Poly ADPR Research Articles

Reading ADP-ribosylation signaling using chemical biology and interaction proteomics

ADP-ribose (ADPr) readers are essential components of ADP-ribosylation signaling, which regulates genome maintenance and immunity. The identification and discrimination between monoADPr (MAR) and polyADPr (PAR) readers is difficult because of a lack of suitable affinity-enrichment reagents. We synthesized well-defined ADPr probes and used these for affinity purifications combined with relative and absolute quantitative mass spectrometry to generate proteome-wide MAR and PAR interactomes, including determination of apparent binding affinities. Among the main findings, MAR and PAR readers regulate various common and distinct processes, such as the DNA-damage response, cellular metabolism, RNA trafficking, and transcription. We monitored the dynamics of PAR interactions upon induction of oxidative DNA damage and uncovered the mechanistic connections between ubiquitin signaling and ADP-ribosylation. Taken together, chemical biology enables exploration of MAR and PAR readers using interaction proteomics. Furthermore, the generated MAR and PAR interaction maps significantly expand our current understanding of ADPr signaling.

DETECTION OF POLY(ADP-RIBOSE) SYNTHETASE ACTIVITY IN ALVEOLAR MACROPHAGES OF RATS EXPOSED TO NITROGEN DIOXIDE AND OZONE

The toxic effects of nitrogen dioxide (NO2) and ozone (O3) are mediated through the formation of free radicals, which can cause DNA strand breaks. The present study demonstrates that exposure to NO2 and O3 causes a stimulation of poly (ADP-ribose) (poly ADPR) synthetase in alveolar macrophages of rats. Three-month-old male Sprague-Dawley rats, specific pathogen free, were exposed to either 1.2 ppm NO2 or 0.3 ppm O3 alone or a combination of these 2 oxidants continuously for 3 days. The control group was exposed to filtered room air. To evaluate whether exposure to these two oxidants (NO2 and O3) caused DNA damage to lung cells, the activity of poly ADPR synthetase was measured. Cellular DNA repair is dependent upon the formation of poly (ADP-ribose) polymerase, which is catalyzed by poly ADPR synthetase. Poly ADPR synthetase is known to be activated in response of DNA damage. The results showed that the enzyme activity was stimulated after exposure to O3 or exposure to NO2 + O3. Ozone exposure caused a 25% increase in the enzyme activity as compared to the control. Combined exposure to NO2 + O3 showed a 53% increase in the enzyme activity. These results were statistically significant as compared to the control and NO2 exposure groups. Other parameters such as total cell count, cell viability, and differential cell count were also determined. The stimulation of poly ADPR synthetase activity after O3 exposure or NO2 + O3 exposure reflects a response to lung cellular DNA repair, which may be used as an indicator for assessing DNA damage caused by oxidant injury.

DETECTION OF POLY(ADP-RIBOSE) SYNTHETASE ACTIVITY IN ALVEOLAR MACROPHAGES OF RATS EXPOSED TO NITROGEN DIOXIDE AND OZONE

The toxic effects of nitrogen dioxide (NO2) and ozone (O3) are mediated through the formation of free radicals, which can cause DNA strand breaks. The present study demonstrates that exposure to NO2 and O3 causes a stimulation of poly (ADP-ribose) (poly ADPR) synthetase in alveolar macrophages of rats. Three-month-old male Sprague-Dawley rats, specific pathogen free, were exposed to either 1.2 ppm NO2 or 0.3 ppm O3 alone or a combination of these 2 oxidants continuously for 3 days. The control group was exposed to filtered room air. To evaluate whether exposure to these two oxidants (NO2 and O3) caused DNA damage to lung cells, the activity of poly ADPR synthetase was measured. Cellular DNA repair is dependent upon the formation of poly (ADP-ribose) polymerase, which is catalyzed by poly ADPR synthetase. Poly ADPR synthetase is known to be activated in response of DNA damage. The results showed that the enzyme activity was stimulated after exposure to O3 or exposure to NO2 + O3. Ozone exposure caused a 25% increase in the enzyme activity as compared to the control. Combined exposure to NO2 + O3 showed a 53% increase in the enzyme activity. These results were statistically significant as compared to the control and NO2 exposure groups. Other parameters such as total cell count, cell viability, and differential cell count were also determined. The stimulation of poly ADPR synthetase activity after O3 exposure or NO2 + O3 exposure reflects a response to lung cellular DNA repair, which may be used as an indicator for assessing DNA damage caused by oxidant injury.

Calcium ionophore A 23187 induces apoptotic cell death in rat thymocytes.

Apoptotic cell death was induced in rat thymocytes on exposure to calcium ionophore A 23187 (100 micron(s)) for 24 h as observed from morphological changes and DNA fragmentation into oligonucleosomal ladder. The cell death was independent of de novo syntheses of protein. However, the involvement of c-Myc, c-Jun, poly ADPR polymerase and antioxidant enzymes CuZn SOD and catalase was observed.

The effect of active oxygen generated by xanthine/xanthine oxidase on genes and signal transduction in mouse epidermal JB6 cells.

To gain insight into the gene regulation and signal transduction effects of active oxygen in tumour promotion and progression, we studied the effect of active oxygen generated extracellularly by xanthine/xanthine oxidase (X/XO) in promotion-insensitive (P-), promotion-sensitive (P+) and transformed (Tx) mouse epidermal JB6 cells. Active oxygen inhibited growth, particularly of P- cells and increased poly ADPR transferase activity and PKC activity more significantly in P- cells. No phenotypic differences in the distribution pattern of PKC isotypes alpha, beta and gamma were seen in JB6 cells. PKC alpha was expressed abundantly, whereas beta and gamma were not detected. Basal levels of the antioxidant enzymes catalase and CuZn. Superoxide dismutase were higher in P+ and Tx cells. X/XO resulted in an initial decrease in the activity of these enzymes, followed by recovery or transient induction in Tx and P+ cells. X/XO induced c-myc and c-fos expression in JB6 cells, with c-fos induction being more pronounced in P- cells, whereas a biphasic increase in c-jun was seen in P+ cells. These early genes may play a role in proliferation whereas post-translational poly ADP-ribosylation and, perhaps, phosphorylation suggest a genetic-epigenetic mechanism in oxidant tumour promotion and progression.

Enhanced poly ADP-ribosylation in human leukemia lymphocytes and ovarian cancers

Freshly isolated human peripheral blood lymphocytes from leukemia (AML, ALL, CML) subjects, showed a 2.5 – 3.5-fold increase in the poly ADPR transferase (poly ADPRT) activity whereas ovarian cancers showed a 2-fold increase. This was accompanied by a drop in NAD levels of 45% – 63% in leukemia cells and 40% in ovarian cancers. Tumour promoters phorbol-12-myristate-13-acetate (PMA) and mezerein produced an increase in poly ADPRT activity in both normal and CML lymphocytes, but the increase was more marked in the case of normals. This was accompanied by a drop in NAD levels. The results presented show a marked increase in poly ADP-ribosylation in malignant cells but normal lymphocytes showed a greater response to tumour promoters as compared to CML lymphocytes.

Poly ADP-ribosylation and DNA strand breakage in SV40 minichromosomes.

Poly ADP-ribosylation and DNA strand breakage in response to treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were studied on SV40 minichromosomes in SV40-infected, permeabilized CV-1 monkey cells. After an initial sharp increase in poly ADP-ribosylation, strand breakage and poly ADPR increased proportionately with increasing dose of MNNG. This suggests a cause-effect relationship between the two reactions. The major poly ADP-ribose acceptor of minichromosomes was core histone H2B. In contrast, H2B, H2A, H1 and protein A24 were poly ADP-ribosylated in the nuclear chromatin of the same cells.

Cell specific response of cardiac poly ADP-R and DNA synthesis to circulatory stress.

Aortic coarctation induces a large increase in poly ADP-R synthetase activity in non-cardiocyte nuclei, and in cardiocyte nuclei inhibition occures, suggesting a differentiation dependent regulation of polymer metabolism. In noncardiocyte nuclei DNA and poly ADP-R (polyadenosine diphosphoribose) synthesis exhibit positive correlation.

Cell specific response of cardiac poly ADP-R to circulatory stress produced by either aortic coarctation or triiodothyronine administration : [formula omitted]. Cardiovascular Research Institute, Univ. California San Francisco, CA 94143

Cell specific response of cardiac poly ADP-R to circulatory stress produced by either aortic coarctation or triiodothyronine administration : [formula omitted]. Cardiovascular Research Institute, Univ. California San Francisco, CA 94143

Studies on poly ADPR polymerase using the specific antibody

Summary Specific antibodies against purified calf thymus poly ADPR polymerase were characterized. A micro complement fixation method was used both to determine the enzyme levels in different tissues, and to investigate the antigenic structure of poly ADPR polymerase.

Adenosine diphosphate ribosylation of histone H1 by purified calf thymus polyadenosine diphosphate ribose polymerase

The mechanism of poly ADPR synthesis and the transfer of poly ADPR to histone H1 molecule by electrophoretically homogenous calf thymus poly ADPR polymerase containing DNA was examined. 1) An acid insoluble radioactive complex (I) was obtained after incubation of purified enzyme with [3H] NAD. The stability of (I) was examined by SDS-polyacrylamide gel electrophoresis. The complex (I) was stable against acid, SDS, urea, DNase and RNase, but labile against pronase, trypsin, alkali and snake venom phosphodiesterase treatment. The molecular weight of (I) was about 130 000 daltons estimated by SDS-gel electrophoresis. The radioactive products of successive alkali, venom phosphodiesterase and Pronase hydrolysis of (I) were PR-AMP and AMP. The mean chain length of poly ADPR of (I) was 20--30. These results suggest that the complex (I) is poly ADP-ribosylated poly ADPR polymerase. 2) Besides (I), a second radioactive peak (II) was observed when acid insoluble products obtained from an incubation mixture containing purified poly ADPR polymerase, [3H] NAD and purified histone H1 were analyzed on SDS-polyacrylamide gel electrophoresis. The molecular weight of (II) was estimated to be about 23 000 daltons. The complex (II) is eluted like histone H1 on CM-cellulose columns and hydrolyzed by alkali, trypsin and snake venom phosphodiesterase but not by DNase, or RNase. The comples (II) was extracted selectively by 5 per cent perchloric acid or 5 per cent trichloroacetic acid from mixture of (I) and (II). The mean chain length of poly ADPR of complex (II) and 5--20; these results suggest that the complex (II) is poly ADP-ribosylated histone H1. 3) Results 1) and 2) indicate that purified DNA containing, thus DNA independent, poly ADPR polymerase catalyzes two different reactions, the ADPR transfer onto the enzyme itself and onto histone H1 and the elongation of ADPR chains. Dimeric forms of ADP-ribosylated histone H1 was not observed. Free poly ADPR was observed only when very small quantities of enzyme were used for incubation.

NAD turnover in microplasmodia of physarum polycephalum.

The rate of NAD turnover in microplasmodia of Physarum polycephalum was investigated using a double labeling technique with (14C)-adenine or adenosine and (3H)-nicotinamide. The half-life of an NAD molecule in Physarum was estimated to be 25 min, which is shorter than in either E. coli or human cell lines. The half-life of NAD in the presence of an inhibitor of NADase and poly ADPR synthase, 5-methylnicotinamide, was also investigated, but found to be indistinguishable from controls. The possible reasons for this and for the rapid turnover is discussed in the light of the known functions for NAD in prokaryotes and eukaryotes.

Magnitude and significance of NAD turnover in human cell line D98/AH2

IN the oxidation–reduction metabolism of a cell the pyridine nucleotide NAD is used catalytically; any NAD that is reduced is reoxidised. But NAD is consumed in certain metabolic reactions in which it serves as a substrate. In eukaryotic cells, the most intriguing of these reactions is the cleavage of NAD to form nicotinamide and a unique polymer, poly adenosine diphosphoribose (poly ADPR) (Fig. 1); the reaction is catalysed by the enzyme poly ADPR synthetase1–4. Other well studied reactions involving the destruction of NAD include the cleavage of NAD to form AMP and NMN by bacterial DNA ligases5,6 and the breakdown of NAD (with the concomitant inactivation of protein synthesis) by diphtheria toxin7,8.

Mode of Action of Rat Liver Phosphodiesterase on a Polymer of Phosphoribosyl Adenosine Monophosphate and Related Compounds

Abstract Pyrophosphatase activity of rat liver phosphodiesterase, which forms nucleoside 5'-monophosphates, was studied. Evidence is presented that NADH, adenosine 5'-diphosphate ribose, and a polymer of phosphoribosyl-AMP (poly (ADPR)) were hydrolyzed by this enzyme. The mode of action of the enzyme on poly ADPR was studied. 1. The enzyme hydrolyzed poly ADPR to produce the same product as was formed by phosphodiesterase from snake venom. 2. Kinetic studies suggest that both the phosphodiester compounds, p-nitrophenyl urinine 5'-monophosphate and poly ADPR, were hydrolyzed by the same site or by overlapping active sites. 3. Poly ADPR was not hydrolyzed randomly, but rather in an exonucleolytic fashion. Little DNA or RNA was hydrolyzed by an amount of enzyme sufficient to decompose half the initial amount of the substrate, poly ADPR.

Myocardial DNA synthesis in experimental cardiac hypertrophy.

Myocardial DNA synthesis in experimental cardiac hypertrophy.