Functional characterization of the poly(ADP-ribose) polymerase activity of tankyrase 1, a potential regulator of telomere length

Factor H (FH) of the complement system acts as a regulatory cofactor for the factor I-mediated cleavage of C3b and binds to polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains. A set of 12 missense mutations in the C-terminal domains between SCR-16 to SCR-20 is associated with haemolytic uraemic syndrome. Recent structural models for intact FH permit the molecular interpretation of these amino acid substitutions. As all nine SCR-20 substitutions correspond to normal amounts of FH in plasma, and were localised in mostly surface-exposed positions, these are inferred to lead to a functional defect in FH. The nine substitutions occur in the same spatial region of SCR-20. As this surface coincides with conserved basic residues in the C-terminal SCR-20 domain, the substitutions provide direct evidence for a polyanionic binding surface. The positions of these conserved basic residues coincide with those of heparin-binding residues in the crystal structure of the acidic fibroblast growth factor-heparin complex. A tenth substitution and another conserved basic residue in SCR-19 are proximate to this binding site. As the remaining FH substitutions could also be correlated with their proximity to conserved basic residues, haemolytic uraemic syndrome may result from a failure of FH to interact with polyanions at cell surfaces in the kidney.     

Rapid regulation of telomere length is mediated by poly(ADP-ribose) polymerase-1

Shelterin/telosome is a multi-protein complex at mammalian telomeres, anchored to the double-stranded region by the telomeric-repeat binding factors-1 and -2. In vitro modification of these proteins by poly(ADP-ribosyl)ation through poly(ADP-ribose) polymerases-5 (tankyrases) and -1/-2, respectively, impairs binding. Thereafter, at least telomeric-repeat binding factor-1 is degraded by the proteasome. We show that pharmacological inhibition of poly(ADP-ribose) polymerase activity in cells from two different species leads to rapid decrease in median telomere length and stabilization at a lower setting. Specific knockdown of poly(ADP-ribose) polymerase-1 by RNA interference had the same effect. The length of the single-stranded telomeric overhang as well as telomerase activity were not affected. Release of inhibition led to a fast re-gain in telomere length to control levels in cells expressing active telomerase. We conclude that poly(ADP-ribose) polymerase-1 activity and probably its interplay with telomeric-repeat binding factor-2 is an important determinant in telomere regulation. Our findings reinforce the link between poly(ADP-ribosyl)ation and aging/longevity and also impact on the use of poly(ADP-ribose) polymerase inhibitors in tumor therapy.     

Functional subdomain in the ankyrin domain of tankyrase 1 required for poly(ADP-ribosyl)ation of TRF1 and telomere elongation

In human cells, telomere elongation by telomerase is repressed in cis by the telomeric protein TRF1. Tankyrase 1 binds TRF1 via its ankyrin domain and poly(ADP-ribosyl)ates it. Overexpression of tankyrase 1 in telomerase-positive cells releases TRF1 from telomeres, resulting in telomere elongation. The tankyrase 1 ankyrin domain is classified into five conserved subdomains, ARCs (ankyrin repeat clusters) I to V. Here, we investigated the biological significance of the ARCs. First, each ARC worked as an independent binding site for TRF1. Second, ARCs II to V recognized the N-terminal acidic domain of TRF1 whereas ARC I bound a discrete site between the homodimerization and the Myb-like domains of TRF1. Inactivation of TRF1 binding in the C-terminal ARC, ARC V, either by deletion or point mutation, significantly reduced the ability of tankyrase 1 to poly(ADP-ribosyl)ate TRF1, release TRF1 from telomeres, and elongate telomeres. In contrast, other ARCs, ARC II and/or IV, inactivated by point mutations still retained the biological function of tankyrase 1. On the other hand, ARC V per se was not sufficient for telomere elongation, suggesting a structural role for multiple ARCs. This work provides evidence that specific ARC-TRF1 interactions play roles in the essential catalytic function of tankyrase 1.     

Telomere elongation by a mutant tankyrase 1 without TRF1 poly(ADP-ribosyl)ation

Telomeres are the capping structures of the eukaryotic chromosome ends. Tankyrase 1 is a poly(ADP-ribose) polymerase that elongates telomeres in a telomerase-dependent manner. This function of tankyrase 1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Namely, tankyrase 1 poly(ADP-ribosyl)ates (PARsylates) TRF1, which in turn dissociates TRF1 from telomeres. The resulting telomeres become better substrates for telomerase-mediated DNA extension. Tankyrase 1 has five independent TRF1 binding sites, ARC (ANK repeat cluster) I to V. Among them, the most C-terminal ARC V is required for TRF1 PARsylation and its release from telomeres. By contrast, functional significance of other four ARCs remains elusive. In this study, we generated a mutant tankyrase 1 that had inactive ARC IV and lacked ARC V but elongated telomeres without TRF1 PARsylation. Consistent with the failure in PARsylation, this mutant only marginally released TRF1 from telomeres. Still, it decreased telomere binding of POT1, a downstream effector of TRF1-mediated telomere length control, and elongated the telomeric 3'-overhang as the wild-type tankyrase 1 did. Thus even without TRF1 PARsylation, this mutant tankyrase 1 seemed to loosen the closed structure of the telomeric heterochromatin. These findings suggest a new role for multiple ARCs in telomere extension by tankyrase 1.     

Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

PARP1 Is a TRF2-associated poly(ADP-ribose)polymerase and protects eroded telomeres

Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.     

GDP-mannose-4,6-dehydratase is a cytosolic partner of tankyrase 1 that inhibits its poly(ADP-ribose) polymerase activity

Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) that participates in a broad range of cellular activities due to interaction with multiple binding partners. Tankyrase 1 recognizes a linear six-amino-acid degenerate motif and, hence, has hundreds of potential target proteins. Binding of partner proteins to tankyrase 1 usually results in their poly(ADP-ribosyl)ation (PARsylation) and can lead to ubiquitylation and proteasomal degradation. However, it is not known how tankyrase 1 PARP activity is regulated. Here we identify GDP-mannose 4,6-dehydratase (GMD) as a binding partner of tankyrase 1. GMD is a cytosolic protein required for the first step of fucose synthesis. We show that GMD is complexed to tankyrase 1 in the cytosol throughout interphase, but its association with tankyrase 1 is reduced upon entry into mitosis, when tankyrase 1 binds to its other partners TRF1 (at telomeres) and NuMA (at spindle poles). In contrast to other binding partners, GMD is not PARsylated by tankyrase 1. Indeed, we show that GMD inhibits tankyrase 1 PARP activity in vitro, dependent on the GMD tankyrase 1 binding motif. In vivo, depletion of GMD led to degradation of tankyrase 1, dependent on the catalytic PARP activity of tankyrase 1. We speculate that association of tankyrase 1 with GMD in the cytosol sequesters tankyrase 1 in an inactive stable form that can be tapped by other target proteins as needed.     

Tankyrase 2 poly(ADP-ribose) polymerase domain-deleted mice exhibit growth defects but have normal telomere length and capping

Regulation of telomere length maintenance and capping are a critical cell functions in both normal and tumor cells. Tankyrase 2 (Tnks2) is a poly(ADP-ribose) polymerase (PARP) that has been shown to modify itself and TRF1, a telomere-binding protein. We show here by overexpression studies that tankyrase 2, like its closely related homolog tankyrase 1, can function as a positive regulator of telomere length in human cells, dependent on its catalytic PARP activity. To study the role of Tnks2 in vivo, we generated mice with the Tnks2 PARP domain deleted. These mice are viable and fertile but display a growth retardation phenotype. Telomere analysis by quantitative fluorescence in situ hybridization (FISH), flow-FISH, and restriction fragment analysis showed no change in telomere length or telomere capping in these mice. To determine the requirement for Tnks2 in long-term maintenance of telomeres, we generated embryonic stem cells with the Tnks2 PARP domain deleted and observed no change, even upon prolonged growth, in telomere length or telomere capping. Together, these results suggest that Tnks2 has a role in normal growth and development but is not essential for telomere length maintenance or telomere capping in mice.     

The telomeric poly(ADP-ribose) polymerase, tankyrase 1, contains multiple binding sites for telomeric repeat binding factor 1 (TRF1) and a novel acceptor, 182-kDa tankyrase-binding protein (TAB182)

Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase, was originally identified through its interaction with TRF1, a negative regulator of telomere length. Tankyrase 1 ADP-ribosylates TRF1 in vitro, and its overexpression induces telomere elongation in human cancer cells. In addition to its telomeric localization, tankyrase 1 resides at multiple subcellular sites, suggesting additional functions for this protein. Here we identify TAB182, a novel tankyrase 1-binding protein of 182 kDa. TAB182 displays a complex pattern of subcellular localization. TAB182 localizes to the nucleus in a heterochromatic staining pattern and to the cytoplasm, where it co-stains with the cortical actin network. TAB182 coimmunoprecipitates with tankyrase 1 from human cells and serves as an acceptor of poly(ADP-ribosyl)ation by tankyrase 1 in vitro. Like TRF1, TAB182 binds to the ankyrin domain (comprising 24 ankyrin repeats) of tankyrase 1. Surprisingly, dissection of this domain reveals multiple discrete and overlapping binding sites for TRF1 and TAB182. Thus, we demonstrate five well conserved ankyrin repeat clusters in tankyrase 1. Although each of the five ankyrin repeat clusters independently binds to TRF1, only three of the five bind toTAB182. These findings suggest that tankyrase 1 may act as a scaffold for large molecular mass complexes made up of multiple binding proteins. We discuss potential roles for tankyrase 1-mediated higher order complexes at telomeres and at other subcellular sites.     

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

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

Detection of poly(ADP-ribose) polymerase and its reaction product poly(ADP-ribose) by immunocytochemistry

Summary Poly(ADP-ribose) polymerase catalyses the formation of ADP-ribose polymers covalently attached to various nuclear proteins, using NAD⁺ as substrate. The activity of this enzyme is strongly stimulated upon binding to DNA single or double strand breaks. Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage and is thought to be involved in DNA repair, genetic recombination, apoptosis and other processes during which DNA strand breaks are formed. In recent years we and others have established cell culture systems with altered poly(ADP-ribose) polymerase activity. Here we describe immunocytochemistry protocols based on the use of antibodies against the DNA-binding domain of human poly(ADP-ribose) polymerase and against its reaction product poly(ADP-ribose). These protocols allow for the convenient mass screening of cell transfectants with overexpression of poly(ADP-ribose) polymerase or of a dominant-negative mutant for this enzyme, i.e. the DNA-binding domain. In addition, the immunocytochemical detection of poly(ADP-ribose) allows screening for cells with altered enzyme activity.     

Control of histone H1 dimer-poly(ADP-ribose) complex formation by poly(ADP-ribose) glycohydrolase

In nuclei incubated in vitro with [³H]NAD to promote poly(ADP-ribose) synthesis, about 6% of the polymer synthesized is differentially extracted into cold 5% PCA along with the H1 histone. Polyacrylamide gel electrophoresis of the extracts revealed large differences in the mobility of the incorporated radioactivity depending on the source of the nuclei used. With rat mammary tumors, the radioactivity co-migrated with the H1 histone on both acid-urea and SDS-urea gels. In contrast, the labeled polymer from HBL-100 mammary cell nuclei co-electrophoresed with a minor protein component which moved more slowly than H1. With lactating mammary glands, an intermediate profile was seen. The difference in mobility on the gels was found to be due to differences in the chain lengths of the poly(ADP-ribose) attached in the H1 protein. The difference in chain length produced was inversely related to the level of poly(ADP-ribose) degrading activity in the various nuclear preparations.     

Crucial role of poly (ADP-ribose) polymerase (PARP-1) in cellular proliferation of Dictyostelium discoideum

The unicellular, as well as multicellular stages of Dictyostelium discoideum’s life cycle, make it an excellent model system for cell type determination, differentiation, development, and cell death studies. Our preliminary results show the involvement of poly (ADP-ribose) polymerase-1 (PARP-1) during D. discoideum growth by its constitutive downregulation as well as by its ortholog overexpression. The current study now analyzes and strengthens the role of the PARP-1 ortholog in cellular proliferation of D. discoideum. ADPRT1A was knocked out (KO) from D. discoideum and studied for its effect on cell growth, cell cycle, morphology, and oxidative stress. The present findings show that ADPRT1A KO ( A KO) cells exhibited reduced cellular proliferation, stressed phenotype, and cell cycle arrest in G2-M phase. Under oxidative stress, A KO cells exhibited slower growth and DNA damage. This is the first report where the involvement of ADPRT1A in growth in D. discoideum is established.     

Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP-ribose) glycohydrolase was detected by the liberation of ADP-ribose from poly(ADP-ribose). Poly(ADP-ribose) polymerase was proved by (a) demonstration of phosphoribosyl-AMP in the phosphodiesterase digest of the reaction product, (b) demonstration of ADP-ribose oligomers by fractionation of the reaction product on DEAE-Sephadex. The (ADP-ribose)-protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein-(ADP-ribose bond is susceptible to 0.1 M NaOH (70%) and 0.4 M NH2OH (33%). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., III (1976) J. Protozool. 23, 13--28]; poly(ADP-ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP-ribose) during evolution.     

Diabetic endothelial dysfunction: the role of poly(ADP-ribose) polymerase activation

Diabetic patients frequently suffer from retinopathy, nephropathy, neuropathy and accelerated atherosclerosis. The loss of endothelial function precedes these vascular alterations. Here we report that activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of endothelial dysfunction in diabetes. Destruction of islet cells with streptozotocin in mice induced hyperglycemia, intravascular oxidant production, DNA strand breakage, PARP activation and a selective loss of endothelium-dependent vasodilation. Treatment with a novel potent PARP inhibitor, starting after the time of islet destruction, maintained normal vascular responsiveness, despite the persistence of severe hyperglycemia. Endothelial cells incubated in high glucose exhibited production of reactive nitrogen and oxygen species, consequent single-strand DNA breakage, PARP activation and associated metabolic and functional impairment. Basal and high-glucose-induced nuclear factor-kappaB activation were suppressed in the PARP-deficient cells. Our results indicate that PARP may be a novel drug target for the therapy of diabetic endothelial dysfunction.     

Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A⁺) mRNA detection by hybridization to [³H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21000–120000 range. The Mr 120000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21000 protein acceptor is abundant in PMS and a Mr 34000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.Abbreviations ADP-ribose adenosine (5) diphospho(5)--D ribose - poly(ADP-ribose) polymer of ADP-ribose - mRNP messenger ribonucleoprotein particles - PMSF phenylmethylsulfonyl fluoride - LDS lithium dodecyl sulfate - TCA trichloroacetic acid     

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health.     

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

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