Biological Functions of PARP-1

The higher-order structure and density of chromatin compaction has determining influence on the accessibility of DNA-associated factors, thus controlling repair and transcription. The degree of chromatin packing is defined by the composition of factors associated with the DNA (histones and non-histones). As one of the earliest sensors of DNA damage and mediator of DNA repair, PARP-1 is involved in the modulation of chromatin packing by three distinct modes ¹⁶⁹.

First, PARP-1 covalently and non-covalently modifies chromatin-associated factors, such as histones, high-mobility group proteins (HMG), heterochromatin protein 1 (HP1) and macroH2A ^92,170-172. Modification with the negative polymer may alter the proteins affinity to DNA and remove them from the site of PARylation. By doing so, PARP-1 activity directly controls chromatin architecture, and grants or denies chromatin accesses for the DNA repair machinery or transcription factors ^173-175. Second, PARP-1 competes with the histone linker H1 for binding to nucleosomes. PARP-1 binding to nucleosomes results in local compaction of the chromatin and transcriptional repression. Upon PARP-1 activation, the protein automodifies itself and dissociates from the chromatin. This results in chromatin decondensation and restoration of transcription ^67,176,177.

Third, PARP-1 recruits chromatin remodelers, such as ALC1 or NURD to the sites of PARylation.

Here, these factors deploy their nucleosome sliding activity, thus facilitating the binding and activity of DNA repair factors ^178-180.

Transcriptional Regulation

Through its role in chromatin regulation, PARP-1 is involved in gene transcription, granting and denying excess of transcription factors by altering compaction status. Beside this, a direct impact on several central transcription factors is known ¹⁸¹.

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The tumor suppressor p53 plays a key role in the cellular response to genotoxic stress. The decision if a cell undergoes DNA repair, cell cycle arrest, senescence or cell death is mainly mediated by p53 ¹⁸². PARP-1 and p53 have been shown to interact in-vitro and in-vivo ^183-185. p53 was reported to be covalently PARylated and interacts with PAR via one of its PBMs 147,186,187. Non-covalent interaction between PAR and p53, influences p53’s DNA-binding affinity and is believed to be involved in nuclear localization and regulation of p53’s transcriptional activity ^154,188. PARP-1 further modulates inflammation and is essential for the transcription of proinflammatory genes, such as TNFα, by binding to NF-κB. This PARylation-independent interaction increases NF-κB’s DNA-binding affinity and transcriptional activity. Translocation of NF-κB from its cytoplasmic detention was further shown to be PARP-activity dependent ¹⁸⁹. A negative regulation was shown for the TATA-binding protein (TBP).

PARylation of this protein blocks the formation of the preinitiation complex (PIC) necessary for general transcription start ^190,191.

Besides this direct influence on transcription, PARP-1 is implicated in DNMT1 (DNA (cytosine-5)-methyltransferase 1)-mediated DNA methylation. PARP-1 forms a complex with CTCF (CCCTC-binding factor) and DNMT1 and modifies both proteins either covalently or non-covalently with PAR polymer ^192,193. PARP inhibition causes a DNMT1-dependent hypermethylation of DNA, while PARP activity is associated with hypomethylation, thus influencing epigenetic transcriptional regulation

194-197.

Protein Stability and Turnover

The observation that one of the non-covalent PAR-binding modules, the WWE domain, is predominantly found in E3-ubiquitin ligases hints for a tight cross-talk between PARylation and ubiquitination ⁶⁰. Indeed, a PAR-dependent ubiquitination and subsequent proteasomal degradation was revealed in the context of Wnt signaling. The E3-ubiquitin ligase Iduna/RNF146 is known to bind and become activated by both, short chains of polymer (transferred by tankyrases) as well as long polymer chains (PARP-1). Upon PARylation of axin by tankyrases, Iduna/RNF146 binds via its WWE domain and marks the protein for degradation by ubiquitination. As a result β-catenin-dependent transcription takes place 126,166,198,199. Besides this pathway, Iduna/RNF146 is implicated in the modification of a number of DNA repair factors, such as PARP-1 and 2, XRCC1, Ku70/80 and DNA ligase III and also herein its activity is PAR-dependent ¹⁶⁶.

The majority of patients suffering of cherubism carry a mutation in the protein 3BP2, thus disturbing the interaction with tankyrase 2. Without the PARylation by tankyrase 2 this protein evades ubiquitination by Iduna/RNF146 and subsequent proteasomal degradation. This ultimately leads to systemic inflammation and the cherubism phenotype ^200,201.

NAD⁺ Metabolism

NAD⁺ is a molecule of vital importance, placed in the midst of several fundamental cellular processes.

The relevance of this molecule is highlighted by the vitamin deficiency disease pellagra (disease of the four Ds; dermatitis, diarrhea, dementia, death). Pellagra is caused by malnutrition in form of lacking intake of niacin (vitamin B3, nicotinic acid) and tryptophan, precursors for NAD⁺ synthesis ²⁰².

NAD⁺ is probably best known for its role as a coenzyme for several reduction-oxidation reactions.

NAD⁺ and its reduced form NADH serve as key intermediates for energy transfer from different metabolic pathways and link the fundamental catabolic pathways of glycolysis and Krebs cycle (TCA) to the electron-transfer chain and oxidative phosphorylation (ATP synthesis). In these redox reactions NAD⁺ and NADH are interconverted but not consumed ¹⁰.

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However, NAD⁺ serves as a cofactor in several other cellular processes in which the molecule is consumed. In each of these processes NAD⁺ is hydrolyzed, nicotinamide is released and the free energy is used to drive the ensuing reaction with ADP-ribose ¹⁹. CD38 and CD157 consume NAD⁺ in order to synthesize cyclic ADP-ribose for calcium signaling ²⁰³. Enzymes of the ART and PARP families transfer ADP-ribose moieties to target substrates, further elongated to long chains by some PARP members. Sirtuins utilize NAD⁺ for deacetylation of proteins, forming the by-product o-acetyl-ADP-ribose.

PARylation as a result of genotoxic stress is probably the major challenger of NAD⁺ pools, transiently reducing concentrations up to 80 % within minutes. Although this only depicts a considerable degree of DNA damage and at more moderate challenges, only 5-10 % of the cellular NAD⁺ is depleted, it is conceivable that mild but continuous PARylation still can jeopardize the cellular machinery ¹⁹.

To maintain NAD⁺ concentrations despite the continuous depletion by NAD⁺-consuming enzymes, several synthesis pathways are available with different NAD⁺ precursors.

The major dietary source of precursors for NAD⁺ synthesis is nicotinic acid. This molecule can be transformed to NAD⁺ in three steps via the Preiss-Handler pathway. De-novo synthesis is performed from tryptophan, the pathway merging at a point with the Preiss-Handler pathway. The salvage pathway is probably the most important one in maintaining cellular NAD⁺ levels. It recycles the byproduct nicotinamide to form NAD⁺ and herby conserves the energetic potential but also relieves the cell from the inhibitory influence which nicotinamide has on NAD⁺-consuming enzymes ^10,204.

NAD⁺ levels and NAD⁺/NADH ratios have been reported to be important elements and determine life and health span of yeast, worms and mammalian cells ^205-208. These observations are tightly linked to the protein family of sirtuins. SIRT1 and SIRT2 have been shown to positively affect aging in mice in a NAD⁺-dependent manner

209,210. Although both are involved in cellular homeostasis and wellbeing, sirtuins and PARPs do show antagonizing influences to a certain extent. Both compete for NAD⁺ with comparable Km values (PARP-1<SIRT1) and upon PARP-1 stimulation SIRT1 activity was reported to be reduced ¹⁰. On the other hand, PARP-1 is inhibited by SIRT1 via direct deacetylation as well as by transcriptional regulation ^211,212. The severe consequences of dysregulation of the PARP-1 and SIRT1 NAD⁺ metabolism are shown by a series of recent studies. The deficiency of the DNA repair factor XPA causes the clinical phenotype of Xeroderma Pigmentosum. This disease is linked to increased disposition of cancer and neurological degeneration. Together with other neurodegenerative disorders (CS and AT) caused by defects in

Figure 1.6: NAD⁺ metabolism in different cellular compartments.

Several different NAD⁺ precursors can be taken up by the cell and metabolized by different pathways to NAD⁺. This is on the one hand an essential coenzyme for energy metabolism and redox systems and links glycolysis and Krebs cycle to the respiratory chain. On the other hand it is consumed by central key enzymes, such as ARTs, PARPs and sirtuins and thus needs to be resynthesized constantly. Adapted from ¹⁰.

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certain DNA repair pathways, this disease was phenotypically clustered with mitochondrial disorders, suggesting shared pathological mechanisms ^213,214. In cells of these patients a hyperactivation of PARP-1 could be observed, probably due to the inability of proper genomic maintenance. It was assumed that sustained PARylation leads to depletion of cellular NAD⁺ pools and decreased SIRT1 activity. This was accompanied by mitochondrial abnormalities as a result of the disturbed NAD⁺-SIRT1-PGC1α axis ²¹⁵. Inhibition of PARP activity or supplementation with NAD⁺ precursors could rescue this phenotype to some degree ²¹⁵. Similar results were obtained in a Cockayne Syndrome mouse model, in which NAD⁺ precursors improved life span and reduced mitochondrial abnormalities and neuronal damage ²¹⁶. Metabolism and Energy Maintenance

The stimulation of PARP-1 activity has severe consequences on cellular metabolism and energy balance, both acute and on a long-term. As a direct result of PARylation NAD⁺ levels can decrease by 80-90% within minutes ²¹⁷. Accompanied by the depletion of NAD⁺, a decrease of ATP levels was observed ²¹⁸. It was assumed that NAD⁺ consumption directly impairs glycolysis and Krebs cycle in which it works as a coenzyme and thus causes energetic catastrophe 217,219,220.

This assumption is challenged by recent studies showing the independency of the energy loss from NAD⁺ depletion. Additionally, the decrease of ATP levels precedes the NAD⁺ depletion ^221-224. Ensuing PARP-1 activity, PAR-degrading enzymes cleave the polymer to monomers, which can be further processed to AMP. This molecule is known to interfere with ADP uptake by the mitochondrion, thus interfering with ATP resynthesis ²²⁵. Further, PARylation directly blocks glycolysis by inhibiting hexokinases ²²⁴. These enzymes are initial factors of the glycolysis, catalyzing the reaction from glucose to glucose-6-phosphate. Non-covalent interaction with PAR via its PBM inhibits hexokinases enzymatic activity ^224,226. Of note, hexokinases are as well involved in the pentose phosphate pathway, essential for the formation of NADPH and the reduction of the antioxidant glutathione ^227-229. Anyhow, it cannot be excluded that NAD⁺ depletion does contribute to the arising and maintenance of the PARylation-induced energy crisis ²²⁴.

Inflammation and NF-κB Signalling

PARP-1 has been shown to have a proinflammatory influence and deficiency in PARP activity was reported to protect from several inflammation-associated pathologies 189,218,230,231. Genotoxic stress triggers PARP-1 activity, which in turn facilitates the assembly of the signalosome (PIASγ, IKKγ and ATM). IKKγ becomes sumoylated and translocates to the cytoplasm, where it phosphorylates IκB, the inhibitor of NF-κB. As a result, IκB becomes ubiquitinated and degraded, which frees NF-κB to enter the nucleus and act as a proinflamatory transcription factor ^232-236. Further, the physically interaction of PARP-1 with NF-κB enhances its transcriptional activity, leading to the expression of inflammatory factors, such as iNOS, IL-6 and TNFα ^236-239.

Finally, Parthanatos is a PARP-dependent form of cell death. It is assigned to the cell death class of regulated necrosis, thus sharing some characteristics with this pathway ²⁴⁰. In contrast to apoptosis, Parthanatos is accompanied by the loss of cell membrane integrity. The uncontrolled release of cell content is a strong inflammatory trigger ¹⁷⁰. Furthermore, PARylation of HMGB1 reduces its affinity for chromatin and facilitates its release. HMGB1 then binds to phosphatidylserine in the cell membrane.

In apoptotic cells presentation of phosphatidylserine on the outer leaflet of the cell membrane serves as a ‘eat me’ signal for macrophages in order to remove apoptotic bodies and reduce immunogenic signaling ²⁴¹.

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Due to the need of primers for each round of DNA replication, chromosomes become shortened. To prevent the loss of coding sequence, repetitive sequences (TTAGGG), the telomeres, can be found at the end of chromosomes. These sequences form an odd DNA structure (t-loop) and are covered by a protein complex (sheltrin). This complex is crucial to protect the chromosomal ends from unscheduled DNA double-strand break repair, and further regulates the elongation of deteriorated telomeres in the presence of active telomerase ²⁴².

PARylation has been shown to play an essential role in the process of telomere elongation. PARP-1 deficiency or inhibition caused a 30 % reduction in telomere length in mice in only one generation ²⁴³. Restoration of PARP activity restored telomere length again ²⁴⁴. PARP-1 is recruited to deteriorated telomeres and interacts with one of the sheltrin complex proteins, the telomeric repeat binding factor 2 (TRF2). This protein is a relevant factor for telomere stability, length regulation and protection from the DNA repair machinery. The modification of TRF2 with the PAR polymer reduces its affinity for telomeric DNA, resulting in a relaxation of the t-loop ^245,246.

DNA Damage Signalling and Mediation of DNA Repair

PARP-1 facilitates the repair of DNA lesions by a wide array of functions. It rapidly senses DNA aberrations, becomes activated and PARylates itself and surrounding chromatin associated factors. By doing so, it locally opens the chromatin, thereby increasing accessibility for DNA repair factors.

Further, a subcellular organization by local liquid demixing of intrinsically disordered proteins to generate a transient compartmentalization at the site of PARylation was recently suggested ²⁴⁷. Local PARylation forms a chromatin-based platform to facilitate the recruitment and assembly of DNA repair complexes, organizes access and removal of repair factors and influences their enzymatic activity.

PARP-1 has been reported to be involved in almost all major DNA repair pathways, such as base excision repair (BER), single-strand break repair (SSBR), double-strand break repair (DSBR), mismatch repair (MMR) and nucleotide excision repair (NER). Details of the role of PARP-1 in the different aspects of DNA repair will be addressed in the following chapters.

Parthanatos and Cell Death

Early on, the activity of PARP-1 was identified to play a crucial role in DNA repair and genomic surveillance. Further, a strong correlation between PARP-1 activity and maximal life span can be observed in mammalian species ²⁴⁸. Thorough maintenance of the genome and the prevention of mutations is believed to be essential for a long and healthy life span. On the other hand, at times the genomic maintenance machinery fails, for instance in case of DNA damage overload, it is favorable for the organism to remove cells with enhanced genomic instability, thus forestalling cancer formation. To date, the ‘National Committee of Cell Death’ lists 12 forms of cell death. Although probably not being completely exclusive, each of them acts independently and displays individual characteristics. In 2012 a PAR-dependent mode of cell death, Parthanatos, was introduced as distinct pathway to death ²⁴⁰. First reports on cell death triggered by excessive PARP-1 activity in response to DNA damaging agents came from the early 1980s ²⁴⁹. Initially, a ‘suicide hypothesis’ was suggested. Heavy exposure of cells to genotoxic agents results in high numbers of DNA lesions. Herein, PARP-1 serves as a sensor of damage load. Upon mild to medium genotoxic encounter its activity facilitates DNA repair and restoration of genomic integrity. In times of massive DNA damage however, PARP-1 becomes over-activated and progressively deprives NAD⁺ from the cellular pools. NAD⁺ depletion (reduction by 80 % within minutes) results in energetic crisis and metabolic catastrophe ²⁵⁰. PARP inhibition,

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supplementation with NAD⁺ precursors as well as intermediates from glycolysis and Krebs cycle were shown to rescue these effects ^219,251.

However, the ‘suicide hypothesis’ as driving mechanism in PARP-dependent cell death is progressively challenged. Increasing numbers of studies reported that Parthanatos was not dependent on NAD⁺ depletion but sole on PAR formation 222,224,252. The rescue observed upon NAD⁺ supplementation is probably attributed to the increased activity of SIRT1. While the rescue of the observed energy crisis with glycolysis and Krebs cycle intermediates can be explained with the bypass of the PAR-dependent inhibition of hexokinase1/2 (initiation of glycolysis) 19,224,253,254.

Parthanatos was described as a consequence of the exposure to several genotoxic agents (MNNG, H2O2, NO and NOO^-) in several mammalian cells. Anyhow, this mode of cell death seems to play a special role in neuronal cells and NMDA-induced excitotoxicity 250,255-259. It shares several features with both, apoptosis and necrosis, which makes it conceivable to be placed among the cell death class of regulated necrosis. Common features with apoptosis are externalization of phosphatidylserine, loss of mitochondrial membrane potential, chromatin condensation and shrinkage. On the other hand, Parthanatos is accompanied by necrosis characteristics such as the loss of cell membrane integrity, lack of energy dependency and caspase-independent large scale fragmentation of the genome (~50 kb)

256,260-262. Besides these overlaps with apoptosis and necrosis, Parthanatos-specific events take place. Upon DNA damage or NMDA-induced excitotoxicity, PARP-1 becomes active and attaches long chains of PAR to itself and other proteins. By a so far unresolved mechanism, free or protein-bound polymer translocates from the nucleus to the mitochondria where it triggers the release of the apoptosis inducing factor (AIF). AIF in return translocates to the nucleus and facilitates chromatin condensation, DNA fragmentation and finally cell death 129,130,256,262. In the mitochondria AIF is located at the inner membrane serving as NADH oxidase involved in oxidative phosphorylation. A smaller portion of the protein was also reported to be associated with the cytoplasmic side of the outer membrane, probably being responsible for the rapid translocation to the nucleus ²⁶³. The determining step in this process is the non-covalent interaction of AIF with preferably long and branched PAR polymer 130,151,264. Mutation studies in AIF’s PBM neither interfered with its mitochondrial function nor with its DNA-binding ability, but prevented the PAR-dependent release from the mitochondria and thus Parthanatos. Similar results were obtained by suppressing PARP-1 activity with a PARP inhibitor ^256,264. This opens a therapeutic window for the treatment or containment of several neuronal disorders showing signs of Parthanatos, like Parkinson Disease, focal cerebral ischemia and stroke ^265-270.