DNA maintenance following bleomycin-induced strand breaks does not require poly(ADP-ribosyl)ation activation in Drosophila S2 cells

BackgroundPoly-ADP ribosylation (PARylation) is a post translational modification, catalyzed by Poly(ADP-ribose)polymerase (PARP) family. In Drosophila, PARP-I (human PARP-1 ortholog) is considered to be the only enzymatically active isoform. PARylation is involved in various cellular processes such as DNA repair in case of base excision and strand-breaks.ObservationsStrand-breaks (SSB and DSB) are detrimental to cell viability and, in Drosophila, that has a unique PARP family organization, little is known on PARP involvement in the control of strand-breaks repair process. In our study, strands-breaks (SSB and DSB) are chemically induced in S2 Drosophila cells using bleomycin. These breaks are efficiently repaired in S2 cells. During the bleomycin treatment, changes in PARylation levels are only detectable in a few cells, and an increase in PARP-I and PARP-II mRNAs is only observed during the recovery period. These results differ strongly from those obtained with Human cells, where PARylation is strongly activating when DNA breaks are generated. Finally, in PARP knock-down cells, DNA stability is altered but no change in strand-breaks repair can be observed.ConclusionsPARP responses in DNA strands-breaks context are functional in Drosophila model as demonstrated by PARP-I and PARP-II mRNA increases. However, no modification of the global PARylation profile is observed during strand-breaks generation, only changes at cellular levels are detectable. Taking together, these results demonstrate that PARylation process in Drosophila is tightly regulated in the context of strands-breaks repair and that PARP is essential during the maintenance of DNA integrity but dispensable in the DNA repair process.     

Ecdysone receptor-dependent gene regulation mediates histone poly(ADP-ribosyl)ation

While the ecdysone dependency of puff formation in giant polytene chromosomes from fly salivary glands has been well documented, the molecular mechanisms underlying this process remain unknown. However, it does appear to involve chromatin remodeling and modification mediated by ecdysone receptor (EcR). As Drosophila poly(ADP-ribose) polymerase (dPARP) has recently been reported to be involved in ecdysone-induced puff formation, we decided to test the possible role of dPARP in ligand-induced dEcR transactivation in an insect system. dPARP co-activated the ligand-induced transactivation function of EcR in the insect cell line S2, and appeared to physically interact with EcR in a ligand-dependent manner. ChIP analysis of an EcR target gene promoter revealed ligand-dependent recruitment of dPARP with poly(ADP-ribosyl)ation of histones in the EcR binding site and, surprisingly, also in a distal region of the promoter. Our results indicated that EcR-mediated gene regulation may be coupled with chromatin modification through poly(ADP-ribosyl)ation.     

Haploinsufficiency of poly(ADP-ribose) polymerase-1-mediated poly(ADP-ribosyl)ation for centrosome duplication

The centrosome plays a vital role in maintaining chromosomal stability. Known as the microtubule organizing center, the centrosome is involved in the formation of spindle poles during mitosis, which ensures the distribution of the correct number of chromosomes to daughter cells. Aberrant centrosome duplication could cause centrosome amplification and chromosomal instability. We have previously shown that poly(ADP-ribose) polymerase-1 (PARP-1) is important for centrosome function and chromosomal stability. In this study, we used PARP-1(+/+), PARP-1(+/-) and PARP-1(-/-) primary mouse embryonic fibroblasts and found that the level of PARP-1 gene dosage correlates with PARP activity and the in vivo level of poly(ADP-ribosyl)ation, which could explain the mechanism by which PARP-1 haploinsufficiency affects centrosome duplication and chromosomal stability. Our results emphasize that correct regulation of poly(ADP-ribosyl)ation levels in vivo is important for maintenance of proper centrosome duplication and chromosomal stability.     

50 Years of poly(ADP-ribosyl)ation

The seminal paper published in 1963 by Chambon, Weil and Mandel reporting a new NAD-dependent protein modification now known as poly(ADP-ribosyl)ation (PARylation) marked the launch of a new era in both protein research and cell biology. In the coming decades, the identity, biochemical characteristics and regulation of enzymes responsible for the synthesis and degradation of protein-bound poly(ADP-ribose) have been discovered and the surprisingly multifarious biological roles of PARylation have not ceased to amaze cell and molecular biologists ever since. The review series on PARylation following this preface is comprised of ten papers written by great experts of the field and aims to provide practicing physicians and basic scientists with the state-of-the-art on the “writers, readers and erasers” of poly(ADP-ribose), some recent paradigm shifts of the field and its translational potential.     

Micropeptide PACMP inhibition elicits synthetic lethal effects by decreasing CtIP and poly(ADP-ribosyl)ation

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

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 providing the basis for selection of the appropriate genetic model organisms to study the physiology of the specific human PARP proteins.     

Human poly(ADP-ribose) glycohydrolase is expressed in alternative splice variants yielding isoforms that localize to different cell compartments

Poly(ADP-ribose) glycohydrolase (PARG) is the only protein known to catalyze hydrolysis of ADP-ribose (ADPR) polymers to free ADP-ribose. While numerous genes encode different poly(ADP-ribose) polymerases (PARPs) that all synthesize ADP-ribose polymer, only a single gene coding for PARG has been detected in mammalian cells. Here, we describe two splice variants of human PARG mRNA, which lead to expression of PARG isoforms of 102 kDa (hPARG102) and 99 kDa (hPARG99) in addition to the full-length PARG protein (hPARG111). These splice variants differ from hPARG111 by the lack of exon 1 (hPARG102) or exons 1 and 2 (hPARG99). They are generated by the utilization of ambiguous splice donor sites in the PARG gene 5' untranslated region. The hPARG111 isoform localizes to the nucleus, whereas hPARG102 and hPARG99 are cytoplasmic proteins. The nuclear targeting of hPARG111 is due to a nuclear localization signal (NLS) in exon 1 that was mapped to the amino acids (aa) (10)CTKRPRW(16). Immunocytochemistry, immunoblotting, and PARG enzyme activity measurements show that the cytoplasmic isoforms of PARG account for most of the PARG activity in cells in the absence and presence of genotoxic stress. The predominantly cytoplasmic location of cellular PARG is intriguing as most known cellular PARPs have a nuclear localization.     

Blockade of PARP activity attenuates poly(ADP-ribosyl)ation but offers only partial neuroprotection against NMDA-induced cell death in the rat retina

Recent reports have linked neuronal cell death by necrosis to poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation. It is believed that under stress, the activity of this enzyme is up-regulated, resulting in extensive poly(ADP-ribosyl)ation of nuclear proteins, using NAD(+) as its substrate, which, in turn, leads to the depletion of NAD(+). In efforts to restore the level of NAD(+), depletion of ATP occurs, resulting in the shutdown of ATP-dependent ionic pumps. This results in cell swelling and eventual loss of membrane selectivity, hallmarks of necrosis. Reports from in vitro and in vivo studies in the brain have shown that NMDA receptor activation stimulates PARP activity and that blockade of the enzyme provides substantial neuroprotection. The present study was undertaken to determine whether PARP activity is regulated by NMDA in the rat retina, and whether blockade of PARP activity provides protection against toxic effects of NMDA. Rat retinas exposed to intravitreal injections containing NMDA, with or without the PARP inhibitor N-(6-oxo-5, 6-dihydrophenanthridin-2-yl)-(N,-dimethylamino) acetamide hydrochloride (PJ-34), were assessed for changes in PARP-1 activity as evidenced by poly(ADP-ribosyl)ation (PAR), loss of membrane integrity, morphological indicators of apoptosis and necrosis, and ganglion cell loss. Results showed that: NMDA increased PAR formation in a concentration-dependent manner and caused a decline in retinal ATP levels; PJ-34 blockade attenuated the NMDA-induced formation of PAR and decline in ATP; NMDA induced the loss of membrane selectivity to ethidium bromide (EtBr) in inner retinal neurons, but loss of membrane selectivity was not prevented by blocking PARP activity; cells stained with EtBr, or reacted for TUNEL-labeling, displayed features characteristic of both apoptosis and necrosis. In the presence of PJ-34, greater numbers of cells exhibited apoptotic features; PJ-34 provided partial neuroprotection against NMDA-induced ganglion cell loss. These findings suggest that although blockade of PARP activity fully attenuates NMDA-induced PAR formation and loss of retinal ATP content, and improves the survival of select populations of ganglion cells, this approach does not provide full neuroprotection. In contrast, blockade of PARP activity promotes apoptotic-like cell death in the majority of cells undergoing cell death. Furthermore, these studies show that the loss of membrane selectivity is not dependent upon PAR formation or the resulting decline of ATP, and suggests that an alternative pathway, other than PARP activation, exists to mediate this event.     

Misregulation of poly(ADP-ribose) polymerase-1 activity and cell type-specific loss of poly(ADP-ribose) synthesis in the cerebellum of aged rats

Protein modification by ADP-ribose polymers is a common regulatory mechanism in eukaryotic cells and is involved in several aspects of brain physiology and physiopathology, including neurotransmission, memory formation, neurotoxicity, ageing and age-associated diseases. Here we show age-related misregulation of poly(ADP-ribose) synthesis in rat cerebellum as revealed by: (i) reduced poly(ADP-ribose) polymerase-1 (PARP-1) activation in response to enzymatic DNA cleavage, (ii) altered protein poly(ADP-ribosyl)ation profiles in isolated nuclei, and (iii) cell type-specific loss of poly(ADP-ribosyl)ation capacity in granule cell layer and Purkinje cells in vivo. In particular, although PARP-1 could be detected in virtually all granule cells, only a fraction of them appeared to be actively engaged in poly(ADP-ribose) synthesis and this fraction was reduced in old rat cerebellum. NAD(+), quantified in tissue homogenates, was essentially the same in the cerebellum of young and old rats suggesting that in vivo factors other than PARP-1 content and/or NAD(+) levels may be responsible for the age-associated lowering of poly(ADP-ribose) synthesis. Moreover, PARP-1 expression was substantially down-regulated in Purkinje cells of senescent rats.     

The role of poly(ADP-ribose) polymerases in manganese exposed Caenorhabditis elegans

Background and aimWhen exceeding the homeostatic range, manganese (Mn) might cause neurotoxicity, characteristic of the pathophysiology of several neurological diseases. Although the underlying mechanism of its neurotoxicity remains unclear, Mn-induced oxidative stress contributes to disease etiology. DNA damage caused by oxidative stress may further trigger dysregulation of DNA-damage-induced poly(ADP-ribosyl)ation (PARylation), which is of central importance especially for neuronal homeostasis. Accordingly, this study was designed to assess in the genetically traceable in vivo model Caenorhabditis elegans the role of PARylation as well as the consequences of loss of pme-1 or pme-2 (orthologues of PARP1 and PARP2) in Mn-induced toxicity.MethodsA specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed to quantify PARylation in worms. Next to monitoring the PAR level, pme-1 and pme-2 gene expression as well as Mn-induced oxidative stress was studied in wildtype worms and the pme deletion mutants.Results and conclusionWhile Mn failed to induce PARylation in wildtype worms, toxic doses of Mn led to PAR-induction in pme-1-deficient worms, due to an increased gene expression of pme-2 in the pme-1 deletion mutants. However, this effect could not be observed at sub-toxic Mn doses as well as upon longer incubation times. Regarding Mn-induced oxidative stress, the deletion mutants did not show hypersensitivity. Taken together, this study characterizes worms to model PAR inhibition and addresses the consequences for Mn-induced oxidative stress in genetically manipulated worms.     

Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V_max) and the michaelis constant (k_m) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.     

Design and synthesis of potent inhibitors of the mono(ADP-ribosyl)transferase,PARP14

A series of (Z)-4-(3-carbamoylphenylamino)-4-oxobut-2-enyl amides were synthesized and tested for their ability to inhibit the mono-(ADP-ribosyl)transferase, PARP14 (a.k.a. BAL-2; ARTD-8). Two synthetic routes were established for this series and several compounds were identified as sub-micromolar inhibitors of PARP14, the most potent of which was compound 4t, IC₅₀ = 160 nM. Furthermore, profiling other members of this series identified compounds with >20-fold selectivity over PARP5a/TNKS1, and modest selectivity over PARP10, a closely related mono-(ADP-ribosyl)transferase.     

Role of poly(ADP-ribose) glycohydrolase silencing in DNA hypomethylation induced by benzo(a)pyrene

Benzo(a)pyrene (BaP) is a known carcinogen cytotoxic which can trigger extensive cellular responses. Many evidences suggest that inhibitors of poly(ADP-ribose) glycohydrolase (PARG) are potent anticancer drug candidates. However, the role of PARG in BaP carcinogenesis is less understood. Here we used PARG-deficient human bronchial epithelial cell line (shPARG cell) as an in vitro model, and investigated the role of PARG silencing in DNA methylation pattern changed by BaP. Our study shows, BaP treatment decreased global DNA methylation levels in 16HBE cells in a dose-dependent manner, but no dramatic changes were observed in shPARG cells. Further investigation revealed PARG silencing protected DNA methyltransferases (DNMTs) activity from change by BaP exposure. Interestingly, Dnmt1 is PARylated in PARG-null cells after BaP exposure. The results show a role for PARG silencing in DNA hypomethylation induced by BaP that may provide new clue for cancer therapy.     

Telomere elongation by a mutant tankyrase 1 without TRF1 poly(ADP-ribosyl)ation

Telomeres are the capping structures of the eukaryotic chromosome ends. Tankyrase 1 is a poly(ADP-ribose) polymerase that elongates telomeres in a telomerase-dependent manner. This function of tankyrase 1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Namely, tankyrase 1 poly(ADP-ribosyl)ates (PARsylates) TRF1, which in turn dissociates TRF1 from telomeres. The resulting telomeres become better substrates for telomerase-mediated DNA extension. Tankyrase 1 has five independent TRF1 binding sites, ARC (ANK repeat cluster) I to V. Among them, the most C-terminal ARC V is required for TRF1 PARsylation and its release from telomeres. By contrast, functional significance of other four ARCs remains elusive. In this study, we generated a mutant tankyrase 1 that had inactive ARC IV and lacked ARC V but elongated telomeres without TRF1 PARsylation. Consistent with the failure in PARsylation, this mutant only marginally released TRF1 from telomeres. Still, it decreased telomere binding of POT1, a downstream effector of TRF1-mediated telomere length control, and elongated the telomeric 3'-overhang as the wild-type tankyrase 1 did. Thus even without TRF1 PARsylation, this mutant tankyrase 1 seemed to loosen the closed structure of the telomeric heterochromatin. These findings suggest a new role for multiple ARCs in telomere extension by tankyrase 1.     

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

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.     

Inhibition of poly(ADP-ribosyl)ation in cancer: Old and new paradigms revisited

Inhibitors of poly(ADP-ribose) polymerases actualized the biological concept of synthetic lethality in the clinical practice, yielding a paradigmatic example of translational medicine. The profound sensitivity of tumors with germline BRCA mutations to PARP1/2 blockade owes to inherent defects of the BRCA-dependent homologous recombination machinery, which are unleashed by interruption of PARP DNA repair activity and lead to DNA damage overload and cell death. Conversely, aspirant BRCA-like tumors harboring somatic DNA repair dysfunctions (a vast entity of genetic and epigenetic defects known as “BRCAness”) not always align with the familial counterpart and appear not to be equally sensitive to PARP inhibition. The acquisition of secondary resistance in initially responsive patients and the lack of standardized biomarkers to identify “BRCAness” pose serious threats to the clinical advance of PARP inhibitors; a feeling is also emerging that a BRCA-centered perspective might have missed the influence of additional, not negligible and DNA repair-independent PARP contributions onto therapy outcome. While regulatory approval for PARP1/2 inhibitors is still pending, novel therapeutic opportunities are sprouting from different branches of the PARP family, although they remain immature for clinical extrapolation. This review is an endeavor to provide a comprehensive appraisal of the multifaceted biology of PARPs and their evolving impact on cancer therapeutics.     

The role of poly(ADP-ribosyl)ation in the adaptive response

An involvement of the poly(ADP-ribosyl)ation system in the expression of the adaptive response has been demonstrated with inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase. This enzyme is a key component of a reaction cycle in chromatin, involving dynamic synthesis and degradation of variably sized ADP-ribose polymers in response to DNA strand breaks. The present report reviews recent work focussing on the response of the poly(ADP-ribosyl)ation system in low dose adaptation. The results suggest that adaptation of human cells to minute concentrations of an alkylating agent involves a different activation mechanism for poly(ADP-ribose) polymerase than DNA break-mediated stimulation after high dose treatment. Moreover, adaptation induces the formation of branched polymers with a very high binding affinity for histone tails and selected other proteins. High dose challenge treatment of adapted cells further enhances formation of branched polymers. We propose that apart from sensing DNA nicks, poly(ADP-ribose) polymerase may be part of pathway protecting cells from downstream events of DNA damage.