Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death.
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ABSTRACT:
SUBMITTER: Blenn C
PROVIDER: S-EPMC1482814 | biostudies-other | 2006 Jun
REPOSITORIES: biostudies-other
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Project description:Benzo(a)pyrene (BaP) is a ubiquitously distributed environmental pollutant and known carcinogen, which can induce malignant transformation in rodent and human cells. Poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme that catalyzes the degradation of poly(ADP-ribose) (PAR), has been known to play an important role in regulating DNA damage repair and maintaining genomic stability. Although PARG has been shown to be a downstream effector of BaP, the role of PARG in BaP induced carcinogenesis remains unclear. In this study, we used the PARG-deficient human bronchial epithelial cell line (shPARG) as a model to examine how PARG contributed to the carcinogenesis induced by chronic BaP exposure under various concentrations (0, 10, 20 and 40 ?M). Our results showed that PARG silencing dramatically reduced DNA damages, chromosome abnormalities, and micronuclei formations in the PARG-deficient human bronchial epithelial cells compared to the control cells (16HBE cells). Meanwhile, the wound healing assay showed that PARG silencing significantly inhibited BaP-induced cell migration. Furthermore, silencing of PARG significantly reduced the volume and weight of tumors in Balb/c nude mice injected with BaP induced transformed human bronchial epithelial cells. This was the first study that reported evidences to support an oncogenic role of PARG in BaP induced carcinogenesis, which provided a new perspective for our understanding in BaP exposure induced cancer.
| S-EPMC4803271 | 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: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:Poly(ADP-ribose)polymerases (PARPs) are a family of NAD+ consuming enzymes that play a crucial role in many cellular processes, most clearly in maintaining genome integrity. Here, we present an extensive analysis of the alteration of mitochondrial morphology and the relationship to PARPs activity after oxidative stress using an in vitro model of human hepatic cells. The following outcomes were observed: reactive oxygen species (ROS) induced by oxidative treatment quickly stimulated PARPs activation, promoted changes in mitochondrial morphology associated with early mitochondrial fragmentation and energy dysfunction and finally triggered apoptotic cell death. Pharmacological treatment with specific PARP-1 (the major NAD+ consuming poly(ADP-ribose)polymerases) and PARP-1/PARP-2 inhibitors after the oxidant insult recovered normal mitochondrial morphology and, hence, increased the viability of human hepatic cells. As the PARP-1 and PARP-1/PARP-2 inhibitors achieved similar outcomes, we conclude that most of the PARPs effects were due to PARP-1 activation. NAD+ supplementation had similar effects to those of the PARPs inhibitors. Therefore, PARPs activation and the subsequent NAD+ depletion are crucial events in decreased cell survival (and increased apoptosis) in hepatic cells subjected to oxidative stress. These results suggest that the alterations in mitochondrial morphology and function seem to be related to NAD+ depletion, and show for the first time that PARPs inhibition abrogates mitochondrial fragmentation. In conclusion, the inhibition of PARPs may be a valuable therapeutic approach for treating liver diseases, by reducing the cell death associated with oxidative stress.
| S-EPMC5658148 | biostudies-literature
Project description:BackgroundThe formation of ADP-ribose polymers on target proteins by poly(ADP-ribose) polymerases serves a variety of cell signaling functions. In addition, extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1) is a dominant cause of cell death in ischemia-reperfusion, trauma, and other conditions. Poly(ADP-ribose) glycohydrolase (PARG) degrades the ADP-ribose polymers formed on acceptor proteins by PARP-1 and other PARP family members. PARG exists as multiple isoforms with differing subcellular localizations, but the functional significance of these isoforms is uncertain.Methods / principal findingsPrimary mouse astrocytes were treated with an antisense phosphorodiamidate morpholino oligonucleotide (PMO) targeted to exon 1 of full-length PARG to suppress expression of this nuclear-specific PARG isoform. The antisense-treated cells showed down-regulation of both nuclear PARG immunoreactivity and nuclear PARG enzymatic activity, without significant alteration in cytoplasmic PARG activity. When treated with the genotoxic agent MNNG to induced PARP-1 activation, the antisense-treated cells showed a delayed rate of nuclear PAR degradation, reduced nuclear condensation, and reduced cell death.Conclusions/significanceThese results support a preferentially nuclear localization for full-length PARG, and suggest a key role for this isoform in the PARP-1 cell death pathway.
| S-EPMC2655720 | biostudies-literature
Project description:Oxidative DNA damage to cells activates poly(ADP-ribose)polymerase-1 (PARP-1) and the poly(ADP-ribose) formed is rapidly degraded to ADP-ribose by poly(ADP-ribose)glycohydrolase (PARG). Here we show that PARP-1 and PARG control extracellular Ca(2+) fluxes through melastatin-like transient receptor potential 2 channels (TRPM2) in a cell death signaling pathway. TRPM2 activation accounts for essentially the entire Ca(2+) influx into the cytosol, activating caspases and causing the translocation of apoptosis inducing factor (AIF) from the inner mitochondrial membrane to the nucleus followed by cell death. Abrogation of PARP-1 or PARG function disrupts these signals and reduces cell death. ADP-ribose-loading of cells induces Ca(2+) fluxes in the absence of oxidative damage, suggesting that ADP-ribose is the key metabolite of the PARP-1/PARG system regulating TRPM2. We conclude that PARP-1/PARG control a cell death signal pathway that operates between five different cell compartments and communicates via three types of chemical messengers: a nucleotide, a cation, and proteins.
| S-EPMC3064896 | biostudies-literature
Project description:Coordinate regulation of PARP-1 and -2 and PARG is required for cellular responses to genotoxic stress. While PARP-1 and -2 are regulated by DNA breaks and covalent modifications, mechanisms of PARG regulation are poorly understood. We report here discovery of a PARG regulatory segment far removed linearly from residues involved in catalysis. Expression and analysis of human PARG segments identified a minimal catalytically active C-terminal PARG (hPARG59) containing a 16-residue N-terminal mitochondrial targeting sequence (MTS). Deletion analysis and site-directed mutagenesis revealed that the MTS, specifically hydrophobic residues L473 and L474, was required for PARG activity. This region of PARG was termed the "regulatory segment/MTS" (REG/MTS). The overall alpha-helical composition of hPARG59, determined by circular dichroism (CD), was unaffected by mutation of the REG/MTS leucine residues, suggesting that activity loss was not due to incorrect protein folding. REG/MTS was predicted to be in a loop conformation because the CD spectra of mutant Delta1-16 lacking the REG/MTS showed a higher alpha-helical content than hPARG59, indicating a secondary structure other than alpha-helix for this segment. Deletion of the REG/MTS from full-length hPARG111 also resulted in a complete loss of activity, indicating that all PARG isoforms are subject to regulation at this site. The presence of the REG/MTS raises the possibility that PARG activity is regulated by interactions of PARP-1 and -2 and other proteins at this site, raises interesting questions concerning mitochondrial PARG because MTS residues are often removed after transport, and offers a potentially novel site for drug targeting of PARG.
| S-EPMC2939713 | biostudies-literature
Project description:The poly(ADP-ribose) (PAR) post-translational modification is essential for diverse cellular functions, including regulation of transcription, response to DNA damage, and mitosis. Cellular PAR is predominantly synthesized by the enzyme poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a critical node in the DNA damage response pathway, and multiple potent PARP-1 inhibitors have been described, some of which show considerable promise in the clinic for the treatment of certain cancers. Cellular PAR is efficiently degraded by poly(ADP-ribose) glycohydrolase (PARG), an enzyme for which no potent, readily accessible, and specific inhibitors exist. Herein we report the discovery of small molecules that effectively inhibit PARG in vitro and in cellular lysates. These potent PARG inhibitors can be produced in two chemical steps from commercial starting materials and have complete specificity for PARG over the other known PAR glycohydrolase (ADP-ribosylhydrolase 3, ARH3) and over PARP-1 and thus will be useful tools for studying the biochemistry of PAR signaling.
| S-EPMC3306470 | 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:Protein poly(ADP-ribosyl)ation (PARylation) regulates a number of important cellular processes. Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for hydrolyzing the poly(ADP-ribose) (PAR) polymer in vivo. Here we report crystal structures of the mouse PARG (mPARG) catalytic domain, its complexes with ADP-ribose (ADPr) and a PARG inhibitor ADP-HPD, as well as four PARG catalytic residues mutants. With these structures and biochemical analysis of 20 mPARG mutants, we provide a structural basis for understanding how the PAR polymer is recognized and hydrolyzed by mPARG. The structures and activity complementation experiment also suggest how the N-terminal flexible peptide preceding the PARG catalytic domain may regulate the enzymatic activity of PARG. This study contributes to our understanding of PARG catalytic and regulatory mechanisms as well as the rational design of PARG inhibitors.
| S-EPMC3897571 | biostudies-literature