteins and 3403 unique chromatin fraction proteins. The present proteomics experiment corroborated our previous experiment-based results that poly ADP-ribose polymerase has a positive association with mutant p53 on the chromatin. Here, for the first time we report that the heterohexomeric minichromosome maintenance complex that participates in DNA replication initiation ranked as a high mutant p53-chromatin associated pathway. Enrichment analysis identified the minichromosome maintenance members 2-7. To validate this mutant p53- poly ADP-ribose polymerase-minichromosome maintenance functional axis, we experimentally depleted R273H mutant p53 and found a large reduction of the amount of minichromosome maintenance complex proteins on the chromatin. Furthermore a mutant p53-minichromosome maintenance 2 direct interaction was detected. Overexpressed mutant p53, but not wild type p53, showed a protein-protein interaction with minichromosome maintenance 2 and minichromosome maintenance 4. To target the mutant p53- poly ADP-ribose polymerase-minichromosome maintenance axis we treated cells with the poly ADP-ribose polymerase inhibitor talazoparib and the alkylating agent temozolomide and detected synergistic activation of apoptosis only in the presence of mutant p53. Furthermore when minichromosome maintenance 2-7 activity was inhibited the synergistic activation of apoptosis was blocked

  6. D-ribose ameliorates cisplatin-induced nephrotoxicity by inhibiting renal inflammation in mice.

    PubMed

    Ueki, Masaaki; Ueno, Masaki; Morishita, Jun; Maekawa, Nobuhiro

    2013-01-01

    Cisplatin is one of the most potent chemotherapeutic anticancer drugs, but it can produce side effects such as nephrotoxicity. Inflammatory cytokines, chemokines and adhesion molecules have important roles in the pathogenesis of cisplatin-induced nephrotoxicity. D-Ribose is a naturally occurring five-carbon monosaccharide that is found in all living cells, and has anti-inflammatory effects in renal ischemia/reperfusion injury. The purpose of this study was to determine the protective effects of D-ribose on cisplatin-induced nephrotoxicity. Forty-eight mice were randomly divided into four groups: control, cisplatin, cisplatin + ribose, and ribose. Mice were given cisplatin (20 mg/kg body weight, intraperitoneally) with or without D-ribose (400 mg/kg body weight, intraperitoneally, immediately after cisplatin injection). At 72 h after cisplatin injection, we measured serum and renal tumor necrosis factor (TNF)-α and renal monocyte chemoattractant protein (MCP)-1 concentrations by enzyme-linked immunosorbent assay; renal expression of intercellular adhesion molecule (ICAM)-1 mRNA by real-time polymerase chain reaction; serum blood urea nitrogen and creatinine; and histological changes. Cisplatin increased serum and renal TNF-α concentrations, renal MCP-1 concentration, and renal ICAM-1 mRNA expression. Treatment with D-ribose attenuated the increase in serum and renal TNF-α concentrations, renal MCP-1 concentration, and renal ICAM-1 mRNA expression. Consequently, cisplatin-induced renal dysfunction and renal tubular necrosis were attenuated by D-ribose treatment. This is believed to be the first time that protective effects of D-ribose on cisplatin-induced nephrotoxicity via inhibition of inflammatory reactions have been investigated. Thus, D-ribose may become a new therapeutic candidate for the treatment of cisplatin-induced nephrotoxicity.

  7. Selective Stabilization of Ribose by Borate

    NASA Astrophysics Data System (ADS)

    Furukawa, Yoshihiro; Horiuchi, Mana; Kakegawa, Takeshi

    2013-10-01

    In this study, borate was found to selectively increase the stability of ribose over other aldopentoses. Ribose is the only sugar present in both early RNA-based biochemistry and contemporary DNA-based life, and the stability of ribose is of fundamental concern for determining the origin of early RNA-based biochemistry. The formose reaction is a potential process in the prebiotic synthesis of ribose and its stereoisomers arabinose, xylose, and lyxose. Ribose is the least stable of these aldopentoses, raising the fundamental question of whether it was originally a component of primitive RNA or was selected through biotic processes. Borate is known to increase the stability of aldopentoses, but the specific differences in the stabilization achieved among different stereoisomers remain unclear. In this study, it was found that the stabilities of all of the tested pentoses increased with the concentration of added borate, but notably, the stability of ribose increased the most. The predominant formation of complexes between borate and ribose was verified, in agreement with previous studies. This borate complex formation might have sequestered ribose from the isomerization and decomposition reactions, resulting in its selective stabilization. These findings indicate that ribose could have accumulated in borate-rich environments on the early Earth and suggest that ribose-based nucleotides combined with phosphate and nucleobases formed abiotically.

  8. 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

  9. Chronic PARP-1 inhibition reduces carotid vessel remodeling and oxidative damage of the dorsal hippocampus in spontaneously hypertensive rats

    PubMed Central

    Eros, Krisztian; Magyar, Klara; Deres, Laszlo; Skazel, Arpad; Riba, Adam; Vamos, Zoltan; Kalai, Tamas; Gallyas, Ferenc; Sumegi, Balazs; Toth, Kalman

    2017-01-01

    Vascular remodeling during chronic hypertension may impair the supply of tissues with oxygen, glucose and other compounds, potentially unleashing deleterious effects. In this study, we used Spontaneously Hypertensive Rats and normotensive Wistar-Kyoto rats with or without pharmacological inhibition of poly(ADP-ribose)polymerase-1 by an experimental compound L-2286, to evaluate carotid artery remodeling and consequent damage of neuronal tissue during hypertension. We observed elevated oxidative stress and profound thickening of the vascular wall with fibrotic tissue accumulation induced by elevated blood pressure. 32 weeks of L-2286 treatment attenuated these processes by modulating mitogen activated protein kinase phosphatase-1 cellular levels in carotid arteries. In hypertensive animals, vascular inflammation and endothelial dysfunction was observed by NF-κB nuclear accumulation and impaired vasodilation to acetylcholine, respectively. Pharmacological poly(ADP-ribose)polymerase-1 inhibition interfered in these processes and mitigated Apoptosis Inducing Factor dependent cell death events, thus improved structural and functional alterations of carotid arteries, without affecting blood pressure. Chronic poly(ADP-ribose)polymerase-1 inhibition protected neuronal tissue against oxidative damage, assessed by nitrotyrosine, 4-hydroxinonenal and 8-oxoguanosine immunohistochemistry in the area of Cornu ammonis 1 of the dorsal hippocampus in hypertensive rats. In this area, extensive pyramidal cell loss was also attenuated by treatment with lowered poly(ADP-ribose)polymer formation. It also preserved the structure of fissural arteries and attenuated perivascular white matter lesions and reactive astrogliosis in hypertensive rats. These data support the premise in which chronic poly(ADP-ribose)polymerase-1 inhibition has beneficial effects on hypertension related tissue damage both in vascular tissue and in the hippocampus by altering signaling events, reducing oxidative

  10. Dimethylnitrosamine-induced reduction in the level of poly-ADP-ribosylation of histone proteins of blood lymphocytes--a sensitive and reliable biomarker for early detection of cancer.

    PubMed

    Kma, Lakhan; Sharan, Rajeshwar Nath

    2014-01-01

    Poly-ADP-ribosylation (PAR) is a post-translational modification of mainly chromosomal proteins. It is known to be strongly involved in several molecular events, including nucleosome-remodelling and carcinogenesis. In this investigation, it was attempted to evaluate PAR level as a reliable biomarker for early detection of cancer in blood lymphocyte histones. PAR of isolated histone proteins was monitored in normal and dimethylnitrosamine (DMN)-exposed mice tissues using a novel ELISA-based immuno-probe assay developed in our laboratory. An inverse relationship was found between the level of PAR and period of DMN exposure in various histone proteins of blood lymphocytes and spleen cells. With the increase in the DMN exposure period, there was reduction in the PAR level of individual histones in both cases. It was also observed that the decrease in the level of PAR of histones resulted in progressive relaxation of genomic DNA, perhaps triggering activation of genes that are involved in initiation of transformation. The observed effect of carcinogen on the PAR of blood lymphocyte histones provided us with a handy tool for monitoring biochemical or physiological status of individuals exposed to carcinogens without obtaining biopsies of cancerous tissues, which involves several medical and ethical issues. Obtaining blood from any patient and separating blood lymphocytes are routine medical practices involving virtually no medical intervention, post-procedure medical care or trauma to a patient. Moreover, the immuno-probe assay is very simple, sensitive, reliable and cost-effective. Therefore, combined with the ease of preparation of blood lymphocytes and the simplicity of the technique, immuno-probe assay of PAR has the potential to be applied for mass screening of cancer. It appears to be a promising step in the ultimate goal of making cancer detection simple, sensitive and reliable in the near future.

  11. Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions.

    PubMed

    Debiak, Malgorzata; Lex, Kirsten; Ponath, Viviane; Burckhardt-Boer, Waltraud; Thiermann, Horst; Steinritz, Dirk; Schmidt, Annette; Mangerich, Aswin; Bürkle, Alexander

    2016-02-26

    Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard-induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy. PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP-ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2-chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence-based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose- and time-dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES-induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the

  12. Function of ZFAND3 in the DNA Damage Response

    DTIC Science & Technology

    2011-06-01

    replication fork, such as polymerase epsilon. Therefore, iPOND provides improved sensitivity of detection for replisome components compared to the...enrichment of DSB repair proteins Mre11, KU70/80 and the recombinase Rad51 at stalled forks. Our data suggest that persistently stalled forks collapse...approach: Poly(ADP) Ribose Polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair. J Clin Oncol 26(22

  13. Apoptosis of Theileria-infected lymphocytes induced upon parasite death involves activation of caspases 9 and 3.

    PubMed

    Guergnon, Julien; Dessauge, Frédéric; Langsley, Gordon; Garcia, Alphonse

    2003-08-01

    The intracellular parasite Theileria parva (T. parva) can infect bovine B and T-lymphocytes. T. parva-infected cells become transformed, and they survive and proliferate independently of exogenous growth factors. In vivo the uncontrolled cellular proliferation associated with lymphocyte transformation underlies the pathogenesis of the disease called East Coast Fever. The transformed state of parasitised cells can be reversed upon elimination of the parasite by specific theilericide drugs. In this study we found that elimination of the parasite by buparvaquone induces apoptosis of transformed B and CD8(+) T-lymphocytes. Apoptosis is accompanied by the activation of caspase 9 and caspase 3 and processing of poly(ADP ribose) polymerase and is inhibited by Z-VAD a general caspase inhibitor. Based on these observations, we propose that the lack of activation of a caspase 9 > caspase 3 > poly(ADP ribose) polymerase pathway is important and protects T. parva-transformed cells from spontaneous apoptosis.

  14. A randomized Phase II study of veliparib with temozolomide or carboplatin/paclitaxel versus placebo with carboplatin/paclitaxel in BRCA1/2 metastatic breast cancer: design and rationale.

    PubMed

    Isakoff, Steven J; Puhalla, Shannon; Domchek, Susan M; Friedlander, Michael; Kaufman, Bella; Robson, Mark; Telli, Melinda L; Diéras, Véronique; Han, Hyo Sook; Garber, Judy E; Johnson, Eric F; Maag, David; Qin, Qin; Giranda, Vincent L; Shepherd, Stacie P

    2017-02-01

    Veliparib is an orally administered poly(ADP-ribose) polymerase inhibitor that is being studied in Phase I-III clinical trials, including Phase III studies in non-small-cell lung cancer, ovarian cancer and breast cancer. Tumor cells with deleterious BRCA1 or BRCA2 mutations are deficient in homologous recombination DNA repair and are intrinsically sensitive to platinum therapy and poly(ADP-ribose) polymerase inhibitors. We describe herein the design and rationale of a Phase II trial investigating whether the addition of veliparib to temozolomide or carboplatin/paclitaxel provides clinical benefit over carboplatin/paclitaxel with placebo in patients with locally recurrent or metastatic breast cancer harboring a deleterious BRCA1 or BRCA2 germline mutation (Trial registration: EudraCT 2011-002913-12, NCT01506609).

  15. (-)-Botryodiplodin, A Unique Ribose Analog Toxin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many toxins owe their mechanisms of action to being structural analogs of essential metabolites, messengers or structural components. Examples range from tubo-curare to penicillin. Ribose plays a unique role in the metabolism of living organisms, whether prokaryotes or eukaryotes. It and its deri...

  16. Synthesis and biological activities of novel furo[2,3,4-jk][2]benzazepin-4(3H)-one derivatives.

    PubMed

    Ando, Kumiko; Akai, Yukiko; Kunitomo, Jun-Ichi; Yokomizo, Takehiko; Nakajima, Hidemitsu; Takeuchi, Tadayoshi; Yamashita, Masayuki; Ohta, Shunsaku; Ohishi, Takahiro; Ohishi, Yoshitaka

    2007-02-21

    A novel seven-membered lactam formation method has been established by intramolecular ring closure reaction of 4-bromo-(E)-3-[(2-alkylvinyl)carbonylamino]benzo[b]furans under Heck coupling conditions. A number of furo[2,3,4-jk][2]benzazepin-4(3H)-ones, tricyclicbenzo[b]furans, have been prepared by this method and evaluated for their leukotriene B(4) (LTB(4)) receptor and poly(ADP-ribose)polymerase-1 (PARP-1) inhibitory activities.

  17. Nicotinamide and the skin.

    PubMed

    Chen, Andrew C; Damian, Diona L

    2014-08-01

    Nicotinamide, an amide form of vitamin B3, boosts cellular energy and regulates poly-ADP-ribose-polymerase 1, an enzyme with important roles in DNA repair and the expression of inflammatory cytokines. Nicotinamide shows promise for the treatment of a wide range of dermatological conditions, including autoimmune blistering disorders, acne, rosacea, ageing skin and atopic dermatitis. In particular, recent studies have also shown it to be a potential agent for reducing actinic keratoses and preventing skin cancers.

  18. Oxidative Stress Increases the Blood Brain Barrier Permeability Resulting in Increased Incidence of Brain Metastasis in BRCA Mutation Carriers

    DTIC Science & Technology

    2012-02-01

    inhibitors and/or selenium in preventing BBB-induced damage by oxidative stress, and in inhibiting breast cancer metastasis to the brain. Further...since selenium has anti- cancer properties that are linked with protection against oxidative stress and Poly (ADP-ribose) polymerase (PARP) inhibitors...BRCA1 in breast cancer cells resulted in an increase in cell proliferation, anchorage-independent growth, cell migration, invasion and a loss of p21

  19. Effect of Her-2/neu Signaling on Sensitivity to TRAIL in Prostate Cancer

    DTIC Science & Technology

    2005-06-01

    understand the of free glucose [1], a high concentration of lactic acid [2, 3], role of tumor microenvironment in the biochemical functions low-oxygen...be sensitized first to become responsive to TRAIL. In this study, we observed that pretreatment of acetyl salicylic acid (ASA) augmented TRAIL...ASA followed by TRAIL treatment activated caspases (8, 9, and 3) and cleaved PARP ( poly (ADP-ribose) polymerase), the hallmark feature of apoptosis

  20. Evaluation of DNA Repair Function as a Predictor of Response in a Clinical Trial of PARP Inhibitor Monotherapy for Recurrent Ovarian Carcinoma

    DTIC Science & Technology

    2014-10-01

    recombination (HR) pathway of DNA repair. Previous work had shown that cancer cells with deleterious FA/HR pathway mutations are hypersensitive to poly...homologous recombination , nonhomologous end-joining (NHEJ), immunohistochemistry, poly(ADP-ribose) polymerase, Ku70, Ku80, PARP1, 53BP1, DNA -PK, Artemis...regulates five different DNA repair pathways (1, 2). Building on preclinical observations that defects in homologous recombination (HR) repair, which

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

    PubMed

    Shirai, Hidenori; Fujimori, Hiroaki; Gunji, Akemi; Maeda, Daisuke; Hirai, Takahisa; Poetsch, Anna R; Harada, Hiromi; Yoshida, Tomoko; Sasai, Keisuke; Okayasu, Ryuichi; Masutani, Mitsuko

    2013-05-24

    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(-/-) and poly(ADP-ribose) polymerase-1 deficient (Parp-1(-/-)) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg(-/-) cells were more sensitive to γ-irradiation than Parp-1(-/-) cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg(-/-) cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg(-/-) ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1(-/-) cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg(-/-) 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(-/-) cells were more sensitive to LET 70 keV/μm carbon-ion irradiation than Parp-1(-/-) cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24h. The induction level of p53 phophorylation at ser18 was not different between wild-type and Parg(-/-) cells. The augmented level of poly(ADP-ribose) accumulation was noted after carbon-ion irradiation compared to γ-irradiation even in the wild-type cells. An enhanced poly(ADP-ribose) accumulation was further observed in Parg(-/-) cells. Both Parg(-/-) cells and Parp-1(-/-) cells did not show sensitization to Fe-ion irradiation. Parg deficiency sensitizes mouse ES cells to a wide therapeutic range of LET radiation through the effects on

  2. Adrenomedullin in inflammatory process associated with experimental pulmonary fibrosis

    PubMed Central

    2011-01-01

    Background Adrenomedullin (AM), a 52-amino acid ringed-structure peptide with C-terminal amidation, was originally isolated from human pheochromocytoma. AM are widely distributed in various tissues and acts as a local vasoactive hormone in various conditions. Methods In the present study, we investigated the efficacy of AM on the animal model of bleomycin (BLM)-induced lung injury. Mice were subjected to intratracheal administration of BLM and were assigned to receive AM daily by an intraperitoneal injection of 200 ngr/kg. Results and Discussion Myeloperoxidase activity, lung histology, immunohistochemical analyses for cytokines and adhesion molecules expression, inducible nitric oxide synthase (iNOS), nitrotyrosine, and poly (ADP-ribose) polymerase (PARP) were performed one week after fibrosis induction. Lung histology and transforming growth factor beta (TGF-β) were performed 14 and 21 days after treatments. After bleomycin administration, AM-treated mice exhibited a reduced degree of lung damage and inflammation compared with BLM-treated mice, as shown by the reduction of (1) myeloperoxidase activity (MPO), (2) cytokines and adhesion molecules expression, (3) nitric oxide synthase expression, (4) the nitration of tyrosine residues, (5) poly (ADP-ribose) (PAR) formation, a product of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (6) transforming growth factor beta (TGF-β) (7)and the degree of lung injury. Conclusions Our results indicate that AM administration is able to prevent bleomycin induced lung injury through the down regulation of proinflammatory factors. PMID:21477302

  3. Synthesis of phosphonate and phostone analogues of ribose-1-phosphates

    PubMed Central

    Nasomjai, Pitak; Slawin, Alexandra M Z

    2009-01-01

    Summary The synthesis of phosphonate analogues of ribose-1-phosphate and 5-fluoro-5-deoxyribose-1-phosphate is described. Preparations of both the α- and β-phosphonate anomers are reported for the ribose and 5-fluoro-5-deoxyribose series and a synthesis of the corresponding cyclic phostones of each α-ribose is also reported. These compounds have been prepared as tools to probe the details of fluorometabolism in S. cattleya. PMID:19777136

  4. Identification, validation, and targeting of the mutant p53-PARP-MCM chromatin axis in triple negative breast cancer

    PubMed Central

    Qiu, Wei-Gang; Polotskaia, Alla; Xiao, Gu; Di, Lia; Zhao, Yuhan; Hu, Wenwei; Philip, John; Hendrickson, Ronald C.; Bargonetti, Jill

    2017-01-01

    Over 80% of triple negative breast cancers express mutant p53. Mutant p53 often gains oncogenic function suggesting that triple negative breast cancers may be driven by p53 protein type. To determine the chromatin targets of this gain-of-function mutant p53 we used inducible knockdown of endogenous gain-of-function mtp53 in MDA-MB-468 cells in conjunction with stable isotope labeling with amino acids in cell culture and subcellular fractionation. We sequenced over 70,000 total peptides for each corresponding reciprocal data set and were able to identify 3010 unique cytoplasmic fraction proteins and 3403 unique chromatin fraction proteins. The present proteomics experiment corroborated our previous experiment-based results that poly ADP-ribose polymerase has a positive association with mutant p53 on the chromatin. Here, for the first time we report that the heterohexomeric minichromosome maintenance complex that participates in DNA replication initiation ranked as a high mutant p53-chromatin associated pathway. Enrichment analysis identified the minichromosome maintenance members 2–7. To validate this mutant p53- poly ADP-ribose polymerase-minichromosome maintenance functional axis, we experimentally depleted R273H mutant p53 and found a large reduction of the amount of minichromosome maintenance complex proteins on the chromatin. Furthermore a mutant p53-minichromosome maintenance 2 direct interaction was detected. Overexpressed mutant p53, but not wild type p53, showed a protein-protein interaction with minichromosome maintenance 2 and minichromosome maintenance 4. To target the mutant p53- poly ADP-ribose polymerase-minichromosome maintenance axis we treated cells with the poly ADP-ribose polymerase inhibitor talazoparib and the alkylating agent temozolomide and detected synergistic activation of apoptosis only in the presence of mutant p53. Furthermore when minichromosome maintenance 2–7 activity was inhibited the synergistic activation of apoptosis was

  5. [Synthesis and activity evaluation of PARP-1 inhibitors with azaindole skeleton].

    PubMed

    Zhou, Jie; Zhu, Zhi-Xiang; Chen, Xiao-Guang; Xu, Bai-Ling

    2013-12-01

    PARP [poly(ADP-ribose)polymerase] represents a novel potential target in cancer therapy. It is involved in a DNA repair process by catalyzing the transfer of ADP-ribose units from NAD to a number of its substrate proteins. In this work, a series of novel azaindole derivatives was designed and synthesized. Moreover, 16 target molecules were screened and 8 compounds displayed inhibitory activity against PARP-1. It has been demonstrated that these azaindoles bearing cycloamine substituents at 2-position were active to both PARP-1 and PARP-2.

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

    SciTech Connect

    Zaalishvili, Giorgi; Margiani, Dina; Kutalia, Ketevan; Suladze, Saba; Zaalishvili, Tengiz

    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.

  7. PARP1 promotes nucleotide excision repair through DDB2 stabilization and recruitment of ALC1

    PubMed Central

    Vrouwe, Mischa G.; Marteijn, Jurgen A.; Typas, Dimitris; Luijsterburg, Martijn S.; Cansoy, Medine; Hensbergen, Paul; Deelder, André; de Groot, Anton; Matsumoto, Syota; Sugasawa, Kaoru; Thoma, Nicolas; Vermeulen, Wim; Vrieling, Harry

    2012-01-01

    The WD40-repeat protein DDB2 is essential for efficient recognition and subsequent removal of ultraviolet (UV)-induced DNA lesions by nucleotide excision repair (NER). However, how DDB2 promotes NER in chromatin is poorly understood. Here, we identify poly(ADP-ribose) polymerase 1 (PARP1) as a novel DDB2-associated factor. We demonstrate that DDB2 facilitated poly(ADP-ribosyl)ation of UV-damaged chromatin through the activity of PARP1, resulting in the recruitment of the chromatin-remodeling enzyme ALC1. Depletion of ALC1 rendered cells sensitive to UV and impaired repair of UV-induced DNA lesions. Additionally, DDB2 itself was targeted by poly(ADP-ribosyl)ation, resulting in increased protein stability and a prolonged chromatin retention time. Our in vitro and in vivo data support a model in which poly(ADP-ribosyl)ation of DDB2 suppresses DDB2 ubiquitylation and outline a molecular mechanism for PARP1-mediated regulation of NER through DDB2 stabilization and recruitment of the chromatin remodeler ALC1. PMID:23045548

  8. Ribose Accelerates Gut Motility and Suppresses Mouse Body Weight Gaining

    PubMed Central

    Liu, Yan; Li, Tong-Ruei R; Xu, Cong; Xu, Tian

    2016-01-01

    The increasing prevalence of obesity is closely related to excessive energy consumption. Clinical intervention of energy intake is an attractive strategy to fight obesity. However, the current FDA-approved weight-loss drugs all have significant side effects. Here we show that ribose upregulates gut motility and suppresses mice body weight gain. Ribokinase, which is encoded by Rbks gene, is the first enzyme for ribose metabolism in vivo. Rbks mutation resulted in ribose accumulation in the small intestine, which accelerated gut movement. Ribose oral treatment in wild type mice also enhanced bowel motility and rendered mice resistance to high fat diets. The suppressed weight gain was resulted from enhanced ingested food excretion. In addition, the effective dose of ribose didn't cause any known side effects (i.e. diarrhea and hypoglycemia). Overall, our results show that ribose can regulate gut motility and energy homeostasis in mice, and suggest that administration of ribose and its analogs could regulate gastrointestinal motility, providing a novel therapeutic approach for gastrointestinal dysfunction and weight control. PMID:27194947

  9. 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.

  10. Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs.

    PubMed

    Gagné, Jean-Philippe; Ethier, Chantal; Defoy, Daniel; Bourassa, Sylvie; Langelier, Marie-France; Riccio, Amanda A; Pascal, John M; Moon, Kyung-Mee; Foster, Leonard J; Ning, Zhibin; Figeys, Daniel; Droit, Arnaud; Poirier, Guy G

    2015-06-01

    An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process.

  11. L-Ribose isomerase and mannose-6-phosphate isomerase: properties and applications for L-ribose production.

    PubMed

    Xu, Zheng; Sha, Yuanyuan; Liu, Chao; Li, Sha; Liang, Jinfeng; Zhou, Jiahai; Xu, Hong

    2016-11-01

    L-Ribose is a synthetic L-form monosaccharide. It is a building block of many novel nucleotide analog anti-viral drugs. Bio-production of L-ribose relies on a two-step reaction: (i) conversion of L-arabinose to L-ribulose by the catalytic action of L-arabinose isomerase (L-AI) and (ii) conversion of L-ribulose to L-ribose by the catalytic action of L-ribose isomerase (L-RI, EC 5.3.1.B3) or mannose-6-phosphate isomerase (MPI, EC 5.3.1.8, alternately named as phosphomannose isomerase). Between the two enzymes, L-RI is a rare enzyme that was discovered in 1996 by Professor Izumori's group, whereas MPI is an essential enzyme in metabolic pathways in humans and microorganisms. Recent studies have focused on their potentials for industrial production of L-ribose. This review summarizes the applications of L-RI and MPI for L-ribose production.

  12. Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition.

    PubMed

    Berti, Matteo; Ray Chaudhuri, Arnab; Thangavel, Saravanabhavan; Gomathinayagam, Shivasankari; Kenig, Sasa; Vujanovic, Marko; Odreman, Federico; Glatter, Timo; Graziano, Simona; Mendoza-Maldonado, Ramiro; Marino, Francesca; Lucic, Bojana; Biasin, Valentina; Gstaiger, Matthias; Aebersold, Ruedi; Sidorova, Julia M; Monnat, Raymond J; Lopes, Massimo; Vindigni, Alessandro

    2013-03-01

    Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal through a process that requires poly(ADP-ribose) polymerase (PARP) activity. Whether regressed forks can efficiently restart and what factors are required to restart fork progression after fork reversal are still unknown. We have combined biochemical and EM approaches with single-molecule DNA fiber analysis to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition that is not shared by other human RecQ proteins. We show that the poly(ADP-ribosyl)ation activity of PARP1 stabilizes forks in the regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition.

  13. Molecular Toxicology of Chromatin

    DTIC Science & Technology

    1982-09-01

    Role of Poly ADP-ribose. Poly ADP-ribosylated proteins were quantitatively isolated from livers of normal and dimethylnitrosamine treated Syrian hamsters...histone proteins (r180-200 kd) were poly ADP-ribosylated at least 6-9 fold over controls, in livers of dimethylnitrosamine treated Syrian hamsters (ref...34Quantitative Isolation of Oligo- and Polyadenosine-diphosphorylated Proteins by Affinity Chromatography from Livers of Normal and Dimethylnitrosamine - treated

  14. On the Stabilization of Ribose by Silicate Minerals

    NASA Astrophysics Data System (ADS)

    Vázquez-Mayagoitia, Álvaro; Horton, Scott R.; Sumpter, Bobby G.; Šponer, Jiří; Šponer, Judit E.; Fuentes-Cabrera, Miguel

    2011-03-01

    The RNA-world theory hypothesizes that early Earth life was based on the RNA molecule. However, the notion that ribose, the sugar in RNA, is unstable still casts a serious doubt over this theory. Recently, it has been found that the silicate-mediated formose reaction facilitates the stabilization of ribose. Using accurate quantum chemical calculations, we determined the relative stability of the silicate complexes of arabinose, lyxose, ribose, and xylose with the intent to determine which would form predominantly from a formose-like reaction. Five stereoisomers were investigated for each complex. The stereoisomers of 2:1 ribose-silicate are the more stable ones, to the extent that the least stable of these is even more stable than the most stable stereoisomer of the other 2:1 sugar-silicate complexes. Thus, thermodynamically, a formose-like reaction in the presence of silicate minerals should preferentially form the silicate complex of ribose over the silicate complex of arabinose, lyxose, and xylose.

  15. Glycation and glycoxidation of histones by ADP-ribose.

    PubMed

    Cervantes-Laurean, D; Jacobson, E L; Jacobson, M K

    1996-05-03

    The reaction of long lived proteins with reducing sugars has been implicated in the pathophysiology of aging and age-related diseases. A likely intranuclear source of reducing sugar is ADP-ribose, which is generated following DNA damage from the turnover of ADP-ribose polymers. In this study, ADP-ribose has been shown to be a potent histone glycation and glycoxidation agent in vitro. Incubation of ADP-ribose with histones H1, H2A, H2B, and H4 at pH 7.5 resulted in the formation of ketoamine glycation conjugates. Incubation of histone H1 with ADP-ribose also rapidly resulted in the formation of protein carboxymethyllysine residues, protein-protein cross-links, and highly fluorescent products with properties similar to the advanced glycosylation end product pentosidine. The formation of glycoxidation products was related to the degradation of ketoamine glycation conjugates by two different pathways. One pathway resulted in the formation of protein carboxymethyllysine residues and release of an ADP moiety containing a glyceric acid fragment. A second pathway resulted in the release of ADP, and it is postulated that this pathway is involved in the formation of histone-histone cross-links and fluorescent advanced glycosylation end products.

  16. Positron emission tomography probe demonstrates a striking concentration of ribose salvage in the liver.

    PubMed

    Clark, Peter M; Flores, Graciela; Evdokimov, Nikolai M; McCracken, Melissa N; Chai, Timothy; Nair-Gill, Evan; O'Mahony, Fiona; Beaven, Simon W; Faull, Kym F; Phelps, Michael E; Jung, Michael E; Witte, Owen N

    2014-07-15

    PET is a powerful technique for quantifying and visualizing biochemical pathways in vivo. Here, we develop and validate a novel PET probe, [(18)F]-2-deoxy-2-fluoroarabinose ([(18)F]DFA), for in vivo imaging of ribose salvage. DFA mimics ribose in vivo and accumulates in cells following phosphorylation by ribokinase and further metabolism by transketolase. We use [(18)F]DFA to show that ribose preferentially accumulates in the liver, suggesting a striking tissue specificity for ribose metabolism. We demonstrate that solute carrier family 2, member 2 (also known as GLUT2), a glucose transporter expressed in the liver, is one ribose transporter, but we do not know if others exist. [(18)F]DFA accumulation is attenuated in several mouse models of metabolic syndrome, suggesting an association between ribose salvage and glucose and lipid metabolism. These results describe a tool for studying ribose salvage and suggest that plasma ribose is preferentially metabolized in the liver.

  17. 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…

  18. 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…

  19. Targeting DNA Repair in Cancer: Beyond PARP Inhibitors.

    PubMed

    Brown, Jessica S; O'Carrigan, Brent; Jackson, Stephen P; Yap, Timothy A

    2017-01-01

    Germline aberrations in critical DNA-repair and DNA damage-response (DDR) genes cause cancer predisposition, whereas various tumors harbor somatic mutations causing defective DDR/DNA repair. The concept of synthetic lethality can be exploited in such malignancies, as exemplified by approval of poly(ADP-ribose) polymerase inhibitors for treating BRCA1/2-mutated ovarian cancers. Herein, we detail how cellular DDR processes engage various proteins that sense DNA damage, initiate signaling pathways to promote cell-cycle checkpoint activation, trigger apoptosis, and coordinate DNA repair. We focus on novel therapeutic strategies targeting promising DDR targets and discuss challenges of patient selection and the development of rational drug combinations.

  20. Present and future evolution of advanced breast cancer therapy

    PubMed Central

    2010-01-01

    Although the introduction of novel therapies and drug combinations has improved the prognosis of metastatic breast cancer, the disease remains incurable. Increased knowledge of the biology and the molecular alterations in breast cancer has facilitated the design of targeted therapies. These agents include receptor and nonreceptor tyrosine kinase inhibitors (epidermal growth factor receptor family), intracellular signaling pathways (phosphatidylinositol-3-kinase, AKT, mammalian target of rapamycin) angiogenesis inhibitors and agents that interfere with DNA repair (poly(ADP-ribose) polymerase inhibitors). In the present review, we present the most promising studies of these new targeted therapies and novel combinations of targeted therapies with cytotoxic agents. PMID:21050422

  1. Molecular Basis for BRCA2-mediated DNA Repair and Breast Tumor Suppression

    DTIC Science & Technology

    2009-10-01

    breaks are mediated by the RAD51 recombinase . In catalyzing recombination reactions, RAD51 must first form a right-handed helical filament, termed the...analogs and poly(ADP-ribose) polymerase inhibitors, exploit the incapability of BRCA2-deficient cells to rely on HR for the repair of DSBs. Apparently...Instability and DNA repair in Taos, New Mexico (March 1-5, 2009). I am a coauthor on manuscript “Enhancement of the RAD51 Recombinase by the Tumor Suppressor

  2. N-methyl-2-pyridone-5-carboxamide (2PY)—Major Metabolite of Nicotinamide: An Update on an Old Uremic Toxin

    PubMed Central

    Lenglet, Aurélie; Liabeuf, Sophie; Bodeau, Sandra; Louvet, Loïc; Mary, Aurélien; Boullier, Agnès; Lemaire-Hurtel, Anne Sophie; Jonet, Alexia; Sonnet, Pascal; Kamel, Said; Massy, Ziad A.

    2016-01-01

    N-methyl-2-pyridone-5-carboxamide (2PY, a major metabolite of nicotinamide, NAM) was recently identified as a uremic toxin. Recent interventional trials using NAM to treat high levels of phosphorus in end-stage renal disease have highlighted new potential uremic toxicities of 2PY. In the context of uremia, the accumulation of 2PY could be harmful—perhaps by inhibiting poly (ADP-ribose) polymerase-1 activity. Here, we review recently published data on 2PY’s metabolism and toxicological profile. PMID:27854278

  3. Lanostanoid triterpenes from Laetiporus sulphureus and apoptosis induction on HL-60 human myeloid leukemia cells.

    PubMed

    León, Francisco; Quintana, José; Rivera, Augusto; Estévez, Francisco; Bermejo, Jaime

    2004-12-01

    A new lanostanoid triterpene, 3-oxosulfurenic acid (1), together with three known triterpenes (3, 4, and 7) were isolated from the fruit bodies of Laetiporus sulphureus. Cytotoxicity of these compounds and their derivatives (2, 5, and 6) was evaluated on HL-60 cells. Further studies revealed that acetyl eburicoic acid (5) was the most potent apoptosis inducer. Apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase-1 and was also associated with an early release of cytochrome c from the mitochondria.

  4. Isolation, structure elucidation, total synthesis, and evaluation of new natural and synthetic ceramides on human SK-MEL-1 melanoma cells.

    PubMed

    León, Francisco; Brouard, Ignacio; Rivera, Augusto; Torres, Fernando; Rubio, Sara; Quintana, José; Estévez, Francisco; Bermejo, Jaime

    2006-09-21

    Two new long-chain ceramides, trametenamides A (1) and B (2), were isolated from the methanolic extract of the fruiting body of the fungus Trametes menziesii. The structures were elucidated by spectroscopic analyses and chemical transformations, and the absolute stereochemistry of trametenamide B (2) was determined by stereoselective total synthesis of four possible diastereomers. The acetyl derivative of the natural ceramide (1a) and synthetic ceramides (24-27) showed cytotoxicity on the human melanoma cell line SK-MEL-1, which was caused by induction of apoptosis as determined by DNA fragmentation, poly(ADP-ribose) polymerase cleavage, and procaspase-9 and -8 processing.

  5. Genetic and epigenetic heterogeneity of epithelial ovarian cancer and the clinical implications for molecular targeted therapy.

    PubMed

    Bai, Huimin; Cao, Dongyan; Yang, Jiaxin; Li, Menghui; Zhang, Zhenyu; Shen, Keng

    2016-04-01

    Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy, and tumoural heterogeneity (TH) has been blamed for treatment failure. The genomic and epigenomic atlas of EOC varies significantly with tumour histotype, grade, stage, sensitivity to chemotherapy and prognosis. Rapidly accumulating knowledge about the genetic and epigenetic events that control TH in EOC has facilitated the development of molecular-targeted therapy. Poly (ADP-ribose) polymerase (PARP) inhibitors, designed to target homologous recombination, are poised to change how breast cancer susceptibility gene (BRCA)-related ovarian cancer is treated. Epigenetic treatment regimens being tested in clinical or preclinical studies could provide promising novel treatment approaches and hope for improving patient survival.

  6. Viral Macro Domains Reverse Protein ADP-Ribosylation

    PubMed Central

    Li, Changqing; Debing, Yannick; Jankevicius, Gytis; Neyts, Johan; Ahel, Ivan

    2016-01-01

    ABSTRACT ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD+ to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. IMPORTANCE Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular

  7. 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

  8. Phosphorylation of Ribose-Borate Complexes at Convergent Margins?

    NASA Astrophysics Data System (ADS)

    Holm, N. G.

    2008-12-01

    The potential of pyrophosphate formation upon heating of hydrogenated orthophosphates like whitlockite ((Ca18Mg2H2(PO4)14) to a few hundred °C in geological environments with low water to rock ratio has probably been underestimated. Once pyrophosphate is available, phosphorylation of pentoses, ribose in particular, may occur. Experiments involving heating of sodium dihydrogen phosphate have even shown high yields of trimetaphosphate. This compound is an even better phosphorylating agent than pyrophosphate and has been identified in volcanic fumaroles. Ribose may be formed from formaldehyde and glycolaldehyde, because the ribose molecule is stabilized by borate that binds to the 2' and 3' positions. Mechanistically, aldehydes can be formed directly from elemental carbon present in mafic rocks in contact with water. The initial reaction of elemental carbon with water gives hydroxymethylene, which can rearrange to formaldehyde. A new hydroxymethylene molecule can then add onto the formaldehyde (and larger aldehyde molecules) and form glycolaldehyde. In this way, the known lag in the formation of glycolaldehyde from formaldehyde is avoided. This lag has previously been a drawback and a reason that the formose reaction was for a while outdated as a possible mechanism for abiotic synthesis of carbohydrates. The reason why pentoses are stabilized by borate is that borate forms trigonal and tetrahedral complexes with oxygen groups and, therefore, has a strong affinity for organic material. Boric acid and borate readily form complexes with a wide variety of sugars, particularly the furanose form of pentoses, and other compounds containing cis-hydroxyl groups like humic substances. Borate is continuously scavenged from seawater by secondary layer minerals of oceanic lithosphere and is released again at moderate heating of the subducting plate at convergent margins. The Mariana back-arc is a good example of this process. The fact that ribose is stabilized by borate may

  9. The patented uses of D-ribose in cardiovascular diseases.

    PubMed

    Shecterle, Linda M; Terry, Kathleen R; St Cyr, John A

    2010-06-01

    Cardiovascular diseases account for more deaths worldwide than any other illness. Myocardial ischemia, a common finding in cardiovascular diseases, lowers cellular energy levels, which affects a cell's integrity and function. Pre-clinical animal studies have reported lower cellular energy levels with an associated decreased function following myocardial ischemia. Recently, scientists have reported that the failing heart is energy starved and yet no pharmaceuticals have been able to address this issue with satisfactory results. Over decades, researchers have explored the use of various metabolites to replenish deficient cellular energy levels following induced ischemia with mixed results. However, D-ribose, a natural occurring carbohydrate, has demonstrated significant enhancing abilities in replenishing deficient cellular energy levels following myocardial ischemia, as well as improving depressed function in numerous animal investigations. Subsequent clinical trials have further substantiated these benefits of D-ribose in patients afflicted with ischemic cardiovascular disease and those carrying the diagnosis of congestive heart failure. The future of effective therapies for ischemic heart disease and congestive heart failure must strongly consider novel pharmaceuticals directed at replenishing cellular energy levels. Intellectual property and the represented patents in this paper emphasize the use of D-ribose for its cellular energy enhancing potential, reflected in both objective and subjective clinical improvements; therefore, substantiating its value in patients with ischemic cardiovascular diseases.

  10. Escherchia coli ribose binding protein based bioreporters revisited

    PubMed Central

    Reimer, Artur; Yagur-Kroll, Sharon; Belkin, Shimshon; Roy, Shantanu; van der Meer, Jan Roelof

    2014-01-01

    Bioreporter bacteria, i.e., strains engineered to respond to chemical exposure by production of reporter proteins, have attracted wide interest because of their potential to offer cheap and simple alternative analytics for specified compounds or conditions. Bioreporter construction has mostly exploited the natural variation of sensory proteins, but it has been proposed that computational design of new substrate binding properties could lead to completely novel detection specificities at very low affinities. Here we reconstruct a bioreporter system based on the native Escherichia coli ribose binding protein RbsB and one of its computationally designed variants, reported to be capable of binding 2,4,6-trinitrotoluene (TNT). Our results show in vivo reporter induction at 50 nM ribose, and a 125 nM affinity constant for in vitro ribose binding to RbsB. In contrast, the purified published TNT-binding variant did not bind TNT nor did TNT cause induction of the E. coli reporter system. PMID:25005019

  11. Thymidine phosphorylase, 2-deoxy-D-ribose and angiogenesis.

    PubMed Central

    Brown, N S; Bicknell, R

    1998-01-01

    Angiogenesis is the term used to describe the formation of new blood vessels from the existing vasculature. In order to attract new vessels, a tissue must release an endothelial-cell chemoattractant. 2-Deoxy-D-ribose is produced in vivo by the catalytic action of thymidine phosphorylase (TP) on thymidine and has recently been identified as an endothelial-cell chemoattractant and angiogenesis-inducing factor. TP, previously known only for its role in nucleotide salvage, is now known to be angiogenic. TP expression is elevated in many solid tumours and in chronically inflamed tissues, both known areas of active angiogenesis. There is evidence that TP is also involved in physiological angiogenesis such as endometrial angiogenesis during the menstrual cycle. The majority of known endothelial-cell chemoattractants are polypeptides that bind to endothelial-cell-surface receptors. In contrast, 2-deoxy-D-ribose appears to lack a cell-surface receptor. Glucose is another sugar that acts as an endothelial-cell chemoattractant. The migratory activity of glucose is blocked by ouabain. It is possible that 2-deoxy-D-ribose and glucose stimulate endothelial-cell migration via a similar mechanistic pathway. PMID:9693094

  12. NAD+ metabolism in health and disease.

    PubMed

    Belenky, Peter; Bogan, Katrina L; Brenner, Charles

    2007-01-01

    Nicotinamide adenine dinucleotide (NAD(+)) is both a coenzyme for hydride-transfer enzymes and a substrate for NAD(+)-consuming enzymes, which include ADP-ribose transferases, poly(ADP-ribose) polymerases, cADP-ribose synthases and sirtuins. Recent results establish protective roles for NAD(+) that might be applicable therapeutically to prevent neurodegenerative conditions and to fight Candida glabrata infection. In addition, the contribution that NAD(+) metabolism makes to lifespan extension in model systems indicates that therapies to boost NAD(+) might promote some of the beneficial effects of calorie restriction. Nicotinamide riboside, the recently discovered nucleoside precursor of NAD(+) in eukaryotic systems, might have advantages as a therapy to elevate NAD(+) without inhibiting sirtuins, which is associated with high-dose nicotinamide, or incurring the unpleasant side-effects of high-dose nicotinic acid.

  13. Global transcriptome response in Lactobacillus sakei during growth on ribose

    PubMed Central

    2011-01-01

    Background Lactobacillus sakei is valuable in the fermentation of meat products and exhibits properties that allow for better preservation of meat and fish. On these substrates, glucose and ribose are the main carbon sources available for growth. We used a whole-genome microarray based on the genome sequence of L. sakei strain 23K to investigate the global transcriptome response of three L. sakei strains when grown on ribose compared with glucose. Results The function of the common regulated genes was mostly related to carbohydrate metabolism and transport. Decreased transcription of genes encoding enzymes involved in glucose metabolism and the L-lactate dehydrogenase was observed, but most of the genes showing differential expression were up-regulated. Especially transcription of genes directly involved in ribose catabolism, the phosphoketolase pathway, and in alternative fates of pyruvate increased. Interestingly, the methylglyoxal synthase gene, which encodes an enzyme unique for L. sakei among lactobacilli, was up-regulated. Ribose catabolism seems closely linked with catabolism of nucleosides. The deoxyribonucleoside synthesis operon transcriptional regulator gene was strongly up-regulated, as well as two gene clusters involved in nucleoside catabolism. One of the clusters included a ribokinase gene. Moreover, hprK encoding the HPr kinase/phosphatase, which plays a major role in the regulation of carbon metabolism and sugar transport, was up-regulated, as were genes encoding the general PTS enzyme I and the mannose-specific enzyme II complex (EIIman). Putative catabolite-responsive element (cre) sites were found in proximity to the promoter of several genes and operons affected by the change of carbon source. This could indicate regulation by a catabolite control protein A (CcpA)-mediated carbon catabolite repression (CCR) mechanism, possibly with the EIIman being indirectly involved. Conclusions Our data shows that the ribose uptake and catabolic machinery in

  14. Progress in the function and regulation of ADP-Ribosylation.

    PubMed

    Hottiger, Michael O; Boothby, Mark; Koch-Nolte, Friedrich; Lüscher, Bernhard; Martin, Niall M B; Plummer, Ruth; Wang, Zhao-Qi; Ziegler, Mathias

    2011-05-24

    Adenosine 5'-diphosphate (ADP)-ribosylation is a protein posttranslational modification that is catalyzed by ADP-ribosyltransferases (ARTs), using nicotinamide adenine dinucleotide (NAD(+)) as a substrate. Mono-ribosylation can be extended into polymers of ADP-ribose (PAR). Poly(ADP-ribosyl)polymerase (PARP) 1, the best-characterized cellular enzyme catalyzing this process, is the prototypical member of a family of mono- and poly(ADP-ribosyl)transferases. The physiological consequences of ADP-ribosylation are inadequately understood. PARP2010, the 18th International Conference on ADP-Ribosylation, attracted scientists from all over the world to Zurich, Switzerland. Highlights from this meeting include promising clinical trials with PARP inhibitors and new insights into cell, structural, and developmental biology of ARTs and the (glyco)hydrolase proteins that catalyze de-ADP-ribosylation of mono- or poly-ADP-ribosylated proteins. Moreover, potential links to the NAD-dependent sirtuin family were explored on the basis of a shared dependence on cellular NAD(+) concentrations and the relationship of ADP-ribosylation with intermediary metabolism and cellular energetics.

  15. T7-RNA Polymerase

    NASA Technical Reports Server (NTRS)

    1997-01-01

    T7-RNA Polymerase grown on STS-81. Structure-Function Relationships of RNA Polymerase: DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. Principal Investigator's include Dr. Dan Carter, Dr. B.C. Wang, and Dr. John Rose of New Century Pharmaceuticals.

  16. 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.

  17. Expanding functions of ADP-ribosylation in the maintenance of genome integrity.

    PubMed

    Martin-Hernandez, K; Rodriguez-Vargas, J-M; Schreiber, V; Dantzer, F

    2017-03-01

    Cell response to genotoxic stress requires a complex network of sensors and effectors from numerous signaling and repair pathways, among them the nuclear poly(ADP-ribose) polymerase 1 (PARP1) plays a central role. PARP1 is catalytically activated in the setting of DNA breaks. It uses NAD(+) as a donor and catalyses the synthesis and subsequent covalent attachment of branched ADP-ribose polymers onto itself and various acceptor proteins to promote repair. Its inhibition is now considered as an efficient therapeutic strategy to potentiate the cytotoxic effect of chemotherapy and radiation or to exploit synthetic lethality in tumours with defective homologous recombination mediated repair. Still, efforts made on understanding the role of PARylation in DNA repair continues to yield novel discoveries. Over the last years, our knowledge in this field has been particularly advanced by the discovery of novel biochemical and functional properties featuring PARP1, by the characterization of the other PARP family members and by the identification of a panel of enzymes capable of erasing poly(ADP-ribose). The aim of this review is to provide an overview of these newest findings and their relevance in genome surveillance.

  18. 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 Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km 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

  19. 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

    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 Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km 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.

  20. Synthetic study on carbocyclic analogs of cyclic ADP-ribose, a novel second messenger: an efficient synthesis of cyclic IDP-carbocyclic-ribose.

    PubMed

    Fukuoka, M; Shuto, S; Minakawa, N; Ueno, Y; Matsuda, A

    1999-01-01

    An efficient synthesis of cyclic IDP-carbocyclic-ribose, as a stable mimic for cyclic ADP-ribose, was achieved. 8-Bromo-N1-carbocyclic-ribosylinosine derivative 10, prepared from N1-(2,4-dinitrophenyl)inosine derivative 5 and an optically active carbocyclic amine 6, was converted to 8-bromo-N1-carbocyclic-ribosylinosine bisphosphate derivative 15. Treatment of 15 with I2 in the presence of molecular sieves in pyridine gave the desired cyclic product 16 quantitatively, which was deprotected and reductively debrominated to give the target cyclic IDP-carbocyclic-ribose (3).

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

    SciTech Connect

    Shirai, Hidenori; Fujimori, Hiroaki; Gunji, Akemi; Maeda, Daisuke; Hirai, Takahisa; Poetsch, Anna R.; Harada, Hiromi; Yoshida, Tomoko; Sasai, Keisuke; Okayasu, Ryuichi; Masutani, Mitsuko

    2013-05-24

    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 responses 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 ser18 was

  2. Targeting DNA Repair: The Role of PARP Inhibition in the Treatment of Castration-Resistant Prostate Cancer.

    PubMed

    Castro, Elena; Mateo, Joaquin; Olmos, David; de Bono, Johann S

    Several genomic studies have identified DNA repair gene defects in prostate cancer in the last 5 years. The mechanisms by which these DNA repair defects promote carcinogenesis and tumor progression in the prostate have not been fully elucidated, but their presence in at least 20-25% of metastatic castration-resistant prostate cancers (CRPCs) provides an opportunity for a therapeutic strategy that turns a tumor strength into its weakness and may lead to arguably the first molecularly stratified treatment for this disease.Poly(ADP-ribose) polymerase (PARP) inhibitors have been developed as an anticancer synthetic lethal therapeutic strategy for tumors with impaired homologous recombination DNA repair, based on a synthetic lethal effect. Poly(ADP-ribose) polymerase inhibitors have shown to induce significant tumor responses in cancer patients carrying germline BRCA1/2 mutations. Recent evidence from a phase II clinical trial supports further testing of PARP inhibitors for the treatment of metastatic CRPC with either germline or somatic defects in BRCA2, ATM, PALB2, and other DNA repair genes.We review the current evidence of how this strategy is relevant for the treatment of advanced prostate cancers, the available data from trials with PARP inhibitors in metastatic CRPC, and the ongoing studies analyzing combinations of these drugs with other therapies.

  3. Ribose and related sugars from ultraviolet irradiation of interstellar ice analogs

    NASA Astrophysics Data System (ADS)

    Meinert, Cornelia; Myrgorodska, Iuliia; de Marcellus, Pierre; Buhse, Thomas; Nahon, Laurent; Hoffmann, Søren V.; d'Hendecourt, Louis Le Sergeant; Meierhenrich, Uwe J.

    2016-04-01

    Ribose is the central molecular subunit in RNA, but the prebiotic origin of ribose remains unknown. We observed the formation of substantial quantities of ribose and a diversity of structurally related sugar molecules such as arabinose, xylose, and lyxose in the room-temperature organic residues of photo-processed interstellar ice analogs initially composed of H2O, CH3OH, and NH3. Our results suggest that the generation of numerous sugar molecules, including the aldopentose ribose, may be possible from photochemical and thermal treatment of cosmic ices in the late stages of the solar nebula. Our detection of ribose provides plausible insights into the chemical processes that could lead to formation of biologically relevant molecules in suitable planetary environments.

  4. Ribose and related sugars from ultraviolet irradiation of interstellar ice analogs.

    PubMed

    Meinert, Cornelia; Myrgorodska, Iuliia; de Marcellus, Pierre; Buhse, Thomas; Nahon, Laurent; Hoffmann, Søren V; d'Hendecourt, Louis Le Sergeant; Meierhenrich, Uwe J

    2016-04-08

    Ribose is the central molecular subunit in RNA, but the prebiotic origin of ribose remains unknown. We observed the formation of substantial quantities of ribose and a diversity of structurally related sugar molecules such as arabinose, xylose, and lyxose in the room-temperature organic residues of photo-processed interstellar ice analogs initially composed of H2O, CH3OH, and NH3 Our results suggest that the generation of numerous sugar molecules, including the aldopentose ribose, may be possible from photochemical and thermal treatment of cosmic ices in the late stages of the solar nebula. Our detection of ribose provides plausible insights into the chemical processes that could lead to formation of biologically relevant molecules in suitable planetary environments.

  5. Enzymatic synthesis of 2'-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates.

    PubMed Central

    Conrad, F; Hanne, A; Gaur, R K; Krupp, G

    1995-01-01

    For the first time mosaic nucleic acids composed of 50% RNA and 50% DNA can be obtained as transcripts with T7 RNA polymerase. Two NTPs could be replaced simultaneously in a transcription reaction. This means more than 40 deoxynucleotides were inserted in one transcript. Previously, a maximum of two deoxynucleotides could be incorporated and 2'-O-methyl-NTPs were not substrates at all. We obtained reasonable transcript yields with a maximal level of 99% 2'-O-methyl-NTPs, and the products contained up to 58% 2'-O-methylnucleotides at more than 20 positions. Sequence-specific nucleotide incorporation was monitored by sequence ladders (partial alkali or iodine cleavage). No base misincorporations were detected with 100% dGTP, dCTP and dTTP, and with partial incorporation of dATP alpha S, 2'-O-methyl-GTP alpha S and 2'-O-methyl-CTP alpha S, whereas they were found with dATP, 2'-O-methyl-ATP alpha S and 2'-O-methyl-UTP alpha S. Quantitative data allow predetermined modification levels of partially modified transcripts. Highly modified transcripts can be used for structural and functional studies, in modification interference approaches and for in vitro evolution procedures. Modification interference studies revealed a small number of important phosphate and ribose moieties in RNase P substrates. The conversion of T7 RNA polymerase to a DNA polymerase extends the observation that there is no absolute distinction between RNA and DNA polymerases. Accordingly, an adapted concept of a primordial RNA world is presented. Images PMID:7541130

  6. Directed Polymerase Evolution

    PubMed Central

    Chen, Tingjian; Romesberg, Floyd E.

    2014-01-01

    Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity. PMID:24211837

  7. The expanding polymerase universe.

    PubMed

    Goodman, M F; Tippin, B

    2000-11-01

    Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

  8. 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

  9. Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin.

    PubMed

    Robert, Carine; Nagaria, Pratik K; Pawar, Nisha; Adewuyi, Adeoluwa; Gojo, Ivana; Meyers, David J; Cole, Philip A; Rassool, Feyruz V

    2016-06-01

    Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP "trapping". Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP "trapping", which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells.

  10. PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks.

    PubMed

    Krietsch, Jana; Caron, Marie-Christine; Gagné, Jean-Philippe; Ethier, Chantal; Vignard, Julien; Vincent, Michel; Rouleau, Michèle; Hendzel, Michael J; Poirier, Guy G; Masson, Jean-Yves

    2012-11-01

    After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood.

  11. Rat germinal cells require PARP for repair of DNA damage induced by gamma-irradiation and H2O2 treatment.

    PubMed

    Atorino, L; Di Meglio, S; Farina, B; Jones, R; Quesada, P

    2001-03-01

    The ability of rat germinal cells to recover from genotoxic stress has been investigated using isolated populations of primary spermatocytes and round spermatids. Using a comet assay at pH 10.0 to assess single strand breakage (SSB) in DNA, it was found that a high level of damage was induced by 5 Gy gamma-irradiation and acute exposure to 50 microM H2O2. This damage was effectively repaired during a subsequent recovery period of 1-3 hours culture in vitro but repair was significantly delayed in the presence of the poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide (3-ABA). Immunofluorescence detection of PARP with specific antibodies localised the protein to discrete foci within the nucleus of both spermatocytes and spermatids. Poly(ADP-ribose) (pADPR) could also be detected in spermatid nuclei following gamma-irradiation or H2O2 treatment. Moreover, PARP activation occurs both in spermatocytes and spermatids left to recover after both genotoxic stresses. The NO donors, 3-morpholino-sydnonimine (SIN-1) and S-nitrosoglutathione (SNOG), caused significant SSBs in both spermatocytes and spermatids. The effects of SIN-1 could be prevented by exogenous catalase (CAT), but not superoxide dismutase (SOD), in the cell suspensions. SNOG-induced SSBs were insensitive to both CAT and SOD. It is concluded that DNA in spermatocytes and spermatids is sensitive to damage by gamma-irradiation and H2O2 and that efficient repair of SSBs requires PARP activity.

  12. PARP-2 domain requirements for DNA damage-dependent activation and localization to sites of DNA damage.

    PubMed

    Riccio, Amanda A; Cingolani, Gino; Pascal, John M

    2016-02-29

    Poly(ADP-ribose) polymerase-2 (PARP-2) is one of three human PARP enzymes that are potently activated during the cellular DNA damage response (DDR). DDR-PARPs detect DNA strand breaks, leading to a dramatic increase in their catalytic production of the posttranslational modification poly(ADP-ribose) (PAR) to facilitate repair. There are limited biochemical and structural insights into the functional domains of PARP-2, which has restricted our understanding of how PARP-2 is specialized toward specific repair pathways. PARP-2 has a modular architecture composed of a C-terminal catalytic domain (CAT), a central Trp-Gly-Arg (WGR) domain and an N-terminal region (NTR). Although the NTR is generally considered the key DNA-binding domain of PARP-2, we report here that all three domains of PARP-2 collectively contribute to interaction with DNA damage. Biophysical, structural and biochemical analyses indicate that the NTR is natively disordered, and is only required for activation on specific types of DNA damage. Interestingly, the NTR is not essential for PARP-2 localization to sites of DNA damage. Rather, the WGR and CAT domains function together to recruit PARP-2 to sites of DNA breaks. Our study differentiates the functions of PARP-2 domains from those of PARP-1, the other major DDR-PARP, and highlights the specialization of the multi-domain architectures of DDR-PARPs.

  13. Structural Basis of Detection and Signaling of DNA Single-Strand Breaks by Human PARP-1

    PubMed Central

    Eustermann, Sebastian; Wu, Wing-Fung; Langelier, Marie-France; Yang, Ji-Chun; Easton, Laura E.; Riccio, Amanda A.; Pascal, John M.; Neuhaus, David

    2015-01-01

    Summary 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 deformability 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. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins. PMID:26626479

  14. PARP-2 domain requirements for DNA damage-dependent activation and localization to sites of DNA damage

    PubMed Central

    Riccio, Amanda A.; Cingolani, Gino; Pascal, John M.

    2016-01-01

    Poly(ADP-ribose) polymerase-2 (PARP-2) is one of three human PARP enzymes that are potently activated during the cellular DNA damage response (DDR). DDR-PARPs detect DNA strand breaks, leading to a dramatic increase in their catalytic production of the posttranslational modification poly(ADP-ribose) (PAR) to facilitate repair. There are limited biochemical and structural insights into the functional domains of PARP-2, which has restricted our understanding of how PARP-2 is specialized toward specific repair pathways. PARP-2 has a modular architecture composed of a C-terminal catalytic domain (CAT), a central Trp-Gly-Arg (WGR) domain and an N-terminal region (NTR). Although the NTR is generally considered the key DNA-binding domain of PARP-2, we report here that all three domains of PARP-2 collectively contribute to interaction with DNA damage. Biophysical, structural and biochemical analyses indicate that the NTR is natively disordered, and is only required for activation on specific types of DNA damage. Interestingly, the NTR is not essential for PARP-2 localization to sites of DNA damage. Rather, the WGR and CAT domains function together to recruit PARP-2 to sites of DNA breaks. Our study differentiates the functions of PARP-2 domains from those of PARP-1, the other major DDR-PARP, and highlights the specialization of the multi-domain architectures of DDR-PARPs. PMID:26704974

  15. Structural Basis of Detection and Signaling of DNA Single-Strand Breaks by Human PARP-1.

    PubMed

    Eustermann, Sebastian; Wu, Wing-Fung; Langelier, Marie-France; Yang, Ji-Chun; Easton, Laura E; Riccio, Amanda A; Pascal, John M; Neuhaus, David

    2015-12-03

    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 deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodification in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.

  16. Tankyrase Sterile α Motif Domain Polymerization Is Required for Its Role in Wnt Signaling.

    PubMed

    Riccio, Amanda A; McCauley, Michael; Langelier, Marie-France; Pascal, John M

    2016-09-06

    Tankyrase-1 (TNKS1/PARP-5a) is a poly(ADP-ribose) polymerase (PARP) enzyme that regulates multiple cellular processes creating a poly(ADP-ribose) posttranslational modification that can lead to target protein turnover. TNKS1 thereby controls protein levels of key components of signaling pathways, including Axin1, the limiting component of the destruction complex in canonical Wnt signaling that degrades β-catenin to prevent its coactivator function in gene expression. There are limited molecular level insights into TNKS1 regulation in cell signaling pathways. TNKS1 has a sterile α motif (SAM) domain that is known to mediate polymerization, but the functional requirement for SAM polymerization has not been assessed. We have determined the crystal structure of wild-type human TNKS1 SAM domain and used structure-based mutagenesis to disrupt polymer formation and assess the consequences on TNKS1 regulation of β-catenin-dependent transcription. Our data indicate the SAM polymer is critical for TNKS1 catalytic activity and allows TNKS1 to efficiently access cytoplasmic signaling complexes.

  17. The C. elegans gene pme-5: molecular cloning and role in the DNA-damage response of a tankyrase orthologue.

    PubMed

    Gravel, Catherine; Stergiou, Lilli; Gagnon, Steve N; Desnoyers, Serge

    2004-02-03

    Tankyrases are recently identified proteins characterized by ankyrin repeats and a poly(ADP-ribose) polymerase (PARP) signature motif. In vertebrates, tankyrases mediate protein-protein interactions via the ankyrin domain. Many partners have been identified that could function in telomere maintenance, signal transduction in vesicular transport, and cell death. To further our knowledge of tankyrases and to study their function in development, we sought and found a tankyrase-related gene in Caenorhabditis elegans that we named pme-5 (poly(ADP-ribose) metabolism enzyme-5). The protein encoded includes a large ankyrin domain and a catalytic PARP domain containing the well-conserved PARP signature sequence and the regulatory region. Unlike other tankyrases, PME-5 lacks a sterile-alpha module (SAM), but has a coiled coil domain which may mediate oligomerization. We also found that pme-5 mRNA is alternatively spliced at the fifth exon, producing a long (PME-5L) and a short (PME-5S) transcript. Both isoforms are constitutively expressed during the life cycle of C. elegans. We also show DNA damage increases expression of pme-5, a response that requires the DNA damage checkpoint gene hus-1. Moreover, DNA damage-induced germ cell apoptosis was slightly increased in pme-5(RNAi) hermaphrodites. Altogether, these data indicate that pme-5 is part of a DNA damage response pathway which leads to apoptosis in C. elegans.

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

    SciTech Connect

    Henrie, Melinda S.; Kurimasa, Akihiro; Burma, Sandeep; Menissier-de Murcia, Josiane; de Murcia, Gilbert; Li, Gloria C.; Chen,David J.

    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 significance 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.

  19. Iduna Protects the Brain from Glutamate Excitotoxicity and Stroke by Interfering with Parthanatos

    PubMed Central

    Andrabi, Shaida A.; Kang, Ho Chul; Haince, Jean-François; Lee, Yun-Il; Zhang, Jian; Chi, Zhikai; West, Andrew B.; Koehler, Raymond C.; Poirier, Guy G.; Dawson, Ted M.; Dawson, Valina L.

    2013-01-01

    Glutamate acting on N-methyl-D-aspartate (NMDA) receptors plays an important role in neurodegenerative diseases and neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a novel NMDA receptor-induced survival gene that is neuroprotective against glutamate NMDA receptor mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer induced cell death (parthanatos). Iduna’s protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer binding protein and mutations at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion (MCAO)-induced stroke in mice. These results define Iduna as the first endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling offers a new therapeutic strategy for the treatment of neurologic disorders. PMID:21602803

  20. Parthanatos: mitochondrial-linked mechanisms and therapeutic opportunities

    PubMed Central

    Fatokun, Amos A; Dawson, Valina L; Dawson, Ted M

    2014-01-01

    Cells die by a variety of mechanisms. Terminally differentiated cells such as neurones die in a variety of disorders, in part, via parthanatos, a process dependent on the activity of poly (ADP-ribose)-polymerase (PARP). Parthanatos does not require the mediation of caspases for its execution, but is clearly mechanistically dependent on the nuclear translocation of the mitochondrial-associated apoptosis-inducing factor (AIF). The nuclear translocation of this otherwise beneficial mitochondrial protein, occasioned by poly (ADP-ribose) (PAR) produced through PARP overactivation, causes large-scale DNA fragmentation and chromatin condensation, leading to cell death. This review describes the multistep course of parthanatos and its dependence on PAR signalling and nuclear AIF translocation. The review also discusses potential targets in the parthanatos cascade as promising avenues for the development of novel, disease-modifying, therapeutic agents. LINKED ARTICLES This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8 PMID:24684389

  1. Automodification switches PARP-1 function from chromatin architectural protein to histone chaperone.

    PubMed

    Muthurajan, Uma M; Hepler, Maggie R D; Hieb, Aaron R; Clark, Nicholas J; Kramer, Michael; Yao, Tingting; Luger, Karolin

    2014-09-02

    Poly [ADP-ribose] polymerase 1 (PARP-1) is a highly abundant chromatin-associated enzyme. It catalyzes the NAD(+)-dependent polymerization of long chains of poly-ADP ribose (PAR) onto itself in response to DNA damage and other cues. More recently, the enzymatic activity of PARP-1 has also been implicated in the regulation of gene expression. The molecular basis for the functional switch from chromatin architectural protein to transcription factor and DNA damage responder, triggered by PARP-1 automodification, is unknown. Here, we show that unmodified PARP-1 engages in at least two high-affinity binding modes with chromatin, one of which does not involve free DNA ends, consistent with its role as a chromatin architectural protein. Automodification reduces PARP-1 affinity for intact chromatin but not for nucleosomes with exposed DNA ends. Automodified (AM) PARP-1 has the ability to sequester histones (both in vitro and in cells) and to assemble nucleosomes efficiently in vitro. This unanticipated nucleosome assembly activity of AM-PARP-1, coupled with the fast turnover of the modification, suggests a model in which DNA damage or transcription events trigger transient histone chaperone activity.

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

    SciTech Connect

    Negi, Geeta; Kumar, Ashutosh; Sharma, Shyam S.

    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 oxidative 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).

  3. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation*

    PubMed Central

    Watanabe, Yukihide; Papoutsoglou, Panagiotis; Maturi, Varun; Tsubakihara, Yutaro; Hottiger, Michael O.; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation. PMID:27129221

  4. 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

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

    SciTech Connect

    Eustermann, Sebastian; Wu, Wing -Fung; Langelier, Marie -France; Yang, Ji -Chun; Easton, Laura E.; Riccio, Amanda A.; Pascal, John M.; Neuhaus, David

    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 deformability 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.

  6. Partial Purification and Characterization of d-Ribose-5-phosphate Reductase from Adonis vernalis L. Leaves

    PubMed Central

    Negm, Fayek B.; Marlow, Gary C.

    1985-01-01

    This study presents evidence for a new enzyme, d-ribose-5-P reductase, which catalyzes the reaction: d-ribose-5-P + NADPH + H+ → d-ribitol-5-P + NADP+. The enzyme was isolated from Adonis vernalis L. leaves in 38% yield and was purified 71-fold. The reductase was NADPH specific and had a pH optimum in the range of 5.5 to 6.0. The Michaelis constant value for d-ribose-5-P reduction was 1.35 millimolar. The enzyme also reduced d-erythrose-4-P, d-erythrose, dl-glyceraldehyde, and the aromatic aldehyde 3-pyridinecarboxaldehyde. Hexoses, hexose phosphates, pentoses, and dihydroxyacetone did not serve as substrates. d-Ribose-5-P reductase is distinct from the other known ribitol synthesizing enzymes detected in bacteria and yeast, and may be responsible for ribitol synthesis in Adonis vernalis. PMID:16664320

  7. Priority of pentose utilization at the level of transcription: arabinose, xylose, and ribose operons.

    PubMed

    Kang, H Y; Song, S; Park, C

    1998-06-30

    When E. coli cells were grown in minimal medium supplemented with D-ribose and D-xylose, a diauxic growth preferring D-xylose was observed. Transcription of the ribose (rbs) operon was repressed in the presence of D-xylose, phenotypically similar to catabolite repression by D-glucose, although D-ribose did not affect transcription of the xylose (xyl) operon. Complementation analysis with xylR revealed that the repression of the rbs operon by D-xylose is exerted at the transcriptional level through XylR, suggesting a novel mechanism for catabolite repression. Furthermore, it was shown that L-arabinose reduced transcriptions of both xyl and rbs operons, whereas the arabinose operon was not affected by D-xylose or D-ribose, suggesting a priority mechanism for pentose utilization.

  8. Metabolism of RNA-ribose by Bdellovibrio bacteriovorus during intraperiplasmic growth on Escherichia coli.

    PubMed Central

    Hespell, R B; Odelson, D A

    1978-01-01

    During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on Escherichia coli some 30 to 60% of the initial E. coli RNA-ribose disappeared as cell-associated orcinol-positive material. The levels of RNA-ribose in the suspending buffer after growth together with the RNA-ribose used for bdellovibrio DNA synthesis accounted for 50% or less of the missing RNA-ribose. With intraperiplasmic growth in the presence of added U-14C-labeled CMP, GMP, or UMP, radioactivity was found both in the respired CO2 and incorporated into the bdellovibrio cell components. The addition of exogenous unlabeled ribonucleotides markedly reduced the amounts of both the 14CO2 and 14C incorporated into the progeny bdellovibrios. During intraperiplasmic growth of B. bacteriovorus on [U-14C]ribose-labeled E. coli BJ565, ca. 74% and ca. 19% of the initial 14C was incorporated into the progeny bdellovibrios and respired CO2, respectively. Under similar growth conditions, the addition of glutamate substantially reduced only the 14CO2; however, added ribonucleotides reduced both the 14CO2 and the 14C incorporated into the progeny bdellovibrios. No similar effects were found with added ribose-5-phosphate. The distribution of 14C in the major cell components was similar in progeny bdellovibrios whether obtained from growth on [U-14C]ribose-labeled E. coli BJ565 or from E. coli plus added U-14C-labeled ribonucleotides. After intraperiplasmic growth of B. bacteriovorus on [5,6-3H-]uracil-[U-14C]ribose-labeled E. coli BJ565 (normal or heat treated), the whole-cell 14C/3H ratio of the progeny bdellovibrios was some 50% greater and reflected the higher 14C/3H ratios found in the cell fractions. B. bacteriovorus and E. coli cell extracts both contained 5'-nucleotidase, uridine phosphorylase, purine phosphorylase, deoxyribose-5-phosphate aldolase, transketolase, thymidine phosphorylase, phosphodeoxyribomutase, and transaldolase enzyme activities. The latter three enzyme activities were either absent

  9. myo-Inositol and d-Ribose Ligand Discrimination in an ABC Periplasmic Binding Protein

    PubMed Central

    Herrou, Julien

    2013-01-01

    The periplasmic binding protein (PBP) IbpA mediates the uptake of myo-inositol by the IatP-IatA ATP-binding cassette transmembrane transporter. We report a crystal structure of Caulobacter crescentus IbpA bound to myo-inositol at 1.45 Å resolution. This constitutes the first structure of a PBP bound to inositol. IbpA adopts a type I PBP fold consisting of two α-β lobes that surround a central hinge. A pocket positioned between the lobes contains the myo-inositol ligand, which binds with submicromolar affinity (0.76 ± 0.08 μM). IbpA is homologous to ribose-binding proteins and binds d-ribose with low affinity (50.8 ± 3.4 μM). On the basis of IbpA and ribose-binding protein structures, we have designed variants of IbpA with inverted binding specificity for myo-inositol and d-ribose. Five mutations in the ligand-binding pocket are sufficient to increase the affinity of IbpA for d-ribose by 10-fold while completely abolishing binding to myo-inositol. Replacement of ibpA with these mutant alleles unable to bind myo-inositol abolishes C. crescentus growth in medium containing myo-inositol as the sole carbon source. Neither deletion of ibpA nor replacement of ibpA with the high-affinity ribose binding allele affected C. crescentus growth on d-ribose as a carbon source, providing evidence that the IatP-IatA transporter is specific for myo-inositol. This study outlines the evolutionary relationship between ribose- and inositol-binding proteins and provides insight into the molecular basis upon which these two related, but functionally distinct, classes of periplasmic proteins specifically bind carbohydrate ligands. PMID:23504019

  10. Polymeric enzyme mimics: catalytic activity of ribose-containing polymers for a phosphate substrate.

    PubMed

    Han, Man Jung; Yoo, Kyung Soo; Kim, Young Heui; Chang, Ji Young

    2003-07-07

    The polymers containing ribose rings: poly(5'-acrylamido-5'-deoxy-1',2'-O-isopropylidene-alpha-D-ribose) (11), poly(5'-acrylamido-5'-deoxy-alpha-D-ribose) (12) and poly(5'-acrylamido-5'-deoxy-1'-O-methyl-D-ribose) (13) were prepared as enzyme mimics. Polymers 12 and 13 with free vic-cis-diol groups catalyzed the hydrolysis of phosphodiester (ethyl p-nitrophenyl phosphate and N-methylpyridinium 4-tert-butylcatechol cyclic phosphate) and phosphomonoester substrates with a rate acceleration of 10 approximately equal to 10(3) compared with the uncatalyzed reaction. They also catalyzed the reverse reactions, i.e., the esterification of phosphomonoester to phosphodiester and the phosphorylation of alcohols with phosphate ions. The catalytic activity was attributable to the vic-cis-diols of riboses on polymer chains, which formed hydrogen bonds with two phosphoryl oxygen atoms of phosphates so as to activate the phosphorus atoms to be attacked by nucleophiles. The catalytic activity was negligible for polymer 11 where vic-cis-diol groups were blocked with isopropylidene groups. The catalytic activity was attributable to the vic-cis-diols of riboses on polymer chains, which formed hydrogen bonds with two phosphoryl oxygen atoms of phosphates so as to activate the phosphorus atoms to be attacked by nucleophiles.

  11. The polymerase chain reaction.

    PubMed

    Welch, Hazel M

    2012-01-01

    The polymerase chain reaction (PCR) has had a significant impact on all aspects of the molecular biosciences, from cancer research to forensic science. The sensitivity and specificity inherent in the technique allow minute quantities of genetic material to be detected while the unique properties of thermostable DNA polymerase ensure that abundant copies are reliably reproduced to levels that can be visualized and/or used for further applications. This chapter describes applications of PCR and PCR-RT to investigate primary cancer and metastatic disease at both the DNA and mRNA expression levels.

  12. HPLC analysis of cyclic adenosine diphosphate ribose and adenosine diphosphate ribose: determination of NAD+ metabolites in hippocampal membranes.

    PubMed

    Casabona, G; Sturiale, L; L'Episcopo, M R; Raciti, G; Fazzio, A; Sarpietro, M G; Genazzani, A A; Cambria, A; Nicoletti, F

    1995-01-01

    Cyclic adenosine diphosphate-ribose (cADPR) and ADPR were separated by high-performance liquid chromatography (HPLC) on a CarboPac PA-1 column at strong basic pH and quantitated by a pulsed amperometric detector. Although this HPLC method was quite sensitive and highly reproducible, it did not allow the separation of cADPR from guanosine monophosphate (GMP) which, when present, could be removed by ion-affinity chromatography, using gel-immobilized Fe3+ columns. Crude synaptic membranes from rat hippocampi were incubated with nicotinamide adenine dinucleotide (NAD) and acidic extracts were subject to HPLC analysis after neutralization. Incubation led to a time-dependent formation of ADPR, which was amplified when membranes were incubated in the presence of guanosine trisphosphate (GTP), guanosine-5'-0-(3-thiotrisphosphate) (GTP-gamma-S) or AlF3. cADPR did not accumulate in detectable amounts and only a minimal proportion (< 5%) of radioactivity originating from [3H]NAD co-eluted with authentic cADPR in extracts from hippocampal membranes. The simultaneous detection of cADPR and ADPR we have described may help the search for inhibitors of cADPR metabolism, which will allow to measure the cADPR that accumulates under basal conditions or in response to extracellular signals.

  13. 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.

  14. Polymerase chain reaction system

    DOEpatents

    Benett, William J.; Richards, James B.; Stratton, Paul L.; Hadley, Dean R.; Milanovich, Fred P.; Belgrader, Phil; Meyer, Peter L.

    2004-03-02

    A portable polymerase chain reaction DNA amplification and detection system includes one or more chamber modules. Each module supports a duplex assay of a biological sample. Each module has two parallel interrogation ports with a linear optical system. The system is capable of being handheld.

  15. Concerted Proton Transfer Mechanism of Clostridium thermocellum Ribose-5-phosphate Isomerase

    PubMed Central

    Wang, Jun; Yang, Weitao

    2013-01-01

    Ribose-5-phosphate isomerase (Rpi) catalyzes the interconversion of D-ribose-5-phosphate and D-ribulose-5-phosphate and plays an essential role in the pentose phosphate pathway and the Calvin cycle of photosynthesis. RpiB, one of the two isoforms of Rpi, is also a potential drug target for some pathogenic bacteria. Clostridium thermocellum ribose-5-phosphate isomerase (CtRpi), belonging to RpiB family, has recently been employed to the industrial production of rare sugars because of it fast reactions kinetics and narrow substrate specificity. It is known this enzyme adopts proton transfer mechanism. It was suggested that the deprotonated Cys65 attracts the proton at C2 of substrate to initiate the isomerization reaction and this step is the rate-limiting step. However the elaborate catalytic mechanism is still unclear. We have performed quantum mechanical/molecular mechanical simulations of this rate-limiting step of the reaction catalyzed by CtRpi with the substrate D-ribose. Our results demonstrate that the deprotonated Cys65 is not a stable reactant. Instead, our calculations revealed a concerted proton-transfer mechanism: Asp8, a highly conserved residue in the RpiB family performs as the base to abstract the proton at Cys65 and Cys65 in turn abstracts the proton of the D-ribose simultaneously. Moreover, we found Thr67 cannot catalyze the proton transfer from O2 to O1 of the D-ribose alone. Water molecule(s) may assist this proton transfer with Thr67. Our findings lead to a clear understanding of the catalysis mechanism of RpiB family and should guide the experiments to increase the catalysis efficiency. This study also highlights the importance of initial protonation states of cysteines. PMID:23875675

  16. [The action of ADP ribose on the mechanical and bioelectrical activity of the frog heart].

    PubMed

    Sosulina, L Iu; Sukhova, G S; Chudnyĭ, M N; Ashmarin, I P

    1999-04-01

    In the frog isolated heart, cyclic perfusion of ADP-ribose induced a dose-dependent decrease in the heart rate and the contraction force, a decrease in the AP duration as well as in the rate of rise in the sinus node. It also shortened the atrial AP and exerted no significant effect upon multicellular ventricular preparations. In conditions of systemic administration in unanesthetised frogs, the ADP-ribose induced a reversible increase in the heart rate due, probably, to a sympathetic effect.

  17. Elucidation of Molecular Mechanism(s) of Cognition Enhancing Activity of Bacomind®: A Standardized Extract of Bacopa Monnieri

    PubMed Central

    Dethe, Shekhar; Deepak, M; Agarwal, Amit

    2016-01-01

    Background: Bacopa monnieri (L.) Wettst., commonly known as Brahmi, is renowned in Indian traditional system for its potent memory enhancing activity, which has been validated by various scientific studies. Objective: The objective of this study was to understand the molecular mechanism of memory enhancing activity of BacoMind® (BM), a standardized extract of B. monnieri. Materials and Methods: BM was screened in vitro in a panel of cell-free and receptor-transfected cell assays. The purified enzymes/membrane homogenates/cells were incubated with substrate/standard ligand in the absence or presence of the test compound. The IC50 values and EC50 values were determined by nonlinear regression analysis of the concentration–response curves generated with mean replicate values using Hill equation curve fitting. Results: BM was found to inhibit three enzymes; Catechol-O-methyl transferase (COMT), Prolyl endopeptidase (PEP), and Poly (ADP-ribose) polymerase (PARP). It also had an antagonistic effect on serotonin 6 and 2A (5-HT6 and 5-HT2A) receptors, known to influence the different neurological pathways, associated with memory and learning disorders, age-associated memory impairment. Conclusion: BM was found to inhibit three enzymes namely, Catechol-O-methyl transferase (COMT), Prolyl endopeptidase (PEP), and Poly (ADP-ribose) polymerase (PARP). It also exhibited an antagonistic effect on 5-HT6 and 5-HT2A receptors. SUMMARY This study was conducted to understand the molecular mechanism of memory enhancing activity of a standardized extract of B. monnieri by was screening it in vitro in a panel of cell-free and receptor-transfected cell assays. The purified enzymes/membrane homogenates/cells were incubated with substrate/standard ligand in the absence or presence of the test compound. BM was found to inhibit three enzymes; Catechol-O-methyl transferase (COMT), Prolyl endopeptidase (PEP), and Poly (ADP-ribose) polymerase (PARP). It also had an antagonistic effect on

  18. Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis in Escherichia coli.

    PubMed

    Shimada, Tomohiro; Kori, Ayako; Ishihama, Akira

    2013-07-01

    Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway.

  19. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    DOE PAGES

    McInerney, Peter; Adams, Paul; Hadi, Masood Z.

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Errormore » rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu , Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.« less

  20. Efficient production of L-ribose with a recombinant Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new synthetic platform with potential for the production of several rare sugars, with L-ribose being the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression...

  1. Response to Comment on "Ribose and related sugars from ultraviolet irradiation of interstellar ice analogs".

    PubMed

    Meinert, Cornelia; Myrgorodska, Iuliia; de Marcellus, Pierre; Buhse, Thomas; Nahon, Laurent; Hoffmann, Søren V; d'Hendecourt, Louis Le Sergeant; Meierhenrich, Uwe J

    2017-01-13

    We detected ribose and related sugars in the organic residues of simulated interstellar ices using multidimensional gas chromatography. Kawai questions the formation of sugar compounds in the ices and suggests that they arise from a classical formose reaction during sample workup for analysis. We disagree with this hypothesis and present additional data to argue that Kawai's criticism does not apply.

  2. In vitro mitigation of arsenic toxicity by tea polyphenols in human lymphocytes.

    PubMed

    Sinha, Dona; Dey, Subhabrata; Bhattacharya, Rathindra Kumar; Roy, Madhumita

    2007-01-01

    The groundwater arsenicals have brought dreadful misery for the people residing in the endemic regions of West Bengal, India. Arsenic-related anomalies include arsenicosis, hyperkera-tosis, gastric complications, liver fibrosis, peripheral neuropathy, and cancer. Some of these diseases have been frequently associated with overproduction of reactive oxygen species that cause DNA damage and improper functioning of body's antioxidant defense mechanism. Natural polyphenols present in tea serve as excellent antioxidants. In the present study, an attempt has been made to elucidate the role of representative polyphenols and extracts of green and black tea in modulating sodium arsenite (As III)-induced DNA damage in normal human lymphocytes. Comet assay was used to detect the DNA damage. Arsenic-induced oxidative stress was measured with generation of reactive oxygen species, lipid peroxidation, and activity of some antioxidant enzymes. Expression of some repair enzymes such as poly(ADP-ribose) polymerase and DNA polymerase beta was measured to assess the effect of tea on DNA repair. Tea afforded efficient reduction of As III-induced DNA damage in human lymphocytes. Tea also quenched the excessive production of reactive oxygen species by arsenic, reduced the elevated levels of lipid peroxidation, and increased the activity of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase. Furthermore, tea enhanced recovery of DNA damage, which was indicative of repair as confirmed by unscheduled DNA synthesis and pronounced expression of DNA repair enzyme poly(ADP-ribose) polymerase. It is speculated that the antioxidant potential and repair-inducing capacity of tea might help in combating the severe genotoxic effects induced by arsenic in the human population.

  3. Caspase-14: A novel caspase in the retina with a potential role in diabetic retinopathy

    PubMed Central

    Ahmad, Saif; Megyerdi, Sylvia; Othman, Amira; Baban, Babak; Palenski, Tammy L.; Shin, Eui Seok; Gurel, Zafer; Hsu, Stephen; Sheibani, Nader

    2012-01-01

    Purpose The purpose of this study was to evaluate caspase-14 expression in the retina under normal and diabetic conditions, and to determine whether caspase-14 contributes to retinal microvascular cell death under high glucose conditions. Methods Quantitative real-time polymerase chain reaction and western blot analysis were used to evaluate caspase-14 expression in retinal cells, including pericytes (PCs), endothelial cells (ECs), astrocytes (ACs), choroidal ECs, and retinal pigment epithelium (RPE) cells. We also determined caspase-14 expression in the retinas of human subjects with or without diabetic retinopathy (DR) and in experimental diabetic mice. Retinal ECs and PCs were infected with adenoviruses expressing human caspase-14 or green fluorescent protein. Caspase-14 expression was also assessed in retinal vascular cells cultured under high glucose conditions. The number of apoptotic cells was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling staining and confirmed by determining the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3. Results Our experiments demonstrated that retinal ECs, PCs, ACs, choroidal ECs, and RPE cells expressed caspase-14, and DR was associated with upregulation and/or activation of caspase-14 particularly in retinal vasculature. High glucose induced marked elevation of the caspase-14 level in retinal vascular cells. There was a significant increase in the apoptosis rate and the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3 in retinal ECs and PCs overexpressing caspase-14. Conclusions Our findings indicate that caspase-14 might play a significant role in the pathogenesis of DR by accelerating retinal PC and EC death. Further investigations are required to elaborate the underlying mechanisms. PMID:22876114

  4. PARP INHIBITION ALLEVIATES DIABETES-INDUCED SYSTEMIC OXIDATIVE STRESS AND NEURAL TISSUE 4-HYDROXYNONENAL ADDUCT ACCUMULATION: CORRELATION WITH PERIPHERAL NERVE FUNCTION

    PubMed Central

    Lupachyk, Sergey; Shevalye, Hanna; Maksimchyk, Yury; Drel, Viktor R.; Obrosova, Irina G.

    2011-01-01

    This study evaluated the role of poly(ADP-ribose) polymerase in systemic oxidative stress and 4-hydoxynonenal adduct accumulation in diabetic peripheral neuropathy. Control and streptozotocin-diabetic rats were maintained with or without treatment with the PARP inhibitor, 1,5-isoquinolinediol, 3 mg kg−1d−1, for 10 weeks after initial 2 weeks. Treatment efficacy was evaluated by poly(ADP-ribosyl)ated protein content in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons and non-neuronal cells (fluorescent immunohistochemistry), as well as by indices of peripheral nerve function. Diabetic rats displayed increased urinary isoprostane and 8-hydroxy-2'-deoxyguanosine excretion (ELISA), 4-hydroxynonenal adduct accumulation in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord, and neurons and glial cells of the dorsal root ganglia (double-label fluorescent immunohistochemistry) as well as motor and sensory nerve conduction velocity deficits, thermal hypoalgesia, and tactile allodynia. PARP inhibition counteracted diabetes-induced systemic oxidative stress and 4-hydroxynonenal adduct accumulation in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons (perikarya, fluorescent immunohistochemistry) which correlated with improvement of large and small nerve fiber function. The findings reveal the important role of PARP activation in systemic oxidative stress and 4-hydroxynonenal adduct accumulation in diabetic peripheral neuropathy. PMID:21300148

  5. PARP1 promotes gene expression at the post-transcriptional level by modulating the RNA-binding protein HuR

    PubMed Central

    Ke, Yueshuang; Han, Yanlong; Guo, Xiaolan; Wen, Jitao; Wang, Ke; Jiang, Xue; Tian, Xue; Ba, Xueqing; Boldogh, Istvan; Zeng, Xianlu

    2017-01-01

    Poly(ADP-ribosyl)ation (PARylation) is mainly catalysed by poly-ADP-ribose polymerase 1 (PARP1), whose role in gene transcription modulation has been well established. Here we show that, in response to LPS exposure, PARP1 interacts with the adenylateuridylate-rich element-binding protein embryonic lethal abnormal vision-like 1 (Elavl1)/human antigen R (HuR), resulting in its PARylation, primarily at site D226. PARP inhibition and the D226 mutation impair HuR's PARylation, nucleocytoplasmic shuttling and mRNA binding. Increases in mRNA level or stability of pro-inflammatory cytokines/chemokines are abolished by PARP1 ablation or inhibition, or blocked in D226A HuR-expressing cells. The present study demonstrates a mechanism to regulate gene expression at the post-transcriptional level, and suggests that blocking the interaction of PARP1 with HuR could be a strategy to treat inflammation-related diseases that involve increased mRNA stability. PMID:28272405

  6. Synergistic Effect of Trabectedin and Olaparib Combination Regimen in Breast Cancer Cell Lines

    PubMed Central

    Ávila-Arroyo, Sonia; Nuñez, Gema Santamaría; García-Fernández, Luis Francisco

    2015-01-01

    Purpose Trabectedin induces synthetic lethality in tumor cells carrying defects in homologous recombinant DNA repair. We evaluated the effect of concomitant inhibition of nucleotide-excision repair and poly (ADP-ribose) polymerase (PARP) activity with trabectedin and PARP inhibitors, respectively, and whether the synthetic lethality effect had the potential for a synergistic effect in breast cancer cell lines. Additionally, we investigated if this approach remained effective in BRCA1-positive breast tumor cells. Methods We have evaluated the in vitro synergistic effect of combinations of trabectedin and three different PARP inhibitors (veliparib, olaparib, and iniparib) in four breast cancer cell lines, each presenting a different BRCA1 genetic background. Antiproliferative activity, DNA damage, cell cycle perturbations and poly(ADP-ribosyl)ation were assessed by MTT assay, comet assay, flow cytometry and western blot, respectively. Results The combination of trabectedin and olaparib was synergistic in all the breast cancer cell lines tested. Our data indicated that the synergy persisted regardless of the BRCA1 status of the tumor cells. Combination treatment was associated with a strong accumulation of double-stranded DNA breaks, G2/M arrest, and apoptotic cell death. Synergistic effects were not observed when trabectedin was combined with veliparib or iniparib. Conclusion Collectively, our results indicate that the combination of trabectedin and olaparib induces an artificial synthetic lethality effect that can be used to kill breast cancer cells, independent of BRCA1 status. PMID:26770239

  7. Olaparib significantly delays photoreceptor loss in a model for hereditary retinal degeneration

    PubMed Central

    Sahaboglu, Ayse; Barth, Melanie; Secer, Enver; Amo, Eva M. del; Urtti, Arto; Arsenijevic, Yvan; Zrenner, Eberhart; Paquet-Durand, François

    2016-01-01

    The enzyme poly-ADP-ribose-polymerase (PARP) mediates DNA-repair and rearrangements of the nuclear chromatin. Generally, PARP activity is thought to promote cell survival and in recent years a number of PARP inhibitors have been clinically developed for cancer treatment. Paradoxically, PARP activity is also connected to many diseases including the untreatable blinding disease Retinitis Pigmentosa (RP), where PARP activity appears to drive the pathogenesis of photoreceptor loss. We tested the efficacy of three different PARP inhibitors to prevent photoreceptor loss in the rd1 mouse model for RP. In retinal explant cultures in vitro, olaparib had strong and long-lasting photoreceptor neuroprotective capacities. We demonstrated target engagement by showing that olaparib reduced photoreceptor accumulation of poly-ADP-ribosylated proteins. Remarkably, olaparib also reduced accumulation of cyclic-guanosine-monophosphate (cGMP), a characteristic marker for photoreceptor degeneration. Moreover, intravitreal injection of olaparib in rd1 animals diminished PARP activity and increased photoreceptor survival, confirming in vivo neuroprotection. This study affirms the role of PARP in inherited retinal degeneration and for the first time shows that a clinically approved PARP inhibitor can prevent photoreceptor degeneration in an RP model. The wealth of human clinical data available for olaparib highlights its strong potential for a rapid clinical translation into a novel RP treatment. PMID:28004814

  8. Understanding specific functions of PARP-2: new lessons for cancer therapy

    PubMed Central

    Ali, Syed O; Khan, Farhaan A; Galindo-Campos, Miguel A; Yélamos, José

    2016-01-01

    Poly(ADP-ribosyl)ation (PARylation) is a widespread and highly conserved post-translational modification catalysed by a large family of enzymes called poly(ADP-ribose) polymerases (PARPs). PARylation plays an essential role in various cardinal processes of cellular physiology and recent approvals and breakthrough therapy designations for PARP inhibitors in cancer therapy have sparked great interest in pharmacological targeting of PARP proteins. Although, many PARP inhibitors have been developed, existing compounds display promiscuous inhibition across the PARP superfamily which could lead to unwanted off-target effects. Thus the prospect of isoform-selective inhibition is being increasingly explored and research is now focusing on understanding specific roles of PARP family members. PARP-2, alongside PARP-1 and PARP-3 are the only known DNA damage-dependent PARPs and play critical roles in the DNA damage response, DNA metabolism and chromatin architecture. However, growing evidence shows that PARP-2 plays specific and diverse regulatory roles in cellular physiology, ranging from genomic stability and epigenetics to proliferative signalling and inflammation. The emerging network of PARP-2 target proteins has uncovered wide-ranging functions of the molecule in many cellular processes commonly dysregulated in carcinogenesis. Here, we review novel PARP-2-specific functions in the hallmarks of cancer and consider the implications for the development of isoform-selective inhibitors in chemotherapy. By considering the roles of PARP-2 through the lens of tumorigenesis, we propose PARP-2-selective inhibition as a potentially multipronged attack on cancer physiology. PMID:27725894

  9. Identification of nuclear proteins in soybean under flooding stress using proteomic technique.

    PubMed

    Oh, Myeong Won; Nanjo, Yohei; Komatsu, Setsuko

    2014-05-01

    Flooding stress restricts soybean growth, it results in decrease the production. In this report, to understand how nuclear proteins in soybean affected by flooding, abundance changes of those proteins was analyzed. Nuclear proteins were extracted from the root tips of soybean treated with or without flooding stress. The extracted proteins were analyzed using a label-free quantitative proteomic technique. Of a total of 94 nuclear proteins that were found to be responsive to flooding, the 19 and 75 proteins were increased and decreased, respectively. The identified flooding-responsive proteins were functionally classified, revealing that 8 increased proteins changed in protein synthesis, posttranslational modification, and protein degradation, while 34 decreased proteins were involved in transcription, RNA processing, DNA synthesis, and chromatin structure maintenance. Among these proteins, those whose levels changed more than 10 fold included two poly ADP-ribose polymerases and a novel G-domain-containing protein that might be involved in RNA binding. The mRNA expression levels of these three proteins indicated a similar tendency to their protein abundance changes. These results suggest that acceleration of protein poly-ADP-ribosylation and suppression of RNA metabolism may be involved in root tip of soybean under flooding stress.

  10. 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.

  11. Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

    PubMed Central

    Shang, Fenqing; Zhang, Jiao; Li, Zhao; Zhang, Jin; Yin, Yanjun; Wang, Yaqiong; Marin, Traci L.; Gongol, Brendan; Xiao, Han; Zhang, You-yi; Chen, Zhen; Shyy, John Y-J; Lei, Ting

    2016-01-01

    Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction. PMID:26986624

  12. 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 (MgCl2 , CaCl2 , SrCl2 , CuCl2 , FeCl2 , FeCl3 , ZnCl2 ). 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.

  13. 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

  14. Exploiting polymerase promiscuity: A simple colorimetric RNA polymerase assay.

    PubMed

    Vassiliou, W; Epp, J B; Wang, B B; Del Vecchio, A M; Widlanski, T; Kao, C C

    2000-09-01

    We developed a convenient colorimetric assay for monitoring RNA synthesis from DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp). ATP and GTP with a p-nitrophenyl moiety attached to the gamma-phosphate were synthesized (PNP-NTPs). These PNP-NTPs can be used for RNA synthesis by several RNA polymerases, including the RdRps from brome mosaic virus and bovine viral diarrhea virus and the DdRps from bacteriophage T7 and SP6. When the polymerase reactions were performed in the presence of alkaline phosphatase, which digests the p-nitrophenylpyrophosphate side-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was observed. These nucleotide analogues were used in continuous colorimetric monitoring of polymerase activity. Furthermore, the PNP-NTPs were found to be stable and utilized by RNA polymerases in the presence of human plasma. This simple colorimetric polymerase assay can be performed in a standard laboratory spectrophotometer and will be useful in screens for inhibitors of viral RNA synthesis.

  15. 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

  16. Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

    PubMed Central

    Kempf, Brian J.; Peersen, Olve B.

    2016-01-01

    ABSTRACT RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a

  17. Structural studies of large nucleoprotein particles, vaults

    PubMed Central

    TANAKA, Hideaki; TSUKIHARA, Tomitake

    2012-01-01

    Vault is the largest nonicosahedral cytosolic nucleoprotein particle ever described. The widespread presence and evolutionary conservation of vaults suggest important biologic roles, although their functions have not been fully elucidated. X-ray structure of vault from rat liver was determined at 3.5 Å resolution. It exhibits an ovoid shape with a size of 40 × 40 × 67 nm3. The cage structure of vault consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The other components of vaults, telomerase-associated proteins, poly(ADP-ribose) polymerases and small RNAs, are in location in the vault particle by electron microscopy. PMID:23060231

  18. Construction and Application of a Rh–Pt DNA Metalloinsertor Conjugate

    PubMed Central

    2015-01-01

    We report the synthesis and characterization of a bimetallic conjugate (RhPt) in which an oxaliplatin derivative is tethered to a rhodium metalloinsertor through an aminomalonate leaving group ligand. The complex interacts with DNA through metalloinsertion at a base pair mismatch followed by formation of a covalent Pt–DNA adduct. Characterization of RhPt in mismatch repair-deficient HCT116O cells reveals increased cytotoxicity compared to cisplatin and oxaliplatin as well as relative to the unconjugated rhodium and platinum counterparts. Caspase and poly-ADP ribose polymerase inhibition assays indicate that RhPt induces apoptotic cell death. Inductively coupled plasma mass spectrometry (ICP-MS) experiments reveal that RhPt exhibits enhanced cellular uptake properties that contribute to its increased efficacy. PMID:25032512

  19. Novel Neuroprotective Strategies in Ischemic Retinal Lesions

    PubMed Central

    Szabadfi, Krisztina; Mester, Laszlo; Reglodi, Dora; Kiss, Peter; Babai, Norbert; Racz, Boglarka; Kovacs, Krisztina; Szabo, Aliz; Tamas, Andrea; Gabriel, Robert; Atlasz, Tamas

    2010-01-01

    Retinal ischemia can be effectively modeled by permanent bilateral common carotid artery occlusion, which leads to chronic hypoperfusion-induced degeneration in the entire rat retina. The complex pathways leading to retinal cell death offer a complex approach of neuroprotective strategies. In the present review we summarize recent findings with different neuroprotective candidate molecules. We describe the protective effects of intravitreal treatment with: (i) urocortin 2; (ii) a mitochondrial ATP-sensitive K+ channel opener, diazoxide; (iii) a neurotrophic factor, pituitary adenylate cyclase activating polypeptide; and (iv) a novel poly(ADP-ribose) polymerase inhibitor (HO3089). The retinoprotective effects are demonstrated with morphological description and effects on apoptotic pathways using molecular biological techniques. PMID:20386654

  20. Breast cancer in BRCA mutation carriers: medical treatment.

    PubMed

    Milani, Andrea; Geuna, Elena; Zucchini, Giorgia; Aversa, Caterina; Martinello, Rossella; Montemurro, Filippo

    2016-10-01

    About 10% of breast cancers are associated with the inheritance of autosomal dominant breast cancer susceptibility alleles BRCA1 and BRCA2. Until recently, the medical management of BRCA mutation-associated breast cancer has not differed from that of the sporadic breast cancer counterpart. However, there is mounting evidence that this molecular alteration confers sensitivity or resistance to systemic therapies that can be exploited in terms of medical management. For example, studies support the use of platinum salts chemotherapy in BRCA mutated cancers. Moreover, a number of targeted therapies are showing activity in BRCA mutation carriers. Above all, BRCA defective tumor cells are particularly sensitive to Poly(ADP-ribose) polymerase (PARP) inhibitors. This review will summarize the state of the art of the medical treatment of breast cancer in BRCA mutation carriers, with a particular focus on chemotherapies and targeted therapies.

  1. BRCA somatic mutations and epigenetic BRCA modifications in serous ovarian cancer.

    PubMed

    Moschetta, M; George, A; Kaye, S B; Banerjee, S

    2016-08-01

    The significant activity of poly(ADP-ribose)polymerase (PARP) inhibitors in the treatment of germline BRCA mutation-associated ovarian cancer, which represents ∼15% of HGS cases, has recently led to European Medicines Agency and food and drug administration approval of olaparib. Accumulating evidence suggests that PARP inhibitors may have a wider application in the treatment of sporadic ovarian cancers. Up to 50% of HGS ovarian cancer patients may exhibit homologous recombination deficiency (HRD) through mechanisms including germline BRCA mutations, somatic BRCA mutations, and BRCA promoter methylation. In this review, we discuss the role of somatic BRCA mutations and BRCA methylation in ovarian cancer. There is accumulating evidence for routine somatic BRCA mutation testing, but the relevance of BRCA epigenetic modifications is less clear. We explore the challenges that need to be addressed if the full potential of these markers of HRD is to be utilised in clinical practice.

  2. Enhancing NAD+ salvage metabolism is neuroprotective in a PINK1 model of Parkinson's disease

    PubMed Central

    Lehmann, Susann; Loh, Samantha H. Y.

    2017-01-01

    ABSTRACT Familial forms of Parkinson's disease (PD) caused by mutations in PINK1 are linked to mitochondrial impairment. Defective mitochondria are also found in Drosophila models of PD with pink1 mutations. The co-enzyme nicotinamide adenine dinucleotide (NAD+) is essential for both generating energy in mitochondria and nuclear DNA repair through NAD+-consuming poly(ADP-ribose) polymerases (PARPs). We found alterations in NAD+ salvage metabolism in Drosophila pink1 mutants and showed that a diet supplemented with the NAD+ precursor nicotinamide rescued mitochondrial defects and protected neurons from degeneration. Additionally, a mutation of Parp improved mitochondrial function and was neuroprotective in the pink1 mutants. We conclude that enhancing the availability of NAD+ by either the use of a diet supplemented with NAD+ precursors or the inhibition of NAD+-dependent enzymes, such as PARPs, which compete with mitochondria for NAD+, is a viable approach to preventing neurotoxicity associated with mitochondrial defects. PMID:28011627

  3. Cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated acute epileptic neuronal death in vitro.

    PubMed

    Wang, Shengjun; Yang, Xue; Lin, Youting; Qiu, Xiaoxue; Li, Hui; Zhao, Xiuhe; Cao, Lili; Liu, Xuewu; Pang, Yuejiu; Wang, Xuping; Chi, Zhaofu

    2013-10-16

    Intense poly(ADP-ribose) polymerase-1 (PARP-1) activation was implicated as a major cause of caspase-independent cell death in the hippocampal neuronal culture (HNC) model of acute acquired epilepsy (AE). The molecular mechanisms are quite complicated. The linkage among neuronal death, cellular nicotinamide adenine dinucleotide (NAD) levels, apoptosis-inducing factor (AIF) translocation, SIRT1 expression and activity were investigated here. The results showed that PARP-1 over-activation caused by Mg²⁺-free stimuli led to cellular NAD depletion which could block AIF translocation from mitochondria to nucleus and attenuate neuronal death. Also, SIRT1 deacetylase activity was reduced by Mg²⁺-free treatment, accompanied by elevated ratio of neuronal death, which could be rescued by NAD repletion. These data demonstrated that cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated epileptic neuronal death in the HNC model of acute AE.

  4. NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease.

    PubMed

    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

    2014-06-03

    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.

  5. Tankyrase is necessary for canonical Wnt signaling during kidney development

    PubMed Central

    Karner, Courtney M.; Merkel, Calli E; Dodge, Michael; Ma, Zhiqiang; Lu, Jianming; Chen, Chuo; Lum, Lawrence; Carroll, Thomas J.

    2010-01-01

    Recent studies utilizing small molecule antagonists have revealed that the poly(ADP-ribose) polymerases (PARPs) Tankyrase 1 and 2 are critical regulators of canonical Wnt signaling in some cellular contexts. However, the absence of any activity during zebrafish embryogenesis suggested that the tankyrases may not be general/core components of the Wnt pathway. Here we show that Tnks1 and 2 are broadly expressed during mouse development and are essential during kidney and lung development. In the kidney, blockage of tankyrase activity phenocopies the effect of blocking production of all Wnt ligands. Tankyrase inhibition can be rescued by activation of β-catenin demonstrating its specificity for the Wnt pathway. In addition, treatment with tankyrase inhibitors appears to be completely reversible in some cell types. These studies suggest that the tankyrases are core components of the canonical Wnt pathway and their inhibitors should enjoy broad usage as antagonists of Wnt signaling. PMID:20549720

  6. 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

    2017-03-02

    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.

  7. Apoptosis induced by the Tibetan herbal remedy PADMA 28 in the T cell-derived lymphocytic leukaemia cell line CEM-C7H2

    PubMed Central

    Jenny, Marcel; Schwaiger, Wolfgang; Bernhard, David; Wrulich, Oliver A; Cosaceanu, Daria; Fuchs, Dietmar; Ueberall, Florian

    2005-01-01

    The Tibetan herbal remedy PADMA 28 revealed promising results to support treatment of atherosclerosis, Charot syndrome (intermittent claudication), chronic active hepatitis and infection of the respiratory tract. The remedy was confirmed to be closely linked with anti- and pro-oxidative properties in vitro. In this study, apoptogenic and survival effects of PADMA 28 were investigated in the T cell-derived lymphocytic leukaemia cell line CEM-C7H2. PADMA 28 led to a concentration-dependent inhibition of cell proliferation accompanied by the accumulation of CEM-C7H2 cells in subG1 phase, fragmentation of poly (ADP-ribose) polymerase (PARP) and nuclear body formation. Treatment with PADMA 28 rescued to some extent cells over-expressing Bcl-2 from apoptosis. This finding suggests that the mechanism of action of PADMA 28 may be via interference with Bcl-2 triggered survival pathways. PMID:16138918

  8. Genome-wide barcoded transposon screen for cancer drug sensitivity in haploid mouse embryonic stem cells

    PubMed Central

    Pettitt, Stephen J.; Krastev, Dragomir B.; Pemberton, Helen N.; Fontebasso, Yari; Frankum, Jessica; Rehman, Farah L.; Brough, Rachel; Song, Feifei; Bajrami, Ilirjana; Rafiq, Rumana; Wallberg, Fredrik; Kozarewa, Iwanka; Fenwick, Kerry; Armisen-Garrido, Javier; Swain, Amanda; Gulati, Aditi; Campbell, James; Ashworth, Alan; Lord, Christopher J.

    2017-01-01

    We describe a screen for cellular response to drugs that makes use of haploid embryonic stem cells. We generated ten libraries of mutants with piggyBac gene trap transposon integrations, totalling approximately 100,000 mutant clones. Random barcode sequences were inserted into the transposon vector to allow the number of cells bearing each insertion to be measured by amplifying and sequencing the barcodes. These barcodes were associated with their integration sites by inverse PCR. We exposed these libraries to commonly used cancer drugs and profiled changes in barcode abundance by Ion Torrent sequencing in order to identify mutations that conferred sensitivity. Drugs tested included conventional chemotherapeutics as well as targeted inhibitors of topoisomerases, poly(ADP-ribose) polymerase (PARP), Hsp90 and WEE1. PMID:28248920

  9. Kynurenine pathway metabolism and neuroinflammatory disease

    PubMed Central

    Braidy, Nady; Grant, Ross

    2017-01-01

    Immune-mediated activation of tryptophan (TRYP) catabolism via the kynurenine pathway (KP) is a consistent finding in all inflammatory disorders. Several studies by our group and others have examined the neurotoxic potential of neuroreactive TRYP metabolites, including quinolinic acid (QUIN) in neuroinflammatory neurological disorders, including Alzheimer's disease (AD), multiple sclerosis, amylotropic lateral sclerosis (ALS), and AIDS related dementia complex (ADC). Our current work aims to determine whether there is any benefit to the affected individuals in enhancing the catabolism of TRYP via the KP during an immune response. Under physiological conditions, QUIN is metabolized to the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD+), which represents an important metabolic cofactor and electron transporter. NAD+ also serves as a substrate for the DNA ‘nick sensor’ and putative nuclear repair enzyme, poly(ADP-ribose) polymerase (PARP). Free radical initiated DNA damage, PARP activation and NAD+ depletion may contribute to brain dysfunction and cell death in neuroinflammatory disease. PMID:28250737

  10. Radiation-inducible immunotherapy for cancer: senescent tumor cells as a cancer vaccine.

    PubMed

    Meng, Yuru; Efimova, Elena V; Hamzeh, Khaled W; Darga, Thomas E; Mauceri, Helena J; Fu, Yang-Xin; Kron, Stephen J; Weichselbaum, Ralph R

    2012-05-01

    Radiotherapy offers an effective treatment for advanced cancer but local and distant failures remain a significant challenge. Here, we treated melanoma and pancreatic carcinoma in syngeneic mice with ionizing radiation (IR) combined with the poly(ADP-ribose) polymerase inhibitor (PARPi) veliparib to inhibit DNA repair and promote accelerated senescence. Based on prior work implicating cytotoxic T lymphocytes (CTLs) as key mediators of radiation effects, we discovered that senescent tumor cells induced by radiation and veliparib express immunostimulatory cytokines to activate CTLs that mediate an effective antitumor response. When these senescent tumor cells were injected into tumor-bearing mice, an antitumor CTL response was induced which potentiated the effects of radiation, resulting in elimination of established tumors. Applied to human cancers, radiation-inducible immunotherapy may enhance radiotherapy responses to prevent local recurrence and distant metastasis.

  11. 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

  12. In vivo imaging of specific drug target binding at subcellular resolution

    PubMed Central

    Dubach, J.M.; Vinegoni, C.; Mazitschek, R.; Fumene Feruglio, P.; Cameron, L.A.; Weissleder, R.

    2015-01-01

    The possibility to measure binding of small molecule drugs to desired targets in live cells could provide a better understanding of drug action. However, current approaches mostly yield static data, require lysis or rely on indirect assays and thus often provide an incomplete understanding of drug action. Here, we present a multiphoton fluorescence anisotropy microscopy live cell imaging technique to measure and map drug-target interaction in real time at subcellular resolution. This approach is generally applicable using any fluorescently labeled drug and enables high resolution spatial and temporal mapping of bound and unbound drug distribution. To illustrate our approach we measure intracellular target engagement of the chemotherapeutic Olaparib, a poly(ADP-ribose) polymerase inhibitor, in live cells and within a tumor in vivo. These results are the first generalizable approach to directly measure drug-target binding in vivo and present a promising tool to enhance understanding of drug activity. PMID:24867710

  13. Lantana camara Induces Apoptosis by Bcl-2 Family and Caspases Activation.

    PubMed

    Han, Eun Byeol; Chang, Bo Yoon; Jung, Young Suk; Kim, Sung Yeon

    2015-04-01

    Breast cancer is one of the most common cancers worldwide, and the second most fatal cancer in women after lung cancer. Because there are instances of cancer resistance to existing therapies, studies focused on the identification of novel therapeutic drugs are very important. In this study, we identified a natural anticancer agent from Lantana camara, a flowering plant species of the genus Verbena. The extract obtained from the L. camara exhibited cell death properties in the human breast cancer cell line, MCF-7. We found that the apoptosis induced by treatment with the L. camara extract was regulated by the Bcl-2 family. Bid and Bax was increased and Bcl-2 was decreased by L. camara extract. L. camara extract modulated cleavage of caspase-8, and caspase-9, as well as poly (ADP-ribose) polymerase (PARP). Our results support the potential use of the L. camara extract as an anti-breast cancer drug.

  14. Development of synthetic lethality anticancer therapeutics.

    PubMed

    Fang, Bingliang

    2014-10-09

    The concept of synthetic lethality (the creation of a lethal phenotype from the combined effects of mutations in two or more genes) has recently been exploited in various efforts to develop new genotype-selective anticancer therapeutics. These efforts include screening for novel anticancer agents, identifying novel therapeutic targets, characterizing mechanisms of resistance to targeted therapy, and improving efficacies through the rational design of combination therapy. This review discusses recent developments in synthetic lethality anticancer therapeutics, including poly ADP-ribose polymerase inhibitors for BRCA1- and BRCA2-mutant cancers, checkpoint inhibitors for p53 mutant cancers, and small molecule agents targeting RAS gene mutant cancers. Because cancers are caused by mutations in multiple genes and abnormalities in multiple signaling pathways, synthetic lethality for a specific tumor suppressor gene or oncogene is likely cell context-dependent. Delineation of the mechanisms underlying synthetic lethality and identification of treatment response biomarkers will be critical for the success of synthetic lethality anticancer therapy.

  15. Olaparib in the management of ovarian cancer

    PubMed Central

    Bixel, Kristin; Hays, John L

    2015-01-01

    Alterations in the homologous repair pathway are thought to occur in 30%–50% of epithelial ovarian cancers. Cells deficient in homologous recombination rely on alternative pathways for DNA repair in order to survive, thereby providing a potential target for therapy. Olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, capitalizes on this concept and is the first drug in its class approved for patients with ovarian cancer. This review article will provide an overview of the BRCA genes and homologous recombination, the role of PARP in DNA repair and the biological rationale for the use of PARP inhibitors as cancer therapy, and ultimately will focus on the use of olaparib in the management of ovarian cancer. PMID:26309417

  16. Apoptosis-inducing effect of a palladium(II) saccharinate complex of terpyridine on human breast cancer cells in vitro and in vivo.

    PubMed

    Ari, Ferda; Cevatemre, Buse; Armutak, Elif Ilkay Ikitimur; Aztopal, Nazlihan; Yilmaz, Veysel T; Ulukaya, Engin

    2014-09-01

    The anti-growth effect of a palladium(II) complex-[PdCl(terpy)](sac)·2H2O] (sac=saccharinate, and terpy=2,2':6',2″-terpyridine)-was tested against human breast cancer cell lines, MCF-7 and MDA-MB-231. Anti-growth effect was assayed by the MTT and ATP viability assays in vitro and then confirmed on Balb/c mice in vivo. The mode of cell death was determined by both histological and biochemical methods. The Pd(II) complex had anti-growth effect on a dose dependent manner in vitro and in vivo. The cells died by apoptosis as evidenced by the pyknotic nucleus, cleavage of poly-(ADP-ribose) polymerase (PARP) and induction of active caspase-3. These results suggest that the palladium(II) saccharinate complex of terpyridine represents a potentially active novel complex for the breast cancer treatment, thus warrants further studies.

  17. PARP-1 activation requires local unfolding of an autoinhibitory domain

    PubMed Central

    Dawicki-McKenna, Jennine M.; Langelier, Marie-France; DeNizio, Jamie E.; Riccio, Amanda A.; Cao, Connie D.; Karch, Kelly R.; McCauley, Michael; Steffen, Jamin D.; Black, Ben E.; Pascal, John M.

    2015-01-01

    SUMMARY Poly(ADP-ribose) polymerase-1 (PARP-1) creates the posttranslational modification PAR from substrate NAD+ to regulate multiple cellular processes. DNA breaks sharply elevate PARP-1 catalytic activity to mount a cell survival repair response, whereas persistent PARP-1 hyperactivation during severe genotoxic stress is associated with cell death. The mechanism for tight control of the robust catalytic potential of PARP-1 remains unclear. By monitoring PARP-1 dynamics using hydrogen/deuterium exchange-mass spectrometry (HXMS), we unexpectedly find that a specific portion of the helical subdomain (HD) of the catalytic domain rapidly unfolds when PARP-1 encounters a DNA break. Together with biochemical and crystallographic analysis of HD deletion mutants, we show that the HD is an autoinhibitory domain that blocks productive NAD+ binding. Our molecular model explains how PARP-1 DNA damage detection leads to local unfolding of the HD that relieves autoinhibition, and has important implications for the design of PARP inhibitors. PMID:26626480

  18. PARP-1 Activation Requires Local Unfolding of an Autoinhibitory Domain.

    PubMed

    Dawicki-McKenna, Jennine M; Langelier, Marie-France; DeNizio, Jamie E; Riccio, Amanda A; Cao, Connie D; Karch, Kelly R; McCauley, Michael; Steffen, Jamin D; Black, Ben E; Pascal, John M

    2015-12-03

    Poly(ADP-ribose) polymerase-1 (PARP-1) creates the posttranslational modification PAR from substrate NAD(+) to regulate multiple cellular processes. DNA breaks sharply elevate PARP-1 catalytic activity to mount a cell survival repair response, whereas persistent PARP-1 hyperactivation during severe genotoxic stress is associated with cell death. The mechanism for tight control of the robust catalytic potential of PARP-1 remains unclear. By monitoring PARP-1 dynamics using hydrogen/deuterium exchange-mass spectrometry (HXMS), we unexpectedly find that a specific portion of the helical subdomain (HD) of the catalytic domain rapidly unfolds when PARP-1 encounters a DNA break. Together with biochemical and crystallographic analysis of HD deletion mutants, we show that the HD is an autoinhibitory domain that blocks productive NAD(+) binding. Our molecular model explains how PARP-1 DNA damage detection leads to local unfolding of the HD that relieves autoinhibition, and has important implications for the design of PARP inhibitors.

  19. 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

  20. Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

    SciTech Connect

    Chen Haibing; Jia Weiping; Xu Xun; Fan Ying; Zhu Dongqing; Wu Haixiang; Xie Zhenggao; Zheng Zhi

    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 represent a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent.

  1. A polysaccharide from the alkaline extract of Glycyrrhiza inflata induces apoptosis of human oral cancer SCC-25 cells via mitochondrial pathway.

    PubMed

    Zeng, Guang; Shen, Huan; Tang, Guo; Cai, Xingwei; Bi, Lixia; Sun, Bin; Yang, Yongjin; Xun, Wenxing

    2015-09-01

    In the present study, we isolated and characterized a homogenous polysaccharide (GIAP1) from the alkaline extract of the roots of Glycyrrhiza inflata. The anti-tumor activity of GIAP1 toward human oral cancer SCC-25 cells and the underlying mechanisms were also examined in vitro. GIAP1 dose-dependently inhibited the proliferation of SCC-25 cells via inducing apoptosis. Moreover, GIAP1 downregulated Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), and caused the release of cytochrome c to cytosol. Besides, GIAP1 triggered activation of capase-3 and caspase-9, as well as the degradation of poly (ADP-ribose) polymerase (PARP). In addition, the caspase-3 or caspase-9 inhibitor significantly inhibited GIAP1-induced apoptosis in SCC-25 cells. Collectively, we can conclude that the GIAP1 induces apoptosis in SCC-25 cells via a mitochondrial pathway.

  2. NAD+ and Sirtuins in Aging and Disease

    PubMed Central

    Imai, Shin-ichiro; Guarente, Leonard

    2014-01-01

    Nicotinamide adenine dinucleotide (NAD+) is a classical coenzyme mediating many redox reactions. NAD+ also plays an important role in the regulation of NAD+-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD+ biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD+ levels decline during the aging process and may be an Achilles’ heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD+ by supplementing NAD+ intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD+ intermediate supplementation may be an effective anti-aging intervention, providing hope to aging societies worldwide. PMID:24786309

  3. Niacinamide - mechanisms of action and its topical use in dermatology.

    PubMed

    Wohlrab, Johannes; Kreft, Daniela

    2014-01-01

    Niacinamide, an amide of vitamin B3 (niacin), is a hydrophilic endogenous substance. Its effects after epicutaneous application have long been described in the literature. Given a sufficient bioavailability, niacinamide has antipruritic, antimicrobial, vasoactive, photo-protective, sebostatic and lightening effects depending on its concentration. Within a complex metabolic system niacinamide controls the NFκB-mediated transcription of signalling molecules by inhibiting the nuclear poly (ADP-ribose) polymerase-1 (PARP-1). Niacinamide is a well-tolerated and safe substance often used in cosmetics. Clinical data for its therapeutic use in various dermatoses can increasingly be found in the literature. Although the existing data are not sufficient for a scientifically founded evaluation, it can be stated that the use of niacinamide in galenic preparations for epicutaneous application offers most interesting prospects.

  4. Parp mutations protect against mitochondrial dysfunction and neurodegeneration in a PARKIN model of Parkinson's disease

    PubMed Central

    Lehmann, S; Costa, A C; Celardo, I; Loh, S H Y; Martins, L M

    2016-01-01

    The co-enzyme nicotinamide adenine dinucleotide (NAD+) is an essential co-factor for cellular energy generation in mitochondria as well as for DNA repair mechanisms in the cell nucleus involving NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Mitochondrial function is compromised in animal models of Parkinson's disease (PD) associated with PARKIN mutations. Here, we uncovered alterations in NAD+ salvage metabolism in Drosophila parkin mutants. We show that a dietary supplementation with the NAD+ precursor nicotinamide rescues mitochondrial function and is neuroprotective. Further, by mutating Parp in parkin mutants, we show that this increases levels of NAD+ and its salvage metabolites. This also rescues mitochondrial function and suppresses dopaminergic neurodegeneration. We conclude that strategies to enhance NAD+ levels by administration of dietary precursors or the inhibition of NAD+-dependent enzymes, such as PARP, that compete with mitochondria for NAD+ could be used to delay neuronal death associated with mitochondrial dysfunction. PMID:27031963

  5. PARP Inhibitors for the Treatment and Prevention of Breast Cancer.

    PubMed

    Vinayak, Shaveta; Ford, James M

    2010-12-01

    Poly (ADP-ribose) polymerase (PARP) inhibitors, a novel class of drugs that target tumors with DNA repair defects, have received tremendous enthusiasm. Early preclinical studies identified BRCA1 and BRCA2 tumors to be highly sensitive to PARP inhibitors as a result of homologous recombination defect. Based on this premise, PARP inhibitors have been tested in early phase clinical trials as a single agent in BRCA1 or BRCA2 mutation carriers and in combination with chemotherapy in triple-negative breast cancer patients. For high-risk populations, use of PARP inhibition as a prevention agent has been postulated, but no robust preclinical or clinical studies exist yet. We review the preclinical and clinical studies in treatment of breast cancer and rationale for use of PARP inhibitors as a prevention agent for high-risk populations. Of significance, PARP inhibitors vary significantly in mechanism of action, dosing intervals, and toxicities, which are highlighted in this review.

  6. Enhancing the Cytotoxic Effects of PARP Inhibitors with DNA Demethylating Agents - A Potential Therapy for Cancer.

    PubMed

    Muvarak, Nidal E; Chowdhury, Khadiza; Xia, Limin; Robert, Carine; Choi, Eun Yong; Cai, Yi; Bellani, Marina; Zou, Ying; Singh, Zeba N; Duong, Vu H; Rutherford, Tyler; Nagaria, Pratik; Bentzen, Søren M; Seidman, Michael M; Baer, Maria R; Lapidus, Rena G; Baylin, Stephen B; Rassool, Feyruz V

    2016-10-10

    Poly (ADP-ribose) polymerase inhibitors (PARPis) are clinically effective predominantly for BRCA-mutant tumors. We introduce a mechanism-based strategy to enhance PARPi efficacy based on DNA damage-related binding between DNA methyltransferases (DNMTs) and PARP1. In acute myeloid leukemia (AML) and breast cancer cells, DNMT inhibitors (DNMTis) alone covalently bind DNMTs into DNA and increase PARP1 tightly bound into chromatin. Low doses of DNMTis plus PARPis, versus each drug alone, increase PARPi efficacy, increasing amplitude and retention of PARP1 directly at laser-induced DNA damage sites. This correlates with increased DNA damage, synergistic tumor cytotoxicity, blunting of self-renewal, and strong anti-tumor responses, in vivo in unfavorable AML subtypes and BRCA wild-type breast cancer cells. Our combinatorial approach introduces a strategy to enhance efficacy of PARPis in treating cancer.

  7. Design, synthesis and biological activity of novel molecules designed to target PARP and DNA.

    PubMed

    Goodfellow, Elliot; Senhaji Mouhri, Zhor; Williams, Christopher; Jean-Claude, Bertrand J

    2017-02-01

    In order to enhance the cytotoxic potential of poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1 or 2 deficient tumours, we designed a series of molecules containing a 1,2,3-triazene moiety tethered to a PARP targeting scaffold. A cell-based selectivity assay involving a BRCA2-deficient Chinese hamster cell line and its corresponding BRCA2 wild type transfectant, was used to predict the PARP targeting potential of the latter agents. The results showed that adding a DNA damaging function to the PARP inhibitors decreased but did not abrogate the selective targeting of the BRCA2-deficient cells. The DNA damaging moiety augmented the potency in BRCA2 deficient cells by 2-20 fold. The most selective dual PARP-DNA targeting agent 14b was found to possess dual DNA and PARP targeting properties.

  8. PARP inhibitors: Synthetic lethality in the clinic.

    PubMed

    Lord, Christopher J; Ashworth, Alan

    2017-03-17

    PARP inhibitors (PARPi), a cancer therapy targeting poly(ADP-ribose) polymerase, are the first clinically approved drugs designed to exploit synthetic lethality, a genetic concept proposed nearly a century ago. Tumors arising in patients who carry germline mutations in either BRCA1 or BRCA2 are sensitive to PARPi because they have a specific type of DNA repair defect. PARPi also show promising activity in more common cancers that share this repair defect. However, as with other targeted therapies, resistance to PARPi arises in advanced disease. In addition, determining the optimal use of PARPi within drug combination approaches has been challenging. Nevertheless, the preclinical discovery of PARPi synthetic lethality and the route to clinical approval provide interesting lessons for the development of other therapies. Here, we discuss current knowledge of PARP inhibitors and potential ways to maximize their clinical effectiveness.

  9. 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.

  10. Targeting the DNA Damage Response in Cancer.

    PubMed

    O'Connor, Mark J

    2015-11-19

    An underlying hallmark of cancers is their genomic instability, which is associated with a greater propensity to accumulate DNA damage. Historical treatment of cancer by radiotherapy and DNA-damaging chemotherapy is based on this principle, yet it is accompanied by significant collateral damage to normal tissue and unwanted side effects. Targeted therapy based on inhibiting the DNA damage response (DDR) in cancers offers the potential for a greater therapeutic window by tailoring treatment to patients with tumors lacking specific DDR functions. The recent approval of olaparib (Lynparza), the poly (ADP-ribose) polymerase (PARP) inhibitor for treating tumors harboring BRCA1 or BRCA2 mutations, represents the first medicine based on this principle, exploiting an underlying cause of tumor formation that also represents an Achilles' heel. This review highlights the different concepts behind targeting DDR in cancer and how this can provide significant opportunities for DDR-based therapies in the future.

  11. Bak is a key molecule in apoptosis induced by methanol extracts of Codonopsis lanceolata and Tricholoma matsutake in HSC-2 human oral cancer cells

    PubMed Central

    SHIN, JI-AE; KIM, JUN SUNG; HONG, IN-SUN; CHO, SUNG-DAE

    2012-01-01

    Since the 5-year survival rate of oral cancer remains low, more effective and non-toxic therapeutic and preventive strategies are required. Certain natural products possess anti-cancer properties. The present study investigated the effects of the methanol extracts of Codonopsis lanceolata (MECI) and Tricholoma matsutake (METM) and identified the molecular target in HSC-2 human oral cancer cells. The results revealed that MECI and METM inhibited growth and induced apoptosis, as demonstrated by poly (ADP-ribose) polymerase (PARP) cleavage and nuclear condensation and fragmentation. The compounds also increased Bak protein expression, while Bax, Bcl-XL and Mcl-1 were not affected. The results of the present study show that MECI and METM induce apoptosis to inhibit tumor growth of HSC-2 cells by modulating the Bak protein and suggest that Codonopsis lanceolata and Tricholoma matsutake are potential anticancer drug candidates for oral cancer. PMID:23205139

  12. The caspase 3-dependent apoptotic effect of pycnogenol in human oral squamous cell carcinoma HSC-3 cells

    PubMed Central

    Yang, In-Hyoung; Shin, Ji-Ae; Kim, Lee-Han; Kwon, Ki Han; Cho, Sung-Dae

    2016-01-01

    In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer. PMID:26798196

  13. The caspase 3-dependent apoptotic effect of pycnogenol in human oral squamous cell carcinoma HSC-3 cells.

    PubMed

    Yang, In-Hyoung; Shin, Ji-Ae; Kim, Lee-Han; Kwon, Ki Han; Cho, Sung-Dae

    2016-01-01

    In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer.

  14. Poliovirus protease 3C(pro) kills cells by apoptosis.

    PubMed

    Barco, A; Feduchi, E; Carrasco, L

    2000-01-20

    The tetracycline-based Tet-Off expression system has been used to analyze the effects of poliovirus protease 3C(pro) on human cells. Stable HeLa cell clones that express this poliovirus protease under the control of an inducible, tightly regulated promoter were obtained. Tetracycline removal induces synthesis of 3C protease, followed by drastic morphological alterations and cellular death. Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies are observed from the second day after 3C(pro) induction. The cleavage of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, occurs after induction of 3C(pro), indicating caspase activation by this poliovirus protease. The 3C(pro)-induced apoptosis is blocked by the caspase inhibitor z-VAD-fmk. Our findings suggest that the protease 3C is responsible for triggering apoptosis in poliovirus-infected cells by a mechanism that involves caspase activation.

  15. Repair of Topoisomerase I-Mediated DNA Damage

    PubMed Central

    Pommier, Yves; Barcelo, Juana; Rao, V. Ashutosh; Sordet, Olivier; Jobson, Andrew G.; Thibaut, Laurent; Miao, Zheyong; Seiler, Jennifer; Zhang, Hongliang; Marchand, Christophe; Agama, Keli; Redon, Christophe

    2008-01-01

    Topoisomerase I (Top1) is an abundant and essential enzyme. Top1 is the selective target of camptothecins, which are effective anticancer agents. Top1-DNA cleavage complexes can also be trapped by various endogenous and exogenous DNA lesions including mismatches, abasic sites and carcinogenic adducts. Tyrosyl-DNA phosphodiesterase (Tdp1) is one of the repair enzymes for Top1-DNA covalent complexes. Tdp1 forms a multiprotein complex that includes poly(ADP) ribose polymerase (PARP). PARP-deficient cells are hypersensitive to camptothecins and functionally deficient for Tdp1. We will review recent developments in several pathways involved in the repair of Top1 cleavage complexes and the role of Chk1 and Chk2 checkpoint kinases in the cellular responses to Top1 inhibitors. The genes conferring camptothecin hypersensitivity are compiled for humans, budding yeast and fission yeast. PMID:16891172

  16. Reverse the Resistance to PARP Inhibitors

    PubMed Central

    Kim, Yevgeniy; Kim, Aleksei; Sharip, Ainur; Sharip, Aigul; Jiang, Juhong; Yang, Qing; Xie, Yingqiu

    2017-01-01

    One of the DNA repair machineries is activated by Poly (ADP-ribose) Polymerase (PARP) enzyme. Particularly, this enzyme is involved in repair of damages to single-strand DNA, thus decreasing the chances of generating double-strand breaks in the genome. Therefore, the concept to block PARP enzymes by PARP inhibitor (PARPi) was appreciated in cancer treatment. PARPi has been designed and tested for many years and became a potential supplement for the conventional chemotherapy. However, increasing evidence indicates the appearance of the resistance to this treatment. Specifically, cancer cells may acquire new mutations or events that overcome the positive effect of these drugs. This paper describes several molecular mechanisms of PARPi resistance which were reported most recently, and summarizes some strategies to reverse this type of drug resistance. PMID:28255272

  17. Activation of caspase-3 in permanent and transient brain ischaemia in man.

    PubMed

    Love, S; Barber, R; Srinivasan, A; Wilcock, G K

    2000-08-03

    Animal studies have shown brain ischaemia to cause oxidative damage to DNA and activation of caspase-3, leading to apoptosis. These changes may be exacerbated by reperfusion. To assess caspase-3 activation after transient and permanent brain ischaemia in man, we examined brain tissue from patients who had experienced a cardiac arrest with resuscitation or an atherothrombotic brain infarct, and died 12 h to 9 days later. Sections were immunostained for activated caspase-3 or the 89 kDa caspase-3-mediated cleavage product of poly(ADP-ribose) polymerase. Brain ischaemia caused activation of caspase-3 in macrophages/microglia. Some neurons showed delayed activation of caspase-3 after cardiac arrest, but very few in atherothrombotic infarcts. In man, activation of caspase-3 plays little part in neuronal death in atherothrombotic infarcts but may contribute to delayed death of neurons after cardiac arrest.

  18. Minocycline Improves the Efficacy of EGFR Inhibitor Therapy: A Hypothesis

    PubMed Central

    Venniyoor, Ajit; Al Bahrani, Bassim

    2016-01-01

    Skin rash is a side effect of drugs that inhibit epithelial growth factor receptor (EGFR) as a part of targeted therapy of cancer. Its appearance and severity correlates with survival. Minocycline, an oral tetracycline antibiotic, is recommended as treatment (and increasingly, for prevention) of the rash, though infection is seen in only one-third of the patients. Minocycline has additional anticancer properties such as poly(ADP-ribose) polymerase inhibition. It is proposed that such properties contribute to the efficacy of EGFR inhibitors and can also explain the positive correlation between grade of rash and survival as patients with higher grades of rash are more likely to receive minocycline. Early concurrent administration of minocycline is recommended in patients planned for EGFR therapy while awaiting trials proving this hypothesis. PMID:27833902

  19. Crosstalk between components of circadian and metabolic cycles in mammals.

    PubMed

    Asher, Gad; Schibler, Ueli

    2011-02-02

    In mammals, most metabolic processes are influenced by biological clocks and feeding rhythms. The mechanisms that couple metabolism to circadian oscillators are just emerging. NAD-dependent enzymes (e.g., Sirtuins and poly[ADP-ribose] polymerases), redox- and/or temperature-dependent transcription factors (e.g., CLOCK, NPAS2, and HSF1), nutrient-sensing transcriptional regulatory proteins (e.g., CREB-CBP-CRCT2, FOXO-p300, nuclear receptors, PGC-1, and SP1 family members) and protein kinases (e.g., AMPK), are plausible candidates for conveying a cell's metabolic state to the core clock circuitry. The intertwining between these acute regulators and circadian clock components is so tight that the discrimination between metabolic and circadian oscillations may be somewhat arbitrary.

  20. Proteasomes play an essential role in thymocyte apoptosis.

    PubMed Central

    Grimm, L M; Goldberg, A L; Poirier, G G; Schwartz, L M; Osborne, B A

    1996-01-01

    Cell death in many different organisms requires the activation of proteolytic cascades involving cytosolic proteases. Here we describe a novel requirement in thymocyte cell death for the 20S proteasome, a highly conserved multicatalytic protease found in all eukaryotes. Specific inhibitors of proteasome function blocked cell death induced by ionizing radiation, glucocorticoids or phorbol ester. In addition to inhibiting apoptosis, these signals prevented the cleavage of poly(ADP-ribose) polymerase that accompanies many cell deaths. Since overall rates of protein degradation were not altered significantly during cell death in thymocytes, these results suggest that the proteasome may either degrade regulatory protein(s) that normally inhibit the apoptotic pathway or may proteolytically activate protein(s) than promote cell death. Images PMID:8670888

  1. Response of BRCA1-mutated gallbladder cancer to olaparib: A case report

    PubMed Central

    Xie, Yuan; Jiang, Yan; Yang, Xiao-Bo; Wang, An-Qiang; Zheng, Yong-Chang; Wan, Xue-Shuai; Sang, Xin-Ting; Wang, Kai; Zhang, Da-Dong; Xu, Jia-Jia; Li, Fu-Gen; Zhao, Hai-Tao

    2016-01-01

    Gallbladder cancer (GBC), although considered as a relatively rare malignancy, is the most common neoplasm of the biliary tract system. The late diagnosis and abysmal prognosis present challenges to treatment. The overall 5-year survival rate for metastatic GBC patients is extremely low. BRCA1 and BRCA2 are the breast cancer susceptibility genes and their mutation carriers are at a high risk for cancer development, both in men and women. Olaparib, an oral poly ADP-ribose polymerase inhibitor, has been approved by the Food and Drug Administration and the European Commission for the treatment of ovarian cancer with any BRCA1/2 mutations. The first case of a BRCA1-mutated GBC patient who responded to olaparib treatment is reported here. PMID:28028375

  2. Biochemical and cellular toxicology of peroxynitrite: implications in cell death and autoimmune phenomenon.

    PubMed

    Ahmad, Rizwan; Rasheed, Zafar; Ahsan, Haseeb

    2009-01-01

    Reactive nitrogen species include nitric oxide (.NO), peroxynitrite (ONOO(-)) and nitrogen dioxide radical (NO2*). Peroxynitrite is a reactive oxidant, produced from nitric oxide (*NO) and superoxide anion (O(2*-), that reacts with a variety of biological macromolecules. It is produced in the body in response to physiological stress and environmental toxins. It is a potent trigger of oxidative protein and DNA damage-including DNA strand breakage and base modification. It activates the nuclear enzyme poly-ADP ribose polymerase (PARP) resulting in energy depletion and apoptosis/necrosis of cells. Peroxynitrite generation is a crucial pathological mechanism in stroke, diabetes, inflammation, neurodegeneration, cancer, etc. Peroxynitrite modified DNA may also lead to the generation of autoantibodies in various autoimmune disorders such as systemic lupus erythematosus (SLE). In chronic inflammatory diseases, peroxynitrite formed by phagocytic cells may cause damage to DNA, generating neoepitopes leading to the production of autoantibodies. Hence, understanding the pathophysiology of peroxynitrite could lead to important therapeutic interventions.

  3. Predictors and Modulators of Synthetic Lethality: An Update on PARP Inhibitors and Personalized Medicine

    PubMed Central

    Murata, Stephen; Zhang, Catherine; Finch, Nathan; Zhang, Kevin; Campo, Loredana

    2016-01-01

    Poly(ADP-ribose) polymerase (PARP) inhibitors have proven to be successful agents in inducing synthetic lethality in several malignancies. Several PARP inhibitors have reached clinical trial testing for treatment in different cancers, and, recently, Olaparib (AZD2281) has gained both United States Food and Drug Administration (USFDA) and the European Commission (EC) approval for use in BRCA-mutated advanced ovarian cancer treatment. The need to identify biomarkers, their interactions in DNA damage repair pathways, and their potential utility in identifying patients who are candidates for PARP inhibitor treatment is well recognized. In this review, we detail many of the biomarkers that have been investigated for their ability to predict both PARP inhibitor sensitivity and resistance in preclinical studies as well as the results of several clinical trials that have tested the safety and efficacy of different PARP inhibitor agents in BRCA and non-BRCA-mutated cancers. PMID:27642590

  4. PARP3 interacts with FoxM1 to confer glioblastoma cell radioresistance.

    PubMed

    Quan, Jun-Jie; Song, Jin-Ning; Qu, Jian-Qiang

    2015-11-01

    Poly(ADP-ribose) polymerase 3 (PARP3), a critical player in cellular response to DNA double-strand breaks (DSBs), plays an essential role in the maintenance of genome integrity. However, the role of PARP3 in tumorigenesis especially in glioblastoma remains largely unknown. In the present study, we found that the mRNA and protein levels of PARP3 were upregulated in primary glioblastoma tissues. Knockdown of PARP3 expression by lentivirus-based shRNA decreased cell glioblastoma proliferation and inhibited tumor growth in vivo by using a xenograft mouse model. Furthermore, we found that silencing the expression of PARP3 resulted in a synergistic radiosensitizing effect when combined with radiotherapy in glioblastoma cell lines. At the molecular level, we found that PARP3 interacted with FoxM1 to enhance its transcriptional activity and conferred glioblastoma cell radioresistance. Thus, our data suggest that PARP3 could be a therapeutic target to overcome radioresistance in glioblastoma.

  5. Novel targeted therapeutics for mantle cell lymphoma--what's on the horizon?

    PubMed

    El Halabi, Layal; Ghez, David; Ribrag, Vincent

    2016-03-01

    Major advances have significantly improved the outcome of mantle cell lymphoma (MCL). Incorporation of rituximab to CHOP regimen, the adoption of high dose cytarabine with frontline autologous stem cell transplantation in young patients, maintenance rituximab or bortezomib based chemotherapy in elderly patients, improved the disease outcome. Bortezomib, lenalidomide, temsirolimus and ibrutinib have proven their efficacy and are approved for the use in refractory or relapsed MCL patients. Several other molecules are currently being evaluated such as cyclin dependent kinase 4/6 (CDK4/6), phosphoinositide 3-kinase (PI3K), B cell lymphoma-2 (BCL2) and Poly ADP-ribose polymerase (PARP) inhibitors. Unfortunately, we don't have specific biomarkers that could reveal which of the underlying pathways or genetic alterations are mostly involved in each individual case of MCL. Efforts should be done in this field aiming to an optimal personalized therapy.

  6. NAD+ and sirtuins in aging and disease.

    PubMed

    Imai, Shin-ichiro; Guarente, Leonard

    2014-08-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a classical coenzyme mediating many redox reactions. NAD(+) also plays an important role in the regulation of NAD(+)-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD(+) biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD(+) levels decline during the aging process and may be an Achilles' heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD(+) by supplementing NAD(+) intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD(+) intermediate supplementation may be an effective antiaging intervention, providing hope to aging societies worldwide.

  7. Theophylline prevents NAD{sup +} depletion via PARP-1 inhibition in human pulmonary epithelial cells

    SciTech Connect

    Moonen, Harald J.J. . E-mail: h.moonen@grat.unimaas.nl; Geraets, Liesbeth; Vaarhorst, Anika; Bast, Aalt; Wouters, Emiel F.M.; Hageman, Geja J.

    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 enzyme 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.

  8. 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.

  9. The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive strand RNA viruses function as mono-ADP-ribosylhydrolases.

    PubMed

    Eckei, Laura; Krieg, Sarah; Bütepage, Mareike; Lehmann, Anne; Gross, Annika; Lippok, Barbara; Grimm, Alexander R; Kümmerer, Beate M; Rossetti, Giulia; Lüscher, Bernhard; Verheugd, Patricia

    2017-02-02

    Human pathogenic positive single strand RNA ((+)ssRNA) viruses, including Chikungunya virus, pose severe health problems as for many neither efficient vaccines nor therapeutic strategies exist. To interfere with propagation, viral enzymatic activities are considered potential targets. Here we addressed the function of the viral macrodomains, conserved folds of non-structural proteins of many (+)ssRNA viruses. Macrodomains are closely associated with ADP-ribose function and metabolism. ADP-ribosylation is a post-translational modification controlling various cellular processes, including DNA repair, transcription and stress response. We found that the viral macrodomains possess broad hydrolase activity towards mono-ADP-ribosylated substrates of the mono-ADP-ribosyltransferases ARTD7, ARTD8 and ARTD10 (aka PARP15, PARP14 and PARP10, respectively), reverting this post-translational modification both in vitro and in cells. In contrast, the viral macrodomains possess only weak activity towards poly-ADP-ribose chains synthesized by ARTD1 (aka PARP1). Unlike poly-ADP-ribosylglycohydrolase, which hydrolyzes poly-ADP-ribose chains to individual ADP-ribose units but cannot cleave the amino acid side chain - ADP-ribose bond, the different viral macrodomains release poly-ADP-ribose chains with distinct efficiency. Mutational and structural analyses identified key amino acids for hydrolase activity of the Chikungunya viral macrodomain. Moreover, ARTD8 and ARTD10 are induced by innate immune mechanisms, suggesting that the control of mono-ADP-ribosylation is part of a host-pathogen conflict.

  10. The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive strand RNA viruses function as mono-ADP-ribosylhydrolases

    PubMed Central

    Eckei, Laura; Krieg, Sarah; Bütepage, Mareike; Lehmann, Anne; Gross, Annika; Lippok, Barbara; Grimm, Alexander R.; Kümmerer, Beate M.; Rossetti, Giulia; Lüscher, Bernhard; Verheugd, Patricia

    2017-01-01

    Human pathogenic positive single strand RNA ((+)ssRNA) viruses, including Chikungunya virus, pose severe health problems as for many neither efficient vaccines nor therapeutic strategies exist. To interfere with propagation, viral enzymatic activities are considered potential targets. Here we addressed the function of the viral macrodomains, conserved folds of non-structural proteins of many (+)ssRNA viruses. Macrodomains are closely associated with ADP-ribose function and metabolism. ADP-ribosylation is a post-translational modification controlling various cellular processes, including DNA repair, transcription and stress response. We found that the viral macrodomains possess broad hydrolase activity towards mono-ADP-ribosylated substrates of the mono-ADP-ribosyltransferases ARTD7, ARTD8 and ARTD10 (aka PARP15, PARP14 and PARP10, respectively), reverting this post-translational modification both in vitro and in cells. In contrast, the viral macrodomains possess only weak activity towards poly-ADP-ribose chains synthesized by ARTD1 (aka PARP1). Unlike poly-ADP-ribosylglycohydrolase, which hydrolyzes poly-ADP-ribose chains to individual ADP-ribose units but cannot cleave the amino acid side chain - ADP-ribose bond, the different viral macrodomains release poly-ADP-ribose chains with distinct efficiency. Mutational and structural analyses identified key amino acids for hydrolase activity of the Chikungunya viral macrodomain. Moreover, ARTD8 and ARTD10 are induced by innate immune mechanisms, suggesting that the control of mono-ADP-ribosylation is part of a host-pathogen conflict. PMID:28150709

  11. FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

    SciTech Connect

    Marshall, Kathryn E.; Robinson, E. W.; Hengel, Shawna M.; Pasa-Tolic, Ljiljana; Roesijadi, Guritno

    2012-03-21

    Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification that enables localization of recombinant proteins in the biosilica cell wall. Our objective was to functionalize diatom biosilica with a reagent-less biosensor with FRET-based imaging capabilities for signaling. The design of the fusion protein conferring these 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 recombinant chimeric protein was first confirmed in E. coli-expressed proteins, prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of CRY showed 95% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica targeting exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 {mu}M and 142.8 {mu}M, respectively. The addition of the silaffin tag for biosilica localization did not influence 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 biosilica 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.

  12. 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

  13. Induction of DNA strand breaks by H/sub 2/O/sub 2/ and PMA

    SciTech Connect

    Schraufstaetter, I.U.; Hyslop, P.A.; Jackson, J.; Cochrane, C.G.

    1986-03-01

    The authors have previously reported that oxidants induce cell lysis in P388Dl cells and lymphocytes which is caused by activation of poly-ADP-ribose polymerase leading to NAD and ATP depletion. Generally, poly-ADP-ribose polymerase is activated in the presence of single stranded DNA. Here they report that small concentrations of H/sub 2/O/sub 2/ (1-2 nmoles/min/ 2 x 10/sup 6/ cells produced by glucose oxidase-glucose) caused DNA strand breaks in human peripheral lymphocytes, P388Dl cells and freshly isolated rabbit pulmonary endothelial cells. DNA strand breaks were determined by an alkaline unwinding technique. To induce DNA strand breaks in rabbit alveolar macrophages or human monocytes higher concentrations of H/sub 2/O/sub 2/ (about 100 nmoles/ml) were necessary, presumably due to high activity of H/sub 2/O/sub 2/ catabolizing enzymes. Peripheral lymphocytes, which do not produce H/sub 2/O/sub 2/, did not form DNA strand breaks in the presence of 100 ng/ml phorbol myristate acetate (PMA). Few strand breaks were observed in monocytes (1.25 x 10/sup 6/ cells/ml) stimulated with PMA, since the concentration of H/sub 2/O/sub 2/ present under these conditions (20-30 nmoles) was insufficient to induce strand breaks in the cells. However, when lymphocytes and monocytes were mixed together in a ratio of 3:1, and exposed to PMA, DNA strand breaks occurred equivalent to those seen with 40 nmoles H/sub 2/O/sub 2/. Their results indicate that oxidants produced by stimulated leukocytes can induce DNA damage in neighboring cells.

  14. The RST and PARP-like domain containing SRO protein family: analysis of protein structure, function and conservation in land plants

    PubMed Central

    2010-01-01

    Background The SROs (SIMILAR TO RCD-ONE) are a group of plant-specific proteins which have important functions in stress adaptation and development. They contain the catalytic core of the poly(ADP-ribose) polymerase (PARP) domain and a C-terminal RST (RCD-SRO-TAF4) domain. In addition to these domains, several, but not all, SROs contain an N-terminal WWE domain. Results SROs are present in all analyzed land plants and sequence analysis differentiates between two structurally distinct groups; cryptogams and monocots possess only group I SROs whereas eudicots also contain group II. Group I SROs possess an N-terminal WWE domain (PS50918) but the WWE domain is lacking in group II SROs. Group I domain structure is widely represented in organisms as distant as humans (for example, HsPARP11). We propose a unified nomenclature for the SRO family. The SROs are able to interact with transcription factors through the C-terminal RST domain but themselves are generally not regulated at the transcriptional level. The most conserved feature of the SROs is the catalytic core of the poly(ADP-ribose) polymerase (PS51059) domain. However, bioinformatic analysis of the SRO PARP domain fold-structure and biochemical assays of AtRCD1 suggested that SROs do not possess ADP-ribosyl transferase activity. Conclusions The SROs are a highly conserved family of plant specific proteins. Sequence analysis of the RST domain implicates a highly preserved protein structure in that region. This might have implications for functional conservation. We suggest that, despite the presence of the catalytic core of the PARP domain, the SROs do not possess ADP-ribosyl transferase activity. Nevertheless, the function of SROs is critical for plants and might be related to transcription factor regulation and complex formation. PMID:20226034

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

    SciTech Connect

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

    2013-06-01

    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. - Highlights: • Low levels of arsenite enhance UV-induced DNA damage in human keratinocytes. • UV-initiated HPRT mutation frequency is enhanced by arsenite. • Zinc supplementation offsets DNA damage and mutation frequency enhanced by arsenite. • Zinc-dependent reduction of arsenite enhanced DNA damage is confirmed in vivo.

  16. The Key Involvement of Poly(ADP-Ribosylation) in Defense Against Toxic Agents: Molecular Biology Studies

    DTIC Science & Technology

    1993-04-29

    Jacobson, M.K., Janzon, L., SeidegArd, J., Smulson, M.E., and Troll, W. The Malmt5 Diet and Cancer Study: The Biomarker Program. Submitted for publication in...ribosylate proteins which bind to the regulatory regions of glucocorticoid responsive genes. (2). TCDD, dioxin , a potent environmental contaminant...like glucocorticoid is mediated by an aromatic hydrocarbon rereptor which a!’c binds to specific DNA sequences (i.e. dioxin -responsive elements

  17. Structures of revertant signal sequences of Escherichia coli ribose binding protein.

    PubMed Central

    Chi, S W; Yi, G S; Suh, J Y; Choi, B S; Kim, H

    1995-01-01

    Recently we reported (Yi et al., 1994) that the alpha-helical content of the signal peptide of Escherichia coli ribose binding protein, when determined by circular dichroism (CD) and two-dimensional NMR in trifluoroethanol/water solvent, is higher than that of its nonfunctional mutant signal peptide. In the present investigation, the structures of the signal peptides of two revertant ribose binding proteins in the same solvent were also determined with CD and two-dimensional 1H NMR spectroscopy. According to the CD results, both of these revertant signal peptides showed an intermediate helicity between those of wild-type and mutant signal peptides, the helical content of the revertant peptide with higher recovery of the translocation capability being higher. On the other hand, the alpha-helix regions of the wild-type and the revertant peptides as determined by NMR were shown to be the same. This discrepancy may be due to the difference in stability between identical alpha-helical stretches in wild-type and revertant peptides. A good correlation was observed between the helical content of these four ribose binding protein signal peptides in TFE/water as studied by CD and their in vivo translocation activities. It appears, therefore, that both the proper length of the helix and the stability are of functional significance. PMID:8599676

  18. Crystal structures and enzyme mechanisms of a dual fucose mutarotase/ribose pyranase.

    PubMed

    Lee, Kwang-Hoon; Ryu, Kyoung-Seok; Kim, Min-Sung; Suh, Hye-Young; Ku, Bonsu; Song, Young-Lan; Ko, Sunggeon; Lee, Weontae; Oh, Byung-Ha

    2009-08-07

    Escherichia coli FucU (Fucose Unknown) is a dual fucose mutarotase and ribose pyranase, which shares 44% sequence identity with its human counterpart. Herein, we report the structures of E. coli FucU and mouse FucU bound to L-fucose and delineate the catalytic mechanisms underlying the interconversion between stereoisomers of fucose and ribose. E. coli FucU forms a decameric toroid with each active site formed by two adjacent subunits. While one subunit provides most of the fucose-interacting residues including a catalytic tyrosine residue, the other subunit provides a catalytic His-Asp dyad. This active-site feature is critical not only for the mutarotase activity toward L-fucose but also for the pyranase activity toward D-ribose. Structural and biochemical analyses pointed that mouse FucU assembles into four different oligomeric forms, among which the smallest homodimeric form is most abundant and would be the predominant species under physiological conditions. This homodimer has two fucose-binding sites that are devoid of the His-Asp dyad and catalytically inactive, indicating that the mutarotase and the pyranase activities appear dispensable in vertebrates. The defective assembly of the mouse FucU homodimer into the decameric form is due to an insertion of two residues at the N-terminal extreme, which is a common aspect of all the known vertebrate FucU proteins. Therefore, vertebrate FucU appears to serve for as yet unknown function through the quaternary structural alteration.

  19. Metabolic flexibility of D-ribose producer strain of Bacillus pumilus under environmental perturbations.

    PubMed

    Srivastava, Rajesh K; Maiti, Soumen K; Das, Debasish; Bapat, Prashant M; Batta, Kritika; Bhushan, Mani; Wangikar, Pramod P

    2012-08-01

    The metabolic reaction rate vector is a bridge that links gene and protein expression alterations to the phenotypic endpoint. We present a simple approach for the estimation of flux distribution at key branch points in the metabolic network by using substrate uptake, metabolite secretion rate, and biomass growth rate for transketolase (tkt) deficient Bacillus pumilus ATCC 21951. We find that the glucose-6-phosphate (G6P) and pseudo catabolic/anabolic branch points are flexible in the D: -ribose-producing tkt deficient strain of B. pumilus. The normalized flux through the pentose phosphate pathway (PPP) varied from 1.5 to 86 % under different growth conditions, thereby enabling substantial extracellular accumulation of D: -ribose under certain conditions. Interestingly, the flux through PPP was affected by the extracellular phosphate concentration and dissolved oxygen concentration. This metabolic flexibility may have been the underlying reason for this strain being selected from thousands of others in a screening for D: -ribose producers conducted in the 1970s.

  20. Sugar-metal ion interactions: the complicated coordination structures of cesium ion with D-ribose and myo-inositol.

    PubMed

    Hu, Haijian; Xue, Junhui; Wen, Xiaodong; Li, Weihong; Zhang, Chao; Yang, Limin; Xu, Yizhuang; Zhao, Guozhong; Bu, Xiaoxia; Liu, Kexin; Chen, Jia'er; Wu, Jinguang

    2013-11-18

    The novel cesium chloride-D-ribose complex (CsCl·C5H10O5; Cs-R) and cesium chloride-myo-inositol complex (CsCl·C6H12O6; Cs-I) have been synthesized and characterized using X-ray diffraction and FTIR, FIR, THz, and Raman spectroscopy. Cs(+) is eight-coordinated to three chloride ions, O1 and O2 from one D-ribose molecule, O1 from another D-ribose molecule, and O4 and O5 from the third D-ribose molecule in Cs-R. For one D-ribose molecule, the oxygen atom O1 in the ring is coordinated to two cesium ions as an oxygen bridge, O2 is cocoordinated with O1 to one of the two cesium ions, and O4 and O5 are coordinated to the third cesium ion, respectively. O3 does not coordinate to metal ions and only takes part in forming hydrogen bonds. One chloride ion is connected to three cesium ions. Thus, a complicated structure of Cs-D-ribose forms. For Cs-I, Cs(+) is 10-coordinated to three chloride ions, O1 and O2 from one myo-inositol molecule, O3 and O4 from another myo-inositol molecule, O5 and O6 from the third myo-inositol molecule, and O6 from the fourth myo-inositol molecule. One metal ion is connected to four ligands, and one myo-inositol is coordinated to four Cs(+) ions, which is also a complicated coordination structure. Crystal structure results, FTIR, FIR, THz, and Raman spectra provide detailed information on the structure and coordination of hydroxyl groups to metal ions in the cesium chloride-D-ribose and cesium chloride-myo-inositol complexes.

  1. Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

    PubMed Central

    2011-01-01

    Background Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis. Results Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD) phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB. Conclusion The C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether. PMID:21995815

  2. No effects of oral ribose supplementation on repeated maximal exercise and de novo ATP resynthesis.

    PubMed

    Eijnde, B O; Van Leemputte, M; Brouns, F; Van Der Vusse, G J; Labarque, V; Ramaekers, M; Van Schuylenberg, R; Verbessem, P; Wijnen, H; Hespel, P

    2001-11-01

    A double-blind randomized study was performed to evaluate the effect of oral ribose supplementation on repeated maximal exercise and ATP recovery after intermittent maximal muscle contractions. Muscle power output was measured during dynamic knee extensions with the right leg on an isokinetic dynamometer before (pretest) and after (posttest) a 6-day training period in conjunction with ribose (R, 4 doses/day at 4 g/dose, n = 10) or placebo (P, n = 9) intake. The exercise protocol consisted of two bouts (A and B) of maximal contractions, separated by 15 s of rest. Bouts A and B consisted of 15 series of 12 contractions each, separated by a 60-min rest period. During the training period, the subjects performed the same exercise protocol twice per day, with 3-5 h of rest between exercise sessions. Blood samples were collected before and after bouts A and B and 24 h after bout B. Knee-extension power outputs were approximately 10% higher in the posttest than in the pretest but were similar between P and R for all contraction series. The exercise increased blood lactate and plasma ammonia concentrations (P < 0.05), with no significant differences between P and R at any time. After a 6-wk washout period, in a subgroup of subjects (n = 8), needle-biopsy samples were taken from the vastus lateralis before, immediately after, and 24 h after an exercise bout similar to the pretest. ATP and total adenine nucleotide content were decreased by approximately 25 and 20% immediately after and 24 h after exercise in P and R. Oral ribose supplementation with 4-g doses four times a day does not beneficially impact on postexercise muscle ATP recovery and maximal intermittent exercise performance.

  3. A Phase I Study of Veliparib in Combination with Metronomic Cyclophosphamide in Adults with Refractory Solid Tumors and Lymphomas

    PubMed Central

    Kummar, Shivaani; Ji, Jiuping; Morgan, Robert; Lenz, Heinz-Josef; Puhalla, Shannon L.; Belani, Chandra P.; Gandara, David R.; Allen, Deborah; Kiesel, Brian; Beumer, Jan H.; Newman, Edward M.; Rubinstein, Larry; Chen, Alice; Zhang, Yiping; Wang, Lihua; Kinders, Robert J.; Parchment, Ralph E.; Tomaszewski, Joseph E.; Doroshow, James H.

    2012-01-01

    Purpose Oral administration of the alkylating agent cyclophosphamide at low doses, metronomic dosing, is well tolerated, with efficacy in multiple tumor types. Poly(ADP-ribose) polymerase (PARP) inhibition potentiates effects of cyclophosphamide in preclinical models. We conducted a phase I trial of the PARP inhibitor veliparib and metronomic cyclophosphamide in patients with refractory solid tumors and lymphoid malignancies. Experimental Design Objectives were to establish the safety and maximum tolerated dose (MTD) of the combination; characterize veliparib pharmacokinetics; measure poly(ADP-ribose) (PAR), a product of PARP, in tumor biopsies and peripheral blood mononuclear cells (PBMCs); and measure the DNA-damage marker γH2AX in PBMCs and circulating tumor cells (CTCs). Cyclophosphamide was administered once daily in 21-day cycles in combination with veliparib administered once daily for 7, 14, or 21 days. Results Thirty-five patients were enrolled. The study treatment was well tolerated, and the MTD was established as veliparib 60 mg with cyclophosphamide 50 mg given once daily. Seven patients had partial responses; an additional six patients had disease stabilization for at least six cycles. PAR was significantly decreased in PBMCs (by at least 50%) and tumor biopsies (by at least 80%) across dose levels; γH2AX levels were increased in CTCs from seven of nine patients evaluated after drug administration. Conclusions The combination of veliparib with metronomic cyclophosphamide is well tolerated and shows promising activity in a subset of patients with BRCA mutations. A phase II trial of the combination compared to single-agent cyclophosphamide is ongoing in BRCA-positive ovarian cancer, triple-negative breast cancer, and low-grade lymphoma. PMID:22307137

  4. NAMPT inhibition sensitizes pancreatic adenocarcinoma cells to tumor-selective, PAR-independent metabolic catastrophe and cell death induced by β-lapachone.

    PubMed

    Moore, Z; Chakrabarti, G; Luo, X; Ali, A; Hu, Z; Fattah, F J; Vemireddy, R; DeBerardinis, R J; Brekken, R A; Boothman, D A

    2015-01-15

    Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (e.g., FK866) target the most active pathway of NAD(+) synthesis in tumor cells, but lack tumor-selectivity for use as a single agent. Reducing NAD(+) pools by inhibiting NAMPT primed pancreatic ductal adenocarcinoma (PDA) cells for poly(ADP ribose) polymerase (PARP1)-dependent cell death induced by the targeted cancer therapeutic, β-lapachone (β-lap, ARQ761), independent of poly(ADP ribose) (PAR) accumulation. β-Lap is bioactivated by NADPH:quinone oxidoreductase 1 (NQO1) in a futile redox cycle that consumes oxygen and generates high levels of reactive oxygen species (ROS) that cause extensive DNA damage and rapid PARP1-mediated NAD(+) consumption. Synergy with FK866+β-lap was tumor-selective, only occurring in NQO1-overexpressing cancer cells, which is noted in a majority (∼85%) of PDA cases. This treatment strategy simultaneously decreases NAD(+) synthesis while increasing NAD(+) consumption, reducing required doses and treatment times for both drugs and increasing potency. These complementary mechanisms caused profound NAD(P)(+) depletion and inhibited glycolysis, driving down adenosine triphosphate levels and preventing recovery normally observed with either agent alone. Cancer cells died through an ROS-induced, μ-calpain-mediated programmed cell death process that kills independent of caspase activation and is not driven by PAR accumulation, which we call NAD(+)-Keresis. Non-overlapping specificities of FK866 for PDA tumors that rely heavily on NAMPT-catalyzed NAD(+) synthesis and β-lap for cancer cells with elevated NQO1 levels affords high tumor-selectivity. The concept of reducing NAD(+) pools in cancer cells to sensitize them to ROS-mediated cell death by β-lap is a novel strategy with potential application for pancreatic and other types of NQO1+ solid tumors.

  5. 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.

  6. In vivo anti-tumor activity of the PARP inhibitor niraparib in homologous recombination deficient and proficient ovarian carcinoma☆,☆☆

    PubMed Central

    AlHilli, Mariam M.; Becker, Marc A.; Weroha, S. John; Flatten, Karen S.; Hurley, Rachel M.; Harrell, Maria I.; Oberg, Ann L.; Maurer, Matt J.; Hawthorne, Kieran M.; Hou, Xiaonan; Harrington, Sean C.; McKinstry, Sarah; Meng, X. Wei; Wilcoxen, Keith M.; Kalli, Kimberly R.; Swisher, Elizabeth M.; Kaufmann, Scott H.; Haluska, Paul

    2017-01-01

    Objective Poly(ADP-ribose) polymerase (PARP) inhibitors have yielded encouraging responses in high-grade serous ovarian carcinomas (HGSOCs), but the optimal treatment setting remains unknown. We assessed the effect of niraparib on HGSOC patient-derived xenograft (PDX) models as well as the relationship between certain markers of homologous recombination (HR) status, including BRCA1/2 mutations and formation of RAD51 foci after DNA damage, and response of these PDXs to niraparib in vivo. Methods Massively parallel sequencing was performed on HGSOCs to identify mutations contributing to HR deficiency. HR pathway integrity was assessed using fluorescence microscopy-based RAD51 focus formation assays. Effects of niraparib (MK-4827) on treatment-naïve PDX tumor growth as monotherapy, in combination with carboplatin/paclitaxel, and as maintenance therapy were assessed by transabdominal ultrasound. Niraparib responses were correlated with changes in levels of poly(ADP-ribose), PARP1, and repair proteins by western blotting. Results Five PDX models were evaluated in vivo. Tumor regressions were induced by single-agent niraparib in one of two PDX models with deleterious BRCA2 mutations and in a PDX with RAD51C promoter methylation. Diminished formation of RAD51 foci failed to predict response, but Artemis loss was associated with resistance. Niraparib generally failed to enhance responses to carboplatin/paclitaxel chemotherapy, but maintenance niraparib therapy delayed progression in a BRCA2-deficient PDX. Conclusions Mutations in HR genes are neither necessary nor sufficient to predict response to niraparib. Assessment of repair status through multiple complementary assays is needed to guide PARP inhibitor therapy, design future clinical trials and identify ovarian cancer patients most likely to benefit from PARP inhibition. PMID:27614696

  7. The topical antimicrobial zinc pyrithione is a heat shock response inducer that causes DNA damage and PARP-dependent energy crisis in human skin cells.

    PubMed

    Lamore, Sarah D; Cabello, Christopher M; Wondrak, Georg T

    2010-05-01

    The differentiated epidermis of human skin serves as an essential barrier against environmental insults from physical, chemical, and biological sources. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in clinical antiseptic products, over-the-counter topical antimicrobials, and cosmetic consumer products including antidandruff shampoos. Here we demonstrate for the first time that cultured primary human skin keratinocytes and melanocytes display an exquisite vulnerability to nanomolar concentrations of ZnPT resulting in pronounced induction of heat shock response gene expression and impaired genomic integrity. In keratinocytes treated with nanomolar concentrations of ZnPT, expression array analysis revealed massive upregulation of genes encoding heat shock proteins (HSPA6, HSPA1A, HSPB5, HMOX1, HSPA1L, and DNAJA1) further confirmed by immunodetection. Moreover, ZnPT treatment induced rapid depletion of cellular ATP levels and formation of poly(ADP-ribose) polymers. Consistent with an involvement of poly(ADP-ribose) polymerase (PARP) in ZnPT-induced energy crisis, ATP depletion could be antagonized by pharmacological inhibition of PARP. This result was independently confirmed using PARP-1 knockout mouse embryonic fibroblasts that were resistant to ATP depletion and cytotoxicity resulting from ZnPT exposure. In keratinocytes and melanocytes, single-cell gel electrophoresis and flow cytometric detection of gamma-H2A.X revealed rapid induction of DNA damage in response to ZnPT detectable before general loss of cell viability occurred through caspase-independent pathways. Combined with earlier experimental evidence that documents penetration of ZnPT through mammalian skin, our findings raise the possibility that this topical antimicrobial may target and compromise keratinocytes and melanocytes in intact human skin.

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

    SciTech Connect

    Wu, Ping; Zhu, Xueping; Jin, Wei; Hao, Shumei; Liu, Qi; Zhang, Linjie

    2015-05-01

    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 oxygen 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

  9. PARP-1 hyperactivation and reciprocal elevations in intracellular Ca2+ during ROS-induced nonapoptotic cell death.

    PubMed

    Zhang, Fengjiao; Xie, Ruiye; Munoz, Frances M; Lau, Serrine S; Monks, Terrence J

    2014-07-01

    The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia/reperfusion injury, and many other pathological conditions. DNA strand breaks caused by ROS lead to the activation of poly(ADP-ribose)polymerase-1 (PARP-1), the excessive activation of which can result in cell death. We have utilized a model in which 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, causes ROS-dependent cell death in human renal proximal tubule epithelial cells (HK-2), to further elucidate the role of PARP-1 in ROS-dependent cell death. TGHQ-induced ROS generation, DNA strand breaks, hyperactivation of PARP-1, rapid depletion of nicotinamide adenine dinucleotide (NAD), elevations in intracellular Ca(2+) concentrations, and subsequent nonapoptotic cell death in both a PARP- and Ca(2+)-dependent manner. Thus, inhibition of PARP-1 with PJ34 completely blocked TGHQ-mediated accumulation of poly(ADP-ribose) polymers and NAD consumption, and delayed HK-2 cell death. In contrast, chelation of intracellular Ca(2+) with BAPTA completely abrogated TGHQ-induced cell death. Ca(2+) chelation also attenuated PARP-1 hyperactivation. Conversely, inhibition of PARP-1 modulated TGHQ-mediated changes in Ca(2+) homeostasis. Interestingly, PARP-1 hyperactivation was not accompanied by the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP-dependent cell death. Thus, pathways coupling PARP-1 hyperactivation to cell death are likely to be context-dependent, and therapeutic strategies designed to target PARP-1 need to recognize such variability. Our studies provide new insights into PARP-1-mediated nonapoptotic cell death, during which PARP-1 hyperactivation and elevations in intracellular Ca(2+) are reciprocally coupled to amplify ROS-induced nonapoptotic cell death.

  10. The topical antimicrobial zinc pyrithione is a heat shock response inducer that causes DNA damage and PARP-dependent energy crisis in human skin cells

    PubMed Central

    Lamore, Sarah D.; Cabello, Christopher M.

    2009-01-01

    The differentiated epidermis of human skin serves as an essential barrier against environmental insults from physical, chemical, and biological sources. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in clinical antiseptic products, over-the-counter topical antimicrobials, and cosmetic consumer products including antidandruff shampoos. Here we demonstrate for the first time that cultured primary human skin keratinocytes and melanocytes display an exquisite vulnerability to nanomolar concentrations of ZnPT resulting in pronounced induction of heat shock response gene expression and impaired genomic integrity. In keratinocytes treated with nanomolar concentrations of ZnPT, expression array analysis revealed massive upregulation of genes encoding heat shock proteins (HSPA6, HSPA1A, HSPB5, HMOX1, HSPA1L, and DNAJA1) further confirmed by immunodetection. Moreover, ZnPT treatment induced rapid depletion of cellular ATP levels and formation of poly(ADP-ribose) polymers. Consistent with an involvement of poly(ADP-ribose) polymerase (PARP) in ZnPT-induced energy crisis, ATP depletion could be antagonized by pharmacological inhibition of PARP. This result was independently confirmed using PARP-1 knockout mouse embryonic fibroblasts that were resistant to ATP depletion and cytotoxicity resulting from ZnPT exposure. In keratinocytes and melanocytes, single-cell gel electrophoresis and flow cytometric detection of γ-H2A.X revealed rapid induction of DNA damage in response to ZnPT detectable before general loss of cell viability occurred through caspase-independent pathways. Combined with earlier experimental evidence that documents penetration of ZnPT through mammalian skin, our findings raise the possibility that this topical antimicrobial may target and compromise keratinocytes and melanocytes in intact human skin. PMID:19809895

  11. 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.

  12. Recycling of 5'-methylthioadenosine-ribose carbon atoms into methionine in tomato tissue in relation to ethylene production.

    PubMed

    Wang, S Y; Adams, D O; Lieberman, M

    1982-07-01

    The ribose moiety of 5'-methylthioadenosine (MTA) is metabolized to form the four-carbon unit (2-aminobutyrate) of methionine in tomato tissue (Lycopersicon esculentum Mill., cv. Pik Red). When [U-(14)C-adenosine] MTA was administered to tomato tissue slices, label was recovered in 5-methylthioribose (MTR), methionine, 1-aminocyclopropane-1-carboxylic acid (ACC), C(2)H(4) and other unidentified compounds. However, when [U-(14)C-ribose]MTR was administered, radioactivities were recovered in methionine, ACC and C(2)H(4), but not MTA. This suggests that C(2)H(4) formed in tomato pericarp tissue may be derived from the ribose portion of MTA via MTR, methionine and ACC. The conversion of MTR to methionine is not inhibited by aminoethoxyvinylglycine (AVG), but is O(2) dependent. These data present a new salvage pathway for methionine biosynthesis which may be important in relation to polyamine and ethylene biosynthesis in tomato tissue.

  13. Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids

    PubMed Central

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Cecílio, Pedro; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null mutant generation in Leishmania infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was retained both in vitro and in vivo in the absence of drug pressure. This suggests the gene is essential for parasite survival. Importantly, the inability to remove the second allele of RPIB gene in sKO mutants complemented with an episomal copy of RPIB carrying a mutation that abolishes isomerase activity suggests the essentiality is due to its metabolic function. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophage infection, however, an impairment in intracellular amastigotes’ replication was observed. Additionally, mice infected with sKO mutants rescued by RPIB complementation had a reduced parasite burden in the liver. Likewise, Trypanosoma brucei is resistant to complete RPIB gene removal and mice infected with sKO mutants showed prolonged survival upon infection. Taken together our results genetically validate RPIB as a potential drug target in trypanosomatids. PMID:27230471

  14. D-ribose competitively reverses inhibition by D-psicose of larval growth in Caenorhabditis elegans.

    PubMed

    Sato, Masashi; Yokoi, Nobutoshi; Kurose, Hiroyuki; Yamasaki, Toru

    2009-05-01

    D-Psicose inhibits the growth of L1 stage Caenorhabditis elegans. Sugars, involved in the pentose phosphate pathway, were examined for their ability to reverse the inhibition. Among these sugars, D-ribose specifically exerted reversing activity in a competitive manner. The ingested sugars are probably phosphorylated, although it remains to be seen whether D-psicose is phosphorylated. The structural similarity of D-psicofuranose 6-phosphate (Pf6P) or D-psicofuranose (Pf) to D-ribofuranose 5-phosphate (Rf5P) suggests that Pf6P or Pf is reversibly docked in the active site(s) of ribose-5-phosphate isomerase(s) to act as an antimetabolite to Rf5P, leading to inhibition of the biosynthesis of nucleic acids. D-Psicose was much less potent against the L4 stage than against the L1 stage. This is probably because in the L4 stage the somatic cell lineages come to an end and the number of germ-line nuclei increases to about 1000.

  15. 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

    Permeation of molecules through membranes is a fundamental process in biological systems, which not only involves mass and signal transfers between the interior of a contemporary cell and its environment, but was also of crucial importance in the origin of life. In the absence of complex protein transporters, nutrients and building blocks of biopolymers must have been able to permeate membranes at sufficient rates to support primordial metabolism and cel-lular reproduction. From this perspective one class of solutes that is of special interest are monosaccharides, which serve not only as nutritional molecules but also as building blocks for information molecules. In particular, ribose is a part of the RNA backbone, but RNA analogs containing a number of other sugars have also been shown to form stable duplexes. Why, among these possibilities, ribose (and, subsequently, deoxyribose) was selected for the backbone of information polymers is still poorly understood. It was recently found that ribose permeates membranes an order of magnitude faster than its diastereomers, arabinose and xylose [1]. On this basis it was hypothesized that differences in membrane permeability to aldopentoses provide a mechanism for preferential delivery of ribose to primitive cells for subsequent, selective incorporation into nucleotides and their polymers. However, the origins of these unusually large differences had not been well understood. We addressed this issue in molecular dynamics simulations combined with free energy calculations. It was found that the free energy barrier for transferring ribose from water to the bilayer is lower by 1.5-2 kcal/mol than the barrier for transferring the other two aldopentoses. The calculated [2] and measured [1] permeability coefficients are in an excellent agreement. The sugar structures that permeate the membrane are -pyranoses, with a possible contribution of the -anomer for arabinose. The furanoid form of ribose is not substantially involved in

  16. Ectocellular in vitro and in vivo metabolism of cADP-ribose in cerebellum.

    PubMed Central

    De Flora, A; Guida, L; Franco, L; Zocchi, E; Pestarino, M; Usai, C; Marchetti, C; Fedele, E; Fontana, G; Raiteri, M

    1996-01-01

    CD38, a type II transmembrane glycoprotein predominantly expressed in blood cells, is a bifunctional ectoenzyme directly involved in the metabolism of cADP-ribose (cADPR). This is a potent Ca2+ mobilizer in several types of cells. The relationship between the ectocellular site of cADPR production and its intracellular calcium-related functions is poorly understood. Cultured rat cerebellar granule cells showed both enzymic activities of CD38, ADP-ribosyl cyclase and cADPR hydrolase, at a ratio of 16 to 1 respectively, and were immunostained by the anti-(human CD38) monoclonal antibody IB4. In these cells externally added cADPR and beta-NAD+ (the precursor of cADPR), but not alpha-NAD+ or ADP-ribose, enhanced the peak of the depolarization-induced rise in intracellular Ca2+ concentration. This effect was inhibited by 1 microM ryanodine, suggesting a potentiation of calcium-induced calcium release by cADPR. CD38 ectoenzyme activities, ADP-ribosyl cyclase and cADPR hydrolase, were also demonstrated in vivo by microdialysis of adult rat cerebellum, where IB4 bound to granule neurons selectively. Trace amounts (11.5 +/- 3.8 nM) of NAD+ were detected by microdialysis sampling and sensitive assays in the basal interstitial fluid of the cerebellum. These results provide a link between ectocellular cADPR turnover and intracellular calcium mobilization in cerebellum. PMID:8973582

  17. FRET Response of a Modified Ribose Receptor Expressed in the Diatom Thalassiosira pseudonana

    SciTech Connect

    Miller, Hanna

    2011-08-26

    The ability to insert complex proteins into silica has many applications including biosensing. Previous research has demonstrated how to direct proteins to the biosilica of diatoms [1]. Here, we show that a complex fusion protein that includes an enzyme, a bacterial ribose periplasmic binding protein, flanked by fluorescent proteins constituting a FRET pair can remain functional in the frustules of living diatoms. A Sil3 tag is attached to the N-terminal end to localize the fusion protein to frustules of the diatom Thalassiosira pseudonana. When ribose was applied, a larger decrease in FRET response was seen in transformed cells than in untransformed cells. Multiple forms of the expression vector were tested to find the optimal system; specifically, a one-vector system was compared to a two-vector system and the gDNA version of the Sil3 localization tag was compared to the cDNA version. The optimal system was found to be a one-vector system with the genomic version of the Sil3 tag to direct the protein to the frustules. Localization of the enzyme to the frustules was further confirmed through cell fluorescence imaging.

  18. Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD(+)-dependent Sir2 histone/protein deacetylases.

    PubMed

    Zhao, Kehao; Harshaw, Robyn; Chai, Xiaomei; Marmorstein, Ronen

    2004-06-08

    Sir2 enzymes are broadly conserved from bacteria to humans and have been implicated to play roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. These enzymes bind NAD(+) and acetyllysine within protein targets and generate lysine, 2'-O-acetyl-ADP-ribose, and nicotinamide products. To provide structural insights into the chemistry catalyzed by Sir2 proteins we report the high-resolution ternary structure of yeast Hst2 (homologue of Sir two 2) with an acetyllysine histone H4 peptide and a nonhydrolyzable NAD(+) analogue, carba-NAD(+), as well as an analogous ternary complex with a reaction intermediate analog formed immediately after nicotinamide hydrolysis, ADP-ribose. The ternary complex with carba-NAD(+) reveals that the nicotinamide group makes stabilizing interactions within a binding pocket harboring conserved Sir2 residues. Moreover, an asparagine residue, N116, strictly conserved within Sir2 proteins and shown to be essential for nicotinamide exchange, is in position to stabilize the oxocarbenium intermediate that has been proposed to proceed the hydrolysis of nicotinamide. A comparison of this structure with the ADP-ribose ternary complex and a previously reported ternary complex with the 2'-O-acetyl-ADP-ribose reaction product reveals that the ribose ring of the cofactor and the highly conserved beta1-alpha2 loop of the protein undergo significant structural rearrangements to facilitate the ordered NAD(+) reactions of nicotinamide cleavage and ADP-ribose transfer to acetate. Together, these studies provide insights into the chemistry of NAD(+) cleavage and acetylation by Sir2 proteins and have implications for the design of Sir2-specific regulatory molecules.

  19. Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K-12.

    PubMed Central

    Iida, A; Harayama, S; Iino, T; Hazelbauer, G L

    1984-01-01

    We isolated spontaneous and transposon insertion mutants of Escherichia coli K-12 that were specifically defective in utilization or in high-affinity transport of D-ribose (or in both). Cotransduction studies located all of the mutations near ilv, at the same position as previously identified mutations causing defects in ribokinase ( rbsK ) or ribose transport ( rbsP ). Plasmids that complemented the rbs mutations were isolated from the collection of ColE1 hybrid plasmids constructed by Clarke and Carbon. Analysis of those plasmids as well as of fragments cloned into pBR322 and pACYC184 allowed definition of the rbs region. Products of rbs genes were identified by examination of the proteins produced in minicells containing various rbs plasmids. We identified four rbs genes: rbsB , which codes for the 29-kilodalton ribose-binding protein; rbsK , which codes for the 34-kilodalton ribokinase ; rbsA , which codes for a 50-kilodalton protein required for high-affinity transport; and rbsC , which codes for a 27-kilodalton protein likely to be a transport system component. Our studies showed that these genes are transcribed from a common promoter in the order rbsA rbsC rbsB rbsK . It appears that the high-affinity transport system for ribose consists of the three components, ribose-binding protein, the 50-kilodalton RbsA protein, and the 27-kilodalton RbsC protein, although a fourth, unidentified component could exist. Mutants defective in this transport system, but normal for ribokinase , are able to grow normally on high concentrations of the sugar, indicating that there is at least a second, low-affinity transport system for ribose in E. coli K-12. Images PMID:6327617

  20. Formation of novel nucleosides from free base and sugar phosphate: aqueous reaction of 2-aminopyrimidine and ribose-5-phosphate.

    PubMed

    Mace, D C

    1983-11-30

    The facile formation of glycosylamines suggests that a base liberated by depurination might react at the free C1 position of the sugar phosphate from which it had been hydrolyzed, effectively repurinating the site. Model experiments testing this hypothesis demonstrate that such a reaction does take place. The primary product of a reaction between 2-aminopyrimidine (a model for guanine) and ribose-5-phosphate is characterized by enzymatic and chemical degradation, and UV spectra. It is shown to be a novel nucleoside with the base attached via its exocyclic amino group to the C1 of the ribose-5-phosphate.

  1. Box C/D RNA guides for the ribose methylation of archaeal tRNAs. The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp

    PubMed Central

    d’Orval, Béatrice Clouet; Bortolin, Marie-Line; Gaspin, Christine; Bachellerie, Jean-Pierre

    2001-01-01

    Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2′-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5′ exon but also onto position 39 (universal tRNA numbering) in the 3′ exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon–intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs. PMID:11713301

  2. The Molecular Toxicology of Chromatin.

    DTIC Science & Technology

    1983-09-09

    critical -. observation was made that in an early exposure time to dimethylnitrosamine significant increase in hepatic poly(ADP-ribose)-NHP-s occurred...treated conditions dimethylnitrosamine at low doses acts also as an S phase augmenting mitogen) it seemed plausible to explain the hitherto obscure

  3. Luring BRCA1 to the scene of the crime.

    PubMed

    Baer, Richard

    2013-05-13

    To preserve genome stability, BRCA1 must be recruited to sites of DNA damage, where BRCA1 facilitates repair of double-strand DNA breaks (DSBs). In this issue of Cancer Cell, Li and Yu report that BRCA1 recruitment involves a novel interaction between its partner protein BARD1 and poly(ADP-ribose) chains at the DSB.

  4. 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.

  5. Cytoplasmic 3' poly(A) addition induces 5' cap ribose methylation: implications for translational control of maternal mRNA.

    PubMed Central

    Kuge, H; Richter, J D

    1995-01-01

    During the early development of many animal species, the expression of new genetic information is governed by selective translation of stored maternal mRNAs. In many cases, this translational activation requires cytoplasmic poly(A) elongation. However, how this modification at the 3' end of an mRNA stimulates translation from the 5' end is unknown. Here we show that cytoplasmic polyadenylation stimulates cap ribose methylation during progesterone-induced oocyte maturation in Xenopus laevis. Translational recruitment of a chimeric reporter mRNA that is controlled by cytoplasmic polyadenylation coincides temporally with cap ribose methylation during this period. In addition, the inhibition of cap ribose methylation by S-isobutyladenosine significantly reduces translational activation of a reporter mRNA without affecting the increase of general protein synthesis or polyadenylation during maturation. These results provide evidence for a functional interaction between the termini of an mRNA molecule and suggest that 2'-O-ribose cap methylation mediates the translational recruitment of maternal mRNA. Images PMID:8557049

  6. 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

  7. Cyclic ADP-ribose, the ryanodine receptor and Ca2+ release.

    PubMed

    Sitsapesan, R; McGarry, S J; Williams, A J

    1995-11-01

    In a variety of vertebrate and invertebrate tissues the ryanodine-sensitive Ca2+ channel is the pathway for Ca2+ release from intracellular stores. The mechanism for activation of the ryanodine receptor-channel complex appears to depend both on the ryanodine receptor isoform and the cell type. In addition, a complex combination of endogenous intracellular compounds regulates channel gating. In this article, Rebecca Sitsapesan, Stephen McGarry and Alan Williams review the mechanisms involved in cyclic ADP-ribose (cADPR)-induced Ca2+ release and discuss the likelihood that cADPR-activated Ca2+ release is mediated by one of the recognized isoforms of the ryanodine receptor-Ca2+ channel complex.

  8. Nucleobase and Ribose Modifications Control Immunostimulation by a MicroRNA-122-mimetic RNA

    PubMed Central

    Peacock, Hayden; Fucini, Raymond V.; Jayalath, Prasanna; Ibarra-Soza, José M.; Haringsma, Henry J.; Flanagan, W. Michael; Willingham, Aarron; Beal, Peter A.

    2011-01-01

    Immune stimulation is a significant hurdle in the development of effective and safe RNA interference therapeutics. Here, we address this problem in the context of a mimic of microRNA-122 by employing novel nucleobase and known 2′-ribose modifications. The nucleobase modifications are analogues of adenosine and guanosine that contain cyclopentyl and propyl minor-groove projections. Via a site-by-site chemical modification analysis, we identify several immunostimulatory ‘hot spots’ within the miRNA guide strand at which single base modifications significantly reduce immune stimulation. A duplex containing one base modification on each strand proved to be most effective in preventing immune stimulation. PMID:21612237

  9. [Role of the ribose residue of substrates in reactions catalyzed by ribonuclease].

    PubMed

    Avramova, Z V; Dudkin, S M

    1978-01-01

    The rate constants and Km for the hydrolysis of the optically active nonglycosidic analogues of the CpA and C greater than p catalysed by RNase A and RNase BS-I were measured. The rate of hydrolysis of the model substrates in 10(5) and 10(3) slower that for the appropriate dinucleoside phosphate and nucleoside cyclophosphate. However, substitution of the relatively rigid ribofuranose ring with flexible alifatic chains is accompanied by little variation in binding constants. The analyses based on the single substrate system indicate that the observed difference in rate constants must be accounted for by a difference between the binding of the substrates in the transition state to the RNase active site. Consequently, the "rigidity" of the ribose rings in RNA leads to large decreases in the free energy of activation for the reactions catalysed by RNases.

  10. Synthesis of Thienamycin methyl ester from 2-deoxy-D-ribose via Kinugasa reaction.

    PubMed

    Soluch, Magdalena; Grzeszczyk, Barbara; Staszewska-Krajewska, Olga; Chmielewski, Marek; Furman, Bartłomiej

    2016-03-01

    A novel synthesis of thienamycin is described. The crucial step of the synthesis is based on Cu(I)-mediated Kinugasa cycloaddition/rearrangement cascade reaction between terminal acetylene derived from D-lactic acid and suitable, partially protected, five-membered cyclic nitrone obtained from 2-deoxy-D-ribose. The reaction was performed in the presence of tetramethylguanidine as a base to provide 5,6-trans substituted carbapenam as the main product. Thus obtained carbapenam 11 with (5R,6S) configuration at the azetidinone ring was subsequently subjected to oxidation/deprotection/oxidation reaction sequence to afford the β-keto ester 20, which was directly transformed into N,O-protected methyl ester of thienamycin.

  11. Comparison of Ca2+ mobilizing activities of cyclic ADP-ribose and inositol trisphosphate.

    PubMed Central

    Dargie, P J; Agre, M C; Lee, H C

    1990-01-01

    We have previously shown that a metabolite of NAD+ generated by an enzyme present in sea urchin eggs and mammalian tissues can mobilize intracellular Ca2+ in the eggs. Structural determination established it to be a cyclized ADP-ribose, and the name cyclic ADP-ribose (cADPR) has been proposed. In this study, Ca2+ mobilizations induced by cADPR and inositol trisphosphate (IP3) in sea urchin egg homogenates were monitored with Ca2+ indicators and Ca2(+)-specific electrodes. Both methods showed that cADPR can release Ca2+ from egg homogenates. Evidence indicated that it did not act as a nonspecific Ca2(+)-ionophore or as a blocker of the microsomal Ca2(+)-transport; instead, it was likely to be operating through a specific receptor system. This was supported by its half-maximal effective concentration of 18 nM, which was 7 times lower than that of IP3. The receptor for cADPR appeared to be different from that of IP3 because heparin, an inhibitor of IP3 binding, had no effect on the cADPR action. The Ca2+ releases induced by cADPR and IP3 were not additive and had an inverse relationship, indicating overlapping stores were mobilized. Microinjection of cADPR into intact eggs induced transient intracellular Ca2+ changes and activated the cortical reaction. The in vivo effectiveness of cADPR was directly comparable with IP3 and neither required external Ca2+. In addition, both were effective in activating the eggs to undergo multiple nuclear cycles and DNA synthesis. These results suggest that cADPR could function as a second messenger in sea urchin eggs. Images PMID:2100201

  12. Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes

    NASA Technical Reports Server (NTRS)

    Egli, M.; Usman, N.; Rich, A.

    1993-01-01

    We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

  13. Ligand-induced conformational changes in a thermophilic ribose-binding protein

    SciTech Connect

    Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.

    2009-05-21

    Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments. Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation (appTm value is 108 C) than the mesophilic Escherichia coli homolog (ecRBP) (appTm value is 56 C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved. Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different

  14. Theoretical Study on the Factors Controlling the Stability of the Borate Complexes of Ribose, Arabinose, Lyxose and Xylose

    SciTech Connect

    Sumpter, Bobby G; Fuentes-Cabrera, Miguel A; Leszczynski, Jerzy; Sponer, Judit; Sponer, Jiri

    2008-01-01

    Recent views on the origin of the "RNA world" suggest that complexation with borate minerals had an indispensable role at stabilizing the cyclic form of aldopentoses that are the potential building blocks of RNA. Experimental investigations by Li Q, Ricardo A, Benner SA, Winefordner JD, Powell DH (2005) Anal Chem 77:4503-4508 and Chapelle S, Verchere J-F (1988) Tetrahedron 44:4469-4482 have shown that stability of the 2:1 complexes formed between ribose and borate is superior to those of the analogous compounds of the other three aldopentoses (xylose, arabinose and lyxose). The distinct stability of the ribose-borate 2:1 complexes is thought to be one of the basic reasons why evolution selected ribose (out of the four aldopentoses) to build up RNA molecules. Here we disclose the factors governing the stability of the aldopentose-borate 2:1 complexes using Density Functional Theory electronic structure calculations with inclusion of solvation effects using a continuum solvent approach. Our results show that the strong electrostatic field of the borate anion leads to the reorientation of the hydroxyl groups of the aldopentoses relative to the geometry adopted in the non-complexed form. The reasons why complex formation between borate and ribose is clearly preferred over the other three aldopentoses is (i) the ribose 3-OH is involved in a H-bond with one of the borate-oxygens and (ii) its 5-CH2OH group is well separated in space from the negatively charged region of the complex and ensures favorable contact with the aqueous medium.

  15. [Influence of ADP-ribose, AMP and adenosine on bioelectric activity of hibernating ground squirrel atrium and papillary muscle].

    PubMed

    Kuz'min, V S; Abramochkin, D V; Sukhova, G S; Rozenshtraukh, L V

    2008-01-01

    The aim of work was to investigate effects of adenosine, AMP and ADP-ribose (1x10(-5)) on bioelectric activity of atrium and papillary muscle of nonhibernating (rat) and hibernating (Yakutian ground squirrel) animals. Action potential (AP) was registered with use of standard microelectrode technique. AP duration (APD) at level of 90% repolarisation in rat atrium in control experiments was 30+/-5 ms, APD at level of 50% repolarisation was 12+/-2 ms. APD at level of 90% repolarisation in rat papillary muscle was 56+/-7 ms, at level of 50% repolarisation was 18+/-2 ms. APD at level of 90% repolarisation in ground squirrel atrium was 77+/-6, APD at level of 50% repolarisation was 38+/-6 ms. APD at level of 90% repolarisation in ground squirrel papillary muscle was 105+/-9 ms, APD at level of 50% repolarisation was 42+/-8 ms. Purine nucleotides and nucleoside, that were tested in work, except ADP-ribose, act as inhibitory factors and decrease APD both in rat and hibernating ground squirrel heart. ADP-ribose decreases APD in papillary muscle of hibernator but did not in its atrium. In ground squirrel atrium AMP and adenosine decrease APD at level of 50% repolarisation by 10+/-3% and 18+/-3% respectively. AMP and adenosine decrease APD at level of 90% repolarisation by 9+/-2% and 11+/-2% respectively. In ground squirrel papillary muscle ADP-ribose, AMP and adenosine decrease APD at level of 50% repolarisation by 26+/-8%, 23+/-8% and 26+/-7%. ADP-ribose, AMP and adenosine decrease APD at level of 90% repolarisation by 12+/-3%, 10+/-3%, 13+/-3%. Thus, decrease of APD in ground squirrel papillary muscle at level of 90% repolarisation during nucleotides and adenosine action was 2-2.5 fold less, than the rat.

  16. Alphavirus polymerase and RNA replication.

    PubMed

    Pietilä, Maija K; Hellström, Kirsi; Ahola, Tero

    2017-01-16

    Alphaviruses are typically arthropod-borne, and many are important pathogens such as chikungunya virus. Alphaviruses encode four nonstructural proteins (nsP1-4), initially produced as a polyprotein P1234. nsP4 is the core RNA-dependent RNA polymerase but all four nsPs are required for RNA synthesis. The early replication complex (RC) formed by the polyprotein P123 and nsP4 synthesizes minus RNA strands, and the late RC composed of fully processed nsP1-nsP4 is responsible for the production of genomic and subgenomic plus strands. Different parts of nsP4 recognize the promoters for minus and plus strands but the binding also requires the other nsPs. The alphavirus polymerase has been purified and is capable of de novo RNA synthesis only in the presence of the other nsPs. The purified nsP4 also has terminal adenylyltransferase activity, which may generate the poly(A) tail at the 3' end of the genome. Membrane association of the nsPs is vital for replication, and alphaviruses induce membrane invaginations called spherules, which form a microenvironment for RNA synthesis by concentrating replication components and protecting double-stranded RNA intermediates. The RCs isolated as crude membrane preparations are active in RNA synthesis in vitro, but high-resolution structure of the RC has not been achieved, and thus the arrangement of viral and possible host components remains unknown. For some alphaviruses, Ras-GTPase-activating protein (Src-homology 3 (SH3) domain)-binding proteins (G3BPs) and amphiphysins have been shown to be essential for RNA replication and are present in the RCs. Host factors offer an additional target for antivirals, as only few alphavirus polymerase inhibitors have been described.

  17. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-10-04

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases.

  18. The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

    PubMed Central

    Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J. M.

    2014-01-01

    ABSTRACT Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. IMPORTANCE The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated

  19. Structure of escherichia coli ribose-5-phosphate isomerase : a ubiquitous enzyme of the pentose phosphate pathway and the Calvin cycle.

    SciTech Connect

    Zhang, R.; Andersson, C. E.; Savchenko, A.; Skarina, T.; Evdokimova, E.; Beasley, S.; Arrowsmith, C. H.; Edwards, A.; Joachimiak, A.; Mowbray, S. L.; Biosciences Division; Uppsala Univ.; Univ. Health Network; Univ. of Toronto; Swedish Univ. of Agricultural Sciences

    2003-01-01

    Ribose-5-phosphate isomerase A (RpiA; EC 5.3.1.6) interconverts ribose-5-phosphate and ribulose-5-phosphate. This enzyme plays essential roles in carbohydrate anabolism and catabolism; it is ubiquitous and highly conserved. The structure of RpiA from Escherichia coli was solved by multiwavelength anomalous diffraction (MAD) phasing, and refined to 1.5 Angstroms resolution (R factor 22.4%, R{sub free} 23.7%). RpiA exhibits an {alpha}/{beta}/({alpha}/{beta})/{beta}/{alpha} fold, some portions of which are similar to proteins of t