Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity.

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.

Synergistically acting PLA2: peptide hemorrhagic complex from Daboia russelii venom.

Snake venoms are complex mixture of enzymatic and non-enzymatic proteins. Non-covalent protein-protein interaction leads to protein complexes, which bring about enhanced pharmacological injuries by their synergistic action. Here we report identification and characterization of a new Daboia russelii hemorrhagic complex I (DR-HC-I) containing phospholipase A2 (PLA2) and non-enzymatic peptide. DR-HC-I was isolated from the venom of D. russelii by CM-Shepadex-C25 and gel permeation chromatography. Individual components were purified and identified by RP-HPL chromatography, mass spectrometry and N-terminal amino acid sequencing. DR-HC-I complex was lethal to mice with the LD50 dose of 0.7 mg/kg body weight with hemorrhagic and neurotoxic properties. DR-HC-I complex consists of non-hemorrhagic PLA2 and neurotoxic non-enzymatic peptide. The non-enzymatic peptide quenched the intrinsic fluorescence of PLA2 in a dose dependent manner, signifying the synergistic interaction between two proteins. PLA2 and peptide toxin in a 5:2 M ratio induced skin hemorrhage in mice with MHD 20 µg. However, addition of ANS (1-Anilino-8-naphthalene sulfonate) to DR-HC-I complex inhibited skin hemorrhagic effect and also synergic interaction. But there was no impact on PLA2 due to this synergistic interaction, and indirect hemolytic or plasma re-calcification activity. However, the synergistic interaction of PLA2 and non-enzymatic peptide contributes to the enhanced venom-induced hemorrhage and toxicity of Daboia russellii venom.

Molecular assembly of botulinum neurotoxin progenitor complexes.

Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum and associates with nontoxic neurotoxin-associated proteins to form high-molecular weight progenitor complexes (PCs). The PCs are required for the oral toxicity of BoNT in the context of food-borne botulism and are thought to protect BoNT from destruction in the gastrointestinal tract and aid in absorption from the gut lumen. The PC can differ in size and protein content depending on the C. botulinum strain. The oral toxicity of the BoNT PC increases as the size of the PC increases, but the molecular architecture of these large complexes and how they contribute to BoNT toxicity have not been elucidated. We have generated 2D images of PCs from strains producing BoNT serotypes A1, B, and E using negative stain electron microscopy and single-particle averaging. The BoNT/A1 and BoNT/B PCs were observed as ovoid-shaped bodies with three appendages, whereas the BoNT/E PC was observed as an ovoid body. Both the BoNT/A1 and BoNT/B PCs showed significant flexibility, and the BoNT/B PC was documented as a heterogeneous population of assembly/disassembly intermediates. We have also determined 3D structures for each serotype using the random conical tilt approach. Crystal structures of the individual proteins were placed into the BoNT/A1 and BoNT/B PC electron density maps to generate unique detailed models of the BoNT PCs. The structures highlight an effective platform that can be engineered for the development of mucosal vaccines and the intestinal absorption of oral biologics.

The enhancing effect of homocysteine thiolactone on insulin fibrillation and cytotoxicity of insulin fibril.

In the current study both structural alteration and fibrillation of insulin were studied in the presence of homocysteine thiolactone (HCTL). The spectroscopic studies revealed that HCTL increases rate of insulin unfolding, giving rise to the appearance of solvent-exposed hydrophobic regions and induces a transition from α-helix into predominantly ß-sheet structures. Thioflavin-T fluorescence studies revealed that HCTL markedly enhanced the quantity of insulin fibril formation in both agitating and non-agitating systems. Also gel electrophoresis results suggest that HCTL accelerates the process of formation of high molecular weight insulin aggregates. Moreover, insulin fibrils obtained in the presence of HCTL and those collected earlier in the pathway of insulin fibrillation displayed improved cytotoxicity against cancer cells. The enhancement of insulin fibril formation with elevated cytotoxic properties as occurred in the presence of HCTL, may suggest this homocysteine derivative as a possible contributing factor in the pathology of insulin fibrils.

Preparation and characterization of toxic Abeta aggregates for structural and functional studies in Alzheimer's disease research.

The amyloid cascade hypothesis, supported by strong evidence from genetics, pathology and studies using animal models, implicates amyloid-beta (Abeta) oligomerization and fibrillogenesis as central causative events in the pathogenesis of Alzheimer's disease (AD). Today, significant efforts in academia, biotechnology and the pharmaceutical industry are devoted to identifying the mechanisms by which the process of Abeta aggregation contributes to neurodegeneration in AD and to the identity of the toxic Abeta species. In this paper, we describe methods and detailed protocols for reproducibly preparing Abeta aggregates of defined size distribution and morphology, including monomers, protofibrils and fibrils, using size exclusion chromatography. In addition, we describe detailed biophysical procedures for elucidating the structural features, aggregation kinetics and toxic properties of the different Abeta aggregation states, with special emphasis on protofibrillar intermediates. The information provided by this approach allows for consistent correlation between the properties of the aggregates and their toxicity toward primary neurons and/or cell lines. A better understanding of the molecular and structural basis of Abeta aggregation and toxicity is crucial for the development of effective strategies aimed at prevention and/or treatment of AD. Furthermore, the identification of specific aggregation states, which correlate with neurodegeneration in AD, could lead to the development of diagnostic tools to detect and monitor disease progression. The procedures described can be performed in as little as 1 day, or may take longer, depending on the exact toxicity assays used.

Complement 5b-9 complex-induced alterations in human RPE cell physiology.

BACKGROUND: Recent evidence shows that complements are closely related to the occurrence of choroidal neovascularization (CNV). We studied the effect of complement 5b-9 complex (C5b-9) on membrane permeability and molecular biological behavior in cultured human retinal pigment epithelium (RPE) cells and considered the role of C5b-9 in CNV. MATERIAL/METHODS: Human RPE cells were exposed to different concentrations of C5b-9 for 24 hours, then observed through light and electron microscopy. The dynamics of calcium ion change in cells exposed to sublysis C5b-9 were analyzed by confocal laser scanning microscope, and the amount of VEGF and TGF-beta2 mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR) RESULTS: RPE cells were destroyed when exposed to 80 microg/ml and 40 microg/ml C5b-9. The structure of RPE cells was not obviously changed when exposed to 20 microg/ml or less C5b-9; however, pigment granules are released from the cell membrane when observed using electron microscopy. In most of the cells, calcium fluorescence intensity increased rapidly after the deposition of C5b-9, to a peak at 4 min, lasted for about 6 min, and then began to decrease. The expression of VEGF and TGF- beta2 mRNA in RPE cells with C5b-9 was increased at 4 h and decreased at 24 h, but they were higher than in the control group. CONCLUSIONS: These observations suggest C5b-9 can induce a change in membrane permeability, an increase in cytoplasmic calcium ion concentration, and significant up-regulation of angiogenic factors in cultured RPE cells, which may be one of many potential mechanisms of CNV formation.

Formation of cytotoxic transthyretin is not dependent on inter-molecular disulphide bridges commonly found within the amyloid form.

Familial amyloidotic polyneuropathy (FAP) is linked to destabilising point mutations in the human plasma protein transthyretin (TTR). Consistent with similar amyloid disorders, low molecular weight TTR oligomers have been shown to exert the major cytotoxic effect. The amyloid structure of TTR contains non-native inter-molecular disulphide linkages via the cysteine at position 10 (Cys10). Moreover, substitution of Cys10 in a mouse model for TTR-amyloidosis abolishes TTR deposits, indicating an important role of Cys10 in FAP pathogenesis. However, the role of disulphide bridges in TTR cytotoxicity has not been elucidated. By probing Cys10Ser TTR variants to the human neuroblastoma SH-SY5Y cell line, we have addressed this question, and our results clearly show that formation of an inter-molecular disulphide bridge is not a pre-requisite for TTR cytotoxicity. This finding suggests that prevention of inter-molecular TTR disulphide bridges as a therapeutic intervention will not impair the cytotoxic potential of TTR.

Insights into stability and toxicity of amyloid-like oligomers by replica exchange molecular dynamics analyses.

Deposition of insoluble amyloid plaques is frequently associated with a large variety of neurodegenerative diseases. However, data collected in the last decade have suggested that the neurotoxic action is exerted by prefibrillar, soluble assemblies of amyloid-forming proteins and peptides. The scarcity of structural data available for both amyloid-like fibrils and soluble oligomers is a major limitation for the definition of the molecular mechanisms linked to the onset of these diseases. Recently, the structural characterization of GNNQQNY and other peptides has shown a general feature of amyloid-like fibers, the so-called steric zipper motif. However, very little is known still about the prefibrillar oligomeric forms. By using replica exchange molecular dynamics we carried out extensive analyses of the properties of several small and medium GNNQQNY aggregates arranged through the steric zipper motif. Our data show that the assembly formed by two sheets, each made of two strands, arranged as in the crystalline states are highly unstable. Conformational free energy surfaces indicate that the instability of the model can be ascribed to the high reactivity of edge backbone hydrogen bonding donors/acceptors. On the other hand, data on larger models show that steric zipper interactions may keep small oligomeric forms in a stable state. These models simultaneously display two peculiar structural motifs: a tightly packed steric zipper interface and a large number of potentially reactive exposed strands. The presence of highly reactive groups on these assemblies likely generates two distinct evolutions. On one side the reactive groups quickly lead, through self-association, to the formation of ordered fibrils, on the other they may interfere with several cellular components thereby generating toxic effects. In this scenario, fiber formation propensity and toxicity of oligomeric states are two different manifestations of the same property: the hyper-reactivity of the exposed strands.