Engineering of a peptide probe for β-amyloid aggregates

Abstract

Aggregation of β-amyloid (Aβ) is central to the pathogenesis of Alzheimer's disease (AD). Aβ aggregation produces amyloid assemblies, such as oligomers and fibrils. In contrast to non-toxic Aβ monomers, Aβ oligomers and fibrils can act directly as major toxic agents and indirectly as pools of the toxic entities, respectively. Thus, the detection of Aβ aggregates is of diagnostic interest and should benefit enhanced molecular understanding of AD. Among many molecular platforms, peptide-based ligands hold promise as Aβ probes due to their relative simplicity, ease of optimization and facile conjugation to other molecular contexts. In this regard, Aβ hydrophobic segments (critical in Aβ self-assembly) or variants thereof can serve as lead molecules for Aβ probe development. Unfortunately, the resulting peptides are either highly self-aggregation-prone or their probe potential has not been thoroughly examined. In the present study, we characterized a novel peptide ligand, KLVFWAK, which was created by simple point mutations of an Aβ hydrophobic segment ((16)KLVFFAE(22)). We found that KLVFWAK displayed low self-aggregation propensity and was preferentially bound to Aβ oligomers and fibrils relative to Aβ monomers. Interestingly, binding of KLVFWAK to Aβ aggregates occurred at a non-homologous Aβ segment (e.g., Aβ C-terminal domain) rather than the homologous (16)KLVFFAE(22). We also show that detection of Aβ aggregates during incubation of fresh Aβ was possible with KLVFWAK, further supporting KLVFWAK's high probe potential for Aβ aggregates. In short, this study presents creation of a non-self-aggregating peptide ligand for Aβ aggregates through simple point mutation of an Aβ-derived segment.

Effects of Aβ-derived peptide fragments on fibrillogenesis of Aβ

“…The first potentially disease-modifying AD drug aducanumab was recently approved by the Food and Drug Administration based on its ability to clear Aβ from brain parenchyma. Short peptides, related or unrelated to Aβ sequence, have been employed to inhibit Aβ aggregation or disperse preformed oligomers and fibrils and impede Aβ-induced cytotoxicity 54 – 67 . The inhibitory effect of peptides on Aβ aggregation and toxicity stems from their ability to intercalate into Aβ assemblies and prevent toxic oligomer formation, which may or may not correlate with their own self-aggregation propensities.…”

Amyloid Aggregation of Bacillus circulans Xylanase under Native Conditions and its Modulation by β‐Amyloid‐Derived Peptide Fragments

“…During the 14‐day incubation, the CD signals of BCX‐KLVFWAK were gradually weakened, which is accounting for by the reduction in soluble protein fractions and/or the loss of secondary structure (Figure B), accompanied by the formation of fibrils in BCX‐KLVFWAK samples (Figure C). As reported previously, KLVFWAK itself was shown to display limited amyloid‐assembly under the incubation conditions—ThT fluorescence of KLVFWAK remained negligible during the 14‐day incubation (Figure A)—this is confirmed by the lack of amyloid fibrils (Figure D) due to electrostatic repulsion caused by the terminal lysine residues . The expression level of a BCX variant with an N‐terminal fusion of KLVFWAK was significantly low and, thus, the fusion was not studied further.…”

“…Aβ40 and αS contain 40 and 140 residues, respectively, and display no significant sequence similarity to each other or to BCX. Our previous studies have demonstrated that the amyloid aggregation of Aβ40 and αS is readily detectable by ThT fluorescence …”

“…C‐terminal fusion of KLVFWAK, however, accelerated the amyloid aggregation of BCX and lowered BCX′s kinetic stability, which could result from local rather than global structural alteration under native conditions. Interestingly, despite the limited self‐aggregation of KLVFWAK as described above (Figure A) and previously, a part of this fragment, in particular LVFWA, is still highly amyloidogenic, as judged by Tango score of ≈80 (Figure S8). Thus, one possible explanation for the observed reduction in lag phase for BCX amyloid aggregation by C‐terminal fusion of KLVFWAK (Figure A) might be the LVFWA sequence, which could contribute to assembly among BCX‐KLVFWAK.…”

Amyloid ß-Targeted Inhibitory Peptides for Alzheimer’s Disease: Current State and Future Perspectives

“…The designed motif exhibited a lower self-aggregation tendency in comparison to other KLVFF-related sequences. Moreover, it demonstrated a higher binding affinity to Aβ aggregates and fibrils than monomers (65).…”