Biomedical subjects
Publications and source records attributed to K Aktories.
Transient intracellular expression of ExoT in CHO cells stimulated cell rounding and actin reorganization. Biochemical studies showed that ExoT was a GTPase-activating protein for RhoA, Rac1, and Cdc42. Together, these data show that ExoT interferes with Rho signal transduction pathways, which regulate actin organization, exocytosis, cell cycle progression, and phagocytosis.
Clostridium novyi alpha-toxin belongs to the family of large clostridial cytotoxins which act on cells through the modification of small GTP-binding proteins. We present here an analysis of the catalytic domain of alpha-toxin. A NH(2)-terminal 551-amino-acid fragment, alpha 551, was found to contain the full enzyme activity of the holotoxin, whereas a slightly shortened fragment encompassing 509 amino acids showed no detectable enzyme activity. Further characterization of the enzymatically active fragment alpha 551 revealed a substrate specificity for both UDP-N-acetylglucosamine and UDP-glucose. A Michaelis-Menten constant of 17 microM was determined for the substrate UDP-N-acetylglucosamine, while that for UDP-glucose was about 20 times higher, indicating a weaker affinity of the toxin for the latter substrate. Mutation of the aspartic acids of a conserved motif DXD within alpha 551 reduced enzyme activity >700-fold and inhibited cytotoxicity after microinjection in cells. Inhibition of enzyme activity of the DXD mutant could be partially overcome by increased concentrations of manganese ions, suggesting the involvement of these aspartic acids in Mn(2+) binding. By construction of chimeras of enzymatically active fragments of C. sordellii lethal toxin and C. novyi alpha-toxin, we located the region involved in nucleotide-sugar specificity to amino acids 133 through 517.
The family of the large clostridial cytotoxins, encompassing Clostridium difficile toxins A and B as well as the lethal and hemorrhagic toxins from Clostridium sordellii, monoglucosylate the Rho GTPases by transferring a glucose moiety from the cosubstrate UDP-glucose. Here we present a new detoxification procedure to block the enzyme activity by treatment with the reactive UDP-2', 3'-dialdehyde to result in alkylation of toxin A and B. Alkylation is likely to occur in the catalytic domain, because the native cosubstrate UDP-glucose completely protected the toxins from inactivation and the alkylated toxin competes with the native toxin at the cell receptor. Alkylated toxins are good antigens resulting in antibodies recognizing only the C-terminally located receptor binding domain, whereas formaldehyde treatment resulted in antibodies recognizing both the receptor binding domain and the catalytic domain, indicating that the catalytic domain is concealed under native conditions. Antibodies against the native catalytic domain (amino acids 1 through 546) and those holotoxin antibodies recognizing the catalytic domain inhibited enzyme activity. However, only antibodies against the receptor binding domain protected intact cells from the cytotoxic activity of toxin B, whereas antibodies against the catalytic domain were protective only when inside the cell.
The binary Clostridium botulinum C2 toxin consists of two separate proteins, the binding component C2II (80.5 kDa) and the actin-ADP-ribosylating enzyme component C2I (49.4 kDa). For its cytotoxic action, C2II binds to a cell membrane receptor and induces cell entry of C2I via receptor-mediated endocytosis. Here we studied the structure-function relationship of C2II by constructing truncated C2II proteins and producing polyclonal antisera against selective regions of C2II. An antibody raised against the C terminus (amino acids 592 to 721) of C2II inhibited binding of C2II to cells. The antibody prevented pore formation by C2II oligomers in artificial membranes but did not influence the properties of existing channels. To further define the region responsible for receptor binding, we constructed proteins with deletions in C2II; specifically, they lacked amino acid residues 592 to 721 and the 7 C-terminal amino acid residues. The truncated proteins still formed sodium dodecyl sulfate-stable oligomers but were unable to bind to cells. Our data indicate that the C terminus of C2II mediates binding of the protein to cells and that the 7 C-terminal amino acids are structurally important for receptor binding.
Pseudomonas aeruginosa exoenzyme S (ExoS) is a bifunctional cytotoxin. The ADP-ribosyltransferase domain is located within the C terminus part of ExoS. Recent studies showed that the N terminus part of ExoS (amino acid residues 1-234, ExoS(1-234)), which does not possess ADP-ribosyltransferase activity, stimulates cell rounding when transfected or microinjected into eukaryotic cells. Here we studied the effects of ExoS(1-234) on nucleotide binding and hydrolysis by Rho GTPases. ExoS(1-234) (100-500 nM) did not influence nucleotide exchange of Rho, Rac, and Cdc42 but increased GTP hydrolysis. A similar increase in GTPase activity was stimulated by full-length ExoS. Half-maximal stimulation of GTP hydrolysis by Rho, Rac, and Cdc42 was observed at 10-11 nM ExoS(1-234), respectively. We identified arginine 146 of ExoS to be essential for the stimulation of GTPase activity of Rho proteins. These data identify ExoS as a GTPase-activating protein for Rho GTPases.
Bordetella dermonecrotic toxin (DNT) causes the deamidation of glutamine 63 of Rho. Here we identified the region of DNT harboring the enzyme activity and compared the toxin with the cytotoxic necrotizing factor 1, which also deamidates Rho. The DNT fragment (DeltaDNT) covering amino acid residues 1136-1451 caused deamidation of RhoA at glutamine 63 as determined by mass spectrometric analysis and by the release of ammonia. In the presence of dansylcadaverine or ethylenediamine, DeltaDNT caused transglutamination of Rho. Deamidase and transglutaminase activities were blocked in the mutant proteins Cys(1292) --> Ala, His(1307) --> Ala, and Lys(1310) --> Ala of DeltaDNT. Deamidation and transglutamination induced by DeltaDNT blocked intrinsic and Rho- GTPase-activating protein-stimulated GTPase activity of RhoA. DeltaDNT deamidated and transglutaminated Rac and Cdc42 in the absence and presence of ethylenediamine, respectively. Modification of Rho proteins by DeltaDNT was nucleotide-dependent and did not occur with GTPgammaS-loaded GTPases. In contrast to cytotoxic necrotizing factor, which caused the same kinetics of ammonia release in the absence and presence of ethylenediamine, ammonia release by DeltaDNT was largely increased in the presence of ethylenediamine, indicating that DeltaDNT acts primarily as a transglutaminase.
The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Gln(63) of RhoA (Gln(61) of Cdc42 and Rac). In addition, both toxins possess in vitro transglutaminase activity in the presence of primary amines. Here we characterized the region of Rho essential for substrate recognition by the toxins using Rho/Ras chimeras as protein substrates. The chimeric protein Ras55Rho was deamidated or transglutaminated by CNF1. Rat pheochromocytoma PC12 cells microinjected with Ras55Rho developed formation of neurite-like structures after treatment with the CNF1 holotoxin indicating activation of the Ha-Ras chimera and Ras-like effects in intact cells. The Ras59Rho78Ras chimera protein contained the minimal Rho sequence allowing deamidation or transglutamination by CNF1. A peptide covering mainly the switch II region and consisting of amino acid residues Asp(59) through Asp(78) of RhoA was substrate for CNF1. Changes of amino acid residues Arg(68) or Leu(72) of RhoA into the corresponding residues of Ras (R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of the mutants by CNF1. In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp(59) through Asp(78)). Glucosylation of RhoA at Thr(37) blocked deamidation by DNT but not by CNF. The data indicate that CNF1 recognizes Rho GTPases exclusively in the switch II region, whereas the substrate recognition by DNT is characterized by additional structural requirements.
The small GTPases Rho, Rac, and Cdc42 are monoglucosylated at effector domain amino acid threonine 37/35 by Clostridium difficile toxins A and B. Glucosylation renders the Rho proteins inactive by inhibiting effector coupling. To understand the functional consequences, effects of glucosylation on subcellular distribution and cycling of Rho GTPases between cytosol and membranes were analyzed. In intact cells and in cell lysates, glucosylation leads to a translocation of the majority of RhoA GTPase to the membranes whereas a minor fraction is monomeric in the cytosol without being complexed with the guanine nucleotide dissociation inhibitor (GDI-1). Rho complexed with GDI-1 is not substrate for glucosylation, and modified Rho does not bind to GDI-1. However, a membranous factor inducing release of Rho from the GDI complex makes cytosolic Rho available as a substrate for glucosylation. The binding of glucosylated RhoA to the plasma membranes is saturable, competable with unmodified Rho-GTPgammaS guanosine 5'-O-(3-thiotriphosphate), and takes place at a membrane protein with a molecular mass of about 70 kDa. Membrane-bound glucosylated Rho is not extractable by GDI-1 as unmodified Rho is, leading to accumulation of modified Rho at membranous binding sites. Thus, in addition to effector coupling inhibition, glucosylation also inhibits Rho cycling between cytosol and membranes, a prerequisite for Rho activation.
Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln(63), caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30-60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.
Studies with cultured cells indicate that small GTPases of the Rho family may be involved in cell proliferation, differentiation, as well as migration. Therefore, we have studied the expression of four members of this protein family, i.e., RhoA, RhoB, the ubiquitous Cdc42u, and brain specific Cdc42b, during the embryonic and early postnatal development of rat neocortex. A clear isoform specificity of expression was found during the prenatal development. Thus, RhoA and Cdc42u were present in the proliferation zone while RhoB and Cdc42b were expressed only in the cortical plate where neural cells settle and differentiate. After birth, this isoform specificity quickly disappeared so that already at postnatal day 8 the adult pattern of expression was present. Our findings of a differential expression of the small GTP-binding proteins RhoA, RhoB, Cdc42u and Cdc42b in developing brain neocortex suggest isoform specific functions during neurogenesis and differentiation.
1. We have studied the modulation of volume-regulated anion channels (VRACs) by the small GTPase Rho and by one of its targets, Rho kinase, in calf pulmonary artery endothelial (CPAE) cells. 2. RT-PCR and immunoblot analysis showed that both RhoA and Rho kinase are expressed in CPAE cells. 3. ICl,swell, the chloride current through VRACs, was activated by challenging CPAE cells with a 25 % hypotonic extracellular solution (HTS) or by intracellular perfusion with a pipette solution containing 100 microM GTPgammaS. 4. Pretreatment of CPAE cells with the Clostridium C2IN-C3 fusion toxin, which inactivates Rho by ADP ribosylation, significantly impaired the activation of ICl,swell in response to the HTS. The current density at +100 mV was 49 +/- 13 pA pF-1 (n = 17) in pretreated cells compared with 172 +/- 17 pA pF-1 (n = 21) in control cells. 5. The volume-independent activation of ICl,swell by intracellular perfusion with GTPgammaS was also impaired in C2IN-C3-pretreated cells (31 +/- 7 pA pF-1, n = 11) compared with non-treated cells (132 +/- 21 pA pF-1, n = 15). 6. Activation of ICl,swell was pertussis toxin (PTX) insensitive. 7. Y-27632, a blocker of Rho kinase, inhibited ICl,swell and delayed its activation. 8. Inhibition of Rho and of Rho kinase by the above-described treatments did not affect the extent of cell swelling in response to HTS. 9. These experiments provide strong evidence that the Rho-Rho kinase pathway is involved in the VRAC activation cascade.
Actin filament (F-actin) depolymerization leads to the use-dependent rundown of N-methyl-D-aspartate (NMDA) receptor activity in rat hippocampal neurones. Depolymerization is promoted by Ca2+ which enters the cells via NMDA receptor channels. The ras homologue (Rho) GTPases (RhoA, Rac1 and Cdc42) promote actin polymerization and thus control the actin cytoskeleton. We have investigated, by means of the whole-cell patch clamp technique, whether the actin fibres which interact with NMDA receptors are controlled by Rho GTPases. In the presence of intracellular ATP which attenuates rundown, the C3 toxin from Clostridium (C.) botulinum was used to inactivate RhoA. Indeed, it enhanced the use-dependent rundown of NMDA-evoked inward currents to a level similar to that obtained in the absence of ATP. Lethal toxin from Clostridium sordellii which inactivates Rac1 and Cdc42 lacked this effect. We suggest that the function of somatodendritic NMDA receptor channels in rat hippocampal neurones can be modulated by RhoA via its action on F-actin.
Pseudomonas aeruginosa delivers exoenzyme S (ExoS) into the intracellular compartment of eukaryotic cells via a type III secretion pathway. Intracellular delivery of ExoS is cytotoxic for eukaryotic cells and has been shown to ADP-ribosylate Ras in vivo and uncouple a Ras-mediated signal transduction pathway. Functional mapping has localized the FAS-dependent ADP-ribosyltransferase domain to the carboxyl-terminus of ExoS. A transient transfection system was used to examine cellular responses to the amino-terminal 234 amino acids of ExoS (DeltaC234). Intracellular expression of DeltaC234 elicited the rounding of Chinese hamster ovary (CHO) cells and the disruption of actin filaments in a dose-dependent manner. Expression of DeltaC234 did not inhibit the expression of two independent reporter proteins, GFP and luciferase, or induce trypan blue uptake, which indicated that expression of DeltaC234 was not cytotoxic to CHO cells. Carboxyl-terminal deletion proteins of DeltaC234 were less efficient in the elicitation of CHO cell rounding than DeltaC234. Cytoskeleton rearrangement elicited by DeltaC234 was blocked and reversed by the addition of cytotoxic necrotizing factor 1 (CNF-1). CNF-1 catalyses the deamidation of Gln-63 of members of the Rho subfamily of small-molecular-weight GTP-binding proteins, resulting in protein activation. This implies a role for small-molecular-weight GTP-binding proteins in the disruption of actin by DeltaC234. Together, these data identify ExoS as a cytotoxin that possesses two functional domains. Intracellular expression of the amino-terminal domain of ExoS elicits the disruption of actin, while expression of the carboxyl-terminal domain of ExoS possesses FAS-dependent ADP-ribosyltransferase activity and is cytotoxic to eukaryotic cells.
The Clostridium botulinum C2 toxin ADP-ribosylates monomeric actin, thereby inducing disassembly of actin filaments, alteration of focal adhesions, and rounding of cells. After treatment with C2 toxin, cells stop to proliferate but remain viable for about 2 days. In view of reported correlations between the structure of the actin cytoskeleton and cell cycle transition, the effects of C2 toxin on the G(2)/M phase transition of the cell division cycle were studied. Since C2 toxin delayed entry into mitosis in HeLa cells, those enzymes which control entry into mitosis, the cyclin-dependent protein kinase mitosis-promoting factor (MPF) and the phosphatase cdc25-C were examined after treatment of synchronized cells with C2 toxin. MPF is composed of the regulatory cyclin B and the enzymatic p34cdc2 kinase subunits. For its activation at the G2/M border, p34cdc2 needs to be associated with cyclin B and additionally dephosphorylated at Tyr-15 by the specific phosphatase cdc25-C. Treatment of synchronized cells in S or G2 phase with C. botulinum C2 toxin prevented p34cdc2 protein kinase activation by inhibiting its tyrosine dephosphorylation at the G2/M border. Furthermore, the activity of cdc25-C phosphatase was decreased after treatment of cells with C2 toxin. Our results suggest that the prevented activation of the mitotic inducers p34cdc2 kinase and cdc25-C phosphatase represents the final downstream events in the action of C2 toxin resulting in a G(2) phase cell cycle delay in synchronized HeLa cells.
Recently, Escherichia coli cytotoxic necrotizing factor 1 (CNF1) was shown to activate the low-molecular-mass GTPase RhoA by deamidation of Gln63, thereby inhibiting intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (G. Schmidt, P. Sehr, M. Wilm, J. Selzer, M. Mann, and K. Aktories, Nature 387:725-729, 1997; G. Flatau, E. Lemichez, M. Gauthier, P. Chardin, S. Paris, C. Fiorentini, and P. Boquet, Nature 387:729-733, 1997). Here we report that in addition to RhoA, Cdc42 and Rac also are targets for CNF1 in vitro and in intact cells. Treatment of HeLa cells with CNF1 induced a transient formation of microspikes and formation of membrane ruffles. CNF1 caused a transient 10- to 50-fold increase in the activity of the c-Jun N-terminal kinase. Tryptic peptides of Cdc42 obtained from CNF1-treated cells by immunoprecipitation exhibited an increase in mass of 1 Da compared to control peptides, indicating the deamidation of glutamine 61 by the toxin. The same increase in mass was observed with the respective peptides obtained from CNF1-modified recombinant Cdc42 and Rac1. Modification of recombinant Cdc42 and Rac1 by CNF1 inhibited intrinsic and GAP-stimulated GTPase activities and retarded binding of 2'(3')-O-(N-methylanthraniloyl)GDP. The data suggest that recombinant as well as cellular Cdc42 and Rac are substrates for CNF1.
Alveolar epithelial type II cells (AET2) respond with exocytosis of surfactant containing lamellar bodies to stimulation with mechanical stretch and secretagogues, a process that is fundamental for maintaining alveolar stability and lung gas exchange. In the present study in cultured rat AET2, we employed botulinum C2 toxin, a binary toxin which ADP ribosylates nonmuscle G-actin, as a specific tool to probe the role of the actin microfilament system in the surfactant secretory process. Incubation of AET2 with C2 toxin caused a dose-dependent decay of the cellular F-actin content to a minimum of 20% of baseline, concomitant with an increase in monomeric actin. In parallel, a significant augmentation of baseline surfactant secretion up to twofold elevated levels above control was noted, as assessed by the release of prelabeled phosphatidylcholine. Pretreatment with phalloidin, which stabilized F-actin and reduced the level of G-actin, prevented the C2 toxin-elicited enhancement of baseline surfactant secretion. Even low C2 toxin concentrations, resulting in a reduction of total cellular F-actin content of approximately 10%, sufficed to augment secretagogue (ATP) and, more impressively, mechanical stress elicited an increase in surfactant secretion; the response to the biophysical challenge more than doubled. When investigated in the absence of toxin, different secretagogues (ATP, phorbol ester, betamimetics) caused a rapid-onset, transient reduction of F-actin in the range between 15 and 25% as a consistent part of their secretory response pattern. These data suggest that the state of actin polymerization is intimately linked to the exocytosis process underlying surfactant secretion in AET2. Microfilament system-related compartmentalization effects and/or or the impact of the state of actin assembly on signaling events may be considered as underlying events.
The actin ADP-ribosylating Clostridium botulinum C2 toxin is a binary toxin composed of the binding component C2II and the enzyme component C2I. C2I ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we studied the structure-function relationship of C2I by site-directed mutagenesis. Exchange of Glu389 to glutamine caused the complete loss of ADP-ribosyltransferase and NAD-glycohydrolase activities of C2I. In contrast, exchange of Glu387 to glutamine blocked ADP-ribosyltransferase but not NAD-glycohydrolase activity. Whereas photoaffinity labeling of the double mutant E387Q/E389Q C2I with [carbonyl-14C]NAD was blocked, labeling of the single C2I mutants was reduced (E389Q) or not changed (E387Q). Exchange of the STS motif (amino acid residues 348-350) of C2I caused a decrease in transferase activity by more than 99 (S348A) and 90% (T349V), or did not affect activity (S350A). Exchange of Arg299 and Arg300 to lysine reduced transferase activity to <0.1 and approximately 35% of wild-type activity. The data indicate that the amino acid residues Glu389, Glu387, Ser348, and Arg299, which are conserved in various prokaryotic and eukaryotic arginine-modifying ADP-ribosyltransferases, are essential for ADP-ribosyltransferase activity of the enzyme component of C. botulinum C2 toxin.
The exact mechanism of the spatial organization of the insulin signaling pathway leading to nuclear events remains unknown. Here, we investigated the involvement of the actin cytoskeleton in propagation of insulin signaling events leading to DNA synthesis and expression of the immediate early genes c-fos and c-jun in L6 muscle cells. Insulin reorganized the cellular actin network and increased the rate of DNA synthesis and the levels of c-fos mRNA, but not those of c-jun mRNA, in undifferentiated L6 myoblasts. Similarly, insulin markedly elevated the levels of c-fos mRNA but not of c-jun mRNA in differentiated L6 myotubes. Disassembly of the actin filaments by cytochalasin D, latrunculin B, or botulinum C2 toxin significantly inhibited insulin-mediated DNA synthesis in myoblasts and abolished stimulation of c-fos expression by the hormone in myoblasts and myotubes. Actin disassembly abolished insulin-induced phosphorylation and activation of extracellulor signal-regulated kinases, activation of a 65-kda member of the p21-activated kinases, and phosphorylation of p38 mitogen-activated protein kinases but did not prevent activation of phosphatidylinositol 3-kinase and p70(S6k). Under these conditions, insulin-induced Ras activation was also abolished, and Grb2 association with the Src and collogen homologous (Shc) molecule was inhibited without inhibition of the tyrosine phosphorylation of Shc. We conclude that the actin filament network plays an essential role in insulin regulation of Shc-dependent signaling events governing gene expression by facilitating the interaction of Shc with Grb2.