Tankyrase-1 regulates RBP-mediated mRNA turnover to promote muscle fiber formation.

Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.

Poly(ADP-ribosyl)ation enhances nucleosome dynamics and organizes DNA damage repair components within biomolecular condensates.

Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3. PARylation disrupted interactions between nucleosomes, preventing self-association. Addition of LIG3 and XRCC1 to PARylated nucleosomes generated condensates that selectively partition DNA repair-associated proteins in a PAR- and phosphorylation-dependent manner in vitro. Our results establish that PARylation influences nucleosomes across different length scales, extending from the atom-level motions of histone tails to the mesoscale formation of condensates with selective compositions.

[Poly(ADP-Ribose) Polymerases 1 and 2: Classical Functions and Interaction with New Histone Poly(ADP-Ribosyl)ation Factor HPF1].

Poly(ADP-ribose) (PAR) is a negatively charged polymer, linear or branched, that consists of ADP-ribose monomers. PAR is synthesized by poly(ADP-ribose)polymerase (PARP) enzymes, which are activated upon DNA damage and use nicotinamide adenine dinucleotide (NAD^(+)) as a substrate. The best-studied members of the PARP family, PARP1 and PARP2, are the most important nuclear proteins involved in many cell processes, including the regulation of DNA repair. PARP1 and PARP2 catalyze PAR synthesis and transfer to amino acid residues of target proteins, including autoPARylation. PARP1 and PARP2 are promising targets for chemotherapy in view of their key role in regulating DNA repair. A novel histone PARylation factor (HPF1) was recently discovered to modulate PARP1/2 activity by forming a transient joint active site with PARP1/2. Histones are modified at serine residues in the presence of HPF1. The general mechanism of the interaction between HPF1 and PARP1/2 is a subject of intense research now. The review considers the discovery and classical mechanism of PARylation in higher eukaryotes and the role of HPF1 in the process.

A Double-Edged Sword: The Two Faces of PARylation.

Poly ADP-ribosylation (PARylation) is a post-translational modification process. Following the discovery of PARP-1, numerous studies have demonstrated the role of PARylation in the DNA damage and repair responses for cellular stress and DNA damage. Originally, studies on PARylation were confined to PARP-1 activation in the DNA repair pathway. However, the interplay between PARylation and DNA repair suggests that PARylation is important for the efficiency and accuracy of DNA repair. PARylation has contradicting roles; however, recent evidence implicates its importance in inflammation, metabolism, and cell death. These differences might be dependent on specific cellular conditions or experimental models used, and suggest that PARylation may play two opposing roles in cellular homeostasis. Understanding the role of PARylation in cellular function is not only important for identifying novel therapeutic approaches; it is also essential for gaining insight into the mechanisms of unexplored diseases. In this review, we discuss recent reports on the role of PARylation in mediating diverse cellular functions and homeostasis, such as DNA repair, inflammation, metabolism, and cell death.

Mechanistic insights into the three steps of poly(ADP-ribosylation) reversal.

Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.

Why structure and chain length matter: on the biological significance underlying the structural heterogeneity of poly(ADP-ribose).

Poly(ADP-ribosyl)ation (PARylation) is a multifaceted post-translational modification, carried out by poly(ADP-ribosyl)transferases (poly-ARTs, PARPs), which play essential roles in (patho-) physiology, as well as cancer therapy. Using NAD+ as a substrate, acceptors, such as proteins and nucleic acids, can be modified with either single ADP-ribose units or polymers, varying considerably in length and branching. Recently, the importance of PAR structural heterogeneity with regards to chain length and branching came into focus. Here, we provide a concise overview on the current knowledge of the biochemical and physiological significance of such differently structured PAR. There is increasing evidence revealing that PAR's structural diversity influences the binding characteristics of its readers, PAR catabolism, and the dynamics of biomolecular condensates. Thereby, it shapes various cellular processes, such as DNA damage response and cell cycle regulation. Contrary to the knowledge on the consequences of PAR's structural diversity, insight into its determinants is just emerging, pointing to specific roles of different PARP members and accessory factors. In the future, it will be interesting to study the interplay with other post-translational modifications, the contribution of natural PARP variants, and the regulatory role of accessory molecules. This has the exciting potential for new therapeutic approaches, with the targeted modulation and tuning of PARPs' enzymatic functions, rather than their complete inhibition, as a central premise.

Increased PARylation impacts the DNA methylation process in type 2 diabetes mellitus.

BACKGROUND: Epigenetic modifications, such as DNA methylation, can influence the genetic susceptibility to type 2 diabetes mellitus (T2DM) and the progression of the disease. Our previous studies demonstrated that the regulation of the DNA methylation pattern involves the poly(ADP-ribosyl)ation (PARylation) process, a post-translational modification of proteins catalysed by the poly(ADP-ribose) polymerase (PARP) enzymes. Experimental data showed that the hyperactivation of PARylation is associated with impaired glucose metabolism and the development of T2DM. Aims of this case-control study were to investigate the association between PARylation and global and site-specific DNA methylation in T2DM and to evaluate metabolic correlates. RESULTS: Data were collected from 61 subjects affected by T2DM and 48 healthy individuals, recruited as controls. Global levels of poly(ADP-ribose) (PAR, a surrogate of PARP activity), cytosine methylation (5-methylcytosine, 5mC) and de-methylation intermediates 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were determined in peripheral blood cells by ELISA-based methodologies. Site-specific DNA methylation profiling of SOCS3, SREBF1 and TXNIP candidate genes was performed by mass spectrometry-based bisulfite sequencing, methyl-sensitive endonucleases digestion and by DNA immuno-precipitation. T2DM subjects presented higher PAR levels than controls. In T2DM individuals, increased PAR levels were significantly associated with higher HbA1c levels and the accumulation of the de-methylation intermediates 5hmC and 5fC in the genome. In addition, T2DM patients with higher PAR levels showed reduced methylation with increased 5hmC and 5fC levels in specific SOCS3 sites, up-regulated SOCS3 expression compared to both T2DM subjects with low PAR levels and controls. CONCLUSIONS: This study demonstrates the activation of PARylation processes in patients with T2DM, particularly in those with poor glycaemic control. PARylation is linked to dysregulation of DNA methylation pattern via activation of the DNA de-methylation cascade and may be at the basis of the differential gene expression observed in presence of diabetes.

Functional Roles of PARP2 in Assembling Protein-Protein Complexes Involved in Base Excision DNA Repair.

Poly(ADP-ribose) polymerase 2 (PARP2) participates in base excision repair (BER) alongside PARP1, but its functions are still under study. Here, we characterize binding affinities of PARP2 for other BER proteins (PARP1, APE1, Polß, and XRCC1) and oligomerization states of the homo- and hetero-associated complexes using fluorescence-based and light scattering techniques. To compare PARP2 and PARP1 in the efficiency of PAR synthesis, in the absence and presence of protein partners, the size of PARP2 PARylated in various reaction conditions was measured. Unlike PARP1, PARP2 forms more dynamic complexes with common protein partners, and their stability is effectively modulated by DNA intermediates. Apparent binding affinity constants determined for homo- and hetero-oligomerized PARP1 and PARP2 provide evidence that the major form of PARP2 at excessive PARP1 level is their heterocomplex. Autoregulation of PAR elongation at high PARP and NAD+ concentrations is stronger for PARP2 than for PARP1, and the activity of PARP2 is more effectively inhibited by XRCC1. Moreover, the activity of both PARP1 and PARP2 is suppressed upon their heteroPARylation. Taken together, our findings suggest that PARP2 can function differently in BER, promoting XRCC1-dependent repair (similarly to PARP1) or an alternative XRCC1-independent mechanism via hetero-oligomerization with PARP1.

HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase.

Poly(ADP-ribose) polymerase 1 (PARP1) is an important player in the response to DNA damage. Recently, Histone PARylation Factor (HPF1) was shown to be a critical modulator of the activity of PARP1 by facilitating PARylation of histones and redirecting the target amino acid specificity from acidic to serine residues. Here, we investigate the mechanism and specific consequences of HPF1-mediated PARylation using nucleosomes as both activators and substrates for PARP1. HPF1 provides that catalytic base Glu284 to substantially redirect PARylation by PARP1 such that the histones in nucleosomes become the primary recipients of PAR chains. Surprisingly, HPF1 partitions most of the reaction product to free ADP-ribose (ADPR), resulting in much shorter PAR chains compared to reactions in the absence of HPF1. This HPF1-mediated switch from polymerase to hydrolase has important implications for the PARP1-mediated response to DNA damage and raises interesting new questions about the role of intracellular ADPR and depletion of NAD+.

MARTs and MARylation in the Cytosol: Biological Functions, Mechanisms of Action, and Therapeutic Potential.

Mono(ADP-ribosyl)ation (MARylation) is a regulatory post-translational modification of proteins that controls their functions through a variety of mechanisms. MARylation is catalyzed by mono(ADP-ribosyl) transferase (MART) enzymes, a subclass of the poly(ADP-ribosyl) polymerase (PARP) family of enzymes. Although the role of PARPs and poly(ADP-ribosyl)ation (PARylation) in cellular pathways, such as DNA repair and transcription, is well studied, the role of MARylation and MARTs (i.e., the PARP 'monoenzymes') are not well understood. Moreover, compared to PARPs, the development of MART-targeted therapeutics is in its infancy. Recent studies are beginning to shed light on the structural features, catalytic targets, and biological functions of MARTs. The development of new technologies to study MARTs have uncovered essential roles for these enzymes in the regulation of cellular processes, such as RNA metabolism, cellular transport, focal adhesion, and stress responses. These insights have increased our understanding of the biological functions of MARTs in cancers, neuronal development, and immune responses. Furthermore, several novel inhibitors of MARTs have been developed and are nearing clinical utility. In this review, we summarize the biological functions and molecular mechanisms of MARTs and MARylation, as well as recent advances in technology that have enabled detection and inhibition of their activity. We emphasize PARP-7, which is at the forefront of the MART subfamily with respect to understanding its biological roles and the development of therapeutically useful inhibitors. Collectively, the available studies reveal a growing understanding of the biochemistry, chemical biology, physiology, and pathology of MARTs.

Poly(ADP-ribose) polymerase 1 regulates mitochondrial DNA repair in an NAD-dependent manner.

Mitochondrial DNA is located in organelle that house essential metabolic reactions and contains high reactive oxygen species. Therefore, mitochondrial DNA suffers more oxidative damage than its nuclear counterpart. Formation of a repair enzyme complex is beneficial to DNA repair. Recent studies have shown that mitochondrial DNA polymerase (Pol γ) and poly(ADP-ribose) polymerase 1 (PARP1) were found in the same complex along with other mitochondrial DNA repair enzymes, and mitochondrial PARP1 level is correlated with mtDNA integrity. However, the molecular basis for the functional connection between Pol γ and PARP1 has not yet been elucidated because cellular functions of PARP1 in DNA repair are intertwined with metabolism via NAD+ (nicotinamide adenosine dinucleotide), the substrate of PARP1, and a metabolic cofactor. To dissect the direct effect of PARP1 on mtDNA from the secondary perturbation of metabolism, we report here biochemical studies that recapitulated Pol γ PARylation observed in cells and showed that PARP1 regulates Pol γ activity during DNA repair in a metabolic cofactor NAD+ (nicotinamide adenosine dinucleotide)-dependent manner. In the absence of NAD+, PARP1 completely inhibits Pol γ, while increasing NAD+ levels to a physiological concentration that enables Pol γ to resume maximum repair activity. Because cellular NAD+ levels are linked to metabolism and to ATP production via oxidative phosphorylation, our results suggest that mtDNA damage repair is coupled to cellular metabolic state and the integrity of the respiratory chain.

Poly(ADP-ribosyl)ation enhances HuR oligomerization and contributes to pro-inflammatory gene mRNA stabilization.

Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification mainly catalyzed by poly-ADP-ribose polymerase 1 (PARP1). In addition to having important roles in DNA damage detection and repair, it functions in gene expression regulation, especially at the posttranscriptional level. Embryonic lethal abnormal vision-like 1/human antigen R (ELAVL/HuR), a canonical 3' untranslated region AU-rich element-binding protein, is a crucial mRNA-stabilizing protein that protects target mRNAs from RNA-destabilizing protein- or microRNA-induced silencing complex (miRISC)-mediated degradation. Additionally, in some cases, HuR itself either promotes or suppresses translation. Here, we demonstrated that in response to inflammatory stimuli, the PARylation of HuR, mostly at the conserved D226 site, by PARP1 increased the formation of the HuR oligomer/multimer, and HuR oligomerization promoted the disassociation of miRISC and stabilized the pro-inflammatory gene mRNAs. The prevention of PARP1 activation or HuR oligomerization attenuated lipopolysaccharide-induced inflammatory gene expression and the airway recruitment of neutrophils in mouse lungs. The present study verified a novel mechanism of PARP1 and HuR PARylation in the RNA stability regulation, increasing our understanding of how PARP1 regulates gene expression.

Mechanisms governing PARP expression, localization, and activity in cells.

Poly-(ADP)-ribose polymerases (PARPs) are a family of 17 enzymes in humans that have diverse roles in cell physiology including DNA damage repair, transcription, innate immunity, and regulation of signaling pathways. The modular domain architecture of PARPs gives rise to this functional diversity. PARPs catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to targets-proteins and poly-nucleic acids. This enigmatic post-translational modification comes in two varieties: the transfer of a single unit of ADP-ribose, known as mono-ADP-ribosylation (MARylation) or the transfer of multiple units of ADP-ribose, known as poly-ADP-ribosylation (PARylation). Emerging data shows that PARPs are regulated at multiple levels to control when and where PARP-mediated M/PARylation occurs in cells. In this review, we will discuss the latest knowledge regarding the regulation of PARPs in cells: from transcription and protein stability to subcellular localization and modulation of catalytic activity.

Real-time monitoring of PARP1-dependent PARylation by ATR-FTIR spectroscopy.

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD+ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3' phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD+ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.

Poly(ADP-ribosyl)ation mediates early phase histone eviction at DNA lesions.

Nucleosomal histones are barriers to the DNA repair process particularly at DNA double-strand breaks (DSBs). However, the molecular mechanism by which these histone barriers are removed from the sites of DNA damage remains elusive. Here, we have generated a single specific inducible DSB in the cells and systematically examined the histone removal process at the DNA lesion. We found that histone removal occurred immediately following DNA damage and could extend up to a range of few kilobases from the lesion. To examine the molecular mechanism underlying DNA damage-induced histone removal, we screened histone modifications and found that histone ADP-ribosylation was associated with histone removal at DNA lesions. PARP inhibitor treatment suppressed the immediate histone eviction at DNA lesions. Moreover, we examined histone chaperones and found that the FACT complex recognized ADP-ribosylated histones and mediated the removal of histones in response to DNA damage. Taken together, our results reveal a pathway that regulates early histone barrier removal at DNA lesions. It may also explain the mechanism by which PARP inhibitor regulates early DNA damage repair.

Forced Self-Modification Assays as a Strategy to Screen MonoPARP Enzymes.

Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are posttranslational modifications found on multiple amino acids. There are 12 enzymatically active mono(ADP-ribose) polymerase (monoPARP) enzymes and 4 enzymatically active poly(ADP-ribose) polymerase (polyPARP) enzymes that use nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate to generate these modifications. While there are approved drugs and clinical trials ongoing for the enzymes that perform PARylation, MARylation is gaining recognition for its role in immune function, inflammation, and cancer. However, there is a lack of chemical probes to study the function of monoPARPs in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop biochemical assays to enable hit finding, and determination of the potency and selectivity of inhibitors. Complicating the development of enzymatic assays is that it is poorly understood how monoPARPs engage their substrates. To overcome this, we have developed a family-wide approach to developing robust high-throughput monoPARP assays where the enzymes are immobilized and forced to self-modify using biotinylated-NAD+, which is detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. Herein we describe the development of assays for 12 monoPARPs and 3 polyPARPs and apply them to understand the potency and selectivity of a focused library of inhibitors across this family.

PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome.

The poly(ADP-ribose) polymerase, PARP1, plays a key role in maintaining genomic integrity by detecting DNA damage and mediating repair. γH2A.X is the primary histone marker for DNA double-strand breaks and PARP1 localizes to H2A.X-enriched chromatin damage sites, but the basis for this association is not clear. We characterize the kinetics of PARP1 binding to a variety of nucleosomes harbouring DNA double-strand breaks, which reveal that PARP1 associates faster with (γ)H2A.X- versus H2A-nucleosomes, resulting in a higher affinity for the former, which is maximal for γH2A.X-nucleosome that is also the activator eliciting the greatest poly-ADP-ribosylation catalytic efficiency. The enhanced activities with γH2A.X-nucleosome coincide with increased accessibility of the DNA termini resulting from the H2A.X-Ser139 phosphorylation. Indeed, H2A- and (γ)H2A.X-nucleosomes have distinct stability characteristics, which are rationalized by mutational analysis and (γ)H2A.X-nucleosome core crystal structures. This suggests that the γH2A.X epigenetic marker directly facilitates DNA repair by stabilizing PARP1 association and promoting catalysis.

Poly-ADP ribosylation in DNA damage response and cancer therapy.

Poly(ADP-ribosyl)ation (aka PARylation) is a unique protein post-translational modification (PTM) first described over 50 years ago. PARylation regulates a number of biological processes including chromatin remodeling, the DNA damage response (DDR), transcription, apoptosis, and mitosis. The subsequent discovery of poly(ADP-ribose) polymerase-1 (PARP-1) catalyzing DNA-dependent PARylation spearheaded the field of DDR. The expanding knowledge about the poly ADP-ribose (PAR) recognition domains prompted the discovery of novel DDR factors and revealed crosstalk with other protein PTMs including phosphorylation, ubiquitination, methylation and acetylation. In this review, we highlight the current knowledge on PAR-regulated DDR, PAR recognition domain, and PARP inhibition in cancer therapy.

PARP1 and Poly(ADP-ribosyl)ation Signaling during Autophagy in Response to Nutrient Deprivation.

Autophagy is considered to be the primary degradative pathway that takes place in all eukaryotic cells. Morphologically, the autophagy pathway refers to a process by which cytoplasmic portions are delivered to double-membrane organelles, called autophagosomes, to fuse with lysosomes for bulk degradation. Autophagy, as a prosurvival mechanism, can be stimulated by different types of cellular stress such as nutrient deprivation, hypoxia, ROS, pH, DNA damage, or ER stress, promoting adaptation of the cell to the changing and hostile environment. The functional relevance of autophagy in many diseases such as cancer or neurodegenerative diseases remains controversial, preserving organelle function and detoxification and promoting cell growth, although in other contexts, autophagy could suppress cell expansion. Poly(ADP-ribosyl)ation (PARylation) is a covalent and reversible posttranslational modification (PTM) of proteins mediated by Poly(ADP-ribose) polymerases (PARPs) with well-described functions in DNA repair, replication, genome integrity, cell cycle, and metabolism. Herein, we review the current state of PARP1 activation and PARylation in starvation-induced autophagy.