Biomedical subjects

I Just

Publications and source records attributed to I Just.

At least 37 records · Page 2Linked to original sources

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

Toxins from anaerobic bacteria: specificity and molecular mechanisms of action.

Major advances have been made in the past five years in the identification of cellular targets of toxins produced by anaerobic bacteria. These targets include the vesicular membrane docking and fusion apparatus, the actin cytoskeleton, the signal transduction machinery and the cell membrane. The recent discovery that large clostridial toxins (Clostridium difficile A and B toxins, C. sordellii lethal and hemorrhagic toxins, and alpha C. novyi toxin) are monoglucosyltransferases, together with the establishment of the perfringolysin crystal structure, has led to new insights in the field of toxins from anaerobic bacteria.

Bacterial Toxins↗

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↗

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

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

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↗

PubMed1997

Glucosylation of small GTP-binding Rho proteins disrupts endothelial barrier function.

The endothelial cytoskeleton is important for the regulation of endothelial barrier function. Small GTP-binding Rho proteins play a central role in the organization of the microfilament system. Clostridium difficile toxin B (TcdB) inactivates Rho proteins by glucosylation at Thr-37. We used TcdB as a probe to study the role of Rho proteins in the regulation of endothelial barrier function. TcdB time (50-170 min) and dose (10-100 ng/ml) dependently increased the hydraulic conductivity of cultured porcine pulmonary artery endothelial cell monolayers approximately 10-fold. Simultaneously, the albumin reflection coefficient decreased substantially from 0.8 to 0.15. Before endothelial hyperpermeability, TcdB reduced F-actin content in a dose-dependent manner, whereas G-actin content remained unchanged. Finally, we proved that TcdB caused dose (5-100 ng/ml)- and time-dependent glucosylation of Rho proteins in endothelial cells. Phalloidin, which stabilizes filamentous actin, prevented the effect of TcdB on endothelial permeability. In contrast to thrombin-, hydrogen peroxide-, or Escherichia coli hemolysin-induced hyperpermeability, the elevation of cyclic nucleotides did not block TcdB-related permeability. The data demonstrate a central role of small GTP-binding Rho proteins for the control of endothelial barrier function.

Actins↗

PubMed1997

ADP-ribosylation of Rho-proteins with botulinum C3 exoenzyme inhibits invasion and shape changes of T-lymphoma cells.

C3 exoenzyme from Clostridium botulinum ADP-ribosylates the small GTP-binding protein Rho with a high specificity. The use of C3 has shown that Rho-mediated signaling is involved in the regulation of actin-dependent processes in various cell types. In order to investigate the role of Rho-proteins in lymphocyte crawling, we have analyzed the effects of C3 on a T-cell line derived from the murine BW5147 lymphoma. Pretreatment of the lymphoma cells with C3, in conditions where Rho was actually ADP-ribosylated, strongly inhibited the characteristic shape changes resulting from extension and retraction of pseudopodia. Concomitantly, invasion of the cells through a monolayer of fibroblast-like cells was also inhibited. C3-treatment did not affect the total F-actin content of the cells, as measured by flow cytometry of cells stained with phalloidin. Yet, microscopical observation revealed that the accumulations of F-actin, which were seen in the pseudopodia of untreated cells, were absent after treatment with C3. This suggests that C3 may affect actin polymerization locally. The inhibitory effect of C3 on invasion was not restricted to the murine BW5147 lymphoma cell line, as it occurred also with CCRF-CEM, a human T-cell lymphoma line. Our results demonstrate that invasion-bound motility of lymphocytes depends on a Rho-mediated signal transduction pathway.

ADP Ribose Transferases↗

PubMed1996

Difference in protein substrate specificity between hemorrhagic toxin and lethal toxin from Clostridium sordellii.

The hemorrhagic toxin (HT) from Clostridium sordellii is pharmacologically related to Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin which have been recently identified as mono-glucosyl-transferases. Here we report that HT, which is coexpressed with lethal toxin, is also a glucosyltransferase. Whereas lethal toxin glucosylates the Rho subfamily proteins Rac and Cdc42 and the Ras subfamily proteins H-Ras and Rap, the substrate specificity of HT is strictly confined to the Rho subfamily proteins Rho, Rac and Cdc42. Comparable to lethal toxin, transferase activity of HT is stimulated by Mn2+. Acceptor amino acid in Rho was identified by mutagenesis as threonine-37. C. sordellii HT is a novel member of the family of clostridial mono-glucosyl-transferases, a family which modifies the Rho and Ras GTPases.

3T3 Cells↗

PubMed1996

Microinjection of ADP-ribosylated actin inhibits actin synthesis in hepatocyte-hepatoma hybrid cells.

Treatment of hepatocyte-hepatoma hybrid cells with Clostridium botulinum C2 toxin led to a 167% increase in monomeric globular actin (G-actin) and to a 57% decrease in filamentous actin (F-actin) within 2 h. Simultaneously, the level of actin mRNA was specifically decreased to 49% and actin synthesis was significantly diminished. In contrast, treatment of hybrid cells with phalloidin led to a decrease in G-actin to 55% and to a reciprocal increase in actin mRNA to 244% and an increase in actin synthesis. These alterations of actin synthesis depending on the G-actin/F-actin ratio corresponded to the autoregulation of actin synthesis observed in primary cultures of rat hepatocytes. Microinjection of C2 toxin or of phalloidin into hepatocyte-hepatoma hybrid cells had the same effects on actin synthesis as incubation with either toxin in the culture medium. Microinjection of nonpolymerizable ADP-ribosylated G-actin into hepatocyte-hepatoma hybrid cells specifically decreased the incorporation of [35S]methionine into newly synthesized actin within 1 h. This decrease continued for at least 19 h. Microinjection of ADP-ribosylated actin led to rounding of cells and obvious disaggregation of actin filaments, which might be due to capping of actin filaments by the ADP-ribosylated actin. Because stabilization of actin filaments by phalloidin before microinjection of ADP-ribosylated actin also resulted in decreased actin synthesis, the concentration of monomeric G-actin seems to be responsible for the regulation of actin synthesis in hepatocyte-hepatoma hybrid cells, which can be regarded as immortalized hepatocytes.

Actins↗

PubMed1996

Role of Rho proteins in carbachol-induced contractions in intact and permeabilized guinea-pig intestinal smooth muscle.

1. The aim of this study was to determine whether the low molecular mass GTPase RhoA or related proteins are involved in carbachol- and high-K(+)-induced contractions in intact intestinal smooth muscle as well as the carbachol-induced increase in Ca2+ sensitivity of the myofilaments in permeabilized preparations. 2. The carbachol-induced increase in the Ca2+ sensitivity of force production in beta-escin-permeabilized intestinal smooth muscle was enhanced in preparations that were loaded with the constitutively active mutant of RhoA, Val14RhoA, and was inhibited by exoenzyme C3 from Clostridium botulinum, which ADP-ribosylates and inactivates small GTPases of the Rho family. The effect of C3 on Ca2+ sensitivity in the absence of the agonist was negligible, while the maximal Ca(2+)-activated force was inhibited by about 20%. 3. Inhibition of carbachol-induced force was associated with an increase in ADP-ribosylation of a protein band with a molecular mass of approximately 22 kDa, corresponding to Rho, and was partially reversed in the presence of Ile41RhoA, which is not a substrate for C3. Val14RhoA did not restore carbachol-induced Ca2+ sensitization in C3-treated smooth muscle. 4. In intact intestinal smooth muscle, toxin B from Clostridium difficile, which monoglucosylates members of the Rho family, inhibited high-K(+)-induced contractions and the initial phasic response to carbachol by about 30%. The delayed contractile response to carbachol was completely inhibited. 5. In smooth muscle preparations that were permeabilized with beta-escin after treatment with toxin B, carbachol-and GTP gamma S-induced Ca2+ sensitization was significantly inhibited. 6. These findings are consistent with a role for Rho or Rho-like proteins in agonist-induced increase in Ca2+ sensitivity of force production in intact and permeabilized intestinal smooth muscle.

Adenosine Diphosphate Ribose↗

PubMed1996

Clostridium novyi alpha-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins.

The lethal and edema-inducing alpha-toxin from Clostridium novyi causes rounding up of cultured cell lines by redistribution of the actin cytoskeleton. alpha-Toxin belongs to the family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A and B and the lethal toxin from Clostridium sordellii. Toxin A and toxin B have been recently identified as monoglucosyltransferases to modify the low molecular mass GTPases of the Rho subfamily (Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503 and Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) J. Biol. Chem. 270, 13932-13936). We report here the identification of the alpha-toxin-catalyzed modification of Rho. Using electrospray mass spectrometry, the mass of the modification was determined as 203 Da, consistent with a N-acetyl-hexosamine moiety. UDP-N-acetyl-glucosamine selectively served as cosubstrate for alpha-toxin-catalyzed modification into the Rho subfamily proteins Rho, Rac, Cdc42, and RhoG. The acceptor amino acid of N-acetyl-glucosaminylation was identified by mutagenesis as Thr-37 in Rho (equivalent to Thr-35 in Rac/Cdc42), which is located in the effector domain of the GTPases. C. novyi alpha-toxin seems to mediate its cytotoxic effects on cells by mimicking endogenous post-translational modification of cellular proteins.

3T3 Cells↗

PubMed1996

The ras-related protein Ral is monoglucosylated by Clostridium sordellii lethal toxin.

Clostridium sordellii lethal toxin (LT), a cytotoxin which causes preferential destruction of the actin cytoskeleton, has been recently identified as glucosyltransferase to modify the low molecular mass GTPases Rac, Ras and Rap. We report here on LT produced by C. sordellii strain 6018 which glucosylates in addition to Rac, Ras and Rap the Ral protein. LT from strain VPI9048 however does not glucosylate Ral. Besides recombinant Ral, cellular Ral is also substrate. In the GDP-bound form, Ral is a superior substrate to the GTP form. Acceptor amino acid for glucose is threonine-46 which is equivalent to threonine-35 in H-Ras located in the effector region. The Ral-glucosylating toxin is a novel isoform of Ras-modifying clostridial cytotoxins.

Animals↗

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 actin by Clostridium perfringens iota toxin and turkey erythrocyte ADP-ribosyltransferase A: effects on profilin-regulated nucleotide exchange and ATPase activity.

Effects of ADP-ribosylation of skeletal muscle alpha-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 Arg 177 by Clostridium perfringens iota toxin increased the nucleotide dissociation rate from 2.2 x 10(-3) s-1 to 4.5 x 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 x 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.

ADP Ribose Transferases↗