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K Aktories

Publications and source records attributed to K Aktories.

At least 73 records · Page 4Linked to original sources
PubMed1998

A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins.

A fragment of the N-terminal 546 amino acid residues of Clostridium sordellii lethal toxin possesses full enzyme activity and glucosylates Rho and Ras GTPases in vitro. Here we identified several amino acid residues in C. sordellii lethal toxin that are essential for the enzyme activity of the active toxin fragment. Exchange of aspartic acid at position 286 or 288 with alanine or asparagine decreased glucosyltransferase activity by about 5000-fold and completely blocked glucohydrolase activity. No enzyme activity was detected with the double mutant D286A/D288A. Whereas the wild-type fragment of C. sordellii lethal toxin was labeled by azido-UDP-glucose after UV irradiation, mutation of the DXD motif prevented radiolabeling. At high concentrations (10 mM) of manganese ions, the transferase activities of the D286A and D288A mutants but not that of wild-type fragment were increased by about 20-fold. The exchange of Asp270 and Arg273 reduced glucosyltransferase activity by about 200-fold and blocked glucohydrolase activity. The data indicate that the DXD motif, which is highly conserved in all large clostridial cytotoxins and also in a large number of glycosyltransferases, is functionally essential for the enzyme activity of the toxins and may participate in coordination of the divalent cation and/or in the binding of UDP-glucose.

Amino Acid Sequence↗
PubMed1998

The Rho-deamidating cytotoxic necrotizing factor 1 from Escherichia coli possesses transglutaminase activity. Cysteine 866 and histidine 881 are essential for enzyme activity.

Recently, it has been reported that cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli induces formation of stress fibers by deamidation of glutamine 63 of RhoA (Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., and Aktories, K. (1997) Nature 387, 725-729); Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C., and Boquet, P. (1997) Nature 387, 729-733). By using mass spectrometric analysis, we show now that the toxin transfers ethylenediamine, putrescine, and dansylcadaverine specifically onto glutamine 63 of RhoA. RhoA was also a substrate for guinea pig liver transglutaminase, which modified not only glutamine 63, but also glutamine residues at positions 52 and 136. Treatment of the fully active N-terminal fragment of CNF1 (amino acid residues 709-1014) with iodoacetamide inhibited both deamidation and transglutamination activities. Moreover, exchange of cysteine 866 with serine blocked the enzyme activity of the N-terminal CNF1 fragment. In addition, we identified histidine 881 to be essential for the enzyme activity of CNF1. The data indicate that CNF1 shares a catalytic dyad of cysteine and histidine residues with eukaryotic transglutaminases and cysteine proteases.

Amides↗
PubMed1998

Glucosylation and ADP ribosylation of rho proteins: effects on nucleotide binding, GTPase activity, and effector coupling.

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 GTPgammaS release from Rho proteins was increased 3-6-fold by glucosylation. ADP ribosylation decreased the rate of GTPgammaS 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 ( approximately 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 diminished its ability to support the activation of the superoxide-generating NADPH oxidase of phagocytes. Glucosylated Rac1 did not interfere with NADPH oxidase activation by unmodified Rac1, even when present in marked molar excess, indicating that it was incapable of competing for a common effector. The data indicate that the functional inactivation of small GTPases by glucosylation is mainly caused by inhibition of GTPase-effector protein interaction.

ADP Ribose Transferases↗
PubMed1998

Clostridium difficile toxin B inhibits carbachol-induced force and myosin light chain phosphorylation in guinea-pig smooth muscle: role of Rho proteins.

1. Clostridium difficile toxin B glucosylates the Ras-related low molecular mass GTPases of the Rho subfamily thereby inactivating them. In the present report, toxin B was applied as a tool to test whether Rho proteins participate in the carbachol-induced increase in the Ca2+ sensitivity of force and myosin light chain (MLC) phosphorylation in intact intestinal smooth muscle. 2. Small strips of the longitudinal muscle of guinea-pig small intestine were incubated in toxin B (40 ng ml-1) overnight. Carbachol-induced force and intracellular [Ca2+], and, in a separate series, force and MLC phosphorylation, were determined. 3. Carbachol induced a biphasic contraction: an initial rapid increase in force (peak 1) followed by a partial relaxation and a second delayed increase in force (peak 2). The peak of the Ca2+ signal measured with fura-2 preceded peak 1 of force and then declined to a lower suprabasal steady-state level. Peak 2 was not associated with a significant increase in [Ca2+]. Toxin B nearly completely inhibited peak 2 while peak 1 was not significantly inhibited. Toxin B had no effect on the Ca2+ transient. 4. In control strips, MLC phosphorylation at peak 2 was 27.7% which was significantly higher than the resting value (18.6%). The inhibition of the second, delayed, rise in force induced by toxin B was associated with complete inhibition of the increase in MLC phosphorylation. The resting MLC phosphorylation was not significantly different from that of the control strips. 5. The initial increase in MLC phosphorylation determined 3 s after exposure to carbachol was 54% in the control strips. Toxin B also inhibited this initial phosphorylation peak despite the fact that the Ca2+ transient and the initial increase in force were not inhibited by toxin B. This suggests that Rho proteins play an important role in setting the balance between MLC phosphorylation and dephosphorylation reactions even at high levels of intracellular Ca2+. 6. These findings are consistent with the hypothesis that the delayed rise in force elicited by carbachol is due to an increase in the Ca2+ sensitivity of MLC phosphorylation mediated by Rho proteins.

Adenosine Diphosphate Ribose↗
PubMed1998

Influence of Clostridium botulinum C2 toxin on Fc epsilonRI-mediated secretion and tyrosine phosphorylation in RBL cells.

We studied the effects of the binary Clostridium botulinum C2 toxin on stimulated [3H]serotonin release and protein tyrosine phosphorylation in RBL 2H3 hm1 cells. Actin was specifically ADP-ribosylated by C2 toxin in intact cells resulting in a 2-3 fold increase in antigen- or calcium ionophore (A23187)-induced degranulation. The effects of C2 toxin were time- and concentration-dependent. Toxin treatment, which dramatically changes the morphology of RBL cells, was not sufficient to induce mediator release in the absence of activators of secretion. Antigen- and A23187-stimulated tyrosine phosphorylation of 60-80 kDa and 110-120 kDa proteins was reduced or blocked after C2 toxin incubation. Treatment of RBL cells with the tyrosine phosphatase inhibitor pervanadate reversed the inhibitory effect of C2 toxin on stimulated protein tyrosine phosphorylation indicating activation of phosphatases by C2 toxin. The data indicate that disassembly of the actin cytoskeleton by C2 toxin facilitates Fc epsilonRI-mediated signal-secretion coupling and suggest a role of the actin cytoskeleton in phosphatase regulation in RBL cells.

Actins↗
PubMed1998

Inhibition of insulin-stimulated glucose transport in 3T3-L1 cells by Clostridium difficile toxin B, Clostridium sordellii lethal toxin, and Clostridium botulinum C2 toxin.

The role of the actin cytoskeleton and/or GTPases of the Rho/Rac-family in glucose transport regulation was investigated in 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho/Rac (Clostridium difficile toxin B and Clostiridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake rates in 3T3-L1 fibroblasts. In parallel, the toxins produced morphological alterations of the cells reflecting disruption of the actin cytoskeleton. Both toxins reduced the maximum response to insulin but failed to alter the half-maximally stimulating concentrations of insulin. In 3T3-L1 adipocytes, the lethal toxin reduced the effect of insulin on 2-DOG uptake, whereas toxin B and C2 failed to affect glucose transport or cell morphology. When cells were exposed to the toxins after treatment with insulin, both toxin B and the lethal toxin, in contrast to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, failed to reduce the 2-DOG uptake rates. Thus, both translocation to the plasma membrane and internalization of glucose transporters were inhibited by the toxins, whereas the PI 3-kinase inhibitor selectively affects translocation. The data suggest that the effects of the clostridial toxins on trafficking of glucose transporters are mediated by the depolymerization of the actin cytoskeleton and are an indirect consequence of Rho or Rac inactivation. It is suggested that pathways signalling through Rac or Rho may play a modulatory role in glucose transport regulation through their effects on the actin network.

3T3 Cells↗
PubMed1998

Bacterial cytotoxins target Rho GTPases.

Low molecular mass GTPases of the Rho family, which are involved in the regulation of the actin cytoskeleton and in various signal transduction processes, are the eukaryotic targets of bacterial protein toxins. The toxins covalently modify Rho proteins by ADP ribosylation, glucosylation, and deamidation, thereby inactivating and activating the GTPases.

Animals↗
PubMed1998

Activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor 1 increases intestinal permeability in Caco-2 cells.

The cytotoxic necrotizing factor 1 (CNF1) activates Rho GTPases by deamidation of glutamine-63 and thereby induces redistribution of the actin cytoskeleton and formation of stress fibers. Here, we have studied the effects of CNF1 on the transepithelial resistance of Caco-2 cells, a human intestinal epithelial cell line, in comparison with the Rho-inactivating toxin B of Clostridium difficile. Whereas toxin B decreased the transepithelial resistance of Caco-2 cells by about 80% after 4 h, CNF1 reduced it by about 40%. Significant changes of the transepithelial resistance induced by CNF1 were detected after 3 h of incubation. Half-maximal effects were observed with 10 and 41 ng of CNF1 and toxin B per ml, respectively. Flux measurement revealed no CNF1-induced increase of fluorescein isothiocyanate-dextran permeation within the first 4 h of incubation and a 2.9-fold increase after 24 h of incubation. In contrast, toxin B induced a 28-fold increase of permeation after 24 h. As detected by rhodamine-phalloidin staining, CNF1 increased polymerization of F actin at focal contacts of adjacent cells and induced formation of stress fibers. The data indicate that not only depolymerization but also polymerization of actin and subsequent reorganization of the actin cytoskeleton alter the barrier function of intestinal tight junctions.

Bacterial Toxins↗
PubMed1998

Chimeric clostridial cytotoxins: identification of the N-terminal region involved in protein substrate recognition.

Clostridium sordellii lethal toxin is a member of the family of large clostridial cytotoxins that glucosylate small GTPases. In contrast to Clostridium difficile toxins A and B, which exclusively modify Rho subfamily proteins, C. sordellii lethal toxin also glucosylates Ras subfamily proteins. By deletion analysis and construction of chimeric fusion proteins of C. sordellii lethal toxin and C. difficile toxin B, we localized the enzyme activity of the lethal toxin to the N terminus of the holotoxin and identified the region involved in protein substrate specificity. The toxin fragment of the N-terminal 546 amino acid residues of C. sordellii lethal toxin glucosylated Rho and Ras subfamily proteins, as the holotoxin did. Deletion of a further 30 amino acid residues from the C terminus of this active fragment drastically reduced glucotransferase activity and blocked glucohydrolase activity. Exchange of amino acid residues 364 through 516 of lethal toxin for those in the active toxin B fragment (1 to 546) allowed glucosylation of Ras subfamily proteins. In contrast, the chimera with amino acids 1 to 364 from toxin B, 365 to 468 from lethal toxin, and 469 to 546 from toxin B exhibited markedly reduced modification of Ras subfamily proteins, whereas modification of Rac and Cdc42 was hardly changed. The data indicate that the region of amino acid residues 364 through 516 primarily defines the substrate specificity of C. sordellii lethal toxin.

Bacterial Proteins↗
PubMed1998

The N-terminal part of the enzyme component (C2I) of the binary Clostridium botulinum C2 toxin interacts with the binding component C2II and functions as a carrier system for a Rho ADP-ribosylating C3-like fusion toxin.

The binary actin-ADP-ribosylating Clostridium botulinum C2 toxin consists of the enzyme component C2I and the binding component C2II, which are separate proteins. The active component C2I enters cells through C2II by receptor-mediated endocytosis and membrane translocation. The N-terminal part of C2I (C2IN), which consists of 225 amino acid residues but lacks ADP-ribosyltransferase activity, was identified as the C2II contact site. A fusion protein (C2IN-C3) of C2IN and the full-length C3-like ADP-ribosyltransferase from Clostridium limosum was constructed. The fusion protein C2IN-C3 ADP-ribosylated Rho but not actin in CHO cell lysates. Together with C2II, C2IN-C3 induced complete rounding up of CHO and HeLa cells after incubation for 3 h. No cell rounding was observed without C2II or with the original C3-like transferase from C. limosum. The data indicate that the N-terminal 225 amino acid residues of C2I are sufficient to cause the cellular uptake of C. limosum transferase via the binding component of C2II, thereby increasing the cytotoxicity of the C3-like exoenzyme several hundred-fold.

ADP Ribose Transferases↗
PubMed1998

Agents that inhibit Rho, Rac, and Cdc42 do not block formation of actin pedestals in HeLa cells infected with enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.

Actins↗
PubMed1997

Gene expression of the small GTP-binding proteins RhoA, RhoB, Rac1, and Cdc42 in adult rat brain.

GTPases of the Rho subfamily, i.e. Rho, Rac and Cdc42, are molecular switches in various signaling pathways. Best characterized are their functions in the regulation of the actin cytoskeleton. In neuronal cell lines they are involved in the mechanisms leading to synapse formation and plasticity. It is still unknown whether they have respective functions in the mammalian CNS. In this case, they should be present in the adult brain, especially in areas known for their synaptic remodeling. We have studied the expression of the Rho GTPases in adult rat brain with in situ hybridization and Western blot analysis. High amounts of RhoA, RhoB, Rac1 and Cdc42 mRNAs were detected in neurons of the hippocampus, i.e. in pyramidal cells of the CA1-CA4 regions as well as in granule cells of the dentate gyrus and in hilar cells. Also in cerebellum, Purkinje and granular cells expressed the four mRNAs. Strong gene expression was also found in brainstem, thalamus and neocortex. Using Western blot analysis, RhoA and Cdc42 proteins were detected in hippocampus, cerebellum, thalamus and neocortex. It is concluded that GTPases of the Rho family play a role in the regulation of cellular functions in the adult brain.

Animals↗
PubMed1997

Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta cells.

We utilized clostridial toxins (with known specificities for inhibition of GTPases) to ascertain the contribution of candidate GTPases in physiologic insulin secretion from beta cells. Exposure of normal rat islets or isolated beta (HIT-T15) cells to Clostridium difficile toxins A and B catalyzed the glucosylation (and thereby the inactivation) of Rac, Cdc42, and Rho endogenous to beta cells; concomitantly, either toxin reduced glucose- or potassium-induced insulin secretion from rat islets and HIT cells. Treatment of beta cells with Clostridium sordellii lethal toxin (LT; which modified only Ras, Rap, and Rac) also reduced glucose- or potassium-induced secretion. However, clostridial toxin C3-exoenzyme (which ADP-ribosylates and inactivates only Rho) was without any effect on either glucose- or potassium-induced insulin secretion. These data suggest that Cdc42, Rac, Ras, and/or Rap (but not Rho) may be needed for glucose- or potassium-mediated secretion. The effects of these toxins appear to be specific on stimulus-secretion coupling, since no difference in metabolic viability (assessed colorimetrically by quantitating the conversion of the tetrazolium salt into a formazan in a reduction reaction driven by nutrient metabolism) was demonstrable between control and toxin (A or LT)-treated beta cells. Toxin (A or LT) treatment also did not alter glucose- or potassium-mediated rises in cytosolic free calcium concentrations ([Ca2+]i), suggesting that these GTPases are involved in steps distal to elevations in [Ca2+]i. Recent findings indicate that the carboxyl methylation of Cdc42 is stimulated by only glucose, whereas that of Rap (Kowluru et al., J Clin Invest 98: 540-555, 1996) and Rac (present study) are regulated by glucose or potassium. Together, these findings provide direct evidence, for the first time, that the Rho subfamily of GTPases plays a key regulatory role(s) in insulin secretion, and they suggest that Cdc42 may be required for early steps in glucose stimulation of insulin release, whereas Rap and/or Rac may be required for a later step(s) in the stimulus-secretion coupling cascade (i.e. Ca2+-induced exocytosis of insulin).

ADP Ribose Transferases↗
PubMed1997

Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1.

The actin cytoskeleton is regulated by GTP-hydrolysing proteins, the Rho GTPases, which act as molecular switches in diverse signal-transduction processes. Various bacterial toxins can inactivate Rho GTPases by ADP-ribosylation or glucosylation. Previous research has identified Rho proteins as putative targets for Escherichia coli cytotoxic necrotizing factors 1 and 2 (CNF1 and 2). These toxins induce actin assembly and multinucleation in culture cells. Here we show that treatment of RhoA with CNF1 inhibits the intrinsic GTPase activity of RhoA and completely blocks GTPase activity stimulated by the Rho-GTPase-activating protein (rhoGAP). Analysis by mass spectrometry and amino-acid sequencing of proteolytic peptides derived from CNF1-treated RhoA indicate that CNF1 induces deamidation of a glutamine residue at position 63 (Gln 63) to give constitutively active Rho protein.

3T3 Cells↗
PubMed1997

Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin.

Clostridium difficile toxin B that is one of the largest cytotoxins (270 kDa) known acts on Rho subfamily proteins by monoglucosylation (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). By deletion analysis we identified the enzyme and cytotoxic activity of the toxin to be located at the N terminus of the holotoxin. A 63-kDa fragment of toxin B covering the first 546 amino acid residues glucosylated Rho, Rac, and Cdc42, but not Ras, by using UDP-glucose as a cosubstrate. As known for the holotoxin, glucosylation by the toxin fragment was favored with the GDP-bound form of the low molecular mass GTPases. Microinjection of the toxin fragment into NIH-3T3 cells induced rounding up of cells and redistribution of the actin cytoskeleton. In contrast, a toxin fragment encompassing the first 516 amino acid residues was at least 1000-fold less active than toxin fragment 1-546 and cytotoxically inactive. The data give direct evidence for location of the enzyme activity of C. difficile toxin B at the N-terminal 546 amino acids residues and indicate a functionally and/or structurally important role of the region from amino acid residues 516 through 546 for enzyme and cytotoxic activities.

3T3 Cells↗
PubMed1997

Complex regulation of human neutrophil activation by actin filaments: dihydrocytochalasin B and botulinum C2 toxin uncover the existence of multiple cation entry pathways.

In human neutrophils, the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenalalanine (fMLP), the Ca(2+)-ATPase inhibitor, thapsigargin, and the lectins, concanavalin A (Con A) and mistletoe lectin I (ML I), stimulate the entry of Ca2+ and Na+ with subsequent activation of exocytosis and superoxide anion (O2-) formation. We studied the role of actin in neutrophil activation. The actin filament-disrupting substances, dihydrocytochalasin B (dhCB) and botulinum C2 toxin (C2 toxin) potentiated fMLP- and lectin-stimulated Ca(2+)- and Na+ entry. Lectin-induced Mn2+ entry was enhanced by actin disruption, whereas fMLP-triggered Mn2+ entry was unaffected. dhCB and C2 toxin inhibited fMLP- and lectin-stimulated Ba2+ influx. The actin disrupters also inhibited fMLP- and ML I-induced Sr2+ influx, whereas Con A-stimulated Sr2+ entry was not influenced by dhCB and C2 toxin. Thapsigargin-stimulated cation entry was not altered by actin disruption. DhCB and botulinum C2 toxin potentiated lysozyme release induced by all four stimuli. Con A and ML I per se activated O2- formation only in the presence and not in the absence of dhCB. Con A potentiated the stimulatory effects of ML I on O2- formation in the presence of dhCB and primed neutrophils to respond to ML I in the absence of dhCB. Our data indicate the following: (1) dhCB and C2 toxin uncover the existence of multiple cation entry pathways in neutrophils; (2) actin disruption facilitates exocytosis and O2- formation by enhancement of Ca(2+)- and Na+ entry and by altering the function of proteins involved in activation of secretion and O2- formation; and (3) Con A and ML I, which possess different sugar specificities, activate different signaling pathways in neutrophils.

Actins↗
PubMed1997

ADP-ribosylating and glucosylating toxins as tools to study secretion in RBL cells.

The influence of different ADP-ribosylating and glucosylating cytotoxins on stimulated protein tyrosine phosphorylation and secretion in rat basophilic leukemia (RBL) cells was studied. Treatment of RBL cells with Clostridium botulinum C2 toxin, which specifically ADP-ribosylated monomeric G-actin and caused complete depolymerization of the actin cytoskeleton in intact cells, inhibited Fc epsilon RI receptor-mediated tyrosine phosphorylation of various proteins in a time- and concentration-dependent manner with maximal effects at 100 ng/ml C2I and 200 ng/ml C2I. C2 toxin (10 ng/ml C2I and 20 ng/ml C2II) increased antigen- or calcium ionophore (A23187)-stimulated [3H]serotonin release maximally by about 3 fold. Clostridium botulinum C3, which ADP-ribosylated Rho in intact RBL cells, had no effect on protein tyrosine phosphorylation and stimulated secretion. In contrast, the cytotoxic Clostridium difficile toxin B (ToxB), which glucosylated the Rho-subtype family members RhoA and Cdc42, blocked or reduced antigen- or calcium ionophore-mediated [3H]serotonin release, respectively, and decreased tyrosine phosphorylation of a 110 kDa protein. The data indicate that different actin pools control tyrosine phosphorylation and secretion in RBL cells and suggest that Rho subfamily proteins regulate secretion independently of the actin cytoskeleton.

ADP Ribose Transferases↗