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1.
ADP-ribosylation of actin causes increase in the rate of ATP exchange and inhibition of ATP hydrolysis 总被引:4,自引:0,他引:4
ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin increased the rate of exchange of actin-bound [gamma-32P]ATP by unlabelled ATP about twofold. Increased exchange rates were observed with ATP and ATP[gamma S], much less with ADP but not with AMP or NAD. ADP-ribosylation of skeletal muscle actin reduced "basal" and Mg2+ (1 mM)-induced ATP hydrolysis by about 80%. Similar inhibition of ATP hydrolysis was observed with liver actin ADP-ribosylated by Clostridium botulinum C2 toxin. The data indicate that ADP-ribosylation of actin at Arg-177 largely affects the ATP-binding and ATPase activity. 相似文献
2.
Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin belong to a novel family of actin ADP-ribosylating toxins. ADP-ribosylation of actin inhibits actin polymerization and G-actin-associated ATPase activity. The ADP-form of actin is ADP-ribosylated at a higher rate than actin with bound ATP. ADP-ribosylation of actin is reversible, a reaction, which is accompanied by reconstitution of actin ATPase activity. 相似文献
3.
ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin 总被引:9,自引:0,他引:9
B Schering M B?rmann G S Chhatwal U Geipel K Aktories 《European journal of biochemistry》1988,171(1-2):225-229
The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize. 相似文献
4.
ADP-ribosylation of the 1:1 (G-A) and 1:2 (G-A-A) gelsolin-actin complexes by Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin was studied. Iota toxin ADP-ribosylated actin in the G-A complex from human platelets as effectively as skeletal muscle actin. The Km for NAD (4 microM) was identical for both substrates. C2 toxin ADP-ribosylated actin in the G-A complex with lower efficacy than nonmuscle actin from platelet cytosol. In the G-A-A complex both actin molecules were ADP-ribosylated by iota toxin. The G-A complex bound ADP-ribosylated actin (Ar) to form the G-A-Ar complex in which the weakly bound actin is ADP-ribosylated. Vice versa, ADP-ribosylated 1:1 gelsolin-actin complex (G-Ar) was able to bind unmodified actin to yield the G-Ar-A complex. ADP-ribosylation did not change the nucleation activity of either the G-Ar complex or the G-Ar-A complex. When monomeric actin was added to the G-A-Ar complex, polymerization of actin was delayed by about 10 min. According to a quantitative kinetic analysis, the delay of polymerization corresponded to the rate of dissociation of ADP-ribosylated actin from the G-A-Ar complex. This suggests that the nucleation activity of the G-A-A complex is inhibited by ADP-ribosylation of the weakly bound actin and that the inhibition can be removed by dissociation of ADP-ribosylated actin from the G-A-Ar complex. 相似文献
5.
ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin 总被引:1,自引:0,他引:1
S Mauss C Chaponnier I Just K Aktories G Gabbiani 《European journal of biochemistry》1990,194(1):237-241
The substrate specificities of the actin-ADP-ribosylating toxins, Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin were studied by using five different preparations of actin isoforms: alpha-skeletal muscle actin, alpha-cardiac muscle actin, gizzard gamma-smooth muscle actin, spleen beta- and gamma-cytoplasmic actin, and aortic smooth muscle actin containing alpha- and gamma-smooth muscle actin isoforms. C. perfringens iota toxin ADP-ribosylated all actin isoforms tested, whereas C. botulinum C2 toxin did not modify alpha-skeletal muscle actin or alpha-cardiac muscle actin. Spleen beta/gamma-cytoplasmic actin and gizzard gamma-smooth muscle actin were substrates of C. botulinum C2 toxin. In the aortic smooth muscle actin preparation, gamma-smooth muscle actin but not alpha-smooth muscle actin was ADP-ribosylated by C. botulinum C2 toxin. The data indicate that, in contrast to C. perfringens iota toxin, C. botulinum C2 toxin ADP-ribosylates only beta/gamma-cytoplasmic and gamma-smooth muscle actin and suggest that the N-terminal region of actin isoforms define the substrate specificity for ADP-ribosylation by C. botulinum C2 toxin. 相似文献
6.
Clostridium perfringens iota toxin ADP-ribosylates skeletal muscle actin in Arg-177 总被引:12,自引:0,他引:12
Clostridium perfringens iota toxin ADP-ribosylates actin. Substrates of C. perfringens toxin are both non-muscle beta/gamma-actin and skeletal muscle actin. This finding suggests that C. perfringens iota ADP-ribosylates the same amino acid in skeletal muscle and non-muscle actin as does C. botulinum C2 toxin in non-muscle actin. Protein chemical analysis involving thermolysin cleavage on [32P]ADP-ribosylated actin or tryptic digestion followed by a secondary thermolysin cleavage of the radiolabelled fragments showed one major site of ADP-ribosylation. From its amino acid composition and sequence, the radiolabelled peptide was identified as peptide 175-177, locating the acceptor ADP-ribosyl amino acid as Arg-177. 相似文献
7.
Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins. 相似文献
8.
The reverse reaction of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota-toxin was studied. In the presence of nicotinamide (30-50 mM) C2 toxin and iota-toxin decreased the radioactive labeling of [32P]ADP-ribosylated actin and catalyzed the formation of [32P]NAD. The pH optima for both reactions were 5.5-6.0. Concomitant with the removal of ADP-ribose, the ability of actin to polymerize was restored and actin ATPase activity increased. Neither ADP-ribosylation nor removal of ADP-ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions. ADP-ribosylation of platelet actin by C2 toxin was reversed by iota-toxin, confirming recent reports that both toxins modify the same amino acid in actin. However, C. botulinum C2 toxin was not able to cleave ADP-ribose from skeletal muscle actin which had been incorporated by iota-toxin, corroborating the different substrate specificities of both toxins. 相似文献
9.
P Hofman L d'Andrea E Guzman E Selva G Le Negrate D F Far E Lemichez P Boquet B Rossi 《European cytokine network》1999,10(2):227-236
The role of the polymorphonuclear leukocyte (PMN) cytoskeleton during the transmigration across colonic epithelial cells is not very well understood. In order to study the role of different components of the PMN cytoskeleton during transepithelial migration across a colonic epithelial cell monolayer (T84), PMN were preincubated with drugs affecting either the actin cytoskeleton (cytochalasin B, iota toxin of Clostridium perfringens, and phalloidin) or the microtubules (colchicine and taxol). The role of PMN myosin during transepithelial migration was investigated using the inhibitor 2,3-butanedione monoxime (BDM) and DC3B toxin. PMN intracellular Ca2+, during neutrophil adhesion and translocation across the epithelium, was assessed by the Ca2+ chelator 1, 2bis-(2-aminophenoxy)-ethane-N,N,N', N'-tetra-acetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM). Transmigration of PMN was initiated by applying either interleukin-8 or formyl-met-leu-phe (fMLP). While colchicine and taxol preexposure did not influence PMN transepithelial migration, treatment with cytochalasin B, iota toxin, phalloidin, BDM, DC3B toxin and BAPTA-AM greatly diminished migration of PMN across T84 monolayers. Similarly, cell-cell contacts established between PMN and epithelial cells during the transmigration were diminished after treatment of PMN with iota toxin or cytochalasin B. These data show that the neutrophil actin cytokeleton and myosin, but not the microtubules, evoke a Ca2+ -dependent motility that facilitates migration across the colonic epithelial barrier. 相似文献
10.
ADP-ribosylated actin caps the barbed ends of actin filaments 总被引:13,自引:0,他引:13
The mode of action on actin polymerization of skeletal muscle actin ADP-ribosylated on arginine 177 by perfringens iota toxin was investigated. ADP-ribosylated actin decreased the rate of nucleated actin polymerization at substoichiometric ratios of ADP-ribosylated actin to monomeric actin. ADP-ribosylated actin did not tend to copolymerize with actin. Actin filaments were depolymerized by the addition of ADP-ribosylated actin. The maximal monomer concentration reached by addition of ADP-ribosylated actin was similar to the critical concentration of the pointed ends of actin filaments. ADP-ribosylated actin had no effect on the rate of polymerization of gelsolin-capped actin filaments which polymerize at the pointed ends. The results suggest that ADP-ribosylated actin acts as a capping protein which binds to the barbed ends of actin filaments to inhibit polymerization. Based on an analysis of the depolymerizing effect of ADP-ribosylated actin, the equilibrium constant for binding of ADP-ribosylated actin to the barbed ends of actin filaments was determined to be about 10(8) M-1. As actin is ADP-ribosylated by perfringens iota toxin and by botulinum C2 toxin, it appears that conversion of actin into a capping protein by ADP-ribosylation is a pathophysiological reaction catalyzed by bacterial toxins which ultimately leads to inhibition of actin assembly. 相似文献
11.
Marvaud JC Stiles BG Chenal A Gillet D Gibert M Smith LA Popoff MR 《The Journal of biological chemistry》2002,277(46):43659-43666
Clostridium perfringens iota toxin consists of two unlinked proteins. The binding component (Ib) is required to internalize into cells an enzymatic component (Ia) that ADP-ribosylates G-actin. To characterize the Ia domain that interacts with Ib, fusion proteins were constructed between the C. botulinum C3 enzyme, which ADP-ribosylates Rho, and various truncated versions of Ia. These chimeric molecules retained the wild type ADP-ribosyltransferase activity specific for Rho and were recognized by antibodies against C3 enzyme and Ia. Internalization of each chimera into Vero cells was assessed by measuring the disorganization of the actin cytoskeleton and intracellular ADP-ribosylation of Rho. Fusion proteins containing C3 linked to the C terminus of Ia were transported most efficiently into cells like wild type Ia in an Ib-dependent manner that was blocked by bafilomycin A1. The minimal Ia fragment that promoted translocation of Ia-C3 chimeras into cells consisted of 128 central residues (129-257). These findings revealed that iota toxin is a suitable system for mediating the entry of heterologous proteins such as C3 into cells. 相似文献
12.
N B El Hadj M R Popoff J C Marvaud B Payrastre P Boquet B Geny 《The Journal of biological chemistry》1999,274(20):14021-14031
Lethal toxin (LT) from Clostridium sordellii has been shown in HeLa cells to glucosylate and inactivate Ras and Rac and, hence, to disorganize the actin cytoskeleton. In the present work, we demonstrate that LT treatment provokes the same effects in HL-60 cells. We show that guanosine 5'-O-(3-thiotriphosphate)-stimulated phospholipase D (PLD) activity is inhibited in a time- and dose-dependent manner after an overnight treatment with LT. A similar dose response to the toxin was found when PLD activity was stimulated by phorbol 12-myristate 13-acetate via the protein kinase C pathway. The toxin effect on actin organization seemed unlikely to account directly for PLD inhibition as cytochalasin D and iota toxin from Clostridium perfringens E disorganize the actin cytoskeleton without modifying PLD activity. However, the enzyme inhibition and actin cytoskeleton disorganization could both be related to a major decrease observed in phosphatidylinositol 4,5-bisphosphate (PtdIns(4, 5)P2). Likely in a relationship with this decrease, recombinant ADP-ribosylation factor, RhoA, Rac, and RalA were not able to reconstitute PLD activity in LT-treated cells permeabilized and depleted of cytosol. Studies of phosphoinositide kinase activities did not allow us to attribute the decrease in PtdIns(4,5)P2 to inactivation of PtdIns4P 5-kinase. LT was also found to provoke a major inhibition in phosphatidylinositol 3-kinase that could not account for the inhibition of PLD activity because wortmannin, at doses that fully inhibit phosphatidylinositol 3-kinase, had no effect on the phospholipase activity. Among the three small G-proteins, Ras, Rac, and RalA, inactivated by LT and involved in PLD regulation, inactivation of Ral proteins appeared to be responsible for PLD inhibition as LT toxin (strain 9048) unable to glucosylate Ral proteins did not modify PLD activity. In HL-60 cells, LT treatment appeared also to modify cytosol components in relationship with PLD inhibition as a cytosol prepared from LT-treated cells was less efficient than one from control HL-60 cells in stimulating PLD activity. Phosphatidylinositol transfer proteins involved in the regulation of polyphosphoinositides and ADP-ribosylation factor, a major cytosolic PLD activator in HL-60 cells, were unchanged, whereas the level of cytosolic protein kinase Calpha was decreased after LT treatment. We conclude that in HL-60 cells, lethal toxin from C. sordellii, in inactivating small G-proteins involved in PLD regulation, provokes major modifications at the membrane and the cytosol levels that participate in the inhibition of PLD activity. Although Ral appeared to play an essential role in PLD activity, we discuss the role of other small G-proteins inactivated by LT in the different modifications observed in HL-60 cells. 相似文献
13.
Cytochalasin B stimulated polymerization and decreased the concentration of G-actin remaining in equilibrium with F-actin filaments. Polymerization in the presence of cytochalasin B gave rise to a smaller increase of viscosity but to the same increase in light scattering, compared to polymerization in the absence of cytochalasin B. Cytochalasin B reduced the viscosity of F-actin and caused the appearance of ATP hydrolysis by F-actin. The cytochalasin B-induced ATPase activity was inhibited by concentrations of KCl higher than 50 mM. The cytochalasin B-induced ATPase activity was enhanced by ethyleneglycol bis(alpha-aminoethyl ether)-N,N'-tetraacetic acid and reduced by MgCl2 at concentrations higher than 0.75 mM. The findings suggest that the stability of actin filaments is reduced by cytochalasin B. 相似文献
14.
Klaus Aktories 《Molecular and cellular biochemistry》1994,138(1-2):167-176
The actin cytoskeleton appears to be as the cellular target of various clostridial ADP-ribosyltransferases which have been described during recent years.Clostridium botulinum C2 toxin,Clostridium perfringens iota toxin andClostridium spiroforme toxin ADP-ribosylate actin monomers and inhibit actin polymerization.Clostridium botulium exoenzyme C3 andClostridium limosum exoenzyme ADP-ribosylate the low-molecular-mass GTP-binding proteins of the Rho family, which participate in the regulation of the actin cytoskeleton. ADP-ribosylation inactivates the regulatory Rho proteins and disturbs the organization of the actin cytoskeleton. 相似文献
15.
The SpvB protein from Salmonella enterica was recently discovered as an actin-ADP-ribosylating toxin. SpvB is most likely delivered via a type-III secretion system into eukaryotic cells and does not have a binding/translocation component. This is in contrast to the family of binary actin-ADP-ribosylating toxins from various Bacillus and Clostridium species. However, there are homologies in amino acid sequences between the C-terminal domain of SpvB and the catalytic domains of the actin-ADP-ribosylating toxins such as C2 toxin from Clostridium botulinum and iota toxin from Clostridium perfringens. We compared the biochemical properties of the catalytic C-terminal domain of SpvB (C/SpvB) with the enzyme components of C2 toxin and iota toxin. The specificity of C/SpvB concerning the modification of G- or F-actin was comparable to the C2 and iota toxins, although there were distinct differences regarding the recognition of actin isoforms. C/SpvB and iota toxin modify both muscle alpha-actin and nonmuscle beta/gamma-actin, whereas C2 toxin only modifies beta/gamma-actin. In contrast to the iota and C2 toxins, C/SpvB possessed no detectable glycohydrolase activity in the absence of a protein substrate. The maximal reaction rates were comparable for all toxins, whereas variable K(m) values for NAD were evident. We identified arginine-177 as the modification site for C/SpvB with the actin homologue protein Act88F from Drosophila. 相似文献
16.
Cytochalasin B stimulated polymerization and decreased the concentration of G-actin remaining in equilibrium with F-actin filaments. Polymerization in the presence of cytochalasin B gave rise to a smaller increase of viscosity but to the same increase in light scattering, compared to polymerization in the absence of cytochalasin B. Cytochalasin B reduced the viscosity of F-actin and caused the appearance of ATP hydrolysis by F-actin. The cytochalasin B-induced ATPase activity was inhibited by concentrations of KCl higher than 50 mM. The cytochalasin B-induced ATPase activity was enhanced by ethyleneglycol bis(α-aminoethyl ether)-N,N′-tetraacetic acid and reduced by MgCl2 at concentrations higher than 0.75 mM. The findings suggest that the stability of actin filaments is reduced by cytochalasin B. 相似文献
17.
The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization 总被引:17,自引:0,他引:17
Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2, increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl, however, accelerated the rate of actin polymerization, although still lowering the final steady state viscosity. Cytochalasin B, at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl, accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2, cytochalasin D uncoupled the actin ATPase activity from actin polymerization, increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and, to a much greater extent, stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D, the initial rate of actin ATPase activity, when little or no polymerization had occurred, was directly proportional to the actin concentration and, therefore, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP, A . ATP, to monomeric actin-bound ADP and Pi, A* . ADP . Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins. 相似文献
18.
Alexander Belyy Irina Tabakova Alexander E. Lang Thomas Jank Yury Belyi Klaus Aktories 《PloS one》2015,10(12)
Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. 相似文献
19.
Summary The ability of two bacterial toxins to modify a plant actin by covalent ADP-ribosylation was tested in the green algaChara corallina. Using [32P]NAD, bothClostridium botulinum C2 toxin andClostridium perfringens iota toxin labelled a protein of Mr 42 kDa which comigrated with actin and was immunoprecipitated by a monoclonal anti-actin antibody. ADP-ribosylation ofChara actin was more efficient with iota toxin than with C2 toxin. The actin bundles in perfusedChara cells were not affected by toxin-containing media competent for ADP-ribosylation. The data indicate that monomeric plant actin is substrate for ADP-ribosylation by the bacterial toxins.Abbreviations ADP
adenosine-diphosphate
- EGTA
ethyleneglycol-bis-(-aminoethyl)N,N,N,N-tetraacetic acid
- NAD
nicotinamide dinucleotide
- pCA -log
[Ca2+]
- PIPES
piperazine-N,N-bis(2-ethanesulfonic acid)
Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement 相似文献
20.
Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin 总被引:13,自引:0,他引:13
We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota. 相似文献