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Mammalian N1-adenosine PARylation is a reversible DNA modification.

ABSTRACT: Poly-ADP-ribosylation (PARylation) is regarded as a protein-specific modification. However, some PARPs were recently shown to modify DNA termini in vitro. Here, we use ultrasensitive mass spectrometry (LC-MS/MS), anti-PAR antibodies, and anti-PAR reagents to show that mammalian DNA is physiologically PARylated and to different levels in primary tissues. Inhibition of PAR glycohydrolase (PARG) increases DNA PARylation, supporting that the modification is reversible. DNA PARylation requires PARP1 and in vitro PARP1 PARylates single-stranded DNA, while PARG reverts the modification. DNA PARylation occurs at the N1-position of adenosine residues to form N1-Poly(ADP-ribosyl)-deoxyadenosine. Through partial hydrolysis of mammalian gDNA we identify PAR-DNA via the diagnostic deamination product N1-ribosyl-deoxyinosine to occur in vivo. The discovery of N1-adenosine PARylation as a DNA modification establishes the conceptual and methodological framework to elucidate its biological relevance and extends the role of PARP enzymes.

SUBMITTER: Musheev MU

PROVIDER: S-EPMC9576699 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Mammalian N1-adenosine PARylation is a reversible DNA modification.

Musheev Michael U MU Schomacher Lars L Basu Amitava A Han Dandan D Krebs Laura L Scholz Carola C Niehrs Christof C

Nature communications 20221017 1

Poly-ADP-ribosylation (PARylation) is regarded as a protein-specific modification. However, some PARPs were recently shown to modify DNA termini in vitro. Here, we use ultrasensitive mass spectrometry (LC-MS/MS), anti-PAR antibodies, and anti-PAR reagents to show that mammalian DNA is physiologically PARylated and to different levels in primary tissues. Inhibition of PAR glycohydrolase (PARG) increases DNA PARylation, supporting that the modification is reversible. DNA PARylation requires PARP1 ...[more]

PMID: 36253381

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Project description:BackgroundAdenosine is a potent endogenous anti-inflammatory and immunoregulatory molecule. Despite its promise, adenosine's extremely short half-life in blood limits its clinical application. Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly. ANO is an oxidized product of adenosine at the N1 position of the adenine base moiety. We found that it is refractory to adenosine deaminase-mediated conversion to inosine. We further examined the anti-inflammatory activities of ANO in vitro and in vivo.MethodsThe effect of ANO on pro-inflammatory cytokine secretion was examined in mouse peritoneal macrophages and the human monocytic cell line THP-1, and compared with that of adenosine, synthetic adenosine receptor (AR)-selective agonists and dipotassium glycyrrhizate (GK2). The anti-inflammatory activity of ANO in vivo was examined in an LPS-induced endotoxin shock model in mice.ResultsANO inhibited secretion of inflammatory mediators at much lower concentrations than adenosine and GK2 when used with peritoneal macrophages and THP-1 cells that were stimulated by LPS plus IFN-γ. The potent anti-inflammatory activity of ANO could not be solely accounted for by its refractoriness to adenosine deaminase. ANO was superior to the synthetic A1 AR-selective agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), A2A AR-selective agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamideadenosine hydrochloride (CGS21680), and A3 AR-selective agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), in suppressing the secretion of a broad spectrum of pro-inflammatory cytokines by peritoneal macrophages. The capacities of ANO to inhibit pro-inflammatory cytokine production by THP-1 cells were comparable with those of CCPA and IB-MECA. Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock. A significant increase in survival rate was also observed by oral administration of ANO. Mechanistic analysis suggested that the up-regulation of the anti-inflammatory transcription factor c-Fos was, at least in part, involved in the ANO-induced suppression of pro-inflammatory cytokine secretion.ConclusionsOur data suggest that ANO, a naturally occurring molecule that is structurally close to adenosine but is functionally more potent, presents potential strategies for the treatment of inflammatory disorders.

Project description:BackgroundEpigenetic modifications, such as DNA methylation, can influence the genetic susceptibility to type 2 diabetes mellitus (T2DM) and the progression of the disease. Our previous studies demonstrated that the regulation of the DNA methylation pattern involves the poly(ADP-ribosyl)ation (PARylation) process, a post-translational modification of proteins catalysed by the poly(ADP-ribose) polymerase (PARP) enzymes. Experimental data showed that the hyperactivation of PARylation is associated with impaired glucose metabolism and the development of T2DM. Aims of this case-control study were to investigate the association between PARylation and global and site-specific DNA methylation in T2DM and to evaluate metabolic correlates.ResultsData were collected from 61 subjects affected by T2DM and 48 healthy individuals, recruited as controls. Global levels of poly(ADP-ribose) (PAR, a surrogate of PARP activity), cytosine methylation (5-methylcytosine, 5mC) and de-methylation intermediates 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were determined in peripheral blood cells by ELISA-based methodologies. Site-specific DNA methylation profiling of SOCS3, SREBF1 and TXNIP candidate genes was performed by mass spectrometry-based bisulfite sequencing, methyl-sensitive endonucleases digestion and by DNA immuno-precipitation. T2DM subjects presented higher PAR levels than controls. In T2DM individuals, increased PAR levels were significantly associated with higher HbA1c levels and the accumulation of the de-methylation intermediates 5hmC and 5fC in the genome. In addition, T2DM patients with higher PAR levels showed reduced methylation with increased 5hmC and 5fC levels in specific SOCS3 sites, up-regulated SOCS3 expression compared to both T2DM subjects with low PAR levels and controls.ConclusionsThis study demonstrates the activation of PARylation processes in patients with T2DM, particularly in those with poor glycaemic control. PARylation is linked to dysregulation of DNA methylation pattern via activation of the DNA de-methylation cascade and may be at the basis of the differential gene expression observed in presence of diabetes.

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Mammalian N1-adenosine PARylation is a reversible DNA modification.

Publications

Mammalian N1-adenosine PARylation is a reversible DNA modification.

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