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Shuh Narumiya - One of the best experts on this subject based on the ideXlab platform.
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synergistic activation of rat brain phospholipase d by adp ribosylation factor and rhoa p21 and its inhibition by clostridium botulinum c3 Exoenzyme
Journal of Biological Chemistry, 1995Co-Authors: Hideo Kuribara, Narito Morii, Shuh Narumiya, Kenji Tago, Takeaki Yokozeki, Takuya Sasaki, Yoshimi Takai, Toshiaki Katada, Yasunori KanahoAbstract:Abstract An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), was partially purified from bovine brain cytosol by a series of chromatographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 Exoenzyme ADP-ribosyltransferase, which strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially purified PLD-activating factor with both C3 Exoenzyme and NAD significantly inhibited the PLD-stimulating activity. These results suggest that rhoA p21 is, at least in part, responsible for the PLD-stimulating activity in the preparation. Recombinant isoprenylated rhoA p21 expressed in and purified from Sf9 cells activated rat brain PLD in a concentration- and GTPS (guanosine 5′-O-(3-thiotriphosphate))-dependent manner. In contrast, recombinant non-isoprenylated rhoA p21 (fused to glutathione S-transferase) expressed in Escherichia coli failed to activate the PLD. This difference cannot be explained by a lower affinity of non-isoprenylated rhoA p21 for GTPS, as the rates of [S]GTPS binding were very similar for both recombinant preparations and the GTPS-bound form of non-isoprenylated rhoA p21 did not induce PLD activation. Interestingly, recombinant isoprenylated rhoA p21 and ARF synergistically activated rat brain PLD; a similar pattern was seen with the partially purified PLD-activating factor. The synergistic activation was inhibited by C3 Exoenzyme-catalyzed ADP-ribosylation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner. Inhibition correlated with the extent of ADP-ribosylation. These findings suggest that rhoA p21 regulates rat brain PLD in concert with ARF, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.
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identification of glu173 as the critical amino acid residue for the adp ribosyltransferase activity of clostridium botulinum c3 Exoenzyme
FEBS Letters, 1995Co-Authors: Y Saito, Toshimasa Ishizaki, Narito Morii, Yasuo Nemoto, Naoki Watanabe, Shuh NarumiyaAbstract:Clostridium botulinum C3 Exoenzyme specifically ADP-ribosylates rho-p21 in eukaryotic cells. Trp18 and Glu173 of this enzyme were substituted with other amino acids via site-directed mutagenesis. All substitutions at Glu173 caused a significant reduction in affinity for NAD and diminished ADP-ribosyltransferase activity. On the other hand, the activity of enzymes with the substitution at Trp18 remained intact. Swiss 3T3 cells treated with the enzyme with the Trp18 substitution showed the typical morphologic changes of the C3 Exoenzyme phenotype. In contrast, no changes were found in cells incubated with the Glu173-substituted enzyme. These results indicate that the Glu173 residue of the C3 Exoenzyme plays a key role in interacting with NAD and in expression of ADP-ribosyltransferase activity, which is essential for the phenotypic change by C3 Exoenzyme treatment.
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botulinum c3 Exoenzyme blocks the tyrosine phosphorylation of p125fak and paxillin induced by bombesin and endothelin
FEBS Letters, 1994Co-Authors: Sara Rankin, Narito Morii, Shuh Narumiya, Enrique RozengurtAbstract:In this study we examined the role of rho p21 in neuropeptide-stimulated tyrosine phosphorylation. Intact Swiss 3T3 cells were treated with the Clostridium botulinum C3 Exoenzyme which specifically ADP ribosylates and inactivates rho p21. C3 Exoenzyme treatment of cells caused a marked decrease in both bombesin- and endothelin-stimulated tyrosine phosphorylation of multiple proteins, including p125 focal adhesion kinase (FAK) and paxillin. Our results suggest that rho p21 is a component of the signal transduction pathway linking seven transmembrane domain receptors with tyrosine phosphorylation and cytoskeletal events.
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adp ribosylation of rho p21 inhibits lysophosphatidic acid induced protein tyrosine phosphorylation and phosphatidylinositol 3 kinase activation in cultured swiss 3t3 cells
Journal of Biological Chemistry, 1993Co-Authors: Naokazu Kumagai, Narito Morii, Yasuo Nemoto, Kazuko Fujisawa, Shuh NarumiyaAbstract:Botulinum C3 Exoenzyme was used to specifically ADP-ribosylate and inactivate rho p21, and the effects of rho p21 inactivation on lysophosphatidic acid (LPA)-induced tyrosine phosphorylation were examined in cultured Swiss 3T3 cells. LPA induced a rapid increase in the tyrosine phosphorylation of a number of proteins. Pretreatment of the cells with the C3 Exoenzyme caused ADP-ribosylation of rho p21 in the cells and selectively attenuated the phosphorylation of several proteins, including p43 mitogen-activated protein kinase, p125 focal adhesion kinase, and two proteins of 72 and 88 kDa. C3 Exoenzyme pretreatment did not block the initial phosphorylation and activation of mitogen-activated protein kinase but suppressed its subsequent rise. In contrast, the enzyme treatment inhibited the induction of phosphorylation of the 72- and 88-kDa proteins and suppressed the basal and LPA-induced tyrosine phosphorylation of p125 focal adhesion kinase. In addition, immunoprecipitation of cell lysates with an antibody directed against the 85-kDa subunit of phosphatidylinositol 3-kinase (PI 3-kinase) co-precipitated a tyrosine-phosphorylated band of 180 kDa. C3 Exoenzyme pretreatment suppressed both the phosphorylation of this band and PI 3-kinase activation associated with LPA stimulation. These findings suggest that rho p21 works as a link between the LPA receptor signal and the subsequent tyrosine phosphorylation and PI 3-kinase activation in these cells.
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a rho like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs
Zygote, 1993Co-Authors: Issei Mabuchi, Narito Morii, Yukihisa Hamaguchi, Hirotaka Fujimoto, Masanori Mishima, Shuh NarumiyaAbstract:Sand dollar eggs were microinjected with botulinum C3 Exoenzyme, an ADP-ribosyltransferase from Clostridium botulinum that specifically ADP-ribosylates and inactivates rho proteins. C3 Exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation. No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining. When microinjected into furrowing eggs, C3 Exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the cleavage furrows. C3 Exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs. By contrast, C3 Exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation. Only one protein (molecular weight 22,000) was ADP-ribosylated by C3 Exoenzyme in the isolated cleavage furrow. This protein co-migrated with ADP-ribosylated rhoA derived from human platelets when analysed by two-dimensional gel electrophoresis. These results strongly suggest that a rho-like, small GTP-binding protein is selectively involved in the organisation and maintenance of the contractile ring.
Dara W Frank - One of the best experts on this subject based on the ideXlab platform.
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identification of type iii secreted products of the pseudomonas aeruginosa Exoenzyme s regulon
Journal of Bacteriology, 1997Co-Authors: Timothy L Yahr, Liane M Mendemueller, Matthew B Friese, Dara W FrankAbstract:Extracellular protein profiles from wild-type and regulatory or secretory isogenic mutants of the Pseudomonas aeruginosa Exoenzyme S regulon were compared to identify proteins coordinately secreted with ExoS. Data from amino-terminal sequence analysis of purified extracellular proteins were combined with data from nucleotide sequence analysis of loci linked to Exoenzyme S production. We report the identification of P. aeruginosa homologs to proteins of Yersinia spp. that function as regulators of the low calcium response, regulators of secretion, and mediators of the type III translocation mechanism.
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The Exoenzyme S regulon of Pseudomonas aeruginosa.
Molecular Microbiology, 1997Co-Authors: Dara W FrankAbstract:Pseudomonas aeruginosa can cause severe life-threatening infections in which the bacterium disseminates rapidly from epithelial colonization sites to the bloodstream. In experimental models, the ability of P. aeruginosa to disseminate is linked to epithelial injury, in vitro cytotoxicity and expression of the Exoenzyme S regulon. Using the expression of ExoS as a model, a series of genes that are important for regulation, secretion and, perhaps, intoxication of eukaryotic cells have been identified. Proteins encoded by the Exoenzyme S regulon and the Yersinia Yop virulon show a high level of amino acid homology, suggesting that P. aeruginosa may use a contact-mediated translocation mechanism to transfer anti-host factors directly into eukaryotic cells. Potential anti-host factors that may disrupt eukaryotic signal transduction through ADP-ribosylation include ExoS and ExoT. Expression of ExoU, another candidate anti-host factor, has been correlated with acute cytotoxicity and lung epithelial injury. Members of the Exoenzyme S regulon represent only a portion of the virulence factor arsenal possessed by P. aeruginosa. It will be important to understand how the Exoenzyme S regulon contributes to pathogenesis and whether these factors could serve as potential therapeutic targets.
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Exoenzyme s of pseudomonas aeruginosa is secreted by a type iii pathway
Molecular Microbiology, 1996Co-Authors: Timothy L Yahr, Joanne Goranson, Dara W FrankAbstract:Exoenzyme S is an extracellular ADP-ribosyltransferase of Pseudomonas aeruginosa. Transposon mutagenesis of P. aeruginosa 388 was used to identify genes required for Exoenzyme S production. Five Tn5Tc insertion mutants were isolated which exhibited an Exoenzyme S-deficient phenotype (388::Tn5Tc 469, 550, 3453, 4885, and 5590). Mapping experiments demonstrated that 388::Tn5Tc 3453, 4885, and 5590 possessed insertions within a 5.0 kb EcoRI fragment that is not contiguous with the Exoenzyme S trans-regulatory operon. 388::Tn5Tc 469 and 550 mapped to a region downstream of the trans-regulatory operon which has been previously shown to contain a promoter region that is co-ordinately regulated with Exoenzyme S synthesis. Nucleotide sequence analysis of a 7.2 kb region flanking the 388::Tn5Tc 469 and 550 insertions, identified 12 contiguous open reading frames (ORFs). Database searches indicated that the first ORF, ExsD, is unique. The other 11 ORFs demonstrated high homology to the YscB-L proteins of the yersiniae Yop type III export apparatus. RNase-protection analysis of wild-type and mutant strains indicated that exsD and pscB-L form an operon. To determine whether ExoS was exported by a type III mechanism, derivatives consisting of internal deletions or lacking amino- or carboxy-terminal residues were expressed in P. aeruginosa. Deletion analyses indicated that the amino-terminal nine residues are required for ExoS export. Combined data from mutagenesis, regulatory, expression, and sequence analyses provide strong evidence that P. aeruginosa possesses a type III secretion apparatus which is required for the export of Exoenzyme S and potentially other co-ordinately regulated proteins.
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genetic relationship between the 53 and 49 kilodalton forms of Exoenzyme s from pseudomonas aeruginosa
Journal of Bacteriology, 1996Co-Authors: Timothy L Yahr, Joseph T Barbieri, Dara W FrankAbstract:Exoenzyme S is an ADP-ribosylating extracellular protein of Pseudomonas aeruginosa that is produced as two immunologically related forms, a 49-kDa enzymatically active form and a 53-kDa inactive form. The postulated relationship between the two proteins involves a carboxy-terminal proteolytic cleavage of the 53-kDa precursor to produce an enzymatically active 49-kDa protein. To determine the genetic relationship between the two forms of Exoenzyme S, exoS (encoding the 49-kDa form) was used as a probe in Southern blot analyses of P. aeruginosa chromosomal digests. Cross-hybridizing bands were detected in chromosomal digests of a strain of P. aeruginosa in which exoS had been deleted by allelic exchange. A chromosomal bank was prepared from the exoS deletion strain, 388deltaexoS::TC, and screened with a probe internal to exoS. Thirteen clones that cross-hybridized with the exoS probe were identified. One representative clone contained the open reading frame exoT; this open reading frame encoded a protein of 457 amino acids which showed 75% amino acid identity to ExoS. The exoT open reading frame, cloned into a T7 expression system, produced a 53-kDa protein in Escherichia coli, termed Exo53, which reacted to antisera against Exoenzyme S. A histidine-tagged derivative of recombinant Exo53 possessed approximately 0.2% of the ADP-ribosyltransferase activity of recombinant ExoS. Inactivation of exoT in an allelic-replacement strain resulted in an Exo53-deficient phenotype without modifying the expression of ExoS. These studies prove that the 53- and 49-kDa forms of Exoenzyme S are encoded by separate genes. In addition, this is the first report of the factor-activating-Exoenzyme-S-dependent ADP-ribosyltransferase activity of the 53-kDa form of Exoenzyme S.
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analyses of the dna binding and transcriptional activation properties of exsa the transcriptional activator of the pseudomonas aeruginosa Exoenzyme s regulon
Journal of Bacteriology, 1995Co-Authors: A K Hovey, Dara W FrankAbstract:ExsA has been implicated as a central regulator of Exoenzyme S production by Pseudomonas aeruginosa. In this study, the DNA-binding and transcriptional activation properties of ExsA were investigated. ExsA was produced and purified as a fusion protein, MALA3A2, which was shown to bind specifically to promoter regions that regulated transcription of the Exoenzyme S trans-regulatory locus (pC) and a locus located directly downstream of exsA (pD). Previously, MALA3A2 was shown to bind the exoS 5' PstI-NsiI region, which contained two independent but coordinately regulated (ExsA-mediated) promoters, pS' (now termed pORF1) and pS. DNase I footprint analysis of the promoter regions bound by ExsA revealed a common protected consensus sequence of TXAAAAXA. The consensus sequence was located -51 to -52 bp upstream of the transcriptional start sites for pD, pS, and pORF1. Promoter fusion, DNA-binding, and mutagenesis analysis indicated that the consensus sequence was important for transcriptional activation. Each ExsA-controlled promoter region contained at least two consensus sites in close proximity, similar to the arrangement of half-sites seen in AraC-controlled (Escherichia coli) or VirF-controlled (Yersinia enterocolitica) promoters. However, the results of this study suggested that only one consensus site was required in the Exoenzyme S (pS) or ORF1 promoter (pORF1) to initiate transcription. These data suggest that members of the Exoenzyme S regulon can be defined as possessing an ExsA consensus element which maps at bp -51 or -52 relative to the transcriptional start site.
Narito Morii - One of the best experts on this subject based on the ideXlab platform.
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synergistic activation of rat brain phospholipase d by adp ribosylation factor and rhoa p21 and its inhibition by clostridium botulinum c3 Exoenzyme
Journal of Biological Chemistry, 1995Co-Authors: Hideo Kuribara, Narito Morii, Shuh Narumiya, Kenji Tago, Takeaki Yokozeki, Takuya Sasaki, Yoshimi Takai, Toshiaki Katada, Yasunori KanahoAbstract:Abstract An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), was partially purified from bovine brain cytosol by a series of chromatographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 Exoenzyme ADP-ribosyltransferase, which strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially purified PLD-activating factor with both C3 Exoenzyme and NAD significantly inhibited the PLD-stimulating activity. These results suggest that rhoA p21 is, at least in part, responsible for the PLD-stimulating activity in the preparation. Recombinant isoprenylated rhoA p21 expressed in and purified from Sf9 cells activated rat brain PLD in a concentration- and GTPS (guanosine 5′-O-(3-thiotriphosphate))-dependent manner. In contrast, recombinant non-isoprenylated rhoA p21 (fused to glutathione S-transferase) expressed in Escherichia coli failed to activate the PLD. This difference cannot be explained by a lower affinity of non-isoprenylated rhoA p21 for GTPS, as the rates of [S]GTPS binding were very similar for both recombinant preparations and the GTPS-bound form of non-isoprenylated rhoA p21 did not induce PLD activation. Interestingly, recombinant isoprenylated rhoA p21 and ARF synergistically activated rat brain PLD; a similar pattern was seen with the partially purified PLD-activating factor. The synergistic activation was inhibited by C3 Exoenzyme-catalyzed ADP-ribosylation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner. Inhibition correlated with the extent of ADP-ribosylation. These findings suggest that rhoA p21 regulates rat brain PLD in concert with ARF, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.
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identification of glu173 as the critical amino acid residue for the adp ribosyltransferase activity of clostridium botulinum c3 Exoenzyme
FEBS Letters, 1995Co-Authors: Y Saito, Toshimasa Ishizaki, Narito Morii, Yasuo Nemoto, Naoki Watanabe, Shuh NarumiyaAbstract:Clostridium botulinum C3 Exoenzyme specifically ADP-ribosylates rho-p21 in eukaryotic cells. Trp18 and Glu173 of this enzyme were substituted with other amino acids via site-directed mutagenesis. All substitutions at Glu173 caused a significant reduction in affinity for NAD and diminished ADP-ribosyltransferase activity. On the other hand, the activity of enzymes with the substitution at Trp18 remained intact. Swiss 3T3 cells treated with the enzyme with the Trp18 substitution showed the typical morphologic changes of the C3 Exoenzyme phenotype. In contrast, no changes were found in cells incubated with the Glu173-substituted enzyme. These results indicate that the Glu173 residue of the C3 Exoenzyme plays a key role in interacting with NAD and in expression of ADP-ribosyltransferase activity, which is essential for the phenotypic change by C3 Exoenzyme treatment.
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botulinum c3 Exoenzyme blocks the tyrosine phosphorylation of p125fak and paxillin induced by bombesin and endothelin
FEBS Letters, 1994Co-Authors: Sara Rankin, Narito Morii, Shuh Narumiya, Enrique RozengurtAbstract:In this study we examined the role of rho p21 in neuropeptide-stimulated tyrosine phosphorylation. Intact Swiss 3T3 cells were treated with the Clostridium botulinum C3 Exoenzyme which specifically ADP ribosylates and inactivates rho p21. C3 Exoenzyme treatment of cells caused a marked decrease in both bombesin- and endothelin-stimulated tyrosine phosphorylation of multiple proteins, including p125 focal adhesion kinase (FAK) and paxillin. Our results suggest that rho p21 is a component of the signal transduction pathway linking seven transmembrane domain receptors with tyrosine phosphorylation and cytoskeletal events.
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effect of botulinum c3 Exoenzyme on cell growth and cytoskeleton organization in transformed human epidermal cells in culture a possible role for rho protein in epidermal cells
Journal of Dermatological Science, 1994Co-Authors: Masamitsu Yamamoto, Narito Morii, Kouichi Ikai, Sadao ImamuraAbstract:Abstract We examined the role of rho gene products ( rho proteins) on cell growth and cytoskeleton organization in transformed human epidermal cells in culture (HSC-1), using recombinant botulinum C3 Exoenzyme which specifically ADP-ribosylates rho proteins. Incubation of HSC-1 cell lysates with C3 Exoenzyme revealed a single [ 32 P]ADP-ribosylated protein with a molecular weight of 23 000. This protein was identified as rhoA protein by isoelectric focusing (pI 6.0). Addition of C3 Exoenzyme to the culture medium of HSC-1 cells changed the shape of HSC-1 cells to a round form with beaded processes in a time- and dose-dependent manner. Moreover, C3 treatment reduced the cell growth rate; 72-h treatment with C3 Exoenzyme at 1, 3, 10, 30 and 60 μg/ml culture medium resulted in 9.0 ± 1.8%, 20 ± 2.9%, 26 ± 2.3%, 50 ± 1.4% and 40 ± 2.0% inhibition of the growth rate relative to controls, respectively. Under this condition, actin stress fibers were disassembled, as revealed using fluorescent-labeled phallacidin, whereas keratin intermediate filaments were not affected, visualized by immunofluorescence using anti-keratin antibody. These results suggest that rho proteins are closely-related to cell growth and that these proteins regulate, at least in part, the assembly of actin stress fibers in transformed human epidermal cells.
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adp ribosylation of rho p21 inhibits lysophosphatidic acid induced protein tyrosine phosphorylation and phosphatidylinositol 3 kinase activation in cultured swiss 3t3 cells
Journal of Biological Chemistry, 1993Co-Authors: Naokazu Kumagai, Narito Morii, Yasuo Nemoto, Kazuko Fujisawa, Shuh NarumiyaAbstract:Botulinum C3 Exoenzyme was used to specifically ADP-ribosylate and inactivate rho p21, and the effects of rho p21 inactivation on lysophosphatidic acid (LPA)-induced tyrosine phosphorylation were examined in cultured Swiss 3T3 cells. LPA induced a rapid increase in the tyrosine phosphorylation of a number of proteins. Pretreatment of the cells with the C3 Exoenzyme caused ADP-ribosylation of rho p21 in the cells and selectively attenuated the phosphorylation of several proteins, including p43 mitogen-activated protein kinase, p125 focal adhesion kinase, and two proteins of 72 and 88 kDa. C3 Exoenzyme pretreatment did not block the initial phosphorylation and activation of mitogen-activated protein kinase but suppressed its subsequent rise. In contrast, the enzyme treatment inhibited the induction of phosphorylation of the 72- and 88-kDa proteins and suppressed the basal and LPA-induced tyrosine phosphorylation of p125 focal adhesion kinase. In addition, immunoprecipitation of cell lysates with an antibody directed against the 85-kDa subunit of phosphatidylinositol 3-kinase (PI 3-kinase) co-precipitated a tyrosine-phosphorylated band of 180 kDa. C3 Exoenzyme pretreatment suppressed both the phosphorylation of this band and PI 3-kinase activation associated with LPA stimulation. These findings suggest that rho p21 works as a link between the LPA receptor signal and the subsequent tyrosine phosphorylation and PI 3-kinase activation in these cells.
Ingo Just - One of the best experts on this subject based on the ideXlab platform.
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potentiation of brain derived neurotrophic factor induced protection of spiral ganglion neurons by c3 Exoenzyme rho inhibitor
Frontiers in Cellular Neuroscience, 2021Co-Authors: Jennifer Harre, Ingo Just, Laura Heinkele, Melanie Steffens, Athanasia Warnecke, Thomas Lenarz, Astrid RohrbeckAbstract:Preservation of the excitability of spiral ganglion neurons (SGN) may contribute to an improved speech perception after cochlear implantation. Thus, the application of exogenous neurotrophic factors such as the neurotrophin brain-derived neurotrophic factor (BDNF) to increase SGN survival in vitro and in vivo is a promising pharmacological approach in cochlear implant (CI) research. Due to the difficult pharmacokinetic profile of proteins such as BDNF, there is a quest for small molecules to mediate the survival of SGN or to increase the efficacy of BDNF. The C3 Exoenzyme from Clostridium botulinum could be a potential new candidate for the protection and regeneration of SGN. Inhibition of the RhoA GTPase pathway which can be mediated by C3 is described as a promising strategy to enhance axonal regeneration and to exert pro-survival signals in neurons. Nanomolar concentrations of C3, its enzymatically inactive form C3E174Q, and a 26mer C-terminal peptide fragment covering amino acid 156-181 (C3156-181) potentiated the neuroprotective effect on SGN mediated by BDNF in vitro. The neuroprotective effect of C3/BDNF was reduced to the neuroprotective effect of BDNF alone after the treatment with wortmannin, an inhibitor of the phosphatidylinositol-3-kinase (PI3K).The Exoenzyme C3 (wild-type and enzyme-deficient) and the C3 peptide fragment C3154-181 present novel biologically active compounds for the protection of the SGN. The exact underlying intracellular mechanisms that mediate the neuroprotective effect are not clarified yet, but the combination of BDNF (TrkB stimulation) and C3 Exoenzyme (RhoA inhibition) can be used to protect SGN in vitro.
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uptake of clostridium botulinum c3 Exoenzyme into intact ht22 and j774a 1 cells
Toxins, 2015Co-Authors: Astrid Rohrbeck, Sandra Hagemann, Leonie Von Elsner, Ingo JustAbstract:The Clostridium botulinum C3 Exoenzyme selectively ADP-ribosylates low molecular weight GTP-binding proteins RhoA, B and C. This covalent modification inhibits Rho signaling activity, resulting in distinct actin cytoskeleton changes. Although C3 Exoenzyme has no binding, the translocation domain assures that C3 enters cells and acts intracellularly. C3 uptake is thought to occur due to the high concentration of the C3 enzyme. However, recent work indicates that C3 is selectively endocytosed, suggesting a specific endocytotic pathway, which is not yet understood. In this study, we show that the C3 Exoenzyme binds to cell surfaces and is internalized in a time-dependent manner. We show that the intermediate filament, vimentin, is involved in C3 uptake, as indicated by the inhibition of C3 internalization by acrylamide, a known vimentin disruption agent. Inhibition of C3 internalization was not observed by chemical inhibitors, like bafilomycin A, methyl-β-cyclodextrin, nocodazole or latrunculin B. Furthermore, the internalization of C3 Exoenzyme was markedly inhibited in dynasore-treated HT22 cells. Our results indicate that C3 internalization depends on vimentin and does not depend strictly on both clathrin and caveolae.
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vimentin mediates uptake of c3 Exoenzyme
PLOS ONE, 2014Co-Authors: Astrid Rohrbeck, Anke Schröder, Sandra Hagemann, Gudrun Ahnerthilger, Andreas Pich, Markus Höltje, Ingo JustAbstract:: Clostridium botulinum C3 Exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 Exoenzyme.
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upregulation of the immediate early gene product rhob by Exoenzyme c3 from clostridium limosum and toxin b from clostridium difficile
Biochemistry, 2007Co-Authors: Johannes Huelsenbeck, Ingo Just, Stefanie C Dreger, Ralf Gerhard, Gerhard Fritz, Harald GenthAbstract:ADP-ribosylation of Rho(A,B,C) by the family of Exoenzyme C3-like transferases induces reorganization of the actin cytoskeleton based on inactivation of RhoA. No data are available on the role of RhoB in C3-treated cells. In murine fibroblasts treated with the cell-permeable Exoenzyme C3 from Clostridium limosum (C3), an increase in the level of RhoB was observed. This upregulation of RhoB was based on transcriptional activation, as it was responsive to inhibition by actinomycin D and accompanied by activation of the rhoB promoter. Upregulation of RhoB was not observed in cells treated with either the actin ADP-ribosylating C2 toxin from Clostridium botulinum or latrunculin B, suggesting that inactivation of Rho but not actin reorganization was required for the upregulation of RhoB. This notion was confirmed, as the Rho/Rac/Cdc42-glucosylating toxin B from Clostridium difficile (TcdB) but not the Rac/R-Ras-glucosylating variant toxin B from C. difficile strain 1470 serotype F (TcdBF) induced a strong upre...
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interaction of the rho adp ribosylating c3 Exoenzyme with rala
Journal of Biological Chemistry, 2002Co-Authors: Christian Wilde, Ingo Just, Holger Barth, Peter Sehr, Martina Schmidt, Klaus AktoriesAbstract:Abstract RhoA, -B, and -C are ADP-ribosylated and biologically inactivated by Clostridium botulinum C3 Exoenzyme and related C3-like transferases. We report that RalA GTPase, which is not ADP-ribosylated by C3, inhibits ADP-ribosylation of RhoA by C3 from C. botulinum (C3bot), Clostridium limosum (C3lim), and Bacillus cereus (C3cer) but not from Staphylococcus aureus (C3stau) in human platelet membranes and rat brain lysate. Inhibition by RalA occurs with the GDP- and guanosine 5′-3-O-(thio)triphosphate-bound forms of RalA and is overcome by increasing concentrations of C3. A direct interaction of RalA with C3 was verified by precipitation of the transferase with GST-RalA-Sepharose. The affinity constant (K d) of the binding of RalA to C3lim was 12 nm as determined by fluorescence titration. RalA increased the NAD glycohydrolase activity of C3bot by about 5-fold. Although RalA had no effect on glucosylation of Rho GTPases by Clostridium difficile toxin B, C3bot and C3lim inhibited glucosylation of RalA by Clostridium sordellii lethal toxin. Furthermore, C3bot decreased activation of phospholipase D by RalA. The data indicate that several C3 Exoenzymes directly interact with RalA without ADP-ribosylating the GTPase. The interaction is of high affinity and interferes with essential functions of C3 and RalA.
Klaus Aktories - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the c3bot rala complex reveals a novel type of action of a bacterial Exoenzyme
The EMBO Journal, 2005Co-Authors: Alexander Pautsch, Martin Vogelsgesang, Jens Trankle, Christian Herrmann, Klaus AktoriesAbstract:C3 Exoenzymes from bacterial pathogens ADP-ribosylate and inactivate low-molecular-mass GTPases of the Rho subfamily. Ral, a Ras subfamily GTPase, binds the C3 Exoenzymes from Clostridium botulinum and C. limosum with high affinity without being a substrate for ADP ribosylation. In the complex, the ADP-ribosyltransferase activity of C3 is blocked, while binding of NAD and NAD-glycohydrolase activity remain. Here we report the crystal structure of C3 from C. botulinum in a complex with GDP-bound RalA at 1.8 A resolution. C3 binds RalA with a helix-loop-helix motif that is adjacent to the active site. A quaternary complex with NAD suggests a mode for ADP-ribosyltransferase inhibition. Interaction of C3 with RalA occurs at a unique interface formed by the switch-II region, helix alpha3 and the P loop of the GTPase. C3-binding stabilizes the GDP-bound conformation of RalA and blocks nucleotide release. Our data indicate that C. botulinum Exoenzyme C3 is a single-domain toxin with bifunctional properties targeting Rho GTPases by ADP ribosylation and Ral by a guanine nucleotide dissociation inhibitor-like effect, which blocks nucleotide exchange.
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interaction of the rho adp ribosylating c3 Exoenzyme with rala
Journal of Biological Chemistry, 2002Co-Authors: Christian Wilde, Ingo Just, Holger Barth, Peter Sehr, Martina Schmidt, Klaus AktoriesAbstract:Abstract RhoA, -B, and -C are ADP-ribosylated and biologically inactivated by Clostridium botulinum C3 Exoenzyme and related C3-like transferases. We report that RalA GTPase, which is not ADP-ribosylated by C3, inhibits ADP-ribosylation of RhoA by C3 from C. botulinum (C3bot), Clostridium limosum (C3lim), and Bacillus cereus (C3cer) but not from Staphylococcus aureus (C3stau) in human platelet membranes and rat brain lysate. Inhibition by RalA occurs with the GDP- and guanosine 5′-3-O-(thio)triphosphate-bound forms of RalA and is overcome by increasing concentrations of C3. A direct interaction of RalA with C3 was verified by precipitation of the transferase with GST-RalA-Sepharose. The affinity constant (K d) of the binding of RalA to C3lim was 12 nm as determined by fluorescence titration. RalA increased the NAD glycohydrolase activity of C3bot by about 5-fold. Although RalA had no effect on glucosylation of Rho GTPases by Clostridium difficile toxin B, C3bot and C3lim inhibited glucosylation of RalA by Clostridium sordellii lethal toxin. Furthermore, C3bot decreased activation of phospholipase D by RalA. The data indicate that several C3 Exoenzymes directly interact with RalA without ADP-ribosylating the GTPase. The interaction is of high affinity and interferes with essential functions of C3 and RalA.
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clostridium botulinum c3 Exoenzyme and c3 like transferases
Handbook of experimental pharmacology, 2000Co-Authors: Klaus Aktories, Holger Barth, Ingo JustAbstract:Clostridium botulinum adenosine diphosphate (ADP)—ribosyltransferase C3 was the first bacterial Exoenzyme/toxin found to act on small guanosine triphosphate (GTP)ases of the Rho GTPase family. Subsequently, it has been realised that C3 is the prototype of a family of related ADP—ribosyltransferases (C3-like transferases) produced by a diverse group of bacteria, such as C. botulinum, C. limosum, Bacillus cereus and Staphylococcus aureus. Whereas the role of all these agents as virulence factors is still unclear, their importance as pharmacological tools is beyond question. Because C3-like Exoenzymes specifically inactive Rho proteins, they are essential for the elucidation of the cellular functions of Rho GTPases. Here, we will review our current knowledge about C. botulinum C3 transferase and other C3-like transferases and will describe their origins, structures and functions. Moreover, another topic of this chapter will be the application of C3 as a pharmacological and cell-biological tool. Other bacterial agents targeting Rho GTPases are the large clostridia) cytotoxins and the group of deamidating toxins from Escherichia coli and Bordetella species, which are discussed in detail in other chapters of this volume.
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A role for Rho in receptor- and G protein-stimulated phospholipase C Reduction in phosphatidylinositol 4,5-bisphosphate by Clostridium difficile toxin B
Naunyn-Schmiedeberg's Archives of Pharmacology, 1996Co-Authors: Martina Schmidt, Ingo Just, Klaus Aktories, Christine Bienek, Ulrich Rümenapp, Chunyi Zhang, Gerd Lümmen, Karl H. Jakobs, Michael Moos, Christoph Eichel-streiberAbstract:Receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P_2)-hydrolyzing phospholipase C (PLC) enzymes by activated α or free βγ subunits of the relevant G proteins. To study whether low molecular weight G proteins of the Rho family are involved in receptor signalling to PLC, we examined the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates Rho proteins, on the regulation of PLC activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) subtype. Toxin B treatment of HEK cells did not affect basal PLC activity, but potently and efficiently inhibited mAChR-stimulated inositol phosphate formation. PLC activation by the endogenously expressed thrombin receptor and by the direct G protein activators, AlF _inf4 ^sup− and guanosine 5′-[γ-thio]triphosphate (GTPγS), studied in intact and permeabilized cells, respectively, were also inhibited by toxin B treatment. C3 Exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTPγS-stimulated PLC activity. Finally, both toxin B and C3 Exoenzyme significantly reduced, by 40 to 50%, the total level of PtdIns(4,5)P_2 in HEK cells, without affecting the levels of phosphatidylinositol and phosphatidylinositol 4-phosphate. Accordingly, when PLC activity was measured with exogenous PtdIns(4,5)P_2 as enzyme substrate, Ca^2+- as well as GTPγS- or A1F _inf4 ^sup− -stimulated PLC activities were not altered by prior toxin B treatment. In conclusion, evidence is provided that toxin B and C3 Exoenzyme, apparently by inactivating Rho proteins, inhibit G protein-coupled receptor signalling to PLC, most likely by reducing the cellular substrate supply.