ADP-ribosylation of proteins in nuclei and mitochondria from tissues rats of various age exposed gamma-radiation

Constitutive and gamma-induced ADP-ribosylation of nuclei and mitochondrial proteins in 2- and 29-month-old rats was studied. ADP-ribosylation was determined by binding of [3H]-adenin with the proteins after incubation of cellular organells in reaction mixture supplemented with [adenin-2,8-3H]-NAD. It was detected that the level of total protein ADP-ribosylation in the nuclei is 4.5-6.2 times higher than in the mitochondria. By inhibition of poly(ADP-ribose) polymerase (PARP) with 3-aminobenzamidine and treatment of ADP-ribosylated proteins with phosphodiesterase I, it was demonstrated that about 90% of [3H]-adenin bound by proteins in the nuclei and 70% in the mitochondria was the result of PARP activity. The level of total ADP-ribosylation of nuclear and mitochondrial proteins in the tissues of old rats was reliably lower than in young animals. This reduction of ADP-ribosylation in old animals is the result of the lower activity of PARP, not of mono(ADP-ribosyl) transferase (MART). The level of ADP-ribosylation of proteins in the nuclei of brain and spleen cells of 2-month-old rats irradiated with of 5 and 10 Gy was by 49-109% higher than in the control. At the same doses of radiation, the level of ADP-ribosylation of nuclear proteins in brain and spleen of old rats increased only by 29-65% compared to the control. Unlike cell nuclei, the radiation-induced activation of ADP-ribosylation in mitochondria was less expressed: the level of ADP-ribosylation increased by 34-37% in young rats and by 11-27% in old animals. This increased binding of ADP-ribose residues by the proteins of nuclei and mitochondria from tissues of gamma-irradiated rats is exceptionally conditioned by activation of poly(ADP-ribosyl)ation because the level of mono(ADP-ribosyl)ation remains constant. The results of this study enable the suggestion that poly(ADP-ribosyl)ation also occurs in the mitochondria of brain and spleen cells of the gamma-irradiated rats, though less pronounced than in cell the cell nuclei of these tissues. Thus, one of the probable causes of the less efficient repair of radiation-induced DNA damage in old organisms is a decline of both constitutive and induced poly(ADP-ribosyl)ation of proteins in cell nucleus and mitochondria.     

ADP-ribosylation of nuclear proteins in rat ventral prostate during ageing

Poly(ADPR)polymerase activity and poly(ADP-ribosyl)ation of nuclear proteins have been investigated in ventral prostate nuclei of different aged rats (14, 28, 60, 180, 360 day old animals), by reverse-phase HPLC and acetic acid-urea polyacrylamide gel electrophoresis. The major ADP-ribose acceptor proteins were identified as histone H1 and H2b. It is concluded that concomitant with major changes to chromatin organization, poly(ADP-ribosyl)ation reaction is progressively inhibited during aging of rat ventral prostate. These results support the hypothesis that prostatic dysfunction in senescent animals is related to a failure of DNA repair mechanisms and deregulated template activity.     

Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.     

Sequential ADP-ribosylation pattern of nucleosomal histones. ADP-ribosylation of nucleosomal histones

The pattern of nucleosomal histones poly(ADP-ribosyl)ation is changed under conditions which affect the poly(ADP-ribosyl)ation state of the enzyme. At low NAD concentrations the enzyme can poly(ADP-ribosyl)ate histones H1 and H1, H2A, A2A, and H2B. However at NAD concentrations above 10 microM the enzyme preferentially poly(ADP-ribosyl)ates histone H1 to a hyper ADP-ribosylated form. Furthermore we have observed hyper ADP-ribosylation of histone H2B at NAD concentrations of 10 microM suggesting that histone H2B can undergo the same type of ADP-ribosylation pattern as histone H1. Also at higher NAD concentrations an elongation of the polymer attached to the enzyme and other nuclear proteins takes place.     

Acceptor proteins for (ADP-ribose)n in the HeLa S3 cell cycle

The acceptor proteins for (ADP-ribose)n were investigated by using nuclei or chromosomes isolated from specific phases of the cell cycle of HeLa S3 cells. Analysis of HMG proteins and histone H1 by acetic acid/urea polyacrylamide gel electrophoresis demonstrated that the (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 increased by 12- and 5-fold, respectively, in the metaphase chromosomes as compared with that in the G1 phase cell nuclei. The degree of (ADP-ribosyl)n-ation of these proteins in the S phase cell nuclei was as low as that in G1 phase cell nuclei. In the G2 phase cell nuclei, the degrees of (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 were about 5- and 2-fold greater, respectively, as compared with that in the G1 phase cell nuclei. The (ADP-ribosyl)n-ation of HMG 1 and 2 was constant through the cell cycle except for a slight decrease in the S phase. The data may imply that the (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 is linked to chromatin structural changes in mitosis.     

Structural study of the porcine NA+/H+ exchanger NHE1 gene and its 5′-flanking region

The poly(ADP-ribosyl)ation system, associated with different nuclear fractions of rat testis, has been analyzed for both pADPR and pADPR acceptor proteins. The DNase I sensitive and resistant chromatin contain 35% and 40%, respectively, of the total pADPR synthesized in intact nuclei incubated with [³²P]NAD. Moreover, the residual 25% were estimated to be associated with the nuclear matrix.Three different classes of pADPR are present in the nuclei. The longest and branched ADPribose polymers modify proteins present in the DNase I resistant (2 M NaCl extractable) chromatin and in the nuclear matrix, whereas polymers of > 20 residues interact with the components of the DNase I sensitive chromatin and oligomers of 6 ADPribose residues are bound specifically to the acid-soluble chromosomal proteins, present in isolated nuclear matrix. The main pADPR acceptor protein in all the nuclear fractions is represented by the PARP itself (auto-modification reaction). The hetero-modification reaction occurs mostly on histone H1 and core histones, that have been found associated to DNase I sensitive and resistant chromatin, respectively. Moreover, an oligo(ADP-ribosyl)ation occurs on core histones tightly-bound to the matrix associated regions (MARs) of chromatin loops.     

Effect of poly(ADP-ribosyl)ation and Mg2+ ions on chromatin structure revealed by scanning force microscopy

Poly(ADP-ribosyl)ation of nuclear proteins is responsible for major changes in the high-order chromatin structure. The effects of this post-translation modification on nuclear architecture were examined at different Mg2+ concentrations using scanning force microscopy. A quantitative analysis of the internucleosomal distance, the width, and the volume of chromatin fibers imaged in tapping mode reveals that poly(ADP-ribosyl)ation induces a complete relaxation and decondensation of the chromatin structure. Our data, on the center-to-center distance between adjacent nucleosomes and on the fiber width, indicate that the poly(ADP-ribosyl)ated fibers remain significantly decondensed even in the presence of Mg2+. Our results also show that the Mg2+ assumes an important role in the folding of chromatin structure, but Mg2+ is not able to restore the native feature of chromatin, when the fibers are depleted of H1/H5 histones. The combined effect of post-translation modification and cation ions on the chromatin structure shows that poly(ADP-ribosyl)ation could promote accessibility to DNA even in those nuclear processes that require Mg2+.     

Reduction of mono(ADP-ribosyl)ation of 20 kDa protein with maturation in rat testis: involvement of guanine nucleotides

When the homogenate prepared from immature rat testes was incubated with [32P]NAD, several proteins (90, 39 and 20 kDa) were ADP-ribosylated in the absence of bacterial toxins. This observation suggested the existence of an endogenous ADP-ribosyltransferase and substrates. The data that the digested product by phosphodiesterase of ADP-ribosylated 20 kDa protein was 5'-AMP suggested that 20 kDa protein was mono(ADP-ribosyl)ated. In addition, the mono(ADP-ribosyl)ation of 20 kDa protein was enhanced by guanine nucleotides such as GTP, GDP and GTP[gamma S], and decreased by the concentrations of 10 mM Mg2+. In contrast, the incorporation of ADP-ribose moiety from NAD to both 90 and 39 kDa proteins was not changed by guanine nucleotides. On the other hand, mono(ADP-ribosyl)ation of 20 kDa protein was not observed in the homogenate prepared from other tissues of the same rats. Furthermore, we found that mono(ADP-ribosyl)ation of 20 kDa protein was decreased with the maturation of the rats and that an endogenous mono(ADP-ribosyl)transferase and 20 kDa protein were located in the nuclei.     

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin. A role of poly(ADP-ribosyl)ation on core nucleosome structure

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin was analyzed by gel electrophoresis, electron microscopy, and velocity sedimentation. In parallel, the interaction of automodified poly(ADP-ribose) polymerase with native and H1-depleted chromatin was analyzed. In H1-depleted chromatin histone H2B becomes the major poly(ADP-ribose) histone acceptor protein, whereas in native chromatin histone H1 was the major histone acceptor. Poly(ADP-ribosyl)ation of H1-depleted chromatin prevented the recondensation of polynucleosomes reconstituted with exogenous histone H1. This is probably due to the presence of modified poly(ADP-ribose) polymerase and hyper(ADP-ribosyl)ated histone H2B. Indeed, about 40% of the modified enzyme remained associated with H1-depleted chromatin, while less than 1% of the modified enzyme was bound to native chromatin. The influence of poly(ADP-ribosyl)ation on the chromatin conformation was also studied at the level of nucleosome in using monoclonal and polyclonal antibodies specific for individual histones and synthetic peptides of histones. In native chromatin incubated in the presence of Mg2+ there was a drop in the accessibility of histone epitopes to monoclonal and polyclonal antibodies whereas upon poly(ADP-ribosyl)ation their accessibility was found to remain even in the presence of Mg2+. In poly(ADP-ribosyl)ated H1-depleted chromatin an increased accessibility of some histone tails to antibodies was observed.     

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.     

Characterization of poly(ADP-ribosyl)ated domains of rat pachytene chromatin.

Poly(ADP-ribosyl)ation of nuclear proteins was several-fold higher in the pachytene spermatocytes than in the premeiotic germ cells of the rat. Among the histones of the pachytene nucleus, histone subtypes H2A, H1 and H3 were poly(ADP-ribosyl)ated. Based on the immunoaffinity fractionation procedure of Malik, Miwa, Sugimara & Smulson [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] we have fractionated DNAase-II-solubilized chromatin into poly(ADP-ribosyl)ated chromatin (PAC) and non-poly(ADP-ribosyl)ated chromatin (non-PAC) domains on an anti-[poly(ADP-ribose)] IgG affinity matrix. Approx. 2.5% of the pachytene chromatin represented the PAC domains. A significant amount of [alpha-32P]dATP-labelled pachytene chromatin (labelled in vitro) was bound to the affinity matrix. The DNA of pachytene PAC domains had internal strand breaks, significant length of gaps and ligatable ends, namely 5'-phosphoryl and 3'-hydroxyl termini. On the other hand, the PAC domains from 18 h regenerating liver had very few gaps, if any. The presence of gaps in the pachytene PAC DNA was also evident from thermal denaturation studies. Although many of the polypeptides were common to the PAC domains of both pachytene and regenerating liver, the DNA sequences associated with these domains were quite different. A 20 kDa protein and the testis-specific histone H1t were selectively enriched in the pachytene PAC domains. The pachytene PAC domains also contained approx. 10% of the messenger coding sequences present in the DNAase-II-solubilized chromatin. The pachytene PAC domains, therefore, may represent highly enriched DNA-repair domains of the pachytene nucleus.     

A method for determining oligo- and poly(ADP-ribosyl)ated enzymes and proteins in vitro

A new method to determine oligo- and poly(ADP-ribosyl)ated enzymes and proteins in vitro has been developed. This method is based on the facts that in Mg2+-depleted condition automodification of poly(ADP-ribose)polymerase is minimized and exogenously added acceptor protein is oligo(ADP-ribosyl)ated predominantly, and in Mg2+-fortified conditions the exogenous acceptor can be poly(ADP-ribosyl)ated. When 13 proteins, including several enzymes, were subjected to this system, dimeric bovine seminal RNase and micrococcal nuclease were found to be oligo(ADP-ribosyl)ated under Mg2+-depleted conditions but their activity was unchanged. Under Mg2+-fortified conditions however, the RNase was deactivated concomitantly with its extensive poly(ADP-ribosyl)ation. When dimeric bovine seminal RNase was monomerized in advance by treatment with dithiothreitol and urea, the enzyme lost ADP-ribose-accepting ability in spite of a significant residual enzyme activity. As used here successfully, the Mg2+-depleted and Mg2+-fortified ADP-ribosylation and subsequent chromatographic analysis of various proteins and enzymes might be an useful method for proving their oligo- and poly(ADP-ribosyl)ation.     

Alkylation-induced mono(ADP-ribosyl)-histones H1 and H2B. Hydroxylamine-resistant linkage in hepatoma cells

Treatment of hepatoma AH 7974 cells with dimethyl sulfate led to a marked accumulation in vivo of mono)ADP-ribosyl)-histone H1A, H1B, H1 and H2B, respectively. In these conjugates, most of the modifying groups were linked to the acceptor proteins by an 'unusual' bond not described so far for ADP-ribosyl histone conjugates. It resisted treatment with 3M hydroxylamine, 0.1M picrylsulfonate and mild alkali, which excluded a linkage through carboxyl or guanidino residues. The stability of these conjugates formed endogenously differed also from 'non-enzymic' histone H1 conjugates formed by incubation of free ADP-ribose with the histone. Histone-linked mono(ADP-ribosyl) residues synthesized in hepatoma cells in response to alkylation were located exclusively in the domains that interact with DNA, i.e. in the non-globular C-terminal tail of histone H1 and in the N-terminus of histone H2B. Besides poly(ADP-ribosyl)ation, the modification of histones by single ADP-ribose groups may represent an independent process to modulate DNA/histone interaction.     

PARP1 Val762Ala polymorphism reduces enzymatic activity

Poly(ADP-ribose) polymerase 1 (PARP1) modifies a variety of nuclear proteins by poly(ADP-ribosyl)ation, and plays diverse roles in molecular and cellular processes. A common PARP1 single nucleotide polymorphism (SNP) at codon 762, resulting in the substitution of alanine (Ala) for valine (Val) in the catalytic domain has been implicated in susceptibility to cancer. To characterize the functional effect of this polymorphism on PARP1, we performed in vitro enzymatic analysis on PARP1-Ala762 and PARP1-Val762. We found that PARP1-Ala762 displayed 57.2% of the activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation and 61.9% of the activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1. The kinetic characterization revealed that the K(m) of PARP1-Ala762 was increased to a 1.2-fold of the K(m) of PARP1-Val762 for trans-poly(ADP-ribosyl)ation. Thus, the PARP1 Val762Ala polymorphism reduces the enzymatic activity of PARP1 by increasing K(m). This finding suggests that different levels of poly(ADP-ribosyl)ation by PARP1 might aid in understanding the cancer risk of carriers of the PARP1 Val762Ala polymorphism.     

Nicotinamide deficiency in human lymphocytes prevents the [H]thymidine-induced adaptive response for the repair of X-ray-induced chromosomal damage

Human lymphocytes treated with [3H]thymidine ([3H]dThd) become refractory to the induction of chromosomal aberrations by subsequent doses of X rays. This adaptive response to [3H]dThd does not occur in the presence of 3-aminobenzamide (3AB). 3AB inhibits the synthesis of poly(ADP-ribose) by the enzyme adenosine diphosphate ribosyl transferase (ADPRT), which requires NAD as a substrate. 3AB also prevents chromosomal repair, as measured in X-ray dose-fractionation studies. Because 3AB might interfere with metabolic reactions other than those mediated by ADPRT, experiments were carried out to see if the adaptive response was also inhibited in nicotinamide-free medium, which prevents poly(ADP-ribosyl)ation by depleting cellular NAD. The experiments show that the incorporation of [3H]dThd has no effect on the induction of chromosomal aberrations by subsequent doses of X rays if the cells are cultured in nicotinamide-free medium. Nicotinamide deficiency mimics the effects of 3AB on both the adaptive response and chromosome repair. The results indicate that ADPRT activity itself, and not other metabolic processes affected by inhibitors of this enzyme, plays an essential role in the adaptive response.     

Identification of a novel gene (ADPRTL1) encoding a potential Poly(ADP-ribosyl)transferase protein

Poly(ADP-ribosyl)ation of nuclear proteins plays a significant role in the maintenance of genomic DNA stability. To date, four poly(ADP-ribosyl)ating proteins have been identified in humans. We now report the full-length sequence, expression profile, and chromosomal localization of a novel gene, ADPRTL1, encoding an ADP-ribosyltransferase-like protein. The predicted open reading frame encodes a protein of 1724 amino acids with a molecular mass of 192.8 kDa. The protein contains a region showing homology to the catalytic domains of the nuclear-localized ADP-ribosyltransferase proteins (Adprt), two recently identified Adprt-like proteins (Adprtl2 and Adprtl3), and the telomere-associated protein tankyrase. Key amino acids known to be important for the activity of these enzymes are conserved within this region of the Adprtl1 protein, indicating that Adprtl1 is a functional poly(ADP-ribosyl)transferase. As has been noted for tankyrase, sequence analysis of the Adprtl1 protein suggests that it is not capable of binding DNA directly. Thus, the transferase activity of Adprtl1 may be activated by other factors such as protein-protein interaction mediated by the extensive carboxyl terminus. We have subsequently refined the location of the ADPRTL1 genomic locus to 13q11, close to the recently cloned ZNF198 gene.