Stoichiometric Inhibition of Amyloid β-Protein Aggregation with Peptides Containing Alternating α,α-Disubstituted Amino Acids

Abstract

We have prepared two peptides based on the hydrophobic core (Lys-Leu-Val-Phe-Phe) of amyloid beta-protein (Abeta) that contain alpha,alpha-disubstituted amino acids at alternating positions, but differ in the positioning of the oligolysine chain (AMY-1, C-terminus; AMY-2, N-terminus). We have studied the effects of AMY-1 and AMY-2 on the aggregation of Abeta and find that, at stoichiometric concentrations, both peptides completely stop Abeta fibril growth. Equimolar mixtures of AMY-1 and Abeta form only globular aggregates as imaged by scanning force microscopy and transmission electron microscopy. These samples show no signs of protofibrillar or fibrillar material even after prolonged periods of time (4.5 months). Also, 10 mol % of AMY-1 prevents Abeta self-assembly for long periods of time; aged samples (4.5 months) show only a few protofibrillar or fibrillar aggregates. Circular dichroism spectroscopy of equimolar mixtures of AMY-1 and Abeta show that the secondary structure of the mixture changes over time and progresses to a predominantly beta-sheet structure, which is consistent with the design of these inhibitors preferring a sheet-like conformation. Changing the position of the charged tail on the peptide, AMY-2 interacts with Abeta differently in that equimolar mixtures form large ( approximately 1 mum) globular aggregates which do not progress to fibrils, but precipitate out of solution. The differences in the aggregation mediated by the two peptides is discussed in terms of a model where the inhibitors act as cosurfactants that interfere with the native ability of Abeta to self-assemble by disrupting hydrophobic interactions either at the C-terminus or N-terminus of Abeta.

Effects of Peptides Derived from Terminal Modifications of the Aβ Central Hydrophobic Core on Aβ Fibrillization

“…This indicates that the N-terminal modification enhances aggregation to form larger assemblies. We have previously reported similar results where large sizes of particles formed with N-terminal modification of RRAA-KLVFF compared to C-terminal modification using six oligolysine chains (25). The particle size difference is closely Article associated with the disruption of the hydrophobic C-terminal group (50), which has more impact on aggregation than the hydrophilic N-terminal (51).…”

“…In contrast, when Aβ 1-40 was aged in the presence of equimolar AAMPs, an unusual CD signature with characteristics of random coil and β sheet was observed, consistent with our previous results for an AAMP-1/Aβ 1-40 mixture (see Supporting Information, Figure S3 for another example of CD with different AAMPs with identical results.) (25). This suggests that our AAMPs delay or disrupt Aβ 1-40 aggregation into β-sheet rich assemblies.…”

“…We have previously shown that AAMP-1 disrupted Aβ fibril formation (25). In vivo studies of AAMP-1 on APP transgenic mice displayed elevated toxicity at higher concentrations (32).…”

“…The effect of RRAA-AAMPs on Aβ 1-40 assembly into β-sheet structure was examined using far-UV (25,40). In contrast, when Aβ 1-40 was aged in the presence of equimolar AAMPs, an unusual CD signature with characteristics of random coil and β sheet was observed, consistent with our previous results for an AAMP-1/Aβ 1-40 mixture (see Supporting Information, Figure S3 for another example of CD with different AAMPs with identical results.)…”

“…Both mitigator peptides AMY-1 and AMY-2 disrupted fibril formation and formed spherical aggregates of different heights (25). We have shown that AMY-1 has the ability to reduce Aβ deposits when injected into the hippocampus of 14-month-old APP transgenic mice, a model of amyloid deposition (26).…”