Inhibition of poly(ADP-ribose) formation by 4-hydroxynonenal in primary cultures of rabbit synovial fibroblasts.
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ABSTRACT:
SUBMITTER: Ullrich O
PROVIDER: S-EPMC1217263 | biostudies-other | 1996 May
REPOSITORIES: biostudies-other
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Project description:Poly(ADP-ribose) polymerase (PARP) is implicated in the maintenance of genomic integrity, given that inhibition or depletion of this enzyme increases genomic instability in cells exposed to genotoxic agents. We previously showed that immortalized fibroblasts derived from PARP(-/-) mice exhibit an unstable tetraploid population, and partial chromosomal gains and losses in PARP(-/-) mice and immortalized fibroblasts are accompanied by changes in the expression of p53, Rb, and c-Jun, as well as other proteins. A tetraploid population has also now been detected in primary fibroblasts derived from PARP(-/-) mice. Oligonucleotide microarray analysis was applied to characterize more comprehensively the differences in gene expression between asynchronously dividing primary fibroblasts derived from PARP(-/-) mice and their wild-type littermates. Of the 11,000 genes monitored, 91 differentially expressed genes were identified. The loss of PARP results in down-regulation of the expression of several genes involved in regulation of cell cycle progression or mitosis, DNA replication, or chromosomal processing or assembly. PARP deficiency also up-regulates genes that encode extracellular matrix or cytoskeletal proteins that are implicated in cancer initiation or progression or in normal or premature aging. These results provide insight into the mechanism by which PARP deficiency impairs mitotic function, thereby resulting in the genomic alterations and chromosomal abnormalities as well as in altered expression of genes that may contribute to genomic instability, cancer, and aging.
| S-EPMC17190 | biostudies-literature
Project description:Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli.
| S-EPMC2602769 | biostudies-literature
Project description: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/Ca(2+)/PKCα signaling axis. Mechanistically, H2O2-induced PAR formation correlated with Ca(2+)-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.
| S-EPMC5027479 | biostudies-literature
Project description:Poly(ADP-ribosyl)ation is a common post-translational modification that mediates a wide variety of cellular processes including DNA damage repair, chromatin regulation, transcription, and apoptosis. The difficulty associated with accessing poly(ADP-ribose) (PAR) in a homogeneous form has been an impediment to understanding the interactions of PAR with poly(ADP-ribose) glycohydrolase (PARG) and other binding proteins. Here we describe the chemical synthesis of the ADP-ribose dimer, and we use this compound to obtain the first human PARG substrate-enzyme cocrystal structure. Chemical synthesis of PAR is an attractive alternative to traditional enzymatic synthesis and fractionation, allowing access to products such as dimeric ADP-ribose, which has been detected but never isolated from natural sources. Additionally, we describe the synthesis of an alkynylated dimer and demonstrate that this compound can be used to synthesize PAR probes including biotin and fluorophore-labeled compounds. The fluorescently labeled ADP-ribose dimer was then utilized in a general fluorescence polarization-based PAR-protein binding assay. Finally, we use intermediates of our synthesis to access various PAR fragments, and evaluation of these compounds as substrates for PARG reveals the minimal features for substrate recognition and enzymatic cleavage. Homogeneous PAR oligomers and unnatural variants produced from chemical synthesis will allow for further detailed structural and biochemical studies on the interaction of PAR with its many protein binding partners.
| S-EPMC6089346 | biostudies-literature
Project description:Important cellular processes are regulated by poly(ADP-ribosyl)ation. This protein modification is catalyzed mainly by nuclear poly(ADP-ribose) polymerase (PARP) 1 in response to DNA damage. Cytosolic PARP isoforms have been described, whereas the presence of poly(ADP-ribose) (PAR) metabolism in mitochondria is controversial. PAR is degraded by poly(ADP-ribose) glycohydrolase (PARG). Recently, ADP-ribosylhydrolase 3 (ARH3) was also shown to catalyze PAR-degradation in vitro. PARG is encoded by a single, essential gene. One nuclear and three cytosolic isoforms result from alternative splicing. The presence and origin of a mitochondrial PARG is still unresolved. We establish here the genetic background of a human mitochondrial PARG isoform and investigate the molecular basis for mitochondrial poly(ADP-ribose) degradation. In common with a cytosolic 60-kDa human PARG isoform, the mitochondrial protein did not catalyze PAR degradation because of the absence of exon 5-encoded residues. In mice, we identified a transcript encoding an inactive cytosolic 52-kDa PARG lacking the mitochondrial targeting sequence and a substantial portion of exon 5. Thus, mammalian PARG genes encode isoforms that do not catalyze PAR degradation. On the other hand, embryonic fibroblasts from ARH3(-/-) mice lack most of the mitochondrial PAR degrading activity detected in wild-type cells, demonstrating a potential involvement of ARH3 in PAR metabolism.
| S-EPMC3351285 | biostudies-literature
Project description:Poly (ADP-ribosylation), known as PARylation, is a post-translational modification catalyzed by poly (ADP-ribose) polymerases (PARP) and primarily removed by the enzyme poly (ADP-ribose) glycohydrolase (PARG). While the aberrant removal of post-translation modifications including phosphorylation and methylation has known tumorigenic effects, deregulation of PARylation has not been widely studied. Increased hydrolysis of PARylation chains facilitates cancer growth through enhancing estrogen receptor (ER)-driven proliferation, but oncogenic transformation has not been linked to increased PARG expression. In this study, we find that elevated PARG levels are associated with a poor prognosis in breast cancers, especially in HER2-positive and triple-negative subtypes. Using both in vitro and in vivo models, we demonstrate that heightened expression of catalytically active PARG facilitates cell transformation and invasion of normal mammary epithelial cells. Catalytically inactive PARG mutants did not recapitulate these phenotypes. Consistent with clinical data showing elevated PARG predicts poor outcomes in HER2+ patients, we observed that PARG acts in synergy with HER2 to promote neoplastic growth of immortalized mammary cells. In contrast, PARG depletion significantly impairs the growth and metastasis of triple-negative breast tumors. Mechanistically, we find that PARG interacts with SMAD2/3 and significantly decreases their PARylation in non-transformed cells, leading to enhanced expression of SMAD target genes. Further linking SMAD-mediated transcription to the oncogenicity of PARG, we show that PARG-mediated anchorage-independent growth and invasion are dependent, at least in part, on SMAD expression. Overall, our study underscores the oncogenic impact of aberrant protein PARylation and highlights the therapeutic potential of PARG inhibition in breast cancer.
| S-EPMC6484711 | biostudies-literature
Project description:Zinc-finger antiviral protein (ZAP), also known as poly(ADP-ribose) polymerase 13 (PARP13), is an antiviral factor that selectively targets viral RNA for degradation. ZAP is active against both DNA and RNA viruses, including important human pathogens such as hepatitis B virus and type 1 human immunodeficiency virus (HIV-1). ZAP selectively binds CpG dinucleotides through its N-terminal RNA-binding domain, which consists of four zinc fingers. ZAP also contains a central region that consists of a fifth zinc finger and two WWE domains. Through structural and biochemical studies, we found that the fifth zinc finger and tandem WWEs of ZAP combine into a single integrated domain that binds to poly(ADP-ribose) (PAR), a cellular polynucleotide. PAR binding is mediated by the second WWE module of ZAP and likely involves specific recognition of an adenosine diphosphate-containing unit of PAR. Mutation of the PAR binding site in ZAP abrogates the interaction in vitro and diminishes ZAP activity against a CpG-rich HIV-1 reporter virus and murine leukemia virus. In cells, PAR facilitates formation of non-membranous sub-cellular compartments such as DNA repair foci, spindle poles and cytosolic RNA stress granules. Our results suggest that ZAP-mediated viral mRNA degradation is facilitated by PAR, and provides a biophysical rationale for the reported association of ZAP with RNA stress granules.
| S-EPMC8853533 | biostudies-literature
Project description:PPAR?-dependent gene expression during adipogenesis is facilitated by ADP-ribosyltransferase D-type 1 (ARTD1; PARP1)-catalyzed poly-ADP-ribose (PAR) formation. Adipogenesis is accompanied by a dynamic modulation of the chromatin landscape at PPAR? target genes by ligand-dependent co-factor exchange. However, how endogenous PPAR? ligands, which have a low affinity for the receptor and are present at low levels in the cell, can induce sufficient co-factor exchange is unknown. Moreover, the significance of PAR formation in PPAR?-regulated adipose tissue function is also unknown. Here, we show that inhibition of PAR formation in mice on a high-fat diet reduces weight gain and cell size of adipocytes, as well as PPAR? target gene expression in white adipose tissue. Mechanistically, topoisomerase II activity induces ARTD1 recruitment to PPAR? target genes, and ARTD1 automodification enhances ligand binding to PPAR?, thus promoting sufficient transcriptional co-factor exchange in adipocytes. Thus, ARTD1-mediated PAR formation during adipogenesis is necessary to adequately convey the low signal of endogenous PPAR? ligand to effective gene expression. These results uncover a new regulatory mechanism of ARTD1-induced ADP-ribosylation and highlight its importance for nuclear factor-regulated gene expression.
| S-EPMC4288160 | biostudies-literature
Project description:Macro domains constitute a protein module family found associated with specific histones and proteins involved in chromatin metabolism. In addition, a small number of animal RNA viruses, such as corona- and toroviruses, alphaviruses, and hepatitis E virus, encode macro domains for which, however, structural and functional information is extremely limited. Here, we characterized the macro domains from hepatitis E virus, Semliki Forest virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). The crystal structure of the SARS-CoV macro domain was determined at 1.8-Angstroms resolution in complex with ADP-ribose. Information derived from structural, mutational, and sequence analyses suggests a close phylogenetic and, most probably, functional relationship between viral and cellular macro domain homologs. The data revealed that viral macro domains have relatively poor ADP-ribose 1"-phosphohydrolase activities (which were previously proposed to be their biologically relevant function) but bind efficiently free and poly(ADP-ribose) polymerase 1-bound poly(ADP-ribose) in vitro. Collectively, these results suggest to further evaluate the role of viral macro domains in host response to viral infection.
| S-EPMC1563857 | biostudies-literature
Project description:BackgroundInitiation, amplitude, duration and termination of transforming growth factor ? (TGF?) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGF? signaling.MethodsA robust cell model of TGF? signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGF? stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.ResultsTGF? signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGF? target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGF?-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.ConclusionNuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGF? signaling.
| S-EPMC4136792 | biostudies-literature