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K Aktories

Publications and source records attributed to K Aktories.

At least 19 recordsLinked to original sources
PubMed1996

Inactivation of Ras by Clostridium sordellii lethal toxin-catalyzed glucosylation.

The lethal toxin (LT) from Clostridium sordellii belongs to the family of large clostridial cytotoxins causing morphological alterations in cultured cell lines accompanied by destruction of the actin cytoskeleton. C. sordellii LT exhibits 90% homology to Clostridium difficile toxin B, which has been recently identified as a monoglucosyltransferase (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). We report here that LT too is a glucosyltransferase, which uses UDP-glucose as cosubstrate to modify low molecular mass GTPases. LT selectively modifies Rac and Ras, whereas the substrate specificity of toxin B is confined to the Rho subfamily proteins Rho, Rac, and Cdc42, which participate in the regulation of the actin cytoskeleton. In Rac, both toxin B and LT share the same acceptor amino acid, threonine 35. Glucosylation of Ras by LT results in inhibition of the epidermal growth factor-stimulated p42/p44 MAP-kinase signal pathway. LT is the first bacterial toxin to inactivate Ras in intact cells.

3T3 Cells↗
PubMed1996

Inhibition of Fc epsilon-RI-mediated activation of rat basophilic leukemia cells by Clostridium difficile toxin B (monoglucosyltransferase)

Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [3H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL cells, toxin B 14C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 microg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [3H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.

2,4-Dinitrophenol↗
PubMed1996

Analysis of the catalytic site of the actin ADP-ribosylating Clostridium perfringens iota toxin.

The enzyme component of actin ADP-ribosylating Clostridium perfringens iota toxin was affinity labelled by UV irradiation in the presence of [carbonyl-14C]NAD. A peptide containing the radiolabel was generated by CNBr cleavage and subsequent proteolysis with trypsin. Its amino acid sequence is Gly-Ser-Pro-Gly-Ala-Tyr-Leu-Ser-Ala-Ile-Pro-Gly-Tyr-Ala-Gly-X-Tyr-Glu-Va l-Leu-Leu-Asn-His-Gly-Ser-Lys corresponding with the region Gly-363 through Lys-388 in the C. perfringens iota toxin. Mass spectrometric data as well as results of the PTH-amino acid analysis are in line with a modification of a glutamic acid side chain located at position 378. Therefore, in addition to Glu-380, as could be concluded by analogy with other ADP-ribosyltransferases, Glu-378 may play a pivotal role in the active site of C. perfringens iota toxin.

ADP Ribose Transferases↗
PubMed1996

Active site mutation of the C3-like ADP-ribosyltransferase from Clostridium limosum--analysis of glutamic acid 174.

Clostridium limosum ADP-ribosyltransferase modifies low molecular mass GTP-binding proteins of the Rho subtype family. Here we cloned and sequenced the gene of the transferase and expressed it in Escherichia coli. The gene encodes a protein of 250 amino acids (M(r) = 27,840), with a putative signal peptide of 45 amino acids, that shows about 60-65% identity with C3 transferases from Clostridium botulinum. The mature C. limosum transferase was expressed as a maltose-binding fusion protein in E. coli and purified to apparent homogeneity. To study the functional role of Glu174 of C. limosum transferase, which was recently photoaffinity-labeled with [carbonyl-14C]NAD [Jung, M., et al. (1993) J. Biol. Chem. 268, 23215-23218], two mutants E174D and E174Q were constructed by a polymerase chain reaction-based system. The E174D and E174Q mutants showed a dramatic decrease in kcat, but no major changes in Km,NAD. Furthermore, replacement of Glu174 by aspartic acid and glutamine largely reduced and completely blocked UV-induced incorporation of [carbonyl-14C]NAD into the transferase. The data indicate that Glu174 is an active site residue of C. limosum transferase.

ADP Ribose Transferases↗
PubMed1996

ADP-ribosylation of an approximately 70-kilodalton protein of Klebsiella pneumoniae.

An approximately 70-kDa protein in the culture supernatant of a human pathogenic strain of Klebsiella pneumoniae was labeled in the presence of [32P-adenylate]NAD. Labeling was significantly increased by the addition of dithiothreitol ( > 1 mM) but prevented by treatment of the culture supernatant for 3 min at 56 degrees C. The addition of unlabeled NAD, but not of ADP-ribose, blocked labeling of the approximately 70-kDa protein. The radioactive label was released by formic acid but not by HgCl2 (1 mM) or neutral hydroxylamine (0.5 M). The addition of homogenates of human platelets, human neutrophils, rat brain, rat lung, or rat spleen tissues to the culture supernatant did not induce labeling of eukaryotic proteins. The data indicate that the K. pneumoniae strain produces ADP-ribosyltransferase which modifies an endogenous protein.

Adenosine Diphosphate Ribose↗
PubMed1995

The ras-related small GTP-binding protein RhoB is immediate-early inducible by DNA damaging treatments.

The low molecular weight GTP-binding proteins RhoA, RhoB, and RhoC are characterized as specific substrates for the ADP-ribosyltransferase C3 from Clostridium botulinum and are supposed to be involved in the organization of the microfilamental network and transformation. rhoB is known to be immediate-early inducible by growth factors and protein-tyrosine kinases. Since increasing evidence indicates overlapping of growth factor- and UV-induced signal pathways, we studied the effect of UV light and other genotoxic agents on early rhoB transcription. Within 30 min after UV irradiation of NIH3T3 cells, the amount of rhoB mRNA increased 3-4-fold. Elevated rhoB mRNA was accompanied by an increase in RhoB protein, as detected by C3-mediated [32P]ADP-ribosylation. The transcription inhibitor actinomycin D prevented the UV-induced increase in rhoB mRNA and proved rhoB mRNA to be unstable with a half-life of approximately 20 min. Transcriptional activation of rhoB by UV light was confirmed by run-on analysis. The increase in rhoB mRNA after UV irradiation was prevented by inhibitors of protein kinase A (H9) and C (H7, Gö18). The tyrosine kinase inhibitor genistein did not affect UV induction of rhoB. In addition to UV, N-methyl-N-nitrosourea and the cytostatic drug cisplatin evoked rhoB response. Cycloheximide was likewise effective in increasing the amount of rhoB mRNA, whereas Bt2cAMP, 12-O-tetradecanoylphorbol-13-acetate, and retinoic acid were without effect. Prior down-regulation of signaling by 12-O-tetradecanoylphorbol-13-acetate and serum pretreatment reduced UV-stimulated rhoB expression. The data indicate that rhoB represents a novel DNA damage-inducible function involved in early steps of signal transduction upon genotoxic stress.

3T3 Cells↗
PubMed1995

The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins.

The enterotoxin from Clostridium difficile (ToxA) is one of the causative agents of the antibiotic-associated pseudomembranous colitis. In cultured monolayer cells ToxA exhibits cytotoxic activity to induce disassembly of the actin cytoskeleton, which is accompanied by morphological changes. ToxA-induced depolymerization of actin filaments is correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, K. (1995) J. Clin. Invest. 95, 1026-1031). Here we report on the identification of the ToxA-induced modification of Rho. Applying electrospray mass spectrometry, the mass of the modification was determined as 162 Da, which is consistent with the incorporation of a hexose into Rho. From several hexoses tested UDP-glucose selectively served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated peptide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (RhoA, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, and Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [14C]glucose, indicating previous glucosylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA mediates its cytotoxic effects on cells.

Amino Acids↗
PubMed1995

Glucosylation of Rho proteins by Clostridium difficile toxin B.

Toxin A and B, the major virulence factors of Clostridium difficile, are the causative agents of antibiotic-associated pseudomembranous colitis. In cultured cell lines their potent cytotoxicity results from their ability to induce disaggregation of the microfilament cytoskeleton. Toxin B acts on the low-molecular-mass GTPase RhoA, which is involved in the regulation of the actin cytoskeleton. We report here that toxin B catalyses the incorporation of up to one mole of glucose per mole of RhoA at the amino acid threonine at position 37. The modification was identified and localized by tandem electrospray mass spectrometry. UDP-glucose selectively serves as cosubstrate for the monoglucosylation reaction catalysed by toxin B. Microinjection of RhoA previously glucosylated by toxin B into monolayer cells caused disaggregation of actin filaments, indicating a dominant-negative activity of glucosylated RhoA.

Actins↗
PubMed1995

Nerve growth factor triggers microfilament assembly and paxillin phosphorylation in human B lymphocytes.

Increasing evidence suggests that the nervous system is involved in allergic inflammation. One of the potential regulatory molecules of the neuroimmune system is nerve growth factor (NGF). Recent studies from our group demonstrated the presence of a functional NGF receptor (NGFR) on human B lymphocytes. Moreover, we showed that gp140trk tyrosine kinase, which serves as an NGFR, was involved in transduction of early signaling events in human B lymphocytes. The mechanisms by which NGF initiates the signaling cascade and the link between the neuroimmune systems are unknown. We have focused on the role of the cytoskeleton as a possible mediator for transduction of signals induced by NGF. Polymerized actin (F-actin) content was determined by fluorescent staining and immunoblotting with antiactin antibody. Addition of NGF caused a time- and concentration-dependent increase in F-actin content, and maximum effects were noted after 1 min. These increases in F-actin content and NGF-induced thymidine incorporation could be blocked by incubating the cells with cytochalasin D and botulinum C2 toxin before the addition of NGF. Incubation of human B lymphocytes with 10 nM K252a, an inhibitor of Trk kinase, decreased NGF-induced microfilament assembly by 75%. In immunoprecipitation experiments, addition of NGF to B cells induced a rapid increase in the tyrosine phosphorylation of paxillin, one of a group of focal adhesion proteins involved in linking actin filaments to the plasma membrane. Coimmunoprecipitation studies demonstrated the association between gp140trk kinase and paxillin. Together, these observations suggest that actin assembly is involved in NGF signaling in human B cells, and that paxillin may be essential in this pathway after phosphorylation by gp140trk kinase.

Actin Cytoskeleton↗
PubMed1995

Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton.

Using antigen-specific T cell clones and peptide-pulsed antigen-presenting cells (APCs) we investigated the mechanisms that lead to sustained signaling, known to be required for activation of effector function. Four lines of evidence indicate that the T cell actin cytoskeleton plays a crucial role in T cell activation by antigen-pulsed APCs, but is not required when T cell receptor (TCR) is cross-linked by soluble antibodies. First, addition of antibodies to the major histocompatibility complex molecules recognized by the TCR aborts the ongoing intracellular calcium concentration ([Ca2+]i) increase in performed T-APC conjugates, indicating that the sustained signaling requires the continuous occupancy of TCR. Second, time-lapse image recording shows that T lymphocytes conjugated to peptide-pulsed APCs undergo a sustained [Ca2+]i increase, which is accompanied by the formation of a large and changing area of contact between the two opposing membranes. Third, drugs that disrupt the actin cytoskeleton, Cytochalasin D and and C2 Clostridium botulinum toxin induce a rapid block of [Ca2+]i rise, coincident with a block of the cyclic changes in T cell shape. Finally, the addition of Cytochalasin D or of anti-MHC antibodies to preformed conjugates inhibits interferon gamma production in an 1-antigen dose- and time-dependent fashion. These results identify T cell actin cytoskeleton as a major motor for sustaining signal transduction and possibly for driving TCR cross-linking and offer an explanation for how T cells equipped with low affinity TCR can be triggered by a small number of complexes on APCs.

Actins↗
PubMed1995

ADP-ribosyltransferase type A from turkey erythrocytes modifies actin at Arg-95 and Arg-372.

Turkey erythrocyte ADP-ribosyltransferase A catalyzes the transfer of ADP-ribose from NAD to both monomeric and polymeric skeletal muscle alpha-actin with the incorporation of 2 mol of ADP-ribose per mol of actin. In contrast, Clostridium perfringens iota toxin ADP-ribosylates only G-actin, with modification at arginine-177 [Vandekerckhove, J., et al. (1987) FEBS Lett. 255, 48-42]. Transferase A-catalyzed modifications are sensitive to 0.5 M neutral hydroxylamine, consistent with the arginine side chain modification. Radiolabeled peptides ADP-ribosylated by transferase A were generated by tryptic digestion and purified by reversed phase high-performance liquid chromatography. Amino acid sequence and molecular mass analysis identified the ADP-ribosylation sites as Arg-95 and Arg-372 of actin; both residues are located within subdomain-1 of the actin 3D structure [Kabsch, W., et al. (1990) Nature 347, 37-44]. ADP-ribosylation did not affect cytochalasin D-stimulated G-actin ATPase, the binding of actin to DNase I or to gelsolin, or the ability of actin to polymerize. Following ADP-ribosylation, however, a prolonged delay in polymerization was observed, consistent with a decreased rate of nucleation.

Actins↗
PubMed1995

Rho-ADP-ribosylating exoenzyme from Bacillus cereus. Purification, characterization, and identification of the NAD-binding site.

The ADP-ribosyltransferase produced by a pathogenic strain of Bacillus cereus was purified to near homogeneity. The transferase is a 28,000 Da molecular mass enzyme with a pI of 10.3. The specific enzyme activity is 7.0 nmol of ADP-ribose min-1 mg-1 with a Km for NAD of 0.3 microM. Partial amino acid sequence analysis of the exoenzyme reveals no significant homology to Clostridium botulinum C3 nor to Clostridium limosum exoenzyme. The novel exoenzyme selectively modifies the small GTP-binding proteins of the Rho family presumably at the same acceptor amino acid (Asn-41) as determined for C3. Besides cellular Rho, recombinant RhoA and -B are substrates for the exoenzyme. However, recombinant Rac1 and CDC42, although belonging to the Rho family, are not modified. B. cereus exoenzyme was photolabeled with [carbonyl-14C]NAD resulting in inhibition of ADP-ribosyltransferase and NAD-glycohydrolase activity. A glutamic acid residue was identified as part of the NAD-binding site which corresponds to Glu-174 of C3. This glutamic acid is located in a domain which shows high homology with the C-terminal part of C3 exoenzyme, C. limosum exoenzyme, and Staphylococcus aureus EDIN and which probably represents the catalytic site of the transferases. The data indicate that B. cereus exoenzyme is a novel member of the family of C3-like ADP-ribosyltransferases which share the same substrate protein Rho and which have an identical highly conserved catalytic domain.

ADP Ribose Transferases↗
PubMed1995

Studies on the active-site structure of C3-like exoenzymes: involvement of glutamic acid in catalysis of ADP-ribosylation.

Various C3-like ADP-ribosyltransferases like Clostridium botulinum exoenzyme C3, C limosum transferase, B cereus transferase and a transferase from Staphylococcus aureus (EDIN) selectively modify the low-molecular mass GTP-binding proteins RhoA,B,C. UV-irradiation of C limosum transferase in the presence of [carbonyl-14C]NAD resulted in radiolabeling of Glu-174. Concomitantly, ADP-ribosyltransferase and NAD glycohydrolase activities were inhibited. Site-directed mutagenesis of Glu-174 (E174D, E174Q) which resulted in more than 1000-fold reduction of enzyme activity, suggests that the glutamic acid residue is essentially involved in the catalytic action of C3-like transferases. These findings support the view that all bacterial ADP-ribosyltransferases share a similar active-site structure.

ADP Ribose Transferases↗
PubMed1995

Involvement of the GTP binding protein Rho in constitutive endocytosis in Xenopus laevis oocytes.

To study an endocytotic role of the GTP-binding protein RhoA in Xenopus oocytes, we have monitored changes in the surface expression of sodium pumps, the surface area of the oocyte and the uptake of the fluid-phase marker inulin. Xenopus oocytes possess intracellular sodium pumps that are continuously exchanged for surface sodium pumps by constitutive endo- and exocytosis. Injection of Clostridium botulinum C3 exoenzyme, which inactivates Rho by ADP-ribosylation, induced a redistribution of virtually all intracellular sodium pumps to the plasma membrane and increased the surface area of the oocytes. The identical effects were caused by injection of ADP-ribosylated recombinant RhoA into oocytes. The C3 exoenzyme acts by blocking constitutive endocytosis in oocytes, as determined using a mAb to the beta 1 subunit of the mouse sodium pump as a reporter molecule and oocytes expressing heterologous sodium pumps. In contrast, an increase in endocytosis and a decrease in the surface area was induced by injection of recombinant Val14-RhoA protein or Val14-rhoA cRNA. PMA stimulated sodium pump endocytosis, an effect that was blocked by a specific inhibitor of protein kinase C (Gö 16) or by ADP-ribosylation of Rho by C3. Similarly, the phorbol ester-induced increase in fluid-phase endocytosis in oocytes was inhibited by Gö 16, C3 transferase, or by injection of ADP-ribosylated RhoA. In contrast to C3 transferase, C. botulinum C2 transferase, which ADP-ribosylates actin, had no effect on sodium pump endocytosis or PMA-stimulated fluid-phase endocytosis. The data suggests that RhoA is an essential component of a presumably clathrin-independent endocytic pathway in Xenopus oocytes which can be regulated by protein kinase C.

ADP Ribose Transferases↗
PubMed1995

Tumour necrosis factor-alpha-mediated dyscohesion of epithelial cells is associated with disordered expression of cadherin/beta-catenin and disassembly of actin filaments.

Tumour necrosis factor (TNF)-alpha induced, in a time- and dose-dependent fashion, dyscohesion (cell-cell dissociation) of the endometrial epithelial cells. TNF-alpha impaired the ability of cells to aggregate and to attain compaction. The cell-cell adherent junction is a specialized region of the plasma membrane where cadherin molecules act as adhesion molecules and actin filaments are densely associated with the plasma membrane through a well-developed plasmalemmal undercoat. Dyscohesion induced by TNF-alpha was associated with the disordered expression of cadherin/beta-catenin at the sites of cell-cell contact. In addition, within the time-frame that dyscohesion was induced, TNF-alpha down-regulated the expression of actin mRNA only at 100 ng/ml without modulating the overall amount of actin protein, its beta-isoform or the amount of ribosylated actin. However, TNF-alpha-mediated dyscohesion of epithelial cells was associated with loss of plasmalemmal undercoat as well as intracytoplasmic aggregates of F-actin and a simultaneous increase in G-actin. The effect of cytochalasin-B, which disrupts actin filaments on cell-cell binding, was less pronounced than the effect of TNF-alpha, suggesting that the effect of this cytokine on dyscohesion is not solely dependent on the disassembly of actin filaments. These findings show that the induction of disordered expression of adhesion molecules, as well as disassembly of actin filaments, are implicated in the dyscohesion induced by TNF-alpha.

Actins↗
PubMed1995

Isolation and characterization of a Clostridium botulinum C2 toxin-resistant cell line: evidence for possible involvement of the cellular C2II receptor in growth regulation.

Clostridium botulinum C2 toxin, which consists of the binding component C2II and the enzyme component C2I, acts on eukaryotic cells by selective ADP-ribosylation of G-actin. To obtain C2 toxin-resistant cells, we mutagenized CHO-K1 cells with N-nitroso-N-methylurea and selected for C2 resistance. Cells which survived the selection procedure with 50 ng of C2I and 100 ng of C2II per ml were obtained with a frequency of 30 x 10(-6). The colony-forming ability of CHO wild-type cells was reduced to 50% with 10 ng of C2I and 20 ng of C2II per ml. In contrast, the colony-forming ability of the isolated CHO mutant cells was not influenced by up to 200 ng of C2I and 400 ng of C2II per ml. Toxin-induced ADP-ribosylation of G-actin was not impaired in lysates of mutant cells. The C2 toxin-resistant phenotype remained sensitive to the cell-rounding activities of cytotoxins from C. perfringens (iota-toxin), C. novyi, C. difficile, and C. botulinum (C3) and to cytochalasin D. Binding of component C2II was impaired in resistant CHO cells, suggesting mutation of the toxin cell surface receptor. Serum factors protected wild-type cells against the cytotoxic effect of C2 toxin. Furthermore, the C2-resistant phenotype correlated with an increased serum dependency. The data suggest that the action of C. botulinum C2 toxin is mediated by its binding and uptake via a cell surface receptor which might be involved in growth regulation.

ADP Ribose Transferases↗
PubMed1995

Role of endothelial cytoskeleton in high-permeability edema due to botulinum C2 toxin in perfused rabbit lungs.

The cytoskeleton of the endothelial cell has been suggested to regulate endothelial barrier function. We investigated the role of actin in the maintenance of pulmonary capillary integrity in perfused rabbit lungs. As a tool for selective perturbation of actin, we employed Clostridium botulinum C2 toxin, which is composed of a membrane translocation component (C2II) and a component (C2I) effecting ADP-ribosylation of nonmuscle G-actin. ADP-ribosylated actin no longer capable of polymerization but acts as a barbed end-capping protein, thereby effecting selective loss of the nonmuscle F-actin content. In buffer-perfused rabbit lungs, combined application of both toxin components (range 50 pg/ml-5 ng/ml C2I) resulted in a time- and dose-dependent increase in the capillary filtration coefficient (Kfc) with concomitant edema formation. Only 300:600 pg/ml C2I:II sufficed to induce a > 10-fold rise of Kfc values within 110 min. This severe lung permeability increase occurred in the absence of vasomotor responses and potassium release or lactate dehydrogenase release. Application of each single toxin component displayed markedly reduced efficacy. Similar to the C2 toxin effect, severe permeability increase without concomitant hemodynamic changes was evoked by cytochalasin D, known to possess F-actin-disrupting properties. Preloading of lung cells with phallacidin, which in opposition to C2 toxin decreases F-actin depolymerization, significantly reduced the C2 toxin-induced increase in vascular permeability. Electron microscopic examination of C2 toxin-poisoned lungs showed early, extensive endothelial cell attenuations, followed by disruptions of the endothelial layer and marked interstitial edema formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗