ABSTRACT
DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Polθ interacts with and is PARylated by PARP1 in a HPF1-independent manner. PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ.
Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA Polymerase theta , DNA-Directed DNA Polymerase , Poly (ADP-Ribose) Polymerase-1 , Humans , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , DNA-Directed DNA Polymerase/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , DNA Damage , Animals , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , DNA/metabolism , DNA/genetics , HEK293 Cells , Poly ADP Ribosylation , Poly(ADP-ribose) Polymerases/metabolism , Poly(ADP-ribose) Polymerases/genetics , Carrier Proteins , Glycoside Hydrolases , Nuclear ProteinsABSTRACT
Aging, marked by a gradual decline in physiological function and heightened vulnerability to age-related diseases, remains a complex biological process with multifaceted regulatory mechanisms. Our study elucidates the critical role of poly(ADP-ribose) glycohydrolase (PARG), responsible for catabolizing poly(ADP-ribose) (pADPr) in the aging process by modulating the expression of age-related genes in Drosophila melanogaster. Specifically, we uncover the regulatory function of the uncharacterized PARG C-terminal domain in controlling PARG activity. Flies lacking this domain exhibit a significantly reduced lifespan compared to wild-type counterparts. Furthermore, we observe progressive dysregulation of age-related gene expression during aging, accelerated in the absence of PARG activity, culminating in a premature aging phenotype. Our findings reveal the critical involvement of the pADPr pathway as a key player in the aging process, highlighting its potential as a therapeutic target for mitigating age-related effects.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Glycoside Hydrolases , Longevity , Animals , Longevity/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Glycoside Hydrolases/metabolism , Glycoside Hydrolases/genetics , Aging/genetics , Aging/metabolism , Gene Expression Regulation , Poly Adenosine Diphosphate Ribose/metabolismABSTRACT
Poly(ADP-ribose) polymerases (PARPs) are critical to regulating cellular activities, such as the response to DNA damage and cell death. PARPs catalyze a reversible post-translational modification (PTM) in the form of mono- or poly(ADP-ribosyl)ation. This type of modification is known to form a ubiquitin-ADP-ribose (Ub-ADPR) conjugate that depends on the actions of Deltex family of E3 ubiquitin ligases (DTXs). In particular, DTXs add ubiquitin to the 3'-OH of adenosine ribose' in ADP-ribose, which effectively sequesters ubiquitin and impedes ubiquitin-dependent signaling. Previous work demonstrates DTX function for ubiquitination of protein-free ADPR, mono-ADP-ribosylated peptides, and ADP-ribosylated nucleic acids. However, the dynamics of DTX-mediated ubiquitination of poly(ADP-ribosyl)ation remains to be defined. Here we show that the ADPR ubiquitination function is not found in other PAR-binding E3 ligases and is conserved across DTX family members. Importantly, DTXs specifically target poly(ADP-ribose) chains for ubiquitination that can be cleaved by PARG, the primary eraser of poly(ADP-ribose), leaving the adenosine-terminal ADPR unit conjugated to ubiquitin. Our collective results demonstrate the DTXs' specific ubiquitination of the adenosine terminus of poly(ADP-ribosyl)ation and suggest the unique Ub-ADPR conjugation process as a basis for PARP-DTX control of cellular activities.
Subject(s)
Adenosine Diphosphate Ribose , Ubiquitin-Protein Ligases , Ubiquitination , Ubiquitin-Protein Ligases/metabolism , Humans , Adenosine Diphosphate Ribose/metabolism , Poly ADP Ribosylation , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/genetics , Ubiquitin/metabolism , ADP-Ribosylation , HEK293 CellsABSTRACT
Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic regulation of DNA damage response. PARG, the most active dePARylation enzyme, is recruited to sites of DNA damage via pADPr-dependent and PCNA-dependent mechanisms. Targeting dePARylation is considered an alternative strategy to overcome PARP inhibitor resistance. However, precisely how dePARylation functions in normal unperturbed cells remains elusive. To address this challenge, we conducted multiple CRISPR screens and revealed that dePARylation of S phase pADPr by PARG is essential for cell viability. Loss of dePARylation activity initially induced S-phase-specific pADPr signaling, which resulted from unligated Okazaki fragments and eventually led to uncontrolled pADPr accumulation and PARP1/2-dependent cytotoxicity. Moreover, we demonstrated that proteins involved in Okazaki fragment ligation and/or base excision repair regulate pADPr signaling and cell death induced by PARG inhibition. In addition, we determined that PARG expression is critical for cellular sensitivity to PARG inhibition. Additionally, we revealed that PARG is essential for cell survival by suppressing pADPr. Collectively, our data not only identify an essential role for PARG in normal proliferating cells but also provide a potential biomarker for the further development of PARG inhibitors in cancer therapy.
Subject(s)
Antineoplastic Agents , Poly Adenosine Diphosphate Ribose , Cell Survival , S Phase , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Antineoplastic Agents/pharmacologyABSTRACT
PARP-1 over-activation results in cell death via excessive PAR generation in different cell types, including neurons following brain ischemia. Glycolysis, mitochondrial function, and redox balance are key cellular processes altered in brain ischemia. Studies show that PAR generated after PARP-1 over-activation can bind hexokinase-1 (HK-1) and result in glycolytic defects and subsequent mitochondrial dysfunction. HK-1 is the neuronal hexokinase and catalyzes the first reaction of glycolysis, converting glucose to glucose-6-phosphate (G6P), a common substrate for glycolysis, and the pentose phosphate pathway (PPP). PPP is critical in maintaining NADPH and GSH levels via G6P dehydrogenase activity. Therefore, defects in HK-1 will not only decrease cellular bioenergetics but will also cause redox imbalance due to the depletion of GSH. In brain ischemia, whether PAR-mediated inhibition of HK-1 results in bioenergetics defects and redox imbalance is not known. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons to mimic brain ischemia in neuronal cultures and observed that PARP-1 activation via PAR formation alters glycolysis, mitochondrial function, and redox homeostasis in neurons. We used pharmacological inhibition of PARP-1 and adenoviral-mediated overexpression of wild-type HK-1 (wtHK-1) and PAR-binding mutant HK-1 (pbmHK-1). Our data show that PAR inhibition or overexpression of HK-1 significantly improves glycolysis, mitochondrial function, redox homeostasis, and cell survival in mouse cortical neurons exposed to OGD. These results suggest that PAR binding and inhibition of HK-1 during OGD drive bioenergetic defects in neurons due to inhibition of glycolysis and impairment of mitochondrial function.
Subject(s)
Brain Ischemia , Oxygen , Mice , Animals , Oxygen/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Hexokinase/genetics , Hexokinase/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/metabolism , Glucose/metabolism , Brain Ischemia/metabolism , Glycolysis , Neurons/metabolism , Oxidation-ReductionABSTRACT
Poly-ADP-ribosylation is an important protein post-translational modification with diverse biological consequences. After binding poly-ADP-ribose on axis inhibition protein 1 (AXIN1) through its WWE domain, RING finger protein 146 (RNF146) can ubiquitinate AXIN1 and promote its proteasomal degradation and thus the oncogenic WNT signaling. Therefore, inhibiting the RNF146 WWE domain is a potential antitumor strategy. However, due to a lack of suitable screening methods, no inhibitors for this domain have been reported. Here, we developed a fluorescence polarization (FP)-based competition assay for the screening of RNF146 WWE inhibitors. This assay relies on a fluorescently tagged iso-ADP-ribose tracer compound, TAMRA-isoADPr. We report the design and synthesis of this tracer compound and show that it is a high-affinity tracer for the RNF146 WWE domain. This provides a convenient assay and will facilitate the development of small-molecule inhibitors for the RNF146 WWE domain.
Subject(s)
Adenosine Diphosphate Ribose , Poly Adenosine Diphosphate Ribose , Adenosine Diphosphate Ribose/metabolism , Poly Adenosine Diphosphate Ribose/chemistry , Poly Adenosine Diphosphate Ribose/metabolism , Protein Processing, Post-Translational , Wnt Signaling PathwayABSTRACT
Poly(ADP-ribosyl)ation (PARylation), as a posttranslational modification mediated by poly(ADP-ribose) polymerases (PARPs) catalyzing the transfer of ADP-ribose from NAD+ molecules to acceptor proteins, involves a number of cellular processes. As mice lacking the PARP-1 gene (Parp1) produce more urine, we investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). In biotin-conjugated nicotinamide adenine dinucleotide (biotin-NAD+) pulldown and immunoprecipitation assays of poly(ADP)-ribose in mpkCCDc14 cells, immunoblots demonstrated that 1-deamino-8-D-arginine vasopressin (dDAVP) induced the PARylation of total proteins, associated with an increase in the cleavage of PARP-1 and cleaved caspase-3 expression. By inhibiting PARP-1 with siRNA, the abundance of dDAVP-induced AQP2 mRNA and protein was significantly diminished. In contrast, despite a substantial decrease in PARylation, the PARP-1 inhibitor (PJ34) had no effect on the dDAVP-induced regulation of AQP2 expression. The findings suggest that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. Bioinformatic analysis revealed that 408 proteins interact with PARP-1 in the collecting duct (CD) cells of the kidney. Among them, the signaling pathway of the vasopressin V2 receptor was identified for 49 proteins. In particular, ß-catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein. A significant decrease of ß-catenin phosphorylation (Ser552) in response to dDAVP was associated with siRNA-mediated PARP-1 knockdown. Taken together, PARP-1 is likely to play a role in vasopressin-induced AQP2 expression by interacting with ß-catenin in renal CD cells.NEW & NOTEWORTHY The poly(ADP-ribose) polymerase (PARP) family catalyzes poly(ADP-ribosylation) (PARylation), which is one of the posttranslational modifications of largely undetermined physiological significance. This study investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). The results demonstrated that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. ß-Catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein.
Subject(s)
Aquaporin 2 , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Mice , Aquaporin 2/genetics , beta Catenin/metabolism , Biotin/metabolism , Deamino Arginine Vasopressin/pharmacology , Kidney/metabolism , NAD/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , RNA, Small Interfering , Vasopressins/pharmacology , Vasopressins/metabolismABSTRACT
Poly (ADP-ribose) glycohydrolase (PARG) is an enzyme that mainly degrades poly (ADP-ribose) (PAR) synthesized by poly (ADP-ribose) polymerase (PARP) family proteins. Although PARG is involved in many biological phenomena, including DNA repair, cell differentiation, and cell death, little is known about the relationship between osteoclast differentiation and PARG. It has also not been clarified whether PARG is a valuable target for therapeutic agents in the excessive activity of osteoclast-related bone diseases such as osteoporosis. In the present study, we examined the effects of PARG inhibitor PDD00017273 on osteoclast differentiation in RANKL-induced RAW264 cells. PDD00017273 induced the accumulation of intracellular PAR and suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. PDD00017273 also downregulated osteoclast differentiation marker genes such as Trap, cathepsin K (Ctsk), and dendrocyte expressed seven transmembrane protein (Dcstamp) and protein expression of nuclear factor of activated T cells 1 (NFATc1), a master regulator of osteoclast differentiation. Taken together, our findings suggest that dysfunction of PARG suppresses osteoclast differentiation via the PAR accumulation and partial inactivation of the NFATc1.
Subject(s)
Osteoclasts , Ribose , Glycoside Hydrolases/metabolism , Osteoclasts/metabolism , Poly (ADP-Ribose) Polymerase-1 , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Animals , MiceABSTRACT
To maintain tissue homeostasis, cell proliferation is balanced by cell death. PARP1 is an important protein involved in both processes. Upon sensing DNA damage, PARP1 forms poly(ADP-ribose) (PAR) chains to recruit the repair proteins, ensuring genome integrity and faithful cell proliferation. In addition, PAR also regulates the activity of PARP1. Persistent DNA damage can signal the cell to progress toward programmed cell death, apoptosis. During apoptosis, proteolytic cleavage of PARP1 generates an N-terminal, ZnF1-2PARP1 (DNA binding or regulatory fragment), and C-terminal, PARP1ΔZnF1-2 (catalytic or PAR carrier fragment), which exhibits a basal activity. Regulation of the apoptotic fragments by PAR has not been studied. Here, we report that PAR inhibits the basal level activity of PARP1ΔZnF1-2, and ZnF1-2PARP1 interacts with PARP1ΔZnF1-2 to exhibit DNA-dependent stimulation and partially restores the PAR-dependent stimulation. Interestingly, along with the auto-modification domain of PARP1, the DNA-binding domains, ZnF1-2PARP1, also acts as an acceptor of PARylation; therefore, ZnF1-2PARP1 exhibits a reduced affinity for DNA upon PARylation. Furthermore, we show that ZnF1-2PARP1 shows trans-dominant inhibition of DNA-dependent stimulation of PARP2. Altogether, our study explores the regulation of the catalytic activity of PARP1ΔZnF1-2 and PARP2 by the regulatory apoptotic fragment of PARP1.
Subject(s)
DNA , Poly Adenosine Diphosphate Ribose , Poly (ADP-Ribose) Polymerase-1/metabolism , DNA/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Poly ADP Ribosylation , DNA Repair , DNA DamageABSTRACT
Hepatocellular carcinoma (HCC) is a major global health concern, representing one of the leading causes of cancer-related deaths. Despite various treatment options, the prognosis for HCC patients remains poor, emphasizing the need for a deeper understanding of the factors contributing to HCC development. This study investigates the role of poly(ADP-ribosyl)ation in hepatocyte maturation and its impact on hepatobiliary carcinogenesis. A conditional Parg knockout mouse model was employed, utilizing Cre recombinase under the albumin promoter to target Parg depletion specifically in hepatocytes. The disruption of the poly(ADP-ribosyl)ating pathway in hepatocytes affects the early postnatal liver development. The inability of hepatocytes to finish the late maturation step that occurs early after birth causes intensive apoptosis and acute inflammation, resulting in hypertrophic liver tissue with enlarged hepatocytes. Regeneration nodes with proliferative hepatocytes eventually replace the liver tissue and successfully fulfill the liver function. However, early developmental changes predispose these types of liver to develop pathologies, including with a malignant nature, later in life. In a chemically induced liver cancer model, Parg-depleted livers displayed a higher tendency for hepatocellular carcinoma development. This study underscores the critical role of the poly(ADP-ribosyl)ating pathway in hepatocyte maturation and highlights its involvement in liver pathologies and hepatobiliary carcinogenesis. Understanding these processes may provide valuable insights into liver biology and liver-related diseases, including cancer.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Precancerous Conditions , Animals , Mice , Humans , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Hepatocytes/metabolism , Precancerous Conditions/metabolism , Carcinogenesis/genetics , Carcinogenesis/metabolism , Glycoside Hydrolases/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Mammals/metabolismABSTRACT
Metabolism, known to be temporally regulated to meet evolving energy demands, plays a crucial role in shaping developmental pace. Recent studies have demonstrated that two key proteins PARP1 and PARG play a regulatory role in the transcription of both morphogenic and metabolic genes. Intriguingly, in Drosophila, the depletion of PARP1 or PARG proteins causes a developmental arrest before pupation, resulting in individuals unable to complete their development. This phenotype highlights the critical involvement of poly(ADP-ribosyl)ating enzymes in regulating the metamorphic process. In this study, we provide compelling evidence that these enzymes intricately coordinate transcriptional changes in both developmental and metabolic pathways during metamorphosis. Specifically, they promote the expression of genes crucial for pupation, while simultaneously negatively regulating the expression of metabolic genes before the transition to the pupal stage. Additionally, these enzymes suppress the expression of genes that are no longer required during this transformative period. Our findings shed light on the intricate interplay between poly(ADP-ribosyl)ating enzymes, developmental processes, and metabolic regulation before metamorphosis and highlight a new role of poly(ADP-ribosyl)ating enzymes in the global regulation of transcription.
Subject(s)
Glycoside Hydrolases , Poly(ADP-ribose) Polymerases , Animals , Humans , Glycoside Hydrolases/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Phenotype , Drosophila/genetics , Poly Adenosine Diphosphate Ribose/metabolismABSTRACT
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.
Subject(s)
Carcinoma, Hepatocellular , Glycoside Hydrolases , Liver Neoplasms , Animals , Humans , Mice , Acetylglucosamine , ADP-Ribosylation , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Glycosylation , Protein Processing, Post-TranslationalABSTRACT
ZNF746 was identified as parkin-interacting substrate (PARIS). Investigating its pathophysiological properties, we find that PARIS undergoes liquid-liquid phase separation (LLPS) and amorphous solid formation. The N-terminal low complexity domain 1 (LCD1) of PARIS is required for LLPS, whereas the C-terminal prion-like domain (PrLD) drives the transition from liquid to solid phase. In addition, we observe that poly(ADP-ribose) (PAR) strongly binds to the C-terminus of PARIS near the PrLD, accelerating its LLPS and solidification. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PAR formation leads to PARIS oligomerization in human iPSC-derived dopaminergic neurons that is prevented by the PARP inhibitor, ABT-888. Furthermore, SDS-resistant PARIS species are observed in the substantia nigra (SN) of aged mice overexpressing wild-type PARIS, but not with a PAR binding-deficient PARIS mutant. PARIS solidification is also found in the SN of mice injected with preformed fibrils of α-synuclein (α-syn PFF) and adult mice with a conditional knockout (KO) of parkin, but not if α-syn PFF is injected into mice deficient for PARP1. Herein, we demonstrate that PARIS undergoes LLPS and PAR-mediated solidification in models of Parkinson's disease.
Subject(s)
Parkinson Disease , Poly Adenosine Diphosphate Ribose , Animals , Humans , Mice , Dopaminergic Neurons/metabolism , Parkinson Disease/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Repressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Zinc finger (ZNF) motifs are some of the most frequently occurring domains in the human genome. It was only recently that ZNF proteins emerged as key regulators of genome integrity in mammalian cells. In this study, we report a new role for the Krüppel-type ZNF-containing protein ZNF432 as a novel poly(ADP-ribose) (PAR) reader that regulates the DNA damage response. We show that ZNF432 is recruited to DNA lesions via DNA- and PAR-dependent mechanisms. Remarkably, ZNF432 stimulates PARP-1 activity in vitro and in cellulo. Knockdown of ZNF432 inhibits phospho-DNA-PKcs and increases RAD51 foci formation following irradiation. Moreover, purified ZNF432 preferentially binds single-stranded DNA and impairs EXO1-mediated DNA resection. Consequently, the loss of ZNF432 in a cellular system leads to resistance to PARP inhibitors while its overexpression results in sensitivity. Taken together, our results support the emerging concept that ZNF-containing proteins can modulate PARylation, which can be embodied by the pivotal role of ZNF432 to finely balance the outcome of PARPi response by regulating homologous recombination.
Subject(s)
Poly ADP Ribosylation , Poly Adenosine Diphosphate Ribose , Humans , DNA/genetics , DNA/metabolism , DNA Damage , DNA Repair , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly Adenosine Diphosphate Ribose/metabolismABSTRACT
PARP-1 activation at DNA damage sites leads to the synthesis of long poly(ADP-ribose) (PAR) chains, which serve as a signal for DNA repair. Here we show that FUS, an RNA-binding protein, is specifically directed to PAR through its RNA recognition motif (RRM) to increase PAR synthesis by PARP-1 in HeLa cells after genotoxic stress. Using a structural approach, we also identify specific residues located in the FUS RRM, which can be PARylated by PARP-1 to control the level of PAR synthesis. Based on the results of this work, we propose a model in which, following a transcriptional arrest that releases FUS from nascent mRNA, FUS can be recruited by PARP-1 activated by DNA damage to stimulate PAR synthesis. We anticipate that this model offers new perspectives to understand the role of FET proteins in cancers and in certain neurodegenerative diseases such as amyotrophic lateral sclerosis.
Subject(s)
DNA Damage , Poly Adenosine Diphosphate Ribose , Poly(ADP-ribose) Polymerases , RNA-Binding Protein FUS , Humans , DNA Repair , HeLa Cells , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , RNA Recognition Motif , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolismABSTRACT
Poly(ADP-ribosyl)ation is predominantly catalyzed by Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single-strand (SSB) and double-strand (DSB) DNA breaks are bona fide stimulators of PARP1 activity. However, PAR-mediated PARP1 regulation remains unexplored. Here, we report ZnF3, BRCT, and WGR, hitherto uncharacterized, as PAR reader domains of PARP1. Surprisingly, these domains recognize PARylated protein with a higher affinity compared with PAR but bind with weak or no affinity to DNA breaks as standalone domains. Conversely, ZnF1 and ZnF2 of PARP1 recognize DNA breaks but bind weakly to PAR. In addition, PAR reader domains, together, exhibit a synergy to recognize PAR or PARylated protein. Further competition-binding studies suggest that PAR binding releases DNA from PARP1, and the WGR domain facilitates DNA release. Unexpectedly, PAR showed catalytic stimulation of PARP1 but hampered the DNA-dependent stimulation. Altogether, our work discovers dedicated high-affinity PAR reader domains of PARP1 and uncovers a novel mechanism of allosteric regulation of DNA-dependent and DNA-independent activities of PARP1 by its catalytic product PAR.
Subject(s)
DNA Repair , Poly(ADP-ribose) Polymerases , Poly(ADP-ribose) Polymerases/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , DNA/metabolism , DNA Damage , Poly Adenosine Diphosphate Ribose/metabolismABSTRACT
Atopic dermatitis (AD) is a chronic inflammatory skin disease of very high prevalence, especially in childhood, with no specific treatment or cure. As its pathogenesis is complex, multifactorial and not fully understood, further research is needed to increase knowledge and develop new targeted therapies. We have recently demonstrated the critical role of NAD+ and poly (ADP-ribose) (PAR) metabolism in oxidative stress and skin inflammation. Specifically, we found that hyperactivation of PARP1 in response to DNA damage induced by reactive oxygen species, and fueled by NAMPT-derived NAD+, mediated inflammation through parthanatos cell death in zebrafish and human organotypic 3D skin models of psoriasis. Furthermore, the aberrant induction of NAMPT and PARP activity was observed in the lesional skin of psoriasis patients, supporting the role of these signaling pathways in psoriasis and pointing to NAMPT and PARP1 as potential novel therapeutic targets in treating skin inflammatory disorders. In the present work, we report, for the first time, altered NAD+ and PAR metabolism in the skin of AD patients and a strong correlation between NAMPT and PARP1 expression and the lesional status of AD. Furthermore, using a human 3D organotypic skin model of AD, we demonstrate that the pharmacological inhibition of NAMPT and PARP reduces pathology-associated biomarkers. These results help to understand the complexity of AD and reveal new potential treatments for AD patients.
Subject(s)
Dermatitis, Atopic , Psoriasis , Animals , Humans , Inflammation , NAD/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Poly ADP Ribosylation , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Psoriasis/etiology , Zebrafish/metabolismABSTRACT
Poly(ADPribosyl)ation is a post-translational protein modification, catalyzed by poly(ADP-ribose) polymerase (PARPs) enzymes, responsible for ADP-ribose polymer synthesis (PAR) from NAD+. PAR turnover is assured by poly(ADPR) glycohydrolase (PARGs) enzymes. In our previous study, the altered histology of zebrafish brain tissue, resulting in demyelination and neurodegeneration also with poly(ADPribosyl)ation hyperactivation, was demonstrated after aluminum (Al) exposure for 10 and 15 days. On the basis of this evidence, the aim of the present research was to study the synthesis and degradation of poly(ADP-ribose) in the brain of adult zebrafish exposed to 11 mg/L of Al for 10, 15, and 20 days. For this reason, PARP and PARG expression analyses were carried out, and ADPR polymers were synthesized and digested. The data showed the presence of different PARP isoforms, among which a human PARP1 counterpart was also expressed. Moreover, the highest PARP and PARG activity levels, responsible for the PAR production and its degradation, respectively, were measured after 10 and 15 days of exposure. We suppose that PARP activation is related to DNA damage induced by Al, while PARG activation is needed to avoid PAR accumulation, which is known to inhibit PARP and promote parthanatos. On the contrary, PARP activity decrease at longer exposure times suggests that neuronal cells could adopt the stratagem of reducing polymer synthesis to avoid energy expenditure and allow cell survival.
Subject(s)
Poly Adenosine Diphosphate Ribose , Zebrafish , Animals , Humans , Poly Adenosine Diphosphate Ribose/metabolism , Zebrafish/metabolism , Aluminum/toxicity , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/metabolism , Glycoside Hydrolases/metabolism , Brain/metabolismABSTRACT
PARP1 is a nuclear protein involved in the maintenance of genomic stability. It catalyses the formation of poly(ADP-ribose) (PAR) to recruit repair proteins at the site of DNA lesions, such as double-strand and single-strand breaks. In the process of DNA replication or repair, there could occur stretch of ssDNA, usually protected by ssDNA binding proteins, but when present in abundance can turn into DNA beaks and cause cell death. PARP1 is an extremely sensitive sensor of DNA breaks; however, the interaction of PARP1 with single-stranded DNA (ssDNA) remains unexplored. Here, we report that the two Zn-fingers, ZnF1 and ZnF2, of PARP1, mediate high-affinity recognition of ssDNA. Our studies suggest that although PAR and ssDNA are chemical analogues, they are recognized by a distinct set of domains of PARP1, yet PAR not only induces dislodging of ssDNA from PARP1 but also hampers the ssDNA-dependent PARP1 activity. It is noteworthy that PAR carrier apoptotic fragment PARP1ΔZnF1-2 gets cleaved from PARP1 to facilitate apoptosis, leaving behind the DNA-bound ZnF1-ZnF2PARP1 . Our studies demonstrate that the PARP1ΔZnF1-2 is competent for ssDNA-dependent stimulation only in the presence of another apoptotic fragment ZnF1-ZnF2PARP1 , suggesting the indispensability of DNA-bound ZnF1-ZnF2PARP1 dual domains for the same.