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Poly(ADP-ribose) polymerase 1 (PARP1) is a highly conserved nuclear protein in multicellular organisms that by modulating chromatin opening facilitates gene expression during development. All reported Parp1 null knockout mouse strains are viable with no developmental anomalies. It was believed that functional redundancy with other PARP family members, mainly PARP2, explains such a controversy. However, while PARP2 has similar catalytic domain to PARP1, it lacks other domains, making the absence of developmental problems in Parp1 mice knockouts unlikely. Contrary to prior assumptions, in our analysis of the best-investigated Parp1 knockout mouse strain, we identified persistent mRNA expression, albeit at reduced levels. Transcript analysis revealed an alternatively spliced Parp1 variant lacking exon 2. Subsequent protein analysis confirmed the existence of a truncated PARP1 protein in knockout mice. The decreased level of poly(ADP-ribose) (pADPr) was detected in Parp1 knockout embryonic stem (ES) cells with western blotting analysis, but immunofluorescence staining did not detect any difference in distribution or level of pADPr in nuclei of knockout ES cells. pADPr level in double Parp1 Parg mutant ES cells greatly exceeded its amount in normal and even in hypomorph Parg mutant ES cells, suggesting the presence of functionally active PARP1. Therefore, our findings challenge the conventional understanding of PARP1 depletion effects. [ABSTRACT FROM AUTHOR]

ADP-ribosylation (ADPRylation) is a mechanism which post-translationally modifies proteins in eukaryotes in order to regulate a broad range of biological processes including programmed cell death, cell signaling, DNA repair, and responses to biotic and abiotic stresses. Poly(ADP-ribosyl) polymerases (PARPs) play a key role in the process of ADPRylation, which modifies target proteins by attaching ADP-ribose molecules. Here, we investigated whether and how PARP1 and PARylation modulate responses of Nicotiana benthamiana plants to methyl viologen (MV)-induced oxidative stress. It was found that the burst of reactive oxygen species (ROS), cell death, and loss of tissue viability invoked by MV in N. benthamiana leaves was significantly delayed by both the RNA silencing of the PARP1 gene and by applying the pharmacological inhibitor 3-aminobenzamide (3AB) to inhibit PARylation activity. This in turn reduced the accumulation of PARylated proteins and significantly increased the gene expression of major ROS scavenging enzymes including SOD (NbMnSOD; mitochondrial manganese SOD), CAT (NbCAT2), GR (NbGR), and APX (NbAPX5), and inhibited cell death. This mechanism may be part of a broader network that regulates plant sensitivity to oxidative stress through various genetically programmed pathways. [ABSTRACT FROM AUTHOR]

Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1′s regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins—PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase β, DNA ligase III, and XRCC1—did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either. [ABSTRACT FROM AUTHOR]

ADP-ribosylation (ADPRylation) is a mechanism which post-translationally modifies proteins in eukaryotes in order to regulate a broad range of biological processes including programmed cell death, cell signaling, DNA repair, and responses to biotic and abiotic stresses. Poly(ADP-ribosyl) polymerases (PARPs) play a key role in the process of ADPRylation, which modifies target proteins by attaching ADP-ribose molecules. Here, we investigated whether and how PARP1 and PARylation modulate responses of Nicotiana benthamiana plants to methyl viologen (MV)-induced oxidative stress. It was found that the burst of reactive oxygen species (ROS), cell death, and loss of tissue viability invoked by MV in N. benthamiana leaves was significantly delayed by both the RNA silencing of the PARP1 gene and by applying the pharmacological inhibitor 3-aminobenzamide (3AB) to inhibit PARylation activity. This in turn reduced the accumulation of PARylated proteins and significantly increased the gene expression of major ROS scavenging enzymes including SOD (NbMnSOD; mitochondrial manganese SOD), CAT (NbCAT2), GR (NbGR), and APX (NbAPX5), and inhibited cell death. This mechanism may be part of a broader network that regulates plant sensitivity to oxidative stress through various genetically programmed pathways.

Abstract Background We reported that PARP-1 regulates genes whose products are crucial for asthma, in part, by controlling STAT6 integrity speculatively through a calpain-dependent mechanism. We wished to decipher the PARP-1/STAT6 relationship in the context of intracellular trafficking and promoter occupancy of the transcription factor on target genes, its integrity in the presence of calpains, and its connection to autophagy. Methods This study was conducted using primary splenocytes or fibroblasts derived from wild-type or PARP-1−/− mice and Jurkat T cells to mimic Th2 inflammation. Results We show that the role for PARP-1 in expression of IL-4-induced genes (e.g. gata-3) in splenocytes did not involve effects on STAT6 phosphorylation or its subcellular trafficking, rather, it influenced its occupancy of gata-3 proximal and distal promoters in the early stages of IL-4 stimulation. At later stages, PARP-1 was crucial for STAT6 integrity as its inhibition, pharmacologically or by gene knockout, compromised the fate of the transcription factor. Calpain-1 appeared to preferentially degrade JAK-phosphorylated-STAT6, which was blocked by calpastatin-mediated inhibition or by genetic knockout in mouse fibroblasts. The STAT6/PARP-1 relationship entailed physical interaction and modification by poly(ADP-ribosyl)ation independently of double-strand-DNA breaks. Poly(ADP-ribosyl)ation protected phosphorylated-STAT6 against calpain-1-mediated degradation. Additionally, our results show that STAT6 is a bonafide substrate for chaperone-mediated autophagy in a selective and calpain-dependent manner in the human Jurkat cell-line. The effects were partially blocked by IL-4 treatment and PARP-1 inhibition. Conclusions The results demonstrate that poly(ADP-ribosyl)ation plays a critical role in protecting activated STAT6 during Th2 inflammation, which may be synthetically targeted for degradation by inhibiting PARP-1.

Poly(ADP-ribose) (PAR) is a negatively charged polymer, linear or branched, that consists of ADP-ribose monomers. PAR is synthesized by poly(ADP-ribose)polymerase (PARP) enzymes, which are activated upon DNA damage and use nicotinamide adenine dinucleotide (NAD+) as a substrate. The best-studied members of the PARP family, PARP1 and PARP2, are the most important nuclear proteins involved in many cell processes, including the regulation of DNA repair. PARP1 and PARP2 catalyze PAR synthesis and transfer to amino acid residues of target proteins, including autoPARylation. PARP1 and PARP2 are promising targets for chemotherapy in view of their key role in regulating DNA repair. A novel histone PARylation factor (HPF1) was recently discovered to modulate PARP1/2 activity by forming a transient joint active site with PARP1/2. Histones are modified at serine residues in the presence of HPF1. The general mechanism of the interaction between HPF1 and PARP1/2 is a subject of intense research now. The review considers the discovery and classical mechanism of PARylation in higher eukaryotes and the role of HPF1 in the process. [ABSTRACT FROM AUTHOR]

Elucidation of the cellular changes that occur in degenerating photoreceptors of people with inherited retinal diseases (IRDs) has been a focus for many research teams, leading to numerous theories on how these changes affect the cell death process. What is clearly emerging from these studies is that there are common denominators across multiple models of IRD, regardless of the underlying genetic mutation. These common markers could open avenues for broad neuroprotective therapeutics to prevent photoreceptor loss and preserve functional vision. In recent years, the role of epigenetic modifications contributing to the pathology of IRDs has been a particular point of interest, due to many studies noting changes in these epigenetic modifications, which coincide with photoreceptor cell death. This review will discuss the two broad categories of epigenetic changes, DNA methylation and histone modifications, that have received particular attention in IRD models. We will review the altered epigenetic regulatory events that are believed to contribute to cell death in IRDs and discuss the therapeutic potential of targeting these alterations.

Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1′s regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins—PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase β, DNA ligase III, and XRCC1—did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either.

PARP1 (aka ARTD1) acts as a prime sensor of cellular genotoxic stress response. PARP1 detects DNA strand breaks and subsequently catalyzes the formation of poly(ADP-ribose) (PAR), which leads to the recruitment of the scaffold protein XRCC1 during base excision and single strand break repair and the assembly of multi-protein complexes to promote DNA repair. Here, we reveal that the recruitment of either protein to sites of DNA damage is impeded in the absence of the other, indicating a strong reciprocal relationship between the two DNA repair factors during genotoxic stress response. We further analyzed several cellular and molecular endpoints in HeLa PARP1 KO, XRCC1 KO, and PARP1/XRCC1 double KO (DKO) cells after genotoxic treatments, i.e., PARylation response, NAD+ levels, clonogenic survival, cell cycle progression, cell death, and DNA repair. The analysis of NAD+ levels and cytotoxicity after treatment with the topoisomerase I inhibitor camptothecin revealed a hypersensitivity phenotype of XRCC1 KO cells compared to PARP1 KO cells—an effect that could be rescued by the additional genetic deletion of PARP1 as well as by pharmacological PARP inhibition. Moreover, impaired repair of hydrogen peroxide and CPT-induced DNA damage in XRCC1 KO cells could be partially rescued by additional deletion of PARP1. Our results therefore highlight important reciprocal regulatory functions of XRCC1 and PARP1 during genotoxic stress response. [ABSTRACT FROM AUTHOR]

Simple Summary: PARP-1 and poly(ADP-ribosyl)ation control gene expression, DNA repair pathways, and genomic stability in multiple ways, such as affecting chromatin remodelling. This review article summarises how PARP-1 activity directly modifies histone proteins and the enzymes involved in DNA/histone epigenetic modifications to mould chromatin structure during transcription and DNA damage response. Understanding the role of poly(ADP-ribosyl)ation in the epigenetic regulation of chromatin organisation will help clarify resistance mechanisms to PARP inhibitors and highlight the clinical relevance of a combinatory approach based on epigenetic drugs. The regulation of chromatin state and histone protein eviction have been proven essential during transcription and DNA repair. Poly(ADP-ribose) (PAR) polymerase 1 (PARP-1) and poly(ADP-ribosyl)ation (PARylation) are crucial mediators of these processes by affecting DNA/histone epigenetic events. DNA methylation/hydroxymethylation patterns and histone modifications are established by mutual coordination between all epigenetic modifiers. This review will focus on histones and DNA/histone epigenetic machinery that are direct targets of PARP-1 activity by covalent and non-covalent PARylation. The effects of these modifications on the activity/recruitment of epigenetic enzymes at DNA damage sites or gene regulatory regions will be outlined. Furthermore, based on the achievements made to the present, we will discuss the potential application of epigenetic-based therapy as a novel strategy for boosting the success of PARP inhibitors, improving cell sensitivity or overcoming drug resistance. [ABSTRACT FROM AUTHOR]

The ARH family of ADP-ribose-acceptor hydrolases consists of three 39-kDa members (ARH1-3), with similarities in amino acid sequence. ARH1 was identified based on its ability to cleave ADP-ribosyl-arginine synthesized by cholera toxin. Mammalian ADP-ribosyltransferases (ARTCs) mimicked the toxin reaction, with ARTC1 catalyzing the synthesis of ADP-ribosyl-arginine. ADP-ribosylation of arginine was stereospecific, with β-NAD+ as substrate and, α-anomeric ADP-ribose-arginine the reaction product. ARH1 hydrolyzed α-ADP-ribose-arginine, in addition to α-NAD+ and O-acetyl-ADP-ribose. Thus, ADP-ribose attached to oxygen-containing or nitrogen-containing functional groups was a substrate. Arh1 heterozygous and knockout (KO) mice developed tumors. Arh1-KO mice showed decreased cardiac contractility and developed myocardial fibrosis. In addition to Arh1-KO mice showed increased ADP-ribosylation of tripartite motif-containing protein 72 (TRIM72), a membrane-repair protein. ARH3 cleaved ADP-ribose from ends of the poly(ADP-ribose) (PAR) chain and released the terminal ADP-ribose attached to (serine)protein. ARH3 also hydrolyzed α-NAD+ and O-acetyl-ADP-ribose. Incubation of Arh3-KO cells with H2O2 resulted in activation of poly-ADP-ribose polymerase (PARP)-1, followed by increased nuclear PAR, increased cytoplasmic PAR, leading to release of Apoptosis Inducing Factor (AIF) from mitochondria. AIF, following nuclear translocation, stimulated endonucleases, resulting in cell death by Parthanatos. Human ARH3-deficiency is autosomal recessive, rare, and characterized by neurodegeneration and early death. Arh3-KO mice developed increased brain infarction following ischemia-reperfusion injury, which was reduced by PARP inhibitors. Similarly, PARP inhibitors improved survival of Arh3-KO cells treated with H2O2. ARH2 protein did not show activity in the in vitro assays described above for ARH1 and ARH3. ARH2 has a restricted tissue distribution, with primary involvement of cardiac and skeletal muscle. Overall, the ARH family has unique functions in biological processes and different enzymatic activities. [ABSTRACT FROM AUTHOR]

Recent studies have revealed a key role of PARP1 that catalyzes the poly-ADP-ribosylation (PARylation) of substrates in regulating gene transcription. We show here that HIV-1 transcriptional activation also requires PARP1 activity. Because efficient HIV-1 transactivation is known to depend on the ELL2-containing super elongation complex (SEC), we investigated the functional relationship between PARP1 and ELL2-SEC in HIV-1 transcriptional control. We show that PARP1 elevates ELL2 protein levels to form more ELL2-SEC in cells. This effect is caused by PARP1's suppression of expression of Siah1, an E3 ubiquitin ligase for ELL2, at both mRNA and protein levels. At the mRNA level, PARP1 coordinates with the co-repressor NCoR to suppress Siah1 transcription. At the protein level, PARP1 promotes Siah1 proteolysis, likely through inducing PARylation-dependent ubiquitination (PARdU) of Siah1. Thus, a PARP1-Siah1 axis activates HIV-1 transcription and controls the expression of ELL2 and other Siah1 substrates.

Background: We reported that PARP-1 regulates genes whose products are crucial for asthma, in part, by controlling STAT6 integrity speculatively through a calpain-dependent mechanism. We wished to decipher the PARP-1/STAT6 relationship in the context of intracellular trafficking and promoter occupancy of the transcription factor on target genes, its integrity in the presence of calpains, and its connection to autophagy.Methods: This study was conducted using primary splenocytes or fibroblasts derived from wild-type or PARP-1-/- mice and Jurkat T cells to mimic Th2 inflammation.Results: We show that the role for PARP-1 in expression of IL-4-induced genes (e.g. gata-3) in splenocytes did not involve effects on STAT6 phosphorylation or its subcellular trafficking, rather, it influenced its occupancy of gata-3 proximal and distal promoters in the early stages of IL-4 stimulation. At later stages, PARP-1 was crucial for STAT6 integrity as its inhibition, pharmacologically or by gene knockout, compromised the fate of the transcription factor. Calpain-1 appeared to preferentially degrade JAK-phosphorylated-STAT6, which was blocked by calpastatin-mediated inhibition or by genetic knockout in mouse fibroblasts. The STAT6/PARP-1 relationship entailed physical interaction and modification by poly(ADP-ribosyl)ation independently of double-strand-DNA breaks. Poly(ADP-ribosyl)ation protected phosphorylated-STAT6 against calpain-1-mediated degradation. Additionally, our results show that STAT6 is a bonafide substrate for chaperone-mediated autophagy in a selective and calpain-dependent manner in the human Jurkat cell-line. The effects were partially blocked by IL-4 treatment and PARP-1 inhibition.Conclusions: The results demonstrate that poly(ADP-ribosyl)ation plays a critical role in protecting activated STAT6 during Th2 inflammation, which may be synthetically targeted for degradation by inhibiting PARP-1. [ABSTRACT FROM AUTHOR]

To overcome genotoxicity, cells have evolved powerful and effective mechanisms to detect and respond to DNA lesions. RecQ Like Helicase-4 (RECQL4) plays a vital role in DNA damage responses. RECQL4 is recruited to DNA double-strand break (DSB) sites in a poly(ADP-ribosyl)ation (PARylation)-dependent manner, but the mechanism and significance of this process remain unclear. Here, we showed that the domain of RECQL4 recruited to DSBs in a PARylation-dependent manner directly interacts with poly(ADP-ribose) (PAR) and contains a PAR-binding motif (PBM). By replacing this PBM with a PBM of hnRNPA2 or its mutated form, we demonstrated that the PBM in RECQL4 is required for PARylation-dependent recruitment and the roles of RECQL4 in the DSB response. These results suggest that the direct interaction of RECQL4 with PAR is critical for proper cellular response to DSBs and provide insights to understand PARylation-dependent control of the DSB response and cancer therapeutics using PARylation inhibitors., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

ADP-ribosylation is a highly dynamic and fully reversible post-translational modification performed by poly(ADP-ribose) polymerases (PARPs) that modulates protein function, abundance, localization and turnover. Here we show that influenza A virus infection causes a rapid and dramatic upregulation of global ADP-ribosylation that inhibits viral replication. Mass spectrometry defined for the first time the global ADP-ribosylome during infection, creating an infection-specific profile with almost 4,300 modification sites on ~1,080 host proteins, as well as over 100 modification sites on viral proteins. Our data indicate that the global increase likely reflects a change in the form of ADP-ribosylation rather than modification of new targets. Functional assays demonstrated that modification of the viral replication machinery antagonizes its activity and further revealed that the anti-viral activity of PARPs and ADP-ribosylation is counteracted by the influenza A virus protein NS1, assigning a new activity to the primary viral antagonist of innate immunity. We identified PARP1 as the enzyme producing the majority of poly(ADP-ribose) present during infection. Influenza A virus replicated faster in cells lacking PARP1, linking PARP1 and ADP-ribosylation to the anti-viral phenotype. Together, these data establish ADP-ribosylation as an anti-viral innate immune-like response to viral infection antagonized by a previously unknown activity of NS1., Competing Interests: DECLARATION OF INTERESTS CP is an employee of ARase Therapeutics.

HKDC1 has been shown to play an important role in promoting malignant progression of pancreatic adenocarcinoma (PAAD), but the exact mechanism is unclear. This study aimed to investigate the function of HKDC1 in autophagy activation and cell proliferation. By GSEA analysis of TCGA data of PAAD, we found that HKDC1 was closely associated with autophagy. We evaluated the effects of HKDC1 knockdown and overexpression on the expression of LC3B, an autophagy marker, and Cyclin D1 and PCNA, cell proliferation-associated proteins, by Western blotting (WB) and transmission electron microscopy (TEM) analysis. Knockdown of HKDC1 decreased LC3B expression and led to a decrease in the accumulation of autophagic vesicles and autophagic lysosomes, while overexpression of HKDC1 produced the opposite effect. Meanwhile, HKDC1 overexpression significantly promoted the proliferation of PAAD cells and increased the expression levels of Cyclin D1 and PCNA. Further studies showed that HKDC1 enhanced PARP1's own poly ADP-ribosylation (PARylation) activity by interacting with PARP1, which in turn promoted autophagy. In vivo experiments showed that knockdown of HKDC1 significantly inhibited the growth of pancreatic cancer cells in nude mice in vivo, reduced tumor volume and weight, and down-regulated the expression of PARP1, LC3, Cyclin D1 and PCNA. HKDC1 plays a critical role in the malignant progression of PAAD by activating autophagy and promoting cell proliferation. Our findings suggest that targeting HKDC1 and its downstream signaling pathways may provide novel strategies for PAAD treatment. • HKDC1 promotes autophagy activation in pancreatic adenocarcinoma (PAAD). • Knockdown of HKDC1 inhibits PAAD cell proliferation, while overexpression enhances cell growth. • HKDC1 regulates autophagy through its interaction with PARP1, leading to PARP1 auto-poly ADP-ribosylation. • HKDC1 knockdown reduces tumor growth and proliferation, highlighting its potential as a PAAD target. [ABSTRACT FROM AUTHOR]

Covalent binding of proteins to DNA forms DNA-protein crosslinks (DPCs), which represent cytotoxic DNA lesions that interfere with essential processes such as DNA replication and transcription. Cells possess different enzymatic activities to counteract DPCs. These include enzymes that degrade the adducted proteins, resolve the crosslinks, or incise the DNA to remove the crosslinked proteins. An important question is how DPCs are sensed and targeted for removal via the most suited pathway. Recent advances have shown the inherent role of DNA replication in triggering DPC removal by proteolysis. However, DPCs are also efficiently sensed and removed in the absence of DNA replication. In either scenario, post-translational modifications (PTMs) on DPCs play essential and versatile roles in orchestrating the repair routes. In this review, we summarize the current knowledge of the mechanisms that trigger DPC removal via PTMs, focusing on ubiquitylation, small ubiquitin-related modifier (SUMO) conjugation (SUMOylation), and poly (ADP-ribosyl)ation (PARylation). We also briefly discuss the current knowledge gaps and emerging hypotheses in the field.

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification reversibly catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs) and plays a key role in multiple cellular processes. The molecular mechanisms by which PARylation regulates innate immunity remain largely unknown in eukaryotes. Here we show that Arabidopsis UBC13A and UBC13B, the major drivers of lysine 63 (K63)-linked polyubiquitination, directly interact with PARPs/PARGs. Activation of pathogen-associated molecular pattern (PAMP)-triggered immunity promotes these interactions and enhances PARylation of UBC13. Both parp1 parp2 and ubc13a ubc13b mutants are compromised in immune responses with increased accumulation of total pathogenesis-related (PR) proteins but decreased accumulation of secreted PR proteins. Protein disulfide-isomerases (PDIs), essential components of endoplasmic reticulum quality control (ERQC) that ensure proper folding and maturation of proteins destined for secretion, complex with PARPs/PARGs and are PARylated upon PAMP perception. Significantly, PARylation of UBC13 regulates K63-linked ubiquitination of PDIs, which may further promote their disulfide isomerase activities for correct protein folding and subsequent secretion. Taken together, these results indicate that plant immunity is coordinately regulated by PARylation and K63-linked ubiquitination. Poly(ADP-ribosyl)ation (PARylation) plays a critical role in multiple cellular processes, but how PARylation regulates immunity remains largely unknown. This study shows that Arabidopsis UBC13, the major ubiquitin-conjugating enzyme that catalyzes K63-linked polyubiquitination, is PARylated upon immune activation. PARylation regulates K63-linked ubiquitination of protein disulfide-isomerases (PDIs), which may subsequently promote their activities for proper folding and secretion of pathogenesis-related (PR) proteins. [ABSTRACT FROM AUTHOR]

The ARH family of ADP-ribose-acceptor hydrolases consists of three 39-kDa members (ARH1-3), with similarities in amino acid sequence. ARH1 was identified based on its ability to cleave ADP-ribosyl-arginine synthesized by cholera toxin. Mammalian ADP-ribosyltransferases (ARTCs) mimicked the toxin reaction, with ARTC1 catalyzing the synthesis of ADP-ribosyl-arginine. ADP-ribosylation of arginine was stereospecific, with β-NAD+ as substrate and, α-anomeric ADP-ribose-arginine the reaction product. ARH1 hydrolyzed α-ADP-ribose-arginine, in addition to α-NAD+ and O-acetyl-ADP-ribose. Thus, ADP-ribose attached to oxygen-containing or nitrogen-containing functional groups was a substrate. Arh1 heterozygous and knockout (KO) mice developed tumors. Arh1-KO mice showed decreased cardiac contractility and developed myocardial fibrosis. In addition to Arh1-KO mice showed increased ADP-ribosylation of tripartite motif-containing protein 72 (TRIM72), a membrane-repair protein. ARH3 cleaved ADP-ribose from ends of the poly(ADP-ribose) (PAR) chain and released the terminal ADP-ribose attached to (serine)protein. ARH3 also hydrolyzed α-NAD+ and O-acetyl-ADP-ribose. Incubation of Arh3-KO cells with H2O2 resulted in activation of poly-ADP-ribose polymerase (PARP)-1, followed by increased nuclear PAR, increased cytoplasmic PAR, leading to release of Apoptosis Inducing Factor (AIF) from mitochondria. AIF, following nuclear translocation, stimulated endonucleases, resulting in cell death by Parthanatos. Human ARH3-deficiency is autosomal recessive, rare, and characterized by neurodegeneration and early death. Arh3-KO mice developed increased brain infarction following ischemia-reperfusion injury, which was reduced by PARP inhibitors. Similarly, PARP inhibitors improved survival of Arh3-KO cells treated with H2O2. ARH2 protein did not show activity in the in vitro assays described above for ARH1 and ARH3. ARH2 has a restricted tissue distribution, with primary involvement of cardiac and skeletal muscle. Overall, the ARH family has unique functions in biological processes and different enzymatic activities.

Poly(ADP-ribosyl)ation (PARylation), catalyzed mainly by poly(ADP-ribose) polymerase (PARP)1, is a key posttranslational modification involved in DNA replication and repair. Here, we report that TIMELESS (TIM), an essential scaffold of the replisome, is PARylated, which is linked to its proteolysis. TIM PARylation requires recognition of auto-modified PARP1 via two poly(ADP-ribose)-binding motifs, which primes TIM for proteasome-dependent degradation. Cells expressing the PARylation-refractory TIM mutant or under PARP inhibition accumulate TIM at DNA replication forks, causing replication stress and hyper-resection of stalled forks. Mechanistically, aberrant engagement of TIM with the replicative helicase impedes RAD51 loading and protection of reversed forks. Accordingly, defective TIM degradation hypersensitizes BRCA2-deficient cells to replication damage. Our study defines TIM as a substrate of PARP1 and elucidates how the control of replisome remodeling by PARylation is linked to stalled fork protection. Therefore, we propose a mechanism of PARP inhibition that impinges on the DNA replication fork instability caused by defective TIM turnover. [Display omitted] • TIMELESS (TIM) and PARP1 interact at DNA replication forks • TIM recognizes auto-PARylated PARP1 by two conserved PAR-binding motifs (PBMs) • PBM-dependent TIM PARylation primes TIM for proteasomal degradation • Aberrant TIM accumulation causes replication stress and impairs stalled fork protection Rageul et al. report that TIMELESS harbors poly(ADP-ribose) (PAR)-binding motifs, which are essential for the PARylation of TIMELESS by PARP1. Defects in this modification prevent proteasomal degradation of TIMELESS and cause DNA replication fork instability. The proteotoxic stress caused by dysregulated TIMELESS turnover underlies a mechanism of PARP inhibitor action. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase (TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/β-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/β-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the β-catenin degradation complex, which allows Axin’s ubiquitination and subsequent degradation, thereby activating β-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of β-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/β-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.

Poly(ADP-ribose) belongs to a momentous post-translational modification of eukaryotic nuclear protein. Protein poly(ADP-ribosyl)ation mediates numerous cell signalings and is related to some diseases. Poly(ADPribose) is currently available through enzymatic or chemical synthesis. Herein, we summarize the structure characteristics and recent developments in the synthesis of poly(ADP-ribose) from a chemical point of view. [ABSTRACT FROM AUTHOR]

• Highly sensitive LC–MS/MS method to quantify poly(ADP-ribosyl)ation in C. elegans. • Characterizing worms defective in pme-1 or pme-2 (orthologues of PARP1 and PARP2). • pme-1 (orthologue of PARP1) holding the major worm PARP activity. • Toxic doses of Mn led to PAR-induction in pme-1-deficient C. elegans. • Mn-induced oxidative stress in pme-1 or pme-2 deletion mutants. When exceeding the homeostatic range, manganese (Mn) might cause neurotoxicity, characteristic of the pathophysiology of several neurological diseases. Although the underlying mechanism of its neurotoxicity remains unclear, Mn-induced oxidative stress contributes to disease etiology. DNA damage caused by oxidative stress may further trigger dysregulation of DNA-damage-induced poly(ADP-ribosyl)ation (PARylation), which is of central importance especially for neuronal homeostasis. Accordingly, this study was designed to assess in the genetically traceable in vivo model Caenorhabditis elegans the role of PARylation as well as the consequences of loss of pme-1 or pme-2 (orthologues of PARP1 and PARP2) in Mn-induced toxicity. A specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed to quantify PARylation in worms. Next to monitoring the PAR level, pme-1 and pme-2 gene expression as well as Mn-induced oxidative stress was studied in wildtype worms and the pme deletion mutants. While Mn failed to induce PARylation in wildtype worms, toxic doses of Mn led to PAR-induction in pme-1-deficient worms, due to an increased gene expression of pme-2 in the pme-1 deletion mutants. However, this effect could not be observed at sub-toxic Mn doses as well as upon longer incubation times. Regarding Mn-induced oxidative stress, the deletion mutants did not show hypersensitivity. Taken together, this study characterizes worms to model PAR inhibition and addresses the consequences for Mn-induced oxidative stress in genetically manipulated worms. [ABSTRACT FROM AUTHOR]

Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3. PARylation disrupted interactions between nucleosomes, preventing self-association. Addition of LIG3 and XRCC1 to PARylated nucleosomes generated condensates that selectively partition DNA repair-associated proteins in a PAR- and phosphorylation-dependent manner in vitro. Our results establish that PARylation influences nucleosomes across different length scales, extending from the atom-level motions of histone tails to the mesoscale formation of condensates with selective compositions. [Display omitted] • Nucleosome PARylation enhances H3 tail dynamics without perturbing the histone core • PARylation dramatically enhances ligation of single-strand nicks in nucleosomes • Stacking and self-association of mononucleosomes is inhibited by PARylation • PARylation reorganizes condensates containing nucleosomes and DNA repair factors Nosella et al. establish that poly(ADP-ribosyl)ation of nucleosomes dramatically increases histone H3 tail dynamics and DNA conformational variability while leaving the core structure intact. These changes correlated with enhanced ligation of nucleosome single-strand nicks, disruption of nucleosome oligomers, and colocalization of nucleosomes with DNA repair factors inside biomolecular condensates. [ABSTRACT FROM AUTHOR]

Cell wall reinforcement with callose is a frequent plant response to infection. Poly(ADP-ribosyl)ation is a protein post-translational modification mediated by poly(ADP-ribose) polymerases (PARPs). Poly(ADP-ribosyl)ation has well-known roles in DNA damage repair and has more recently been shown to contribute to plant immune responses. 3-aminobenzamide (3AB) is an established PARP inhibitor and it blocks the callose deposition elicited by flg22 or elf18, two microbe-associated molecular patterns (MAMPs). However, we report that an Arabidopsis parp1parp2parp3 triple mutant does not exhibit loss of flg22-induced callose deposition. Additionally, the more specific PARP inhibitors PJ-34 and INH2BP inhibit PARP activity in Arabidopsis but do not block MAMP-induced callose deposition. These data demonstrate off-target activity of 3AB and indicate that 3AB inhibits callose deposition through a mechanism other than poly(ADP-ribosyl)ation. POWDERY MILDEW RESISTANT 4 (PMR4) is the callose synthase responsible for the majority of MAMP- and wound-induced callose deposition in Arabidopsis. 3AB does not block wound-induced callose deposition, and 3AB does not reduce the PMR4 mRNA abundance increase in response to flg22. Levels of PMR4-HA protein increase in response to flg22, and increase even more in flg22 + 3AB despite no callose being produced. The callose synthase inhibitor 2-deoxy-D-glucose does not cause similar impacts on PMR4-HA protein levels. Beyond MAMPs, we find that 3AB also reduces callose deposition induced by powdery mildew (Golovinomyces cichoracearum) and impairs the penetration resistance of a PMR4 overexpression line. 3AB thus reveals pathogenesis-associated pathways that activate callose synthase enzymatic activity distinct from those that elevate PMR4 mRNA and protein abundance.

Poly(ADP-ribose) polymerase 2 (PARP2) participates in base excision repair (BER) alongside PARP1, but its functions are still under study. Here, we characterize binding affinities of PARP2 for other BER proteins (PARP1, APE1, Polβ, and XRCC1) and oligomerization states of the homo- and hetero-associated complexes using fluorescence-based and light scattering techniques. To compare PARP2 and PARP1 in the efficiency of PAR synthesis, in the absence and presence of protein partners, the size of PARP2 PARylated in various reaction conditions was measured. Unlike PARP1, PARP2 forms more dynamic complexes with common protein partners, and their stability is effectively modulated by DNA intermediates. Apparent binding affinity constants determined for homo- and hetero-oligomerized PARP1 and PARP2 provide evidence that the major form of PARP2 at excessive PARP1 level is their heterocomplex. Autoregulation of PAR elongation at high PARP and NAD+ concentrations is stronger for PARP2 than for PARP1, and the activity of PARP2 is more effectively inhibited by XRCC1. Moreover, the activity of both PARP1 and PARP2 is suppressed upon their heteroPARylation. Taken together, our findings suggest that PARP2 can function differently in BER, promoting XRCC1-dependent repair (similarly to PARP1) or an alternative XRCC1-independent mechanism via hetero-oligomerization with PARP1.

Poly(ADP-ribosyl)ation is a unique post-translational modification of proteins. The metabolism of poly(ADP-ribose) (PAR) is tightly regulated mainly by poly(ADP-ribose) polymerases (PARP) and poly(ADP-ribose) glycohydrolase (PARG). Accumulating evidence has suggested the biological functions of PAR metabolism in control of many cellular processes, such as cell proliferation, differentiation and death by remodeling chromatin structure and regulation of DNA transaction, including DNA repair, replication, recombination and transcription. However, the physiological roles of the catabolism of PAR catalyzed by PARG remain less understood than those of PAR synthesis by PARP. Noteworthy biochemical studies have revealed the importance of PAR catabolic pathway generating nuclear ATP via the coordinated actions of PARG and ADP-ribose pyrophosphorylase (ADPRPPL) for the driving of DNA repair and the maintenance of DNA replication apparatus while repairing DNA damage. Furthermore, genetic studies have shown the value of PARG as a therapeutic molecular target for PAR-mediated diseases, such as cancer, inflammation and many pathological conditions. In this review, we present the current knowledge of de-poly(ADP-ribosyl)ation catalyzed by PARG focusing on its role in DNA repair, replication and apoptosis. Furthermore, the induction of apoptosis code of DNA replication catastrophe by synthetic lethality of PARG inhibition and the recent progresses regarding the development of small molecule PARG inhibitors and their therapeutic potentials in cancer chemotherapy are highlighted in this review. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribosyl)ation (aka PARylation) is a unique protein post-translational modification (PTM) first described over 50 years ago. PARylation regulates a number of biological processes including chromatin remodeling, the DNA damage response (DDR), transcription, apoptosis, and mitosis. The subsequent discovery of poly(ADP-ribose) polymerase-1 (PARP-1) catalyzing DNA-dependent PARylation spearheaded the field of DDR. The expanding knowledge about the poly ADP-ribose (PAR) recognition domains prompted the discovery of novel DDR factors and revealed crosstalk with other protein PTMs including phosphorylation, ubiquitination, methylation and acetylation. In this review, we highlight the current knowledge on PAR-regulated DDR, PAR recognition domain, and PARP inhibition in cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract Base excision repair (BER) involves many enzymes acting in a coordinated fashion at the most common types of DNA damage. The coordination is facilitated by interactions between the enzymes and accessory proteins, X-ray repair cross-complementing protein 1 (XRCC1) and poly(ADP-ribose) polymerase 1 (PARP1). Here we use dynamic light scattering (DLS) technique to determine the hydrodynamic sizes of several BER enzymes and proteins, DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), tyrosyl-DNA phosphodiesterase 1 (TDP1), XRCC1 and PARP1, present alone or in the equimolar mixtures with each other. From the DLS data combined with glutaraldehyde cross-linking experiments and previous quantitative binding data the oligomeric states of BER proteins and their complexes are estimated. All the proteins have been proposed to form homodimers upon their self-association. The most probable oligomerization state of the binary complexes formed by PARP1 with various proteins is a heterotetramer. The oligomerization state of the binary complexes formed by XRCC1 varies from heterodimer to heterotetramer, depending on the partner. The DLS technique is applied for the first time to measure the hydrodynamic sizes of PARP1 molecules covalently bound with poly(ADP-ribose) (PAR) synthesized upon the automodification reaction. PARP1 has been detected to form huge conglomerates stabilized by Mg2+ coordinated bonds with PAR polymers. Highlights • Most proteins involved in BER self-associate via homodimerization. • Complexes of PARP1 with various BER proteins are heterotetramers. • The oligomerization state of XRCC1 heterocomplexes depends on the partner. • PARP1 forms huge conglomerates upon the autoPARylation stabilized by magnesium ions. [ABSTRACT FROM AUTHOR]

Abstract Neural stem/progenitor cells (NSPCs) express higher levels of poly(ADP-ribose) polymerase 1 (PARP1) than mouse embryonic fibroblasts (MEFs). Inhibition of PARP induces the expression of several genes in the p53 signaling pathway, including p21 , which is critical for cell cycle control at the G1/S phase, triggers apoptosis, and suppresses cell cycle progression in NSPCs. However, upon the up-regulation of p21, the cell cycle does not arrest at any specific phase. In the present study, the expression of genes specific to the G1/S and G2/M phases of the cell cycle were analyzed following treatment with PJ34 (N -[6-oxo-5,6-dihydro-phenanthridin-2-yl]- N,N -dimethylacetamide), an inhibitor of PARP. PJ34 treatment dramatically down-regulated cyclin B1 expression in NSPCs, but not in MEFs, which was confirmed by a promoter assay. Down-regulation of FoxM1 and B-MYB revealed that the down-regulation of cyclin B occurs at the transcriptional level. GADD45 was also specifically up-regulated in NSPCs. Taken together, the activation of p53 by PJ34 treatment in NSPCs induced changes in the expression of genes involved in the cell cycle. Fluorescence-activated cell sorting analysis revealed that PJ34 treatment suppressed G2/M to G1 progression in NSPCs, but not in MEFs. These data indicate that PJ34 treatment inhibits cyclin expression at the mRNA level and suppresses cell cycle progression in NSPCs. Highlights • Inhibition of poly(ADP-ribosyl)ation in NSCPs decreases cyclin B1 promoter activity. • Down-regulation of FoxM1 and B-MYB by PJ34 in NSPCs reduces cyclin B gene expression. • Up-regulation of GADD4 5 by PJ34 in NSPCs suppresses cell cycle progression at G2/M. [ABSTRACT FROM AUTHOR]

Cell wall reinforcement with callose is a frequent plant response to infection. Poly(ADP-ribosyl)ation is a protein post-translational modification mediated by poly(ADP-ribose) polymerases (PARPs). Poly(ADP-ribosyl)ation has well-known roles in DNA damage repair and has more recently been shown to contribute to plant immune responses. 3-aminobenzamide (3AB) is an established PARP inhibitor and it blocks the callose deposition elicited by flg22 or elf18, two microbe-associated molecular patterns (MAMPs). However, we report that an Arabidopsis parp1parp2parp3 triple mutant does not exhibit loss of flg22-induced callose deposition. Additionally, the more specific PARP inhibitors PJ-34 and INH2BP inhibit PARP activity in Arabidopsis but do not block MAMP-induced callose deposition. These data demonstrate off-target activity of 3AB and indicate that 3AB inhibits callose deposition through a mechanism other than poly(ADP-ribosyl)ation. POWDERY MILDEW RESISTANT 4 (PMR4) is the callose synthase responsible for the majority of MAMP- and wound-induced callose deposition in Arabidopsis. 3AB does not block wound-induced callose deposition, and 3AB does not reduce the PMR4 mRNA abundance increase in response to flg22. Levels of PMR4-HA protein increase in response to flg22, and increase even more in flg22 + 3AB despite no callose being produced. The callose synthase inhibitor 2-deoxy-D-glucose does not cause similar impacts on PMR4-HA protein levels. Beyond MAMPs, we find that 3AB also reduces callose deposition induced by powdery mildew (Golovinomyces cichoracearum) and impairs the penetration resistance of a PMR4 overexpression line. 3AB thus reveals pathogenesis-associated pathways that activate callose synthase enzymatic activity distinct from those that elevate PMR4 mRNA and protein abundance. [ABSTRACT FROM AUTHOR]

Highlights • RPA poly(ADP-ribosyl)ation efficiency depends on the structure of DNA. • RPA influences PARP1 activity, and the effect depends on DNA structure. • RPA holds PARP1 on DNA in the course of poly(ADP-ribosyl)ation. • RPA can accelerate replacement of modified PARP1 on DNA by unmodified one. Abstract Replication protein A contributes to all major pathways of DNA metabolism and is a target for post-translation modifications, including poly(ADP-ribosyl)ation catalyzed by PARP1. Here we demonstrate that the efficiency of RPA poly(ADP-ribosyl)ation strongly depends on the structure of DNA used for PARP1 activation and on the polarity of RPA binding. Moreover, RPA influences PARP1 activity, and this effect also depends on DNA structure: RPA inhibits PAR synthesis catalyzed by PARP1 in the presence of ssDNA and stimulates it in the presence of a DNA duplex, in particular that containing a nick or a gap. Using fluorescently labeled proteins, we showed their direct interaction and characterized it quantitatively. RPA can accelerate the replacement of poly(ADP-ribosyl)ated PARP1 molecules bound to DNA by the unmodified ones. Thus, our data allow us to suggest that the balance between the affinities of PARP1 and RPA for DNA and the interaction of these proteins with each other are the cornerstone of the modulating effect of RPA on PARP1 activity. This effect might contribute to the regulation of PARP1 activity in various DNA processing mechanisms including DNA replication and repair pathways, where both PARP1 and RPA participate. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribosyl)ation is known to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, genomic stability and cell differentiation by poly(ADP-ribose) polymerase (PARP). While PARP inhibitors are presently under clinical investigation for cancer therapy, little is known about their side effects. However, PARP involvement in mesenchymal stem cell (MSC) differentiation potentiates MSC-related side effects arising from PARP inhibition. In this study, effects of PARP inhibitors on MSCs were examined. MSCs demonstrated suppressed osteogenic differentiation after 1 µM PJ34 treatment without cytotoxicity, while differentiation of MSCs into chondrocytes or adipocytes was unaffected. PJ34 suppressed mRNA induction of osteogenic markers, such as Runx2, Osterix, Bone Morphogenetic Protein-2, Osteocalcin, bone sialoprotein, and Osteopontin, and protein levels of Bone Morphogenetic Protein-2, Osterix and Osteocalcin. PJ34 treatment also inhibited transcription factor regulators such as Smad1, Smad4, Smad5 and Smad8. Extracellular mineralized matrix formation was also diminished. These results strongly suggest that PARP inhibitors are capable of suppressing osteogenic differentiation and poly(ADP-ribosyl)ation may play a physiological role in this process through regulation of BMP-2 signaling. Therefore, PARP inhibition may potentially attenuate osteogenic metabolism, implicating cautious use of PARP inhibitors for cancer treatments and monitoring of patient bone metabolism levels.

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, a prevalent protein post-translational modification (PTM) that occurs intracellularly, has been shown to crosstalk with phosphorylation and ubiquitination. However, it is unclear whether it interplays with other PTMs. Here we studied its relationship with ADP-ribosylation, which involves decorating target proteins with the ADP-ribose moiety. We discovered that the poly(ADP-ribosyl)ation "eraser", ADP-ribose glycohydrolase (PARG), is O-GlcNAcylated at Ser26, which is in close proximity to its nuclear localization signal. O-GlcNAcylation of PARG promotes nuclear localization and chromatin association. Upon DNA damage, O-GlcNAcylation augments the recruitment of PARG to DNA damage sites and interacting with proliferating cell nuclear antigen (PCNA). In hepatocellular carcinoma (HCC) cells, PARG O-GlcNAcylation enhances the poly(ADP-ribosyl)ation of DNA damage-binding protein 1 (DDB1) and attenuates its auto-ubiquitination, thereby stabilizing DDB1 and allowing it to degrade its downstream targets, such as c-Myc. We further demonstrated that PARG-S26A, the O-GlcNAc-deficient mutant, promoted HCC in mouse xenograft models. Our findings thus reveal that PARG O-GlcNAcylation inhibits HCC, and we propose that O-GlcNAc glycosylation may crosstalk with many other PTMs., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Summary The Fanconi anemia (FA) pathway is a pivotal genome maintenance network that orchestrates the repair of DNA interstrand crosslinks (ICLs). The tumor suppressors RUNX1 and RUNX3 were shown to regulate the FA pathway independent of their canonical transcription activities, by controlling the DNA damage-dependent chromatin association of FANCD2. Here, in further biochemical characterization, we demonstrate that RUNX3 is modified by PARP-dependent poly(ADP-ribosyl)ation (PARylation), which in turn allows RUNX binding to DNA repair structures lacking transcription-related RUNX consensus motifs. SILAC-based mass spectrometric analysis revealed significant association of RUNX3 with core DNA repair complexes, including PARP1, even in unstressed cells. After DNA damage, the increased interaction between RUNX3 and BLM facilitates efficient FANCD2 chromatin localization. RUNX-Walker motif mutations from breast cancers are impaired for DNA damage-inducible PARylation, unveiling a potential mechanism for FA pathway inactivation in cancers. Our results reinforce the emerging paradigm that RUNX proteins are tumor suppressors with genome gatekeeper function. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribosyl)ation (PARylation) is a complex and reversible posttranslational modification catalyzed by poly(ADP-ribose)polymerases (PARPs), which orchestrates protein function and subcellular localization. The function of PARP1 in genotoxic stress response upon induction of oxidative DNA lesions and strand breaks is firmly established, but its role in the response to chemical-induced, bulky DNA adducts is understood incompletely. To address the role of PARP1 in the response to bulky DNA adducts, we treated human cancer cells with benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE), which represents the active metabolite of the environmental carcinogen benzo[a]pyrene [B(a)P], in nanomolar to low micromolar concentrations. Using a highly sensitive LC-MS/MS method, we revealed that BPDE induces cellular PAR formation in a time- and dose-dependent manner. Consistently, PARP1 activity significantly contributed to BPDE-induced genotoxic stress response. On one hand, PARP1 ablation rescued BPDE-induced NAD+ depletion and protected cells from BPDE-induced short-term toxicity. On the other hand, strong sensitization effects of PARP inhibition and PARP1 ablation were observed in long-term clonogenic survival assays. Furthermore, PARP1 ablation significantly affected BPDE-induced S- and G2-phase transitions. Together, these results point towards unresolved BPDE-DNA lesions triggering replicative stress. In line with this, BPDE exposure resulted in enhanced formation and persistence of DNA double-strand breaks in PARP1-deficient cells as evaluated by microscopic co-localization studies of 53BP1 and γH2A.X foci. Consistently, an HPRT mutation assay revealed that PARP inhibition potentiated the mutagenicity of BPDE. In conclusion, this study demonstrates a profound role of PARylation in BPDE-induced genotoxic stress response with significant functional consequences and potential relevance with regard to B[a]P-induced cancer risks. [ABSTRACT FROM AUTHOR]

Oxidative stress can cause DNA breaks which induce activation of the DNA nick sensor enzyme poly(ADP-ribose) polymerase-1 (PARP-1), part of the 17 member PARP enzyme family. PARP-1 modifies target proteins by attaching to them several NAD-derived ADP-ribose units forming poly(ADP-ribose) (PAR) polymers. PARylation controls many cellular processes while intense PARylation may also lead to cell death by various mechanisms. Here we summarize the modes of activation, inhibitors and modulators of PARP-1 and review the cellular functions regulated by the enzyme.

Poly(ADP-ribosyl)ation is a rapid and transient post-translational protein modification that was described first in mammalian cells. Activated by the sensing of DNA strand breaks, poly(ADP-ribose)polymerase1 (PARP1) transfers ADP-ribose units onto itself and other target proteins using NAD+ as a substrate. Subsequently, DNA damage responses and other cellular responses are initiated. In plants, poly(ADP-ribose) polymerases (PARPs) have also been implicated in responses to DNA damage. The Arabidopsis genome contains three canonical PARP genes, the nomenclature of which has been uncoordinated in the past. Albeit assumptions concerning the function and roles of PARP proteins in planta have often been inferred from homology and structural conservation between plant PARPs and their mammalian counterparts, plant-specific roles have become apparent. In particular, PARPs have been linked to stress responses of plants. A negative role under abiotic stress has been inferred from studies in which a genetic or, more commonly, pharmacological inhibition of PARP activity improved the performance of stressed plants; in response to pathogen-associated molecular patterns, a positive role has been suggested. However, reports have been inconsistent, and the effects of PARP inhibitors appear to be more robust than the genetic abolition of PARP gene expression, indicating the presence of alternative targets of those drugs. Collectively, recent evidence suggests a conditionality of stress-related phenotypes of parp mutants and calls for a reconsideration of PARP inhibitor studies on plants. This review critically summarizes our current understanding of poly(ADP-ribosylation) and PARP proteins in plants, highlighting similarities and differences to human PARPs, areas of controversy, and requirements for future studies.

It is well known that the number of dividing cells in an organism decreases with age. The average rate of cell division in tissues and organs of a mature organism sharply decreases, which is probably a trigger for accumulation of damage leading to disturbance of genome integrity. This can be a cause for the development of many age-related diseases and appearance of phenotypic and physiological signs of aging. In this connection, the protein poly(ADP-ribosyl)ation system, which is activated in response to appearance of various DNA damage, attracts great interest. This review summarizes and analyzes data on changes in the poly(ADP-ribosyl)ation system during development and aging in vivo and in vitro, and due to restriction of cell proliferation. Special attention is given to methodological aspects of determination of activity of poly(ADP-ribose) polymerases (PARPs). Analysis of relevant publications and our own data has led us to the conclusion that PARP activity upon the addition of free DNA ends (in this review referred to as stimulated PARP activity) is steadily decreasing with age. However, the dynamics of PARP activity measured without additional activation of the enzyme (in this review referred to as unstimulated activity) does not have such a clear trend: in many studies, the presented differences are statistically non-significant, although it is well known that the number of unrepaired DNA lesions steadily increases with aging. Apparently, the cell has additional regulatory systems that limit its own capability of reacting to DNA damage. Special attention is given to the influence of the cell proliferative status on PARP activity. We have systematized and analyzed data on changes in PARP activity during development and aging of an organism, as well as data on differences in the dynamics of this activity in the presence/absence of additional stimulation and on cellular processes that are associated with activation of these enzymes. Moreover, data obtained in different models of cellular aging are compared. [ABSTRACT FROM AUTHOR]

Long-term exposure to arsenic has been known to induce neoplastic initiation and progression in several organs; however, the role of arsenic (As2O3) in oxidative stress-mediated DNA damage remains elusive. One of the immediate cellular responses to DNA damage is poly(ADP-ribosyl)ation (PARylation), which mediates DNA repair and enhances cell survival. In this study, we found that oxidative stress (H2O2)-induced PARylation was suppressed by As2O3 exposure in different human cancer cells. Moreover, As2O3 treatment promoted H2O2-induced DNA damage and apoptosis, leading to increased cell death. We found that N-ethylmaleimide (NEM), an organic compound derived from maleic acid, could reverse As2O3-mediated effects, thus enhancing PARylation with attenuated cell death and increased cell survival. Pharmacologic inhibition of glutathione with l-buthionine-sulfoximine blocked the antagonistic effect of NEM on As2O3, thereby continuing As2O3-mediated suppression of PARylation and causing DNA damage. Our findings identify NEM as a potential antidote against As2O3-mediated DNA damage in a glutathione-dependent manner. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribosyl)ation (PARylation) plays important roles in DNA repair, apoptosis, transcriptional regulation, and cell death, and occurs via the activity of poly(ADP-ribose) polymerases (PARPs). Previous studies have shown that PARylation affects mouse and porcine pre-implantation development and participates in mechanisms of autophagy. However, there have not yet been reported the role of PARylation during in vitro maturation (IVM) of porcine oocytes. Thus, we investigated the effect of PARylation inhibition on this process; cumulus-oocyte complexes (COCs) were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during porcine IVM. Full cumulus expansion was significantly reduced (10.34 ± 1.23 [3-ABA] vs. 48.17 ± 2.03% [control]), but nuclear maturation rates were not changed in the 3-ABA treatment group. Especially, we observed that cumulus cells were little expanded after 22 h in 3-ABA treated COCs. The mRNA expression levels of oocyte maturation- and cumulus expansion-related genes were evaluated at 22 and 44 h. GDF9, BMP15, COX-2, and PTX3 expression were upregulated at 44 h, whereas the levels of HAS2 and TNFAIP6 were downregulated in the 3-ABA treated group. Furthermore, 3-ABA treatment significantly decreased the developmental rate (28.24 ± 1.06 vs. 40.24 ± 3.03%) and total cell number (41.12 ± 2.10 vs. 50.38 ± 2.27), but increased the total apoptotic index (6.44 ± 0.81 vs. 3.08 ± 0.51) in parthenogenetically activated embryos. In conclusion, these results showed that PARylation regulates cumulus expansion through the regulation of gene expression and affects developmental competence and quality in parthenogenetic embryos. [ABSTRACT FROM AUTHOR]

Background: Poly(ADP-ribose) polymerase 1 (PARP-1), which catalyzes poly(ADP-ribosyl)ation of proteins by using NAD+ as a substrate, plays a key role in several nuclear events, including DNA repair, replication, and transcription. Recently, PARP-1 was reported to participate in the somatic cell reprogramming process. Previously, we revealed a role for PARP-1 in the induction of neural apoptosis in a cellular model of cerebral ischemia and suggested the possible use of PARP inhibitors as a new therapeutic intervention. In the present study, we examined the effects of PARP inhibitors on neural stem/progenitor cells (NSPCs) of the mouse brain.Results: PARP-1 was more abundant and demonstrated higher activity in NSPCs than in mouse embryonic fibroblasts. Treatment with PARP inhibitors suppressed the formation of neurospheres by NSPCs through the suppression of cell cycle progression and the induction of apoptosis. In order to identify the genes responsible for these effects, we investigated gene expression profiles by microarray analyses and found that several genes in the p53 signaling pathway were upregulated, including Cdkn1a, which is critical for cell cycle control, and Fas, Pidd, Pmaip1, and Bbc3, which are principal factors in the apoptosis pathway. Inhibition of poly(ADP-ribosyl)ation increased the levels of p53 protein, but not p53 mRNA, and enhanced the phosphorylation of p53 at Ser18. Experiments with specific inhibitors and also shRNA demonstrated that PARP-1, but not PARP-2, has a role in the regulation of p53. The effects of PARP inhibitors on NSPCs were not observed in Trp53 -/- NSPCs, suggesting a key role for p53 in these events.Conclusions: On the basis of the finding that PARP inhibitors facilitated the p53 signaling pathway, we propose that poly(ADP-ribosyl)ation contributes to the proliferation and self-renewal of NSPCs through the suppression of p53 activation. [ABSTRACT FROM AUTHOR]