Publications by authors named "Aswin Mangerich"

52 Publications

The C-Terminal Domain of Y-Box Binding Protein 1 Exhibits Structure-Specific Binding to Poly(ADP-Ribose), Which Regulates PARP1 Activity.

Front Cell Dev Biol 2022 21;10:831741. Epub 2022 Jun 21.

LBCE, Institute Chemical Biology and Fundamental Medicine (ICBFM), Novosibirsk, Russia.

Y-box-binding protein 1 (YB-1) is a multifunctional protein involved in the regulation of gene expression. Recent studies showed that in addition to its role in the RNA and DNA metabolism, YB-1 is involved in the regulation of PARP1 activity, which catalyzes poly(ADP-ribose) [PAR] synthesis under genotoxic stress through auto-poly(ADP-ribosyl)ation or protein trans-poly(ADP-ribosyl)ation. Nonetheless, the exact mechanism by which YB-1 regulates PAR synthesis remains to be determined. YB-1 contains a disordered Ala/Pro-rich N-terminal domain, a cold shock domain, and an intrinsically disordered C-terminal domain (CTD) carrying four clusters of positively charged amino acid residues. Here, we examined the functional role of the disordered CTD of YB-1 in PAR binding and in the regulation of PARP1-driven PAR synthesis . We demonstrated that the rate of PARP1-dependent synthesis of PAR is higher in the presence of YB-1 and is tightly controlled by the interaction between YB-1 CTD and PAR. Moreover, YB-1 acts as an effective cofactor in the PAR synthesis catalyzed by the PARP1 point mutants that generate various PAR polymeric structures, namely, short hypo- or hyperbranched polymers. We showed that either a decrease in chain length or an increase in branching frequency of PAR affect its binding affinity for YB-1 and YB-1-mediated stimulation of PARP1 enzymatic activity. These results provide important insight into the mechanism underlying the regulation of PARP1 activity by PAR-binding proteins containing disordered regions with clusters of positively charged amino acid residues, suggesting that YB-1 CTD-like domains may be considered PAR "readers" just as other known PAR-binding modules.
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http://dx.doi.org/10.3389/fcell.2022.831741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9253770PMC
June 2022

PARP1 and XRCC1 exhibit a reciprocal relationship in genotoxic stress response.

Cell Biol Toxicol 2022 Jul 1. Epub 2022 Jul 1.

Molecular Toxicology Group, Department of Biology, University of Konstanz, 78457, Constance, Germany.

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.
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http://dx.doi.org/10.1007/s10565-022-09739-9DOI Listing
July 2022

Fueling genome maintenance: On the versatile roles of NAD in preserving DNA integrity.

J Biol Chem 2022 06 17;298(6):102037. Epub 2022 May 17.

Molecular Toxicology Group, Department of Biology, University of Konstanz, Konstanz, Germany. Electronic address:

NAD is a versatile biomolecule acting as a master regulator and substrate in various cellular processes, including redox regulation, metabolism, and various signaling pathways. In this article, we concisely and critically review the role of NAD in mechanisms promoting genome maintenance. Numerous NAD-dependent reactions are involved in the preservation of genome stability, the cellular DNA damage response, and other pathways regulating nucleic acid metabolism, such as gene expression and cell proliferation pathways. Of note, NAD serves as a substrate to ADP-ribosyltransferases, sirtuins, and potentially also eukaryotic DNA ligases, all of which regulate various aspects of DNA integrity, damage repair, and gene expression. Finally, we critically analyze recent developments in the field as well as discuss challenges associated with therapeutic actions intended to raise NAD levels.
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http://dx.doi.org/10.1016/j.jbc.2022.102037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9194868PMC
June 2022

ADP-ribosyltransferases, an update on function and nomenclature.

FEBS J 2021 Jul 29. Epub 2021 Jul 29.

Institute of Basic Medical Sciences, University of Oslo, Norway.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.
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http://dx.doi.org/10.1111/febs.16142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9027952PMC
July 2021

Mechanistic insights into the three steps of poly(ADP-ribosylation) reversal.

Nat Commun 2021 07 28;12(1):4581. Epub 2021 Jul 28.

Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.
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http://dx.doi.org/10.1038/s41467-021-24723-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8319183PMC
July 2021

Why structure and chain length matter: on the biological significance underlying the structural heterogeneity of poly(ADP-ribose).

Nucleic Acids Res 2021 09;49(15):8432-8448

Department of Biology, University of Konstanz, 78467 Konstanz, Germany.

Poly(ADP-ribosyl)ation (PARylation) is a multifaceted post-translational modification, carried out by poly(ADP-ribosyl)transferases (poly-ARTs, PARPs), which play essential roles in (patho-) physiology, as well as cancer therapy. Using NAD+ as a substrate, acceptors, such as proteins and nucleic acids, can be modified with either single ADP-ribose units or polymers, varying considerably in length and branching. Recently, the importance of PAR structural heterogeneity with regards to chain length and branching came into focus. Here, we provide a concise overview on the current knowledge of the biochemical and physiological significance of such differently structured PAR. There is increasing evidence revealing that PAR's structural diversity influences the binding characteristics of its readers, PAR catabolism, and the dynamics of biomolecular condensates. Thereby, it shapes various cellular processes, such as DNA damage response and cell cycle regulation. Contrary to the knowledge on the consequences of PAR's structural diversity, insight into its determinants is just emerging, pointing to specific roles of different PARP members and accessory factors. In the future, it will be interesting to study the interplay with other post-translational modifications, the contribution of natural PARP variants, and the regulatory role of accessory molecules. This has the exciting potential for new therapeutic approaches, with the targeted modulation and tuning of PARPs' enzymatic functions, rather than their complete inhibition, as a central premise.
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http://dx.doi.org/10.1093/nar/gkab618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8421145PMC
September 2021

Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.

Mol Cell 2021 06 20;81(12):2640-2655.e8. Epub 2021 May 20.

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK. Electronic address:

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.
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http://dx.doi.org/10.1016/j.molcel.2021.04.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221567PMC
June 2021

Impact of the Cellular Zinc Status on PARP-1 Activity and Genomic Stability in HeLa S3 Cells.

Chem Res Toxicol 2021 03 1;34(3):839-848. Epub 2021 Mar 1.

Department of Food Chemistry and Toxicology, Institute of Applied Biosciences (IAB), Karlsruhe Institute of Technology (KIT), Adenauerring 20a, 76131 Karlsruhe, Germany.

Poly(ADP-ribose) polymerase 1 (PARP-1) is actively involved in several DNA repair pathways, especially in the detection of DNA lesions and DNA damage signaling. However, the mechanisms of PARP-1 activation are not fully understood. PARP-1 contains three zinc finger structures, among which the first zinc finger has a remarkably low affinity toward zinc ions. Within the present study, we investigated the impact of the cellular zinc status on PARP-1 activity and on genomic stability in HeLa S3 cells. Significant impairment of HO-induced poly(ADP-ribosyl)ation and an increase in DNA strand breaks were detected in the case of zinc depletion by the zinc chelator -tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) which reduced the total and labile zinc concentrations. On the contrary, preincubation of cells with ZnCl led to an overload of total as well as labile zinc and resulted in an increased poly(ADP-ribosyl)ation response upon HO treatment. Furthermore, the impact of the cellular zinc status on gene expression profiles was investigated via high-throughput RT-qPCR, analyzing 95 genes related to metal homeostasis, DNA damage and oxidative stress response, cell cycle regulation and proliferation. Genes encoding metallothioneins responded most sensitively on conditions of mild zinc depletion or moderate zinc overload. Zinc depletion induced by higher concentrations of TPEN led to a significant induction of genes encoding DNA repair factors and cell cycle arrest, indicating the induction of DNA damage and genomic instability. Zinc overload provoked an up-regulation of the oxidative stress response. Altogether, the results highlight the potential role of zinc signaling for PARP-1 activation and the maintenance of genomic stability.
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http://dx.doi.org/10.1021/acs.chemrestox.0c00452DOI Listing
March 2021

Chronic senescent human mesenchymal stem cells as possible contributor to the wound healing disorder after exposure to the alkylating agent sulfur mustard.

Arch Toxicol 2021 02 25;95(2):727-747. Epub 2021 Jan 25.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937, Munich, Germany.

Wound healing is a complex process, and disturbance of even a single mechanism can result in chronic ulcers developing after exposure to the alkylating agent sulfur mustard (SM). A possible contributor may be SM-induced chronic senescent mesenchymal stem cells (MSCs), unable to fulfil their regenerative role, by persisting over long time periods and creating a proinflammatory microenvironment. Here we show that senescence induction in human bone marrow derived MSCs was time- and concentration-dependent, and chronic senescence could be verified 3 weeks after exposure to between 10 and 40 µM SM. Morphological changes, reduced clonogenic and migration potential, longer scratch closure times, differences in senescence, motility and DNA damage response associated genes as well as increased levels of proinflammatory cytokines were revealed. Selective removal of these cells by senolytic drugs, in which ABT-263 showed initial potential in vitro, opens the possibility for an innovative treatment strategy for chronic wounds, but also tumors and age-related diseases.
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http://dx.doi.org/10.1007/s00204-020-02946-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870771PMC
February 2021

A Multi-Endpoint Approach to Base Excision Repair Incision Activity Augmented by PARylation and DNA Damage Levels in Mice: Impact of Sex and Age.

Int J Mol Sci 2020 Sep 9;21(18). Epub 2020 Sep 9.

Department of Food Chemistry, Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany.

Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.
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http://dx.doi.org/10.3390/ijms21186600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555950PMC
September 2020

PARP1 catalytic variants reveal branching and chain length-specific functions of poly(ADP-ribose) in cellular physiology and stress response.

Nucleic Acids Res 2020 10;48(18):10015-10033

Department of Biology, University of Konstanz, 78457 Konstanz, Germany.

Poly(ADP-ribosyl)ation regulates numerous cellular processes like genome maintenance and cell death, thus providing protective functions but also contributing to several pathological conditions. Poly(ADP-ribose) (PAR) molecules exhibit a remarkable heterogeneity in chain lengths and branching frequencies, but the biological significance of this is basically unknown. To unravel structure-specific functions of PAR, we used PARP1 mutants producing PAR of different qualities, i.e. short and hypobranched (PARP1\G972R), short and moderately hyperbranched (PARP1\Y986S), or strongly hyperbranched PAR (PARP1\Y986H). By reconstituting HeLa PARP1 knockout cells, we demonstrate that PARP1\G972R negatively affects cellular endpoints, such as viability, cell cycle progression and genotoxic stress resistance. In contrast, PARP1\Y986S elicits only mild effects, suggesting that PAR branching compensates for short polymer length. Interestingly, PARP1\Y986H exhibits moderate beneficial effects on cell physiology. Furthermore, different PARP1 mutants have distinct effects on molecular processes, such as gene expression and protein localization dynamics of PARP1 itself, and of its downstream factor XRCC1. Finally, the biological relevance of PAR branching is emphasized by the fact that branching frequencies vary considerably during different phases of the DNA damage-induced PARylation reaction and between different mouse tissues. Taken together, this study reveals that PAR branching and chain length essentially affect cellular functions, which further supports the notion of a 'PAR code'.
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http://dx.doi.org/10.1093/nar/gkaa590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544232PMC
October 2020

The Nucleolus and PARP1 in Cancer Biology.

Cancers (Basel) 2020 Jul 6;12(7). Epub 2020 Jul 6.

Molecular Toxicology Group, Department of Biology, 78457 Konstanz, Germany.

The nucleolus has been known for a long time to fulfill crucial functions in ribosome biogenesis, of which cancer cells can become addicted to in order to produce sufficient amounts of proteins for cell proliferation. Recently, the nucleolus has emerged as a central regulatory hub in many other cancer-relevant processes, including stress sensing, DNA damage response, cell cycle control, and proteostasis. This fostered the idea that nucleolar processes can be exploited in cancer therapy. Interestingly, a significant proportion of poly(ADP-ribose) polymerase 1 (PARP1) molecules are localized in the nucleolus and PARP1 also plays crucial roles in many processes that are important in cancer biology, including genome maintenance, replication, transcription, and chromatin remodeling. Furthermore, during the last years, PARP1 came into focus in oncology since it represents a promising target of pharmacological PARP inhibitors in various types of cancers. Here, we provide an overview of our current understanding on the role of PARP1 in nucleolar functions and discuss potential implications in cancer biology and therapy.
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http://dx.doi.org/10.3390/cancers12071813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408768PMC
July 2020

Long-term simulation of lead concentrations in agricultural soils in relation to human adverse health effects.

Arch Toxicol 2020 07 5;94(7):2319-2329. Epub 2020 May 5.

Niedersächsisches Landesgesundheitsamt, 30449, Hannover, Germany.

Lead (Pb) exposure of consumers and the environment has been reduced over the past decades. Despite all measures taken, immission of Pb onto agricultural soils still occurs, with fertilizer application, lead shot from hunting activities, and Pb from air deposition representing major sources. Little is known about the intermediate and long-term consequences of these emissions. To gain more insight, we established a mathematical model that considers input from fertilizer, ammunition, deposition from air, uptake of Pb by crops, and wash-out to simulate the resulting Pb concentrations in soil over extended periods. In a further step, human oral exposure by crop-based food was simulated and blood concentrations were derived to estimate the margin of exposure to Pb-induced toxic effects. Simulating current farming scenarios, a new equilibrium concentration of Pb in soil would be established after several centuries. Developmental neurotoxicity represents the most critical toxicological effect of Pb for humans. According to our model, a Pb concentration of ~ 5 mg/kg in agricultural soil leads to an intake of approximately 10 µg Pb per person per day by the consumption of agricultural products, the dose corresponding to the tolerable daily intake (TDI). Therefore, 5 mg Pb/kg represents a critical concentration in soil that should not be exceeded. Starting with a soil concentration of 0.1 mg/kg, the current control level for crop fields, our simulation predicts periods of ~ 50 and ~ 175 years for two Pb immission scenarios for mass of Pb per area and year [scenario 1: ~ 400 g Pb/(ha × a); scenario 2: ~ 175 g Pb/(ha × a)], until the critical concentration of ~ 5 mg/kg Pb in soil would be reached. The two scenarios, which differ in their Pb input via fertilizer, represent relatively high but not unrealistic Pb immissions. From these scenarios, we calculated that the annual deposition of Pb onto soil should remain below ~ 100 g/(ha × a) in order not to exceed the critical soil level of 5 mg/kg. We propose as efficient measures to reduce Pb input into agricultural soil to lower the Pb content of compost and to use alternatives to Pb ammunition for hunting.
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http://dx.doi.org/10.1007/s00204-020-02762-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367917PMC
July 2020

Real-time monitoring of PARP1-dependent PARylation by ATR-FTIR spectroscopy.

Nat Commun 2020 05 1;11(1):2174. Epub 2020 May 1.

Department of Biology, University of Konstanz, Konstanz, 78464, Germany.

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3' phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.
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http://dx.doi.org/10.1038/s41467-020-15858-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195430PMC
May 2020

NAD in sulfur mustard toxicity.

Toxicol Lett 2020 May 1;324:95-103. Epub 2020 Feb 1.

Molecular Toxicology Group, Department of Biology, University of Konstanz, 78457, Konstanz, Germany. Electronic address:

Sulfur mustard (SM) is a toxicant and chemical warfare agent with strong vesicant properties. The mechanisms behind SM-induced toxicity are not fully understood and no antidote or effective therapy against SM exists. Both, the risk of SM release in asymmetric conflicts or terrorist attacks and the usage of SM-derived nitrogen mustards as cancer chemotherapeutics, render the mechanisms of mustard-induced toxicity a highly relevant research subject. Herein, we review a central role of the abundant cellular molecule nicotinamide adenine dinucleotide (NAD) in molecular mechanisms underlying SM toxicity. We also discuss the potential beneficial effects of NAD precursors in counteracting SM-induced damage.
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http://dx.doi.org/10.1016/j.toxlet.2020.01.024DOI Listing
May 2020

NAD augmentation restores mitophagy and limits accelerated aging in Werner syndrome.

Nat Commun 2019 11 21;10(1):5284. Epub 2019 Nov 21.

Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD, 21224, USA.

Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. Here we report impaired mitophagy and depletion of NAD, a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD repletion restores NAD metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD levels counteracts WS phenotypes.
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http://dx.doi.org/10.1038/s41467-019-13172-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872719PMC
November 2019

The role of poly(ADP-ribose) polymerases in manganese exposed Caenorhabditis elegans.

J Trace Elem Med Biol 2020 Jan 14;57:21-27. Epub 2019 Sep 14.

Department of Food Chemistry, Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany; TraceAge - DFG Research Unit FOR 2558, Berlin-Potsdam, Jena, Germany; Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Gaußstraße 20, 42119 Wuppertal, Germany. Electronic address:

Background And Aim: 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.

Methods: 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.

Results And Conclusion: 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.
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http://dx.doi.org/10.1016/j.jtemb.2019.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6878993PMC
January 2020

PARP1 regulates DNA damage-induced nucleolar-nucleoplasmic shuttling of WRN and XRCC1 in a toxicant and protein-specific manner.

Sci Rep 2019 07 11;9(1):10075. Epub 2019 Jul 11.

Molecular Toxicology Group, Department of Biology, University of Konstanz, Konstanz, Germany.

The prime function of nucleoli is ribogenesis, however, several other, non-canonical functions have recently been identified, including a role in genotoxic stress response. Upon DNA damage, numerous proteins shuttle dynamically between the nucleolus and the nucleoplasm, yet the underlying molecular mechanisms are incompletely understood. Here, we demonstrate that PARP1 and PARylation contribute to genotoxic stress-induced nucleolar-nucleoplasmic shuttling of key genome maintenance factors in HeLa cells. Our work revealed that the RECQ helicase, WRN, translocates from nucleoli to the nucleoplasm upon treatment with the oxidizing agent HO, the alkylating agent 2-chloroethyl ethyl sulfide (CEES), and the topoisomerase inhibitor camptothecin (CPT). We show that after treatment with HO and CEES, but not CPT, WRN translocation was dependent on PARP1 protein, yet independent of its enzymatic activity. In contrast, nucleolar-nucleoplasmic translocation of the base excision repair protein, XRCC1, was dependent on both PARP1 protein and its enzymatic activity. Furthermore, gossypol, which inhibits PARP1 activity by disruption of PARP1-protein interactions, abolishes nucleolar-nucleoplasmic shuttling of WRN, XRCC1 and PARP1, indicating the involvement of further upstream factors. In conclusion, this study highlights a prominent role of PARP1 in the DNA damage-induced nucleolar-nucleoplasmic shuttling of genome maintenance factors in HeLa cells in a toxicant and protein-specific manner.
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http://dx.doi.org/10.1038/s41598-019-46358-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624289PMC
July 2019

Restriction of AID activity and somatic hypermutation by PARP-1.

Nucleic Acids Res 2019 08;47(14):7418-7429

Department of Cell Biology, Institute of Biochemistry and Biophysics, School of Biology and Pharmacy, Friedrich Schiller University, 07745 Jena, Germany.

Affinity maturation of the humoral immune response depends on somatic hypermutation (SHM) of immunoglobulin (Ig) genes, which is initiated by targeted lesion introduction by activation-induced deaminase (AID), followed by error-prone DNA repair. Stringent regulation of this process is essential to prevent genetic instability, but no negative feedback control has been identified to date. Here we show that poly(ADP-ribose) polymerase-1 (PARP-1) is a key factor restricting AID activity during somatic hypermutation. Poly(ADP-ribose) (PAR) chains formed at DNA breaks trigger AID-PAR association, thus preventing excessive DNA damage induction at sites of AID action. Accordingly, AID activity and somatic hypermutation at the Ig variable region is decreased by PARP-1 activity. In addition, PARP-1 regulates DNA lesion processing by affecting strand biased A:T mutagenesis. Our study establishes a novel function of the ancestral genome maintenance factor PARP-1 as a critical local feedback regulator of both AID activity and DNA repair during Ig gene diversification.
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http://dx.doi.org/10.1093/nar/gkz466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698665PMC
August 2019

Comparison of Aristolochic acid I derived DNA adduct levels in human renal toxicity models.

Toxicology 2019 05 30;420:29-38. Epub 2019 Mar 30.

Human and Environmental Toxicology, University of Konstanz, Konstanz, Germany. Electronic address:

Aristolochic acid (AA) dependent human nephropathy results either from environmental exposure to Aristolochiaceae plant subspecies or their use in traditional phytotherapy. The toxic components are structurally related nitrophenanthrene carboxylic acids, i.e. Aristolochic acid I (AAI) and II (AAII). AAI is considered to be the major cause of Aristolochic acid nephropathy, characterized by severe renal fibrosis and upper urothelial cancer. Following enzymatic activation in kidney and/or liver, AAI metabolites react with genomic DNA to form persistent DNA adducts with purines. To determine whether AAI can be activated in human renal cells to form DNA adducts, we exposed telomerase immortalized renal proximal tubular epithelial cells (RPTEC/TERT1), the human embryonic kidney (HEK293) cell line, as well as primary human kidney cells (pHKC) to AAI in vitro. We modified an isotope dilution ultra-performance liquid chromatography/tandem mass spectrometry (ID-UPLC-MS/MS) based method for the quantification of dA-AAI adducts in genomic DNA. In addition, time dependent accumulation of adducts in renal cortex and bladder tissue from AAI/II treated Eker rats were used to validate the detection method. AAI-induced toxicity in human renal cells was determined by dA-AAI adduct quantification, the impact on cell viability, and NQO1 expression and activity. Our findings demonstrated adduct formation in all cell lines, although only pHKC and RPTEC/TERT1 expressed NQO1. The highest adduct formation was detected in pHKC despite low NQO1 expression, while we observed much lower adduct levels in NQO1-negative HEK293 cells. Adduct formation and decreased cell viability correlated only weakly. Therefore, our data suggested that i.) enzymes other than NQO1 could be at least equally important for AA bioactivation in human renal proximal tubule cells, and ii.) the suggested correlation between adduct levels and viability appears to be questionable.
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http://dx.doi.org/10.1016/j.tox.2019.03.013DOI Listing
May 2019

Interactions of p53 with poly(ADP-ribose) and DNA induce distinct changes in protein structure as revealed by ATR-FTIR spectroscopy.

Nucleic Acids Res 2019 05;47(9):4843-4858

Department of Biology, University of Konstanz, Konstanz 78464, Germany.

Due to multiple domains and in part intrinsically disordered regions, structural analyses of p53 remain a challenging task, particularly in complex with DNA and other macromolecules. Here, we applied a novel attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic approach to investigate changes in secondary structure of full-length p53 induced by non-covalent interactions with DNA and poly(ADP-ribose) (PAR). To validate our approach, we confirmed a positive regulatory function of p53's C-terminal domain (CTD) with regard to sequence-specific DNA binding and verified that the CTD mediates p53-PAR interaction. Further, we demonstrate that DNA and PAR interactions result in distinct structural changes of p53, indicating specific binding mechanisms via different domains. A time-dependent analysis of the interplay of DNA and PAR binding to p53 revealed that PAR represents p53's preferred binding partner, which efficiently controls p53-DNA interaction. Moreover, we provide infrared spectroscopic data on PAR pointing to the absence of regular secondary structural elements. Finally, temperature-induced melting experiments via CD spectroscopy show that DNA binding stabilizes the structure of p53, while PAR binding can shift the irreversible formation of insoluble p53 aggregates to higher temperatures. In conclusion, this study provides detailed insights into the dynamic interplay of p53 binding to DNA and PAR at a formerly inaccessible molecular level.
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http://dx.doi.org/10.1093/nar/gkz175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511852PMC
May 2019

A novel exposure system generating nebulized aerosol of sulfur mustard in comparison to the standard submerse exposure.

Chem Biol Interact 2019 Jan 28;298:121-128. Epub 2018 Nov 28.

Bundeswehr Institute of Pharmacology and Toxicology, 80937, Munich, Germany; Walther Straub Institute of Pharmacology and Toxicology, University of Munich, 80336, Munich, Germany. Electronic address:

Inhalation of the chemical warfare agent sulfur mustard (SM) is associated with severe acute and long-term pulmonary dysfunctions and health effects. The still not completely elucidated molecular toxicology and a missing targeted therapy emphasize the need for further research. However, appropriate human data are extremely rare. In vivo animal experiments are often regarded as gold standard in toxicology but may exhibit significant differences compared to the human pulmonary anatomy and physiology. Thus, alternative in vitro exposure methods, adapted to the human in vivo situation by exposing cells at the air-liquid interface (ALI), are complimentary approaches at a cellular level. So far, it is unclear whether the enhanced experimental complexity of ALI exposure, that is potentially biologically more meaningful, is superior to submerged exposures which are typically performed. Aim of our study was the evaluation of an appropriate in vitro exposure system (CULTEX Radial Flow System (RFS) equipped with an eFlow membrane nebulizer) for the exposure of cultivated human lung cells (A549) with SM under ALI conditions. Cellular responses (i.e. cell viability) and formation of SM-specific DNA-adducts were investigated and compared between ALI and submerse SM exposures. Our results proved the safe applicability of our ALI exposure system setup. The aerosol generation and subsequent deposition at the ALI were stable and uniform. The technical CULTEX RFS setup is based on ALI exposure with excess of aerosol from that only some is deposited on the cell layer. As expected, a lower cytotoxicity and DNA-adduct formation were detected when identical SM concentrations were used compared to experiments under submerged conditions. A distinct advantage of SM-ALI compared to SM-submerse exposures could not be found in our experiments. Though, the CULTEX RFS was found suitable for SM-ALI exposures.
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http://dx.doi.org/10.1016/j.cbi.2018.11.025DOI Listing
January 2019

A mass spectrometric platform for the quantitation of sulfur mustard-induced nucleic acid adducts as mechanistically relevant biomarkers of exposure.

Arch Toxicol 2019 01 15;93(1):61-79. Epub 2018 Oct 15.

Molecular Toxicology, Department of Biology, University of Konstanz, 78457, Konstanz, Germany.

Despite its worldwide ban, the warfare agent sulfur mustard (SM) still represents a realistic threat, due to potential release in terroristic attacks and asymmetric conflicts. Therefore, the rigorous and quantitative detection of SM exposure is crucial for diagnosis, health risk assessment, and surveillance of international law. Alkylation adducts of nucleic acids can serve as valuable toxicologically relevant 'biomarkers of SM exposure'. Here, we developed a robust and versatile bioanalytical platform based on isotope dilution UPLC-MS/MS to quantify major SM-induced DNA and RNA adducts, as well as adducts induced by the monofunctional mustard 2-chloroethyl ethyl sulfide. We synthesized N/C-labeled standards, which allowed absolute quantitation with full chemical specificity and subfemtomole sensitivities. DNA and RNA mono-alkylation adducts and crosslinks were carefully analyzed in a dose- and time-dependent manner in various matrices, including human cancer and primary cells, derived of the main SM-target tissues. Nucleic acid adducts were detected up to 6 days post-exposure, indicating long persistence, which highlights their toxicological relevance and proves their suitability as forensic and medical biomarkers. Finally, we investigated ex vivo-treated rat skin biopsies and human blood samples, which set the basis for the implementation into the method portfolio of Organization for the Prohibition of Chemical Weapons-designated laboratories to analyze authentic samples from SM-exposed victims.
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http://dx.doi.org/10.1007/s00204-018-2324-7DOI Listing
January 2019

PARP-1 protects against colorectal tumor induction, but promotes inflammation-driven colorectal tumor progression.

Proc Natl Acad Sci U S A 2018 04 9;115(17):E4061-E4070. Epub 2018 Apr 9.

Department of Toxicology, University Medical Center Mainz, 55131 Mainz, Germany;

Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1 mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1 animals were then crossed with -methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1/MGMT double knockout (DKO) mice developed more, but much smaller tumors than MGMT animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar -methylguanine levels. Studies with PARP-1 cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to -methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.
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http://dx.doi.org/10.1073/pnas.1712345115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924876PMC
April 2018

Mass spectrometric analysis of sulfur mustard-induced biomolecular adducts: Are DNA adducts suitable biomarkers of exposure?

Toxicol Lett 2018 Sep 23;293:21-30. Epub 2017 Dec 23.

Molecular Toxicology Group, Department of Biology, University of Konstanz, 78457, Konstanz, Germany. Electronic address:

The bi-functional chemical warfare agent sulfur mustard (SM), whose release in asymmetric conflicts or terrorist attacks represents a realistic threat, induces several kinds of biomolecular adducts, including highly toxic DNA adducts. Isotope dilution liquid chromatographic tandem mass spectrometry (ID-LC-MS/MS) is considered the gold standard for highly accurate, precise, specific and sensitive quantification of DNA adducts in general. Recently, a number of LC-MS/MS approaches have been established to analyze SM-induced protein and DNA adducts in cell culture and rodent animal models. As DNA adducts are mechanism-based biomarkers for SM exposure, results from such studies provide a deeper understanding of the etiology of SM-induced pathologies, especially of long-term effects such as cancer formation. As a result, medical treatment of SM-exposed individuals might be improved. Yet, despite the progress that has been made during the last years, there is still a need for advanced methods of ID-LC-MS/MS for the detection and quantitation of SM adducts.
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http://dx.doi.org/10.1016/j.toxlet.2017.12.014DOI Listing
September 2018

The C-terminal domain of p53 orchestrates the interplay between non-covalent and covalent poly(ADP-ribosyl)ation of p53 by PARP1.

Nucleic Acids Res 2018 01;46(2):804-822

Department of Biology, University of Konstanz, 78457 Konstanz, Germany.

The post-translational modification poly(ADP-ribosyl)ation (PARylation) plays key roles in genome maintenance and transcription. Both non-covalent poly(ADP-ribose) binding and covalent PARylation control protein functions, however, it is unknown how the two modes of modification crosstalk mechanistically. Employing the tumor suppressor p53 as a model substrate, this study provides detailed insights into the interplay between non-covalent and covalent PARylation and unravels its functional significance in the regulation of p53. We reveal that the multifunctional C-terminal domain (CTD) of p53 acts as the central hub in the PARylation-dependent regulation of p53. Specifically, p53 bound to auto-PARylated PARP1 via highly specific non-covalent PAR-CTD interaction, which conveyed target specificity for its covalent PARylation by PARP1. Strikingly, fusing the p53-CTD to a protein that is normally not PARylated, renders this a target for covalent PARylation as well. Functional studies revealed that the p53-PAR interaction had substantial implications on molecular and cellular levels. Thus, PAR significantly influenced the complex p53-DNA binding properties and controlled p53 functions, with major implications on the p53-dependent interactome, transcription, and replication-associated recombination. Remarkably, this mechanism potentially also applies to other PARylation targets, since a bioinformatics analysis revealed that CTD-like regions are highly enriched in the PARylated proteome.
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http://dx.doi.org/10.1093/nar/gkx1205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5778597PMC
January 2018

PARP1 protects from benzo[a]pyrene diol epoxide-induced replication stress and mutagenicity.

Arch Toxicol 2018 Mar 1;92(3):1323-1340. Epub 2017 Dec 1.

Molecular Toxicology Group, Department of Biology, University of Konstanz, 78457, Konstanz, Germany.

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.
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http://dx.doi.org/10.1007/s00204-017-2115-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866831PMC
March 2018

Kinetics of poly(ADP-ribosyl)ation, but not PARP1 itself, determines the cell fate in response to DNA damage in vitro and in vivo.

Nucleic Acids Res 2017 Nov;45(19):11174-11192

Leibniz Institute on Aging - Fritz-Lipmann Institute (FLI), Beutenbergstr. 11, 07745 Jena, Germany.

One of the fastest cellular responses to genotoxic stress is the formation of poly(ADP-ribose) polymers (PAR) by poly(ADP-ribose)polymerase 1 (PARP1, or ARTD1). PARP1 and its enzymatic product PAR regulate diverse biological processes, such as DNA repair, chromatin remodeling, transcription and cell death. However, the inter-dependent function of the PARP1 protein and its enzymatic activity clouds the mechanism underlying the biological response. We generated a PARP1 knock-in mouse model carrying a point mutation in the catalytic domain of PARP1 (D993A), which impairs the kinetics of the PARP1 activity and the PAR chain complexity in vitro and in vivo, designated as hypo-PARylation. PARP1D993A/D993A mice and cells are viable and show no obvious abnormalities. Despite a mild defect in base excision repair (BER), this hypo-PARylation compromises the DNA damage response during DNA replication, leading to cell death or senescence. Strikingly, PARP1D993A/D993A mice are hypersensitive to alkylation in vivo, phenocopying the phenotype of PARP1 knockout mice. Our study thus unravels a novel regulatory mechanism, which could not be revealed by classical loss-of-function studies, on how PAR homeostasis, but not the PARP1 protein, protects cells and organisms from acute DNA damage.
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http://dx.doi.org/10.1093/nar/gkx717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737718PMC
November 2017

Quantitation of Poly(ADP-Ribose) by Isotope Dilution Mass Spectrometry.

Methods Mol Biol 2017 ;1608:3-18

Molecular Toxicology Group, Department of Biology, University of Konstanz, 78457, Konstanz, Germany.

Poly(ADP-ribosyl)ation (PARylation), i.e., the formation of the nucleic acid-like biopolymer poly(ADP-ribose) (PAR), is an essential posttranslational modification carried out by poly(ADP-ribose) polymerases (PARPs). While PAR levels are low under physiological conditions, they can transiently increase more than 100-fold upon induction of genotoxic stress. The accurate quantitation of cellular PAR with high sensitivity is of critical importance to understand the role of PARylation in cellular physiology and pathophysiology and to determine the pharmacodynamic efficiencies of clinically relevant PARP inhibitors, which represent a novel class of promising chemotherapeutics. Previously, we have developed a bioanalytical platform based on isotope dilution mass spectrometry (LC-MS/MS) to quantify cellular PAR with unequivocal chemical specificity in absolute terms with femtomol sensitivity (Martello et al. ACS Chem Biol 8(7):1567-1575, 2013). This method enables the analysis of steady-state levels, as well as stress-induced levels of PAR in various biological systems including cell lines, mouse tissues, and primary human lymphocytes. It has a wide range of potential applications in basic research, as well as in drug development (Martello et al. ACS Chem Biol 8(7):1567-1575, 2013; Mangerich et al. Toxicol Lett 244:56-71, 2016). Here, we present an improved and adjusted version of the original protocol by Martello/Mangerich et al., which uses UPLC-MS/MS instrumentation.
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http://dx.doi.org/10.1007/978-1-4939-6993-7_1DOI Listing
April 2018

Analyzing structure-function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells.

Nucleic Acids Res 2016 Dec 29;44(21):10386-10405. Epub 2016 Sep 29.

Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure-function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1-PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\L713F expression triggered apoptosis, whereas PARP1\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure-function relationships of natural and artificial PARP1 variants.
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http://dx.doi.org/10.1093/nar/gkw859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137445PMC
December 2016
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