Binding kinetics and activity of human poly(ADP‐ribose) polymerase‐1 on oligo‐deoxyribonucleotide substrates

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a mammalian enzyme that attaches long branching chains of ADP‐ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP‐1 plays an important role in DNA strand‐break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single‐stranded to double‐strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single‐strandedness were used for measurements of both PARP‐1/DNA binding kinetics and PARP‐1's enzyme activities. We found that binding correlated with activity, but was unrelated to single‐strandedness of the ODN. Instead, PARP‐1 binding and activity were highest on ODNs that modeled a DNA double‐strand break (DSB). These results provide support for PARP‐1 recognizing and binding DSBs in a manner that is independent of single‐stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP‐1 binding and PARP‐1 auto‐poly(ADP‐ribosyl)ation activities within the same in vitro system. Copyright © 2009 John Wiley & Sons, Ltd.     

Critical role of poly(ADP‐ribose) polymerase‐1 in modulating the mode of cell death caused by continuous oxidative stress

Continuously generated hydrogen peroxide (H₂O₂) inhibits typical apoptosis and instead initiates a caspase‐independent, apoptosis‐inducing factor (AIF)‐mediated pyknotic cell death. This may be related to H₂O₂‐mediated DNA damage and subsequent ATP depletion, although the exact mechanisms by which the mode of cell death is decided after H₂O₂ exposure are still unclear. Accumulated evidence and our previous data led us to hypothesize that continuously generated H₂O₂, not an H₂O₂ bolus, induces severe DNA damage, signaling poly(ADP‐ribose) polymerase‐1 (PARP‐1) activation, ATP depletion, and eventually caspase‐independent cell death. Results from the present study support that H₂O₂ generated continuously by glucose oxidase causes excessive DNA damage and PARP‐1 activation. Blockage of PARP‐1 by a siRNA transfection or by pharmacological inhibitor resulted in the significant inhibition of ATP depletion, loss of mitochondrial membrane potential, nuclear translocation of AIF and endonuclease G, and eventually conversion to caspase‐dependent apoptosis. Overall, the current study demonstrates the different roles of PARP‐1 inhibition in modulation of cell death according to the method of H₂O₂ exposure, that is, continuous generation versus a direct addition. J. Cell. Biochem. 108: 989–997, 2009. © 2009 Wiley‐Liss, Inc.     

Poly (ADP‐ribose) polymerase‐1 binds to BCL2 major breakpoint region and regulates BCL2 expression

BCL2, originally identified as a proto‐oncogene in B‐cell lymphoma, is a key regulator of apoptosis. Although it is more than 200 kb in length, at least 70% of the t(14;18) translocation in follicular lymphomas occurs at the BCL2 major breakpoint region (mbr), located in the 3′‐untranslated region (3'‐UTR). We have previously found that the mbr is a regulatory element which positively regulates BCL2 expression and this regulatory function was closely associated with SATB1, which binds to a 37 bp mbr (37 mbr) in the 3′‐end of the mbr directly. However, the precise molecular mechanisms by which the mbr regulates gene expression are not fully understood. In this study, we purified Poly(ADP‐ribose) polymerase‐1 (PARP‐1) from the DNA–protein complexes formed by 37 mbr in Jurkat cells and demonstrated that PARP‐1 participates in the 37 mbr–protein complex's formation in vitro and in vivo. Functional analysis showed that overexpression of PARP‐1 decreases 37 mbr regulatory function and BCL2 expression. Conversely, knockdown of PARP‐1 with RNAi increases BCL2 expression. Taken together, the present findings indicate that PARP‐1 is a component of BCL2 37 mbr–protein complexes, and PARP‐1 is involved in the regulation of BCL2 expression. These findings are helpful in understanding the regulatory mechanisms of BCL2 expression. J. Cell. Biochem. 110: 1208–1218, 2010. Published 2010 Wiley‐Liss, Inc.     

Geometrical constraints limiting the poly(ADP‐ribose) conformation investigated by molecular dynamics simulation

Poly(ADP‐ribosylation) is a post‐transductional modification that regulates protein's function. Most of the proteins subjected to this control mechanism belong to machineries involved in DNA damage repair, or DNA interacting proteins. Poly(ADP‐ribose) polymers are long chains of even 100 monomer length that can be branched at several positions but, not withstanding its importance, nothing is known concerning its structure. To understand, which are the geometrical parameters that confer to the polymer the structural constraints that determine its interaction with the target proteins, we have performed molecular dynamics of three chains of different length, made by 5, 25, and 30 units, the last one being branched. Analysis of the simulations allowed us to identify the main intra‐ and inter‐monomer dihedral angles that govern the structure of the polymer that however, does not reach a unique definite conformation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 78–86, 2014.     

Overexpressed poly(ADP‐ribose) polymerase delays the release of rat cells from p53‐mediated G1 checkpoint

We have previously reported that in cells ectopically expressing temperature‐sensitive p53^135val mutant, p53 formed tight complexes with poly(ADP‐ribose) polymerase (PARP). At elevated temperatures, p53^135val protein, adopting the mutant phenotype, was localized in the cytoplasm and sequestered the endogenous PARP. To prove whether an excess of p53^135val protein led to this unusual intracellular distribution of PARP, we have established cell lines overexpressing p53^135val + c‐Ha‐ras alone or in combination with PARP. Interestingly, immunostaining revealed that PARP is sequestered in the cytoplasm by mutant p53 in cells overexpressing both proteins. Simultaneous overexpression of PARP had no effect on temperature‐dependent cell proliferation and only negligibly affected the kinetics of p53‐mediated G₁ arrest. However, if the cells were completely growth arrested at 32°C and then shifted up to 37°C, coexpressed PARP dramatically delayed the reentry of transformed cells into the cell cycle. Even after 72 h at 37°C the proportion of S‐phase cells was reduced to 20% compared to those expressing only p53^135val + c‐Ha‐ras. The coexpressed PARP stabilized wt p53 protein and its enzymatic activity was necessary for stabilization. J. Cell. Biochem. 80:85–103, 2000. © 2000 Wiley‐Liss, Inc.     

Comprehensive ADP‐ribosylome analysis identifies tyrosine as an ADP‐ribose acceptor site

Despite recent mass spectrometry (MS)‐based breakthroughs, comprehensive ADP‐ribose (ADPr)‐acceptor amino acid identification and ADPr‐site localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP‐ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1‐dependent in vivo ADPr‐acceptor amino acid. MS analyses of in vitro ADP‐ribosylated proteins confirmed tyrosine as an ADPr‐acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that trans‐modification of RPS3A is dependent on HPF1. We provide an ADPr‐site Localization Spectra Database (ADPr‐LSD), which contains 288 high‐quality ADPr‐modified peptide spectra, to serve as ADPr spectral references for correct ADPr‐site localizations.     

Tocotrienols promote apoptosis in human breast cancer cells by inducing poly(ADP‐ribose) polymerase cleavage and inhibiting nuclear factor kappa‐B activity

Objectives

Tocotrienols and tocopherols are members of the vitamin E family, with similar structures; however, only tocotrienols have been reported to achieve potent anti‐cancer effects. The study described here has evaluated anti‐cancer activity of vitamin E to elucidate mechanisms of cell death, using human breast cancer cells.

Materials and methods

Anti‐cancer activity of a tocotrienol‐rich fraction (TRF) and a tocotrienol‐enriched fraction (TEF) isolated from palm oil, as well as pure vitamin E analogues (α‐tocopherol, α?, δ? and γ?tocotrienols) were studied using highly aggressive triple negative MDA‐MB‐231 cells and oestrogen‐dependent MCF‐7 cells, both of human breast cancer cell lines. Cell population growth was evaluated using a Coulter particle counter. Cell death mechanism, poly(ADP‐ribose) polymerase cleavage and levels of NF‐κB were determined using commercial ELISA kits.

Results

Tocotrienols exerted potent anti‐proliferative effects on both types of cell by inducing apoptosis, the underlying mechanism of cell death being ascertained using respective IC₅₀ concentrations of all test compounds. There was marked induction of apoptosis in both cell lines by tocotrienols compared to treatment with Paclitaxel, which was used as positive control. This activity was found to be associated with cleavage of poly(ADP‐ribose) polymerase (a DNA repair protein), demonstrating involvement of the apoptotic cell death signalling pathway. Tocotrienols also inhibited expression of nuclear factor kappa‐B (NF‐κB), which in turn can increase sensitivity of cancer cells to apoptosis.

Conclusion

Tocotrienols induced anti‐proliferative and apoptotic effects in association with DNA fragmentation, poly(ADP‐ribose) polymerase cleavage and NF‐κB inhibition in the two human breast cancer cell lines.

     

Poly(ADP‐ribose) polymerases regulate cell division and development in Arabidopsis roots

Root organogenesis involves cell division,differentiation and expansion. The molecular mechanisms regulating root development are not fully understood.In this study, we identified poly(adenosine diphosphate(ADP)-ribose) polymerases(PARPs) as new players in root development. PARP catalyzes poly(ADP-ribosyl)ation of proteins by repeatedly adding ADP-ribose units onto proteins using nicotinamide adenine dinucleotide(NADt)as the donor. We found that inhibition of PARP activities by3-aminobenzomide(3-AB) increased the growth rates of both primary and lateral roots, leading to a more developed root system. The double mutant of Arabidopsis PARPs, parp1parp2, showed more rapid primary and lateral root growth. Cyclin genes regulating G1-to-S and G2-to-Mtransition were up-regulated upon treatment by 3-AB.The proportion of 2C cells increased while cells with higher DNA ploidy declined in the roots of treated plants, resulting in an enlarged root meristematic zone. The expression level of PARP2 was very low in the meristematic zone but high in the maturation zone, consistent with a role of PARP in inhibiting mitosis and promoting cell differentiation. Our results suggest that PARPs play an important role in root development by negatively regulating root cell division.     

Poly (ADP‐ribose) polymerase inhibition protects against myocardial ischaemia/reperfusion injury via suppressing mitophagy

Myocardial ischaemia/reperfusion (I/R) injury attenuates the beneficial effects of reperfusion therapy. Poly(ADP‐ribose) polymerase (PARP) is overactivated during myocardial I/R injury. Mitophagy plays a critical role in the development of myocardial I/R injury. However, the effect of PARP activation on mitophagy in cardiomyocytes is unknown. In this study, we found that I/R induced PARP activation and mitophagy in mouse hearts. Poly(ADP‐ribose) polymerase inhibition reduced the infarct size and suppressed mitophagy after myocardial I/R injury. In vitro, hypoxia/reoxygenation (H/R) activated PARP, promoted mitophagy and induced cell apoptosis in cardiomyocytes. Poly(ADP‐ribose) polymerase inhibition suppressed H/R‐induced mitophagy and cell apoptosis. Parkin knockdown with lentivirus vectors inhibited mitophagy and prevented cell apoptosis in H/R‐treated cells. Poly(ADP‐ribose) polymerase inhibition prevented the loss of the mitochondrial membrane potential (ΔΨm). Cyclosporin A maintained ΔΨm and suppressed mitophagy but FCCP reduced the effect of PARP inhibition on ΔΨm and promoted mitophagy, indicating the critical role of ΔΨm in H/R‐induced mitophagy. Furthermore, reactive oxygen species (ROS) and poly(ADP‐ribosylation) of CypD and TSPO might contribute to the regulation of ΔΨm by PARP. Our findings thus suggest that PARP inhibition protects against I/R‐induced cell apoptosis by suppressing excessive mitophagy via the ΔΨm/Parkin pathway.     

The nuclear protein Poly(ADP‐ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP‐ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear‐localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3‐1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col‐0 wild‐type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability.     

Poly(ADP‐ribose) polymerase inhibition with HYDAMTIQ reduces allergen‐induced asthma‐like reaction,bronchial hyper‐reactivity and airway remodelling

Activation of poly(ADP‐ribose) polymerases (PARPs) is considered a key event in the molecular and cellular processes leading from acute asthma attacks to bronchial hyper‐reactivity, leucocyte recruitment, chronic inflammation, airway remodelling and lung damage. The present investigation has been carried out to investigate the action of hydroxyl‐dimethylaminomethyl‐thieno[2,3‐c]isoquinolin‐5(4H)‐one (HYDAMTIQ), a new potent PARP inhibitor, in the process leading from asthma‐like events to airway damage. Ovalbumin‐sensitized guinea pigs exposed two times to allergen inhalation were treated for 8 days with vehicle or HYDAMTIQ. Asthma‐like signs, bronchial hyper‐reactivity to methacholine, cytokine production, histamine release from mast cells, airway remodelling, collagen deposition and lung damage were evaluated. Repeated HYDAMTIQ administration (1‐10 mg/kg/day i.p.) reduced lung PARP activity, delayed the appearance and reduced the severity of allergen‐induced cough and dyspnoea and dampened the increased bronchial responses to methacholine. HYDAMTIQ‐treated animals presented reduced bronchial or alveolar abnormalities, lower number of eosinophils and other leucocytes in the lung and decreased smooth muscle or goblet cell hyperplasia. The treatment also reduced lung oxidative stress markers, such as malondialdehyde or 8‐hydroxy‐2′‐deoxyguanosine and the lung content of pro‐inflammatory cytokines (TNF‐α, interleukin (IL)‐1β, IL‐5, IL‐6 and IL‐18). Finally, mast cells isolated from the peritoneal or pleural cavities of sensitized, HYDAMTIQ‐treated animals had a reduced ability to release histamine when exposed to ovalbumin in vitro. Our findings support the proposal that PARP inhibitors could have a therapeutic potential to reduce chronic lung inflammation, airway damage and remodelling in severe unresponsive asthmatic patients.     

Deficiency of terminal ADP‐ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

Adenosine diphosphate (ADP)‐ribosylation is a post‐translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP‐ribosylation reactions are the poly(ADP‐ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP‐ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP‐ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP‐ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP‐interacting protein that removes mono(ADP‐ribosyl)ation on glutamate amino acid residues in PARP‐modified proteins. X‐ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl‐(ADP‐ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP‐ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.     

In vitro poly(ADP‐ribosyl)ated histones H1a and H1t modulate rat testis chromatin condensation differently

Rat testis H1 proteins were poly(ADP‐ribosyl)ated in vitro. The modifying product, poly(ADP‐ribose), was found covalently bound to each histone variant at various extents and exhibited distinct structural features (linear and short, rather than branched and long chains). Interest was focused on the somatic H1a, particularly abundant in the testis, as compared with other tissues, and the testis‐specific H1t, which appears only at the pachytene spermatocyte stage of germ cell development. These H1s were modified with poly(ADP‐ribose) by means of two in vitro experimental approaches. In the first system, each variant was incubated with purified rat testis poly(ADP‐ribose)polymerase in the presence of [³²P] NAD. In parallel, poly(ADP‐ribosyl)ated H1s were also prepared following incubation of intact rat testis nuclei with [³²P] NAD. In both experiments, the poly(ADP‐ribosyl)ated proteins were purified from the native forms by means of phenyl boronic agarose chromatography. The results from both analyses were in agreement and showed qualitative differences with regard to the poly(ADP‐ribose) covalently associated with H1a and H1t. Comparison of the bound polymers clearly indicated that the oligomers associated with H1a were within 10–12 units long, whereas longer chains (≤20 ADP‐R units) were linked to H1t. Individual poly(ADP‐ribosyl)ated H1s were complexed with homologous H1‐depleted oligonucleosomes (0.5–2.5 kbp) in order to measure their ability to condensate chromatin, in comparison with the native ones. Circular dichroism showed that the negative charges of the oligomeric polyanion, although present in limited numbers, highly influenced the DNA‐binding properties of the analyzed H1s. In particular, the poly(ADP‐ribosyl)ated H1a and H1t had opposite effects on the condensation of H1‐depleted oligonucleosomes. J. Cell. Biochem. 76:20–29, 1999. © 1999 Wiley‐Liss, Inc.