Poly ADP-ribose Polymerase Activity Research Articles

Baicalein attenuates caspase-independent cells death via inhibiting PARP-1 activation and AIF nuclear translocation in cerebral ischemia/reperfusion rats.

It is reported that baicalein can activate PI3K/AKT pathway, inhibit caspase activation and reduce cerebral infarct volume in middle cerebral artery occlusion (MCAO) rats. However, a caspase-independent mechanism initiated by poly (ADP-ribose) polymerase-1 (PARP-1) activation has been reported to make more contribution to cells death after ischemic stroke. In the present study, we established a cerebral ischemia/reperfusion (I/R) rat model through middle cerebral artery occlusion following reperfusion to investigate the mechanisms of ischemic tissue recovery following baicalein treatment. The data showed that baicalein treatment at dose of 100 mg/kg for 7 days significantly inhibited the release of cytokines, activation of PARP-1, nuclear translocation of apoptosis-inducing factor (AIF) and macrophage migration inhibitory factor (MIF) in cerebral I/R rats, therefore decreased cerebral infarct volume and neurological scores. Then, we further investigated the signal transduction mechanisms of ischemic tissue protection by baicalein in vitro. Following oxygen and glucose deprivation (OGD) in SH-SY5Y cells, the mitochondrial AIF was translocated into nucleus after 12 h. The co-immunoprecipitation analysis showed that the interaction between AIF and MIF was activated by OGD and subsequently resulted in MIF nuclear translocation. Also, the baicalein inhibited apoptosis, reduced oxidative stress, protected mitochondrial function and restored mitochondrial membrane potential in OGD cells. The results obtained from both in vivo and in vitro study demonstrated the PARP-1/AIF pathway involved in mechanisms of baicalein to protect the cerebral tissues from ischemic injury.

Kaempferol protects against cadmium chloride-induced hippocampal damage and memory deficits by activation of silent information regulator 1 and inhibition of poly (ADP-Ribose) polymerase-1

The neuroprotective effect of Kaempferol against cadmium chloride (CdCl2) -induced neurotoxicity is well reported. The silent information regulator 1 (SIRT1) and poly (ADP-Ribose) polymerase-1 (PARP1) are two related cellular molecules that can negatively affect the activity of each other to promote or inhibit cell survival, respectively. It is still largely unknown if the neurotoxicity of CdCl2 or the neuroprotection of Kaempferol are mediated by modulating SIRT1 and/or PAPR1 activities. In this study, we tested the hypothesis that CdCl2-induced memory deficit and hippocampal damage are associated with downregulation/inhibition of SIRT1 and activation of PAPR1, an effect that can be reversed by co-treatment with Kaempferol. Rats (n = 12/group) were divided into 4 groups as control, control + Kaempferol (50 mg//kg), CdCl2 (0.5 mg/kg), and CdCl2 + Kaempferol. All treatments were administered orally for 30 days daily. As compared to control rats, CdCl2 reduced rat's final body weights (21.8%) and their food intake (30%), induced oxidative stress and apoptosis in their hippocampi, and impaired their short and long-term recognition memory functions. Besides, the hippocampi of CdCl2-treated rats had higher levels of TNF-α (197%), and IL-6 (190%) with a concomitant increase in nuclear activity and levels of NF-κB p65 (721% & 554%). Besides, they showed reduced nuclear activity (53%) and levels (74%) of SIRT1, higher nuclear activity and levels of PARP1 (292% & 138%), increased nuclear levels of p53 (870%), and higher acetylated levels of NF-κB p65 (513%), p53 (644%), PARP1 (696%), and FOXO-2 (149%). All these events were significantly reversed in the CdCl2 + Kaempferol-treated rats. Of note, Kaempferol also increased levels of MnSOD (73.5%), and GSH (40%), protein levels of Bcl-2 (350%), and nuclear activity (67%) and levels (46%) of SIRT1 in the hippocampi of the control rats. In conclusion, Kaempferol ameliorates CdCl2-induced memory deficits and hippocampal oxidative stress, inflammation, and apoptosis by increasing SIRT1 activity and inhibiting PARP1 activity.

Specific Binding of snoRNAs to PARP-1 Promotes NAD+-Dependent Catalytic Activation.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant and ubiquitous nuclear enzyme that catalyzes the transfer of ADP-ribose from donor NAD+ molecules to specific amino acids on substrate proteins. The catalytic activity of PARP-1 has long been known to be allosterically stimulated by the free ends of DNA, such as those found at double-strand breaks in the genome. A number of studies have also shown that the catalytic activity of PARP-1 can also be stimulated by various types of RNA. A recent study by Nakamoto et al., however, has contradicted these results, concluding that the apparent stimulatory activity of the RNAs was due to contaminating DNA in the RNA preparations used in the biochemical assays. Here we show using a carefully controlled set of biochemical assays that DNA-free, in vitro-transcribed, PARP-1-interacting snoRNAs can stimulate PARP-1 catalytic activity. We confirmed the activation of PARP-1 by snoRNAs using a chemically synthesized snoRNA, as well as CRISPR/Cas9-mediated knockout of snoRNAs in cells. Finally, we provide a set of considerations and experimental conditions for the careful evaluation of RNA-stimulated PARP-1 catalytic activity that will help researchers avoid artifacts.

Therapeutic Strategies and Biomarkers to Modulate PARP Activity for Targeted Cancer Therapy.

Poly-(ADP-ribose) polymerase 1 (PARP1) is commonly known for its vital role in DNA damage response and repair. However, its enzymatic activity has been linked to a plethora of physiological and pathophysiological transactions ranging from cellular proliferation, survival and death. For instance, malignancies with BRCA1/2 mutations heavily rely on PARP activity for survival. Thus, the use of PARP inhibitors is a well-established intervention in these types of tumors. However, recent studies indicate that the therapeutic potential of attenuating PARP1 activity in recalcitrant tumors, especially where PARP1 is aberrantly overexpressed and hyperactivated, may extend its therapeutic utility in wider cancer types beyond BRCA-deficiency. Here, we discuss treatment strategies to expand the tumor-selective therapeutic application of PARP inhibitors and novel approaches with predictive biomarkers to perturb NAD+ levels and hyperPARylation that inactivate PARP in recalcitrant tumors. We also provide an overview of genetic alterations that transform non-BRCA mutant cancers to a state of “BRCAness” as potential biomarkers for synthetic lethality with PARP inhibitors. Finally, we discuss a paradigm shift for the use of novel PARP inhibitors outside of cancer treatment, where it has the potential to rescue normal cells from severe oxidative damage during ischemia-reperfusion injury induced by surgery and radiotherapy.

PARP-1-regulated TNF-α expression in the dorsal root ganglia and spinal dorsal horn contributes to the pathogenesis of neuropathic pain in rats

Emerging evidence has implicated poly-(ADP-ribose) polymerase 1 (PARP-1), a transcriptional coregulator, in a variety of inflammatory diseases. In the current study, the role of PARP-1 in neuropathic pain and the underlying mechanisms were investigated. Neuropathic pain was determined by assessing the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) following lumbar 5 spinal nerve ligation (SNL) in male rates. Western blotting, qRT-PCR, immunohistochemistry, chromatin immunoprecipitation (ChIP), and Co-IP assays were performed to elucidate the mechanisms. The results showed that SNL resulted in a significant increase in the expression and activation of PARP-1 in the ipsilateral L4/5 dorsal root ganglia (DRG) and spinal dorsal horn, which occurred on day one, reached peak on day 7, and persisted more than 2 weeks after surgery. Double immunofluorescence staining revealed that PARP-1 was expressed exclusively in DRG A-type and C-type neurons. In the spinal cord, PARP-1 mainly colocalized with the neuronal marker NeuN and the astrocytic marker GFAP specifically in the superficial lamina. Prior intrathecal (i.t.) injection of PJ-34, a PARPs inhibitor, or Tiq-A, a specific PARP-1 inhibitor, dose-dependently prevented the reductions in PWT and PWL following SNL. Established neuropathic pain-like hypersensitivity was also attenuated with i.t. injection of PJ-34 and Tiq-A starting on day 7 following SNL, a timepoint at which neuropathic pain was fully established. SNL-induced mechanical allodynia and thermal hyperalgesia were also alleviated by i.t. injection of PARP-1 siRNA following a reduction in PARP-1 expression in the dorsal horn. Moreover, the SNL-induced increases in TNF-α protein and mRNA in the dorsal horn and DRG were dramatically suppressed by i.t. injection of Tiq-A or PARP-1 siRNA. The i.t. lipopolysaccharide (LPS)-induced increase in the production of TNF-α in the dorsal horn was also inhibited by prior to i.t. injection of PARP-1 siRNA. Results of ChIP assay showed that SNL-induced PARP-1 activation promoted the binding of NF-κB p65 with the TNF-α promoter in the dorsal horn and that PARP-1 inhibition reduced this binding and suppressed TNF-α expression. Co-IP assay revealed that SNL caused a significant increase in the level of histone H1 poly(ADP)–ribosylation. Together, these results indicate that PARP-1-regulated TNF-α expression in the DRG and spinal dorsal horn following SNL contributes to the development and maintenance of neuropathic pain. Targeting PARP-1 might be a promising therapeutic strategy for the treatment of the chronic pain.

Selective targeting of ubiquitination and degradation of PARP1 by E3 ubiquitin ligase WWP2 regulates isoproterenol-induced cardiac remodeling.

The elevated expression of poly(ADP-ribose) polymerase-1 (PARP1) and increased PARP1 activity, namely, poly(ADP-ribosyl)ation (PARylation), have been observed in cardiac remodeling, leading to extreme energy consumption and myocardial damage. However, the mechanisms underlying the regulation of PARP1 require further study. WWP2, a HECT-type E3 ubiquitin ligase, is highly expressed in the heart, but its function there is largely unknown. Here, we clarified the role of WWP2 in the regulation of PARP1 and the impact of this regulatory process on cardiac remodeling. We determined that the knockout of WWP2 specifically in myocardium decreased the level of PARP1 ubiquitination and increased the effects of isoproterenol (ISO)-induced PARP1 and PARylation, in turn aggravating ISO-induced myocardial hypertrophy, heart failure, and myocardial fibrosis. Similar findings were obtained in a model of ISO-induced H9c2 cells with WWP2 knockdown, while the reexpression of WWP2 significantly increased PARP1 ubiquitination and decreased PAPR1 and PARylation levels. Mechanistically, coimmunoprecipitation results identified that WWP2 is a novel interacting protein of PARP1 and mainly interacts with its BRCT domain, thus mediating the degradation of PARP1 through the ubiquitin-proteasome system. In addition, lysine 418 (K418) and lysine 249 (K249) were shown to be of critical importance in regulating PARP1 ubiquitination and degradation by WWP2. These findings reveal a novel WWP2-PARP1 signal transduction pathway involved in controlling cardiac remodeling and may provide a basis for exploring new strategies for treating heart disorders related to cardiac remodeling.

SIRT3 inhibits cardiac hypertrophy by regulating PARP-1 activity.

Sirtuin 3 (SIRT3) is a type III histone deacetylase that inhibits cardiac hypertrophy. It is mainly localized in the mitochondria and is thus implicated in mitochondrial metabolism. Recent studies have shown that SIRT3 can also accumulate in the nuclear under stressed conditions, and participated in histone deacetylation of target proteins. Poly [ADP-ribose] polymerase 1 (PARP-1) functions as an important PARP isoform that was involved in cardiac hypertrophy. Our experiments showed that SIRT3 accumulated in the nuclear of cardiomyocytes treated with isoproterenol or SIRT3 overexpression. Moreover, overexpression of SIRT3 by adenovirus inhibited the expression of cardiac hypertrophic genes-ANF and BNP, as well as abrogating PARP-1 activation induced by isoproterenol or phenylephrine. In addition, co-immunoprecipitation experiments revealed that SIRT3 could interact with PARP-1, and overexpression of SIRT3 could decrease the acetylation level of PARP-1. Our results indicate that SIRT3 exerts protective effects against cardiac hypertrophy by reducing the level of acetylation and activity of PARP-1, thus providing novel mechanistic insights into SIRT3-mediated cardiprotective actions.

Acid invertase confers heat tolerance in rice plants by maintaining energy homoeostasis of spikelets.

Heat stress impairs both pollen germination and pollen tube elongation, resulting in pollination failure caused by energy imbalance. Invertase plays a critical role in the maintenance of energy homoeostasis; however, few studies investigated this during heat stress. Two rice cultivars with different heat tolerance, namely, TLY83 (heat tolerant) and LLY722 (heat susceptible), were subjected to heat stress. At anthesis, heat stress significantly decreased spikelet fertility, accompanied by notable reductions in pollen germination on stigma and pollen tube elongation in ovule, especially in LLY722. Acid invertase (INV), rather than sucrose synthase, contributed to sucrose metabolism, which explains the different tolerances of both cultivars. Under heat stress, larger enhancements in NAD(H), ATP, and antioxidant capacity were found in TLY83 compared with LLY722, whereas a sharp reduction in poly(ADP-ribose) polymerase (PARP) activity was found in the former compared with the latter. Importantly, exogenous INV, 3-aminobenzamide (a PARP inhibitor), sucrose, glucose, and fructose significantly increased spikelet fertility under heat stress, where INV activity was enhanced and PARP activity was inhibited. Therefore, INV can balance the energy production and consumption to provide sufficient energy for pollen germination and pollen tube growth under heat stress.

Identification of PARP-1, Histone H1 and SIRT-1 as New Regulators of Breast Cancer-Related Aromatase Promoter I.3/II.

Paracrine interactions between malignant estrogen receptor positive (ER+) breast cancer cells and breast adipose fibroblasts (BAFs) stimulate estrogen biosynthesis by aromatase in BAFs. In breast cancer, mainly the cAMP-responsive promoter I.3/II-region mediates excessive aromatase expression. A rare single nucleotide variant (SNV) in this promoter region, which caused 70% reduction in promoter activity, was utilized for the identification of novel regulators of aromatase expression. To this end, normal and mutant promoter activities were measured in luciferase reporter gene assays. DNA-binding proteins were captured by DNA-affinity and identified by mass spectrometry. The DNA binding of proteins was analyzed using electrophoretic mobility shift assays, immunoprecipitation-based in vitro binding assays and by chromatin immunoprecipitation in BAFs in vivo. Protein expression and parylation were analyzed by western blotting. Aromatase activities and RNA-expression were measured in BAFs. Functional consequences of poly (ADP-ribose) polymerase-1 (PARP-1) knock-out, rescue or overexpression, respectively, were analyzed in murine embryonic fibroblasts (MEFs) and the 3T3-L1 cell model. In summary, PARP-1 and histone H1 (H1) were identified as critical regulators of aromatase expression. PARP-1-binding to the SNV-region was crucial for aromatase promoter activation. PARP-1 parylated H1 and competed with H1 for DNA-binding, thereby inhibiting its gene silencing action. In MEFs (PARP-1 knock-out and wild-type) and BAFs, PARP-1-mediated induction of the aromatase promoter showed bi-phasic dose responses in overexpression and inhibitor experiments, respectively. The HDAC-inhibitors butyrate, panobinostat and selisistat enhanced promoter I.3/II-mediated gene expression dependent on PARP-1-activity. Forskolin stimulation of BAFs increased promoter I.3/II-occupancy by PARP-1, whereas SIRT-1 competed with PARP-1 for DNA binding but independently activated the promoter I.3/II. Consistently, the inhibition of both PARP-1 and SIRT-1 increased the NAD+/NADH-ratio in BAFs. This suggests that cellular NAD+/NADH ratios control the complex interactions of PARP-1, H1 and SIRT-1 and regulate the interplay of parylation and acetylation/de-acetylation events with low NAD+/NADH ratios (reverse Warburg effect), promoting PARP-1 activation and estrogen synthesis in BAFs. Therefore, PARP-1 inhibitors could be useful in the treatment of estrogen-dependent breast cancers.

Alkyladenine DNA glycosylase deficiency uncouples alkylation-induced strand break generation from PARP-1 activation and glycolysis inhibition

DNA alkylation damage is repaired by base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG). Despite its role in DNA repair, AAG-initiated BER promotes cytotoxicity in a process dependent on poly (ADP-ribose) polymerase-1 (PARP-1); a NAD+-consuming enzyme activated by strand break intermediates of the AAG-initiated repair process. Importantly, PARP-1 activation has been previously linked to impaired glycolysis and mitochondrial dysfunction. However, whether alkylation affects cellular metabolism in the absence of AAG-mediated BER initiation is unclear. To address this question, we temporally profiled repair and metabolism in wild-type and Aag−/− cells treated with the alkylating agent methyl methanesulfonate (MMS). We show that, although Aag−/− cells display similar levels of alkylation-induced DNA breaks as wild type, PARP-1 activation is undetectable in AAG-deficient cells. Accordingly, Aag−/− cells are protected from MMS-induced NAD+ depletion and glycolysis inhibition. MMS-induced mitochondrial dysfunction, however, is AAG-independent. Furthermore, treatment with FK866, a selective inhibitor of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT), synergizes with MMS to induce cytotoxicity and Aag−/− cells are resistant to this combination FK866 and MMS treatment. Thus, AAG plays an important role in the metabolic response to alkylation that could be exploited in the treatment of conditions associated with NAD+ dysregulation.

Microglial NMDA receptors drive pro-inflammatory responses via PARP-1/TRMP2 signaling.

Chronic neuroinflammation driven by microglia is a characteristic feature associated with numerous neurodegenerative diseases. While acute inflammation can assist with recovery and repair, prolonged microglial pro-inflammatory responses are known to exacerbate neurodegenerative processes. Yet, detrimental outcomes of extended microglial activation are counterbalanced by beneficial outcomes including phagocytosis and release of trophic factors promoting neuronal viability. Our past work has shown that the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is a key signaling hub driving pro-inflammatory microglia responses, but the signaling pathway maintaining PARP-1 activation remains elusive. While best understood for its role in promoting DNA repair, our group has shown that PARP-1 activity can be stimulated via Ca2+ influx-dependent ERK1/2-mediated phosphorylation. However, to date, the route of Ca2+ entry responsible for stimulating PARP-1 has not been identified. A likely candidate is via Ca2+ -permeable transient receptor potential melastatin 2 (TRPM2) channels activated downstream of PARP-1 in a cascade that involves ADP-ribose (ADPR) production by poly(ADP-ribose) glycohydrolase (PARG). Here we demonstrate that NMDA receptor (NMDAR) stimulation in primary cultured microglia induces their proliferation, morphological activation and release of pro-inflammatory mediators. These responses were contingent on the recruitment of PARP-1, PARG and Ca2+ permeable TRPM2 channels. Furthermore, we show that Ca2+ influx is necessary to activate PARP-1/TRPM2 signaling, in an ERK1/2-dependent, but DNA damage independent, manner. Our findings, showing that PARP-1/TRPM2 mediate the pro-inflammatory effects of NMDAR stimulation, provides a unifying mechanism linking elevated glutamate levels to chronic neuroinflammation.

Interplay between compartmentalized NAD+ synthesis and consumption: a focus on the PARP family.

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor for redox enzymes, but also moonlights as a substrate for signaling enzymes. When used as a substrate by signaling enzymes, it is consumed, necessitating the recycling of NAD+ consumption products (i.e., nicotinamide) via a salvage pathway in order to maintain NAD+ homeostasis. A major family of NAD+ consumers in mammalian cells are poly-ADP-ribose-polymerases (PARPs). PARPs comprise a family of 17 enzymes in humans, 16 of which catalyze the transfer of ADP-ribose from NAD+ to macromolecular targets (namely, proteins, but also DNA and RNA). Because PARPs and the NAD+ biosynthetic enzymes are subcellularly localized, an emerging concept is that the activity of PARPs and other NAD+ consumers are regulated in a compartmentalized manner. In this review, I discuss NAD+ metabolism, how different subcellular pools of NAD+ are established and regulated, and how free NAD+ levels can control signaling by PARPs and redox metabolism.

Kinetics of DNA damage repair response accompanying initial hepadnavirus-host genomic integration in woodchuck hepatitis virus infection of hepatocyte

Mechanism of initial hepatitis B virus (HBV) integrations and kinetics of DNA repair immediately after infection remain essentially unknown impairing understanding of hepadnaviral oncogenesis. WCM260 hepatocytes susceptible to HBV-compatible woodchuck hepatitis virus (WHV) were examined from 15 min to 72 h post-infection (p.i.). WHV strongly induced reactive oxygen species (ROS), transiently inducible nitric oxide (iNOS) and DNA damage from 15 min p.i. All initial WHV-host fusions had the head-to-tail format indicating their formation by non-homologous end joining (NHEJ). Transcription of poly(ADP-ribose) polymerase 1 (PARP1) and X-ray repair cross-complementing protein 1 (XRCC1), the PARP1 binding partner, were induced in 30 min p.i. and that of 8-oxyguanine DNA glycosylse (OGG1) responding to oxidative DNA damage at 12 h p.i. Nicotinamide adenine dinucleotide (NAD+), a marker of PARP1 activation, and heme oxygenase-1 (HO1), an indicator of pro-oxidative stress, were significantly augmented from 15-30 min p.i. Additionally, PARP1 cleavage activity was evident from 30 min p.i. confirming that PARP1-mediated DNA repair became operational almost instatly after hepatocyte contact with virus. By applying complementary approaches, the study showed that initial WHV integration was due to virus-induced oxidative DNA damage and implied that the NHEJ PARP1-dependent repair pathway determined format of the first virus-host DNA junctions.

Alternative activation of human macrophages enhances tissue factor expression and production of extracellular vesicles.

Macrophages are versatile cells that can be polarized by the tissue environment to fulfill required needs. Proinflammatory polarization is associated with increased tissue degradation and propagation of inflammation whereas alternative polarization within a Th2 cytokine environment is associated with wound healing and angiogenesis. To understand whether polarization of macrophages can lead to a procoagulant macrophage subset we polarized human monocyte-derived macrophages to proinflammatory and alternative activation states. Alternative polarization with interleukin-4 and interleukin-13 led to a macrophage phenotype characterized by increased tissue factor (TF) production and release and by an increase in extracellular vesicle production. In addition, TF activity was enhanced in extracellular vesicles of alternatively polarized macrophages. This TF induction was dependent on signal transducer and activator of transcription- 6 signaling and poly ADP ribose polymerase activity. In contrast to monocytes, human macrophages did not show increased TF expression upon stimulation with lipopolysaccharide and interferon-γ. Previous polarization to either a proinflammatory or an alternative activation subset did not change the subsequent stimulation of TF. The inability of proinflammatory activated macrophages to respond to lipopolysaccharide and interferon- γ with an increase in TF production seemed to be due to an increase in TF promoter methylation and was reversible when these macrophages were treated with a demethylating agent. In conclusion, we provide evidence that proinflammatory polarization of macrophages does not lead to enhanced procoagulatory function, whereas alternative polarization of macrophages leads to an increased expression of TF and increased production of TF-bearing extracellular vesicles by these cells suggesting a procoagulatory phenotype of alternatively polarized macrophages.

Yin and Yang Regulation of Liver X Receptor α Signaling Control of Cholesterol Metabolism by Poly(ADP-ribose) polymerase 1

Liver X receptor α (LXRα) controls a set of key genes involved in cholesterol metabolism. However, the molecular mechanism of this regulation remains unknown. The regulatory role of poly(ADP-ribose) polymerase 1 (PARP1) in cholesterol metabolism in the liver was examined. Activation of PARP1 in the liver suppressed LXRα sensing and prevented upregulation of genes involved in HCD-induced cholesterol disposal. Mechanistically, LXRα was poly(ADP-ribosyl)ated by activated PARP1, which decreased DNA binding capacity of LXRα, thus preventing its recruitment to the target promoter. Intriguingly, we found that unactivated PARP1 was indispensable for LXRα transactivation and target expression. Further exploration identified unactivated PARP1 as an essential component of the LXRα-promoter complex. Taken together, the results indicate that activated PARP1 suppresses LXRα activation through poly(ADP-ribosyl)ation, while unactivated PARP1 promotes LXRα activation through physical interaction. PARP1 is a pivotal regulator of LXRα signaling and cholesterol metabolism in the liver.

ADP-ribosylation: from molecular mechanisms to human disease.

Post-translational modification of proteins by ADP-ribosylation, catalysed by poly (ADP-ribose) polymerases (PARPs) using NAD+ as a substrate, plays central roles in DNA damage signalling and repair, modulates a range of cellular signalling cascades and initiates programmed cell death by parthanatos. Here, we present mechanistic aspects of ADP-ribose modification, PARP activation and the cellular functions of ADP-ribose signalling, and discuss how this knowledge is uncovering therapeutic avenues for the treatment of increasingly prevalent human diseases such as cancer, ischaemic damage and neurodegeneration.

Cloning of the Arabidopsis thaliana poly (Adp-ribose) polymerase 2 cdna gene in Saccharomyces cerevisiae

 Poly(ADP-ribose)polymerase (PARP) catalyzes the synthesis of covalently attached ADP-ribose polymers to acceptor proteins. The donor of the residues of ADP-ribose is NAD+. The prominent roles of PARP is a DNA damage sensor. PARP1, in poly(ADP-ribosyl)ated form binds to DNA breaks and engages proteins of  DNA repair to this sites. Recently, it has been shown that PARP poly(ADP-ribosyl)ates both proteins and DNA breaks. However, there is no direct evidence of  poly(ADP-ribosyl)ated  DNA adducts in vivo in animals and plants. PARP activity is absent in Saccharomyces cerevisiae cells, despite the fact that it has been identified in organisms ranging from archaebacteria to mammals. Due to the simplicity of manipulation and genetic interpretation S. cerevisiae yeasts have been used to analyze the functions of proteins from animal and plant cells. Therein, the goal of our work is the cloning and heterologous expression of the AtPARP2 gene from the Arabidopsis thaliana in yeasts. By using genetic engineering  methods, we have constructed a recombinant plasmid with cDNA of the AtPARP2 gene under the control of the inducible promoter GAL10 from episomal vector pESC-LEU2. A recombinant S.cerevisiae strain capable for expressing the cDNA of the AtPARP2 gene was obtained by genetic transformation method. By using Western blotting with the applying of polyclonal anti-AtPARP2 antibodies, it was revealed that the product of gene expression in yeast is a globular protein weighing 72kDa, consisting  637 amino acids (pI 5.92). A slight inhibition of growth was shown in FF 18733 yeast cells which express AtPARP2. Induction of  AtPARP2 expression in yeast revealed PARylating  activity of protein in vivo. Auto-poly(ADP-ribosyl)ation activity of AtPARP2 was shown in yeasts in response to double-stranded  DNA breaks induced by bleomycin treatment of cells. 3-AB, inhibitor of PARP suppressed the amount of poly(ADP-ribosyl)ated protein products. Key words: Poly(ADP-ribose)polymerase, AtPARP2, poly(ADP-ribosyl)ation, Arabidopsis thaliana, Saccharomyces cerevisae.

Клонирование кднк гена поли(АДФ-рибоза) полимеразы 2 Arabidopsis thaliana в Saccharomyces cerevisiae

 Poly(ADP-ribose)polymerase (PARP) catalyzes the synthesis of covalently attached ADP-ribose polymers to acceptor proteins. The donor of the residues of ADP-ribose is NAD+. The prominent roles of PARP is a DNA damage sensor. PARP1, in poly(ADP-ribosyl)ated form binds to DNA breaks and engages proteins of  DNA repair to this sites. Recently, it has been shown that PARP poly(ADP-ribosyl)ates both proteins and DNA breaks. However, there is no direct evidence of  poly(ADP-ribosyl)ated  DNA adducts in vivo in animals and plants. PARP activity is absent in Saccharomyces cerevisiae cells, despite the fact that it has been identified in organisms ranging from archaebacteria to mammals. Due to the simplicity of manipulation and genetic interpretation S. cerevisiae yeasts have been used to analyze the functions of proteins from animal and plant cells. Therein, the goal of our work is the cloning and heterologous expression of the AtPARP2 gene from the Arabidopsis thaliana in yeasts. By using genetic engineering  methods, we have constructed a recombinant plasmid with cDNA of the AtPARP2 gene under the control of the inducible promoter GAL10 from episomal vector pESC-LEU2. A recombinant S.cerevisiae strain capable for expressing the cDNA of the AtPARP2 gene was obtained by genetic transformation method. By using Western blotting with the applying of polyclonal anti-AtPARP2 antibodies, it was revealed that the product of gene expression in yeast is a globular protein weighing 72kDa, consisting  637 amino acids (pI 5.92). A slight inhibition of growth was shown in FF 18733 yeast cells which express AtPARP2. Induction of  AtPARP2 expression in yeast revealed PARylating  activity of protein in vivo. Auto-poly(ADP-ribosyl)ation activity of AtPARP2 was shown in yeasts in response to double-stranded  DNA breaks induced by bleomycin treatment of cells. 3-AB, inhibitor of PARP suppressed the amount of poly(ADP-ribosyl)ated protein products. Key words: Poly(ADP-ribose)polymerase, AtPARP2, poly(ADP-ribosyl)ation, Arabidopsis thaliana, Saccharomyces cerevisae.

Apoptosis and regeneration of hepatocytes in rats orally exposed to caffeinated energy drinks

Caffeinated energy drinks (CEDs) have become extremely popular over the last decade. In recent years, many researchers have raised a concern about the safety of CEDs. The aim of our study was to evaluate the morphology of the liver, as well as the features of hepatocyte apoptosis/regeneration, in rats orally exposed to CEDs for 2 weeks. Sixteen WAG rats formed two equal groups. Rats from the experimental group were orally exposed to a CED at a dose of 12 ml/kg of body weight for 2 weeks. The control group consisted of intact animals with the provided access to drinking water instead. The content of apoptosis marker caspase-3 and the activity of poly-(ADP-ribose) polymerase (PARP) were evaluated in liver homogenates. The activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was determined in the blood serum. Hepatic Ki-67 expression was quantitatively assessed immunohistochemically. A statistically significant increase in the level of caspase-3 and a decrease in the activity of PARP in liver homogenates against the background of energy drink consumption by rats indicate the activation of hepatocyte apoptosis, evidenced by chromatin margination of hepatocytes as well. Cytolysis of hepatocytes was confirmed by the elevation of ALT and AST. In response to cell death, liver parenchyma starts proliferating. This fact is supported by the statistically significant increase in the amount of Ki-67+ hepatocytes in rats exposed to CED compared with controls. Oral exposure to a CED during 2 weeks promotes liver damage, apoptosis, and regeneration of hepatocytes.

Tankyrase promotes primary precursor miRNA processing to precursor miRNA

Tankyrases (TNKS and TNKS2) are members of poly(ADP-ribose) polymerase (PARP) family proteins. Tankyrase has multiple ankyrin repeat cluster (ARC) domains, which recognize the tankyrase-binding motifs in proteins including the telomeric protein, TRF1 and Wnt signal regulators, AXINs. However, the functional significance of tankyrase interaction with many other putative binding proteins remains unknown. Here, we found that several proteins involved in microRNA (miRNA) processing have putative tankyrase-binding motifs and their functions are regulated by tankyrase. First, chemical inhibition of tankyrase PARP activity downregulated the expression levels of precursor miRNAs (pre-miRNAs) but not primary precursor miRNAs (pri-miRNAs). A subsequent reporter assay revealed that tankyrase inhibitors or PARP-dead mutant tankyrase overexpression repress pri-miRNA processing to pre-miRNA. Conversely, a PARP-1/2 inhibitor, olaparib, did not affect pri-miRNA processing. Tankyrase ARCs bound to DGCR8 and DROSHA, which are essential components for pri-miRNA processing and have putative tankyrase-binding motifs. These observations indicate that tankyrase binds to Microprocessor, DGCR8 and DROSHA complex and modulates pri-miRNA processing to pre-miRNA.