In vitro and mechanistic studies of an antiamyloidogenic self-assembled cyclic D,L-α-peptide architecture.

Misfolding of the Aß protein and its subsequent aggregation into toxic oligomers are related to Alzheimer's disease. Although peptides of various sequences can self-assemble into amyloid structures, these structures share common three-dimensional features that may promote their cross-reaction. Given the significant similarities between amyloids and the architecture of self-assembled cyclic D,L-α-peptide, we hypothesized that the latter may bind and stabilize a nontoxic form of Aß, thereby preventing its aggregation into toxic forms. By screening a focused library of six-residue cyclic D,L-α-peptides and optimizing the activity of a lead peptide, we found one cyclic D,L-α-peptide (CP-2) that interacts strongly with Aß and inhibits its aggregation. In transmission electron microscopy, optimized thioflavin T and cell survival assays, CP-2 inhibits the formation of Aß aggregates, entirely disassembles preformed aggregated and fibrillar Aß, and protects rat pheochromocytoma PC12 cells from Aß toxicity, without inducing any toxicity by itself. Using various immunoassays, circular dichroism spectroscopy, photoinduced cross-linking of unmodified proteins (PICUP) combined with SDS/PAGE, and NMR, we probed the mechanisms underlying CP-2's antiamyloidogenic activity. NMR spectroscopy indicates that CP-2 interacts with Aß through its self-assembled conformation and induces weak secondary structure in Aß. Upon coincubation, CP-2 changes the aggregation pathway of Aß and alters its oligomer distribution by stabilizing small oligomers (1-3 mers). Our results support studies suggesting that toxic early oligomeric states of Aß may be composed of antiparallel ß-peptide structures and that the interaction of Aß with CP-2 promotes formation of more benign parallel ß-structures. Further studies will show whether these kinds of abiotic cyclic D,L-α-peptides are also beneficial as an intervention in related in vivo models.

Specific binding of a ß-cyclodextrin dimer to the amyloid ß peptide modulates the peptide aggregation process.

Alzheimer's disease involves progressive neuronal loss. Linked to the disease is the amyloid ß (Aß) peptide, a 38-43-amino acid peptide found in extracellular amyloid plaques in the brain. Cyclodextrins are nontoxic, cone-shaped oligosaccharides with a hydrophilic exterior and a hydrophobic cavity making them suitable hosts for aromatic guest molecules in water. ß-Cyclodextrin consists of seven α-d-glucopyranoside units and has been shown to reduce the level of fibrillation and neurotoxicity of Aß. We have studied the interaction between Aß and a ß-cyclodextrin dimer, consisting of two ß-cyclodextrin monomers connected by a flexible linker. The ß-cyclodextrin monomer has been found to interact with Aß(1-40) at sites Y10, F19, and/or F20 with a dissociation constant (K(D)) of 3.9 ± 2.0 mM. Here (1)H-(15)N and (1)H-(13)C heteronuclear single-quantum correlation nuclear magnetic resonance (NMR) spectra show that in addition, the ß-cyclodextrin monomer and dimer bind to the histidines. NMR translational diffusion experiments reveal the increased affinity of the ß-cyclodextrin dimer (apparent K(D) of 1.1 ± 0.5 mM) for Aß(1-40) compared to that of the ß-cyclodextrin monomer. Kinetic aggregation experiments based on thioflavin T fluorescence indicate that the dimer at 0.05-5 mM decreases the lag time of Aß aggregation, while a concentration of 10 mM increases the lag time. The ß-cyclodextrin monomer at a high concentration decreases the lag time of the aggregation. We conclude that cyclodextrin monomers and dimers have specific, modulating effects on the Aß(1-40) aggregation process. Transmission electron microscopy shows that the regular fibrillar aggregates formed by Aß(1-40) alone are replaced by a major fraction of amorphous aggregates in the presence of the ß-cyclodextrin dimer.

pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-ß peptide.

Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-ß (Aß) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with (15)N- and (13)C,(15)N-labeled Aß(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aß may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aß peptide under these conditions.

N-terminal engineering of amyloid-ß-binding Affibody molecules yields improved chemical synthesis and higher binding affinity.

The aggregation of amyloid-ß (Aß) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease. Molecules binding with high affinity and selectivity to Aß-peptides are important tools for investigating the aggregation process. An Aß-binding Affibody molecule, ZAß3 , has earlier been selected by phage display and shown to bind Aß(1-40) with nanomolar affinity and to inhibit Aß-peptide aggregation. In this study, we create truncated functional versions of the ZAß3 Affibody molecule better suited for chemical synthesis production. Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges. The N-terminally truncated Affibody molecules ZAß3 (12-58), ZAß3 (15-58), and ZAß3 (18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule ZAß3 (1-58). Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, ZAß3 (18-58), exhibited complete loss of binding to the Aß(1-40)-peptide, while the ZAß3 (12-58) and ZAß3 (15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the Aß(1-40)-peptide compared to the full-length Affibody molecule. Nuclear magnetic resonance spectroscopy showed that the structure of Aß(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the Aß(1-40)-peptide in complex with the full-length ZAß3 Affibody molecule. This indicates that the N-terminally truncated Affibody molecules ZAß3 (12-58) and ZAß3 (15-58) are highly promising for further engineering and future use as binding agents to monomeric Aß(1-40).

Detergent-like interaction of Congo red with the amyloid beta peptide.

Accumulating evidence links prefibrillar oligomeric species of the amyloid beta peptide (Abeta) to cellular toxicity in Alzheimer's disease, potentially via disruption of biological membranes. Congo red (CR) affects protein aggregation. It is known to self-associate into micelle-like assemblies but still reduces the toxicity of Abeta aggregates in cell cultures and model organisms. We show here that CR interacts with Abeta(1-40) in a manner similar to that of anionic detergents. Although CR promotes beta sheet formation and peptide aggregation, it may also solubilize toxic protein species, making them less harmful to critical cellular components and thereby reducing amyloid toxicity.

Secondary structure conversions of Alzheimer's Abeta(1-40) peptide induced by membrane-mimicking detergents.

The amyloid beta peptide (Abeta) with 39-42 residues is the major component of amyloid plaques found in brains of Alzheimer's disease patients, and soluble oligomeric peptide aggregates mediate toxic effects on neurons. The Abeta aggregation involves a conformational change of the peptide structure to beta-sheet. In the present study, we report on the effect of detergents on the structure transitions of Abeta, to mimic the effects that biomembranes may have. In vitro, monomeric Abeta(1-40) in a dilute aqueous solution is weakly structured. By gradually adding small amounts of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate to a dilute aqueous solution, Abeta(1-40) is converted to beta-sheet, as observed by CD at 3 degrees C and 20 degrees C. The transition is mainly a two-state process, as revealed by approximately isodichroic points in the titrations. Abeta(1-40) loses almost all NMR signals at dodecyl sulfate concentrations giving rise to the optimal beta-sheet content (approximate detergent/peptide ratio = 20). Under these conditions, thioflavin T fluorescence measurements indicate a maximum of aggregated amyloid-like structures. The loss of NMR signals suggests that these are also involved in intermediate chemical exchange. Transverse relaxation optimized spectroscopy NMR spectra indicate that the C-terminal residues are more dynamic than the others. By further addition of SDS or lithium dodecyl sulfate reaching concentrations close to the critical micellar concentration, CD, NMR and FTIR spectra show that the peptide rearranges to form a micelle-bound structure with alpha-helical segments, similar to the secondary structures formed when a high concentration of detergent is added directly to the peptide solution.

Antiprion properties of prion protein-derived cell-penetrating peptides.

In prion diseases, the cellular prion protein (PrP(C)) becomes misfolded into the pathogenic scrapie isoform (PrP(Sc)) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1-1) and scrapie-infected (ScGT1-1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrP(C) or PrP(Sc). Treatment with the prion protein-derived CPPs mouse mPrP(1-28) or bovine bPrP(1-30) significantly reduced PrP(Sc) levels in prion-infected cells but had no effect on PrP(C) levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1-1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP(23-28,) mPrP(19-30,) or mPrP(23-50)) had no effect on PrP(Sc) levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrP(Sc) and disables formation of prions.