Inhibition of Crm1-p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation

Poly(ADP-ribose) polymerase 1 (PARP-1) and p53 are two key proteins in the DNA-damage response. Although PARP-1 is known to poly(ADP-ribosyl)ate p53, the role of this modification remains elusive. Here, we identify the major poly(ADP-ribosyl)ated sites of p53 by PARP-1 and find that PARP-1-mediated poly(ADP-ribosyl)ation blocks the interaction between p53 and the nuclear export receptor Crm1, resulting in nuclear accumulation of p53. These findings molecularly link PARP-1 and p53 in the DNA-damage response, providing the mechanism for how p53 accumulates in the nucleus in response to DNA damage. PARP-1 becomes super-activated by binding to damaged DNA, which in turn poly(ADP-ribosyl)ates p53. The nuclear export machinery is unable to target poly(ADP-ribosyl)ated p53, promoting accumulation of p53 in the nucleus where p53 exerts its transactivational function.     

Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.     

Modulation of chromatin structure by poly(ADP-ribosyl)ation

Poly(ADP-ribose) polymerase is a nuclear enzyme that is highly conserved in eucaryotes. Its activity is totally dependent on the presence of DNA containing single or double stranded breaks. We have shown that this activation results in a decondensation of chromatin superstructure in vitro, which is caused mainly by hyper(ADP-ribosy)ation of histone H1. In core particles, the modification of histone H2B leads to a partial dissociation of DNA from core histones. The conformational change of native chromatin by poly(ADP-ribosyl)ation is reversible upon degradation of the histone H1-bound poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase. We propose that cuts produced in vivo on DNA during DNA repair activate poly(ADP-ribose) polymerase, which then synthesizes poly(ADP-ribose) on histone H1, in particular, and contributes to the opening of the 25-nm chromatin fiber, resulting in the increased accessibility of DNA to excision repair enzymes. This mechanism is fast and reversible.     

Poly(ADP-ribosyl)ation of p53 Contributes to TPEN-Induced Neuronal Apoptosis

Depletion of intracellular zinc by N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.     

Inhibition of poly(ADP-ribosyl)ation by overexpressing the poly(ADP-ribose) polymerase DNA-binding domain in mammalian cells

Poly(ADP-ribose) polymerase specifically recognizes DNA strand breaks by its DNA-binding domain. DNA binding activates the enzyme to catalyze the formation of poly(ADP-ribose) utilizing NAD as substrate. By a molecular genetic approach we set out to inhibit this enzyme activity in a highly specific manner, thus avoiding the inherent side effects of NAD analogs which have been used extensively as enzyme inhibitors. cDNA sequences coding for the human poly(ADP-ribose) polymerase DNA-binding domain were subcloned into eucaryotic expression plasmids and transiently transfected into monkey cells. Cells were fixed with ethanol followed by incubation with NAD. Indirect double immunofluorescence to detect both overexpressed protein and poly(ADP-ribose) in situ revealed that overexpression of the DNA-binding domain greatly inhibited poly(ADP-ribosyl)ation catalyzed by the resident enzyme during NAD postincubation. The same inhibition was observed when transfected cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine to induce DNA strand breaks in vivo and subjected to trichloroacetic acid/ethanol fixation and subsequent immunofluorescence analysis, a novel method we developed for the in situ detection of polymer synthesis in intact cells. This molecular genetic approach may prove to be a selective and efficient tool to investigate possible functions of poly(ADP-ribosyl)ation in living cells.     

p66 an in vivo target for poly(ADP-ribosyl)ation co-purifies with poly(ADP-ribose) polymerase

A direct immunoassay has been applied for the quantitation of poly(ADP-ribose) polymerase and its automodification in BALB/3T3 (A31) cells. As the cell population reached a high density, a 66 kDa protein (designated p66) which co-purified with the enzyme became highly poly(ADP-ribosyl)ated. Pulse-chase experiments as well as a Western blot analysis indicated that the p66 was not a degradation product of poly(ADP-ribose) polymerase.     

Glyoxylic acid promotes poly(ADP-ribosyl)ation of nuclear proteins in K562 cells cultured at limiting dilution

Addition of glyoxylic acid to the culture medium allows survival and proliferation of K562 human erythroleukemic cells cultured at low population density in the absence of serum. Concomitantly, glyoxylic acid induces a remarkable increase in nuclear poly(ADP-ribose) content, as compared to control cells cultured without addition of glyoxylate. The latter effect is reversed by addition of micromolar concentrations of benzamide to the cultures. As glyoxylic acid is metabolized through NADH-dependent reduction to glycolic acid only, the observed effects on cell growth and on nuclear poly(ADP-ribose) content seem to be mediated by an increased cellular ability to oxidize NADH.     

Chromosomal protein poly(ADP-ribosyl)ation in pancreatic nucleosomes

When pancreatic chromatin fragments were prepared and resolved in the presence of 80 mM NaCl, endogenous poly(ADP-ribose) polymerase activity was found to be maximal in nucleosome periodicities of four to five units and did not respond to any further increases in nucleosomal architecture. Furthermore, in nucleosome complexities spanning 1 through 14 and over unit lengths, polyacrylamide gel electrophoresis on acid-urea and acid-urea-Triton gels has shown pancreatic histone H1 to be the only actively ADP-ribosylated histone species. The extent of ADP-ribosylation of histone H1 was also demonstrated to retard the protein's mobility in acid-urea, acid-urea-Triton, and lithium dodecyl sulfate polyacrylamide gels and to consist of at least 12 distinct ADP-ribosylated species extractable in all nucleosome complexities studied. Finally, extraction and subsequent electrophoresis of total chromosomal proteins in the presence of lithium dodecyl sulfate also evidenced heavy ADP-ribosylation at the level of nonhistone chromosomal proteins of the high mobility group comigrating in the core histone region, as well as in the topmost region of the gels where poly(ADP-ribose) polymerase was found to form a poly(ADP-ribosyl)ated aggregate.     

Decline of poly(ADP-ribosyl)ation during in vitro senescence in human diploid fibroblasts

Poly(ADP-ribose) polymerase activity was determined at various times during the in vitro life span of two human diploid fibroblast-like cell lines of different donor ages. The cell lines differed in their ability to transfer ADP-ribose, with cells from an embryonic donor exhibiting 2 to 3 times the activity found in cells obtained from a newborn donor. The activity in both cell lines decreased by 30-60% as the cells moved through their in vitro life spans. The decline could not be attributed to increases in glycohydrolase or the leakage of polymerase from older cell preparations. Enzyme activation with DNase I indicated that similar levels of enzyme were present in both cell lines at all in vitro ages. These results indicate that although poly(ADP-ribosyl)ation is inversely related to donor age as well as in vitro age the decrease is in response to other factors which change with increasing age.     

DNA replication and poly(ADP-ribosyl)ation of chromatin

The role of poly(ADP-ribosyl)ation in chromatin replication and the activity of poly(ADP-ribose) synthetase in the newly synthesized and old chromatin was studied. It was found that 3-aminobenzamide, which is an inhibitor of poly(ADP-ribose) synthetase, had no effect on the initiation of DNA synthesis and only a moderate effect on DNA chain elongation. However, poly(ADP-ribose) synthetase activity in the newly replicated chromatin was two to three times higher than that of the unreplicated chromatin.     

New readers and interpretations of poly(ADP-ribosyl)ation

Poly(ADP-ribosyl)ation (PARylation), a protein post-translational modification that was originally connected to the DNA damage response, is now known to engage in a continuously increasing number of biological processes. Despite extensive research and ceaseless, important findings about its role and mode of action, poly(ADP-ribose) remains an enigma regarding its structural complexity and diversity. The recent identification and structural characterization of four different poly(ADP-ribose) binding motifs represents a quantum leap in the comprehension of how this molecule can be decoded. Moreover, the recent discovery of a direct connection between PARylation and poly-ubiquitylation in targeting proteins for degradation by the proteasome has paved the way for a new interpretation of this protein modification. These two novel aspects, poly(ADP-ribose) recognition and readout by the ubiquitylation/proteasome system are developed here.     

In vitro poly(ADP-ribosyl)ation of seminal ribonuclease

The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase.     

Metabolic changes in the poly(ADP-ribosyl)ation pathway of differentiating rat germinal cells

A sialoglycoprotein fraction was isolated from chicken erythrocytes by two methods based on the phenol extraction or chloroform/2-propanol extraction of differently prepared erythrocyte membranes. Both preparations gave in SDS-PAGE two major PAS-stained bands (GP2 and GP3), which migrated as 60- and 33-kDa species, respectively, compared to reference proteins, or as 44- and 23-kDa molecules, compared to human glycophorins. Some less abundant slower migrating PAS-stained components, antigenically related to GP2 and GP3, also were detected. No evidence for the presence of antigenically distinct glycoproteins of leukosialin type was obtained. Interconversion in SDS-PAGE, similar carbohydrate composition, and similar antigenic properties of GP2 and GP3 indicated that they are a dimer and monomer, respectively, of the same glycoprotein which shows properties that allow it to be classified as a glycophorin. Lectin binding studies and methylation analysis of beta-elimination products of chicken glycophorin preparation showed the presence of O-glycans and N-glycans. The major O-glycans include sialylated Galbeta1-3GalNAc units and more complex GlcNAc-containing chains. Among the N-glycans, there are complex-type biantennary structures with a bisecting GlcNAc residue, accompanied by chains with additional antennas linked to alpha-mannose residues. A characteristic feature of the chicken glycophorin is a relatively high proportion of N-glycans to O-glycans, compared to the glycophorin A from human erythrocytes.