poly(ADP-ribose) Polymerase 1 Interaction Research Articles

PARP-1: a critical regulator in radioprotection and radiotherapy-mechanisms, challenges, and therapeutic opportunities.

Background: Since its discovery, poly (ADP-ribose) polymerase 1 (PARP-1) has been extensively studied due to its regulatory role in numerous biologically crucial pathways. PARP inhibitors have opened new therapeutic avenues for cancer patients and have gained approval as standalone treatments for certain types of cancer. With continued advancements in the research of PARP inhibitors, we can fully realize their potential as therapeutic targets for various diseases. Purpose: To assess the current understanding of PARP-1 mechanisms in radioprotection and radiotherapy based on the literature. Methods: We searched the PubMed database and summarized information on PARP inhibitors, the interaction of PARP-1 with DNA, and the relationships between PARP-1 and p53/ROS, NF-κB/DNA-PK, and caspase3/AIF, respectively. Results: The enzyme PARP-1 plays a crucial role in repairing DNA damage and modifying proteins. Cells exposed to radiation can experience DNA damage, such as single-, intra-, or inter-strand damage. This damage, associated with replication fork stagnation, triggers DNA repair mechanisms, including those involving PARP-1. The activity of PARP-1 increases 500-fold on DNA binding. Studies on PARP-1-knockdown mice have shown that the protein regulates the response to radiation. A lack of PARP-1 also increases the organism's sensitivity to radiation injury. PARP-1 has been found positively or negatively regulate the expression of specific genes through its modulation of key transcription factors and other molecules, including NF-κB, p53, Caspase 3, reactive oxygen species (ROS), and apoptosis-inducing factor (AIF). Conclusion: This review provides a comprehensive analysis of the physiological and pathological roles of PARP-1 and examines the impact of PARP-1 inhibitors under conditions of ionizing radiation exposure. The review also emphasizes the challenges and opportunities for developing PARP-1 inhibitors to improve the clinical outcomes of ionizing radiation damage.

The HPF1-dependent histone PARylation catalyzed by PARP2 is specifically stimulated by an incised AP site-containing BER DNA intermediate

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are DNA-dependent poly(ADP-ribose)transferases localized in nucleus. They have a significant homology in the C-terminal catalytic domain structure but differ in their N-terminal DNA-binding parts. The structural difference has an impact on the interaction of PARP1 and PARP2 with DNA and their DNA-dependent activation. Here, we compare the interaction of PARP1 and PARP2 with free 147 bp nucleosomal DNA and its nucleosome-associated variant (NCP) that contain in one strand a 1-nucleotide gap with 5'-dRP (imitating the intermediate of Base Excision Repair) or no specific damage. The affinity of PARP2 for the DNA strongly depends on the gap presence and to a lesser extent on the association with nucleosomes, while PARP1 interacts primarily with blunt ends of all DNAs and with a lower affinity with the single-strand break. The activities of PARP1 and PARP2 in the autoPARylation reaction and heteromodification of histones are distinctly stimulated by HPF1, depending on the gap presence in activating DNA. The most significant HPF1-induced stimulation of the histone modification in the presence of gapped NCP is a peculiar feature of PARP2. We propose a specific regulatory role of PARP2 in the process of DNA repair in the context of chromatin.

Cell-Penetrating Peptide TAT-HuR-HNS3 Suppresses Proinflammatory Gene Expression via Competitively Blocking Interaction of HuR with Its Partners.

Proinflammatory cytokines/chemokines are commonly regulated by RNA-binding proteins at posttranscriptional levels. Human Ag R (HuR)/embryonic lethal abnormal vision-like 1 (ELAVL1) is one of the well-characterized RNA-binding proteins that increases the stability of short-lived mRNAs, which encode proinflammatory mediators. HuR employs its nucleocytoplasmic shuttling sequence (HNS) domain, interacting with poly(ADP-ribose) polymerase 1 (PARP1), which accounts for the enhanced poly-ADP-ribosylation and cytoplasmic shuttling of HuR. Also by using its HNS domain, HuR undergoes dimerization/oligomerization, underlying the increased binding of HuR with proinflammatory cytokine/chemokine mRNAs and the disassociation of the miRNA-induced silencing complex from the targets. Therefore, competitively blocking the interactions of HuR with its partners may suppress proinflammatory mediator production. In this study, peptides derived from the sequence of the HuR-HNS domain were synthesized, and their effects on interfering HuR interacting with PARP1 and HuR itself were analyzed. Moreover, cell-penetrating TAT-HuR-HNS3 was delivered into human and mouse cells or administered into mouse lungs with or without exposure of TNF-α or LPS. mRNA levels of proinflammatory mediators as well as neutrophil infiltration were evaluated. We showed that TAT-HuR-HNS3 interrupts HuR-PARP1 interaction and therefore results in a lowered poly-ADP-ribosylation level and decreased cytoplasmic distribution of HuR. TAT-HuR-HNS3 also blocks HuR dimerization and promotes Argonaute 2-based miRNA-induced silencing complex binding to the targets. Moreover, TAT-HuR-HNS3 lowers mRNA stability of proinflammatory mediators in TNF-α-treated epithelial cells and macrophages, and it decreases TNF-α-induced inflammatory responses in lungs of experimental animals. Thus, TAT-HuR-HNS3 is a promising lead peptide for the development of inhibitors to treat inflammation-related diseases.

Aldehyde dehydrogenase 2 and PARP1 interaction modulates hepatic HDL biogenesis by LXRα-mediated ABCA1 expression.

HDL cholesterol (HDL-C) predicts risk of cardiovascular disease (CVD), but the factors regulating HDL are incompletely understood. Emerging data link CVD risk to decreased HDL-C in 8% of the world population and 40% of East Asians who carry an SNP of aldehyde dehydrogenase 2 (ALDH2) rs671, responsible for alcohol flushing syndrome; however, the underlying mechanisms remain unknown. We found significantly decreased HDL-C with increased hepatosteatosis in ALDH2-KO (AKO), ALDH2/LDLR–double KO (ALKO), and ALDH2 rs671–knock-in (KI) mice after consumption of a Western diet. Metabolomics identified ADP-ribose as the most significantly increased metabolites in the ALKO mouse liver. Moreover, ALDH2 interacted with poly(ADP-ribose) polymerase 1 (PARP1) and attenuated PARP1 nuclear translocation to downregulate poly(ADP-ribosyl)ation of liver X receptor α (LXRα), leading to an upregulation of ATP-binding cassette transporter A1 (ABCA1) and HDL biogenesis. Conversely, AKO or ALKO mice exhibited lower HDL-C with ABCA1 downregulation due to increased nuclear PARP1 and upregulation of LXRα poly(ADP-ribosyl)ation. Consistently, PARP1 inhibition rescued ALDH2 deficiency–induced fatty liver and elevated HDL-C in AKO mice. Interestingly, KI mouse or human liver tissues showed ABCA1 downregulation with increased nuclear PARP1 and LXRα poly(ADP-ribosyl)ation. Our study uncovered a key role of ALDH2 in HDL biogenesis through the LXRα/PARP1/ABCA1 axis, highlighting a potential therapeutic strategy in CVD.

PARP Traps Rescue the Pro-Inflammatory Response of Human Macrophages in the In Vitro Model of LPS-Induced Tolerance.

Secondary infections cause sepsis that lead to patient disability or death. Contact of macrophages with bacterial components (such as lipopolysaccharide—LPS) activates the intracellular signaling pathway downstream of Toll-like receptors (TLR), which initiate an immune proinflammatory response. However, the expression of nuclear factor-kappa B (NF-κB)-dependent proinflammatory cytokines significantly decreases after single high or multiple LPS stimulations. Knowing that poly(ADP-ribose) polymerase-1 (PARP1) serves as a cofactor of NF-κB, we aimed to verify a hypothesis of the possible contribution of PARP1 to the development of LPS-induced tolerance in human macrophages. Using TNF-α mRNA expression as a readout, we demonstrate that PARP1 interaction with the TNF-α promoter, controls macrophage immunoparalysis. We confirm that PARP1 is extruded from the gene promoter, whereas cell pretreatment with Olaparib maintains macrophage responsiveness to another LPS treatment. Furthermore, cell pretreatment with proteasome inhibitor MG132 completely abrogates the effect of Olaparib, suggesting that PARP1 acts with NF-κB in the same regulatory pathway, which controls pro-inflammatory cytokine transcription. Mechanistically, PARP1 trapping allows for the re-rebinding of p65 to the TNF-α promoter in LPS-stimulated cells. In conclusion, PARP traps prevent PARP1 extrusion from the TNF-α promoter upon macrophage stimulation, thereby maintaining chromatin responsiveness of TLR activation, allowing for the re-binding of p65 and TNF-α transcription.

Kaposi's sarcoma-associated herpesvirus processivity factor (PF-8) recruits cellular E3 ubiquitin ligase CHFR to promote PARP1 degradation and lytic replication.

Kaposi’s sarcoma–associated herpesvirus (KSHV), which belongs to the gammaherpesvirus subfamily, is associated with the pathogenesis of various tumors. Nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) catalyzes the polymerization of ADP-ribose units on target proteins. In KSHV-infected cells, PARP1 inhibits replication and transcription activator (RTA), a molecular switch that initiates lytic replication, through direct interaction. Thus, for efficient replication, KSHV has to overcome the molecular barrier in the form of PARP1. Previously, we have demonstrated that KSHV downregulates the expression of PARP1 through PF-8, a viral processivity factor. PF-8 induces ubiquitin–proteasome system–mediated degradation of PARP1 via direct physical association and enhances RTA transactivation activity. Here, we showed that dimerization domains of PF-8 are crucial not only for PARP1 interaction and degradation but also for enhancement of the RTA transactivation activity. PF-8 recruited CHFR for the PARP1 degradation. A knockdown of CHFR attenuated the PF-8–induced PARP1 degradation and enhancement of the RTA transactivation activity, leading to reduced KSHV lytic replication. These findings reveal a mechanism by which KSHV PF-8 recruits a cellular E3 ligase to curtail the inhibitory effect of PARP1 on KSHV lytic replication.

The poly(ADP-ribosyl)ation of BRD4 mediated by PARP1 promoted pathological cardiac hypertrophy

The bromodomain and extraterminal (BET) family member BRD4 is pivotal in the pathogenesis of cardiac hypertrophy. BRD4 induces hypertrophic gene expression by binding to the acetylated chromatin, facilitating the phosphorylation of RNA polymerases II (Pol II) and leading to transcription elongation. The present study identified a novel post-translational modification of BRD4: poly(ADP-ribosyl)ation (PARylation), that was mediated by poly(ADP-ribose)polymerase-1 (PARP1) in cardiac hypertrophy. BRD4 silencing or BET inhibitors JQ1 and MS417 prevented cardiac hypertrophic responses induced by isoproterenol (ISO), whereas overexpression of BRD4 promoted cardiac hypertrophy, confirming the critical role of BRD4 in pathological cardiac hypertrophy. PARP1 was activated in ISO-induced cardiac hypertrophy and facilitated the development of cardiac hypertrophy. BRD4 was involved in the prohypertrophic effect of PARP1, as implied by the observations that BRD4 inhibition or silencing reversed PARP1-induced hypertrophic responses, and that BRD4 overexpression suppressed the anti-hypertrophic effect of PARP1 inhibitors. Interactions of BRD4 and PARP1 were observed by co-immunoprecipitation and immunofluorescence. PARylation of BRD4 induced by PARP1 was investigated by PARylation assays. In response to hypertrophic stimuli like ISO, PARylation level of BRD4 was elevated, along with enhanced interactions between BRD4 and PARP1. By investigating the PARylation of truncation mutants of BRD4, the C-terminal domain (CTD) was identified as the PARylation modification sites of BRD4. PARylation of BRD4 facilitated its binding to the transcription start sites (TSS) of hypertrophic genes, resulting in enhanced phosphorylation of RNA Pol II and transcription activation of hypertrophic genes. The present findings suggest that strategies targeting inhibition of PARP1-BRD4 might have therapeutic potential for pathological cardiac hypertrophy.

Methylated-UHRF1 and PARP1 Interaction Is Critical for Homologous Recombination

A recent study suggested that methylation of ubiquitin-like with PHD and RING finger domain 1 (UHRF1) is regulated by SET7 and lysine-specific histone demethylase 1A (LSD1) and is essential for homologous recombination (HR). The study demonstrated that SET7-mediated methylation of UHRF1 promotes polyubiquitination of proliferating cell nuclear antigen (PCNA), inducing HR. However, studies on mediators that interact with and recruit UHRF1 to damaged lesions are needed to elucidate the mechanism of UHRF1 methylation-induced HR. Here, we identified that poly [ADP-ribose] polymerase 1 (PARP1) interacts with damage-induced methylated UHRF1 specifically and mediates UHRF1 to induce HR progression. Furthermore, cooperation of UHRF1-PARP1 is essential for cell viability, suggesting the importance of the interaction of UHRF1-PARP1 for damage tolerance in response to damage. Our data revealed that PARP1 mediates the HR mechanism, which is regulated by UHRF1 methylation. The data also indicated the significant role of PARP1 as a mediator of UHRF1 methylation-correlated HR pathway.

Identification of PARP-1, Histone H1 and SIRT-1 as New Regulators of Breast Cancer-Related Aromatase Promoter I.3/II.

Paracrine interactions between malignant estrogen receptor positive (ER+) breast cancer cells and breast adipose fibroblasts (BAFs) stimulate estrogen biosynthesis by aromatase in BAFs. In breast cancer, mainly the cAMP-responsive promoter I.3/II-region mediates excessive aromatase expression. A rare single nucleotide variant (SNV) in this promoter region, which caused 70% reduction in promoter activity, was utilized for the identification of novel regulators of aromatase expression. To this end, normal and mutant promoter activities were measured in luciferase reporter gene assays. DNA-binding proteins were captured by DNA-affinity and identified by mass spectrometry. The DNA binding of proteins was analyzed using electrophoretic mobility shift assays, immunoprecipitation-based in vitro binding assays and by chromatin immunoprecipitation in BAFs in vivo. Protein expression and parylation were analyzed by western blotting. Aromatase activities and RNA-expression were measured in BAFs. Functional consequences of poly (ADP-ribose) polymerase-1 (PARP-1) knock-out, rescue or overexpression, respectively, were analyzed in murine embryonic fibroblasts (MEFs) and the 3T3-L1 cell model. In summary, PARP-1 and histone H1 (H1) were identified as critical regulators of aromatase expression. PARP-1-binding to the SNV-region was crucial for aromatase promoter activation. PARP-1 parylated H1 and competed with H1 for DNA-binding, thereby inhibiting its gene silencing action. In MEFs (PARP-1 knock-out and wild-type) and BAFs, PARP-1-mediated induction of the aromatase promoter showed bi-phasic dose responses in overexpression and inhibitor experiments, respectively. The HDAC-inhibitors butyrate, panobinostat and selisistat enhanced promoter I.3/II-mediated gene expression dependent on PARP-1-activity. Forskolin stimulation of BAFs increased promoter I.3/II-occupancy by PARP-1, whereas SIRT-1 competed with PARP-1 for DNA binding but independently activated the promoter I.3/II. Consistently, the inhibition of both PARP-1 and SIRT-1 increased the NAD+/NADH-ratio in BAFs. This suggests that cellular NAD+/NADH ratios control the complex interactions of PARP-1, H1 and SIRT-1 and regulate the interplay of parylation and acetylation/de-acetylation events with low NAD+/NADH ratios (reverse Warburg effect), promoting PARP-1 activation and estrogen synthesis in BAFs. Therefore, PARP-1 inhibitors could be useful in the treatment of estrogen-dependent breast cancers.

PARP1 Hinders Histone H2B Occupancy at the NFATc1 Promoter to Restrain Osteoclast Differentiation.

Induction of nuclear factor of activated T cell cytoplasmic 1 (NFATc1) by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) is essential for macrophage differentiation into osteoclasts (OCs), but the underlying mechanisms remain unclear. The ability of poly(ADP-ribose) polymerase 1 (PARP1) to poly-ADP-ribosylate NFATc1 in T cells prompted us to investigate the PARP1 and NFATc1 interaction during osteoclastogenesis. However, extensive studies failed to directly link PARP1 to NFATc1. A combination of transcriptomics and proteomics studies was then used to identify PARP1 targets under these conditions. These unbiased approaches in conjunction with site-directed mutagenesis studies revealed that PARP1 inhibited NFATc1 expression and OC formation by ADP-ribosylating histone H2B at serine 7 and decreasing the occupancy of this histone variant at the NFATc1 promoter. The anti-osteoclastogenic function of PARP1 was confirmed in vivo in several mouse models of PARP1 loss-of-function or gain-of-function, including a novel model in which PARP1 was conditionally ablated in myeloid cells. Thus, PARP1 ADP-ribosylates H2B to negatively regulate NFATc1 expression and OC differentiation. © 2019 American Society for Bone and Mineral Research.

G‐Quadruplex Loop Length Regulates PARP‐1 Enzymatic Activation

Guanine‐rich sequences can interact by Hoogsteen‐hydrogen bonding to form planar quartets that stack into four‐stranded structures known as G‐Quadruplexes (G4DNA). G4DNA is non‐random, overrepresented at key regulatory regions of the cell such as promoters of proto‐oncogenes and telomeres. The overrepresentation in such sites suggests G4DNA serves a significant role in the cell. Poly(ADP‐ribose) polymerase 1 (PARP‐1) is an enzyme that mediates production of polymers of ADP‐ribose units (PAR) that serve as a signal for important cellular processes. PARP‐1 plays a role in the regulation of gene expression, transcription, chromatin remodeling, and DNA repair. Interestingly, PARP‐1 interacts with G4DNA. We show that PARP‐1 binds several G4DNA structures with tight affinity. We also confirm previous evidence revealing that PARP‐1 is enzymatically activated by c‐KIT G4DNA in a DNA structure‐specific manner. In addition, our activity assays reveal that while PARP‐1 binds various G4DNA structures only a subset promote PARP‐1‐mediated PARylation. We show that PARP‐1 enzymatic activation is regulated by G4DNA loop length where longer loops, at least >1 nucleotide, confer higher PARP‐1 activation while shorter loops reduce PARP‐1 activation. Our approach supports a PARP‐1 and G4DNA interaction but emphasizes PARP‐1's catalytic activity is G4DNA loop length‐dependent.Support or Funding InformationNational Institutes of Health Grant R35 GM122601 (to K.D.R.) UAMS Initiative for Maximizing Student Development Program (IMSD) Grant R25 GM083247This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Poly(ADP-ribosyl)ation by PARP1: reaction mechanism and regulatory proteins.

Poly(ADP-ribosyl)ation (PARylation) is posttranslational modification of proteins by linear or branched chains of ADP-ribose units, originating from NAD+. The central enzyme for PAR production in cells and the main target of poly(ADP-ribosyl)ation during DNA damage is poly(ADP-ribose) polymerase 1 (PARP1). PARP1 ability to function as a catalytic and acceptor protein simultaneously made a considerable contribution to accumulation of contradictory data. This topic is directly related to other questions, such as the stoichiometry of PARP1 molecules in auto-modification reaction, direction of the chain growth during PAR elongation and functional coupling of PARP1 with PARylation targets. Besides DNA damage necessary for the folding of catalytically active PARP1, other mechanisms appear to be required for the relevant intensity and specificity of PARylation reaction. Indeed, in recent years, PARP research has been enriched by the discovery of novel PARP1 interaction partners modulating its enzymatic activity. Understanding the details of PARP1 catalytic mechanism and its regulation is especially important in light of PARP-targeted therapy and may significantly aid to PARP inhibitors drug design. In this review we summarize old and up-to-date literature to clarify several points concerning PARylation mechanism and discuss different ways for regulation of PAR synthesis by accessory proteins reported thus far.

Abstract 68: Functional interactions of HPV and PARP-1 in head and neck cancer

Abstract Though the overall incidence of head and neck cancer has been declining over the last several decades, there has been a concomitant and worrisome rise in human-papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC). HPV infection is a necessary but not sufficient condition for cancer development. Unlike in cervical cancer, where several co-factors including smoking and co-infection with other agents are well established, how HPV drives malignant transformation in head and neck cancer and which cellular factors are required are not well characterized. Recent evidence from our group has shown that poly (ADP-ribose) polymerase-1 (PARP-1) is overexpressed and overactive in HPV-positive (HPV(+)), but not HPV-negative (HPV(-)), oropharyngeal squamous cell carcinoma (OPSCC). The significance of PARP-1 overexpression and hyperactivation in cancer is only beginning to emerge. High levels of PARP-1 are found in a fraction of hepatocellular and breast cancers and correlate with poor prognosis. Recently, PARP-1 was implicated in prostate cancer progression. This study explores the role of PARP-1 in HPV infection and HNSCC carcinogenesis. Biochemical methods were used to determine PARP activity in vitro, in head and neck tumors, and during infection with HPV16 pseudovirus (PsV) or overexpression of HPV16 proteins in epithelial cells. The response to PARP inhibition was evaluated with clonogenic survival assays. HPV L1 mRNA in HNSCC was detected using quantitative real-time PCR and HPV L1 protein levels were determined by immunohistochemistry. Here, we show that the HPV16 major capsid protein L1 directly binds PARP-1 and activates poly(ADP-ribosyl)ation in the absence of DNA damage in vitro and in HPV(-) cells. Using PARP-1 knockout cells, we found that PARP-1 expression and enzymatic activity supports initial HPV infection. Furthermore, HPV(-) cells were sensitized to PARP inhibition both after L1 overexpression and after infection with HPV16 PsV particles. In conclusion, we report functional interactions between the viral capsid and cellular protein, signifying a vital role played by PARP-1 in HPV infection and carcinogenesis. Importantly, our data provide a mechanistic explanation for pharmacological PARP inhibition as an intriguing therapeutic opportunity for HPV(+) head and neck cancers. Citation Format: Cyril S. Gary, Michael Hajek, Asel Biktasova, Andrew Sewell, Yarbrough G. Wendell, Natalia Issaeva. Functional interactions of HPV and PARP-1 in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 68.

Melatonin regulates PARP1 to control the senescence-associated secretory phenotype (SASP) in human fetal lung fibroblast cells.

Cellular senescence is an important tumor-suppressive mechanism. However, acquisition of a senescence-associated secretory phenotype (SASP) in senescent cells has deleterious effects on the tissue microenvironment and, paradoxically, promotes tumor progression. In a drug screen, we identified melatonin as a novel SASP suppressor in human cells. Strikingly, melatonin blunts global SASP gene expression upon oncogene-induced senescence (OIS). Moreover, poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, was identified as a new melatonin-dependent regulator of SASP gene induction upon OIS. Here, we report two different but potentially coherent epigenetic strategies for melatonin regulation of SASP. The interaction between the telomeric repeat-containing RNA (TERRA) and PARP-1 stimulates the SASP, which was attenuated by 67.9% (illustrated by the case of IL8) by treatment with melatonin. Through binding to macroH2A1.1, PARP-1 recruits CREB-binding protein (CBP) to mediate acetylation of H2BK120, which positively regulates the expression of target SASP genes, and this process is interrupted by melatonin. Consequently, the findings provide novel insight into melatonin's epigenetic role via modulating PARP-1 in suppression of SASP gene expression in OIS-induced senescent cells. Our studies identify melatonin as a novel anti-SASP molecule, define PARP-1 as a new target by which melatonin regulates SASP, and establish a new epigenetic paradigm for a pharmacological mechanism by which melatonin interrupts PARP-1 interaction with the telomeric long noncoding RNA(lncRNA) or chromatin.

PARP1 promotes gene expression at the post-transcriptional level by modulating the RNA-binding protein HuR

Poly(ADP-ribosyl)ation (PARylation) is mainly catalysed by poly-ADP-ribose polymerase 1 (PARP1), whose role in gene transcription modulation has been well established. Here we show that, in response to LPS exposure, PARP1 interacts with the adenylateuridylate-rich element-binding protein embryonic lethal abnormal vision-like 1 (Elavl1)/human antigen R (HuR), resulting in its PARylation, primarily at site D226. PARP inhibition and the D226 mutation impair HuR’s PARylation, nucleocytoplasmic shuttling and mRNA binding. Increases in mRNA level or stability of pro-inflammatory cytokines/chemokines are abolished by PARP1 ablation or inhibition, or blocked in D226A HuR-expressing cells. The present study demonstrates a mechanism to regulate gene expression at the post-transcriptional level, and suggests that blocking the interaction of PARP1 with HuR could be a strategy to treat inflammation-related diseases that involve increased mRNA stability.

PARP-1 Interaction with and Activation by Histones and Nucleosomes.

Poly(ADP-ribose) Polymerase 1 (PARP-1) is an abundant chromatin associated protein, typical for most eukaryotic nuclei. The localization of PARP-1 in chromatin and its enzymatic activation involves multiple interactions of PARP-1 with nucleosomal histones, other proteins, and DNA. We report a set of methods designed to reconstitute PARP-1 regulation in vitro. These methods involve the expression of PARP-1 and PARP-1-regulating proteins using bacterial and eukaryotic systems, purification of these proteins using chromatography, testing of individual interactions in vitro, assembly of active complexes, and reconstitution of PARP-1 regulating reactions in vitro.

The poly(ADP-ribosyl)ation of FoxO3 mediated by PARP1 participates in isoproterenol-induced cardiac hypertrophy

The Forkhead box-containing protein, O subfamily 3 (FoxO3) transcription factor negatively regulates myocardial hypertrophy, and its transcriptional activity is finely conditioned by diverse posttranslational modifications, such as phosphorylation, acetylation, ubiquitination, methylation and glycosylation. Here, we introduce a novel modification of the FoxO3 protein in cardiomyocytes: poly(ADP-ribosyl)ation (PARylation) mediated by poly(ADP-ribose) polymerase-1 (PARP1). This process catalyzes the NAD+-dependent synthesis of polymers of ADP-ribose (PAR) and their subsequent attachment to target proteins by PARPs. Primary-cultured neonatal rat cardiomyocytes were incubated with isoproterenol (ISO) to induce hypertrophy, or were infected with recombinant adenovirus vectors harboring PARP1 cDNA (Ad-PARP1). Sprague-Dawley (SD) rats were treated with ISO to induce cardiac hypertrophy, or were injected with Ad-PARP1 into the anterior and posterior left ventricular walls. Cardiomyocyte surface area, the mRNA expression of hypertrophic biomarkers, echocardiography, morphometry of the hearts were measured. The PARP1 activity was tested by cellular PAR levels. Interactions of PARP1 and FoxO3 were investigated by co-immunoprecipitation and immunofluorescence technique. PARylation of FoxO3 mediated by PARP1 facilitated its phosphorylation at the T32, S252 and S314 sites, triggered its nucleus export and suppressed its transcriptional activity and target genes expression, ultimately inducing cardiac hypertrophy. Additionally, PARP1 silencing or specific inhibition by 3-Aminobenzamide (3AB) and veliparib (ABT-888) alleviated the inhibition of FoxO3 activity by ISO, thus suppressing ISO-induced cardiac hypertrophy. Our data provide the first evidence that PARP1 exacerbates cardiac hypertrophy by PARylation of FoxO3.

Abstract 2754: Antibody-based tools for in vitro and live cell analysis of endogenous PARP1, an essential human DNA repair enzyme

Abstract DNA damage caused by normal cell activity or exogenous genotoxic agents is a constant threat to the genome. The nuclear enzyme Poly(ADP-ribose) polymerase 1 (PARP1) is rapidly activated by DNA lesions such as single-strand breaks and signals their presence by attaching ADP-ribose units to chromatin-associated proteins. To improve our understanding of PARP1 within a disease context, there is an ongoing need to develop novel PARP1 detection systems. Here we describe the development of a VHH directed against human PARP1. The variable domain of single chain antibodies (VHH or nanobody) is a versatile research tool for a variety of applications. The high binding affinity of VHHs, their small size (15 kDa) and their robust expression in various cellular systems make them preferable to conventional antibodies. Moreover, VHH domains can be selected to recognize and bind their target structures within living cells. This highly specific PARP1 VHH was selected by phage display using a VHH library from an immunized alpaca. We characterized its affinity, selectivity and activity both in vitro and in live cells. To validate the in vitro interactions of this PARP1-specific VHH, we developed a PARP1 immunoprecipitation reagent by conjugating the PARP1-specific VHH to an immobilizing matrix (termed PARP1-Trap). This PARP1-Trap was shown to bind with high specificity to human PARP1 and not to other members of the PARP family. Importantly, the binding of the PARP1-Trap to PARP1 leaves the enzymatic activity of PARP1 unaffected. The epitope of the PARP1-Trap was localized to the N-terminal domain of PARP1 and consists of the three-dimensional motif of zinc fingers 2 and 3 together. To determine whether the PARP1 VHH recognizes its target structure also in a cellular environment, the VHH was genetically fused to a fluorescent protein and expressed in living cells (termed Chromobody). The interaction of the PARP1 Chromobody with PARP1 was visualized using a protein-protein interaction assay called fluorescent two-hybrid (F2H). The F2H principle is based on a tethering strategy: a GFP-tagged protein (here GFP-PARP1) is enriched at a protein interaction platform engineered into F2H-BHK cells and serves as bait, whereas the RFP-tagged PARP1 VHH serves as a prey. Using the F2H assay, we could show that the PARP1 Chromobody is functional within living cells and specifically recognizes its antigen in a cellular environment. Moreover, by monitoring the PARP1 Chromobody signal after microirradiation, we were for the first time able to follow the recruitment of endogenous PARP1 to sites of DNA damage in living cells. In summary, we developed a novel PARP1 VHH for both biochemical and live cell analysis of human PARP1. We anticipate that PARP1-VHH based reagents will provide new insights into the PARP1 enzyme. For example, the use of the PARP1-Trap coupled with mass spectrometry analysis may lead to the identification of hitherto unknown PARP1 interaction partners. Citation Format: Andrea Buchfellner, Larisa Yurlova, Stephanie Dennison, Benjamin Ruf, Ulrich Rothbauer, Tina Romer. Antibody-based tools for in vitro and live cell analysis of endogenous PARP1, an essential human DNA repair enzyme. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2754.

Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro

The existing methodologies for studying robust responses of poly (ADP-ribose) polymerase-1 (PARP-1) to DNA damage with strand breaks are often not suitable for examining its subtle responses to altered DNA without strand breaks, such as UV-damaged DNA. Here we describe two novel assays with which we characterized the interaction of PARP-1 with UV-damaged DNA in vivo and in vitro. Using an in situ fractionation technique to selectively remove free PARP-1 while retaining the DNA-bound PARP-1, we demonstrate a direct recruitment of the endogenous or exogenous PARP-1 to the UV-lesion site in vivo after local irradiation. In addition, using the model oligonucleotides with single UV lesion surrounded by multiple restriction enzyme sites, we demonstrate in vitro that DDB2 and PARP-1 can simultaneously bind to UV-damaged DNA and that PARP-1 casts a bilateral asymmetric footprint from −12 to +9 nucleotides on either side of the UV-lesion. These techniques will permit characterization of different roles of PARP-1 in the repair of UV-damaged DNA and also allow the study of normal housekeeping roles of PARP-1 with undamaged DNA.

P88: Multiple mechanisms of DNA minor groove binding ligand epigenetic effects

Purpose DNA minor groove is the main target of small molecules, which noncovalently and to a certain extent site-specifically bind to appropriate nucleotide sequences. Study of these substances can give rise to understanding the mechanistic relationship between sites of interaction and activity of appropriate enzymes with “houskeeping” function including helicases, topoisomerases, methyltransferases, demethylases and DNA/RNA-polymerases. Results We revealed for the first time that AT-specific minor groove binding ligands (MGBLs), in particular bisbenzimidazoles (Hoechst33258 and its derivatives), widely used in molecular and cell biology for DNA-staining, induce loss of heterozygosity at high frequency while point mutations and chromosome deletions at insignificant levels. Moreover, we demonstrated that the agents realized their genotoxic blastomogenic effects via homologous recombination mechanism exclusively. Lately the same mechanism of genotoxicity has been shown for MGBL carbazole derivative Curaxin, which is toxic for a broad range of tumor cell lines in vitro and inhibit tumor growth in different mouse models of cancer in vivo. Moreover, powerful antitumor activity has been demonstrated for Trabectedin, which binds to the DNA’s minor groove and alkylate guanine residues. All this provided a framework for wide-ranging investigation of cell response to MGBLs exposure, molecular mechanisms of their recombinogenic as also their anticancer activity. A special interest is paid to epigenetic mechanisms of MGBs action. Our study aimed to examine the epigenetic effects of recombinogenic (Hoechst33342, Hoechst33258) and non-recombinogenic (DAPI, Diminazene, Pentamidine and Netropsin) MGBLs. After we unmasked MGBLs’ recombinogenic activity, we hypothesized that their molecular mechanism of indirect DNA damage involves poly(ADP-ribose)polymerase-1 (PARP-1) activation. Surprisingly, we found that all AT-specific MGBLs preventing PARP-1 interaction with DNA inhibit its activation, and hence, the DNA-dependent pathway of PARP-1 activation function. These inhibitors effectively block PARP-1 activity in vivo, as it was demonstrated in a Drosophila experimental system and in human breast cancer-derived BT474 cell line. Further epigenetic effects of these indirect genotoxic carcinogens were analyzed using HeLa cell population with epigenetically suppressed GFP-reporter gene as a model. All compounds had strong GFP- reactivation effect. The obtained results confirm scarce data of previous publications on the ability of DNA minor groove ligands to influence gene transcription process. Statistically significant results of changes of DNA methylation level were detected under 5-azaC and Hoechst 33258 treatment, but it was absent after Hoechst 33342 treatment. For the rest compounds significant loss of promoter region methylation was not observed. The common epigenetic marks of transcription include histone H3 trimethylation in lysine 4 (H3K4me3) and histone H3 and H4 acetylation (acH3/acH4) on promoter regions of genes. We showed that TSA, Hoechst 33342 and DAPI treatment of HeLa-TI cells lead to increased level of histone H3 trimethylation of lysines 4 (H3K4me3), but for all that the level of histone H4 acetylation remains without significant changes. On the contrary, histone H3 acetylation level was stably increased at all samples. The modifications are typical for silent genes and decrease of their amount suggests the transcriptional reactivation. Loss of H4K20me3 mark in comparison with untreated control was demonstrated for MGBLs. Conclusion Taking together, findings of the study will lay the fundamental groundwork for the development of novel anticancer strategies and new chemotherapy effects of small molecules. The mechanism of PARP-1 inhibition by MGBLs and its epigenetic influence on silent genes, via DNA methylation and histone modifications make reasonable further study of these compounds in three prospective directions: (1) as self-acting cytotoxic agents; (2) as a component of combined chemotherapy targeting DNA repair by PARP-1, thereby facilitating DNA damage caused by other anticancer drugs; (3) as an agents reactivating epigenetically repressed genes which silencing occurs during the earliest stages of neoplasia and accumulates with progression toward malignancy.