The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Klaus Aktories - One of the best experts on this subject based on the ideXlab platform.
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Activation of phospholipase D1 by ADP-ribosylated RhoA.
Biochemical and Biophysical Research Communications, 2003Co-Authors: Harald Genth, Ralf Gerhard, Klaus Aktories, Martina Schmidt, Ingo JustAbstract: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.
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Glucosylation and ADP Ribosylation of Rho Proteins: Effects on Nucleotide Binding, GTPase Activity, and Effector Coupling†
Biochemistry, 1998Co-Authors: Peter Sehr, Ingo Just, Harald Genth, Gili Joseph, Edgar Pick, Klaus AktoriesAbstract: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...
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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, 1996Co-Authors: Peter Sehr, Ingo Just, Klaus AktoriesAbstract: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.
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differentiation induced increase in clostridium botulinum c3 exoenzyme catalyzed adp Ribosylation of the small gtp binding protein rho
FEBS Journal, 1994Co-Authors: G Fritz, Ingo Just, Peter Wollenberg, Klaus AktoriesAbstract: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.
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adp Ribosylation of rho proteins by clostridium botulinum exoenzyme c3 is influenced by phosphorylation of rho associated factors
Biochemical Journal, 1994Co-Authors: G Fritz, Klaus AktoriesAbstract:Specific [32P]ADP-Ribosylation by Clostridium botulinum exoenzyme C3 was used to study the involvement of phosphorylation in the regulation of the low-molecular-mass GTP-binding protein Rho. Dephosphorylation of CHO cell extracts by alkaline phosphatase treatment resulted in a 80-90% reduction in the C3-catalysed [32P]ADP-Ribosylation of Rho proteins in both cytosolic and membrane fractions. Similar results were obtained after dephosphorylation with protein phosphatase type-1 from bovine retina, whereas type-2B and type-2C phosphatases had no effect on the level of subsequent [32P]ADP-Ribosylation of Rho by C3. Incubation of CHO cell lysate under phosphorylation conditions increased the subsequent C3-mediated [32P]ADP-Ribosylation of Rho proteins. The protein kinase inhibitors H7 and H9 had no effect on [32P]ADP-Ribosylation at concentrations which are specific for inhibition of protein kinase A or C. Recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was phosphorylated by protein kinase A; however, the phosphorylation had no stimulatory effect on the ADP-Ribosylation of GST-RhoA by C3. An approx. 48 kDa phosphoprotein was identified which bound specifically to recombinant GST-RhoA fusion protein. By gel-permeation chromatography, Rho-containing complexes of approx. 50 kDa and 130-170 kDa were detected. The ADP-Ribosylation of Rho in the 130-170 kDa complex was reduced by alkaline phosphatase pretreatment. The data suggest that Rho activity is influenced by phosphorylation of Rho-associated regulatory factors. Phosphorylation/dephosphorylation of these Rho-regulating factors appears to alter the ability of Rho to serve as a substrate for C3-induced [32P]ADP-Ribosylation.
Bernhard Lüscher - One of the best experts on this subject based on the ideXlab platform.
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adp Ribosylation a multifaceted posttranslational modification involved in the control of cell physiology in health and disease
Chemical Reviews, 2017Co-Authors: Bernhard Lüscher, Patricia Verheugd, Mareike Bütepage, Laura Eckei, Sarah Krieg, Brian H ShiltonAbstract: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.
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Players in ADP-Ribosylation: Readers and Erasers
Current protein & peptide science, 2016Co-Authors: Patricia Verheugd, Mareike Bütepage, Laura Eckei, Bernhard LüscherAbstract: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.
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Expanding functions of intracellular resident mono‐ADP‐Ribosylation in cell physiology
The FEBS journal, 2013Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Bernhard LüscherAbstract: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.
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artd10 substrate identification on protein microarrays regulation of gsk3β by mono adp Ribosylation
Cell Communication and Signaling, 2013Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Henning Kleine, Anne K Braczynski, Alexandra H Forst, Nicolas Herzog, Ulrike Linzen, Elisabeth Kremmer, Bernhard LüscherAbstract: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.
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ARTD10 substrate identification on protein microarrays: regulation of GSK3β by mono-ADP-Ribosylation
Cell Communication and Signaling, 2013Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Henning Kleine, Anne K Braczynski, Alexandra H Forst, Nicolas Herzog, Ulrike Linzen, Elisabeth Kremmer, Bernhard LüscherAbstract: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.
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Reversible ADP-Ribosylation of RNA.
Nucleic acids research, 2019Co-Authors: Deeksha Munnur, Michael S. Cohen, Edward Bartlett, Petra Mikolčević, Ilsa T. Kirby, Johannes Gregor Matthias Rack, Andreja Mikoč, Ivan AhelAbstract: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.
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Hydrolysis of ADP-Ribosylation by Macrodomains.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Melanija Posavec Marjanović, Gytis Jankevicius, Ivan AhelAbstract: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.
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reversible mono adp Ribosylation of dna breaks
FEBS Journal, 2017Co-Authors: Deeksha Munnur, Ivan AhelAbstract: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.
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Reversible mono‐ADP‐Ribosylation of DNA breaks
The FEBS journal, 2017Co-Authors: Deeksha Munnur, Ivan AhelAbstract: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.
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hpf1 c4orf27 is a parp 1 interacting protein that regulates parp 1 adp Ribosylation activity
Molecular Cell, 2016Co-Authors: Ian Gibbsseymour, Johannes Gregor Matthias Rack, Pietro Fontana, Ivan AhelAbstract:We report the identification of histone PARylation factor 1 (HPF1; also known as C4orf27) as a regulator of ADP-Ribosylation signaling in the DNA damage response. HPF1/C4orf27 forms a robust protein complex with PARP-1 in cells and is recruited to DNA lesions in a PARP-1-dependent manner, but independently of PARP-1 catalytic ADP-Ribosylation activity. Functionally, HPF1 promotes PARP-1-dependent in trans ADP-Ribosylation of histones and limits DNA damage-induced hyper-automodification of PARP-1. Human cells lacking HPF1 exhibit sensitivity to DNA damaging agents and PARP inhibition, thereby suggesting an important role for HPF1 in genome maintenance and regulating the efficacy of PARP inhibitors. Collectively, our results demonstrate how a fundamental step in PARP-1-dependent ADP-Ribosylation signaling is regulated and suggest that HPF1 functions at the crossroads of histone ADP-Ribosylation and PARP-1 automodification.
Ingo Just - One of the best experts on this subject based on the ideXlab platform.
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MS-based quantification of RhoA/B and RhoC ADP-Ribosylation.
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2018Co-Authors: Anke Schröder, Ingo Just, Anastasia Benski, Anne Oltmanns, Astrid Rohrbeck, Andreas PichAbstract: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.
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Activation of phospholipase D1 by ADP-ribosylated RhoA.
Biochemical and Biophysical Research Communications, 2003Co-Authors: Harald Genth, Ralf Gerhard, Klaus Aktories, Martina Schmidt, Ingo JustAbstract: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.
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Glucosylation and ADP Ribosylation of Rho Proteins: Effects on Nucleotide Binding, GTPase Activity, and Effector Coupling†
Biochemistry, 1998Co-Authors: Peter Sehr, Ingo Just, Harald Genth, Gili Joseph, Edgar Pick, Klaus AktoriesAbstract: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...
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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, 1996Co-Authors: Peter Sehr, Ingo Just, Klaus AktoriesAbstract: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.
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differentiation induced increase in clostridium botulinum c3 exoenzyme catalyzed adp Ribosylation of the small gtp binding protein rho
FEBS Journal, 1994Co-Authors: G Fritz, Ingo Just, Peter Wollenberg, Klaus AktoriesAbstract: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.
Karla L. H. Feijs - One of the best experts on this subject based on the ideXlab platform.
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processing of protein adp Ribosylation by nudix hydrolases
Biochemical Journal, 2015Co-Authors: Luca Palazzo, Karla L. H. Feijs, Benjamin Thomas, Annsofie Jemth, Thomas Colby, Orsolya Leidecker, Roko Zaja, Olga Loseva, Jordi Carreras Puigvert, Ivan MaticAbstract:ADP-Ribosylation is a post-translational modification (PTM) of proteins found in organisms from all kingdoms of life which regulates many important biological functions including DNA repair, chromatin structure, unfolded protein response and apoptosis. Several cellular enzymes, such as macrodomain containing proteins PARG [poly(ADP-ribose) glycohydrolase] and TARG1 [terminal ADP-ribose (ADPr) protein glycohydrolase], reverse protein ADP-Ribosylation. In the present study, we show that human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-Ribosylation in vitro , converting it into ribose-5′-phosphate (R5P) tags covalently attached to the modified proteins. Furthermore, our data show that hNUDT16 enzymatic activity can be used to trim ADP-Ribosylation on proteins in order to facilitate analysis of ADP-Ribosylation sites on proteins by MS.
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Expanding functions of intracellular resident mono‐ADP‐Ribosylation in cell physiology
The FEBS journal, 2013Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Bernhard LüscherAbstract: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.
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artd10 substrate identification on protein microarrays regulation of gsk3β by mono adp Ribosylation
Cell Communication and Signaling, 2013Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Henning Kleine, Anne K Braczynski, Alexandra H Forst, Nicolas Herzog, Ulrike Linzen, Elisabeth Kremmer, Bernhard LüscherAbstract: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.
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ARTD10 substrate identification on protein microarrays: regulation of GSK3β by mono-ADP-Ribosylation
Cell Communication and Signaling, 2013Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Henning Kleine, Anne K Braczynski, Alexandra H Forst, Nicolas Herzog, Ulrike Linzen, Elisabeth Kremmer, Bernhard LüscherAbstract: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.
