Nuclear poly(ADP-ribose) polymerase-1 rapidly triggers mitochondrial dysfunction

To obtain further information on time course and mechanisms of cell death after poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, we used HeLa cells exposed for 1 h to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. This treatment activated PARP-1 and caused a rapid drop of cellular NAD(H) and ATP contents, culminating 8-12 h later in cell death. PARP-1 antagonists fully prevented nucleotide depletion and death. Interestingly, in the early 60 min after challenge with N-methyl-N'-nitro-N-nitrosoguanidine, mitochondrial membrane potential and superoxide production significantly increased, whereas cellular ADP contents decreased. Again, these events were prevented by PARP-1 inhibitors, suggesting that PARP-1 hyperactivity leads to mitochondrial state 4 respiration. Mitochondrial membrane potential collapsed at later time points (3 h), when mitochondria released apoptosis-inducing factor and cytochrome c. Using immunocytochemistry and targeted luciferase transfection, we found that, despite an exclusive localization of PARP-1 and poly(ADP-ribose) in the nucleus, ATP levels first decreased in mitochondria and then in the cytoplasm of cells undergoing PARP-1 activation. PARP-1 inhibitors rescued ATP (but not NAD(H) levels) in cells undergoing hyper-poly(ADP-ribosyl)ation. Glycolysis played a central role in the energy recovery, whereas mitochondria consumed ATP in the early recovery phase and produced ATP in the late phase after PARP-1 inhibition, further indicating that nuclear poly(ADP-ribosyl)ation rapidly modulates mitochondrial functioning. Together, our data provide evidence for rapid nucleus-mitochondria cross-talk during hyper-poly(ADP-ribosyl)ation-dependent cell death.     

Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.     

Poly(ADP-ribosyl)ation of p53 Contributes to TPEN-Induced Neuronal Apoptosis

Depletion of intracellular zinc by N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.     

The sequence-specific DNA binding of NF-kappa B is reversibly regulated by the automodification reaction of poly (ADP-ribose) polymerase 1

Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-kappaB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-kappaB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-kappaB (NF-kappaB-p50) in the presence or absence of betaNAD+ in vitro. Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-kappaB-p50 DNA binding was dependent on the presence of betaNAD+. DNA binding by NF-kappaB-p50 was not efficient in the absence of betaNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-kappaB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-kappaB-p50 with high specificity in the absence of betaNAD+. Because NF-kB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-kappaB-p50 to its DNA by inhibiting the specific protein.protein interactions between NF-kappaB-p50 and PARP-1. We also report the activation of NF-kappaB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H(2)O(2). In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-kappaB activation in vivo as well.     

Haploinsufficiency of poly(ADP-ribose) polymerase-1-mediated poly(ADP-ribosyl)ation for centrosome duplication

The centrosome plays a vital role in maintaining chromosomal stability. Known as the microtubule organizing center, the centrosome is involved in the formation of spindle poles during mitosis, which ensures the distribution of the correct number of chromosomes to daughter cells. Aberrant centrosome duplication could cause centrosome amplification and chromosomal instability. We have previously shown that poly(ADP-ribose) polymerase-1 (PARP-1) is important for centrosome function and chromosomal stability. In this study, we used PARP-1(+/+), PARP-1(+/-) and PARP-1(-/-) primary mouse embryonic fibroblasts and found that the level of PARP-1 gene dosage correlates with PARP activity and the in vivo level of poly(ADP-ribosyl)ation, which could explain the mechanism by which PARP-1 haploinsufficiency affects centrosome duplication and chromosomal stability. Our results emphasize that correct regulation of poly(ADP-ribosyl)ation levels in vivo is important for maintenance of proper centrosome duplication and chromosomal stability.     

Inhibition of Crm1-p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation

Poly(ADP-ribose) polymerase 1 (PARP-1) and p53 are two key proteins in the DNA-damage response. Although PARP-1 is known to poly(ADP-ribosyl)ate p53, the role of this modification remains elusive. Here, we identify the major poly(ADP-ribosyl)ated sites of p53 by PARP-1 and find that PARP-1-mediated poly(ADP-ribosyl)ation blocks the interaction between p53 and the nuclear export receptor Crm1, resulting in nuclear accumulation of p53. These findings molecularly link PARP-1 and p53 in the DNA-damage response, providing the mechanism for how p53 accumulates in the nucleus in response to DNA damage. PARP-1 becomes super-activated by binding to damaged DNA, which in turn poly(ADP-ribosyl)ates p53. The nuclear export machinery is unable to target poly(ADP-ribosyl)ated p53, promoting accumulation of p53 in the nucleus where p53 exerts its transactivational function.     

Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells

Genome integrity is constantly threatened by DNA lesions arising from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity.     

3'-5' phosphoadenosine phosphate is an inhibitor of PARP-1 and a potential mediator of the lithium-dependent inhibition of PARP-1 in vivo

pAp (3'-5' phosphoadenosine phosphate) is a by-product of sulfur and lipid metabolism and has been shown to have strong inhibitory properties on RNA catabolism. In the present paper we report a new target of pAp, PARP-1 [poly(ADP-ribose) polymerase 1], a key enzyme in the detection of DNA single-strand breaks. We show that pAp can interact with PARP-1 and inhibit its poly(ADP-ribosyl)ation activity. In vitro, inhibition of PARP-1 was detectable at micromolar concentrations of pAp and altered both PARP-1 automodification and heteromodification of histones. Analysis of the kinetic parameters revealed that pAp acted as a mixed inhibitor that modulated both the Km and the Vmax of PARP-1. In addition, we showed that upon treatment with lithium, a very potent inhibitor of the enzyme responsible for pAp recycling, HeLa cells exhibited a reduced level of poly(ADP-ribosyl)ation in response to oxidative stress. From these results, we propose that pAp might be a physiological regulator of PARP-1 activity.     

The emerging role of poly(ADP-ribose) polymerase-1 in longevity

In the present paper, the involvement of the family of poly(ADP-ribose) polymerases (PARPs), and especially of PARP-1, in mammalian longevity is reviewed. PARPs catalyse poly(ADP-ribosyl)ation, a covalent post-translational protein modification in eukaryotic cells. PARP-1 and PARP-2 are activated by DNA strand breaks, play a role in DNA base-excision repair (BER) and are survival factors for cells exposed to low doses of ionising radiation or alkylating agents. PARP-1 is the main catalyst of poly(ADP-ribosyl)ation in living cells under conditions of DNA breakage, accounting for about 90% of cellular poly(ADP-ribose). DNA-damage-induced poly(ADP-ribosyl)ation also functions as a negative regulator of DNA damage-induced genomic instability. Cellular poly(ADP-ribosyl)ation capacity in permeabilised mononuclear blood cells (MNC) is positively correlated with life span of mammalian species. Furthermore PARP-1 physically interacts with WRN, the protein deficient in Werner syndrome, a human progeroid disorder, and PARP-1 and WRN functionally cooperate in preventing carcinogenesis in vivo. Some of the other members of the PARP family have also been revealed as important regulators of cellular functions relating to ageing/longevity. In particular, tankyrase-1, tankyrase-2, PARP-2 as well as PARP-1 have been found in association with telomeric DNA and are able to poly(ADP-ribosyl)ate the telomere-binding proteins TRF-1 and TRF-2, thus blocking their DNA-binding activity and controlling telomere extension by telomerase.     

Ataxia telangiectasia mutated (ATM) signaling network is modulated by a novel poly(ADP-ribose)-dependent pathway in the early response to DNA-damaging agents

Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling.     

Central role for the Werner syndrome protein/poly(ADP-ribose) polymerase 1 complex in the poly(ADP-ribosyl)ation pathway after DNA damage

A defect in the Werner syndrome protein (WRN) leads to the premature aging disease Werner syndrome (WS). Hallmark features of cells derived from WS patients include genomic instability and hypersensitivity to certain DNA-damaging agents. WRN contains a highly conserved region, the RecQ conserved domain, that plays a central role in protein interactions. We searched for proteins that bound to this region, and the most prominent direct interaction was with poly(ADP-ribose) polymerase 1 (PARP-1), a nuclear enzyme that protects the genome by responding to DNA damage and facilitating DNA repair. In pursuit of a functional interaction between WRN and PARP-1, we found that WS cells are deficient in the poly(ADP-ribosyl)ation pathway after they are treated with the DNA-damaging agents H2O2 and methyl methanesulfonate. After cellular stress, PARP-1 itself becomes activated, but the poly(ADP-ribosyl)ation of other cellular proteins is severely impaired in WS cells. Overexpression of the PARP-1 binding domain of WRN strongly inhibits the poly(ADP-ribosyl)ation activity in H2O2-treated control cell lines. These results indicate that the WRN/PARP-1 complex plays a key role in the cellular response to oxidative stress and alkylating agents, suggesting a role for these proteins in the base excision DNA repair pathway.