Amyloid beta-protein (Abeta) oligomers may be the proximate neurotoxins in Alzheimer's disease (AD). "Oligomer" is an ill-defined term because many kinds have been reported and they often exist in rapid equilibrium with monomers and higher-order assemblies. We report here results of studies in which specific oligomers have been stabilized structurally, fractionated in pure form, and then studied by using a combination of CD spectroscopy, Thioflavin T fluorescence, EM, atomic force microscopy (AFM), and neurotoxicity assays. Abeta monomers were largely unstructured, but oligomers exhibited order-dependent increases in beta-sheet content. EM and AFM data suggest that dimerization and subsequent monomer addition are processes in which significant and asymmetric monomer conformational changes occur. Oligomer secondary structure and order correlated directly with fibril nucleation activity. Neurotoxic activity increased disproportionately (order dependence >1) with oligomer order. The structure-activity correlations reported here significantly extend our understanding of the conformational dynamics, structure, and relative toxicity of pure Abeta oligomers of specific order.

Alzheimer's disease (AD) is linked to the aberrant assembly of the amyloid β-protein (Aβ). The (21)AEDVGSNKGA(30) segment, Aβ(21-30), forms a turn that acts as a monomer folding nucleus. Amino acid substitutions within this nucleus cause familial forms of AD. To determine the biophysical characteristics of the folding nucleus, we studied the biologically relevant acetyl-Aβ(21-30)-amide peptide using experimental techniques (limited proteolysis, thermal denaturation, urea denaturation followed by pulse proteolysis, and electron microscopy) and computational methods (molecular dynamics). Our results reveal a highly stable foldon and suggest new strategies for therapeutic drug development.

Mutations in the amyloid beta-protein (Abeta) precursor gene cause autosomal dominant Alzheimer disease in a number of kindreds. In two such kindreds, the English and the Tottori, the mutations produce amyloid beta-proteins containing amino acid substitutions, H6R and D7N, respectively, at the peptide N terminus. To elucidate the structural and biological effects of the mutations, we began by examining monomer conformational dynamics and oligomerization. Relative to their wild type homologues, and in both the Abeta40 and Abeta42 systems, the English and Tottori substitutions accelerated the kinetics of secondary structure change from statistical coil --> alpha/beta --> beta and produced oligomer size distributions skewed to higher order. This skewing was reflected in increases in average oligomer size, as measured using electron microscopy and atomic force microscopy. Stabilization of peptide oligomers using in situ chemical cross-linking allowed detailed study of their properties. Each substitution produced an oligomer that displayed substantial beta-strand (H6R) or alpha/beta (D7N) structure, in contrast to the predominately statistical coil structure of wild type Abeta oligomers. Mutant oligomers functioned as fibril seeds, and with efficiencies significantly higher than those of their wild type homologues. Importantly, the mutant forms of both native and chemically stabilized oligomers were significantly more toxic in assays of cell physiology and death. The results show that the English and Tottori mutations alter Abeta assembly at its earliest stages, monomer folding and oligomerization, and produce oligomers that are more toxic to cultured neuronal cells than are wild type oligomers.

Amyloid beta-protein (Abeta) assembly into toxic oligomeric and fibrillar structures is a seminal event in Alzheimer's disease, therefore blocking this process could have significant therapeutic benefit. A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Prior studies have shown that Abeta fibrillogenesis involves conformational changes leading to the formation of extended beta-sheets and that an alpha-helix-containing intermediate may be involved. However, the significance of this intermediate has been a matter of debate. We report here that the formation of an oligomeric, alpha-helix-containing assembly is a key step in Abeta fibrillogenesis. The generality of this phenomenon was supported by conformational studies of 18 different Abeta peptides, including wild-type Abeta(1-40) and Abeta(1-42), biologically relevant truncated and chemically modified Abeta peptides, and Abeta peptides causing familial forms of cerebral amyloid angiopathy. Without exception, fibrillogenesis of these peptides involved an oligomeric alpha-helix-containing intermediate and the kinetics of formation of the intermediate and of fibrils was temporally correlated. The kinetics varied depending on amino acid sequence and the extent of peptide N- and C-terminal truncation. The pH dependence of helix formation suggested that Asp and His exerted significant control over this process and over fibrillogenesis in general. Consistent with this idea, Abeta peptides containing Asp-->Asn or His-->Gln substitutions showed altered fibrillogenesis kinetics. These data emphasize the importance of the dynamic interplay between Abeta monomer conformation and oligomerization state in controlling fibrillogenesis kinetics.

Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer's disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu(2+). Cu(2+) cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu(2+) regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu(2+) cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu(2+) cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu(2+) cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu(2+) cations induce Aβ42 peptide structural changes resulting in N-termini interactions within the dimers. Cu(2+) cations also stabilize the dimers. No effects of Cu(2+) cations on Aβ-Aβ interactions were observed at pH 4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu(2+) cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu(2+) cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu(2+) cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu(2+) cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu(2+).

Formation of toxic oligomeric and fibrillar structures by the amyloid beta-protein (Abeta) is linked to Alzheimer's disease (AD). To facilitate the targeting and design of assembly inhibitors, intrinsic fluorescence was used to probe assembly-dependent changes in Abeta conformation. To do so, Tyr was substituted in Abeta40 or Abeta42 at position 1, 10 (wild type), 20, 30, 40, or 42. Fluorescence then was monitored periodically during peptide monomer folding and assembly. Electron microscopy revealed that all peptides assembled readily into amyloid fibrils. Conformational differences between Abeta40 and Abeta42 were observed in the central hydrophobic cluster (CHC) region, Leu17-Ala21. Tyr20 was partially quenched in unassembled Abeta40 but displayed a significant and rapid increase in intensity coincident with the maturation of an oligomeric, alpha-helix-containing intermediate into amyloid fibrils. This process was not observed during Abeta42 assembly, during which small decreases in fluorescence intensity were observed in the CHC. These data suggest that the structure of the CHC in Abeta42 is relatively constant within unassembled peptide and during the self-association process. Solvent accessibility of the Tyr ring was studied using a mixed solvent (dimethyl sulfoxide/water) system. [Tyr40]Abeta40, [Tyr30]Abeta42, and [Tyr42]Abeta42 all were relatively shielded from solvent. Analysis of the assembly dependence of the site-specific intrinsic fluorescence data suggests that the CHC is particularly important in controlling Abeta40 assembly, whereas the C-terminus plays the more significant role in Abeta42 assembly.

Aβ42 and Aβ40 are the two primary alloforms of human amyloid β-protein (Aβ). The two additional C-terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single-molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C-termini, the N-terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N-terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N-terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments.

A unique lipopeptide antimycotic, termed cryptocandin, is described from Cryptosporiopsis cf. quercina, an endophytic fungus. Cryptocandin, with a molecular mass of 1079 Da, contains equimolar amounts of 3,4-dihydroxyhomotyrosine, 4-hydroxyproline, threonine, glutamine, 3-hydroxy-4-hydroxymethylproline, 4,5-dihydroxyornithine and palmitic acid. Cryptocandin is chemically related to well-known antimycotics, the echinocandins and pneumocandins, which are produced by such fungi as Zalerion arboricola, Pezicula spp. and Aspergillus spp. Cryptocandin has minimal inhibitory concentration values of 0.03-0.07 microgram ml-1 against isolates of Candida albicans, Trichophyton mentagrophytes and Trichophyton rubrum. Cryptocandin is also active against a number of plant-pathogenic fungi including Sclerotinia sclerotiorum and Botrytis cinerea.

In a Flemish kindred, an Ala(692)-->Gly amino acid substitution in the amyloid beta-protein precursor (AbetaPP) causes a form of early-onset Alzheimer's disease (AD) which displays prominent amyloid angiopathy and unusually large senile plaque cores. The mechanistic basis of this Flemish form of AD is unknown. Previous in vitro studies of amyloid beta-protein (Abeta) production in HEK-293 cells transfected with cDNA encoding Flemish AbetaPP have shown that full-length [Abeta(1-40)] and truncated [Abeta(5-40) and Abeta(11-40)] forms of Abeta are produced. In an effort to determine how these peptides might contribute to the pathogenesis of the Flemish disease, comparative biophysical and neurotoxicity studies were performed on wild-type and Flemish Abeta(1-40), Abeta(5-40) and Abeta(11-40). The results revealed that the Flemish amino acid substitution increased the solubility of each form of peptide, decreased the rate of formation of thioflavin-T-positive assemblies, and increased the SDS-stability of peptide oligomers. Although the kinetics of peptide assembly were altered by the Ala(21)-->Gly substitution, all three Flemish variants formed fibrils, as did the wild-type peptides. Importantly, toxicity studies using cultured primary rat cortical cells showed that the Flemish assemblies were as potent a neurotoxin as were the wild-type assemblies. Our results are consistent with a pathogenetic process in which conformational changes in Abeta induced by the Ala(21)-->Gly substitution would facilitate peptide adherence to the vascular endothelium, creating nidi for amyloid growth. Increased peptide solubility and assembly stability would favour formation of larger deposits and inhibit their elimination. In addition, increased concentrations of neurotoxic assemblies would accelerate neuronal injury and death.