ADP-ribosylation Of Nuclear Proteins Research Articles

Synthesis of ADP-Ribosylated Histones Reveals Site-Specific Impacts on Chromatin Structure and Function.

ADP-ribosylation of nuclear proteins is a critical feature of various DNA damage repair pathways. Histones, particularly H3 and H2B, are major targets of ADP-ribosylation and are primarily modified on serine with a single ADP-ribose unit following DNA damage. While the overall impact of PARP1-dependent poly-ADP-ribosylation is heavily investigated, very little is known about the specific roles of histone ADP-ribosylation. Here, we report the development of an efficient and modular semisynthetic route to full-length ADP-ribosylated histones H3 and H2B, chemically installed at specific serine residues. The modified histones were used to generate various chemically defined ADP-ribosylated chromatin substrates, which were employed in biophysical assays. These studies revealed that ADP-ribosylation of serine-6 of histone H2B (H2BS6ADPr) inhibits chromatin folding and higher-order organization; notably, this effect was enhanced by ADP-ribosylation of H3S10. In addition, ADP-ribosylated nucleosomes were utilized in biochemical experiments employing a panel of lysine methyltransferase enzymes, revealing a context-dependent inhibition of histone H3K9 methylation. The availability of designer ADP-ribosylated chromatin described here is expected to facilitate further biochemical and structural studies regarding the roles of histone ADP-ribosylation in the DNA damage response.

Ethanol increases the formation of NADP+ in rat hepatocytes

The acute effects of ethanol on total (bound + free) pyridine dinucleotides were determined in freshly isolated rat hepatocytes. Pyridine dinucleotides and adenine nucleotides were determined by high-performance liquid chromatography. Exposure of the hepatocytes to 8 mmol/L ethanol resulted in a decrease in NAD+ and an increase in NADP+ after 2 min incubation. There were no significant changes in NADH and NADPH. Ethanol decreased ATP and increased AMP after 2 min, whereas an increase in ADP was only apparent after 15 min of incubation. Ethanol 8 mmol/L and 100 mmol/L resulted in an increased incorporation of [32P] into NADP+ from [32P]-prelabeled NAD+ and ATP. Ethanol increased hepatocyte NAD+ kinase activity; this effect was blocked by 4-methylpyrazole but reproduced by 10 mumol acetaldehyde. These observations indicate that ethanol increases the synthesis of NADP+ and that this effect is most likely the result of increased NAD+ kinase activity. The ethanol-induced decrease of NAD+ may limit ADP ribosylation of nuclear proteins, whereas increases in NADP+ may stimulate the pentose phosphate cycle.

Influence of thyroid hormone on ADP-ribosylation of nuclear proteins in cultured GH1 cells

We present evidence that T3 can alter the ADP-ribosylation of chromatin associated proteins. Nuclei from GH1 cells were incubated with [adenylate- 32P]NAD and the radioactivity incorporated into histone and non-histone proteins was quantitated and analyzed by gel electrophoresis and autoradiography. Incubation of GH1 cells for 24 h with T3 lowered by 40–70% the [ 32P]ADP-ribose incorporated into nuclear proteins. However, incubation for 3 h with T3 resulted in a simulation instead of a decrease of in vitro [ 32P]ADP-ribose incorporation. The major ADP-ribosylated component electrophoresed as a 120 000 molecular mass non-histone protein, and radiolabeled histones were also observed. The same protein species were observed for all the experimental groups and T3 affected the extent of ADP-ribosylation but did not alter the sedimentation of the [ 32P]ADP-ribosylated components excised from chromatin after micrococcal nuclease digestion.

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.

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 ageing 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.

ADP-ribosylation in isolated nuclei of Physarum polycephalum.

ADP-ribosylation of histones and non-histone nuclear proteins was studied in isolated nuclei during the naturally synchronous cell cycle of Physarum polycephalum. Aside from ADP-ribosyltransferase (ADPRT) itself, histones and high mobility group-like proteins are the main acceptors for ADP-ribose. The majority of these ADP-ribose residues is NH2OH-labile. ADP-ribosylation of the nuclear proteins is periodic during the cell cycle with maximum incorporation in early to mid G2-phase. In activity gels two enzyme forms with Mr of 115,000 and 75,000 can be identified. Both enzyme forms are present at a constant ratio of 3:1 during the cell cycle. The higher molecular mass form cannot be converted in vitro to the low molecular mass form, excluding an artificial degradation during isolation of nuclei. The ADPRT forms were purified and separated by h.p.l.c. The low molecular mass form is inhibited by different ADPRT inhibitors to a stronger extent and is the main acceptor for auto-ADP-ribosylation. The high molecular mass form is only moderately auto-ADP-ribosylated.

Cell cycle variations in ADP-ribosylation of HeLa nuclear proteins

Changes in ADP-ribosylation of nuclear proteins during the HeLa cell cycle were determined. Portions of synchronized cultures were withdrawn at intervals and cells were permeabilized by resuspension in hypotonic buffer containing detergents. Nuclear proteins were radioactively labeled by incubating samples with [ 32P]NAD. Modified species were resolved using one-dimensional and two-dimensional polyacrylamide gel electrophoresis. Measurements of the incorporation of [ 32P]NAD by permeabilized cells showed that ADP-ribosylation is a significant modification throughout the cell cycle. A twofold increase was detected during S phase. Autoradiograms of one-dimensional sodium dodecyl sulfate-polyacrylamide gels revealed that many nuclear nonhistones are modified, though the major acceptors of 32P were the histones and a 116,000-Da species (poly(ADP-ribose) polymerase). The same modified proteins were present through the cell cycle, but densitometry of autoradiograms demonstrated a general increase in the level of incorporation in S phase. Autoradiograms of two-dimensional gels of nuclear proteins labeled with [ 32P]NAD were consistent with these results. Although nonhistones of isolated metaphase chromosomes show a substantial reduction in ADP-ribosylation, histone modification is essentially unchanged in metaphase.

ADP-ribosylation of nuclear proteins in vivo. Identification of histone H2B as a major acceptor for mono- and poly(ADP-ribose) in dimethyl sulfate-treated hepatoma AH 7974 cells.

Nuclear mono- and poly(ADP-ribosyl) protein conjugates formed in living hepatoma AH 7974 cells in response to treatment with the alkylating agent dimethyl sulfate have been studied. They were isolated from the perchloric acid precipitate of freshly prepared nuclei in a relatively pure form and with an overall yield of more than 80%, utilizing aminophenylboronic acid-agarose chromatography. Exposure of the cells to 400 microM dimethyl sulfate led to a transient rise of ADP-ribosylated proteins. After 20 min, the level of endogenous poly(ADP-ribosyl) residues increased by a factor of 21, amounting to a final value of 772 +/- 57 pmol/mg of DNA while the mono(ADP-ribosyl) residues were raised to even higher concentrations (1864 pmol/mg of DNA), corresponding to a 12-fold stimulation as compared to untreated cells. As a result of dimethyl sulfate treatment, the amount of acceptor protein being modified by (ADP-ribose)n was elevated 15-fold, reaching a final proportion of 2.3 +/- 0.4% of total nuclear protein. The increase in (ADP-ribosyl)n-modified proteins was suppressed by benzamide, a potent inhibitor of poly(ADP-ribose) synthetase. More than half of the nuclear mono- and poly(ADP-ribosyl) residues were linked to histone H2B. The modifying residues could be removed from the major acceptor by treatment with 0.1 M NaOH, but not with neutral hydroxylamine. Minor amounts of other histones, especially of histone H4, were possibly also ADP-ribosylated under the stimulating effect of dimethyl sulfate. In addition, several nonhistone proteins with apparent molecular masses of 100-116 and 170 kDa were found to carry substantial amounts of mono- and poly(ADP-ribose).

ADP-ribosylation of nuclear proteins in mouse testis.

The nuclear acceptor proteins for poly(ADP-ribose) were investigated in mouse liver and testis. In liver, histones are ribosylated preferentially, whereas in testis the major acceptors are non-histone proteins. An analysis of the purified testicular acceptor proteins suggests that they are high- and low-mobility-group-like proteins.

Stimulation of ADP-ribosylation of nuclear proteins in permeabilized cells after treatment of mouse lymphoma (L1210) cells with the alkylating agent, dimethyl sulphate

Stimulation of ADP-ribosylation of nuclear proteins in permeabilized cells after treatment of mouse lymphoma (L1210) cells with the alkylating agent, dimethyl sulphate

Separation of poly(ADP-ribosylated) nuclear proteins by polyacrylamide gel electrophoresis at acidic pH and low temperature

A method for the extraction and electrophoresis of poly(ADP-ribosylated) nuclear proteins is described. An extraction method using lithium dodecyl sulfate as detergent at pH 2.4 and room temperature is shown to fully extract nuclear proteins under conditions where full stability of protein-linked polymer is ensured. The polyacrylamide gel electrophoresis is performed again under conditions where full stability is ensured. This work provides a technique whereby misinterpretation of relative ADP ribosylation of nuclear proteins can be avoided.

ADP-ribosylation of nuclear proteins.

ADP-ribosylation of nuclear proteins.

ADP-ribosylation of nuclear proteins

Covalent modification of nuclear proteins by mono ADP-ribosylation and poly ADP-ribosylation was studied in various tissues and under various growth conditions with the aid of a newly developed radioimmunoassay. Two types of (ADPR) n protein conjugates were found in vitro and in intact tissues which could be differentiated by their sensitivity towards neutral NH 2OH. Analysis of the cell cycle of Physarum polycephalum showed independent synthesis of these two fractions. Widely differing ratios of the two types of conjugates were also found in different hepatic tissues. Yoshida hepatoma cells had very low levels of both types of protein bound ADPR residues (1 ADPR residue per 2,000–3,000 DNA bases), while neonatal liver was characterized by the (near) absence of NH 2OH resistant ADPR protein conjugates. Conjugates carrying poly(ADPR) residues exhibited independent variations. Proliferating tissues consistently had somewhat lower levels of mono(ADPR) protein conjugates than the same tissues in resting conditions. The independent changes of mono and poly(ADPR) protein conjugates under various conditions, and the existence of multiple acceptor proteins in the nucleus point to multiple functions of ADP ribosylation rather than to a single role of this covalent modification reaction. Analysis of (ADPR) n histone H1 conjugates isolated from ( 3H) adenosine labeled HeLa cultures by a new procedure indicated modification of < 1% of total histone H1 by ADP-ribosylation. Most of the conjugates carried single ADPR residues, while (ADPR) n histone H1 conjugates formed by incubation of isolated nuclei with NAD contained mainly poly(ADPR) residues. There were additional basic differences in the degree of ADP- and polyADP-ribosylation as well as in the types of bonds linking ADPR to the histone which seriously limit the conclusions drawn from experiments with isolated nuclei or chromatin. The low levels of ADPR conjugates in vivo, together with the high enzymic capacity for ADPR transfer observed in many cell types, point to a rapid turnover of protein bound ADPR residues being consistent with the occurrence of rapid transient modifications of protein in localized regions of chromatin.

Suppression and stimulation of DNA synthesis by ADP-ribosylation of nuclear proteins from adult hen and chick embryo liver

The stimulation of DNA synthesis by ADP-ribosylation of nuclear proteins was observed in chick embryo liver nuclei. In contrast, a significant decrease in template activity was detected in hen liver nuclei treated with NAD. When a 0.35 M NaCl extract from embryo, but not adult, liver nuclei was treated with NAD and then combined with either adult or embryo liver nuclear residue, the ability to activate the template was greatly enhanced. These results indicate that in the chick embryo liver, the ADP-ribosylation of the nuclei plays an important role as a regulator of DNA synthesis.

Effect of polyamines on ADP-ribosylation of nuclear proteins from rat liver

The effects of polyamines on the in vitro ADP-ribosylation of rat liver nuclei have been found to be dose dependent. These effects were detected in the absence of Mg 2+. Maximal increases occurred at 1 mM spermine, 5 mM spermidine, and 7.5 mM putrescine, respectively. The rate of ADP-ribosylation in the presence of 1 mM spermine was 2-fold higher than that in the presence of 10 mM Mg 2+, a concentration which maximally stimulates ADP-ribosylation. When the nuclei were incubated with NAD in the presence of spermine, ADP-ribosylation occurred predominantly in the non-histone protein fractions, but in the presence of Mg 2+, it occurred predominantly in the histone fractions. These results suggest that polyamines may have some regulatory effect on the ADP-ribosylation of non-histone chromatin proteins in rat liver.

Quantitation of hydroxylamine sensitive mono(adenosine diphosphate ribose) residues in different hepatic tissues

ADP-ribosylation of nuclear proteins in vitro was first shown by Hayaishi’s group [I] , subsequent work from Dietrich et al. [2] placing special emphasis on the existence of mono(ADPR) residues rather than poly(ADPR) in the protein conjugates. However, secondary degradation of oligoor poly (ADPR) residues to the monomer has not been ruled out in these experiments. In vitro experiments using short-term incubation of nuclei followed by immediate inactivation of enzymes revealed considerable differences in tissues with widely differing proliferation rates, in the levels of monoADP-ribosylated nuclear proteins susceptible to NH2 OH [3] . We were interested in finding out whether this in vitro correlation reflected the in vivo situation. Since no method was available for the quantitation of mono(ADPR) residues we have developed an isotope dilution procedure for measuring NH2 OH-sensitive, proteinbound mono(ADPR), and in this communication report on the endogenous levels of such ADPR residues in different tissues. The data show rather low concentrations of mono(ADPR) residues in vivo, reaching only l-5% of the level of its precursor NAD. Proliferating tissues have significantly lower monoADP-ribosylated proteins than adult rat liver.

Poly(ADP-ribose): release of template restriction in HeLa cells.

Evidence is presented to show that ADP-ribosylation of nuclear proteins by poly(ADP-ribose) polymerase enhances template-primer activity of HeLa cell nuclear DNA. We used Escherichia coli DNA polymerase I (EC 2.7.7.7; DNA nucleotidyltransferase) as an exogenous probe of nuclear DNA status. Neither NAD nor free poly(ADP-ribose) acts directly on the bacterial enzyme. The stimulation is specific for formation of ADP-ribosylated proteins and is not a generalized polyanion or nucleotide effect on chromatin. The release of template restriction is proportional to the capacity of a given cell line to synthesize poly(ADP-ribose); both the stimulation and poly(ADP-ribose) formation are coordinately proportional to NAD concentration. Binding studies with DNA polymerase indicate exposure or generation of additional 3'-OH primer sites due to ADP-ribosylation in intact nuclei. With intact cells, there appears a correlation among growth, physiological template restriction, and the above effects of ADP-ribosylation.

Nuclear polyadenosine diphosphoribosylation during restricted macromolecular synthesis of HeLa cells

The relationships of macromolecular synthesis of intact HeLa cells has been correlated with the ability of nuclei to carry out the ADP ribosylation of nuclear proteins. 1. 1. Selective restriction of DNA replication by hydroxyurea and cytosine arabinoside caused an increase in the rate of enzyme activity similar to that observed when DNA synthesis ceased during the asynchronous growth cycle of the cells. 2. 2. Restriction of cellular protein synthesis, by either amino acid deprivation or cycloheximide inhibition did not affect the specific activity of poly(ADP-ribose) polymerase. 3. 3. A significant inhibition of both the rate of poly(ADP-ribose) polymerase and ADP-ribose acceptor activity of nuclei and chromatin was noted when RNA synthesis was inhibited by actinomycin D only in vivo and not in vitro. Response was dose dependent, with a minimum of 1 μg/ml required for inhibition. Inhibition occurred within 30 min indicating the possibility of a labile species of RNA being involved. 4. 4. Inhibition of cellular RNA by cordycepin (3′-deoxyadenosine) also caused inhibition of poly(ADP-ribose) polymerase in purified nuclei. The data further characterize the structural nucleic acid components of chromatin necessary for ADP ribosylation of nuclear proteins.