ABSTRACT
Studies on the molecular mechanisms of heavy metal toxicity in invertebrate reproduction are limited. Given that PARP-catalysed ADP-ribosylation is also involved in counteracting heavy metal toxicity and maintaining genomic integrity, and that PARylation is implicated in chromatin remodelling but its role in sperm chromatin remains to be elucidated, we investigated the effects of chromium(VI) at 1, 10 and 100 nM on the reproductive health of Mytilus galloprovincialis. The damage to the gonads was assessed by morphological analyses and the damage indices PARP and ɣH2A.X were measured. Changes in the binding of protamine-like (PL) to DNA and the possibility of poly(ADP-ribosyl)ation of PL proteins were also investigated. Gonadal chromium accumulation and morphological damage were found, especially when the mussels were exposed to the highest dose of chromium(VI). In addition, the maximum expression of gonadal ɣH2A.X and PARP were obtained at 100 and 10 nM Cr(VI), respectively. Interestingly, for the first time in all exposed conditions, poly(ADP)-ribosylation was detected on PL-II, which, together with PL-III and PL-IV, are the major nuclear basic proteins of Mytilus galloprovincialis sperm chromatin. Since PL-II is involved in the final high level of sperm chromatin compaction, this post-translational modification altered the binding of the PL protein to DNA, favouring the action of micrococcal nuclease on sperm chromatin. This study provides new insights into the effects of chromium(VI) on Mytilus galloprovincialis reproductive system and proposes a molecular mechanism hypothesis describing the toxic effects of this metal on PL-DNA binding, sperm chromatin and gonads.
Subject(s)
Chromium , Mytilus , Protamines , Animals , Mytilus/drug effects , Mytilus/metabolism , Male , Chromium/toxicity , Protamines/metabolism , Poly ADP Ribosylation/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Histones/metabolism , Gonads/drug effects , Gonads/metabolism , Spermatozoa/drug effects , Spermatozoa/metabolism , Reproduction/drug effects , DNA/metabolism , DNA/drug effectsABSTRACT
DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.
Subject(s)
DNA Repair , DNA Replication , DNA Topoisomerases, Type I , Poly (ADP-Ribose) Polymerase-1 , Poly ADP Ribosylation , Animals , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , DNA Topoisomerases, Type I/metabolism , Xenopus laevis , Ubiquitination , Humans , DNA/metabolism , DNA Damage , Camptothecin/pharmacology , Protein Processing, Post-Translational , DNA, Single-Stranded/metabolism , Xenopus Proteins/metabolismABSTRACT
ADP-ribosylation is a prominent and versatile post-translational modification, which regulates a diverse set of cellular processes. Poly-ADP-ribose (PAR) is synthesised by the poly-ADP-ribosyltransferases PARP1, PARP2, tankyrase (TNKS), and tankyrase 2 (TNKS2), all of which are linked to human disease. PARP1/2 inhibitors have entered the clinic to target cancers with deficiencies in DNA damage repair. Conversely, tankyrase inhibitors have continued to face obstacles on their way to clinical use, largely owing to our limited knowledge of their molecular impacts on tankyrase and effector pathways, and linked concerns around their tolerability. Whilst detailed structure-function studies have revealed a comprehensive picture of PARP1/2 regulation, our mechanistic understanding of the tankyrases lags behind, and thereby our appreciation of the molecular consequences of tankyrase inhibition. Despite large differences in their architecture and cellular contexts, recent structure-function work has revealed striking parallels in the regulatory principles that govern these enzymes. This includes low basal activity, activation by intra- or inter-molecular assembly, negative feedback regulation by auto-PARylation, and allosteric communication. Here we compare these poly-ADP-ribosyltransferases and point towards emerging parallels and open questions, whose pursuit will inform future drug development efforts.
Subject(s)
Poly (ADP-Ribose) Polymerase-1 , Tankyrases , Tankyrases/metabolism , Tankyrases/antagonists & inhibitors , Tankyrases/genetics , Tankyrases/chemistry , Humans , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , Poly (ADP-Ribose) Polymerase-1/genetics , Poly(ADP-ribose) Polymerases/metabolism , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/genetics , Animals , Protein Processing, Post-Translational , DNA Repair , ADP-Ribosylation , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly ADP Ribosylation/geneticsABSTRACT
The maintenance of a highly functional metabolic epithelium in vitro is challenging. Metabolic impairments in primary human hepatocytes (PHHs) over time is primarily due to epithelial-to-mesenchymal transitioning (EMT). The immature hepatoma cell line HepG2 was used as an in vitro model to explore strategies for enhancing the hepatic phenotype. The phenotypic characterization includes measuring the urea cycle, lipid storage, tricarboxylic acid-related metabolites, reactive oxygen species, endoplasmic reticulum calcium efflux, mitochondrial membrane potentials, oxygen consumptions rate, and CYP450 biotransformation capacity. Expression studies were performed with transcriptomics, co-immunoprecipitation and proteomics. CRISPR/Cas9 was also employed to genetically engineer HepG2 cells. After confirming that PHHs develop an EMT phenotype, expression of tankyrase1/2 was found to increase over time. EMT was reverted when blocking tankyrases1/2-dependent poly-ADP-ribosylation (PARylation) activity, by biochemical and genetic perturbation. Wnt/ß-catenin inhibitor XAV-939 blocks tankyrase1/2 and treatment elevated several oxygen-consuming reactions (electron-transport chain, OXHPOS, CYP450 mono-oxidase activity, phase I/II xenobiotic biotransformation, and prandial turnover), suggesting that cell metabolism was enhanced. Glutathione-dependent redox homeostasis was also significantly improved in the XAV-939 condition. Oxygen consumption rate and proteomics experiments in tankyrase1/2 double knockout HepG2 cells then uncovered PARylation as master regulator of aerobic-dependent cell respiration. Furthermore, novel tankyrase1/2-dependent PARylation targets, including mitochondrial DLST, and OGDH, were revealed. This work exposed a new mechanistic framework by linking PARylation to respiration and metabolism, thereby broadening the current understanding that underlies these vital processes. XAV-939 poses an immediate and straightforward strategy to improve aerobic activities, and metabolism, in (immature) cell cultures.
Subject(s)
Epithelial-Mesenchymal Transition , Hepatocytes , Tankyrases , Humans , Tankyrases/antagonists & inhibitors , Tankyrases/metabolism , Hep G2 Cells , Hepatocytes/drug effects , Hepatocytes/metabolism , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/physiology , Poly ADP Ribosylation/drug effects , Enzyme Inhibitors/pharmacology , Phenanthrenes/pharmacologyABSTRACT
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
Post-transcriptional regulation of cytokine/chemokine mRNA turnover is critical for immune processes and contributes to the mammalian cellular response to diverse inflammatory stimuli. The ubiquitous RNA-binding protein human antigen R (HuR) is an integral regulator of inflammation-associated mRNA fate. HuR function is regulated by various post-translational modifications that alter its subcellular localization and ability to stabilize target mRNAs. Both poly (ADP-ribose) polymerase 1 (PARP1) and p38 mitogen-activated protein kinases (MAPKs) have been reported to regulate the biological function of HuR, but their specific regulatory and crosstalk mechanisms remain unclear. In this study, we show that PARP1 acts via p38 to synergistically promote cytoplasmic accumulation of HuR and stabilization of inflammation-associated mRNAs in cells under inflammatory conditions. Specifically, p38 binds to auto-poly ADP-ribosylated (PARylated) PARP1 resulting in the covalent PARylation of p38 by PARP1, thereby promoting the retention and activity of p38 in the nucleus. In addition, PARylation of HuR facilitates the phosphorylation of HuR at the serine 197 site mediated by p38, which then increases the translocation of HuR to the cytoplasm, ultimately stabilizing the inflammation-associated mRNA expression at the post-transcriptional level.
Subject(s)
Cytoplasm , ELAV-Like Protein 1 , Inflammation , Poly (ADP-Ribose) Polymerase-1 , RNA, Messenger , p38 Mitogen-Activated Protein Kinases , ELAV-Like Protein 1/metabolism , ELAV-Like Protein 1/genetics , p38 Mitogen-Activated Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/genetics , Humans , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , Cytoplasm/metabolism , Inflammation/metabolism , Inflammation/genetics , Inflammation/pathology , RNA, Messenger/metabolism , RNA, Messenger/genetics , Phosphorylation , Gene Expression Regulation , Animals , Poly ADP Ribosylation/genetics , HEK293 Cells , Cell Nucleus/metabolism , MiceABSTRACT
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-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.
Subject(s)
Muscle Development , Muscle Fibers, Skeletal , Myogenin , RNA, Messenger , Tankyrases , Tankyrases/metabolism , Tankyrases/genetics , Humans , RNA, Messenger/metabolism , RNA, Messenger/genetics , Muscle Development/genetics , Animals , Muscle Fibers, Skeletal/metabolism , Mice , Myogenin/genetics , Myogenin/metabolism , Nucleophosmin , ELAV-Like Protein 1/metabolism , ELAV-Like Protein 1/genetics , RNA Stability/genetics , Poly ADP Ribosylation/genetics , Cell Line , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Cell Differentiation/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , HEK293 CellsABSTRACT
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.
Subject(s)
Poly ADP Ribosylation , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerases/metabolism , DNA Damage , DNA ReplicationABSTRACT
ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents.
Subject(s)
Antineoplastic Agents , Histones , Poly(ADP-ribose) Polymerase Inhibitors , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , DNA Damage , DNA Repair , High-Throughput Screening Assays , Histones/drug effects , Histones/metabolism , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , Poly ADP Ribosylation , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/pharmacologyABSTRACT
Poly (ADP-ribose) polymerase-1 (PARP-1) activity significantly increases during cerebral ischemia/reperfusion. PARP-1 is an NAD+-consumption enzyme. PARP-1 hyperactivity causes intracellular NAD+ deficiency and bioenergetic collapse, contributing to neuronal death. Besides, the powerful trigger of PARP-1 causes the catalyzation of poly (ADP-ribosyl)ation (PARylation), a posttranslational modification of proteins. Here, we found that PARP-1 was activated in the ischemic brain tissue during middle-cerebral-artery occlusion and reperfusion (MCAO/R) for 24 h, and PAR accumulated in the neurons in mice. Using immunoprecipitation, Western blotting, liquid chromatography-mass spectrometry, and 3D-modeling analysis, we revealed that the activation of PARP-1 caused PARylation of hexokinase-1 and lactate dehydrogenase-B, which, therefore, caused the inhibition of these enzyme activities and the resulting cell energy metabolism collapse. PARP-1 inhibition significantly reversed the activity of hexokinase and lactate dehydrogenase, decreased infarct volume, and improved neuronal deficiency. PARP-1 inhibitor combined with pyruvate further alleviated MCAO/R-induced ischemic brain injury in mice. As such, we conclude that PARP-1 inhibitor alleviates neuronal death partly by inhibiting the PARylation of metabolic-related enzymes and reversing metabolism reprogramming during cerebral ischemia/reperfusion injury in mice. PARP-1 inhibitor combined with pyruvate might be a promising therapeutic approach against brain ischemia/reperfusion injury.
Subject(s)
Brain Ischemia , Reperfusion Injury , Mice , Animals , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Poly(ADP-ribose) Polymerases/metabolism , Poly ADP Ribosylation , Hexokinase/metabolism , NAD/metabolism , Reperfusion Injury/drug therapy , Brain Ischemia/drug therapy , Pyruvates , Lactate Dehydrogenases/metabolismABSTRACT
The chromatin remodeler ALC1 is activated by DNA damage-induced poly(ADP-ribose) deposited by PARP1/PARP2 and their co-factor HPF1. ALC1 has emerged as a cancer drug target, but how it is recruited to ADP-ribosylated nucleosomes to affect their positioning near DNA breaks is unknown. Here we find that PARP1/HPF1 preferentially initiates ADP-ribosylation on the histone H2B tail closest to the DNA break. To dissect the consequences of such asymmetry, we generate nucleosomes with a defined ADP-ribosylated H2B tail on one side only. The cryo-electron microscopy structure of ALC1 bound to such an asymmetric nucleosome indicates preferential engagement on one side. Using single-molecule FRET, we demonstrate that this asymmetric recruitment gives rise to directed sliding away from the DNA linker closest to the ADP-ribosylation site. Our data suggest a mechanism by which ALC1 slides nucleosomes away from a DNA break to render it more accessible to repair factors.
Subject(s)
Nucleosomes , Poly ADP Ribosylation , Nucleosomes/genetics , Cryoelectron Microscopy , Poly (ADP-Ribose) Polymerase-1/metabolism , Chromatin , DNA Repair , DNA BreaksABSTRACT
The intracellular ATP-ribosyltransferases PARP1 and PARP2, contribute to DNA base excision repair (BER) and DNA demethylation and have been implicated in epigenetic programming in early mammalian development. Recently, proteomic analyses identified BER proteins to be covalently poly-ADP-ribosylated by PARPs. The role of this posttranslational modification in the BER process is unknown. Here, we show that PARP1 senses AP-sites and SSBs generated during TET-TDG mediated active DNA demethylation and covalently attaches PAR to each BER protein engaged. Covalent PARylation dissociates BER proteins from DNA, which accelerates the completion of the repair process. Consistently, inhibition of PARylation in mESC resulted both in reduced locus-specific TET-TDG-targeted DNA demethylation, and in reduced general repair of random DNA damage. Our findings establish a critical function of covalent protein PARylation in coordinating molecular processes associated with dynamic DNA methylation.
Subject(s)
DNA Repair , Excision Repair , Animals , Poly ADP Ribosylation , DNA Demethylation , Proteomics , Poly (ADP-Ribose) Polymerase-1/metabolism , DNA Damage , DNA/genetics , DNA/metabolism , Mammals/geneticsABSTRACT
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.
Subject(s)
Nucleosomes , Poly ADP Ribosylation , Nucleosomes/genetics , Poly ADP Ribosylation/genetics , Poly(ADP-ribose) Polymerases/metabolism , Cryoelectron Microscopy , Biomolecular Condensates , DNA Repair , Histones/genetics , Histones/metabolism , DNA/genetics , DNA/metabolism , DNA Damage , Poly (ADP-Ribose) Polymerase-1/metabolismABSTRACT
BACKGROUND: Tendon stem/progenitor cells (TSPCs) play a vital role in tendon repair, regeneration and homeostasis. However, the specific mechanism of TSPCs aging is still unclear. OBJECTIVE: This study aims to explore the role and molecular mechanism of HPF1 in the aging of TSPCs. METHODS: Young and aged TSPCs (Y-TSPCs and A-TSPCs) were acquired from 3 to 4 and 24-26-month-old Sprague-Dawley male rats, TSPCs (Y-TSPCs and A-TSPCs) were subjected to senescence-associated ß-galactosidase (SA-ß-Gal))staining and telomerase activity detection, p16, p21, Scx, Tnmd, Col1, Col3HPF1 and PAPR1 expression levels were detected by Western blot or Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), Reciprocal co-immunoprecipitation (co-IP) was used to explore the interaction between HPF1 and PARP1. Ribonucleoprotein immunoprecipitation (RNP-IP) was used to analyze the binding of HuR to the senescence marker gene mRNAs, IP was used to perform HPF1 to the PARylation of HuR, and the half-life of p16 and p21 were detected. Finally, we established an in vivo model, and the tendon tissue was used to perform hematoxylin and eosin (HE) and masson's trichrome staining, as well as the immunohistochemical analysis of Col I and TNMD. RESULTS: Compared with Y-TSPCs, A-TSPCs had significantly enhanced cell senescence and significantly reduced tendon differentiation ability, and significantly increased the expression of HPF1 and PARP1. In addition, HPF1 and PARP1 interacted and coordinated the senescence and differentiation of TSPCs, HPF1 could also regulate the expression of p21 and p21, the interaction of p16 or p21 with HuR, and the poly-ADP ribosylation of PARP1 to HuR. HPF1 overexpression and siHuR co-transfection significantly reduced the half-life of p16 and p21, and HPF1 and PARP1 regulated the mRNA levels of p16 and p21 through HuR. Finally, in vivo experiments have shown that HPF1 or PARP1 overexpression could both inhibit the ability of tendon differentiation and promote cell senescence. CONCLUSIONS: HPF1 promoted the senescence of TSPCs and inhibits the tendon differentiation of TSPCs through PARP1-mediated poly-ADP ribosylation of HuR.
Subject(s)
Cellular Senescence , Poly ADP Ribosylation , Rats , Animals , Male , Rats, Sprague-Dawley , Tendons/metabolism , Stem Cells/metabolismABSTRACT
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
Tankyrases, a versatile protein group within the poly(ADP-ribose) polymerase family, are essential for post-translational poly(ADP-ribosyl)ation, influencing various cellular functions and contributing to diseases, particularly cancer. Consequently, tankyrases have become important targets for anti-cancer drug development. Emerging approaches in drug discovery aim to disrupt interactions between tankyrases and their binding partners, which hinge on tankyrase-binding motifs (TBMs) within partner proteins and ankyrin repeat cluster domains within tankyrases. Our study addresses the challenge of identifying and ranking TBMs. We have conducted a comprehensive review of the existing literature, classifying TBMs into three distinct groups, each with its own scoring system. To facilitate this process, we introduce TBM Hunter-an accessible, web-based tool. This user-friendly platform provides a cost-free and efficient means to screen and assess potential TBMs within any given protein. TBM Hunter can handle individual proteins or lists of proteins simultaneously. Notably, our results demonstrate that TBM Hunter not only identifies known TBMs but also uncovers novel ones. In summary, our study offers an all-encompassing perspective on TBMs and presents an easy-to-use, precise, and free tool for identifying and evaluating potential TBMs in any protein, thereby enhancing research and drug development efforts focused on tankyrases.
Subject(s)
Tankyrases , Tankyrases/metabolism , Ankyrin Repeat , Poly ADP RibosylationABSTRACT
PARP1/2 inhibitors (PARPi) are effective clinically used drugs for the treatment of cancers with BRCA deficiencies. PARPi have had limited success and applicability beyond BRCA deficient cancers, and their effect is diminished by resistance mechanisms. The recent discovery of Histone PARylation Factor (HPF1) and the role it plays in the PARylation reaction by forming a shared active site with PARP1 raises the possibility that novel inhibitors that target the PARP1-HPF1 complex can be identified. Herein we describe a simple and cost-effective high-throughput screening (HTS) method aimed at discovering inhibitors of the PARP1-HPF1 complex. Upon HTS validation, we first applied this method to screen a small PARP-focused library of compounds and then scale up our approach using robotic automation to conduct a pilot screen of 10,000 compounds and validating >100 hits. This work demonstrates for the first time the capacity to discover potent inhibitors of the PARP1-HPF1 complex, which may have utility as probes to better understand the DNA damage response and as therapeutics for cancer.
Subject(s)
Histones , Neoplasms , Poly(ADP-ribose) Polymerase Inhibitors , Humans , Catalytic Domain , Histones/metabolism , Neoplasms/drug therapy , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , Poly ADP Ribosylation , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic useABSTRACT
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
Although only a small number of primordial follicles are known to be selectively activated during female reproductive cycles, the mechanisms that trigger this recruitment remain largely uncharacterized. Misregulated activation of primordial follicles may lead to the exhaustion of the non-renewable pool of primordial follicles, resulting in premature ovarian insufficiency. Here, we found that poly(ADP-ribose) polymerase 1 (PARP1) enzymatic activity in the surrounding granulosa cells (GCs) in follicles determines the subpopulation of the dormant primordial follicles to be awakened. Conversely, specifically inhibiting PARP1 in oocytes in an in vitro mouse follicle reconstitution model does not affect primordial follicle activation. Further analysis revealed that PARP1-catalyzed transcription factor YY1 PARylation at Y185 residue facilitates YY1 occupancy at Grp78 promoter, a key molecular chaperone of endoplasmic reticulum stress (ERS), and promotes Grp78 transcription in GCs, which is required for GCs maintaining proper ERS during primordial follicle activation. Inhibiting PARP1 prevents the loss of primordial follicle pool by attenuating the excessive ERS in GCs under fetal bisphenol A exposure. Together, we demonstrate that PARP1 in GCs acts as a pivotal modulator to determine the fate of the primordial follicles and may represent a novel therapeutic target for the retention of primordial follicle pool in females.