ADP-ribosylation

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Klaus Aktories - One of the best experts on this subject based on the ideXlab platform.

  • Strain-alleviation model of ADP-ribosylation
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: Thomas Jank, Klaus Aktories
    Abstract:

    ADP-ribosylation was one of the first molecular mechanisms described to be used by bacterial protein toxins to target eukaryotic cells. Most potent and devastating toxins belong to this group, including diphtheria toxin and Pseudomonas exotoxins A, which block protein synthesis by ADP-ribosylation of elongation factor 2. Cholera toxin from Vibrio cholerae, which causes several thousand cases of death worldwide every year, is another ADP-ribosylating toxin, of which ADP-ribosylate Gs proteins is involved in receptor signaling (1). All of these toxins modify host proteins by transferring ADP ribose from NAD+ onto specific eukaryotic target proteins, thereby altering their physiological functions. Although ADP-ribosylating toxins share only limited sequence similarity, crystal structures available from various toxins are amazingly similar (2). However, so far the precise molecular reaction by which these toxins catalyze the ADP-ribosylation of their target proteins has remained largely enigmatic. In an exciting study published in PNAS (3), Tsurumura et al. describe several crystal structures of Clostridium perfringens iota toxin, in complex with its protein substrate actin, which are obtained during different phases of the ADP-ribosylation process. The authors’ findings are groundbreaking not only for the understanding of toxin-induced ADP-ribosylation but also for comprehension of the molecular reaction induced by an increasing number of endogenous ADP ribosyltransferases.

  • Activation of phospholipase D1 by ADP-ribosylated RhoA.
    Biochemical and Biophysical Research Communications, 2003
    Co-Authors: Harald Genth, Ralf Gerhard, Klaus Aktories, Martina Schmidt, Ingo Just
    Abstract:

    Abstract Clostridium botulinum exoenzyme C3 exclusively ADP-ribosylates RhoA, B, and C to inactivate them, resulting in disaggregation of the actin filaments in intact cells. The ADP-ribose resides at Asn-41 in the effector binding region, leading to the notion that ADP-ribosylation inactivates Rho by blocking coupling of Rho to its downstream effectors. In a recombinant system, however, ADP-ribosylated Rho bound to effector proteins such as phospholipase D-1 (PLD1), Rho-kinase (ROK), and rhotekin. The ADP-ribose rather mediated binding of Rho-GDP to PLD1. ADP-ribosylation of Rho-GDP followed by GTP-γ-S loading resulted in binding but not in PLD activation. On the other hand, ADP-ribosylation of Rho previously activated by binding to GTP-γ-S resulted in full PLD activation. This finding indicates that ADP-ribosylation seems to prevent GTP-induced change to the active conformation of switch I, the prerequisite of Rho–PLD interaction. In contrast to recombinant systems, ADP-ribosylation in intact cells results in functional inactivation of Rho, indicating other mechanisms of inactivation than blocking effector coupling.

  • Glucosylation and ADP Ribosylation of Rho Proteins: Effects on Nucleotide Binding, GTPase Activity, and Effector Coupling†
    Biochemistry, 1998
    Co-Authors: Peter Sehr, Ingo Just, Harald Genth, Gili Joseph, Edgar Pick, Klaus Aktories
    Abstract:

    We studied the effects of glucosylation of RhoA, Rac1, and Cdc42 at threonine-35 and -37 by Clostridium difficile toxin B on nucleotide binding, GTPase activity, and effector coupling and compared these results with the ADP ribosylation of RhoA at asparagine-41 catalyzed by Clostridium botulinum C3 transferase. Whereas glucosylation and ADP ribosylation had no major effects on GDP release from RhoA, Rac1, and Cdc42, the rate of GTPγS release from Rho proteins was increased 3−6-fold by glucosylation. ADP ribosylation decreased the rate of GTPγS release by about 50%. Glucosylation reduced the intrinsic activities of the GTPases by 3−7-fold and completely blocked GTPase stimulation by Rho-GAP. In contrast, ADP ribosylation slightly increased GTPase activity (∼2-fold) and had no major effect on GAP stimulation of GTPase. Whereas ADP ribosylation did not affect the interaction of RhoA with the binding domain of protein kinase N, glucosylation inhibited this interaction. Glucosylation of Rac1 markedly diminishe...

  • ADP-ribosylation of actin by Clostridium perfringens iota toxin and turkey erythrocyte ADP-ribosyltransferase A: effects on profilin-regulated nucleotide exchange and ATPase activity
    Naunyn-Schmiedeberg's Archives of Pharmacology, 1996
    Co-Authors: Peter Sehr, Ingo Just, Klaus Aktories
    Abstract:

    Effects of ADP-ribosylation of skeletal muscle α-actin by Clostridium perfringens iota toxin and by turkey erythrocyte ADP-ribosyltransferase A on profilin-regulated nucleotide exchange and ATPase activity were compared. ADP-ribosylation of actin at Arg177 by Clostridium perfringens iota toxin increased the nucleotide dissociation rate from 2.2×10^−3 s^−1 to 4.5×10^−3 s^−1 without affecting the profilin-induced stimulation of nucleotide exchange. In contrast, ADP-ribosylation of actin at Arg95/Arg372 induced by turkey erythrocyte transferase decreased the nucleotide dissociation rate to 1.5×10^−3 s^−1 and inhibited the profilin-induced stimulation of nucleotide exchange. Whereas toxin-induced ADP-ribosylation at Arg177 blocked actin ATPase, basal G-actin ATPase was not altered by ADP-ribosylation at Arg95/Arg372 but inhibited profilin effects on actin ATPase.

  • differentiation induced increase in clostridium botulinum c3 exoenzyme catalyzed adp ribosylation of the small gtp binding protein rho
    FEBS Journal, 1994
    Co-Authors: G Fritz, Ingo Just, Peter Wollenberg, Klaus Aktories
    Abstract:

    The specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study differentiation-dependent changes in the regulation of the low-molecular-mass GTP-binding protein Rho. Differentiation of F9 teratocarcinoma cells to neuronal-like cells by treatment with retinoic acid and dibutyryl-adenosine 3′,5′-monophosphate [(Bt)2cAMP] increased the C3-catalyzed ADP-ribosylation of RhoA proteins in cytosolic and membrane fractions by about threefold and sixfold, respectively. Phenotypical differentiation of F9 cells was not required for increase in ADP-ribosylation. Increase in ADP-ribosylation after (Bt)2cAMP and retinoic acid treatments was blocked by cycloheximide, indicating the requirement of protein biosynthesis. As deduced from specific rho mRNA amounts and from Western analysis with a monoclonal RhoA antibody, the stimulation in the [32P]ADP-ribosylation of Rho was not caused by an increased de-novo synthesis of Rho proteins. GDP increased the ADP-ribosylation of membrane-associated Rho from non-differentiated, but not from differentiated F9 cells. GTP[S] decreased ADP-ribosylation of membranous Rho from differentiated and much less from non-differentiated F9 cells. Differentiation-dependent increase in ADP-ribosylation of cytosolic Rho was reversed by protein phosphatase type-1. Treatment with SDS (0.01%) which releases Rho from complexation with guanine nucleotide dissociation inhibitor, increased ADP-ribosylation both in differentiated and non-differentiated cells, indicating no differentiation-specific change of such complexes. In total, our data indicate that the induction of the differentiation process in F9 cells is accompanied by changes in the regulation of cytosolic and membrane-associated Rho proteins.

Michael O. Hottiger - One of the best experts on this subject based on the ideXlab platform.

  • Uncovering the Invisible: Mono-ADP-ribosylation Moved into the Spotlight.
    Cells, 2021
    Co-Authors: Ann-katrin Hopp, Michael O. Hottiger
    Abstract:

    Adenosine diphosphate (ADP)-ribosylation is a nicotinamide adenine dinucleotide (NAD+)-dependent post-translational modification that is found on proteins as well as on nucleic acids. While ARTD1/PARP1-mediated poly-ADP-ribosylation has extensively been studied in the past 60 years, comparably little is known about the physiological function of mono-ADP-ribosylation and the enzymes involved in its turnover. Promising technological advances have enabled the development of innovative tools to detect NAD+ and NAD+/NADH (H for hydrogen) ratios as well as ADP-ribosylation. These tools have significantly enhanced our current understanding of how intracellular NAD dynamics contribute to the regulation of ADP-ribosylation as well as to how mono-ADP-ribosylation integrates into various cellular processes. Here, we discuss the recent technological advances, as well as associated new biological findings and concepts.

  • A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP‑1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-ribosylation
    2019
    Co-Authors: Hideyuki Higashi, Michael O. Hottiger, Takashi Maejima, Lang Ho Lee, Yukiyoshi Yamazaki, Sasha A. Singh, Masanori Aikawa
    Abstract:

    ADP-ribosylation is a post-translational modification that, until recently, has remained elusive to study at the cellular level. Previously dependent on radioactive tracers to identify ADP-ribosylation targets, several advances in mass spectrometric workflows now permit global identification of ADP-ribosylated substrates. In this study, we capitalized on two ADP-ribosylation enrichment strategies, and multiple activation methods performed on the Orbitrap Fusion Lumos, to identify IFN-γ-induced ADP-ribosylation substrates in macrophages. The ADP-ribosyl binding protein, Af1521, was used to enrich ADP-ribosylated peptides, and the antipoly-ADP-ribosyl antibody, 10H, was used to enrich ADP-ribosylated proteins. ADP-ribosyl-specific mass spectra were further enriched by an ADP-ribose product ion triggered EThcD and HCD activation strategy, in combination with multiple acquisitions that segmented the survey scan into smaller ranges. HCD and EThcD resulted in overlapping and unique ADP-ribosyl peptide identifications, with HCD providing more peptide identifications but EThcD providing more reliable ADP-ribosyl acceptor sites. Our acquisition strategies also resulted in the first ever characterization of ADP-ribosyl on three poly-ADP-ribose polymerases, ARTD9/PARP9, ARTD10/PARP10, and ARTD8/PARP14. IFN-γ increased the ADP-ribosylation status of ARTD9/PARP9, ARTD8/PARP14, and proteins involved in RNA processes. This study therefore summarizes specific molecular pathways at the intersection of IFN-γ and ADP-ribosylation signaling pathways

  • ADP-ribose-specific chromatin-affinity purification for investigating genome-wide or locus-specific chromatin ADP-ribosylation
    Nature Protocols, 2017
    Co-Authors: Lavinia Bisceglie, Giody Bartolomei, Michael O. Hottiger
    Abstract:

    This protocol describes how to investigate genome-wide or locus-specific chromatin ADP-ribosylation using ADPr-ChAP. Protein ADP-ribosylation is a structurally heterogeneous post-translational modification (PTM) that influences the physicochemical and biological properties of the modified protein. ADP-ribosylation of chromatin changes its structural properties, thereby regulating important nuclear functions. A lack of suitable antibodies for chromatin immunoprecipitation (ChIP) has so far prevented a comprehensive analysis of DNA-associated protein ADP-ribosylation. To analyze chromatin ADP-ribosylation, we recently developed a novel ADP-ribose-specific chromatin-affinity purification (ADPr-ChAP) methodology that uses the recently identified ADP-ribose-binding domains RNF146 WWE and Af1521. In this protocol, we describe how to use this robust and versatile method for genome-wide and loci-specific localization of chromatin ADP-ribosylation. ADPr-ChAP enables bioinformatic comparisons of ADP-ribosylation with other chromatin modifications and is useful for understanding how ADP-ribosylation regulates biologically important cellular processes. ADPr-ChAP takes 1 week and requires standard skills in molecular biology and biochemistry. Although not covered in detail here, this technique can also be combined with conventional ChIP or DNA analysis to define the histone marks specifically associated with the ADP-ribosylated chromatin fractions and dissect the molecular mechanism and functional role of chromatin ADP-ribosylation.

  • Cell fate regulation by chromatin ADP-ribosylation.
    Seminars in cell & developmental biology, 2016
    Co-Authors: Jeannette Abplanalp, Michael O. Hottiger
    Abstract:

    ADP-ribosylation is an evolutionarily conserved complex posttranslational modification that alters protein function and/or interaction. Intracellularly, it is mainly catalyzed by diphtheria toxin-like ADP-ribosyltransferases (ARTDs), which attach one or several ADP-ribose residues onto target proteins. Several specific mono- and poly-ADP-ribosylation binding modules exist; hydrolases reverse the modification. The best-characterized ARTD family member, ARTD1, regulates various DNA-associated processes. Here, we focus on the role of ARTD1-mediated chromatin ADP-ribosylation in development, differentiation, and pluripotency, and the recent development of new methodologies that will enable more insight into these processes.

  • SnapShot: ADP-ribosylation Signaling.
    Molecular cell, 2016
    Co-Authors: Michael O. Hottiger
    Abstract:

    Intracellular protein ADP-ribosylation is catalyzed by diphteria toxin-like ADP-ribosyltransferases (ARTDs, formerly PARPs) ("writers"), which use NAD(+) for the modification of different amino acids. While some ARTD members catalyze protein poly-ADP-ribosylation, most of them are mono-ADP-ribosyltransferases. ADP-ribosylation is recognized by protein domains ("readers") and reversed by different enzymes ("erasers"). ADP-ribosylation signaling regulates several key cellular processes during health and disease.

Ingo Just - One of the best experts on this subject based on the ideXlab platform.

  • MS-based quantification of RhoA/B and RhoC ADP-ribosylation.
    Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2018
    Co-Authors: Anke Schröder, Ingo Just, Anastasia Benski, Anne Oltmanns, Astrid Rohrbeck, Andreas Pich
    Abstract:

    Abstract Mono ADP-ribosylation is a common characteristic of bacterial toxins resulting to aberrant activation or inactivation of target proteins. The C3 exoenzyme of Clostridium botulinum (C3bot) ADP-ribosylates the small GTPases RhoA, RhoB and RhoC, leading to inactivation of these important regulators and impaired down-stream signaling. Quantification of ADP-ribosylation using gel migration assays, antibodies, and radioactivity-based methods are limited. Therefore a novel LC-MS-based method to specifically determine and quantify ADP-ribosylation of Rho GTPases was established. A heavy labeled protein standard that contained ADP-ribosylation specific peptides in similar amounts in ADP ribosylated and non ADP ribosylated form was used for relative quantification in vivo. In a proof of principle experiment HT22 cells were treated with C3bot and the kinetics of RhoA/B and RhoC ADP-ribosylation were determined in vivo.

  • Activation of phospholipase D1 by ADP-ribosylated RhoA.
    Biochemical and Biophysical Research Communications, 2003
    Co-Authors: Harald Genth, Ralf Gerhard, Klaus Aktories, Martina Schmidt, Ingo Just
    Abstract:

    Abstract Clostridium botulinum exoenzyme C3 exclusively ADP-ribosylates RhoA, B, and C to inactivate them, resulting in disaggregation of the actin filaments in intact cells. The ADP-ribose resides at Asn-41 in the effector binding region, leading to the notion that ADP-ribosylation inactivates Rho by blocking coupling of Rho to its downstream effectors. In a recombinant system, however, ADP-ribosylated Rho bound to effector proteins such as phospholipase D-1 (PLD1), Rho-kinase (ROK), and rhotekin. The ADP-ribose rather mediated binding of Rho-GDP to PLD1. ADP-ribosylation of Rho-GDP followed by GTP-γ-S loading resulted in binding but not in PLD activation. On the other hand, ADP-ribosylation of Rho previously activated by binding to GTP-γ-S resulted in full PLD activation. This finding indicates that ADP-ribosylation seems to prevent GTP-induced change to the active conformation of switch I, the prerequisite of Rho–PLD interaction. In contrast to recombinant systems, ADP-ribosylation in intact cells results in functional inactivation of Rho, indicating other mechanisms of inactivation than blocking effector coupling.

  • Glucosylation and ADP Ribosylation of Rho Proteins: Effects on Nucleotide Binding, GTPase Activity, and Effector Coupling†
    Biochemistry, 1998
    Co-Authors: Peter Sehr, Ingo Just, Harald Genth, Gili Joseph, Edgar Pick, Klaus Aktories
    Abstract:

    We studied the effects of glucosylation of RhoA, Rac1, and Cdc42 at threonine-35 and -37 by Clostridium difficile toxin B on nucleotide binding, GTPase activity, and effector coupling and compared these results with the ADP ribosylation of RhoA at asparagine-41 catalyzed by Clostridium botulinum C3 transferase. Whereas glucosylation and ADP ribosylation had no major effects on GDP release from RhoA, Rac1, and Cdc42, the rate of GTPγS release from Rho proteins was increased 3−6-fold by glucosylation. ADP ribosylation decreased the rate of GTPγS release by about 50%. Glucosylation reduced the intrinsic activities of the GTPases by 3−7-fold and completely blocked GTPase stimulation by Rho-GAP. In contrast, ADP ribosylation slightly increased GTPase activity (∼2-fold) and had no major effect on GAP stimulation of GTPase. Whereas ADP ribosylation did not affect the interaction of RhoA with the binding domain of protein kinase N, glucosylation inhibited this interaction. Glucosylation of Rac1 markedly diminishe...

  • ADP-ribosylation of actin by Clostridium perfringens iota toxin and turkey erythrocyte ADP-ribosyltransferase A: effects on profilin-regulated nucleotide exchange and ATPase activity
    Naunyn-Schmiedeberg's Archives of Pharmacology, 1996
    Co-Authors: Peter Sehr, Ingo Just, Klaus Aktories
    Abstract:

    Effects of ADP-ribosylation of skeletal muscle α-actin by Clostridium perfringens iota toxin and by turkey erythrocyte ADP-ribosyltransferase A on profilin-regulated nucleotide exchange and ATPase activity were compared. ADP-ribosylation of actin at Arg177 by Clostridium perfringens iota toxin increased the nucleotide dissociation rate from 2.2×10^−3 s^−1 to 4.5×10^−3 s^−1 without affecting the profilin-induced stimulation of nucleotide exchange. In contrast, ADP-ribosylation of actin at Arg95/Arg372 induced by turkey erythrocyte transferase decreased the nucleotide dissociation rate to 1.5×10^−3 s^−1 and inhibited the profilin-induced stimulation of nucleotide exchange. Whereas toxin-induced ADP-ribosylation at Arg177 blocked actin ATPase, basal G-actin ATPase was not altered by ADP-ribosylation at Arg95/Arg372 but inhibited profilin effects on actin ATPase.

  • differentiation induced increase in clostridium botulinum c3 exoenzyme catalyzed adp ribosylation of the small gtp binding protein rho
    FEBS Journal, 1994
    Co-Authors: G Fritz, Ingo Just, Peter Wollenberg, Klaus Aktories
    Abstract:

    The specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study differentiation-dependent changes in the regulation of the low-molecular-mass GTP-binding protein Rho. Differentiation of F9 teratocarcinoma cells to neuronal-like cells by treatment with retinoic acid and dibutyryl-adenosine 3′,5′-monophosphate [(Bt)2cAMP] increased the C3-catalyzed ADP-ribosylation of RhoA proteins in cytosolic and membrane fractions by about threefold and sixfold, respectively. Phenotypical differentiation of F9 cells was not required for increase in ADP-ribosylation. Increase in ADP-ribosylation after (Bt)2cAMP and retinoic acid treatments was blocked by cycloheximide, indicating the requirement of protein biosynthesis. As deduced from specific rho mRNA amounts and from Western analysis with a monoclonal RhoA antibody, the stimulation in the [32P]ADP-ribosylation of Rho was not caused by an increased de-novo synthesis of Rho proteins. GDP increased the ADP-ribosylation of membrane-associated Rho from non-differentiated, but not from differentiated F9 cells. GTP[S] decreased ADP-ribosylation of membranous Rho from differentiated and much less from non-differentiated F9 cells. Differentiation-dependent increase in ADP-ribosylation of cytosolic Rho was reversed by protein phosphatase type-1. Treatment with SDS (0.01%) which releases Rho from complexation with guanine nucleotide dissociation inhibitor, increased ADP-ribosylation both in differentiated and non-differentiated cells, indicating no differentiation-specific change of such complexes. In total, our data indicate that the induction of the differentiation process in F9 cells is accompanied by changes in the regulation of cytosolic and membrane-associated Rho proteins.

Bernhard Lüscher - One of the best experts on this subject based on the ideXlab platform.

  • adp ribosylation a multifaceted posttranslational modification involved in the control of cell physiology in health and disease
    Chemical Reviews, 2017
    Co-Authors: Bernhard Lüscher, Patricia Verheugd, Mareike Bütepage, Laura Eckei, Sarah Krieg, Brian H Shilton
    Abstract:

    Posttranslational modifications (PTMs) regulate protein functions and interactions. ADP-ribosylation is a PTM, in which ADP-ribosyltransferases use nicotinamide adenine dinucleotide (NAD+) to modify target proteins with ADP-ribose. This modification can occur as mono- or poly-ADP-ribosylation. The latter involves the synthesis of long ADP-ribose chains that have specific properties due to the nature of the polymer. ADP-ribosylation is reversed by hydrolases that cleave the glycosidic bonds either between ADP-ribose units or between the protein proximal ADP-ribose and a given amino acid side chain. Here we discuss the properties of the different enzymes associated with ADP-ribosylation and the consequences of this PTM on substrates. Furthermore, the different domains that interpret either mono- or poly-ADP-ribosylation and the implications for cellular processes are described.

  • Players in ADP-ribosylation: Readers and Erasers
    Current protein & peptide science, 2016
    Co-Authors: Patricia Verheugd, Mareike Bütepage, Laura Eckei, Bernhard Lüscher
    Abstract:

    ADP-ribosylation describes an ancient and highly conserved posttranslational modification (PTM) of proteins. Many cellular processes have been identified that are regulated by ADP-ribosylation, including DNA repair, gene transcription and signaling processes. Enzymes catalyzing ADP-ribosylation use NAD+ as a cofactor to transfer ADP-ribose to a substrate under release of nicotinamide. In mammals extracellular and intracellular enzymes have been described. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs) and some Sirtuins. Extracellular and intracellular ARTs belong to the cholera toxin-like (ARTC) and the diphtheria toxin-like (ARTD) subclass, respectively. ARTDs can be further subdivided depending on their ability to either generate poly-ADP-ribose chains, or to mono-ADP-ribosylate substrates. Similar to the latter, ARTCs and Sirtuins are restricted to mono-ADP-ribosylation. Recent findings have provided information about the functional consequences of ADP-ribosylation. Analogous to other PTMs, ADP-ribosylation can exert allosteric effects on enzymes, thereby controlling their catalytic activity. Moreover, this PTM can be read by multiple protein motifs and domains mediating protein-protein interactions. Typically these readers can distinguish between mono- and poly-ADP-ribosylation. Furthermore, with the description of proteins that can erase ADP-ribosylation, this posttranslational modification is fully reversible and thus provides an additional mechanism to transiently control protein functions and networks. In this review we will describe the most recent findings on motifs and domains that are related to ADP-ribosylation processes with a particular focus on readers and erasers. These new findings provide evidence for broad functional roles of ADP-ribosylation and a high diversity of mechanisms that contribute to the downstream consequences of this modification.

  • Expanding functions of intracellular resident mono‐ADP‐ribosylation in cell physiology
    The FEBS journal, 2013
    Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Bernhard Lüscher
    Abstract:

    Poly-ADP-ribosylation functions in diverse signaling pathways, such as Wnt signaling and DNA damage repair, where its role is relatively well characterized. Contrarily, mono-ADP-ribosylation by for example ARTD10/PARP10 is much less understood. Recent developments hint at the involvement of mono-ADP-ribosylation in transcriptional regulation, the unfolded protein response, DNA repair, insulin secretion and immunity. Additionally, macrodomain-containing hydrolases, MacroD1, MacroD2 and C6orf130/TARG1, have been identified that make mono-ADP-ribosylation reversible. Complicating further progress is the lack of tools such as mono-ADP-ribose-specific antibodies. The currently known functions of mono-ADP-ribosylation are summarized here, as well as the available tools such as mass spectrometry to study this modification in vitro and in cells.

  • artd10 substrate identification on protein microarrays regulation of gsk3β by mono adp ribosylation
    Cell Communication and Signaling, 2013
    Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Henning Kleine, Anne K Braczynski, Alexandra H Forst, Nicolas Herzog, Ulrike Linzen, Elisabeth Kremmer, Bernhard Lüscher
    Abstract:

    Although ADP-ribosylation has been described five decades ago, only recently a distinction has been made between eukaryotic intracellular poly- and mono-ADP-ribosylating enzymes. Poly-ADP-ribosylation by ARTD1 (formerly PARP1) is best known for its role in DNA damage repair. Other polymer forming enzymes are ARTD2 (formerly PARP2), ARTD3 (formerly PARP3) and ARTD5/6 (formerly Tankyrase 1/2), the latter being involved in Wnt signaling and regulation of 3BP2. Thus several different functions of poly-ADP-ribosylation have been well described whereas intracellular mono-ADP-ribosylation is currently largely undefined. It is for example not known which proteins function as substrate for the different mono-ARTDs. This is partially due to lack of suitable reagents to study mono-ADP-ribosylation, which limits the current understanding of this post-translational modification. We have optimized a novel screening method employing protein microarrays, ProtoArrays®, applied here for the identification of substrates of ARTD10 (formerly PARP10) and ARTD8 (formerly PARP14). The results of this substrate screen were validated using in vitro ADP-ribosylation assays with recombinant proteins. Further analysis of the novel ARTD10 substrate GSK3β revealed mono-ADP-ribosylation as a regulatory mechanism of kinase activity by non-competitive inhibition in vitro. Additionally, manipulation of the ARTD10 levels in cells accordingly influenced GSK3β activity. Together these data provide the first evidence for a role of endogenous mono-ADP-ribosylation in intracellular signaling. Our findings indicate that substrates of ADP-ribosyltransferases can be identified using protein microarrays. The discovered substrates of ARTD10 and ARTD8 provide the first sets of proteins that are modified by mono-ADP-ribosyltransferases in vitro. By studying one of the ARTD10 substrates more closely, the kinase GSK3β, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.

  • ARTD10 substrate identification on protein microarrays: regulation of GSK3β by mono-ADP-ribosylation
    Cell Communication and Signaling, 2013
    Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Henning Kleine, Anne K Braczynski, Alexandra H Forst, Nicolas Herzog, Ulrike Linzen, Elisabeth Kremmer, Bernhard Lüscher
    Abstract:

    Background Although ADP-ribosylation has been described five decades ago, only recently a distinction has been made between eukaryotic intracellular poly- and mono-ADP-ribosylating enzymes. Poly-ADP-ribosylation by ARTD1 (formerly PARP1) is best known for its role in DNA damage repair. Other polymer forming enzymes are ARTD2 (formerly PARP2), ARTD3 (formerly PARP3) and ARTD5/6 (formerly Tankyrase 1/2), the latter being involved in Wnt signaling and regulation of 3BP2. Thus several different functions of poly-ADP-ribosylation have been well described whereas intracellular mono-ADP-ribosylation is currently largely undefined. It is for example not known which proteins function as substrate for the different mono-ARTDs. This is partially due to lack of suitable reagents to study mono-ADP-ribosylation, which limits the current understanding of this post-translational modification. Results We have optimized a novel screening method employing protein microarrays, ProtoArrays®, applied here for the identification of substrates of ARTD10 (formerly PARP10) and ARTD8 (formerly PARP14). The results of this substrate screen were validated using in vitro ADP-ribosylation assays with recombinant proteins. Further analysis of the novel ARTD10 substrate GSK3β revealed mono-ADP-ribosylation as a regulatory mechanism of kinase activity by non-competitive inhibition in vitro . Additionally, manipulation of the ARTD10 levels in cells accordingly influenced GSK3β activity. Together these data provide the first evidence for a role of endogenous mono-ADP-ribosylation in intracellular signaling. Conclusions Our findings indicate that substrates of ADP-ribosyltransferases can be identified using protein microarrays. The discovered substrates of ARTD10 and ARTD8 provide the first sets of proteins that are modified by mono-ADP-ribosyltransferases in vitro . By studying one of the ARTD10 substrates more closely, the kinase GSK3β, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.

Ivan Ahel - One of the best experts on this subject based on the ideXlab platform.

  • Reversible ADP-ribosylation of RNA.
    Nucleic acids research, 2019
    Co-Authors: Deeksha Munnur, Michael S. Cohen, Edward Bartlett, Petra Mikolčević, Ilsa T. Kirby, Johannes Gregor Matthias Rack, Andreja Mikoč, Ivan Ahel
    Abstract:

    ADP-ribosylation is a reversible chemical modification catalysed by ADP-ribosyltransferases such as PARPs that utilize nicotinamide adenine dinucleotide (NAD+) as a cofactor to transfer monomer or polymers of ADP-ribose nucleotide onto macromolecular targets such as proteins and DNA. ADP-ribosylation plays an important role in several biological processes such as DNA repair, transcription, chromatin remodelling, host-virus interactions, cellular stress response and many more. Using biochemical methods we identify RNA as a novel target of reversible mono-ADP-ribosylation. We demonstrate that the human PARPs - PARP10, PARP11 and PARP15 as well as a highly diverged PARP homologue TRPT1, ADP-ribosylate phosphorylated ends of RNA. We further reveal that ADP-ribosylation of RNA mediated by PARP10 and TRPT1 can be efficiently reversed by several cellular ADP-ribosylhydrolases (PARG, TARG1, MACROD1, MACROD2 and ARH3), as well as by MACROD-like hydrolases from VEEV and SARS viruses. Finally, we show that TRPT1 and MACROD homologues in bacteria possess activities equivalent to the human proteins. Our data suggest that RNA ADP-ribosylation may represent a widespread and physiologically relevant form of reversible ADP-ribosylation signalling.

  • Hydrolysis of ADP-ribosylation by Macrodomains.
    Methods in molecular biology (Clifton N.J.), 2018
    Co-Authors: Melanija Posavec Marjanović, Gytis Jankevicius, Ivan Ahel
    Abstract:

    ADP-ribosylation is the process of transferring the ADP-ribose moiety from NAD+ to a substrate. While a number of proteins represent well described substrates accepting ADP-ribose modification, a recent report demonstrated biological role for DNA ADP-ribosylation as well. The conserved macrodomain fold of several known hydrolyses was found to possess de-ADP-ribosylating activity and the ability to hydrolyze (reverse) ADP-ribosylation. Here we summarize the methods that can be employed to study mono-ADP-ribosylation hydrolysis by macrodomains.

  • reversible mono adp ribosylation of dna breaks
    FEBS Journal, 2017
    Co-Authors: Deeksha Munnur, Ivan Ahel
    Abstract:

    Adenosine diphosphate (ADP)-ribosylation is a chemical modification of macromolecules that plays an important role in regulation of quintessential biological processes such as DNA repair, transcription, chromatin remodelling, stress response, apoptosis, bacterial metabolism and many others. ADP-ribosylation is carried out by ADP-ribosyltransferase proteins, such as poly (ADP-ribose) polymerases (PARPs) that transfer either monomer or polymers of ADP-ribose onto the molecular targets by using nicotinamide adenine dinucleotide (NAD+ ) as a cofactor. Traditionally, proteins have been described as primary targets of ADP-ribosylation; however, there has been growing evidence that DNA may be a common target as well. Here, we show using biochemical studies that PARP3, a DNA damage-activated ADP-ribosyltransferase, can mono-ADP-ribosylate double-stranded DNA ends. ADP-ribosylation of DNA mediated by PARP3 attaches a single mono-ADP-ribose moiety to the phosphate group at the terminal ends of DNA. We further show that mono ADP-ribosylation at DNA ends can be efficiently reversed by several cellular hydrolases (PARG, MACROD2, TARG1 and ARH3). This suggests that mono ADP-ribosylated DNA adducts can be efficiently removed in cells by several mechanisms.

  • Reversible mono‐ADP‐ribosylation of DNA breaks
    The FEBS journal, 2017
    Co-Authors: Deeksha Munnur, Ivan Ahel
    Abstract:

    Adenosine diphosphate (ADP)-ribosylation is a chemical modification of macromolecules that plays an important role in regulation of quintessential biological processes such as DNA repair, transcription, chromatin remodelling, stress response, apoptosis, bacterial metabolism and many others. ADP-ribosylation is carried out by ADP-ribosyltransferase proteins, such as poly (ADP-ribose) polymerases (PARPs) that transfer either monomer or polymers of ADP-ribose onto the molecular targets by using nicotinamide adenine dinucleotide (NAD+ ) as a cofactor. Traditionally, proteins have been described as primary targets of ADP-ribosylation; however, there has been growing evidence that DNA may be a common target as well. Here, we show using biochemical studies that PARP3, a DNA damage-activated ADP-ribosyltransferase, can mono-ADP-ribosylate double-stranded DNA ends. ADP-ribosylation of DNA mediated by PARP3 attaches a single mono-ADP-ribose moiety to the phosphate group at the terminal ends of DNA. We further show that mono ADP-ribosylation at DNA ends can be efficiently reversed by several cellular hydrolases (PARG, MACROD2, TARG1 and ARH3). This suggests that mono ADP-ribosylated DNA adducts can be efficiently removed in cells by several mechanisms.

  • The role of ADP-ribosylation in regulating DNA interstrand crosslink repair
    Journal of Cell Science, 2016
    Co-Authors: Alasdair R. Gunn, Benito Banos-pinero, Peggy Paschke, Luis Sanchez-pulido, Antonio Ariza, Mehera Emrich, David Leys, Chris P. Ponting, Ivan Ahel
    Abstract:

    ABSTRACT ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here, we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches were used to identify a protein that contains a permutated macrodomain (which we call aprataxin/APLF-and-PNKP-like protein; APL). Structural analysis reveals that this permutated macrodomain retains features associated with ADP-ribose interactions and that APL is capable of binding poly(ADP-ribose) through this macrodomain. APL is enriched in chromatin in response to cisplatin treatment, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2− cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells owing to redundant signalling by the double-strand break (DSB)-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify that NHEJ can function to resolve this form of DNA damage in the absence of Adprt2.