Regulation Of poly(ADP-ribose) Polymerase-1 Research Articles

Minocycline Blocks Asthma-associated Inflammation in Part by Interfering with the T Cell Receptor-Nuclear Factor κB-GATA-3-IL-4 Axis without a Prominent Effect on Poly(ADP-ribose) Polymerase

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

BZLF1 Expression of EBV is correlated with PARP1 Regulation on Nasopharyngeal Carcinoma Tissues

Nasopharyngeal carcinomas (NPC) is a cancer that arises in the epithelial tissue that covers the inside of the nasopharyngeal mucosa and nasopharynx. Infected Epstein Barr Virus (EBV) cell in a latent infection associated with the expression of nine latent proteins. Latent Membrane Protein 1 (LMP1) is one of latent proteins, and mayor EBV oncoprotein, with functions including virus growth, and to activate BamHI-Z Leftward Reading Frame 1 (BZLF1)-EBV, which can inhibit p53 to induce apoptotic resistance, metastasis, and immune modulation. The body will respond to the expansion of EBV infection with activation of Poly(ADP-ribose)Polymerase-1 (PARP1). The objective of study is to observe the expression of BZLF1 and determine PARP1 regulation in nasopharyngeal tissues. NPC-T2, NPC-T3 and polyp tissues slides are from Ulin Hospital, Banjarmasin. To characterize the necrotic cells such as pyknosis, karyorrhexsis, and karyolysis, histological slides were stained by HE that the necrotic cells measured by using a BX-53 microscope (Olympus) with CellSens Standard software. Tissues slides were stained by using immunofluorohistochemistry with EBV-BZLF1 antibody-Mouse anti-EBV monoclonal antibody against Goat anti-mouse IgG-FITC and anti-PARP1 antibody (MC-10) against Goat anti-mouse IgG labeled Rhodamin. The expression intensities were measured by Confocal Laser Scanning Microscope (Olympus). The percentage number of necrotic cells and BZLF1 and PARP1 expression intensity were analyzed using SPSS 16.0 by one-way ANOVA test with α = 0.05, beside that we use correlate and regression analyze. The research showed that the amount of karryorhexis higher than pyknosis and karyolysis in both tissues. BZLF1 expression 1.79 INT/sel (in polyp), 2.76 INT/sel (NPC Type 2) and 4.36 INT/sel (NPC Type 3), PARP1 expression 2.25 INT/sel (in polyp), 3.31 INT/sel (NPC Type 2), dan 5.93 INT/sel (NPC Type 3).The high of intensity of expression BZLF1 induced the increasing of PARP1 expression, but not correlated with percentage of necrotic cell. Interestingly, in all tissues (NPC-T1, NPC-T2 and polyp) is increased number of karyolysis cells properly, but not in pyknosis and karryorrhexis cells. The conclusion of this study indicated that the BZLF1 induce PARP1 to repair DNA damage against EBV infection.Keywords: BZLF1, CLSM, EBV, nasopharyngeal carcinoma, PARP1.

The 3'-5' Exonuclease, TREX1, Interacts with Poly(ADP-ribose) Polymerase-1 (PARP1) in Response to DNA Damage

AbstractAbstract 1046 Background:Recognition of nucleic acids plays a key role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). The major 3’-5’ DNA exonuclease, TREX1, degrades single- and double-stranded DNA to minimize potential immune activation by persistent self or retroelement DNA. Mutations in the human TREX1gene have been linked to four diseases: SLE; Aicardi-Goutières syndrome (AGS); retinal vasculopathy and cerebral leukodystrophy (RVCL); and familial chilblain lupus (CBL). SLE, AGS and CBL have overlapping features characterized by expression of interferon-alpha and autoantibody production, but are distinct clinical conditions. RVCL is a non-inflammatory endotheliopathy. Methods:As part of our efforts to determine the molecular mechanisms underlying TREX1 involvement in the pathogenesis of these diseases, we identified a novel TREX1-binding partner protein by Mass Spectrometry and Co-Immunoprecipitation (Co-IP) assays. We examined the interaction domains and the effects of mutations in TREX1 associated with these diseases. Results:We found that TREX1 interacted with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear DNA repair enzyme involved in the DNA damage response. The interaction domain of PARP1 was mapped to the amino-terminal zinc finger domains. Subcellular localization analysis showed that TREX1 normally localized to the cytoplasm and cytomembranes, but translocated to the nucleus (with some bound to chromatin) in response to sublethal DNA damage. Co-IP assays revealed that the interaction was increased following DNA damage in concert with the post-translational PARlation of PARP1. Functional studies showed that TREX1 induced the PARlation of PARP1 in a TREX1 dose-dependent manner. Furthermore, siRNA knockdown of TREX1 promoted PARP1 cleavage and loss of PARP1 enzymatic activity, resulting in the induction of apoptosis. We further analyzed the effects of TREX1-PARP1 interaction in relation to the pathogenesis of AGS or RVCL. Co-IP assays revealed that the RVCL-related TREX1-mutant protein, V235fs, bound to PARP1 as well as wild-type TREX1, whereas the AGS-related R114H mutant protein did not bind PARP1. Conclusion:These findings demonstrate that TREX1 contributes to the regulation of PARP1 in the DNA damage response and that mutation-induced alterations in the function of TREX1 may be a factor in the development or progression of these autoimmune diseases by affecting PARP1 activity. Disclosures:No relevant conflicts of interest to declare.

Vascular endothelial growth factor confers endothelial resistance to apoptosis through poly(ADP‐ribose) polymerase

Vascular endothelial growth factor (VEGF) inhibits the endothelial apoptosis that is induced by caspases during vascular remodeling; however, the underlying mechanisms have not been completely elucidated. We hypothesized that VEGF upregulates poly(ADP-ribose) polymerase-1 (PARP) as a caspase mediator, and sought to investigate the link between apoptosis inhibition by VEGF and PARP regulation in the human vasculature. Human endothelial cells (primary cells, macrovascular/microvascular lines) were incubated with 100 pg mL(-1) to 1 μg mL(-1) VEGF-A(165) in the absence or presence of PARP small interfering RNA (siRNA). Apoptosis induced by integrin inhibition was measured by flow cytometry, trypan blue exclusion, and nuclear morphology. PARP expression and activity were determined by real-time RT-PCR, Western blot, and ribosylation assay. VEGF receptors and signal transduction were analyzed by inhibitor experiments, enzyme assays, western blot, and immunofluorescence microscopy. Immunohistochemistry was applied to a vascular culture model and to 24 atherosclerotic and 10 normal human arteries. VEGF-A(165) induced resistance to apoptosis caused by caspase activation in endothelial cells in a time-dependent manner. VEGF, but not fibroblast growth factor-2 or transforming growth factor-β, time-dependently and dose-dependently induced PARP expression and activity, involving VEGF receptor-2 colocalized with neuropilin-1 as well as the signal transduction molecules c-Jun N-terminal kinase and Akt. The antiapoptotic effect of VEGF was abrogated by PARP siRNA. The relationship between local VEGF influence and endothelial PARP expression was confirmed in human arteries and the vascular culture model. VEGF exerts its antiapoptotic effect on the endothelium through the regulation of PARP expression. PARP has been attributed an ambiguous role in apoptosis; here, we show that PARP promotes vascular endothelial integrity in VEGF-associated processes.

Crystal Structures of Poly(ADP-ribose) Polymerase-1 (PARP-1) Zinc Fingers Bound to DNA: STRUCTURAL AND FUNCTIONAL INSIGHTS INTO DNA-DEPENDENT PARP-1 ACTIVITY

Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNA interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.

217 Testing of a poly(ADP-ribose) polymerase (PARP) inhibitor on human BRCA2 heterozygous cell lines

Poly (ADP-ribose) polymerase 1 (PARP1) is an ADP- ribosylation enzyme and plays important roles in a variety of cellular processes, including DNA damage response and tumor development. However, the post-transcriptional regulation of PARP1 remains largely unknown. In this study, we identified that the mRNA of PARP1 is associated with nuclear factor 90 (NF90) by RNA immunoprecipitation plus sequencing (RIP-seq) assay. The mRNA and protein levels of PARP1 are dramatically decreased in NF90-depleted cells, and NF90 stabilizes PARP1's mRNA through its 3′UTR. Moreover, the expression levels of PARP1 and NF90 are positively correlated in hepatocellular carcinoma (HCC). Finally, we demonstrated that NF90-depleted cells are sensitive to PARP inhibitor Olaparib (AZD2281) and DNA damage agents. Taken together, these results suggest that NF90 regulates PARP1 mRNA stability in hepatocellular carcinoma cells, and NF90 is a potential target to inhibit PARP1 activity.

Proteomic Investigation of Phosphorylation Sites in Poly(ADP-ribose) Polymerase-1 and Poly(ADP-ribose) Glycohydrolase

Phosphorylation is a very common post-translational modification event known to modulate a wide range of biological responses. Beyond the regulation of protein activity, the interrelation of phosphorylation with other post-translational mechanisms is responsible for the control of diverse signaling pathways. Several observations suggest that phosphorylation of poly(ADP-ribose) polymerase-1 (PARP-1) regulates its activity. There is also accumulating evidence to suggest the establishment of phosphorylation-dependent assembly of PARP-1-associated multiprotein complexes. Although it is relatively straightforward to demonstrate phosphorylation of a defined target, identification of the actual amino acids involved still represents a technical challenge for many laboratories. With the use of a combination of bioinformatics-based predictions tools for generic and kinase-specific phosphorylation sites, in vitro phosphorylation assays and mass spectrometry analysis, we investigated the phosphorylation profile of PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), two major enzymes responsible for poly(ADP-ribose) turnover. Mass spectrometry analysis revealed the phosphorylation of several serine/threonine residues within important regulatory domains and motifs of both enzymes. With the use of in vivo microirradiation-induced DNA damage, we show that altered phosphorylation at specific sites can modify the dynamics of assembly and disassembly of PARP-1 at sites of DNA damage. By documenting and annotating a collection of known and newly identified phosphorylation sites, this targeted proteomics study significantly advances our understanding of the roles of phosphorylation in the regulation of PARP-1 and PARG.

Reciprocal regulation of iNOS and PARP-1 during allergen-induced eosinophilia

Inducible nitric oxide synthase (iNOS) inhibition was recently shown to exert no effect on allergen challenge in human asthma, raising serious concerns about the role of the protein in the disease. The present study investigated the role of iNOS in ovalbumin-induced eosinophilia from the perspective of its relationship with poly(ADP-ribose) polymerase-1 (PARP-1) and oxidative DNA damage. A mouse model of ovalbumin-induced eosinophilia was used to conduct the studies. iNOS-associated protein nitration and tissue damage were partially responsible for allergen-induced eosinophilia. iNOS expression was required for oxidative DNA damage and PARP-1 activation upon allergen challenge. PARP-1 was required for iNOS expression and protein nitration, and this requirement was connected to nuclear factor-kappaB. PARP-1 was an important substrate for iNOS-associated by-products after ovalbumin-challenge. PARP-1 nitration blocked its poly(ADP-ribosyl)ation activity. Interleukin-5 re-establishment in ovalbumin-exposed PARP-1(-/-) mice reversed eosinophilia and partial mucus production without a reversal of iNOS expression, concomitant protein nitration or associated DNA damage. The present results demonstrate a reciprocal relationship between inducible nitric oxide synthase and poly(ADP-ribose) polymerase-1 and suggest that expression of inducible nitric oxide synthase may be dispensable for eosinophilia after interleukin-5 production. Inducible nitric oxide synthase may be required for oxidative DNA damage and full manifestation of mucus production. Such dispensability may explain, in part, the reported ineffectiveness of inducible nitric oxide synthase inhibition in preventing allergen-induced inflammation in humans.