Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Werner's syndrome (WS) is an inherited disease characterized by genomic instability and premature aging. The WS gene encodes a protein (WRN) with helicase and exonuclease activities. We have previously reported that WRN interacts with Ku70/80 and this interaction strongly stimulates WRN exonuclease activity. To gain further insight on the function of WRN and its relationship with the Ku heterodimer, we established a cell line expressing tagged WRN(H), a WRN point mutant lacking helicase activity, and used affinity purification, immunoblot analysis and mass spectroscopy to identify WRN-associated proteins. To this end, we identified three proteins that are stably associated with WRN in nuclear extracts. Two of these proteins, Ku70 and Ku80, were identified by immunoblot analysis. The third polypeptide, which was identified by mass spectrometry analysis, is identical to poly(ADP-ribose) polymerase-1(PARP-1), a 113-kDa enzyme that functions as a sensor of DNA damage. Biochemical fractionation studies and immunoprecipitation assays and studies confirmed that endogenous WRN is associated with subpopulations of PARP-1 and Ku70/80 in the cell. Protein interaction assays with purified proteins further indicated that PARP-1 binds directly to WRN and assembles in a complex with WRN and Ku70/80. In the presence of DNA and NAD(+), PARP-1 poly(ADP-ribosyl)ates itself and Ku70/80 but not WRN, and gel-shift assays showed that poly-(ADP-ribosyl)ation of Ku70/80 decreases the DNA-binding affinity of this factor. Significantly, (ADP-ribosyl)ation of Ku70/80 reduces the ability of this factor to stimulate WRN exonuclease, suggesting that covalent modification of Ku70/80 by PARP-1 may play a role in the regulation of the exonucleolytic activity of WRN.
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PMID:Identification and biochemical characterization of a Werner's syndrome protein complex with Ku70/80 and poly(ADP-ribose) polymerase-1. 1473 61

Loss of telomere equilibrium and associated chromosome-genomic instability might effectively promote tumour progression. Telomere function may have contrasting roles: inducing replicative senescence and promoting tumourigenesis and these roles may vary between cell types depending on the expression of the enzyme telomerase, the level of mutations induced, and efficiency/deficiency of related DNA repair pathways. We have identified an alternative telomere maintenance mechanism in mouse embryonic stem cells lacking telomerase RNA unit (mTER) with amplification of non-telomeric sequences adjacent to existing short stretches of telomere repeats. Our quest for identifying telomerase-independent or alternative mechanisms involved in telomere maintenance in mammalian cells has implicated the involvement of potential DNA repair factors in such pathways. We have reported earlier on the telomere equilibrium in scid mouse cells which suggested a potential role of DNA repair proteins in telomere maintenance in mammalian cells. Subsequently, studies by us and others have shown the association between the DNA repair factors and telomere function. Mice deficient in a DNA-break sensing molecule, PARP-1 (poly [ADP]-ribopolymerase), have increased levels of chromosomal instability associated with extensive telomere shortening. Ku80 null cells showed a telomere shortening associated with extensive chromosome end fusions, whereas Ku80+/- cells exhibited an intermediate level of telomere shortening. Inactivation of PARP-1 in p53-/- cells resulted in dysfunctional telomeres and severe chromosome instability leading to advanced onset and increased tumour incidence in mice. Interestingly, haploinsufficiency of PARP-1 in Ku80 null cells causes more severe telomere shortening and chromosome abnormalities compared to either PARP-1 or Ku80 single null cells and Ku80+/-PARP-/- mice develop spontaneous tumours. This overview will focus mainly on the role of DNA repair/recombination and DNA damage signalling molecules such as PARP-1, DNA-PKcs, Ku70/80, XRCC4 and ATM which we have been studying for the last few years. Because the maintenance of telomere function is crucial for genomic stability, our results will provide new insights into the mechanisms of chromosome instability and tumour formation.
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PMID:DNA repair factors and telomere-chromosome integrity in mammalian cells. 1516 24

Thyroid transcription factor 1 (TTF-1/Nkx-2.1) plays a critical role in lung morphogenesis and regulates the expression of lung-specific genes, including the surfactant proteins required for pulmonary function after birth. The activity of TTF-1 is influenced by its interactions with other transcription factors and coactivators, including CBP/p300 and SRC-1. In this study, we have identified poly(ADP-ribose) polymerases (PARP-2 and PARP-1) as TTF-1 interacting proteins that influence its transcriptional activity. Endogenous PARP-2 was coimmunoprecipitated from transformed mouse lung epithelial cell (MLE15) extracts with TTF-1 and was identified by mass spectrometry. PARP-1 and Ku70/Ku80 were also coimmunoprecipitated from the cell extracts with TTF-1. The E domain of PARP-2 interacted via the C-terminal domain of TTF-1. Both PARP-1 and PARP-2 enhanced the activity of the promoter of surfactant protein-B (Sftpb gene) but not other surfactant proteins in vitro. PARP-2 was selectively expressed in epithelial cells of the conducting and peripheral lung tubules of the fetal mouse lung from embryonic day 12.5 and was detected in bronchial epithelial cells in the adult lung at cellular sites consistent with that of surfactant protein B. PARP-2 and PARP-1 interact with TTF-1 and regulate the expression of surfactant protein B, a protein required for lung function.
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PMID:PARP-2 interacts with TTF-1 and regulates expression of surfactant protein-B. 1646 52

Prostate cancer cell lines were examined for proteins that partnered with the transcription factor C/EBPalpha by use of a pull-down assay with S-tagged C/EBPalpha combined with matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis. Ku70, Ku80, and poly(ADP-ribose) polymerase-1 (PARP-1) were identified as proteins that associated with C/EBPalpha. The physical interaction of C/EBPalpha with these partner proteins was further demonstrated by glutathione S-transferase (GST) pull-downs using purified protein expressed in Escherichia coli. The strongest binding was between C/EBPalpha and PARP-1. Immunoprecipitation of C/EBPalpha expressed in prostate cancer cells co-precipitated Ku70, Ku80, and PARP-1. Deletion analysis of C/EBPalpha indicated that the C terminus of C/EBPalpha was essential for the interaction of C/EBPalpha with Ku70, Ku80, and PARP-1. Functional analysis of the interaction between C/EBPalpha and the Ku proteins as well as PARP-1 showed that cells exhibiting these interactions had increased radiation sensitivity and decreased ability to repair double strand DNA breaks. Deficient DNA repair was dependent on the prostate cancer cell line tested, suggesting a complex process. We conclude that the association of C/EBPalpha with Ku proteins and PARP-1 raises the likelihood that C/EBPalpha-expressing prostate cancer cells may be more sensitive to DNA-damaging agents and may be important in the design of new prostate cancer therapies.
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PMID:In prostate cancer cells the interaction of C/EBPalpha with Ku70, Ku80, and poly(ADP-ribose) polymerase-1 increases sensitivity to DNA damage. 1649 Jul 87

The viability of non-homologous end-joining (NHEJ)-defective mice suggests that homologous recombination (HR) might take over its role in DNA repair. To test this hypothesis, we examined gene targeting frequencies (TF) in DNA-PK(cs), Ku80 and poly(ADP-ribose) polymerase (PARP-1) nullizygous cells. We observed a 3-fold TF increase in PARP-1 knockout embryonic stem (ES) cells, which is consistent with the predicted role of PARP-1 as a switch between HR and NHEJ. To a lesser extent, such effect could be reproduced upon chemical inhibition of PARP-1. However, TF was not enhanced in Ku80- or DNA-PK(cs)-defective cells. Our study also suggests an unexpected involvement of DNA-PK(cs) in HR.
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PMID:Down-regulation of PARP-1, but not of Ku80 or DNA-PKcs', results in higher gene targeting efficiency. 1650 47

Poly (ADP-ribose) polymerase (PARP-1), ATM and DNA-dependent protein kinase (DNA-PK) are all involved in responding to DNA damage to activate pathways responsible for cellular survival. Here, we demonstrate that PARP-1-/- cells are sensitive to the ATM inhibitor KU55933 and conversely that AT cells are sensitive to the PARP inhibitor 4-amino-1,8-napthalamide. In addition, PARP-1-/- cells are shown to be sensitive to the DNA-PK inhibitor NU7026 and DNA-PKcs or Ku80 defective cells shown to be sensitive to PARP inhibitors. We believe PARP inhibition results in an increase in unresolved spontaneous DNA single-strand breaks (SSBs), which collapse replication forks and trigger homologous recombination repair (HRR). We show that ATM is activated following inhibition of PARP. Furthermore, PARP inhibitor-induced HRR is abolished in ATM, but not DNA-PK, inhibited cells. ATM and DNA-PK inhibition together give the same sensitivity to PARP inhibitors as ATM alone, indicating that ATM functions in the same pathways as DNA-PK for survival at collapsed forks, likely in non-homologous end joining (NHEJ). Altogether, we suggest that ATM is activated by PARP inhibitor-induced collapsed replication forks and may function upstream of HRR in the repair of certain types of double-strand breaks (DSBs).
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PMID:Inhibition of poly (ADP-ribose) polymerase activates ATM which is required for subsequent homologous recombination repair. 1655 9

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.
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PMID:PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery. 1692 74

Formation of the T-cell factor-4 (TCF-4) and beta-catenin nuclear complex is considered crucial to embryonic development and colorectal carcinogenesis. We previously reported that poly(ADP-ribose) polymerase-1 (PARP-1) interacts with the TCF-4 and beta-catenin complex and enhances its transcriptional activity. However, its biological significance remains unexplained. Using immunoprecipitation and mass spectrometry, we found that two Ku proteins, Ku70 and Ku80, were also associated with the complex. Knockdown of Ku70 by RNA interference increased the amount of beta-catenin associated with TCF-4 and enhanced the transcriptional activity. PARP-1 competed with Ku70 for binding to TCF-4. Treatment with bleomycin, a DNA-damaging alkylating agent, induced polyADP-ribosylation of PARP-1 protein and inhibited its interaction with TCF-4. Bleomycin conversely increased the amounts of Ku70 coimmunoprecipitated with TCF-4 and removed beta-catenin from TCF-4. We propose a working model in which the transcriptional activity of TCF-4 is regulated by the relative amount of Ku70, PARP-1, and beta-catenin proteins binding to TCF-4. Identification of the functional interaction of Ku70 as well as PARP-1 with the TCF-4 and beta-catenin transcriptional complex may provide insights into a novel linkage between DNA damage recognition/repair and Wnt signaling.
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PMID:Ku70 and poly(ADP-ribose) polymerase-1 competitively regulate beta-catenin and T-cell factor-4-mediated gene transactivation: possible linkage of DNA damage recognition and Wnt signaling. 1728 21

Poly(ADP-ribose) polymerases (PARP) is enzyme family repairing single or double DNA strand breaks induced by different alkylating agents, ionizing- or UV-irradiation as well as by oxidative stress. Poly(ADP-ribose) polymerase-1 (PARP-1) is the most studied enzyme involved in a number of pathways including DNA replication and repair, recombination, gene transcription, cell proliferation and death. A positive correlation between the PARP-activity and the life span of different mammalians has been detected. PARP inhibition in vitro with inhibitors of PARP activity (3-aminobenzamide, nicotinamide, picolinamide e.t.c.) in cells from wild type or PARP-1(-/-) mice was followed by high genomic instability (i.e. aneuploidy, gene amplifications and deletions, micronuclei formation, sister chromatic exchange, cell ploidy and centrosome number increase) and increased sensitivity to mutagens. Life span reduction, latency period of spontaneous tumors development shortening and the increase in susceptibility to carcinogens have been observed in PARP-knockout mice. Treatment with PARP inhibitors stimulated chemical and radiation carcinogenesis in animals. The PARP-1(-/-) mice being additionally disrupted in WRN, p53, DNA-PKcs or Ku80 genes the promotion of spontaneous carcinogenesis was observed as compared with a single gene-disrupted mice. Available data suggest a significant role of PARP in maintenance of genomic stability, preventing of aging and carcinogenesis.
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PMID:[Poly(ADP-ribosa)polymerase--the relationships with life span and carcinogenesis]. 1830 94

Roscovitine has been reported to have anti-proliferative properties and is in process of undergoing clinical trials. In addition to its intrinsic anticancer properties, it has recently been suggested that roscovitine may also enhance the activity of traditional chemo- and radio- therapies in certain cancer cell lines. The purpose of this study was to define the activity of roscovitine in increasing radiosensitivity of human non-small cell lung cancer (NSCLC) cell line A549 cells in vitro. A549 cells were exposed to ionizing radiation (IR) of gamma-ray with or without roscovitine pretreatment. Clonogenic assay was performed and cell cycle and apoptosis were analyzed by flow cytometry. Expression of PARP, Ku70 and Ku80 proteins was detected by Western blot. The active form of caspase-3 positive cells were measured by flow cytometry. Our results showed that roscovitine caused dose-dependent apoptosis in A549 cells. Pretreatment with minimally toxic concentration of roscovitine significantly radiosensitized A549 cells by inhibiting colony formation. We then examined potential mechanisms that may contribute to the enhanced radiation response induced by roscovitine. Our results showed that the combination treatment significantly induced apoptosis in A549 cells compared to roscovitine or IR treatment alone. Meanwhile, in the co-treatment group, the percentage of cells with the active form of caspase-3 was markedly increased, while roscovitine or IR alone had little effect. Roscovitine decreased S phase cells when used alone or in sequential combination with IR. Furthermore, this combination treatment blocked DNA repair process after IR, indicated by down regulation of Ku70 and Ku80 proteins, while the singly used treatment did not. Taken together, these results suggest that roscovitine has the potential to act as a radio-sensitizer in A549 cells by promoting caspase-3 activity and increasing apoptosis, affecting cell cycle distribution and impairing DNA repair process.
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PMID:Enhancement of radiosensitivity by roscovitine pretreatment in human non-small cell lung cancer A549 cells. 1872 43


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