Poly ADP-ribosyl Polymerase-1 Research Articles

Ceramide induces macrophage migration inhibitory factor -mediated parthanatos in mouse neurons by increasing ROS levels

Ceramides, the key component of sphingolipid metabolism and second messengers, have been associated with neurodegenerative diseases progression and pathology, and can induce neuronal apoptosis and necrosis, but the effect of ceramide on parthanatos has not been fully elucidated. In this study, we investigated the ceramide-mediated parthanatos pathway and the role of macrophage inhibitory factor (MIF) in parthanatos. We found that ceramide significantly diminished the viability and induced the death of primary cortical neurons. These effects were not prevented by treatment with the pan-caspase inhibitor Z-VAD-FMK treatment; in contrast, treatment with the poly (ADP ribosyl) polymerase-1 (PARP-1) inhibitor ABT-888 prevented these ceramide-mediated effects. Specifically, ceramide induced PARP-1 overactivation, increased PAR polymer levels, facilitated apoptosis-inducing factor (AIF) and MIF nuclear translocation and induced DNA damage. Knockdown of MIF with an adenovirus carrying a MIF short hairpin RNA (shRNA) inhibited ceramide-induced DNA damage and neuronal death, but nuclear translocation of AIF was unaffected. Furthermore, ceramide increased reactive oxygen species (ROS) levels, and N-acetyl cysteine (NAC) significantly inhibited PAR production and neuronal death. These findings suggested that ceramide induced neuronal parthanatos by increasing ROS levels and that MIF might be downstream of AIF in the ceramide-mediated parthanatos pathway. In conclusion, our results suggest that knocking down MIF expression may be a potential therapeutic strategy for nervous system diseases.

Zinc finger protein ZNF384 is an adaptor of Ku to DNA during classical non-homologous end-joining

DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a ‘Ku-adaptor’ that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance.

PARP1-mediated PARylation activity is essential for oligodendroglial differentiation and CNS myelination.

SUMMARYThe function of poly(ADP-ribosyl) polymerase 1 (PARP1) in myelination and remyelination of the central nervous system (CNS) remains enigmatic. Here, we report that PARP1 is an intrinsic driver for oligodendroglial development and myelination. Genetic PARP1 depletion impairs the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes and impedes CNS myelination. Mechanistically, PARP1-mediated PARylation activity is not only necessary but also sufficient for OPC differentiation. At the molecular level, we identify the RNA-binding protein Myef2 as a PARylated target, which controls OPC differentiation through the PARylation-modulated derepression of myelin protein expression. Furthermore, PARP1’s enzymatic activity is necessary for oligodendrocyte and myelin regeneration after demyelination. Together, our findings suggest that PARP1-mediated PARylation activity may be a potential therapeutic target for promoting OPC differentiation and remyelination in neurological disorders characterized by arrested OPC differentiation and remyelination failure such as multiple sclerosis.

Dissecting the molecular determinants of clinical PARP1 inhibitor selectivity for tankyrase1

Poly-ADP-ribosyltransferases play a critical role in DNA repair and cell death, and poly(ADP-ribosyl) polymerase 1 (PARP1) is a particularly important therapeutic target for the treatment of breast cancer because of its synthetic lethal relationship with breast cancer susceptibility proteins 1 and 2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA. Biochemically, talazoparib is also suspected to be a potent inhibitor of PARP5a/b (tankyrase1/2 [TNKS1/2]), which is an important regulator of Wnt/β-catenin pathway. Here we show using competition experiments in cell lysate that, at a clinically relevant concentration, talazoparib can potentially bind and engage TNKS1. Using surface plasmon resonance, we measured the dissociation constants of talazoparib, olaparib, niraparib, and veliparib for their interaction with PARP1 and TNKS1. The results show that talazoparib has strong affinity for PARP1 as well as uniquely strong affinity for TNKS1. Finally, we used crystallography and hydrogen deuterium exchange mass spectroscopy to dissect the molecular mechanism of differential selectivity of these PARP1 inhibitors. From these data, we conclude that subtle differences between the ligand-binding sites of PARP1 and TNKS1, differences in the electrostatic nature of the ligands, protein dynamics, and ligand conformational energetics contribute to the different pharmacology of these PARP1 inhibitors. These results will help in the design of drugs to treat Wnt/β-catenin pathway–related cancers, such as colorectal cancers.

Synthesis of Some Benzimidazole-derived Molecules and their Effects on PARP-1 Activity and MDA-MB-231, MDA-MB-436, MDA-MB-468 Breast Cancer Cell Viability.

Poly (ADP-ribosyl) polymerase-1 (PARP-1) inhibitors are compounds that are used to treat cancers, which are defective in DNA-repair and DNA Damage-Response (DDR) pathways. In this study, a series of potential PARP-1 inhibitor substituted (piperazine-1-carbonyl)phenyl)-1Hbenzo[ d]imidazole-4-carboxamide compounds were synthesised and tested for their PARP-1 inhibitory and anticancer activities. Compounds were tested by cell-free colorimetric PARP-1 activity and MTT assay in MDA-MB-231, MDA-MB-436, MDA-MB-468 breast cancer, and L929 fibroblast cell lines. Our results showed that compound 6a inhibited viability in MDA-MB-231 and MDA-MB-468 cells whereas 8a inhibited viability in MDA-MB-468 cells. Compound 6b significantly inhibited cell viability in tested cancer cells. However, 6b exhibited toxicity in L929 cells, whereas 6a and 8a were found to be non-toxic for L929 cells. Compounds 6a, 6b and 8a exhibited significant inhibition of PARP-1 activity. These three compounds exhibited PARP-1 inhibitory activities and anticancer effects on breast cancer cells, and further research will enlighten the underlying mechanisms of their effects.

SFRP1 has a biphasic effect on doxorubicin-induced cardiotoxicity in a cellular location-dependent manner in NRCMs and Rats.

Doxorubicin (Dox) is an effective anticancer drug, however, its clinical application is restricted by the life-threatening cardiotoxic effects. Secreted Frizzled-related protein 1 (sFRP1) has been reported to participate in both the cancer and cardiovascular diseases and was one of the differential expression genes in normal hearts compared with Dox-treated hearts. Thus, it is important to reveal the potential role of sFRP1 in Dox-induced cardiotoxicity. Here, we show that sFRP1 has a biphasic effect on Dox-induced cardiotoxicity in a location-dependent manner. The secretion of sFRP1 was significantly increased in Dox-treated neonatal rat cardiomyocytes (NRCMs) (1µM) and SD rats (5mg/kg/injection at day 1, 5, and 9, i.p.). Adding the anti-sFRP1 antibody (0.5µg/ml) and inhibiting sFRP1 secretion by caffeine (5mM) both relieved Dox-induced cardiotoxicity through activating Wnt/β-catenin signaling, whereas increasing the secretion of sFRP1 by heparin (100µg/ml) had the opposite effect. The intracellular level of sFRP1 was significantly decreased after Dox treatment both in vitro and in vivo. Knockdown of sFRP1 by sgRNA aggravated Dox-induced cardiotoxicity, while moderate overexpression of sFRP1 by Ad-sFRP1 exhibited protective effect. Besides, poly(ADP-ribosyl) polymerase-1 (PARP1) was screened as an interacting partner of sFRP1 in NRCMs by mass spectrometry. Our results suggested that the intracellular sFRP1 protected NRCMs from Dox-induced cardiotoxicity by interacting with PARP1. Thus, our results provide a novel evidence that sFRP1 has a biphasic effect on Dox-induced cardiotoxicity. In addition, the oversecretion of sFRP1 might be used as a biomarker to indicate the occurrence of cardiotoxicity induced by Dox treatment.

Poly (ADP-Ribosyl) Polymerase 1 Inhibitors: A Patent Review.

Poly (ADP-ribosyl) polymerase 1 (PARP1) is important in maintaining genomic stability, repairing DNA damage, and regulating transcriptional processes. Altered PARP1 activity is associated with a multitude of pathologies especially cancer. The broad application prospects of PARP1 inhibitors attract many well-known pharmaceutical companies, which promotes the development of PARP1 inhibitors. Present review aims to introduce PARP1 inhibitors by their structures and try to point out future development direction of PARP1 inhibitors. Details regarding the PARP1and PARP1 inhibitors are obtained from PubMed literatures and patent databases. The action mode of PARP1 inhibitors developed so far is competing with NAD+ for the catalytic site of PARP1. Using such inhibitors affects multiple NAD+-dependent enzymatic pathways, which results in secondary toxic effects. Designing inhibitors targeting other binding sites on the PARP1 protein is a strategy to bypass this pitfall. Analyzing the structure-activity relationships of active PARP1 inhibitors described in the patents, we conclude that for the binding activity, amide group, aromatic ring or heterocyclic ring with rich electronics and heteroatom-substituted in the meta position of amide group are essential. Big substituents introduced in the heterocyclic ring can enhance inhibitory activity and improve solubility or other physicochemical properties. Clinical trials of PARP1 inhibitor were focused on cancer therapies and have achieved remarkable results.

Increased Susceptibility to Oxidative Death of Lymphocytes from Alzheimer Patients Correlates with Dementia Severity

We previously reported on enhanced susceptibility to death of lymphocytes from Alzheimer's disease (AD) patients when exposed to hydrogen peroxide (H2O2)-induced oxidative stress and an increased resistance to death in those of patients with a history of skin cancer. This is consistent with our hypothesis proposing that the cellular machinery controlling cell death is deregulated in opposite directions in Alzheimer's disease (AD) and cancer, to explain the inverse association observed in epidemiological studies. Here we investigated whether the observed increased susceptibility correlates with the degree of dementia severity. Peripheral lymphocytes from 23 AD patients, classified using the Clinical Dementia Rating (CDR) into severe dementia (CDR 3, n=10) and mild-to-moderate dementia (CDR 1- 2, n=13), and 15 healthy controls (HC) (CDR 0), were exposed to H2O2 for 20 hours. Lymphocyte death was determined by flow cytometry and propidium iodide staining. The greatest susceptibility to H2O2-induced death was observed for lymphocytes from severe dementia patients, whereas those with mild-to-moderate dementia exhibited intermediate values, compared to healthy controls. A significant increase in the apoptosis/necrosis ratio was found in AD patients. Poly (ADP-ribosyl) polymerase-1 (PARP-1) inhibition significantly protected from H2O2-induced death of lymphocytes, whereby a lower degree of protection was observed in severe AD patients. Moreover, inhibition of PARP-1 abolished the differences in apoptosis/necrosis ratios observed between the three groups of patients. These results support the notion that AD is a systemic disorder, whereby enhanced susceptibility to H2O2-induced death in peripheral lymphocytes correlates with dementia severity and enhanced death in AD patients is attributable to a PARP-dependent increase in the apoptosis/necrosis ratio.

β-Lapachone induces programmed necrosis through the RIP1-PARP-AIF-dependent pathway in human hepatocellular carcinoma SK-Hep1 cells.

β-Lapachone activates multiple cell death mechanisms including apoptosis, autophagy and necrotic cell death in cancer cells. In this study, we investigated β-lapachone-induced cell death and the underlying mechanisms in human hepatocellular carcinoma SK-Hep1 cells. β-Lapachone markedly induced cell death without caspase activation. β-Lapachone increased PI uptake and HMGB-1 release to extracellular space, which are markers of necrotic cell death. Necrostatin-1 (a RIP1 kinase inhibitor) markedly inhibited β-lapachone-induced cell death and HMGB-1 release. In addition, β-lapachone activated poly (ADP-ribosyl) polymerase-1(PARP-1) and promoted AIF release, and DPQ (a PARP-1 specific inhibitor) or AIF siRNA blocked β-lapachone-induced cell death. Furthermore, necrostatin-1 blocked PARP-1 activation and cytosolic AIF translocation. We also found that β-lapachone-induced reactive oxygen species (ROS) production has an important role in the activation of the RIP1-PARP1-AIF pathway. Finally, β-lapachone-induced cell death was inhibited by dicoumarol (a NQO-1 inhibitor), and NQO1 expression was correlated with sensitivity to β-lapachone. Taken together, our results demonstrate that β-lapachone induces programmed necrosis through the NQO1-dependent ROS-mediated RIP1-PARP1-AIF pathway.

ATM-dependent chromatin remodeler Rsf-1 facilitates DNA damage checkpoints and homologous recombination repair

As a member of imitation switch (ISWI) family in ATP-dependent chromatin remodeling factors, RSF complex consists of SNF2h ATPase and Rsf-1. Although it has been reported that SNF2h ATPase is recruited to DNA damage sites (DSBs) in a poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner in DNA damage response (DDR), the function of Rsf-1 is still elusive. Here we show that Rsf-1 is recruited to DSBs confirmed by various cellular analyses. Moreover, the initial recruitment of Rsf-1 and SNF2h to DSBs shows faster kinetics than that of γH2AX after micro-irradiation. Signals of Rsf-1 and SNF2h are retained over 30 min after micro-irradiation, whereas γH2AX signals are gradually reduced at 10 min. In addition, Rsf-1 is accumulated at DSBs in ATM-dependent manner, and the putative pSQ motifs of Rsf-1 by ATM are required for its accumulation at DSBs. Furtheremore, depletion of Rsf-1 attenuates the activation of DNA damage checkpoint signals and cell survival upon DNA damage. Finally, we demonstrate that Rsf-1 promotes homologous recombination repair (HRR) by recruiting resection factors RPA32 and Rad51. Thus, these findings reveal a new function of chromatin remodeler Rsf-1 as a guard in DNA damage checkpoints and homologous recombination repair.