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CORD-19:d8c4042441e88788e26135cb83f2510ac050aae7 JSONTXT 12 Projects

Annnotations TAB TSV DIC JSON TextAE Lectin_function IAV-Glycan

Id Subject Object Predicate Lexical cue
T1 0-66 Sentence denotes Reprogramming cellular events by poly(ADP-ribose)-binding proteins
T2 68-76 Sentence denotes Abstract
T3 77-189 Sentence denotes Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADPribose) polymerases (PARPs).
T4 190-350 Sentence denotes These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units.
T5 351-514 Sentence denotes The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities.
T6 515-712 Sentence denotes Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death.
T7 713-860 Sentence denotes In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways.
T8 861-1026 Sentence denotes This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways.
T9 1027-1252 Sentence denotes Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions.
T10 1253-1400 Sentence denotes In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions.
T11 1402-1682 Sentence denotes It was almost 50 years ago that poly(ADP-ribose) (pADPr) was discovered as an adenine-containing RNA-like polymer (Chambon et al., 1963) and early on, there were indications that pADPr turnover is very tightly regulated in mammalian cells Reeder et al., 1967; Ueda et al., 1972) .
T12 1683-1874 Sentence denotes Cellular levels of pADPr are governed by the finely tuned balance of the synthetic poly(ADP-ribose) polymerases (PARPs) and degrading poly(ADP-ribose) glycohydrolase (PARG) enzyme activities.
T13 1875-2023 Sentence denotes In the human genome, 17 proteins share a PARP signature sequence homologous to the catalytic domain of the founding and most described member PARP1.
T14 2024-2161 Sentence denotes In the context of DNA damage, PARP1 generates within minutes approximately 90% of all pADPr, preferentially on itself (automodification).
T15 2162-2264 Sentence denotes However, pADPr accumulation is transient, as it is rapidly degraded by PARG (Davidovic et al., 2001) .
T16 2265-2415 Sentence denotes Notably, the polymerase activity has been demonstrated for only six of the PARP family members (PARP1, PARP2, PARP3, PARP4/vPARP, Tankyrases 1 and 2).
T17 2416-2768 Sentence denotes Based on experimental and structural examinations, it has been proposed that the other PARP family members are either inactive (PARP9/BAL and PARP13/ ZAP) or carry a mono-ADP-ribosyl transferase activity (PARP6, TiPARP, PARP8, PARP10, PARP11, PARP12, PARP14/BAL2, PARP15/BAL3, PARP16) (Goenka et al., 2007; Hottiger et al., 2010; Kleine et al., 2008) .
T18 2769-2878 Sentence denotes Their functions are only starting to emerge, but suggest an important role for these poorly studied proteins.
T19 2879-3199 Sentence denotes The seminal work by Benjamin and Gill (1980a,b) showed that PARP1 activity is highly stimulated by the presence of DNA containing single-and double-strand breaks, a discovery that has been followed up by a succession of studies that linked pADPr metabolism to maintenance of genome stability (Meyer-Ficca et al., 2005) .
T20 3200-3384 Sentence denotes Mono-and poly-(ADP-ribosyl)ation are reversible and phylogenetically ancient posttranslational protein modifications, for which the list of acceptor/target proteins is still expanding.
T21 3385-3464 Sentence denotes Poly(ADP-ribosyl)ation can be achieved covalently or non-covalently (Fig. 1A) .
T22 3465-3685 Sentence denotes The covalent posttranslational modification (PTM) occurs on glutamic, aspartic or lysine residues, while non-covalent interactions between proteins and pADPr add another level for modulating proteins biological activity.
T23 3686-3890 Sentence denotes This PTM has profound physico-chemical implications, as poly(ADP-ribose) bears two negatively charged phosphate groups per ADP-ribose (ADPr) residue, i.e. twice as many charges than DNA or RNA (Fig. 1A) .
T24 3891-4071 Sentence denotes The size and flexibility of pADPr polymers render them capable of mediating multiple contacts with protein surfaces, thus providing a significant enhancement in binding efficiency.
T25 4072-4219 Sentence denotes Such physico-chemical attributes of the pADPr are capable of contributing to the creation of a scaffold for the assembly of multiprotein complexes.
T26 4220-4531 Sentence denotes Accumulating evidence indicates that pADPr actually conveys a broad spectrum of cellular signals through direct binding of a variety of protein motifs to pADPr, such as the DNA damage response, replication, chromatin structure, transcription, telomere homeostasis and cell death (Krishnakumar and Kraus, 2010) .
T27 4532-4747 Sentence denotes Given the structural complexity of the pADPr polymers, it is not surprising to observe that several evolutionary conserved protein domains have emerged to accomplish unique functions through interactions with pADPr.
T28 4748-5093 Sentence denotes Indeed, the average chain length of pADPr synthesized by the PARP family members can range from very short and linear oligomers to extended molecules of up to 200 units and branched at every 20-50 residues (Fig. 1B) (Alvarez-Gonzalez and Jacobson, 1987; D'Amours et al., 1999; Kiehlbauch et al., 1993; Kleine et al., 2008; Tanaka et al., 1977) .
T29 5094-5360 Sentence denotes There are very limited investigations conducted on the physico-chemical properties of pADPr, such as flexibility and conformation, but the formation of helical pADPr structures was postulated upon protein binding (Minaga and Kun, 1983a,b; Schultheisz et al., 2009 ).
T30 5361-5625 Sentence denotes The first experimental lines of evidence for proteins that bind pADPr in a non-covalent, yet specific, manner were given in the late 1960s and early 1970s when it was shown that histones possess high affinity for pADPr Nakaz-awa et al., 1968; Otake et al., 1969) .
T31 5626-5756 Sentence denotes However, whether the chemical nature of the bond was covalent or non-covalent was a highly debated topic (Adamietz et al., 1975) .
T32 5757-5926 Sentence denotes At that time, the prevailing view was that poly(ADP-ribosyl)ation was a covalent protein posttranslational modification on histones as is phosphorylation or methylation.
T33 5927-6154 Sentence denotes This concept persisted for years until the 1980s when studies began to report interactions with PARP1-bound pADPr (Ohashi et al., 1983) and free pADPr (Sauermann and Wesierska-Gadek, 1986; Wesierska-Gadek and Sauermann, 1988) .
T34 6155-6373 Sentence denotes In the last decade, rapid progress has been achieved in the identification of pADPr-binding proteins among some of the chromatin-associated non-histone proteins and proteins involved in extranuclear signaling networks.
T35 6374-6586 Sentence denotes Remarkably, more than four specialized pADPr-binding modules that recognize different structural features of the pADPr are responsible for the functional diversification of the pADPr-responsive cellular pathways.
T36 6587-6980 Sentence denotes This paper first provides an inventory of the predominant techniques currently used to detect and measure non-covalent protein-pADPr interactions, then turns to an in-depth description of the specialized pADPr-binding modules that recognize different structural features of the pADPr, and finally, presents the functional consequences of this association in pADPr-responsive cellular pathways.
T37 6981-7151 Sentence denotes The posttranslational modification of a protein substrate by enzymatic covalent attachment of poly(ADP-ribose) (pADPr) to specific amino acids side chains is represented.
T38 7152-7287 Sentence denotes On the pADPr structure, a collection of modular protein domains non-covalently binds to pADPr through different recognition mechanisms.
T39 7288-7509 Sentence denotes There are currently four pADPr-binding protein modules that have been experimentally characterized: the pADPr-binding motif (PBM); pADPr-binding zinc finger motif (PBZ); the macro domain (Macro) and the WWE domain (WWE) .
T40 7510-7689 Sentence denotes Experimental evidence suggests that other protein modules and sequence motifs can read this modification (see Section 3). (B) Detailed view of the covalent poly(ADP-ribosyl)ation.
T41 7690-7965 Sentence denotes The proximal ADP-ribose (ADPr) is bound by an ester linkage to glutamic (Glu) and aspartic (Asp) amino acid side chains (the asterisk indicates one or two CH 2 units to represent the respective side chains of Asp or Glu) or to lysine (Lys) side chains via a ketamine linkage.
T42 7966-8108 Sentence denotes The mechanism that determines selective modification of specific residues and the functional significance of this heterogeneity are not known.
T43 8109-8221 Sentence denotes Additional ADP-ribose units are subsequently attached by O-glycosidic linkages to form linear or branched pADPr.
T44 8222-8360 Sentence denotes Some components of the pADPr chemical structure recognized by pADPr-binding modules are shown: iso-ADPr, grey shadow, ADPr, yellow shadow.
T45 8361-8363 Sentence denotes 2.
T46 8364-8545 Sentence denotes Affinity of pADPr-binding domains to ADP-ribose metabolites Over the years, several different techniques have been developed to characterize the interaction of pADPr with proteins .
T47 8546-8862 Sentence denotes Polymer-blot and electrophoretic mobility shift assays (EMSA) are currently used to determine whether there is binding or not, while isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and a variation of the EMSA method allow to measure the affinity for pADPr by determining an affinity constant.
T48 8863-8975 Sentence denotes The polymer-blot assay has also been exploited in saturation binding experiments to determine binding affinity .
T49 8976-9092 Sentence denotes Owing to its simplicity, the polymer-blot assay is the most frequently used method to study pADPr-binding proteins .
T50 9093-9265 Sentence denotes Proteins of interest are either hand-spotted, vacuum-aspirated or separated on a polyacrylamide gel prior to being transferred and renatured onto a nitrocellulose membrane.
T51 9266-9428 Sentence denotes The interaction with pADPr is subsequently revealed by incubating the membrane with purified label-free pADPr, [ 32 P]-radiolabeled pADPr or biotin-labeled pADPr.
T52 9429-9551 Sentence denotes Bound pADPr is detected with anti-pADPr antibodies, by autoradiographic exposure or streptavidin conjugates, respectively.
T53 9552-9646 Sentence denotes This technique is also used with peptide arrays to map the pADPr-binding regions of a protein.
T54 9647-9845 Sentence denotes In this assay, peptides are chemically synthesized to represent the sequence of putative pADPr-binding sites, such as those predicted in silico based on consensus motifs (e.g. PBM, see Section 3.1).
T55 9846-9956 Sentence denotes EMSA was developed to characterize pADPr-protein non-covalent interactions in solution (Fahrer et al., 2007) .
T56 9957-10055 Sentence denotes Incubations are made in the presence of pADPr and increasing concentrations of a purified protein.
T57 10056-10129 Sentence denotes Protein-pADPr complexes are subsequently resolved by gel electrophoresis.
T58 10130-10195 Sentence denotes Free and bound pADPr are detected using a streptavidin conjugate.
T59 10196-10287 Sentence denotes A shift in the mobility of the protein indicates the formation of a protein-ligand complex.
T60 10288-10415 Sentence denotes Since all measurements are made at equilibrium, the binding affinities can be calculated using a sigmoidal dose-response curve.
T61 10416-10581 Sentence denotes ITC and SPR are elegant label-free biophysical methods specifically designed to study the interaction kinetics between a ligand (such as pADPr) and a target protein.
T62 10582-10672 Sentence denotes ITC measures the binding affinity and thermodynamics between two biomolecules in solution.
T63 10673-10802 Sentence denotes In this method, a solution that contains a ligand is titrated into a solution of its binding partner until saturation is reached.
T64 10803-10987 Sentence denotes A complete thermodynamic profile of the molecular interaction as well as the binding affinity (K D ) are calculated from the heat released or absorbed over time during the interaction.
T65 10988-11065 Sentence denotes SPR measures in real-time the refractive index changes near a sensor surface.
T66 11066-11258 Sentence denotes A ligand (such as biotinylated pADPr) is immobilized on the surface of a solid support (chip) and the analyte (protein of interest) is passed over the surface to make contacts with the ligand.
T67 11259-11352 Sentence denotes Interactions induce a change in the refractive index proportional to the mass on the surface.
T68 11353-11504 Sentence denotes The data are fitted to a kinetic model to calculate the rate of association (ka), the rate of dissociation (kd) and the binding affinity (K D = kd/ka).
T69 11505-11673 Sentence denotes Each of these methods may also be conducted using fractionated pADPr, allowing further characterization of the binding specificity of a protein for long or short pADPr.
T70 11674-11801 Sentence denotes In addition, SPR and ITC methods are amenable to determine the critical amino acid residues implicated in the binding of pADPr.
T71 11802-11943 Sentence denotes Site-specific mutagenesis of critical residues that mediate pADPrbinding typically leads to at least a 10-fold reduction in binding affinity.
T72 11944-12091 Sentence denotes Collectively, these methods have therefore been critical in characterizing the pADPr binding modules that are described in the following Section 3.
T73 12092-12096 Sentence denotes 3.1.
T74 12097-12101 Sentence denotes PBM:
T75 12102-12136 Sentence denotes The poly(ADP-ribose)-binding motif
T76 12137-12434 Sentence denotes The notion of non-covalent pADPr-binding was originally demonstrated with histones (Sauermann and Wesierska-Gadek, 1986; Wesierska-Gadek and Sauermann, 1988) and later better characterized using pADPr of different lengths and branching frequencies (Panzeter et al., 1992 (Panzeter et al., , 1993 .
T77 12435-12708 Sentence denotes This concept was further extended to non-histone proteins such as p53, DNA-PK or KU70/80 and led to the definition of a common polymer-binding domain of 22-26 amino acids that conveyed the specific affinity for pADPr (Table 1) (Althaus et al., 1999; Malanga et al., 1998) .
T78 12709-12943 Sentence denotes Notably, the Althaus group had a strong intuition when they raised the innovative hypothesis that ''PARP-associated polymers may recruit signal proteins to sites of DNA breakage and reprogram their functions'' (Althaus et al., 1999) .
T79 12944-13185 Sentence denotes The first defined pADPr-binding motif (PBM) was derived from a region of high similarity in a multiple sequence alignment of proteins involved in signaling pathways that control cell cycle progression and DNA damage (Pleschke et al., 2000) .
T80 13186-13343 Sentence denotes This PBM is composed of a property-based sequence motif harboring basic and hydrophobic residues downstream of a lysine-and arginine-rich region ( Fig. 2A) .
T81 13344-13595 Sentence denotes Consistent with their previous observation with histones, the authors reported that long and branched pADPr are the preferred ligands of non-chromatin proteins comprising the PBM (Panzeter et al., 1992 (Panzeter et al., , 1993 Pleschke et al., 2000) .
T82 13596-13750 Sentence denotes To better define and address prediction accuracy of the PBM, we adopted a strategy based on a refinement of the consensus PBM motif (Gagne et al., 2008) .
T83 13751-13854 Sentence denotes We showed that restrictions to specific amino acid types exist for positions within the PBM (Fig. 2B ).
T84 13855-14127 Sentence denotes The previously reported preference for hydrophobic residues [ACGVILMFYW] was recovered, but there was a clear tendency for limited residue types to be allowed (mostly aliphatic residues), especially at position À1, +1 and +2 relative to the central K/R doublet (Fig. 2B) .
T85 14128-14238 Sentence denotes Clearly, the PBM refers to the conservation of a physical property pattern rather than a fixed sequence motif.
T86 14239-14401 Sentence denotes The refined motif offers a more stringent definition of the original motif that decreases the probability of a PBM arising by chance in a protein database search.
T87 14402-14548 Sentence denotes Computational PBM prediction has proven to be a powerful tool for the identification of protein regions that could mediate interaction with pADPr.
T88 14549-14617 Sentence denotes They have been shown to convey important functions in animal models.
T89 14618-14834 Sentence denotes Notably, the PBM discovered in the apoptosis-inducing factor (AIF) is critical for Macro Egloff et al. (2006) the ability of AIF to induce cell death by parthanatos (PARP1-dependent cell death) in cells and in vivo .
T90 14835-15092 Sentence denotes A detailed PBM-pADPr complex has yet to be modeled but a study of the structural features of AIF's PBM showed that it occupies an area on the surface of the protein which could provide stabilizing non-covalent contacts of amino acid side chains with pADPr .
T91 15093-15276 Sentence denotes We can only speculate as to whether all PBMs possess common structural features, but a highly exposed solvent-accessible surface must be present to make contacts with pADPr molecules.
T92 15277-15411 Sentence denotes Based on the helix propensity scale, positively charged amino acids (K/R) have a tendency to form a-helices (Pace and Scholtz, 1998) .
T93 15412-15541 Sentence denotes Since PBMs are located in lysine-and arginine-rich regions, it would be likely to find several of them in a helical conformation.
T94 15542-15657 Sentence denotes A summary of pADPr-binding proteins for which binding affinity constants have been determined is given in Table 2 .
T95 15658-15829 Sentence denotes Of particular interest, several reports have shown that pADPr chain length is a crucial determinant for high affinity non-covalent interactions of PBM proteins with pADPr.
T96 15830-16285 Sentence denotes The binding of the tumor suppressor protein p53, the nucleotide excision repair XPA, and the DEK oncoprotein with long (55-mer) and short (16-mer) pADPr chains were assessed by EMSA and SPR a Listed proteins were retrieved from studies that specifically addressed the direct non-covalent binding to pADPr. b n.d. not determined. c Proteins shown to accumulate at DNA-damage sites in a pADPr-dependent fashion. (Fahrer et al., 2007 (Fahrer et al., , 2010 .
T97 16286-16508 Sentence denotes These experiments revealed the high affinity (10 À7 to 10 À9 M range) of XPA and DEK to long pADPr chains but the lack of binding to short pADPr while p53 bound both short and long chains of pADPr with equivalent affinity.
T98 16509-16711 Sentence denotes Remarkably, PBMs are present in a marked number of proteins involved in the response to DNA damage and other chromatin transactions such as chromatin structure, replication and transcription (Table 1) .
T99 16712-16815 Sentence denotes Furthermore, the PBM often overlaps with important regulatory protein domains (Pleschke et al., 2000) .
T100 16816-17009 Sentence denotes This has triggered the idea that upon binding to pADPr, the PBM could shield a regulatory surface by steric hindrance, thus destabilizing several protein-protein or protein-ligand interactions.
T101 17010-17158 Sentence denotes Alternatively, a highly extended and flexible polymer bound to a protein domain could distort it so that perturbations of the native fold may arise.
T102 17159-17322 Sentence denotes Globally, molecular crowding by the pADPr provides the basis for the concept of ''reprogrammation'' of protein functions as suggested (Malanga and Althaus, 2005) .
T103 17323-17550 Sentence denotes Actually, the affinity of several DNA damage response factors for pADPr can modulate (I) the sensing of DNA lesions; (II) the dynamic chromatin remodeling events and (III) the assembly and functionality of DNA repair complexes.
T104 17551-17761 Sentence denotes We believe that the transient accumulation of pADPr following DNA-dependent PARP activation can result in vast pleiotropic effects on a systems-wide scale that implicates numerous DNA damage response effectors.
T105 17762-17900 Sentence denotes This is supported by the predominant presence of nucleic acid-interacting proteins in the PBM's prediction datasets (Gagne et al., 2008) .
T106 17901-18132 Sentence denotes Indeed, DNA-and RNA-binding modules are significantly over-represented as putative pADPr-binding modules and thus represent a general class of pADPr-targeted proteins with potential for broad implication in the DNA damage response.
T107 18133-18334 Sentence denotes However, in some proteins, the PBM is distinct from the nucleic acid binding domains, such as in AIF, providing the ability of pADPr to modulate protein function in the context of nucleic acid binding.
T108 18335-18479 Sentence denotes Generally, proteins associate in multi-protein complex machineries that execute biological processes that a single protein cannot execute alone.
T109 18480-18698 Sentence denotes Macromolecules that disrupt or stabilize such complexes drive a wide variety of cellular processes. pADPr possesses the biochemical and structural properties to fulfill such functions through non-covalent interactions.
T110 18699-18793 Sentence denotes Similar to DNA and RNA, the pADPr carries a net negative charge due to its phosphate backbone.
T111 18794-18982 Sentence denotes Because these three macromolecules tend to bind positively charged protein domains, some competition exists among DNA, RNA or pADPr for the same binding site in specific cellular contexts.
T112 18983-19109 Sentence denotes Indeed, in addition to the classical PBM, recent studies suggest alternative PBMs located in nucleic acid-interaction domains.
T113 19110-19434 Sentence denotes The glycine-and arginine-rich domain (GAR) lacks hydrophobic amino acids commonly found in PBMs. This region rather accumulates a very high positive charge owing to the presence of a repetition of arginine residues, thus making it an ideal binding surface for a polymer with a high negative charge density such as the pADPr.
T114 19435-19695 Sentence denotes The GAR, also referred to as RGG box and the RG domain, is a protein module typically found in proteins involved in RNA metabolism (Burd and Dreyfuss, 1994) as well as in some chromatin associated proteins (Bernstein and Allis, 2005; Kornblihtt et al., 2009) .
T115 19696-20243 Sentence denotes Selected examples include fibrillarin (FBL), heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), fragile X mental retardation protein 1 (FMRP), small nuclear ribonucleoprotein Sm D1 (SNRPD1), chromatin target of PRMT1 (Protein arginine N-methyltransferase 1) protein (CHTOP), bromodomain and WD repeat-containing protein 3 (BRWD3), cell death and ATM (serine-protein kinase ATM) regulator AVEN, Ras GTPase-activating protein-binding protein 1 (G3BP1), double-strand break repair protein MRE11 and tumor suppressor p53-binding protein 1 (53BP1).
T116 20244-20344 Sentence denotes Evidence that the GAR is a pADPr-binding module came from the study of MRE11 (Haince et al., 2008) .
T117 20345-20625 Sentence denotes MRE11, a core component of the MRN complex (MRE11, RAD50 and NBS1), is responsible for the initial recognition of DNA double-strand breaks (DSBs), mediates end-resection by its exonuclease activity and together with 53BP1 and other DNA damage response factors, facilitates repair.
T118 20626-20857 Sentence denotes The GAR domain of MRE11 is required for its DNA binding activity (Boisvert et al., 2005; Dery et al., 2008) but also mediates its interaction with pADPr as well as its rapid accumulation at DNA strand breaks (Haince et al., 2008) .
T119 20858-21069 Sentence denotes The MRE11 exonuclease activity is inhibited by pADPr in vitro, suggesting that pADPr may regulate MRE11-dependent end-resection at DSBs or at stalled replication forks, as recently reported (Ying et al., 2012) .
T120 21070-21185 Sentence denotes Interestingly, several other GARcontaining proteins participate in the DNA damage response and genome surveillance.
T121 21186-21367 Sentence denotes In view of the high pADPr level that transiently accumulates at sites of damage, it is suspected that the function of some of these GAR-bearing proteins might be regulated by pADPr.
T122 21368-21589 Sentence denotes 53BP1 regulates repair of DSBs, while the nuclear DNA helicase II (RNA Helicase A) interacts and regulates the DSBs biomarker c-H2AX (Mischo et al., 2005) and the Werner syndrome helicase (WRN) (Friedemann et al., 2005) .
T123 21590-21713 Sentence denotes The nucleosome remodeling and histone deacetylase (NuRD) complex comprises several core components with affinity for pADPr.
T124 21714-21978 Sentence denotes Methyl-CpG-binding domain protein 2 (MBD2) has a GAR motif, while CHD4 (chromodomain helicase DNA binding protein 4) and MTA1 (metastasis associated 1) interact with pADPr through a still undefined motif (Chou et al., 2010; Lai and Wade, 2011; Polo et al., 2010) .
T125 21979-22126 Sentence denotes The latter two proteins are involved in the recruitment of the NuRD complex to DNA strand breaks in a pADPr-dependent manner (Lai and Wade, 2011) .
T126 22127-22324 Sentence denotes Our current understanding suggests that the presence of pADPr acts as a recruitment module for the organization of the PARP1-associated DNA repair and chromatin remodeling machinery at DNA lesions.
T127 22325-22519 Sentence denotes On the other hand, pADPr binding to the GAR domain could be considered as a DNA displacement mechanism required to reconfigure the DNA repair protein complexes and provide access to damaged DNA.
T128 22520-22697 Sentence denotes It may also interfere with other DNA damage-induced posttranslational modifications, such as PRMT1-dependent arginine methylation in the GAR domain (Bedford and Richard, 2005) .
T129 22698-22788 Sentence denotes This view supports a concept where pADPr is a key orchestrator of the DNA damage response.
T130 22789-22979 Sentence denotes Recently, it has been suggested that pADPr regulates post-transcriptional gene regulation in the cytoplasm, notably through the assembly of cytoplasmic stress granules (Leung et al., 2012) .
T131 22980-23080 Sentence denotes In support of this, we showed that the stress granule effector G3BP1 binds pADPr by its GAR domain .
T132 23081-23309 Sentence denotes Importantly, G3BP1-mediated stress granule assembly is impaired by PARP inhibition during genotoxic insult, suggesting that pADPr is critical for the reprogrammation of messenger ribonucleoparticles in cellular stress responses.
T133 23310-23465 Sentence denotes This result adds to the experimental evidence that the pADPrbinding protein AIF functions as a negative regulator of stress granules (Cande et al., 2004) .
T134 23466-23697 Sentence denotes These results emphasize the fact that pADPr can perform various functions in several different DNA damage-processing pathways and can enable a crosstalk between the regulation of the early and late steps of the DNA damage response.
T135 23698-23901 Sentence denotes The RNA recognition motif (RRM), also referred to as RNA-binding domain (RBD) or ribonucleoprotein domain (RNP), is the most abundant nucleic acid-binding motif in the human genome (Clery et al., 2008) .
T136 23902-23969 Sentence denotes RRMs are found in a wide variety of RNA and ssDNA-binding proteins.
T137 23970-24032 Sentence denotes RRMs may be found in conjunction with GAR-containing proteins.
T138 24033-24368 Sentence denotes One prominent example is hnRNP A1 that possesses two RRMs in addition to its GAR motif. hnRNPs are highly versatile proteins that can participate in various aspects of nucleic acid metabolism: mRNA stability and splicing, DNA replication, chromatin remodeling, telomere maintenance, DNA repair and genome stability (Han et al., 2010) .
T139 24369-24555 Sentence denotes Based on a proteome-wide screen to identify pADPr-binding proteins, our laboratory was the first to identify hnRNPs as a family of proteins with affinity for pADPr (Gagne et al., 2003) .
T140 24556-24800 Sentence denotes More recently, Ji and Tulin (2009) validated this finding by providing evidence that hrp38 (the Drosophila melanogaster homologue of human hnRNP A1) binds pADPr in a non-covalent way in vivo, with the consequence of reduced RNA-binding ability.
T141 24801-25021 Sentence denotes RNA processing factors (such as NONO and RBMX) recently emerged as guardians of genomic stability with widespread involvement in preventing DNA damage (Adamson et al., 2012; Krietsch et al., 2012; Paulsen et al., 2009) .
T142 25022-25256 Sentence denotes Both NONO (Non-POU domain-containing octamer-binding protein) and RBMX (RNA-binding motif protein, X chromosome) comprise RRMs and are recruited in a pADPr-dependent manner to DNA damage (Adamson et al., 2012; Krietsch et al., 2012) .
T143 25257-25331 Sentence denotes We have recently reported the binding of pADPr to the RRM1 domain of NONO.
T144 25332-25428 Sentence denotes As it was observed for hnRNPA1, pADPr interfered with the interaction of NONO with RNA in vitro.
T145 25429-25579 Sentence denotes Notably, the binding of NONO to pADPr by RRM1 is crucial for the recruitment of NONO to DNA damage sites and influences the outcome of DNA DSB repair.
T146 25580-25654 Sentence denotes The high binding affinity of pADPr to NONO was assessed by SPR (Table 2 ).
T147 25655-25821 Sentence denotes Similar to DEK and XPA described above, NONO showed a strong affinity for non-fractionated and long pADPr chains while no binding was detected for short pADPr chains.
T148 25822-25932 Sentence denotes These observations therefore provide further support for RRMs as biologically relevant pADPr-binding modules .
T149 25933-26145 Sentence denotes Given the frequent occurrence of RRM-containing proteins in the human proteome, interactions with pADPr are likely to have a significant impact on cell signaling through a complex network of biochemical pathways.
T150 26146-26385 Sentence denotes Another large group of RRM-containing proteins that bind to RNAs are the SR (Serine/Arginine repeats) proteins (Long and Caceres, 2009 ) that, along with hnRNPs, contribute to the formation of messenger ribonucleoprotein particles (mRNPs).
T151 26386-26487 Sentence denotes It has been shown that the SR protein ASF/SF2 binds pADPr with high affinity (Malanga et al., 2008) .
T152 26488-26654 Sentence denotes Two domains in ASF/SF2 can mediate the interaction with pADPr: (I) a N-terminal fragment that contains a RRM1 and (II) a C-terminal SR domain (Malanga et al., 2008) .
T153 26655-26918 Sentence denotes Given that the SR domain carries an excess positive charge with the predominance of arginine residues, this pADPr-binding feature resembles that of the GAR which also harbors a basic arginine-rich cluster expected to interact with the phosphate backbone of pADPr.
T154 26919-27077 Sentence denotes Similarly, strong pADPr-binding was observed in lysine-and arginine-rich (K/R-rich) motifs located in the nucleosome remodeler dMi-2 (Murawska et al., 2011) .
T155 27078-27365 Sentence denotes It remains to be determined whether the presence of a basic electrostatic patch on a protein surface could be considered as a general pADPr-protein interface or if additional structural determinants are required (such as the helical conformation of the SR domain (Sellis et al., 2012) ).
T156 27366-27552 Sentence denotes Zinc fingers specifically interacting with pADPr rather than DNA or RNA were discovered in a subset of proteins related directly or indirectly with pADPr metabolism (Ahel et al., 2008) .
T157 27553-27765 Sentence denotes This newly identified C2H2-type ''pADPr-binding zinc finger'' (PBZ) has a consensus sequence defined as [K/R]xxCx[F/Y]GxxCxbbxxxxHxxx[F/Y]xH where b denotes a basic residue and x any residue (Ahel et al., 2008) .
T158 27766-27842 Sentence denotes PBZ domains have been observed only in eukaryotic proteins, excluding yeast.
T159 27843-28023 Sentence denotes The absence of PBZ motifs in prokaryote and yeast proteins parallels the absence of pADPr metabolism in those, suggesting a coevolution of the PBZ motif with the presence of PARPs.
T160 28024-28289 Sentence denotes Only three human proteins appear to carry a PBZ motif: the aprataxin and PNK-like factor (APLF, also called XIP1, PALF), the checkpoint protein with FHA and RING domains (CHFR), and the DNA cross-link repair 1A protein (DCLRE1A/SNM1A) (Fig. 3) (Ahel et al., 2008) .
T161 28290-28419 Sentence denotes Interestingly, the PBZ module was found in some non-human proteins involved in the maintenance of genome integrity or DNA repair:
T162 28420-28554 Sentence denotes Ku70, Rad17, Parp and Chk2 in Dictyostelium discoideum and DNA Ligase in Caenorhabditis elegans corresponding to human DNA Ligase III.
T163 28555-28658 Sentence denotes However, the human orthologues do not contain any PBZ domain (Ahel et al., 2008; Isogai et al., 2010) .
T164 28659-28769 Sentence denotes Human CHFR and DCLRE1A contain a unique PBZ while APLF has two PBZ placed in tandem (PBZ1 and PBZ2 - Fig. 3) .
T165 28770-29126 Sentence denotes Structural studies of the PBZ domains with small molecules that mimic the features of pADPr have revealed that besides the cysteines and histidines coordinating the Zn ion, critical aromatic residues mediate interactions with the adenine ring of ADPr (Ahel et al., 2008; Eustermann et al., 2010; Isogai et al., 2010; Li et al., 2010; Oberoi et al., 2010) .
T166 29127-29332 Sentence denotes The lack of conservation of most of these critical binding residues in the PBZ sequence of DCLRE1A suggests that it may not bind pADPr, but this has not been experimentally assessed (Oberoi et al., 2010) .
T167 29333-29523 Sentence denotes The affinity of CHFR (5 Â 10 À10 M) and APLF (9.5 Â 10 À10 M) for pADPr measured by SPR indicates that this module has the highest affinity for pADPr of all pADPr-binding domains (Table 2) .
T168 29524-29647 Sentence denotes Interestingly, the affinity of APLF for pADPr is in the same range than that of CHFR, despite the fact that it has two PBZ.
T169 29648-29867 Sentence denotes Each PBZ of APLF binds independently pADPr, but with reduced affinity relative to the tandem PBZ and the full length protein (Table 2) (Ahel et al., 2008; Eustermann et al., 2010; Li et al., 2010; Rulten et al., 2008) .
T170 29868-29999 Sentence denotes Moreover, the affinity of PBZ1 for pADPr is 10-fold higher than that of PBZ2 (Table 2) (Eustermann et al., 2010; Li et al., 2010) .
T171 30000-30289 Sentence denotes These observations are in line with the structural details of CHFR and APLF, which strongly suggested that the CHFR PBZ and the PBZ1 domain of APLF are able to interact with two consecutive ADPr moieties in pADPr while the second PBZ of APLF probably binds only one (Oberoi et al., 2010) .
T172 30290-30479 Sentence denotes These observations are also consistent with the more deleterious effects of mutations in PBZ1 than in PBZ2 for the recruitment of APLF to UV-induced DNA strand breaks Rulten et al., 2008) .
T173 30480-30629 Sentence denotes PBZ1 may also interact with PARP1 as well, providing further affinity of PBZ1 for automodified PARP1 (Eustermann et al., 2010; Macrae et al., 2008) .
T174 30630-30910 Sentence denotes The role of APLF in the DNA damage response and repair via the non-homologous end-joining (NHEJ) pathway has been recognized by several research groups (Bekker-Jensen et al., 2007; Iles et al., 2007; Kanno et al., 2007; Li et al., 2010; Macrae et al., 2008; Rulten et al., 2008) .
T175 30911-31048 Sentence denotes APLF comprises a N-terminal FHA domain and displays apurinic-apyrimidinic (AP) endonuclease and 3 0 -5 0 exonuclease activities in vitro.
T176 31049-31158 Sentence denotes APLF is rapidly recruited to DNA strand breaks introduced by UV irradiation via both its FHA and PBZ domains.
T177 31159-31360 Sentence denotes While the FHA domain mediates interactions with the repair proteins XRCC1 and XRCC4, the tandem PBZ domain directs pADPr-dependent recruitment of APLF to DNA strand breaks, where APLF facilitates NHEJ.
T178 31361-31523 Sentence denotes Both PARP1 and PARP3-dependent poly(ADP-ribosyl)ation have been shown to promote APLF responses to DNA strand breaks (Rulten et al., 2008 (Rulten et al., , 2011 .
T179 31524-31821 Sentence denotes Similar to APLF, CHFR comprises a phospho-binding FHA module but also a RING finger domain with E3 ubiquitin ligase activity that plays an essential role in the antephase checkpoint, delaying mitotic entry under certain stress conditions (Chaturvedi et al., 2002; Scolnick and Halazonetis, 2000) .
T180 31822-32102 Sentence denotes This function of CHFR is dependent on its interaction with PARP1 and pADPr, because mutations in the CHFR PBZ that disrupts pADPr-binding as well as PARP inhibitors both abolish the CHFR-dependent mitotic checkpoint (Ahel et al., 2008; Kashima et al., 2012; Oberoi et al., 2010) .
T181 32103-32241 Sentence denotes Recently, a mechanistic interplay behind CHFR and pADPr interactions inducing a mitotic entry delay was uncovered (Kashima et al., 2012) .
T182 32242-32395 Sentence denotes The activation of PARP1 following mitotic stress promotes the pADPr-dependent ubiquitylation of PARP1 by CHFR and its proteasomal degradation (Fig. 4A ).
T183 32396-32500 Sentence denotes This finding further revealed that PARP1 levels must be critically controlled during cell proliferation.
T184 32501-32554 Sentence denotes The binding of DCLRE1A to pADPr has not been studied.
T185 32555-32704 Sentence denotes This protein has an endonuclease function and it is involved in the repair of DNA interstrand cross-links (Hazrati et al., 2008; Yang et al., 2010) .
T186 32705-32868 Sentence denotes However, the putative PBZ does not comprise the aromatic residues needed to contact pADPr, suggesting that its functions are independent of poly(ADP-ribosyl)ation.
T187 32870-33043 Sentence denotes The macro domain was first described in a variant of the histone H2A, as a 130-190 amino acid module on the C-terminal side of the histone domain (Pehrson and Fried, 1992) .
T188 33044-33296 Sentence denotes It was soon recognized as an important protein domain, well conserved throughout all living organisms, as well as in a small subset of single-stranded RNA viruses that infect mammalian cells, including coronaviruses, alphaviruses and hepatitis E virus.
T189 33297-33460 Sentence denotes Determination of the 3D structure of the thermophile Archaeoglobus fulgidus macro protein Af1521 provided the first clues to the potential function of this domain.
T190 33461-33650 Sentence denotes It revealed an organization of helices and sheets reminiscent of the P-loop found in nucleotide hydrolases, suggesting a related enzymatic function for the macro fold (Allen et al., 2003) .
T191 33651-33932 Sentence denotes Additional studies with Af1521 and with the yeast macro domain protein YBR022 W supported this observation as they revealed the ability of these macro domains to hydrolyze ADP-ribose-1''-phosphate into ADPr and inorganic phosphate (Pi) (Karras et al., 2005; Martzen et al., 1999) .
T192 33933-34201 Sentence denotes Subsequent studies indicated that some macro domains could not only interact with ADPr but also with pADPr, making it a novel pADPr-interaction module (Ahel et al., 2009; Gottschalk et al., 2009; Karras et al., 2005; Neuvonen and Ahola, 2009; Timinszky et al., 2009) .
T193 34202-34304 Sentence denotes Based on primary amino acid sequence comparisons, 11 human proteins comprise a macro domain (Fig. 3) .
T194 34305-34639 Sentence denotes This macro domain may exist on its own (macroD1, also called MDO1 and LRP16, macroD2 also called MDO2 and C6orf130) or in association with the histone fold (macrohistones H2A), the PARP catalytic domain (PARP9, 14 and 15), the SNF2/helicase ATPase domain (ALC1/CHD1L), or the Sec14p/CRAL-TRIO protein-lipid interaction module (GDAP2).
T195 34640-34908 Sentence denotes Intriguingly, macroPARPs (also called BAL PARPs) have the unique feature of bearing two (PARP9 and PARP15) or even three (PARP14) macro domains in tandem, which, in PARP14, are further associated with two more putative pADPr binding modules, a RRM and a WWE (Fig. 3) .
T196 34909-35078 Sentence denotes The affinity of most human macro domain proteins for NAD + -derived metabolites permits the assessment of the importance of this module for pADPr binding and metabolism.
T197 35079-35302 Sentence denotes It should be stressed that, out of nine macro domain-containing human proteins so far tested for pADPr binding (PARP14 and PARP15 have not been examined), only four bind pADPr (namely macroH2A1.1, CHD1L, macroD1 and PARP9).
T198 35303-35385 Sentence denotes The others have affinity only for ADPr or O-acetyl-ADP-ribose ( Fig. 1; Table 2 ).
T199 35386-35593 Sentence denotes Furthermore, the detailed structural analysis of macroH2A1.1 revealed that it is able to bind solely the terminal ADPr of the polymer, indicating that its macro domain is in practice an ADPr binding module .
T200 35594-35723 Sentence denotes Therefore, structural details critically affect the ability of the macro fold binding pocket to accommodate ligands such as ADPr.
T201 35724-35846 Sentence denotes For instance, the macroH2A1.2 and macroH2A2 variants as well as GDAP2 do not interact with ADPr Till and Ladurner, 2009 ).
T202 35847-36090 Sentence denotes While the structural characteristics of macroH2A1.2 differ only slightly from those of the macroH2A1.1 variant, three additional amino acid residues in macroH2A1.2 critically fall in the ADPr binding pocket, thereby hindering the interaction .
T203 36091-36334 Sentence denotes The structure of some viral macro domains (also called X-domain) has also been investigated as well as their ability to interact with ADPr and pADPr (Egloff et al., 2006; Malet et al., 2009; Neuvonen and Ahola, 2009; Piotrowski et al., 2009) .
T204 36335-36432 Sentence denotes As it is the case for human proteins, some but not all tested present affinity for ADPr or pADPr.
T205 36433-36732 Sentence denotes The binding of viral macro domains to ADPr is at least 10-fold lower (K D of the severe acute respiratory syndrome (SARS) coronavirus is 24 lM, that of hepatitis E virus above 50 lM) but interactions with pADPr have been shown by polymer blot assays (Egloff et al., 2006; Neuvonen and Ahola, 2009) .
T206 36733-36824 Sentence denotes It remains to be determined whether this interaction is critical for viral host infections.
T207 36825-37015 Sentence denotes Collectively, these observations indicate that the presence of a macro fold hints to a possible interaction with ADPr-related metabolites, which however needs to be experimentally addressed.
T208 37016-37188 Sentence denotes Recent structural analysis of poly(ADP-ribose) glycohydrolase (PARG) revealed the striking finding that its pADPr-interaction module folds in a macro domain-like structure.
T209 37189-37420 Sentence denotes First identified in the distantly PARG-related bacterial protein DUF2263, this finding was then confirmed with the structural analysis of a protozoan PARG and rat PARG (Dunstan et al., 2012; Kim et al., 2012a; Slade et al., 2011) .
T210 37421-37684 Sentence denotes Therefore, despite minimal sequence similarity between the typical macro domain and PARG, part of the PARG catalytic domain adopts this characteristic macro fold organization in which the PARG sequence signature GGG-X 6-8 -QEE lines the ADP-ribose binding pocket.
T211 37685-37901 Sentence denotes However, in the mammalian PARG, additional essential sequences extending beyond the macro domain adopt structural conformations around the macro fold that specify the catalytic pocket and the glycohydrolase activity.
T212 37902-38185 Sentence denotes Importantly, a unique ''tyrosine clasp'' near the catalytic pocket offers an explanation for the exoglycosidic and endoglycosidic activities of mammalian PARG towards pADPr (Brochu et al., 1994) , the latter endoglycosidic activity lacking in the bacterial PARG (Kim et al., 2012a) .
T213 38186-38419 Sentence denotes These recent findings have thus highlighted that some macro domains may only be revealed once the 3D structure is determined, indicating that there may be other pADPr-binding macro proteins in mammalian cells awaiting identification.
T214 38420-38563 Sentence denotes Many of the macro domain-ADPr/pADPr interactions have been examined in vitro, using purified macro domains or proteins and purified ADPr/pADPr.
T215 38564-38768 Sentence denotes Because PARPs catalyze the addition of ADPr onto protein substrates, it will be critical to investigate whether the macro-ADPr interaction can be extended to ADPr covalently attached to acceptor proteins.
T216 38769-38960 Sentence denotes A recent study using synthetic peptides corresponding to mono-ADP-ribosylated histone H2B tail showed that it could be the case, as macroH2A1.1 did bind such peptides (Moyle and Muir, 2010) .
T217 38961-39084 Sentence denotes Recent detailed analysis of the enzymatic activity of the macro domain indicates that some deacetylate O-acetyl-ADP-ribose.
T218 39085-39196 Sentence denotes This molecule is produced by the sirtuin deacetylases, which uses NAD + as a co-factor to deacetylate proteins.
T219 39197-39427 Sentence denotes The three stand-alone macro domain proteins, namely macroD1, macroD2, and C6orf130, appear to form a subgroup of macro domain proteins showing this deacetylase activity by cleaving the ester bond between the acetyl group and ADPr.
T220 39428-39589 Sentence denotes One could envision that some macro domain proteins possessing O-acetyl-ADPr deacetylase activity may be able to hydrolyze the protein-ADPr ester bond (Fig. 1B) .
T221 39590-39675 Sentence denotes Until now, the ability of PARG and ARH3 to fulfill this function has been questioned.
T222 39676-39936 Sentence denotes The existence of a distinct enzyme (an ADP-ribose lyase) able to hydrolyze the ester bond between the glutamic or aspartic acid residue of the acceptor protein and ADPr has been proposed nearly 30 years ago (Oka et al., 1984) , but remains to be characterized.
T223 39937-40163 Sentence denotes However, the recent indications that lysines could also constitute ADPr acceptor sites on PARP1 and histones, forming a ketamine in this case (Fig. 1B) Messner et al., 2010) , suggest that there may be more than one ''lyase''.
T224 40164-40243 Sentence denotes Biological functions of macro domain proteins remain to be examined in details.
T225 40244-40444 Sentence denotes Macrohistones and CHD1L contribute to the structure of chromatin (see Sections 4.4 and 4.5) and regulatory transcriptional functions have been ascribed to PARP9, PARP14, macroD1 and macroH2A variants.
T226 40445-40754 Sentence denotes The latter macrohistones have been generally linked to transcriptional repression as they induce a more condensed chromatin state and impede access to transcription factors, although in some specific cases they can also promote transcription (reviewed by (Gamble and Kraus, 2010) ) (Muthurajan et al., 2011) .
T227 40755-40846 Sentence denotes PARP9 was also shown to repress transcription via its macro domains (Aguiar et al., 2005) .
T228 40847-41152 Sentence denotes In contrast, PARP14, also named CoaSt6 because of its co-activator function for the transcription factor Stat6, promotes interleukin-4 dependent gene activation in a manner dependent on its macrodomains as well on its mono-ADP-ribosyl-transferase activity (Goenka and Boothby, 2006; Goenka et al., 2007) .
T229 41153-41384 Sentence denotes MacroD1, originally named leukemia related protein 16 (LRP16), was identified as a co-activator of androgen and estrogen receptor transcriptional activity as well as of NF-jB (Han et al., 2007; Wu et al., 2011; Yang et al., 2009 ).
T230 41385-41464 Sentence denotes This transcriptional co-activation of macroD1 is dependent on its macro domain.
T231 41465-41580 Sentence denotes Collectively, these examples suggest that pADPr-macro domain interactions contribute to transcriptional regulation.
T232 41581-41817 Sentence denotes The most recently discovered pADPr-binding motif, the so-called WWE domain, is named after its most conserved amino acids (tryptophan (W) and glutamate (E)), which are flanked by an otherwise rather low degree of sequence conservation .
T233 41818-42023 Sentence denotes The 12 human proteins that contain a WWE domain (Fig. 3 ) belong mostly to two functional classes of proteins, namely those associated with ubiquitylation and those associated with poly(ADP-ribosyl)ation .
T234 42024-42260 Sentence denotes The best studied example is the RING finger protein 146 (RNF146) also called Iduna. pADPr binding of Iduna/RNF146 was first ascribed to a PBM which was further defined as part of the WWE domain that mediates the interaction with pADPr .
T235 42261-42451 Sentence denotes The structural characteristics of the Iduna/RNF146 WWE domain and its interaction with pADPr were thoroughly investigated by polymer blot assays, NMR and crystallography Wang et al., 2012) .
T236 42452-42555 Sentence denotes Iso-ADPr rather than ADPr is the smallest unit that can be bound by the Iduna/RNF146 domain (Fig. 1B) .
T237 42556-42746 Sentence denotes The lack of interaction between WWE and ADPr was explained by the need for phosphate groups on either side of the adenine-ribose structure to make extensive contacts with the binding pocket.
T238 42747-42892 Sentence denotes This supported the idea that the WWE domain is a pADPr-binding module because at least two ADPr units are needed to generate the iso-ADPr ligand.
T239 42893-43055 Sentence denotes The proposed structure is compatible with interactions with iso-ADPr within longer pADPr chains , consistent with the co-purification of Iduna/RNF146 with pADPr .
T240 43056-43154 Sentence denotes Four residues have been identified as crucial for iso-ADPr-binding in the Iduna/RNF146 WWE domain.
T241 43155-43374 Sentence denotes These residues are well conserved throughout most human WWE domains , including the putative or demonstrated ubiquitin ligases Deltex 1,2,4, HUWE1 and TRIP12, which have been shown by SPR to also bind pADPr ( Table 2 ).
T242 43375-43537 Sentence denotes The WWE domains of several PARP family members (PARP11, PARP13 and the first WWE of PARP12) also comprise the conserved residues, suggesting that they bind pADPr.
T243 43538-43683 Sentence denotes Only the binding of PARP11 has been examined, and showed interactions with ADPr and pADPr with rather low affinity (Table 2) Wang et al., 2012) .
T244 43684-43941 Sentence denotes In contrast, two of the residues are not conserved in the second WWE of PARP12, in TiPARP, PARP14 and the putative phospholipase DDHD2, suggesting that these may not bind pADPr, as shown for DDHD2 (DDHD domain containing 2) in in vitro binding experiments .
T245 43942-44130 Sentence denotes A common theme among WWE containing proteins is the association with domains of the E3 ligase type, strongly suggesting a functional link between ubiquitylation and poly(ADP-ribosyl)ation.
T246 44131-44281 Sentence denotes This link was uncovered in the regulation of Wnt/b-catenin signaling pathway by tankyrases and Iduna/RNF146 (Huang et al., 2009; Zhang et al., 2011) .
T247 44282-44412 Sentence denotes This pathway, essential for embryonic development and adult tissue homeostasis, is tightly regulated by the concentration of axin.
T248 44413-44541 Sentence denotes It turns out that axin is a substrate for tankyrase such that its poly(ADP-ribosyl)ation allows its recognition by Iduna/RNF146.
T249 44542-44664 Sentence denotes Binding of Iduna/RNF146 to poly(ADP-ribosyl)ated axin triggers axin ubiquitylation and proteasomal degradation (Fig. 4A) .
T250 44665-44903 Sentence denotes Interestingly, we concurrently showed that Iduna/RNF146 plays a prominent role in the context of DNA damage through its pADPr-dependent E3 ligase activity as it also ubiquitylates several DNA repair factors in a way that depends on pADPr.
T251 44904-45060 Sentence denotes PARP1/2, KU70, XRCC1 and DNA ligase III were identified as Iduna/RNF146 substrates when poly(ADP-ribosyl)ated by PARP1 (see Section 4.2 and Fig. 4B and E) .
T252 45061-45208 Sentence denotes The cross-talk between ubiquitylation and poly(ADP-ribosyl)ation may not be restricted to Iduna/RNF146 but awaits further experimental examination.
T253 45209-45298 Sentence denotes For instance, the E3 ligases Deltex1, Deltex2 and Deltex3 play a role in notch signaling.
T254 45299-45437 Sentence denotes The N-terminus of these proteins contains tandem WWE motifs mediating interactions with the ankyrin repeats of Notch intracellular domain.
T255 45438-45663 Sentence denotes Several studies performed in vivo and in cell culture have shown that Notch ubiquitylation is promoted by Deltex expression (Baron, 2012; Hori et al., 2004; Matsuno et al., 2002; Mukherjee et al., 2005; Wilkin et al., 2008) .
T256 45664-45907 Sentence denotes In the context of DNA damage responses, HUWE1 (also called Mule, ARF-BP1, LASU1 and HectH9) and TRIP12 (also called E3 ubiquitin ligase for Arf (ULF)) both belong to the family of HECT domain (homologous to E6-AP carboxyl terminus) E3 ligases.
T257 45908-46098 Sentence denotes HUWE1 participates in the DNA damage response by tightly regulating steady state levels of XRCC1, DNA polymerase b, and DNA ligase III (Khoronenkova and Dianov, 2011; Parsons et al., 2009) .
T258 46099-46192 Sentence denotes Several substrates of HUWE1 for ubiquitylation/ proteosomal degradation have been identified:
T259 46193-46307 Sentence denotes Cdc6, the c-Myc oncogene, histones, as well as p53 (Adhikary et al., 2005; Chen et al., 2005; Hall et al., 2007) .
T260 46308-46617 Sentence denotes TRIP12 is a key regulator of the DNA damage response (Gudjonsson et al., 2012) by acting as a guard against excessive spreading of ubiquitylated chromatin at DNA damage sites as it functionally suppresses RNF168, another E3 ligase, which promotes the concerted accumulation of H2A and H2AX at DNA damage site.
T261 46618-46754 Sentence denotes The importance of pADPr-binding by the WWE motif of HUWE1 and TRIP12 for their recruitment to DNA strand breaks remains to be addressed.
T262 46755-46940 Sentence denotes It is interesting to note that the members of the PARP family that carry WWE domains are most likely mono(ADPribosyl)transferases and unable to produce the iso-ADPr moiety bound by WWE.
T263 46941-46998 Sentence denotes Little is known about these proteins and their functions.
T264 46999-47145 Sentence denotes One aspect that may be of further functional relevance is the presence of classical zinc fingers associated with TiPARP, PARP12, PARP13 (Fig. 3) .
T265 47146-47245 Sentence denotes Of these, PARP13 has been examined as an antiviral protein (also named zinc antiviral protein ZAP).
T266 47246-47371 Sentence denotes PARP13 inhibits the replication of viruses by recruiting the cellular RNA degradation machineries to degrade the viral mRNAs.
T267 47372-47483 Sentence denotes It targets RNA viruses such as the retroviridae human immunodeficiency virus type 1 (HIV-1) Zhu et al., 2011) .
T268 47484-47665 Sentence denotes The mechanistic link between poly(ADP-ribosyl)ation and the regulation of protein degradation is one of the most surprising aspects of the recent advances on poly(ADP-ribosyl)ation.
T269 47666-47874 Sentence denotes Three research groups identified independently the E3ligase Iduna/RNF146 as being a key regulator of protein stability in a pADPr-dependent manner Callow et al., 2011; Kang et al., 2011; Zhang et al., 2011) .
T270 47875-48026 Sentence denotes Remarkably, this pathway appears to function in several biological contexts, regulated not only by PARP1 but also by the tankyrases 1 and 2 (Fig. 4A ).
T271 48027-48170 Sentence denotes In the context of DNA damage, PARP1 automodification triggers its ubiquitylation by Iduna/RNF146 and subsequent degradation by the proteasome .
T272 48171-48418 Sentence denotes In the context of Wnt signaling, it is tankyrase-dependent poly(ADP-ribosyl)ation of axin that induces its Iduna/ RNF146-dependent proteasomal degradation and subsequent b-catenin-dependent transcription (Callow et al., 2011; Zhang et al., 2011) .
T273 48419-48638 Sentence denotes Iduna/RNF146 binds to pADPr of varying lengths and ubiquitylates substrates poly(ADPribosyl)ated with short chains as occurs with tankyrases and substrates poly(ADP-ribosyl)ated with long chains that occurs with PARP1 .
T274 48639-48758 Sentence denotes Thus Iduna/RNF146's dynamic range of protein quality control in the setting of poly(ADP-ribosyl)ation may be extensive.
T275 48759-48915 Sentence denotes Because of this, there are likely to be multiple checkpoints that control Iduna/RNF146's activity and biological actions that require further investigation.
T276 48916-49249 Sentence denotes Interestingly, regulation of protein stability in a pADPr-dependent manner is not restricted to WWE-containing E3-ligases because the PBZ-bearing CHFR E3-ligase has been shown recently to ubiquitylate PARP1 to target it for proteasomal degradation in the context of mitotic stress (see Section 3.3; Fig. 4A ) (Kashima et al., 2012) .
T1 49250-49472 Sentence denotes The clinical importance of the tankyrase-Iduna/RNF146-dependent regulation of protein stability was revealed recently in studies focusing on the cherubism-mutated protein 3BP2 (Guettler et al., 2011; Levaot et al., 2011) .
T2 49473-49656 Sentence denotes Cherubism is a rare autosomal dominant human disorder characterized by inflammatory destructive bone lesions resulting in abnormal fibrous tissue growth in the lower part of the face.
T3 49657-49803 Sentence denotes Approximately 80% of all cherubism patients carry a mutation in the Sh3bp2 gene, which encodes the Src homology 3 domain-binding protein-2 (3BP2).
T4 49804-49974 Sentence denotes Interestingly, most of these mutations lie within a six amino acid sequence (RSPPDG) that corresponds to the tankyrase substrate-recognition motif (Levaot et al., 2011) .
T5 49975-50253 Sentence denotes Upon successful binding of tankyrase 2 to the latter motif in 3BP2 of healthy cells, 3BP2 is poly(ADP-ribosyl)ated, which serves as a signal for its Iduna/RNF146-directed ubiquitylation and proteasome-mediated degradation (Fig. 4A) (Guettler et al., 2011; Levaot et al., 2011) .
T6 50254-50483 Sentence denotes The regulated degradation of 3BP2 is profoundly disturbed in cherubism patients because the interaction and hence poly(ADP-ribosyl)ation of mutated 3BP2 by tankyrase 2 is impaired, abolishing 3BP2 ubiquitylation by Iduna/ RNF146.
T7 50484-50750 Sentence denotes The abnormal accumulation of 3BP2 within cells appears to alter the signaling balance of SRC kinase multiprotein complex to which it is associated, causing systematic inflammation, and leading to the cherubism phenotype (Guettler et al., 2011; Levaot et al., 2011) .
T8 50751-50895 Sentence denotes Hence, understanding the concerted action of poly(ADP-ribosyl)ation and ubiquitylation might improve therapeutic approaches targeting cherubism.
T9 50896-51094 Sentence denotes It is tempting to speculate that the tankyrase-dependent poly(ADP-ribosyl)ation coupled to Iduna/RNF146 ubiquitylation/ degradation pathway is a widespread process to regulate protein steady states.
T10 51095-51326 Sentence denotes By in silico searches for putative tankyrase interacting proteins, Guettler et al. (2011) have identified a very large list of proteins carrying the tankyrase interaction sequence RXX(G/P)DG that could constitute potential targets.
T11 51327-51561 Sentence denotes For instance, the stability of the centrosomal associated protein (CPAP), important for centriole duplication during mitosis, is regulated by tankyrase-dependent poly(ADP-ribosyl)ation and proteasomal degradation (Kim et al., 2012b) .
T12 51562-51695 Sentence denotes Massive activation of PARP1 following a genotoxic stress has been long recognized as a critical event in the induction of cell death.
T13 51696-51932 Sentence denotes However, it is only in recent years that pADPr has been recognized as a death signaling molecule after the identification of In the first view, cytoplasmic ADPr and pADPr are synthesized by tankyrases and PARP12-15 upon stress exposure.
T14 51933-52036 Sentence denotes This triggers the aggregation of RNA-binding proteins (RBP) to ADPr/pADPr and stress granule formation.
T15 52037-52156 Sentence denotes In the second view, the nucleo-cytoplasmic trafficking of pADPr is responsible for its accumulation into the cytoplasm.
T16 52157-52421 Sentence denotes By virtue of its endoglycosidic activity, PARG releases free and protein-bound pADPr following genotoxic stress and PARP activation. pADPr translocated into the cytoplasm is targeted by G3BP1 and RNA-binding proteins to initiate the aggregation of stress granules.
T17 52422-52602 Sentence denotes In both views, pADPr in ribonucleoparticles acts as a scaffold for the recruitment of RNA-binding proteins. (D) pADPr plays regulatory roles in the dynamics of chromatin structure.
T18 52603-52695 Sentence denotes Automodification of PARP1 and poly(ADP-ribosy)ation of histones induce chromatin relaxation.
T19 52696-52800 Sentence denotes This also involves the chromatin remodeling factor CHD1L, which is recruited to specific sites by pADPr.
T20 52801-52885 Sentence denotes The ATPase activity of CHD1L is stimulated by pADPr and triggers nucleosome sliding.
T21 52886-52997 Sentence denotes This possibly facilitates access of the DNA repair machineries. (E) Functions of pADPr in DNA damage responses.
T22 52998-53319 Sentence denotes DNA strand breaks as well as other types of altered DNA structures and DNA adducts activate PARP1. pADPr triggers the recruitment of proteins and enzymes involved in DNA damage signaling, in base excision repair (BER), non-homologous end joining (NHEJ), homologous recombination (HR) and nucleotide excision repair (NER).
T23 53320-53426 Sentence denotes See Section 4.5 for details. pADPr as the trigger of apoptosis inducing factor (AIF)-dependent cell death.
T24 53427-53624 Sentence denotes This insight came from studies attempting to determine the linkage between PARP1 activation during genotoxic stress and translocation of AIF from the mitochondria to the nucleus (Yu et al., 2002) .
T25 53625-53958 Sentence denotes Experiments to determine the mechanism of how PARP1 activation was intimately coupled to AIF translocation led to the discovery that pADPr translocates from the nucleus to mitochondria where it acts as an AIF releasing factor to cause the mitochondrial-nuclear translocation of AIF, initiating cell death (Fig. 4B) Yu et al., 2006) .
T26 53959-54173 Sentence denotes This type of cell death, called parthanatos, occurs in neuronal cells following excitotoxicity as well as other cell types in which DNA damage is induced by specific genotoxic agents (Fig. 4B) (Wang et al., 2009 ).
T27 54174-54268 Sentence denotes As noted above, the PBM within AIF is required for the release of AIF after PARP1 activation .
T28 54269-54366 Sentence denotes It is important to note that the pADPr binding site in AIF is distinct from its DNA binding site.
T29 54367-54523 Sentence denotes Thus, agents could be designed to block this interaction serving as inhibitors of parthanatos or to enhance the release of AIF for cancer chemotherapeutics.
T30 54524-54739 Sentence denotes In a screen for cell survival factors, we identified the E3-ligase Iduna/RNF146 as a pADPr-dependent pro-survival factor, triggered by the same pADPr signal, in this case during non-toxic neuronal stress (Fig. 4B) .
T31 54740-54934 Sentence denotes Iduna/RNF146 protects against parthanatos (pADPr-dependent) cell death mediated by glutamate excitotoxicity both in vitro and in vivo and against middle cerebral artery occlusion-induced stroke.
T32 54935-55094 Sentence denotes Iduna/ RNF146's protective properties are due to its ability to bind pADPr, consistent with the notion that pADPr can act as a death signal during parthanatos.
T33 55095-55236 Sentence denotes PARG also inhibits parthanatos via degradation of the pADPr (Koh et al., 2004) , whereas Iduna/ RNF146 interferes with pADPr death signaling.
T34 55237-55415 Sentence denotes Together both PARG and Iduna/RNF146 function as inhibitors of parthanatos during genotoxic stress providing endogenous controllers of cell death initiated by activation of PARP1.
T35 55416-55601 Sentence denotes Iduna/RNF146 also protects against the N-methyl-N-nitro-N-nitrosoguanidine (MNNG), a DNA damaging agent, and after c-irradiation rescues cells from G1 arrest and promotes cell survival.
T36 55602-55734 Sentence denotes Iduna/RNF146 reduces purinic/apyrimidinic (AP) sites after MNNG exposure and it also facilitates DNA repair following c-irradiation.
T37 55735-55869 Sentence denotes Iduna/RNF146's protective function not only requires a functional pADPr-binding domain, but its ubiquitin E3 ligase activity as well .
T38 55870-56134 Sentence denotes Thus, Iduna/ RNF146 links poly(ADP-ribosyl)ation and protein stability in the DNA damage response by controlling the levels of proteins important in this process through modulating the levels of poly(ADP-ribosyl)ated proteins via ubiquitin proteasomal degradation.
T39 56135-56253 Sentence denotes Identification of Iduna/RNF146's substrates that play roles in parthanatos and DNA repair are the critical next steps.
T40 56254-56393 Sentence denotes A novel function of pADPr has been recently described in the regulation of stress granules (SG) (Leung et al., 2011 (Leung et al., , 2012 .
T41 56394-56491 Sentence denotes These structures are composed of cytoplasmic aggregates of stalled pre-initiation mRNA complexes.
T42 56492-56720 Sentence denotes In addition to a subset of ribosomal proteins and translation initiation factors, the SG contains a variety of RNA-binding proteins that promote its nucleation and participate in the reprogrammation of translation during stress.
T43 56721-56947 Sentence denotes The localization of several PARPs in SG (PARP12, PARP13, PARP14, PARP15 and tankyrase, called here SG-PARPs) uncovered a connection between poly(ADPribosyl)ation reactions and posttranscriptional regulation of gene expression.
T44 56948-57212 Sentence denotes It has been shown that the overexpression of the SG-PARPs promotes the poly(ADP-ribosyl)ation of mRNA-associated proteins and the assembly of SGs, while PARG overexpression inhibits their appearance supporting a key role for pADPr in the assembly of SG (Fig. 4C ).
T45 57213-57375 Sentence denotes This notion was further supported by the identification of G3BP1, one of the primary nucleator of SGs (Kedersha and Anderson, 2007) , as a pADPr-binding protein .
T46 57376-57592 Sentence denotes In contrast to the cytoplasm-restricted poly(ADP-ribosyl)ation reactions of the SG-PARPs, the G3BP1-associated SG are assembled with a pADPr-dependent mechanism that originates from the nuclear activation of PARP1/2.
T47 57593-57645 Sentence denotes These two possibilities are schematized in Fig. 4C .
T48 57646-57752 Sentence denotes PARP activation results in chromatin relaxation (reviewed in Beneke, 2012; Krishnakumar and Kraus, 2010) .
T49 57753-57821 Sentence denotes The mechanism through which this occurs remains to be characterized.
T50 57822-58021 Sentence denotes One model stipulates that histones, which are poly(ADP-ribosyl)ated after PARP activation, are less tightly bound to DNA, thereby inducing relaxation (Ball and Yokomori, 2011; Poirier et al., 1982) .
T51 58022-58121 Sentence denotes Another mechanism has arisen from studies of the non-covalent interaction of ALC1/CHD1L with pADPr.
T52 58122-58341 Sentence denotes PARP1 activation not only recruits this protein to sites of DNA damage, it also stimulates its ATPase activity and induces CHD1Ldependent nucleosome repositioning (Fig. 4D) (Ahel et al., 2009; Gottschalk et al., 2009 ).
T53 58342-58567 Sentence denotes This PARP-dependent function of CHD1L may be of importance during DNA damage signaling and repair of DNA strand breaks, as well as of cyclobutane pyrimidine dimers by nucleotide excision repair (Fig. 4E) Pines et al., 2012) .
T54 58568-58974 Sentence denotes Poly(ADP-ribosyl)ation in the context of DNA damage responses has long been recognized, but the concept is emerging that it is not involved in the DNA repair processes per se. pADPr synthesis at sites of DNA lesions triggers the recruitment of many DNA damage mediators and repair factors as well as chromatin remodeling and may serve as a scaffold for the assembly of repair complexes (Table 1; Fig. 4E ).
T55 58975-59412 Sentence denotes Regardless of the type of DNA damage, be it single-strand breaks repaired by base excision repair, double-strand breaks repaired by non-homologous end joining or homologous recombination, or cyclobutane pyrimidine dimers repaired by nucleotide excision repair, the recent elucidation of large sets of pADPr-binding proteins and associated complexes supports critical functions for pADPr in the early sensing and signaling of DNA damage .
T56 59413-59508 Sentence denotes A number of cancer cells are crucially dependent on the DNA repair pathways regulated by PARP1.
T57 59509-59584 Sentence denotes BRCA1 and 2 are the major breast and ovarian susceptibility genes reported.
T58 59585-59698 Sentence denotes Mutations in the latter genes render cells deficient for DSB repair and exquisitely sensitive to PARP inhibitors.
T59 59699-59881 Sentence denotes This concept is now being extended to other DNA repair factors, including mutations in ATM, p53 and the MRE11-RAD50-NBS1 complex (Oplustilova et al., 2012; Williamson et al., 2012) .
T60 59882-60020 Sentence denotes Interestingly, the exact mechanism of action of PARP inhibitors is still a matter of ongoing debate (Helleday, 2011; Patel et al., 2011) .
T61 60021-60313 Sentence denotes Large-scale sequencing projects of human genomes, such as the ENCODE project consortium, may help to reveal novel sequence variants or mutations in proteins involved in the maintenance of genomic stability with critical implications in the development of human cancers (Dunham et al., 2012) .
T62 60314-60491 Sentence denotes It is also reasonable to think that a critical mutation in a pADPr-binding motif might have deleterious consequences in signaling pathways that comprise the DNA damage response.
T63 60492-60550 Sentence denotes Such information might be positively exploited clinically.
T64 60551-60741 Sentence denotes While defective DNA damage repair pathways are one type of susceptibility to PARP inhibitors, there appear to be others for which mechanistic basis are failing to be explained at the moment.
T65 60742-60946 Sentence denotes For instance, the susceptibility of human epidermal growth factor 2 positive (HER2+) breast cancer cells to PARP inhibitors alone was recently shown, independent of defects in HR (Nowsheen et al., 2012) .
T66 60947-61185 Sentence denotes Similarly intriguing is the sensitivity of cancers and cells bearing gene fusions with ETS transcription factors (mainly ERG), including prostate cancer and Ewing's sarcoma, to PARP inhibitors (Brenner et al., 2011 Garnett et al., 2012) .
T67 61186-61381 Sentence denotes In this case, both the transcriptional and DNA damage signaling functions of PARP1 may be involved to explain the sensitivity (Brenner et al., 2011; Garnett et al., 2012; Schiewer et al., 2012) .
T68 61382-61812 Sentence denotes Based on the latter findings and on the fact that PARP inhibitors have minimal side-effects, they have been in clinical trials for almost a decade, either in combination with chemotherapeutic drugs, as a single-agent or very recently in combination with phosphoinositide 3-kinase (PI3K) inhibitors, that further expand the treatment options for cancer patients Javle and Curtin, 2011; Juvekar et al., 2012; Rouleau et al., 2010) .
T69 61813-62080 Sentence denotes With these examples in mind, and as the list of pADPr-binding proteins and pathways using poly(ADP-ribosyl)ation as signaling mechanisms is still expanding, it becomes crucial to investigate the broad spectrum of biological implications of pADPr-protein interactions.
T70 62081-62214 Sentence denotes It will undoubtedly lead to a better understanding of more applications of PARP inhibitors as single-agent and combination therapies.
T71 62215-62317 Sentence denotes For instance, several pADPr-binding proteins have been linked to cancer progression or aggressiveness.
T72 62318-62546 Sentence denotes PARP9, initially referred to as ''BAL PARP'', was originally identified as a risk-related gene in diffuse large B-cell aggressive lymphoma, being over-expressed in such cancer cells (Aguiar et al., 2000; Takeyama et al., 2003) .
T73 62547-62760 Sentence denotes Similarly, CHD1L was originally named ''amplified in liver cancer 1'' (ALC1) because it was discovered as a candidate oncogene in hepatocellular carcinoma (Chen et al., 2009 (Chen et al., , 2010 Ma et al., 2008) .
T74 62761-63018 Sentence denotes Further investigation of the mechanistic roles of pADPr in the regulation of cancer-associated protein networks and signaling pathways will be fundamental for the development of innovative treatment strategies, and to overcome resistance to such treatments.
T75 63019-63151 Sentence denotes Our understanding of the role of pADPr has remarkably expanded since the original observation that DNA strand breaks activate PARP1.
T76 63152-63494 Sentence denotes As described above, in humans, there are at least four pADPr-binding modules (and others perhaps waiting to be discovered), coupled to a discrete number of additional domains linking poly(ADP-ribosyl)ation to ubiquitylation and chromatin structure in several cellular contexts such as protein degradation, DNA damage responses and cell death.
T77 63495-63621 Sentence denotes Although sufficient to ensure binding to pADPr, these pADPr-binding domains vary widely in their degree of ligand specificity.
T78 63622-63908 Sentence denotes While some seems to have a general affinity for the polyanionic backbone of biomolecules (i.e. DNA, RNA and pADPr) due to the presence of basic patches of amino acid residues, others evolved to perform specialized functions and exhibit a high degree of target specificity towards pADPr.
T79 63909-64056 Sentence denotes Currently, the PBZ domain, which forms a divergent C2H2-type zinc finger fold with specialized functions, possesses the highest affinity for pADPr.
T80 64057-64217 Sentence denotes The C2H2 zinc finger fold is amongst the most prevalent protein motifs in the human proteome and comprises the largest family of regulatory proteins in mammals.
T81 64218-64394 Sentence denotes With this knowledge, we can speculate that uncharacterized variations in finger-like protrusions might provide the specificity required to recognize different pADPr structures.
T82 64395-64693 Sentence denotes Over 60 human proteins have been shown to interact with pADPr (Table 1) , but based on in silico predictions of the occurrence of the PBM sequence, there may be over 500 of them, and many more if we consider that proteins in complexes with pADPr-binding proteins are (indirectly) affected by pADPr.
T83 64694-65020 Sentence denotes Because the PBM represents a short contiguous protein segment, examination of other context criteria, such as protein surface accessibility, evolutionary conservation as well as the determination of three-dimensional PBM-bound pADPr complexes will help to establish the local structural environment required for pADPr-binding.
T84 65021-65206 Sentence denotes Because of this potential that poly(ADP-ribosyl)ation affects a significant portion of the proteome, it is crucial to pursue the extensive examination of the pADPr-protein interactions.
T85 65207-65327 Sentence denotes In this regard, it is very intriguing to consider the high number of pADPr-binding proteins working in a single pathway.
T86 65328-65564 Sentence denotes One may envision that there is a ''pADPr code'' where the length, complexity, and conformation adopted by pADPr covalently linked to a particular protein target, all contribute to favor some non-covalent interactions relative to others.
T87 65565-65720 Sentence denotes The remarkably high affinity and processivity of PARG for long pADPr will certainly have a role to play in the regulation of the non-covalent interactions.
T88 65721-65851 Sentence denotes Equally intriguing is the presence of several pADPr-binding modules within single proteins (i.e. Deltex, macro-PARPs, APLF, etc.).
T89 65852-65963 Sentence denotes Does it confer higher specificity, stronger interactions, or preference for pADPr in a particular conformation?
T90 65964-66053 Sentence denotes Or does it organize pADPr in a scaffold as proposed for the formation of stress granules?
T91 66054-66170 Sentence denotes The functions of pADPr in pathological conditions (i.e. DNA damage, mitotic stress, etc.) are now better understood.
T92 66171-66293 Sentence denotes Still, the mechanisms underlying the relocalization of pADPr to the cytoplasm after specific stresses are largely unknown.
T93 66294-66523 Sentence denotes The physiological aspects of poly(ADP-ribosyl)ation are only starting to emerge as they are more difficult to grasp. pADPr levels often fall below the threshold of detectability using current methods, especially in the cytoplasm.
T94 66524-66636 Sentence denotes The triggers of pADPr synthesis by the damage-independent PARPs, such as tankyrases and SG-PARPs, are undefined.
T95 66637-66827 Sentence denotes Nonetheless, in view of their important functional outcomes in regulating protein stability and posttranslational gene expression in the cytoplasm, no doubt that it must be finely regulated.
T96 66828-67213 Sentence denotes Considering the critical functions of Iduna/RNF146 in directing poly(ADP-ribosyl)ated proteins towards proteasomal degradation, and the high catabolic activity of PARG, it is conceivable that some functional aspects of pADPr-binding proteins may consist in protecting pADPr from degradation, or in shielding the pADPr from Iduna/RNF146/CHFR to protect target proteins from degradation.
T97 67214-67321 Sentence denotes We can also wonder whether there may be pADPr-dependent deubiquitylases that further regulate this process.
T98 67322-67581 Sentence denotes It is now important to critically examine the regulation of pADPr degradation by PARG, ARH3 and possibly some macro domain proteins, in cellular contexts where pADPr-binding proteins also operate, to truly understand the extent of signaling afforded by pADPr.
T99 67582-67734 Sentence denotes Some pathways appear to rely on the generation of free pADPr molecules, requiring an endoglycosidic activity that so far, only PARG is known to display.
T100 67735-67915 Sentence denotes Our understanding of these fundamental questions now depends on the development of sensitive and quantitative methods for the detection of nuclear and cytoplasmic pADPr structures.