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1.
Cesare Colasante Ornella Rossetto Laura Morbiato Marco Pirazzini Jordi Molgó Cesare Montecucco 《Molecular neurobiology》2013,48(1):120-127
Botulinum neurotoxin type A (BoNT/A) is the most frequent cause of human botulism and, at the same time, is largely used in human therapy. Some evidence indicates that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been directly proven. The metalloprotease L chain of the neurotoxin then reaches the cytosol in a process driven by low pH, but the acidic compartment wherefrom it translocates has not been identified. Using immunoelectron microscope, we show that BoNT/A does indeed enter inside synaptic vesicles and that each vesicle contains either one or two toxin molecules. This finding indicates that it is the BoNT/A protein receptor synaptic vesicle protein 2, and not its polysialoganglioside receptor that determines the number of toxin molecules taken up by a single vesicle. In addition, by rapid quenching the vesicle trans-membrane pH gradient, we show that the neurotoxin translocation into the cytosol is a fast process. Taken together, these results strongly indicate that translocation of BoNT/A takes place from synaptic vesicles, and not from endosomal compartments, and that the translocation machinery is operated by no more than two neurotoxin molecules. 相似文献
2.
Puhar A Johnson EA Rossetto O Montecucco C 《Biochemical and biophysical research communications》2004,319(1):66-71
Clostridial neurotoxins are internalized inside acidic compartments, wherefrom the catalytic chain translocates across the membrane into the cytosol in a low pH-driven process, reaching its proteolytic substrates. The pH range in which the structural rearrangement of clostridial neurotoxins takes place was determined by 8-anilinonaphthalene-1-sulfonate and tryptophan fluorescence measurements. Half conformational change was attained at pH 4.55, 4.50, 4.40, 4.60, 4.40, and 4.40 for tetanus neurotoxin and botulinum neurotoxin serotypes /A, /B, /C, /E, and /F, respectively. This similarity indicates the key residues for the conformation transition are strongly conserved. Acidic liposomes support the conformational rearrangement shifting the effect versus higher pH values, whereas zwitterionic liposomes do not. The disulfide bridge linking the light and the heavy chains together needs to be oxidized to allow toxin membrane insertion, indicating that in vivo its reduction follows exposure to the cytosol after penetration of the endosomal membrane. 相似文献
3.
Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal 总被引:4,自引:0,他引:4
Neale EA Bowers LM Jia M Bateman KE Williamson LC 《The Journal of cell biology》1999,147(6):1249-1260
The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval. 相似文献
4.
Marco Pirazzini Ornella Rossetto Cristina Bertasio Fulvio Bordin Clifford C. Shone Thomas Binz Cesare Montecucco 《Biochemical and biophysical research communications》2013,430(1):38-42
Tetanus and botulinum neurotoxins act inside nerve terminals and, therefore, they have to translocate across a membrane to reach their targets. This translocation is driven by a pH gradient, acidic on the cis side and neutral on the cytosol. Recently, a protocol to induce translocation from the plasma membrane was established. Here, we have used this approach to study the temperature dependence and time course of the entry of the L chain of tetanus neurotoxin and of botulinum neurotoxins type C and D across the plasma membrane of cerebellar granular neurons. The time course of translocation of the L chain varies for the three neurotoxins, but it remains in the range of minutes at 37 °C, whilst it takes much longer at 20 °C. BoNT/C does not enter neurons at 20 °C. Translocation also depends on the dimension of the pH gradient. These data are discussed with respect to the contribution of the membrane translocation step to the total time to paralysis and to the low toxicity of these neurotoxins in cold-blood vertebrates. 相似文献
5.
Tetanus neurotoxin and botulinum neurotoxins are the causative agents of tetanus and botulism. They block the release of neurotransmitters from synaptic vesicles in susceptible animals and man and act in nanogram quantities because of their ability to specifically attack motoneurons. They developed an ingenious strategy to enter neurons. This involves a concentration step via complex polysialo gangliosides at the plasma membrane and the uptake and ride in recycling synaptic vesicles initiated by binding to a specific protein receptor. Finally, the neurotoxins shut down the synaptic vesicle cycle, which they had misused before to enter their target cells, via specific cleavage of protein core components of the cellular membrane fusion machinery. The uptake of four out of seven known botulinum neurotoxins into synaptic vesicles has been demonstrated to rely on binding to intravesicular segments of the synaptic vesicle proteins synaptotagmin or synaptic vesicle protein 2. This review summarizes the present knowledge about the cell receptor molecules and the mode of toxin-receptor interaction that enables the toxins' sophisticated access to their site of action. 相似文献
6.
Marco Pirazzini Fulvio Bordin Ornella Rossetto Clifford C. Shone Thomas Binz Cesare Montecucco 《FEBS letters》2013,587(2):150-155
Tetanus and botulinum neurotoxins cause paralysis by cleaving SNARE proteins within the cytosol of nerve terminals. They are endocytosed inside acidic vesicles and the pH gradient across the membrane drives the translocation of their metalloprotease L domain in the cytosol. This domain is linked to the rest of the molecule by a single interchain disulfide bridge that has to be reduced on the cytosolic side of the membrane to free its enzymatic activity. By using specific inhibitors of the various cytosolic protein disulfides reducing systems, we show here that the NADPH-thioredoxin reductase-thioredoxin redox system is the main responsible for this disulfide reduction. In addition, we indicate auranofin, as a possible basis for the design of novel inhibitors of these neurotoxins. 相似文献
7.
Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc. 总被引:10,自引:0,他引:10
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G Schiavo B Poulain O Rossetto F Benfenati L Tauc C Montecucco 《The EMBO journal》1992,11(10):3577-3583
Tetanus and botulinum neurotoxins are the most potent toxins known. They bind to nerve cells, penetrate the cytosol and block neurotransmitter release. Comparison of their predicted amino acid sequences reveals a highly conserved segment that contains the HexxH zinc binding motif of metalloendopeptidases. The metal content of tetanus toxin was then measured and it was found that one atom of zinc is bound to the light chain of tetanus toxin. Zinc could be reversibly removed by incubation with heavy metal chelators. Zn2+ is coordinated by two histidines with no involvement in cysteines, suggesting that it plays a catalytic rather than a structural role. Bound Zn2+ was found to be essential for the tetanus toxin inhibition of neurotransmitter release in Aplysia neurons injected with the light chain. The intracellular activity of the toxin was blocked by phosphoramidon, a very specific inhibitor of zinc endopeptidases. Purified preparations of light chain showed a highly specific proteolytic activity against synaptobrevin, an integral membrane protein of small synaptic vesicles. The present findings indicate that tetanus toxin, and possibly also the botulinum neurotoxins, are metalloproteases and that they block neurotransmitter release via this protease activity. 相似文献
8.
The mode of botulinum neurotoxin action involves binding of its heavy chain for internalization into the presynaptic end of a nerve cell through endocytosis. The low-pH conditions of endosomes trigger translocation of the light chain across the endosomal membrane to the cytosol, where the light chain cleaves specific target proteins involved in the docking and fusion of synaptic vesicles for acetylcholine release. In an effort to model the interaction of botulinum neurotoxin and its subunit chains with lipid bilayer at low pH during the translocation process, we have examined type A botulinum neurotoxin-mediated calcein release from asolectin liposomes. At equimolar concentration (0.1 M), the neurotoxin and its heavy and light chains evoked 23%, 58%, and 28% calcein release, respectively. Calcein release was observed only when the cis-side (the side to which neurotoxin samples were added) pH was lowered to 4. Calcein release activity of the heavy chain was mostly blocked (76%) by a polyclonal antibody raised against the neurotoxin. Additionally, two peptide-specific polyclonal antibodies derived from the N-terminal and C-terminal halves of the heavy chain were also able to block the calcein release activity by 15–20%. In summary, these results suggest that calcein release from liposomes is specifically mediated by the heavy chain, and the light chain also integrates into the membrane. Implications of these results for the molecular mode of neurotoxin light-chain translocation across the endosomal membrane are discussed. 相似文献
9.
Effect of pH on the interaction of botulinum neurotoxins A, B and E with liposomes. 总被引:4,自引:0,他引:4
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The interaction of botulinum neurotoxin serotypes A, B and E with membranes of different lipid compositions was examined by photolabelling with two photoreactive phosphatidylcholine analogues that monitor the polar region and the hydrophobic core of the lipid bilayer. At neutral pH the neurotoxins interacted both with the polar head groups and with fatty acid chains of phospholipids. At acidic pHs the neurotoxins underwent structural changes characterized by a more extensive interaction with lipids. Both the heavy and light chain subunits of the neurotoxins were involved in the process. The change in the nature and extent of toxin-lipid interaction occurred in the pH range 4-6 and was not influenced by the presence of polysialogangliosides. The present data are in agreement with the idea that botulinum neurotoxins enter into nerve cells from a low pH intracellular compartment. 相似文献
10.
Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin. 总被引:34,自引:4,他引:30
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The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc-dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017-1023; Schiavo et al., Nature, 359, 832-835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC-1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC-1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC-1/syntaxin and the toxin light chain which acts as a metallo-endoprotease. Toxin-induced cleavage resulted in the generation of a soluble fragment of HPC-1/syntaxin that is 2-4 kDa smaller than the native protein. When HPC-1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full-length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane-free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC-1/syntaxin, which lacked the putative transmembrane domain at the C-terminus, was soluble and resistant to toxin action. We conclude that HPC-1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
Evidence for a vesicle-mediated maintenance of store-operated calcium channels in a human embryonic kidney cell line 总被引:5,自引:0,他引:5
Direct microinjection of the clostridial neurotoxins botulinum neurotoxin A light chain or tetanus neurotoxin into cells of a human embryonic kidney cell line significantly reduced calcium entry after depletion of internal calcium stores by cyclopiazonic acid, a reversible inhibitor of the sarcoplasmic-endoplasmic reticular calcium-ATPases. Botulinum neurotoxin A light chain specifically hydrolyzes a synaptosomal-associated protein of 25 kilodaltons (SNAP-25), and tetanus neurotoxin specifically hydrolyzes synaptobrevin-2 (vesicle-associated membrane protein 2, VAMP-2) and cellubrevin (vesicle-associated membrane protein 3, VAMP-3). Since these substrate proteins are required for vesicle docking and fusion, inhibition of store-operated calcium entry by botulinum neurotoxin A light chain and tetanus neurotoxin supports a model in which vesicle fusion is a prerequisite for activation of store-operated calcium entry. Brefeldin A, a fungal metabolite that interferes with vesicle traffic, partially reduced calcium entry following store depletion. The size of the reserve pool of vesicles or parallel vesicle recycling pathways employing brefeldin A-sensitive and brefeldin A-insensitive ADP-ribosylation factors may explain the failure of brefeldin A to completely inhibit store-operated calcium entry. 相似文献
12.
Claudia Verderio Silvia Coco Ornella Rossetto Cesare Montecucco Michela Matteoli 《Journal of neurochemistry》1999,73(1):372-379
Tetanus and botulinum toxins bind and are internalized at the neuromuscular junction. Botulinum neurotoxins (BoNTs) enter the cytosol at the motor nerve terminal; tetanus neurotoxin (TeNT) proceeds retroaxonally inside the motor axon to reach the spinal cord inhibitory interneurons. Although the major target of BoNTs is the peripheral cholinergic terminals, CNS neurons are susceptible to intoxication as well. We investigated the route of entry and the proteolytic activity of BoNT/B and BoNT/F in cultured hippocampal neurons and astrocytes. We show that, differently from TeNT, which enters hippocampal neurons via the process of synaptic vesicle (SV) recycling, BoNTs are internalized and cleave the substrate synaptobrevin/VAMP2 via a process independent of synaptic activity. Labeling of living neurons with Texas Red-conjugated BoNTs and fluoresceinated dextran revealed that these toxins enter hippocampal neurons via endocytic processes not mediated by SV recycling. Botulinum toxins also exploit endocytosis to enter cultured astrocytes, where they partially cleave cellubrevin, a ubiquitous synaptobrevin/VAMP isoform. These results indicate that, in spite of their closely related protein structure, TeNT and BoNTs use different routes to penetrate hippocampal neurons. These findings bear important implications for the identification of the protein receptors of clostridial toxins. 相似文献
13.
Clostridium botulinum neurotoxins are the most potent toxins to humans and cause paralysis by blocking neurotransmitter release at the presynaptic nerve terminals. The toxicity involves four steps, viz., binding to neuronal cells, internalization, translocation, and catalytic activity. While the catalytic activity is a zinc endopeptidase activity on the SNARE complex proteins, the translocation is believed to be a pH-dependent process allowing the translocation domain to change its conformation to penetrate the endosomal membrane. Here, we report the crystal structures of botulinum neurotoxin type B at various pHs and of an apo form of the neurotoxin, and discuss the role of metal ions and the effect of pH variation in the biological activity. Except for the perturbation of a few side chains, the conformation of the catalytic domain is unchanged in the zinc-depleted apotoxin, suggesting that zinc's role is catalytic. We have also identified two calcium ions in the molecule and present biochemical evidence to show that they play a role in the translocation of the light chain through the membrane. 相似文献
14.
Phosphorylation of VAMP/Synaptobrevin in Synaptic Vesicles by Endogenous Protein Kinases 总被引:6,自引:0,他引:6
Henk B. Nielander †Franco Onofri ‡Flavia Valtorta §Giampietro Schiavo §Cesare Montecucco Paul Greengard † Fabio Benfenati 《Journal of neurochemistry》1995,65(4):1712-1720
Abstract: VAMP/synaptobrevin (SYB), an integral membrane protein of small synaptic vesicles, is specifically cleaved by tetanus neurotoxin and botulinum neurotoxins B, D, F, and G and is thought to play an important role in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. Potential phosphorylation sites for various kinases are present in SYB sequence. We have studied whether SYB is a substrate for protein kinases that are present in nerve terminals and known to modulate neurotransmitter release. SYB can be phosphorylated within the same vesicle by endogenous Ca2+ /calmodulin-dependent protein kinase II (CaMKII) associated with synaptic vesicles. This phosphorylation reaction occurs rapidly and involves serine and threonine residues in the cytoplasmic region of SYB. Similarly to CaMKII, a casein kinase II (CasKII) activity copurifying with synaptic vesicles is able to phosphorylate SYB selectively on serine residues of the cytoplasmic region. This phosphorylation reaction is markedly stimulated by sphingosine, a sphingolipid known to activate CasKII and to inhibit CaMKII and protein kinase C. The results show that SYB is a potential substrate for protein kinases involved in the regulation of neurotransmitter release and open the possibility that phosphorylation of SYB plays a role in modulating the molecular interactions between synaptic vesicles and the presynaptic membrane. 相似文献
15.
A 50-kDa fragment from the NH2-terminus of the heavy subunit of Clostridium botulinum type A neurotoxin forms channels in lipid vesicles 总被引:5,自引:0,他引:5
1. A 50-kDa fragment representing the NH2-terminus of the heavy subunit of botulinum type A neurotoxin was found, at low pH, to evoke the release of K+ from lipid vesicles loaded with potassium phosphate. Similar K+ release was also observed with the intact neurotoxin, its heavy chain and a fragment consisting of the light subunit linked the 50-kDa NH2-terminal heavy chain fragment. The light subunit alone, however, was inactive. 2. In addition to K+, the channels formed in lipid bilayers by botulinum neurotoxin type A or the NH2-terminal heavy chain fragment were found to be large enough to permit the release of NAD (Mr 665). 3. The optimum pH for the release of K+ was found to be 4.5. Above this value K+ release rapidly decreased and was undetectable above pH 6.0. 4. The binding of radiolabelled botulinum toxin to a variety of phospholipids was assessed. High levels of toxin binding were only observed to lipid vesicles with an overall negative charge; much weaker binding occurred to lipid vesicles composed of electrically neutral phospholipids. 5. A positive correlation between the efficiency of toxin-binding and the efficiency of K+ release from lipid vesicles was not observed. Whereas lipid vesicles containing the lipids cardiolipin or dicetyl phosphate bound the highest levels of neurotoxin, the toxin-evoked release of K+ was low compared to vesicles containing either phosphatidyl glycerol, phosphatidyl serine or phosphatidyl inositol. 6. The implications of these observations to the mechanism by which the toxin molecule is translocated into the nerve ending are discussed. 相似文献
16.
Partial amino acid sequences of the heavy and light chains of botulinum neurotoxin type E 总被引:4,自引:0,他引:4
V Sathyamoorthy B R DasGupta 《Biochemical and biophysical research communications》1985,127(3):768-772
The dichain type E botulinum neurotoxin, a product of nicking the single chain protein by trypsin, is composed of a heavy and light chains. Sequence of the first 13 and 20 N-terminal residues of these two chains were determined. Also, proof is provided here that (i) the light chain of the nicked (dichain) is derived from the N-terminal one-third of the parent single chain neurotoxin, and (ii) molecular events leading to the activation, of the single chain neurotoxin cannot involve tryptic cleavage at or very close to the N-terminal of the single chain protein. The partial amino acid sequence of the light chain of botulinum type E and tetanus neurotoxins show significant similarity between the two clostridial neurotoxins. 相似文献
17.
Yelamanchili SV Reisinger C Becher A Sikorra S Bigalke H Binz T Ahnert-Hilger G 《European journal of cell biology》2005,84(4):467-475
Synaptophysin and synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. In mature, differentiated neurons they form the synaptophysin/synaptobrevin (Syp/Syb) complex. Synaptobrevin also interacts with the plasma membrane-associated proteins syntaxin and SNAP25, thereby forming the SNARE complex necessary for exocytotic membrane fusion. The two complexes are mutually exclusive. Synaptobrevin is a C-terminally membrane-anchored protein with one transmembrane domain. While its interaction with its SNARE partners is mediated exclusively by its N-terminal cytosolic region it has been unclear so far how binding to synaptophysin is accomplished. Here, we show that synaptobrevin can be cleaved in its synaptophysin-bound form by tetanus toxin and botulinum neurotoxin B, or by botulinum neurotoxin D, leaving shorter or longer C-terminal peptide chains bound to synaptophysin, respectively. A recombinant, C-terminally His-tagged synaptobrevin fragment bound to nickel beads specifically bound synaptophysin, syntaxin and SNAP25 from vesicular detergent extracts. After cleavage by tetanus toxin or botulinum toxin D light chain, the remaining C-terminal fragment no longer interacted with syntaxin or SNAP 25. In contrast, synaptophysin was still able to bind to the residual C-terminal synaptobrevin cleavage product. In addition, the His-tagged C-terminal synaptobrevin peptide 68-116 was also able to bind synaptophysin in detergent extracts from adult brain membranes. These data suggest that synaptophysin interacts with the C-terminal transmembrane part of synaptobrevin, thereby allowing the N-terminal cytosolic chain to interact freely with the plasma membrane-associated SNARE proteins. Thus, by binding synaptobrevin, synaptophysin may positively modulate neurotransmission. 相似文献
18.
Interaction of botulinum and tetanus toxins with the lipid bilayer surface. 总被引:7,自引:0,他引:7
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C Montecucco G Schiavo Z Gao E Bauerlein P Boquet B R DasGupta 《The Biochemical journal》1988,251(2):379-383
The interaction of botulinum neurotoxins serotypes A, B and E (from Clostridium botulinum) and of tetanus neurotoxin (from Clostridium tetani) with the surface of liposomes made of different lipid compositions was studied by photolabelling with a radioiodinated photoactive phosphatidylethanolamine analogue [125I-dipalmitoyl (3,4-azidosalicylamido)phosphatidylethanolamine]. When the vesicles were made of negatively charged lipids (asolectin), each of these neurotoxic proteins was radioiodinated, thus providing evidence for their attachment to the membrane surface. The presence of gangliosides on liposome membranes enhanced fixation of the neurotoxic proteins to the lipid vesicle surface. Both the heavy and light chains of the clostridial neurotoxins were involved in the attachment to the lipid bilayer surface. Each of the toxins tested here attached poorly to liposomes made of zwitterionic lipids (egg phosphatidylcholine), even when polysialogangliosides were present. The data suggest that the binding of botulinum and tetanus neurotoxins to their target neuronal cells involves negatively charged lipids and polysialogangliosides on the cell membrane. 相似文献
19.
Doussau F Humeau Y Vitiello F Popoff MR Poulain B 《Journal de la Société de Biologie》1999,193(6):457-467
Several bacterial toxins are powerful and highly specific tools for studying basic mechanisms involved in cell biology. Whereas the clostridial neurotoxins are widely used by neurobiologists, many other toxins (i.e. toxins acting on small G-proteins or actin) are still overlooked. Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT), known under the generic term of clostridial neurotoxins, are characterized by their unique ability to selectively block neurotransmitter release. These proteins are formed of a light (Mr approximately 50) and a heavy (Mr approximately 100) chain which are disulfide linked. The cellular action of BoNT and TeNT involves several steps: heavy chain-mediated binding to the nerve ending membrane, endocytosis, and translocation of the light chain (their catalytic moiety) into the cytosol. The light chains each cleaves one of three, highly conserved, proteins (VAMP/synaptobrevin, syntaxin, and SNAP-25 also termed SNAREs) implicated in fusion of synaptic vesicles with plasma membrane at the release site. Hence, when these neurotoxins are applied extracellularly, they can be used as specific tools to inhibit evoked and spontaneous transmitter release from certain neurones whereas, when the membrane limiting steps are bypassed by the mean of intracellular applications, BoNTs orTeNT can be used to affect regulated secretion in various cell types. Several members of the Rho GTPase family have been involved in intracellular trafficking of synaptic vesicles and secretory organelles. As they are natural targets for several bacterial exoenzymes or cytotoxins, their role in neurotransmitter release can be probed by examining the action of these toxins on neurotransmission. Such toxins include: i) the non permeant C3 exoenzymes from C. botulinum or C. limosum which ADP-ribosylate and thereby inactivate Rho, ii) exoenzyme S from Pseudomonas aeruginosa which ADP-ribosylates different members of the Ras, Rab, Ral and Rap families, iii) toxin B from C. difficile which glucosylates Rho, Rac and CDC42, iv) lethal toxin from C. sordellii which glucosylates Rac, Ras and to a lesser extent, Rap and Ral, but not on Rho or CDC42, and v) CNF deamidases secreted by pathogenic strains of E. coli which activate Rho and, to a lesser extent, CDC42. Since these toxins or exoenzymes have no or little ability to enter into the neurones, they must be applied intraneuronally to bypass the membrane limiting steps. Injection of several of these toxins into Aplysia neurones allowed us to reveal a new role for Rac in the control of exocytosis. ADP-ribosylating enzymes, which specifically act on monomeric actin (C2 binary toxin from C. botulinum and iota toxin from C. perfringens), are potential tools to probe the role of actin filaments during secretion. 相似文献
20.
The botulinum neurotoxins are produced by anaerobic, spore-forming bacteria belonging to the Clostridium genus. They are synthesised as a single chain protein (150 kDa), which is not or weakly active. The active form results from a proteolysis cleaving the precursor in a light chain (about 50 kDa) and a heavy chain (about 100 kDa), which are linked by a disulfide bridge. The heavy chain is involved in the recognition of a specific neuronal surface receptor and mediates the internalization of the light chain into the cytosol. The light chain is responsible for the intracellular activity. It catalyses the proteolysis of SNARE proteins, which are involved in the exocytosis of synaptic vesicles containing acetylcholine. Hence, the release of acetylcholine at the neuromuscular junction is blocked, leading to a flaccid paralysis. Human botulism, usually type A, B or E, is associated with intoxination, ingestion of preformed toxin in food, with digestive toxi-infection, mainly in newborns (infant botulism), or with wound contamination (wound botulism). The treatment of botulism is usually symptomatic. The specific treatment is based on the serotherapy or on the use of purified specific antibodies. The vaccination against botulism is efficient. However, since the botulinum neurotoxins are widely used for the treatment of numerous dystonias, a generalised vaccination is not conceivable. 相似文献