Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells

The clostridial neurotoxins tetanus and botulinum toxin type A are known to block transmitter release from nerve terminals, probably by interfering with some essential process controlling exocytosis after the entry of Ca2+ ions. Although exocytosis occurs in many secretory cells, these toxins show a high specificity for neurones and the secretory response of cultured bovine adrenal medullary cells is not inhibited by exposure to medium containing tetanus or botulinum toxin type A (although it is by botulinum toxin type D). Here we report that when tetanus toxin and botulinum neurotoxin type A are injected intracellularly into chromaffin cells they strongly inhibit secretion, as revealed by the measurement of cell capacitance. These results indicate that these toxins are normally ineffective in chromaffin cells because they are not bound and internalized, so do not reach their site of action. Furthermore, we have localized the secretion-blocking effects of the toxin to a fragment comprising the light chain covalently linked to part of the heavy chain, suggesting that this part of the molecule contains the active site.     

Substrate recognition strategy for botulinum neurotoxin serotype A

Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. CNTs impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs. SNARE assembly into a low-energy ternary complex is believed to catalyse membrane fusion, precipitating neurotransmitter release; this process is attenuated in response to SNARE proteolysis. Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases. The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation; it is thought to use one or more regions of enzyme-substrate interaction remote from the active site (exosites). Here we report the first structure of a CNT endopeptidase in complex with its target SNARE at a resolution of 2.1 A: botulinum neurotoxin serotype A (BoNT/A) protease bound to human SNAP-25. The structure, together with enzyme kinetic data, reveals an array of exosites that determine substrate specificity. Substrate orientation is similar to that of the general zinc-dependent metalloprotease thermolysin. We observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. The novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.     

Inhibition of exocytosis in bovine adrenal medullary cells by botulinum toxin type D

Botulinum toxins are known to block transmitter release at peripheral cholinergic synapses, producing muscular weakness and paralysis. The toxins may also block adrenergic transmission, although this effect is less well understood. The mechanisms by which toxins act are unclear. They are proteins of relative molecular mass approximately 150,000 and are structurally similar to tetanus toxin. It is generally accepted that a rise in intracellular calcium concentration is sufficient to trigger secretion by exocytosis, but it is not known whether the toxins block secretion by preventing this Ca transient or whether they act downstream from Ca entry by interfering with the process of exocytosis itself. We have attempted to resolve these questions in the case of the adrenergic system by studying the effects of botulinum toxins (types A, B, D and E) on the secretory response of isolated bovine adrenal medullary cells maintained in culture. The cells were either challenged with various secretagogues or rendered leaky and challenged directly with Ca buffers. We report here that botulinum toxin type D inhibits secretion in a time- and dose-dependent manner, the results being entirely consistent with the idea that the toxin acts at or near the site of exocytosis rather than at the sites controlling the rise in free Ca.     

Botulinum C2 toxin ADP-ribosylates actin

ADP-ribosylation of regulatory proteins is an important pathological mechanism by which various bacterial toxins affect eukaryotic cell functions. While diphtheria toxin catalyses the ADP-ribosylation of elongation factor 2, which results in inhibition of protein synthesis, cholera toxin and pertussis toxin ADP-ribosylate Ns and Ni, respectively, the GTP-binding regulatory components of the adenylate cyclase system, thereby modulating the bidirectional hormonal regulation of the adenylate cyclase. Botulinum C2 toxin is another toxin which has been reported to possess ADP-ribosyltransferase activity. This extremely toxic agent is produced by certain strains of Clostridium botulinum and induces hypotension, an increase in intestinal secretion, vascular permeability and haemorrhaging in the lungs. In contrast to botulinum neurotoxins, the botulinum C2 toxin apparently lacks any neurotoxic effects. Here we report that botulinum C2 toxin ADP-ribosylates a protein of relative molecular mass 43,000 (43K) in intact cells and in cell-free preparations. We present evidence that the 43K protein substrate is actin, which is apparently mono-ADP-ribosylated by the toxin. Botulinum C2 toxin also ADP-ribosylated purified liver G-actin, whereas liver F-actin was only poorly ADP-ribosylated and skeletal muscle actin was not ADP-ribosylated in either its G form or its F form. ADP-ribosylation of liver G-actin by botulinum C2 toxin resulted in a drastic reduction in viscosity of actin polymerized in vitro.     

Structural basis of cell surface receptor recognition by botulinum neurotoxin B

Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking neurotransmitter release. A 'double receptor' model has been proposed in which BoNTs recognize nerve terminals via interactions with both gangliosides and protein receptors that mediate their entry. Of seven BoNTs (subtypes A-G), the putative receptors for BoNT/A, BoNT/B and BoNT/G have been identified, but the molecular details that govern recognition remain undefined. Here we report the crystal structure of full-length BoNT/B in complex with the synaptotagmin II (Syt-II) recognition domain at 2.6 A resolution. The structure of the complex reveals that Syt-II forms a short helix that binds to a hydrophobic groove within the binding domain of BoNT/B. In addition, mutagenesis of amino acid residues within this interface on Syt-II affects binding of BoNT/B. Structural and sequence analysis reveals that this hydrophobic groove is conserved in the BoNT/G and BoNT/B subtypes, but varies in other clostridial neurotoxins. Furthermore, molecular docking studies using the ganglioside G(T1b) indicate that its binding site is more extensive than previously proposed and might form contacts with both BoNT/B and synaptotagmin. The results provide structural insights into how BoNTs recognize protein receptors and reveal a promising target for blocking toxin-receptor recognition.     

Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.     

常见食物中毒菌毒素的研究进展

食物中毒是食品卫生中经常遇到的问题,而细菌(毒素)性食物中毒又是最常见的形式之一,其中O157:H7大肠杆菌的Vero毒素、金黄色葡萄球菌肠毒素和肉毒梭菌神经毒素是经常引起食物中毒的细菌毒素.对这3种食物中毒菌毒素的产生、中毒情况、毒素类型、基因特性、致病机理、检测方法、应用研究及发展趋势进行了综述.     

Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization

Botulinum neurotoxin (BoNY) type A, a causative agent of botulism, is a di-chain protein (molecular weight 140,000) from Clostridium botulinum, and the most neurotoxic substance known. Some cases of sudden infant cot deaths have been attributed to such a neuroparalytic condition. BoNT inhibits irreversibly the release of acetylcholine from peripheral nerves in a highly selective manner. Hence, it is potentially an invaluable probe for studying the mechanism of transmitter release. Here we demonstrate specific labelling of murine motor nerve terminals with neurotoxic, 125I-labelled BoNT (type A) by autoradiography. We observed saturable, temperature-sensitive binding of BoNT to sites which reside solely on the nerve terminal membrane; these were distributed on all unmyelinated areas, at an average density of 150-500 per micron2 of membrane. The binding was mediated by the larger subunit of the toxin and was inhibited partially by tetanus toxin, another microbial protein. No specific binding was detectable on any other cell types examined, including noradrenergic terminals. Following binding, internalization of radioactivity was observed; this process was energy-dependent as it could be prevented totally by azide or dinitrophenol (DNP). This direct demonstration of separable steps, including highly selective binding and acceptor-mediated internalization, is reconcilable with the unique potency and the multiphasic inhibitory action of BoNT on transmitter release, as shown electrophysiologically.     

具有排除体积的两维高分子链的静态标度律

运用四位置模型对具有排除体积的两维高分子链进行ＭｏｎｔｅＣａｒｌｏ模拟和标度分析,为了提高长链的抽样效率,还提出了被称为“链段伸展法”的抽样方法。对单个自避行走链的模拟结果表明,两维ＳＡＷ链有育明显不同于夫规飞行链以及三维ＳＡＷ链的特点。     

肉毒毒素肠溶微球灭鼠剂的制备及性质研究

以肉毒毒素作为主体灭鼠药物,用丙烯酸树脂Ⅱ号为肠溶材料,通过相分离法制备肠溶微球;考察了微球的形态特征、包封率、载体回收率、抗酸能力、体外溶出度以及包封蛋白的活性保留度,并经拌饵投食试验,表明该制剂能有效地提高毒蛋白在肠胃中的稳定性,使其毒力(LD50)提高100倍以上,并且有较强的缓释作用．     

AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.     

Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands

The diseases caused by Shiga and cholera toxins account for the loss of millions of lives each year. Both belong to the clinically significant subset of bacterial AB5 toxins consisting of an enzymatically active A subunit that gains entry to susceptible mammalian cells after oligosaccharide recognition by the B5 homopentamer. Therapies might target the obligatory oligosaccharide-toxin recognition event, but the low intrinsic affinity of carbohydrate-protein interactions hampers the development of low-molecular-weight inhibitors. The toxins circumvent low affinity by binding simultaneously to five or more cell-surface carbohydrates. Here we demonstrate the use of the crystal structure of the B5 subunit of Escherichia coli O157:H7 Shiga-like toxin I (SLT-I) in complex with an analogue of its carbohydrate receptor to design an oligovalent, water-soluble carbohydrate ligand (named STARFISH), with subnanomolar inhibitory activity. The in vitro inhibitory activity is 1-10-million-fold higher than that of univalent ligands and is by far the highest molar activity of any inhibitor yet reported for Shiga-like toxins I and II. Crystallography of the STARFISH/Shiga-like toxin I complex explains this activity. Two trisaccharide receptors at the tips of each of five spacer arms simultaneously engage all five B subunits of two toxin molecules.     

Lactose binding to heat-labile enterotoxin revealed by X-ray crystallography.

Recognition of the oligosaccharide portion of ganglioside GM1 in membranes of target cells by the heat-labile enterotoxin from Escherichia coli is the crucial first step in its pathogenesis, as it is for the closely related cholera toxin. These toxins have five B subunits, which are essential for GM1 binding, and a single A subunit, which needs to be nicked by proteolysis and reduced, yielding an A1-'enzyme' and an A2-'linker' peptide. A1 is translocated across the membrane of intestinal epithelial cells, possibly after endocytosis, upon which it ADP-ribosylates the G protein Gs alpha. The mechanism of binding and translocation of these toxins has been extensively investigated, but how the protein is orientated on binding is still not clear. Knowing the precise arrangement of the ganglioside binding sites of the toxins will be useful for designing drugs against the diarrhoeal diseases caused by organisms secreting these toxins and in the development of oral vaccines against them. We present here the three-dimensional structure of the E. coli heat-labile enterotoxin complexed with lactose. This reveals the location of the binding site of the terminal galactose of GM1, which is consistent with toxin binding to the target cell with its A1 fragment pointing away from the membrane. A small helix is identified at the carboxy terminus of A2 which emerges through the central pore of the B subunits and probably comes into contact with the membrane upon binding, whereas the A1 subunit is flexible with respect to the B pentamer.     

Brevetoxicosis: red tides and marine mammal mortalities

Potent marine neurotoxins known as brevetoxins are produced by the 'red tide' dinoflagellate Karenia brevis. They kill large numbers of fish and cause illness in humans who ingest toxic filter-feeding shellfish or inhale toxic aerosols. The toxins are also suspected of having been involved in events in which many manatees and dolphins died, but this has usually not been verified owing to limited confirmation of toxin exposure, unexplained intoxication mechanisms and complicating pathologies. Here we show that fish and seagrass can accumulate high concentrations of brevetoxins and that these have acted as toxin vectors during recent deaths of dolphins and manatees, respectively. Our results challenge claims that the deleterious effects of a brevetoxin on fish (ichthyotoxicity) preclude its accumulation in live fish, and they reveal a new vector mechanism for brevetoxin spread through food webs that poses a threat to upper trophic levels.     

Induction of light chain expression in a pre-B cell line by fusion to myeloma cells

Pre-B cells, the first cells in the B-lymphocyte differentiation pathway which express immunoglobulin, have recently been shown to express cytoplasmic mu heavy chain (H) but not light chain (L). If, as is believed, pre-B cells are the precursors of immature B lymphocytes, which express surface IgM, the differentiation of pre-B cells to immature B lymphocytes must be accompanied by the expression of light chains. In this case, it should be possible for the progeny of a single pre-B cell to express a variety of light chains in association with the same heavy chain. We have tested this hypothesis by hybridizing a pre-B cell line 18-81 expressing only cytoplasmic mu chains with variant myeloma cells which do not express light chains. Hybridization of B-lymphoma cells with myeloma cells usually produces a hybrid with the phenotype of the more differentiated parent. In this case, the fusion resulted in the induction of light chain expression from the 18-81 genes and we have been able to demonstrate that independent hybrids express different light chains, in accordance with the hypothesis that a pre-B cell committed to expression of a single mu heavy chain can generate progeny expressing different slight chains.     

Clostridium botulinum can grow and form toxin at pH values lower than 4.6.

It is generally accepted that in Clostridium botulinum both growth and toxin formation are completely inhibited at pH values below 4.6. This critical pH value has been confirmed by many investigators using food as substrate or culture media. Occasionally growth of C. botulinum and toxin formation at pH values lower than 4.6 have been reported. In these cases the authors ascribed the unexpected outgrowth and toxin formation to local pH differences in inhomogeneous media and growth of C. botulinum before pH equilibration, or to the fact that fungi created microenvironments within or adjacent to the mycelial mat, where the pH was higher than 4.6 as was demonstrated by Odlaug and Pflug. We show here that the general assumption that C. botulinum does not grow below pH 4.6 is incorrect. We have observed that growth and toxin formation by C. botulinum can take place in homogeneous protein rich substrates (containing 3% or more soya or milk protein) at pH values lower than 4.6.     

A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin

Antibodies and growth factors have been chemically coupled to different toxins to produce cytotoxic molecules that selectively kill cells bearing appropriate antigens or receptors. Antibody-toxin conjugates (immunotoxins) produced using conventional chemical coupling techniques have several undesirable characteristics. The smallest binding unit of an antibody is an Fv fragment which consists of a light and heavy chain variable domain. Recently, active single chain Fv fragments of antibodies have been produced in Escherichia coli by attaching the light and heavy chain variable domains together with a peptide linker. Here we describe the construction and expression in E. coli of a single chain antibody toxin fusion protein, anti-Tac(Fv)-PE40, in which the variable regions of anti-Tac, a monoclonal antibody to the p55 subunit of the human interleukin-2 receptor, are joined in peptide linkage to PE40, a modified form of Pseudomonas exotoxin lacking its binding domain. Anti-Tac(Fv)-PE40 was very cytotoxic to two interleukin-2 receptor-bearing human cell lines but was not cytotoxic to receptor-negative cells.     

Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic alpha-helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.     

Autocatalytic cleavage of Clostridium difficile toxin B

Clostridium difficile, the causative agent of nosocomial antibiotic-associated diarrhoea and pseudomembranous colitis, possesses two main virulence factors: the large clostridial cytotoxins A and B. It has been proposed that toxin B is cleaved by a cytosolic factor of the eukaryotic target cell during its cellular uptake. Here we report that cleavage of not only toxin B, but also all other large clostridial cytotoxins, is an autocatalytic process dependent on host cytosolic inositolphosphate cofactors. A covalent inhibitor of aspartate proteases, 1,2-epoxy-3-(p-nitrophenoxy)propane, completely blocked toxin B function on cultured cells and was used to identify its catalytically active protease site. To our knowledge this is the first report on a bacterial toxin that uses eukaryotic signals for induced autoproteolysis to deliver its toxic domain into the cytosol of target cells. On the basis of our data, we present an integrated model for the uptake and inositolphosphate-induced activation of toxin B.     

聚丙烯腈PAN结构链的密度泛函理论研究

在HF/STO-3g基础上,利用密度泛函B3P86方法,选用6-311g基组,对聚丙烯腈PAN的结构链进行了理论研究.在结构优化方面,主链CC平均键长0.154 1 nm,属于sp3型杂化.支链CC平均键长0.146 0 nm.CN平均键长0.115 7 nm.电荷分布方面,计算得出,主链C原子带较强的负电荷,N原子带负电荷,相对较弱.H原子带较强的正电荷,是和N原子相连的C原子所带正电荷的3倍多.最后分析了结构链的振动模式.