2023-10-02, Ruszkiewicz, Joanna A., Papatheodorou, Ylea, Jäck, Nathalie, Melzig, Jasmin, Eble, Franziska, Pirker, Annika, Thomann, Marius, Haberer, Andreas, Rothmiller, Simone, Bürkle, Alexander, Mangerich, Aswin

Sulfur mustard (SM) and its derivatives are potent genotoxic agents, which have been shown to trigger the activation of poly (ADP-ribose) polymerases (PARPs) and the depletion of their substrate, nicotinamide adenine dinucleotide (NAD⁺). NAD⁺ is an essential molecule involved in numerous cellular pathways, including genome integrity and DNA repair, and thus, NAD⁺ supplementation might be beneficial for mitigating mustard-induced (geno)toxicity. In this study, the role of NAD⁺ depletion and elevation in the genotoxic stress response to SM derivatives, i.e., the monofunctional agent 2-chloroethyl-ethyl sulfide (CEES) and the crosslinking agent mechlorethamine (HN2), was investigated with the use of NAD⁺ booster nicotinamide riboside (NR) and NAD⁺ synthesis inhibitor FK866. The effects were analyzed in immortalized human keratinocytes (HaCaT) or monocyte-like cell line THP-1. In HaCaT cells, NR supplementation, increased NAD⁺ levels, and elevated PAR response, however, did not affect ATP levels or DNA damage repair, nor did it attenuate long- and short-term cytotoxicities. On the other hand, the depletion of cellular NAD⁺ via FK866 sensitized HaCaT cells to genotoxic stress, particularly CEES exposure, whereas NR supplementation, by increasing cellular NAD⁺ levels, rescued the sensitizing FK866 effect. Intriguingly, in THP-1 cells, the NR-induced elevation of cellular NAD⁺ levels did attenuate toxicity of the mustard compounds, especially upon CEES exposure. Together, our results reveal that NAD⁺ is an important molecule in the pathomechanism of SM derivatives, exhibiting compound-specificity. Moreover, the cell line-dependent protective effects of NR are indicative of system-specificity of the application of this NAD⁺ booster.

2022-05-17, Ruszkiewicz, Joanna A., Bürkle, Alexander, Mangerich, Aswin

Nicotinamide adenine dinucleotide (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 (ARTs), sirtuins (SIRTs), 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.

2020-05-01, Krüger, Annika, Bürkle, Alexander, Hauser, Karin, Mangerich, Aswin

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.

2019-12, Veith, Sebastian, Schink, Andrea, Engbrecht, Marina, Mack, Matthias, Rank, Lisa, Rossatti, Pascal, Hakobyan, Mariam, Goly, Denise, Hefele, Tanja, Frensch, Marco, Fischbach, Arthur, Bürkle, Alexander, Mangerich, Aswin

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 H₂O₂, the alkylating agent 2-chloroethyl ethyl sulfide (CEES), and the topoisomerase inhibitor camptothecin (CPT). We show that after treatment with H₂O₂ 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.

2023, Reber, Julia M., Božić-Petković, Jovana, Lippmann, Michelle, Mazzardo, Marvin, Dilger, Asisa, Warmers, Rebecca, Bürkle, Alexander, Mangerich, Aswin

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.

2021-02, Rothmiller, Simone, Jäger, Niklas, Meier, Nicole, Meyer, Thimo, Neu, Adrian, Steinritz, Dirk, Thiermann, Horst, Mangerich, Aswin, Bürkle, Alexander, Schmidt, Annette

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.

2020-05, Ruszkiewicz, Joanna A., Bürkle, Alexander, Mangerich, Aswin

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.

2023, Krassnig, Sarah C., Mäser, Marina, Probst, Nicola Anna, Werner, Jens, Schlett, Charlotte, Schumann, Nina, von Scheven, Gudrun, Mangerich, Aswin, Bürkle, Alexander

Chlorambucil (CLB) belongs to the class of nitrogen mustards (NMs), which are highly reactive bifunctional alkylating agents and were the first chemotherapeutic agents developed. They form DNA interstrand crosslinks (ICLs), which cause a blockage of DNA strand separation, inhibiting essential processes in DNA metabolism like replication and transcription. In fast replicating cells, e.g., tumor cells, this can induce cell death. The upregulation of ICL repair is thought to be a key factor for the resistance of tumor cells to ICL-inducing cytostatic agents including NMs. To monitor induction and repair of CLB-induced ICLs, we adjusted the automated reversed fluorometric analysis of alkaline DNA unwinding assay (rFADU) for the detection of ICLs in adherent cells. For the detection of monoalkylated DNA bases we established an LC-MS/MS method. We performed a comparative analysis of adduct formation and removal in five human cell lines and in peripheral blood mononuclear cells (PBMCs) after treatment with CLB. Dose-dependent increases in adduct formation were observed, and suitable treatment concentrations were identified for each cell line, which were then used for monitoring the kinetics of adduct formation. We observed significant differences in the repair kinetics of the cell lines tested. For example, in A2780 cells, hTERT immortalized VH10 cells, and in PBMCs a time-dependent repair of the two main monoalkylated DNA-adducts was confirmed. Regarding ICLs, repair was observed in all cell systems except for PBMCs. In conclusion, LC-MS/MS analyses combined with the rFADU technique are powerful tools to study the molecular mechanisms of NM-induced DNA damage and repair. By applying these methods to a spectrum of human cell systems of different origin and transformation status, we obtained insight into the cell-type specific repair of different CLB-induced DNA lesions, which may help identify novel resistance mechanisms of tumors and define molecular targets for therapeutic interventions.

2020-10-09, Aberle, Lisa, Krüger, Annika, Reber, Julia M., Lippmann, Michelle, Schmalz, Michael, Trussina, Irmela R.E.A., Schlesiger, Sarah, Zubel, Tabea, Schütz, Karina, Marx, Andreas, Ferrando-May, Elisa, Bürkle, Alexander, Mangerich, Aswin

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'.

2020-01, Neumann, Catherine, Baesler, Jessica, Steffen, Gereon, Nicolai, Merle Marie, Zubel, Tabea, Aschner, Michael, Bürkle, Alexander, Mangerich, Aswin, Schwerdtle, Tanja, Bornhorst, Julia

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.