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Biomedical subjects

I Just

Publications and source records attributed to I Just.

At least 55 records · Page 3Linked to original sources
PubMed1996

A role for Rho in receptor- and G protein-stimulated phospholipase C. Reduction in phosphatidylinositol 4,5-bisphosphate by Clostridium difficile toxin B.

Receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-hydrolyzing phospholipase C (PLC) enzymes by activated alpha of free beta gamma subunits of the relevant G proteins. To study whether low molecular weight G proteins of the Rho family are involved in receptor signaling 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, A1F-4 and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), 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 GTP gamma S-stimulated PLC activity. Finally both toxin B and C3 exoenzyme significantly reduced, by 40 to 50%, the total level of PtdIns(4,5)P2 in HEK cells, without affecting the levels of phosphatidylinositol and phosphatidylinositol 4-phosphate. Accordingly, When PLC activity was measured with exogenous PtdIns(4,5)P2 as enzyme substrate, Ca(2+)- as well as GTP gamma S- or A1F-4-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.

ADP Ribose Transferases↗
PubMed1996

Cytotoxic effects by microinjection of ADP-ribosylated skeletal muscle G-actin in PtK2 cells in the absence of Clostridium perfringens iota toxin.

The ADP-ribosylating toxins Clostridium botulinum C2 toxin and C. perfringens iota toxin, which ADP-ribosylate monomeric G-actin at Arg-177 but not the polymeric F-actin, induce depolymerization of the actin cytoskeleton in cultured cells. Since ADP-ribosylated G-actin has properties of a barbed-end-capping protein, we studied whether the ADP-ribosylated actin affects the actin cytoskeleton of PtK2 cells even in the absence of ADP-ribosylating toxin. Skeletal muscle actin was ADP-ribosylated by C. perfringens iota toxin and the toxin was removed using an anti-iota toxin antibody. Microinjection of ADP-ribosylated actin caused retraction of the cell body, redistribution and depolymerization of the actin cytoskeleton in a concentration- and time-dependent manner. The finding that ADP-ribosylated actin affects per se the actin cytoskeleton explains the cytopathic effects of ADP-ribosylating toxins on microfilaments, although F-actin is not directly modified by the toxins.

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 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

Autoregulation of actin synthesis in hepatocytes by transcriptional and posttranscriptional mechanisms.

Treatment of rat hepatocytes with the filamentous-actin-stabilizing toxin phalloidin decreased the amount of globular actin by 77% in the cytosol and by 80% in the nucleus within 12 h. Simultaneously, actin mRNA was specifically increased by 230%. The de-novo synthesis of actin mRNA, as measured by nuclear run-on transcription, was enhanced by 250%. Treatment of cells with actinomycin D blocked the increase of actin mRNA. The apparent half-life of actin mRNA was not significantly altered during treatment with phalloidin. In contrast, the globular-actin-stabilizing botulinum C2 toxin increased the amount of cytosolic globular actin by 50% within 12 h. Simultaneously, the actin mRNA level was decreased by 62%. However, de-novo synthesis of actin mRNA was not impaired. The apparent half-life of actin mRNA was decreased by approximately 60% during treatment with C2 toxin. The data strongly suggest an autoregulatory control of actin synthesis on the basis of the globular/filamentous actin ratio in rat hepatocytes at the transcriptional as well as at the posttranscriptional levels.

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

Monoglucosylation of low-molecular-mass GTP-binding Rho proteins by clostridial cytotoxins.

Rho proteins, which are involved in receptor-mediated regulation of the actin cytoskeleton, are substrates for ADP-ribosylation by Clostridium botulinum C3 toxins. Recently, it was shown that Rho and other members of the Rho subfamily of low-molecular-mass GTP-binding proteins are glucosylated by C. difficile toxins A and B. Glucosylation occurs at threonine-37, which is a crucial amino acid residue for the regulatory functions of the small GTP-binding proteins. These toxins should prove useful as tools for studying the functions of Rho proteins.

Journal Article↗
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

The low molecular mass GTP-binding protein Rho is affected by toxin A from Clostridium difficile.

Enterotoxin A is one of the major virulence factors of Clostridium difficile, and the causative agent of antibiotic-associated pseudomembranous colitis. In cell culture (NIH-3T3, rat basophilic leukemia cells) toxin A inhibits Clostridium botulinum ADP-ribosyltransferase C3 (C3)-catalyzed ADP-ribosylation of the low molecular mass GTP-binding Rho proteins. Rho participates in the regulation of the microfilament cytoskeleton. Decrease in ADP-ribosylation of Rho occurs in a time- and concentration-dependent manner and precedes the toxin A-induced destruction of the actin cytoskeleton. Action of toxin A is not due to proteolytical degradation of Rho or to an inherent ADP-ribosyltransferase activity of toxin A. Toxin A-induced decrease in ADP-ribosylation is observed also in cell lysates and with recombinant RhoA protein. A heat stable low molecular mass cytosolic factor is essential for the toxin effect on Rho. Thus, the enterotoxin (toxin A) resembles the effects of the C. difficile cytotoxin (toxin B) on Rho proteins (Just, I., G. Fritz, K. Aktories, M. Giry, M. R. Popoff, P. Boquet, S. Hegenbath, and C. Von Eichel-Streiber. 1994. J. Biol. Chem. 269:10706-10712). The data indicate that despite different in vivo effects, toxin A and toxin B act on the same cellular target protein Rho to elicit their toxic effects.

ADP Ribose Transferases↗
PubMed1995

Autoregulation of actin synthesis by physiological alterations of the G-actin level in hepatocytes.

Hypotonic treatment of cultured rat hepatocytes significantly decreased the monomeric G-actin level by 18% after 120 min while the level of filamentous F-actin remained essentially unchanged. Simultaneously the level of cellular actin mRNA was increased by 53%. Incubation of hepatocytes for 120 min with the F-actin stabilizing toxin phalloidin from Amanita phalloides led to a decrease of G-actin by 70% and an increase of F-actin by 55%. Although the toxin dependent decrease of G-actin was much more pronounced than the decrease after hypotonic treatment, the increase of actin mRNA was similar under both conditions. Simultaneous treatment with hypotonic medium did not result in a further decrease of the G-actin level. On the other hand, the G-actin elevating C2 toxin from Clostridium botulinum completely blocked the effects of osmotic stress on G-actin and actin-mRNA content. The results demonstrate that already an essentially physiological decrease of G-actin without alterations of F-actin results in a substantial enhancement of the actin mRNA level, indicating the physiological significance of this autoregulation.

Actins↗
PubMed1995

Effects of Clostridium botulinum C2 toxin and cytochalasin D on in vitro invasiveness, motility and F-actin content of a murine T-lymphoma cell line.

In order to investigate the role of microfilaments in the crawling movements of lymphoid cells, we have analyzed the effects of botulinum C2 toxin and of cytochalasin D (cytoD) on the actin cytoskeleton and on the motility of a BW5147 T-lymphoma-derived cell line. Actin was ADP-ribosylated by C2 toxin in the living cells, and this resulted in a time and dose-dependent disappearance of F-actin, as assessed by staining with labeled phalloidin. CytoD did not affect the amount of polymerized actin, but rather changed its distribution from a diffuse peripheral network to focal accumulations on one side of the cell. Both treatments affected the motility of the lymphoma cells in two assay systems. Fourier analysis was used to quantify shape changes performed by the cells. C2 toxin as well as CytoD caused the cessation of pseudopodal protrusion. Invasion of the lymphoma cells through a monolayer of fibroblast-like cells was also inhibited by the treatments, in a dose-dependent way. C2 toxin significantly inhibited invasion at concentrations at which only part of the actin pool had been ADP-ribosylated. We conclude that partial depolymerization, as well as disorganization, of the microfilament network impairs the active cellular deformations that are involved in the crawling movements of the lymphoma cells. From previous work, there is evidence to state that the monolayer invasion assay to some extent mimics tissue infiltration by hematopoietic cells. The present study is the first to analyze the role of actin polymerization in a model system that is relevant for the migration of lymphoid cells in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Differentiation-induced increase in Clostridium botulinum C3 exoenzyme-catalyzed ADP-ribosylation of the small GTP-binding protein Rho.

The specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study differentiation-dependent changes in the regulation of the low-molecular-mass GTP-binding protein Rho. Differentiation of F9 teratocarcinoma cells to neuronal-like cells by treatment with retinoic acid and dibutyryl-adenosine 3',5'-monophosphate [(Bt)2cAMP] increased the C3-catalyzed ADP-ribosylation of RhoA proteins in cytosolic and membrane fractions by about threefold and sixfold, respectively. Phenotypical differentiation of F9 cells was not required for increase in ADP-ribosylation. Increase in ADP-ribosylation after (Bt)2cAMP and retinoic acid treatments was blocked by cycloheximide, indicating the requirement of protein biosynthesis. As deduced from specific rho mRNA amounts and from Western analysis with a monoclonal RhoA antibody, the stimulation in the [32P]ADP-ribosylation of Rho was not caused by an increased de-novo synthesis of Rho proteins. GDP increased the ADP-ribosylation of membrane-associated Rho from non-differentiated, but not from differentiated F9 cells. GTP[S] decreased ADP-ribosylation of membranous Rho from differentiated and much less from non-differentiated F9 cells. Differentiation-dependent increase in ADP-ribosylation of cytosolic Rho was reversed by protein phosphatase type-1. Treatment with SDS (0.01%) which releases Rho from complexation with guanine nucleotide dissociation inhibitor, increased ADP-ribosylation both in differentiated and non-differentiated cells, indicating no differentiation-specific change of such complexes. In total, our data indicate that the induction of the differentiation process in F9 cells is accompanied by changes in the regulation of cytosolic and membrane-associated Rho proteins.

ADP Ribose Transferases↗
PubMed1994

Tissue-specific variations in the expression and regulation of the small GTP-binding protein Rho.

Rho proteins are involved in the regulation of the assembly of the microfilamental cellular network and are known to be specific substrates for the ADP-ribosyltransferase C3 from Clostridium botulinum. Here, we studied the distribution of Rho and Rho-regulating proteins in extracts from various rabbit tissues. The highest amounts of [32P]ADP-ribosylated proteins were detected in cell extracts from lung and kidney. Compared to these tissues, 50-95% reduced labeling of Rho proteins was observed in extracts from liver, spleen, brain, heart and muscle. The level of the C3-mediated [32P]ADP-ribosylation of Rho did not correlate with the amount of RhoA proteins detected by Western analysis. The relative amounts of [32P]ADP-ribosylated proteins located in cytosolic or membrane fractions, respectively, depended on the type of tissue investigated, indicating a tissue-specific variation in the subcellular distribution of Rho proteins. The same was true for the complexation of Rho with other factors and the expression of diverse Rho species. In respect to Rho-regulating proteins, extracts from lung and brain contained the highest amounts of guanine nucleotide dissociation-inhibitor proteins (Rho-GDI). The association of Rho with Rho-GDI however showed tissue specificity and did not correlate with Rho-GDI amounts. The highest Rho-GAP (GAP = GTPase-activating protein) activities were observed in extracts from lung, kidney and spleen, the lowest ones in extracts from muscle and heart. In total, our data demonstrate tissue-specific differences in the expression of RhoA, [32P]ADP-ribosylated proteins and Rho-regulating factors, indicating a tissue-specific variation in the activity and regulation of Rho proteins.

Animals↗