Study of nuclear poly(ADP-ribose)polymerase and DNA-topoisomerase II of brain cells during postnatal development of rats

The nuclear poly(ADP-ribose)polymerase activity of neuronal and glial cells during postnatal development of rats was studied. It was shown that the poly(ADP-ribose)polymerase activity of nuclei and nuclear matrix of neuronal cells during postnatal development of rats is increased, whereas the polymerase activity of glial cell nuclei and nuclear matrix in newborn and adult rats is higher than in 14-day-old animals. The DNA-topoisomerase II activity of neuronal nuclear matrix during the postnatal development of rats does not change, whereas the topoisomerase activity of glial nuclear matrix decreases but is always higher than the DNA-topoisomerase II activity of neuronal cell matrix during the postnatal development of rats. It is suggested that ADP-ribosylation in the nuclear matrix of neuronal cells causes the inhibition of the DNA-topoisomerase II activity of nuclear matrix.     

ADP-ribosylation intensifies cleavage of DNA loops in the nuclear matrix

Using DNA pulse field electrophoresis it has been shown that ADP-ribosylation in the nucleoids of human mononuclear leukocytes and rat brain cortex neurons stimulates cleavage of DNA loops at their attachmant sites to the nuclear matrix. The conclusion has been drawn suggesting possible participation of ADP-ribosylation in DNA-topoisomerase II activity modulation in the nuclear matrix of eukaryotic cells.     

Changes in the level of poly ADP-ribosylation during a cell cycle

In order to analyze the fluctuation of the poly ADP-ribosylation level during the cell cycle of synchronously growing He La S3 cells, we have developed three different assay systems; intact and disrupted nuclear systems, and poly(ADP-ribose) polymerase in vitro system. The optimum conditions for poly ADP-ribosylation in each assay system were similar except the pH optimum. Under the conditions favoring poly ADP-ribosylation, little radioactivity incorporated into poly(ADP-ribose) was lost after termination of the poly ADP-ribosylation by addition of nicotinamide which inhibits the reactions by more than 90% in any system. In the intact nuclear system, the level of poly ADP-ribosylation increased slightly subsequent to late G2 phase with a peak at M phase. The high level of poly ADP-ribosylation in M phase was also confirmed by using selectively collected mitotic cells which were arrested in M phase by Colcemid. The level in mitotic chromosomes was 5.1-fold higher than that in the nuclei from logarithmically growing cells. Colcemid has no effect on the poly ADP-ribosylation. In the disrupted nuclear system, a relatively high level of poly ADP-ribosylation was observed during mid S-G2 phase. When poly(ADP-ribose) polymerase was extracted from the nuclei with a buffer solution containing 0.3 M KCl, more than 90% of the enzyme activity was recovered. The poly(ADP-ribose) polymerase in vitro system was dependent on both DNA and histone—10 μg each. In the enzyme system, enzyme activity was detected throughout the cell cycle and was observed to be highest in G2 phase. The high level at M phase observed in the intact nuclear system was not seen in the other two systems. Under the assay conditions, little influence of poly(ADP-ribose) degrading enzymes was noted on the level of poly ADP-ribosylation in any of the three systems. This was confirmed at various stages during the cell cycle through pulse-labeling and “chasing” by adding nicotinamide.     

Immunochemical detection of guanine nucleotide binding proteins mono-ADP-ribosylated by bacterial toxins

Rabbits immunized with ADP-ribose chemically conjugated to carrier proteins developed antibodies reactive against guanine nucleotide binding proteins (G proteins) that had been mono-ADP-ribosylated by bacterial toxins. Antibody reactivity on immunoblots was strictly dependent on incubation of substrate proteins with both toxin and NAD and was quantitatively related to the extent of ADP-ribosylation. Gi, Go, and transducin (ADP-ribosylated by pertussis toxin) and elongation factor II (EF-II) (ADP-ribosylated by pseudomonas exotoxin) all reacted with ADP-ribose antibodies. ADP-ribose antibodies detected the ADP-ribosylation of an approximately 40-kilodalton (kDa) membrane protein related to Gi in intact human neutrophils incubated with pertussis toxin and the ADP-ribosylation of an approximately 90-kDa cytosolic protein, presumably EF-II, in intact HUT-102 cells incubated with pseudomonas exotoxin. ADP-ribose antibodies represent a novel tool for the identification and study of G proteins and other substrates for bacterial toxin ADP-ribosylation.     

Evidence of poly(ADP-ribosylation) in the cockroach Periplaneta americana

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins typical of most eukaryotic cells. This process participates in DNA replication and repair and is mainly regulated by two enzymes, poly(ADP-ribose) polymerase, which is responsible for the synthesis of polymers of ADP-ribose, and poly(ADP-ribose) glycohydrolase, which performs polymer degradation. The aim of this work was to investigate in the cockroach Periplaneta americana L. (Blattaria: Blattidae) the behaviour of poly(ADP-ribosylation). In particular, we addressed: (i) the possible modulation of poly(ADP-ribosylation) during the embryonic development; (ii) the expression of poly(ADP-ribose) polymerase and glycohydrolase in different tissues; and (iii) the role of poly(ADP-ribosylation) during spermatogenesis. In this work we demonstrated that: (i) as revealed by specific biochemical assays, active poly(ADP-ribose) polymerase and glycohydrolase are present exclusively in P. americana embryos at early stages of development; (ii) an activity carrying out poly(ADP-ribose) synthesis was found in extracts from testes; and (iii) the synthesis of poly(ADP-ribose) occurs preferentially in differentiating spermatids/spermatozoa. Collectively, our results indicate that the poly(ADP-ribosylation) process in P. americana, which is a hemimetabolous insect, displays catalytical and structural features similar to those described in the holometabolous insects and in mammalian cells. Furthermore, this process appears to be modulated during embryonic development and spermatogenesis.     

NAD-dependent ADP-ribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (labile toxin, LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the ADP-ribosylation of arginine (Moss, J., and Richardson, S.H. (1978) J. Clin. Invest. 62, 281-285). Analysis of the product of the ADP-ribosylation of arginine by nuclear magnetic resonance spectroscopy indicated that the reaction was stereospecific and resulted in the formation of alpha-ADP-ribosyl-L-arginine. This reaction product rapidly anomerized to yield a mixture of the alpha and beta forms. In the presence of [adenine-U-14C]NAD, E. coli enterotoxin catalyzed the transfer of the radiolabel to proteins; the ADP-ribosylation of proteins was inhibited by arginine methyl ester, an alternative substrate. Digestion of the 14C-protein with snake venom phosphodiesterase released predominantly 5'-AMP. No product was obtained with a mobility similar to that of 2'-(5'-phosphoribosyl)-5'-AMP. This result is consistent with the covalent attachment by the enterotoxin of ADP-ribose rather than poly(ADP-ribose) to protein. Thus, LT is catalytically equivalent to choleragen, an enterotoxin of Vibrio cholerae, and activates adenylate cyclase through a similar stereospecific ADP-ribosylation reaction.     

Participation of Poly(ADP-ribose)-polymerase of nuclear matrix in DNA repair

The poly(ADP-ribose)-polymerase activity of brain and liver cell nuclei is changed during X-irradiation of rats. In the nuclear matrix, poly(ADP-ribose)-polymerase activity increases at a low dose of irradiation (1.7 Gy) and decreases at a high dose (6.7 Gy). A significant part of the activity of nuclear NMN-adenylyltransferase, a key enzyme for biosynthesis of NAD (the substrate of poly(ADP-ribose)-polymerase), has been found in the nuclear matrix. An interrelation between ADP-ribosylation taking place on the matrix level and eukaryotic cell DNA repair is suggested.     

Poly(ADP-ribosylated) histones in chromatin replication

Poly(ADP-ribosylation) of histones and several other nuclear proteins seem to participate in nuclear processes involving DNA strand breaks like repair, replication, or recombination. This is suggested from the fact that the enzyme poly(ADP-ribose) polymerase responsible for this modification is activated by DNA strand breaks produced in these nuclear processes. In this article I provide three lines of evidence supporting the idea that histone poly(ADP-ribosylation) is involved in chromatin replication. First, cellular lysates from rapidly dividing mouse or human cells in culture synthesize a significant number of oligo- in addition to mono(ADP-ribosylated) histones. Blocking the cells by treatment of cultures with 5 mM butyrate for 24 h or by serum or nutrient depletion results in the synthesis of only mono- but not of oligo(ADP-ribosylated) histones under the same conditions. Thus, the presence of oligo(ADP-ribosylated) histones is related to cell proliferation. Second, cellular lysates or nuclei isolated under mild conditions in the presence of spermine and spermidine and devoid of DNA strand breaks mainly synthesize mono(ADP-ribosylated) histones; introduction of a small number of cuts by DNase I or micrococcal nuclease results in a dramatic increase in the length of poly(ADP-ribose) attached to histones presumably by activation of poly(ADP-ribose) polymerase. Free ends of DNA that could stimulate poly(ADP-ribosylation) of histones are present at the replication fork. Third, putatively acetylated species of histone H4 are more frequently ADP-ribosylated than nonacetylated H4; the number of ADP-ribose groups on histone H4 was found to be equal or exceed by one the number of acetyl groups on this molecule. Since one recognized role of tetraacetylated H4 is its participation in the assembly of new nucleosomes, oligo(ADP-ribosylation) of H4 (and by extension of other histones) may function in new nucleosome formation. Based on these results I propose that poly(ADP-ribosylated) histones are employed for the assembly of histone complexes and their deposition on DNA during replication. Modified histones arise at the replication fork by activation of poly(ADP-ribose) polymerase by unligated Okazaki fragments.     

Poly(ADP-ribosylation) of nuclear proteins in rat thymocytes

Specific antibodies to poly(ADP-ribose) were obtained and characterized. Using these antibodies, the tissue specificity of poly(ADP-ribose) modified nuclear proteins from rat thymocytes and hepatocytes was studied. The differences in the levels of poly(ADP-ribosylation) of nuclear proteins from both tissues were found to be quantitative rather than qualitative. Analysis of intranuclear distribution of poly(ADP-ribose) acceptor proteins revealed that the bulk of them is localized in the nuclear sap and matrix. A comparison of spectral properties of poly(ADP-ribosylated) proteins, using specific antibodies and label incorporation from [14C]NAD showed the existence of two protein groups. Some of those were modified in a great degree but exchange poly(ADP-ribose) at a slow rate, whereas others (e.g., histones and HMG proteins) modified in a small degree exchanged poly(ADP-ribose) at a much higher rate. The results obtained by different methods are discussed.     

The effect of gamma-irradiation on the poly(ADP-ribosylation) of nuclear proteins in rat thymocytes

Differences in the spectra of modified nuclear proteins of thymocytes of control and irradiated rats were investigated using antibodies specific for poly(ADP-ribose) and incorporation of a label from 14C-NAD in vitro. Two classes of modified proteins were identified differing in the rate of the polymer metabolism and the degree of poly(ADP-ribosylation). No postirradiation changes were detected in poly(ADP-ribosylation) of the nuclear sap proteins and chromatin. A pronounced increase in modification of proteins with the molecular mass of 72 and 83 kD and a sharp decrease in poly(ADP-ribosylation) of a protein group with the molecular mass of 47 to 65 kD were detected within the nuclear matrix by the second hour following irradiation. A study was made of the localization of modified proteins in polydeoxynucleotide fractions of different sizes (mononucleosomes and their oligomers).     

Selected nuclear matrix proteins are targets for poly(ADP-ribose)-binding

Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system. J. Cell. Biochem. 70:596–603. © 1998 Wiley-Liss, Inc.     

Polypeptide Hormones and Chromatin-Associated Proteins Act as Acceptors for Cholera Toxin-Catalyzed ADP-Ribosylation

Abstract: Cholera toxin catalyzed the ADP-ribosylation of the pituitary protein hormones thyrotropin (TSH), lutropin (LH), follitropin (FSH), human chorionic gonadotropin (hCG). and corticotropin (ACTH)_1–24, and ADP-ribosylation of the basic proteins histone subfraction H₁ and protamine. Casein and phosvitin, acidic nuclear proteins, did not act as acceptors for toxin-catalyzed ADP-ribosylation. The isolated TSH A and B subunits were tested for their ADP-ribose acceptor activity. The TSH A subunit showed fourfold greater ADP-ribose acceptor activity than the TSH B subunit. The ADP-ribose acceptor protein protamine was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis following incubation with cholera toxin under ADP-ribosylating conditions. [³H]ADP-ribose incorporated into protein from [³H]NAD migrated with the acceptor protein protamine. In the absence of added acceptor protein, the [³H]ADP-ribose incorporated into protein migrated with the A₁ fragment of cholera toxin. Cholera toxin A and B subunits were isolated and tested for their ability to catalyze the transfer of ADP-ribose to protamine. The cholera toxin A subunit showed 50-fold greater ADP-ribosyltransferase activity than the B subunit. Our data indicate that a variety of adenohypophyseal hormones and regulatory proteins act as acceptors for toxin-catalyzed ADP-ribosylation. These studies may help in understanding the role of endogenous ADP-ribosyltransferases and the physiological effects of this modification of protein.     

Poly ADP-ribosylation of histones in tumor promoter phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts C3H10T1/2

The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the poly ADP-ribosylation of acid extractable (0.2N H2SO4) nuclear proteins in mouse embryo fibroblasts C3H10T1/2. Catalase suppresses the reaction by approximately 50%. Polyacrylamide gel electrophoresis reveals that the core histones H2B, A24 and H3d serve as major poly ADP-ribose acceptors. Smaller amounts of poly ADP-ribose are associated with histones H2A/H3 and H1. Poly ADP-ribosylation of histones may change the nucleosomal structure and function and play a role in PMA induced modulation of gene expression in promotion.     

Unexpected stimulation of mitochondrial ADP-ribosylation by cyanide

Cyanide, the classical inhibitor of the mitochondrial respiratory chain at site III, stimulates ADP-ribosylation of a number of mitochondrial proteins, the major protein being the 50-55 kDa band. Sodium azide, sharing the same inhibitory site, does not have the same effect. Rotenone or antimycin A have no influence on mitochondrial ADP-ribosylation. Data suggest that no apparent correlation exists between oxidoreductase function and protein ADP-ribosylation. Purified nuclear poly(ADP-ribose) polymerase activity was not affected by cyanide. The cyanide effect on mitochondrial ADP-ribosylation seems intriguing and may be attributed to NAD+-CN complex formation, since NAD reacts with cyanide at pH greater than 8 with N-substituted nicotinamide which may prevent inhibition of ADP-ribosylation.     

Functional interaction of poly(ADP-ribose) with the 20S proteasome in vitro.

The nuclear enzyme poly(ADP-ribosyl) transferase (pADPRT) catalyzes the formation of poly(ADP-ribose) from NAD+. Several nuclear proteins and pADPRT itself are targets for the modification by poly(ADP-ribosyl)ation. It is demonstrated here that poly(ADP-ribose) or pADPRT automodified with poly(ADP-ribose) interacts noncovalently with the 20S proteasome in vitro. The interaction of pADPRT with the 20S proteasome requires the long ADP-ribose polymers formed by automodification of the pADPRT with poly(ADP-ribose). As a result pADPRT automodified with short ADP-ribose oligomers is unable to associate with the 20S proteasome. The interaction with poly(ADP-ribose) causes a specific stimulation of the peptidase activity of the 20S proteasome. Modified pADPRT does not serve as a substrate for the degradation by the 20S proteasome. No covalent modification of the 20S proteasome by ADP-ribosylation was observed. The results may point to a functional relationship between pADPRT and the 20S proteasome in a pathway protecting the cell from oxidative damage.     

Increased ionic strength: effects on DNA fragmentation and poly(ADP-ribose) polymerase activity in HeLa nuclei

Poly(ADP-ribose) polymerase activity was measured in a crude nuclear fraction isolated from HeLa cells. It was found that the addition of ammonium sulfate or other salts to the standard incubation medium inhibited the formation of poly(ADP-ribose). Through the use of alkaline sucrose density gradients it was also noted that this same increase in ionic strength inhibited the in vitro breakdown of the HeLa DNA. Additional experiments with alkaline sucrose density gradients and deoxyribonuclease I showed that the in vitro activity of poly(ADP-ribose) polymerase is largely dependent upon DNA fragmentation but that DNA fragmentation at least in vitro is not dependent upon the formation of poly(ADP-ribose). These observations imply that this nuclear enzyme is not extremely sensitive to changes in the ionic strength of the reaction media but is affected indirectly, supposedly through changes in the endonuclease activity of the HeLa nuclei. If this proves to be true, then the addition of salt to the incubation medium for poly(ADP-ribose) polymerase could prove to be a valuable tool for the study of ADP-ribosylation reactions.     

Sensitivity in vitro of mature and immature mouse thymocytes to dexamethasone cytotoxicity and its correlation to poly ADP-ribosylation.

Mouse thymocytes were fractionated into heavy (subtype I, 79% of total cell number), medium (subtype II, 18%) and light (subtype III, 3%) ones by Percoll density centrifugation and they were identified as immature (subtype I and II) and mature (subtype III) thymocytes based on their proliferative response to mitogens. Whereas the nuclear activity of poly (ADP-ribose) polymerase (EC 2.4.2.30) in the subtype III was only one half that of denser subtypes, it increased two-fold upon mitogen stimulation. The sensitivity of three thymocyte subtypes to the dexamethasone cytotoxicity, as judged by the extent of the DNA cleavage, depletion of NAD and cell viability, was highest in the subtype I and lowest in the subtype III. The possible involvement of poly ADP-ribosylation in the apoptotic (programmed) cell death during intrathymic development of immature to mature thymocytes is discussed.     

Poly(adenosine diphosphate-ribosylation) of nuclear matrix proteins in alkylating agent resistant and sensitive cell lines

Using Walker 256 breast carcinoma cell lines either with or without acquired resistance to alkylating agents, the structural framework proteins of the nucleus, the nuclear matrix proteins, were found to be effective acceptors for poly(ADP-ribose). Incubation of isolated nuclei with nicotinamide adenine [32P] dinucleotide ([32P] NAD), followed by the isolation of the nuclear matrix, demonstrated that two polypeptides of approximate molecular weight (Mr) 105 000 and 116 000 were extensively poly(ADP-ribosylated). By an in vitro [32P] NAD assay, the nuclear matrix fraction was found to maintain approx. 15% of the total nuclear matrix activity of poly(ADP-ribose) polymerase. Confirmation that the trichloroacetic acid (TCA) precipitable material represented ADP-ribose units was achieved by enzymatic digestion of the nuclear matrix preparation with snake venom phosphodiesterase (SVP). Within 15 min, greater than 85% of the 32P label was digested by SVP and the final digestion products were found to be phosphoribosyl-AMP (PR-AMP) and adenosine 5'-monophosphate (5'-AMP) by thin layer chromatographic analysis. The average polymer chain length was estimated to be 6-7 ADP-ribose units. Because poly(ADP-ribose) polymerase has a putative role in DNA repair, a comparison of the nuclear matrix fractions from Walker resistant and sensitive tumor cell lines was made. In both cell lines, the quantitative and qualitative patterns of the nuclear matrix associated poly(ADP-ribosylation) were similar.     

Increase in histone poly (ADP-ribosylation) in mitogen-activated lymphoid cells

Poly (ADP-ribosylated) histones appear to be intermediates in nuclear processes that involve DNA strand breaks. We have studied histone ADP-ribosylation in cellular lysates from activated human lymphoid cells in culture. Modified histones differing in the number of ADP-ribose groups gave separate bands upon two-dimensional gel electrophoresis. Cellular lysates from control cells contained histones modified with 1 to 15 ADP-ribose groups. Stimulation of the cells during culture with phytohemagglutinin (PHA) or a phorbol ester (TPA) as well as combinations of these two reagents led to a significant increase in the upper limit number of ADP-ribose groups attached to histones in the presence of divalent metal ions. Hyper (ADP-ribosylated) H2B carrying at least 32 ADP-ribose groups gave a distinctly characteristic pattern on two-dimensional gels showing that highly ordered enzymatic steps are followed for its synthesis. Moreover, it was found that PHA and/or TPA induces branching of the poly (ADP-ribose) on H2B. The increase in histone poly (ADP-ribosylation) following lymphocyte activation was less dramatic during incubation of cellular lysates in the absence of divalent metal ions. The increased histone modification observed in this study may result from an increase in cell proliferation during activation of lymphoid cells. The finding that the number of ADP-ribose groups on H4 equals or exceeds by one the number of acetyl groups suggests that the two modifications may share common functions.     

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.