Poly(ADP-Ribose) Polymerase-3 Regulates Regeneration in Planarians.

Protein ADP-ribosylation is a reversible post-translational modification (PTM) process that plays fundamental roles in cell signaling. The covalent attachment of ADP ribose polymers is executed by PAR polymerases (PARP) and it is essential for chromatin organization, DNA repair, cell cycle, transcription, and replication, among other critical cellular events. The process of PARylation or polyADP-ribosylation is dynamic and takes place across many tissues undergoing renewal and repair, but the molecular mechanisms regulating this PTM remain mostly unknown. Here, we introduce the use of the planarian Schmidtea mediterranea as a tractable model to study PARylation in the complexity of the adult body that is under constant renewal and is capable of regenerating damaged tissues. We identified the evolutionary conservation of PARP signaling that is expressed in planarian stem cells and differentiated tissues. We also demonstrate that Smed-PARP-3 homolog is required for proper regeneration of tissues in the anterior region of the animal. Furthermore, our results demonstrate, Smed-PARP-3(RNAi) disrupts the timely location of injury-induced cell death near the anterior facing wounds and also affects the regeneration of the central nervous system. Our work reveals novel roles for PARylation in large-scale regeneration and provides a simplified platform to investigate PARP signaling in the complexity of the adult body.

Plant PARPs, PARGs and PARP-like Proteins.

Poly(ADP-ribos)ylation, originally described as a mechanism of DNA break repair, is now considered as part of a complex regulatory system involved in dynamic reorganization of chromatin structure, transcriptional control of gene expression and regulation of metabolism. In plants poly(ADP-ribos)ylation has received surprisingly little attention. It has been implicated in abiotic and biotic stress responses, cell cycle control and development; however, the molecular mechanisms and proteins involved are largely unknown. In this review we summarize current knowledge on plant PARP, PARG and PARP-like domain containing proteins and discuss their possible roles in plant development, immune responses, programmed cell death and stress responses in general. The genome of the model plant Arabidopsis contains three genes encoding PARP proteins, two of which have been shown to be active PARPs, and two genes encoding PARG proteins, one of which was shown to possess enzymatic activity. In addition, SROs (Similar to RCD One) represent a plant specific family of proteins containing a PARP-like domain. Although bioinformatics and biochemical data suggest that the PARP-like domain in SRO proteins does not have PARP activity, these proteins play a significant role in stress response as revealed by mutant analyses. SRO proteins interact with transcription factors involved in various stress and developmental responses and are suggested to serve as hubs in many signaling pathways. Altogether current data imply that poly(ADP-ribos)ylation plays significant regulatory role in many aspects of plant biology.

Evolutionary history of the poly(ADP-ribose) polymerase gene family in eukaryotes.

BACKGROUND: The poly(ADP-ribose) polymerase (PARP) superfamily was originally identified as enzymes that catalyze the attachment of ADP-ribose subunits to target proteins using NAD+ as a substrate. The family is characterized by the catalytic site, termed the PARP signature. While these proteins can be found in a range of eukaryotes, they have been best studied in mammals. In these organisms, PARPs have key functions in DNA repair, genome integrity and epigenetic regulation. More recently it has been found that proteins within the PARP superfamily have altered catalytic sites, and have mono(ADP-ribose) transferase (mART) activity or are enzymatically inactive. These findings suggest that the PARP signature has a broader range of functions that initially predicted. In this study, we investigate the evolutionary history of PARP genes across the eukaryotes. RESULTS: We identified in silico 236 PARP proteins from 77 species across five of the six eukaryotic supergroups. We performed extensive phylogenetic analyses of the identified PARPs. They are found in all eukaryotic supergroups for which sequence is available, but some individual lineages within supergroups have independently lost these genes. The PARP superfamily can be subdivided into six clades. Two of these clades were likely found in the last common eukaryotic ancestor. In addition, we have identified PARPs in organisms in which they have not previously been described. CONCLUSIONS: Three main conclusions can be drawn from our study. First, the broad distribution and pattern of representation of PARP genes indicates that the ancestor of all extant eukaryotes encoded proteins of this type. Second, the ancestral PARP proteins had different functions and activities. One of these proteins was similar to human PARP1 and likely functioned in DNA damage response. The second of the ancestral PARPs had already evolved differences in its catalytic domain that suggest that these proteins may not have possessed poly(ADP-ribosyl)ation activity. Third, the diversity of the PARP superfamily is larger than previously documented, suggesting as more eukaryotic genomes become available, this gene family will grow in both number and type.

Toward a unified nomenclature for mammalian ADP-ribosyltransferases.

ADP-ribosylation is a post-translational modification of proteins catalyzed by ADP-ribosyltransferases. It comprises the transfer of the ADP-ribose moiety from NAD+ to specific amino acid residues on substrate proteins or to ADP-ribose itself. Currently, 22 human genes encoding proteins that possess an ADP-ribosyltransferase catalytic domain are known. Recent structural and enzymological evidence of poly(ADP-ribose)polymerase (PARP) family members demonstrate that earlier proposed names and classifications of these proteins are no longer accurate. Here we summarize these new findings and propose a new consensus nomenclature for all ADP-ribosyltransferases (ARTs) based on the catalyzed reaction and on structural features. A unified nomenclature would facilitate communication between researchers both inside and outside the ADP-ribosylation field.

Structural recognition of DNA by poly(ADP-ribose)polymerase-like zinc finger families.

PARP-like zinc fingers (zf-PARPs) are protein domains apt to the recognition of multiple DNA secondary structures. They were initially described as the DNA-binding, nick-sensor domains of poly(ADP-ribose)polymerases (PARPs). It now appears that zf-PARPs are evolutionary conserved in the eukaryotic lineage and associated with various enzymes implicated in nucleic acid transactions. In the present study, we discuss the functional and structural data of zf-PARPSs in the light of a comparative analysis of the protein family. Sequence and structural analyses allow the definition of the conserved features of the zf-PARP domain and the identification of five distinct phylogenetic groups. Differences among the groups accumulate on the putative DNA binding surface of the PARP zinc-finger fold. These observations suggest that different zf-PARP types have distinctive recognition properties for DNA secondary structures. A comparison of various functional studies confirms that the different finger types can accomplish a selective recognition of DNA structures.

BRCA2-deficient CAPAN-1 cells are extremely sensitive to the inhibition of Poly (ADP-Ribose) polymerase: an issue of potency.

We have previously demonstrated that deficiency of either the BRCA1 or BRCA2 breast cancer susceptibility proteins confers substantial cellular sensitivity to the inhibition of Poly(ADP-Ribose) polymerase (PARP). PARP is a key enzyme in the repair of single strand DNA damage via the Base Excision Repair pathway. We suggested that PARP inhibition produces persistent single-strand DNA breaks or gaps which degenerate into stalled replication forks and double-strand breaks, which may be repaired by homologous recombination, a process partially dependent on BRCA1 and BRCA2. It has recently been suggested that our results might be limited to certain BRCA2 mutations as the CAPAN-1 cell line, which carries a naturally occurring 6174delT mutation in one BRCA2 allele accompanied by loss of the wild-type allele, is apparently insensitive to two PARP inhibitors 3-aminobenzamide (IC50 33 microM) and NU1025 (IC50 400 nM). Here we show that CAPAN-1 cells are in fact very sensitive to the potent PARP inhibitors KU0058684 (IC50 3.2 nM) and KU0058948 (IC50 3.4 nM). In contrast, our results reveal much less sensitivity to a chemically related but much less active compound KU0051529 (IC50 730 nM) and to NU1025. These results confirm that treatment with potent PARP inhibitors remains an exciting potential therapy for cancers involving BRCA1 or BRCA2 deficiency.

The PARP superfamily.

Poly(ADP-ribosyl)ation is an immediate DNA-damage-dependent post-translational modification of histones and other nuclear proteins that contributes to the survival of injured proliferating cells. Poly(ADP-ribose) polymerases (PARPs) now constitute a large family of 18 proteins, encoded by different genes and displaying a conserved catalytic domain in which PARP-1 (113 kDa), the founding member, and PARP-2 (62 kDa) are so far the sole enzymes whose catalytic activity has been shown to be immediately stimulated by DNA strand breaks. A large repertoire of sequences encoding novel PARPs now extends considerably the field of poly(ADP-ribosyl)ation reactions to various aspects of the cell biology including cell proliferation and cell death. Some of these new members interact with each other, share common partners and common subcellular localizations suggesting possible fine tuning in the regulation of this post-translational modification of proteins. This review summarizes our present knowledge of this emerging superfamily, which might ultimately improve pharmacological strategies to enhance both antitumor efficacy and the treatment of a number of inflammatory and neurodegenerative disorders. A provisional nomenclature is proposed.

Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex.

A member of the Bacillus-produced vegetative insecticidal proteins (VIPs) possesses high specificity against the major insect pest, corn rootworms, and belongs to a class of binary toxins and regulators of biological pathways distinct from classical A-B toxins. The 1.5 A resolution crystal structure of the enzymatic ADP-ribosyltransferase component, VIP2, from Bacillus cereus reveals structurally homologous N- and C-terminal alpha/beta domains likely representing the entire class of binary toxins and implying evolutionary relationships between families of ADP-ribosylating toxins. The crystal structure of the kinetically trapped VIP2-NAD complex identifies the NAD binding cleft within the C-terminal enzymatic domain and provides a structural basis for understanding the targeting and catalysis of the medically and environmentally important binary toxins. These structures furthermore provide specific experimental results to help resolve paradoxes regarding the specific mechanism of ADP-ribosylation of actin by implicating ground state destabilization and nicotinamide product sequestration as the major driving forces for catalysis.

Sequence and structural links between distant ADP-ribosyltransferase families.

The low resolution structure of the Pseudomonas aeroginosa exotoxin A (ETA) presented in 1986 provided the first tantalizing three-dimensional view of an ADP-ribosyl-transferase (ADPRT) catalytic domain. The major features of this protein fold have recurred in the more recently solved crystal structures of the cholera toxin-related heat-labile enterotoxin (LT), diphtheria toxin (DT) and pertussis toxin (PT). A core set of alpha + beta elements define a minimal, conserved scaffold with remarkably plastic sequence requirements-only a single glutamic acid residue critical to catalytic activity is invariant. Other interchangeable residues in locations important for catalysis and binding are suggested by the cocrystal structures of DT with the inhibitor ApUp, ETA with bound AMP and nicotinamide, and DT with substrate NAD-in close accord with labeling and mutagenic data. Faint sequence resemblances that were earlier noticed among prokaryotic ADPRTs have now been securely extended by the structural concordance between toxin folds; more recently, eukaryotic ADPRTs have surfaced and their sequences can be reliably threaded into the conserved core fold. We will briefly summarize efforts in Palo Alto and Hamburg to explore these latter relationships, and to mount a rigorous search for new ADPRT families in the growing sequence databases.