Transthyretin-derived peptides as β-amyloid inhibitors

Abstract

Self-association of β-amyloid (Aβ) into soluble oligomers and fibrillar aggregates is associated with Alzheimer's disease pathology, motivating the search for compounds that selectively bind to and inhibit Aβ oligomerization and/or neurotoxicity. Numerous small-molecule inhibitors of Aβ aggregation or toxicity have been reported in the literature. However, because of their greater size and complexity, peptides and peptidomimetics may afford improved specificity and affinity as Aβ aggregation modulators compared to small molecules. Two divergent strategies have been employed in the search for peptides that bind Aβ: (i) using recognition domains corresponding to sequences in Aβ itself (such as KLVFF) and (ii) screening random peptide-based libraries. In this study, we propose a third strategy, specifically, designing peptides that mimic binding domains of Aβ-binding proteins. Transthyretin, a plasma transport protein that is also relatively abundant in cerebrospinal fluid, has been shown to bind to Aβ, inhibit aggregation, and reduce its toxicity. Previously, we identified strand G of transthyretin as a specific Aβ binding domain. In this work we further explore and define the necessary features of this binding domain. We demonstrate that peptides derived from transthyretin bind Aβ and inhibit its toxicity. We also show that, although both transthyretin and transthyretin-derived peptides bind Aβ and inhibit toxicity, they differ significantly in their effect on Aβ aggregation.

Figure 1

Ribbon structure of transthyretin (PDB entry 1DVQ) tetramer, showing residues corresponding to peptide G16 (residues 102–117, yellow) and peptide EFh (residues 74–83, blue). Leucine side chains for L82 (blue) and L110 (yellow) are shown explicitly. Each monomer contains two four-stranded β-sheets and a single helix.

Figure 2

Aβ binding to G-derived peptides. (A) Peptide sequences corresponding to each spot on the membrane. (B) Aβ was bound to the peptide array; then bound Aβ was transferred to a PVDF membrane and imaged by Western blot analysis. (C) Comparison of amyloidogenicity (TANGO, solid bars) and hydrophobicity (GRAVY, open bars) scores for binders and nonbinders. The TANGO Score was calculated by analyzing each sequence for amyloidogenicity in TANGO, summing up the total score for each position, and then dividing by the number of residues. The GRAVY score is the grand average of hydropathy and was calculated using an online calculator www.gravy-calculator.de/.

Figure 3

(A) Photoinduced cross-linking of Aβ incubated without (−) or with peptides G16, Gsc, GLA, EFh, and G12. Position of molecular weight markers (in kDa) is shown on left. Aβ concentration was 24 μM, and peptide concentration was 2.4 μM. Aβ was detected by Western blot with anti-Aβ antibody 4G8. (B) Photoinduced cross-linking of G16, Gsc, GLA, EFh, and G12. Peptide concentration was 24 μM, and peptides were visualized by silver staining.

Figure 4

Proteolytic fragmentation assay of Aβ incubated with or without TTR-derived peptides G16, Gsc, GLA, G12, and EFh. Aβ concentration was 20 μM, and peptide concentration was 40 μM.

Figure 5

Effect of TTR-derived peptides and mTTR on Aβ aggregate growth kinetics. The mean hydrodynamic diameter (a) and average scattering intensity (b) were measured by dynamic light scattering. Samples contained 140 μM Aβ alone (○) or Aβ with peptide G16 (△), with peptide Gsc (●), and with mTTR (▲). Concentration for G16, Gsc, and mTTR was 12 μM. At the conditions of these experiments, concentrations of G16, Gsc, and mTTR were too low to contribute to the scattering signal.

Figure 6

TEM images, taken after 20 h incubation at 37 °C: (a) 140 μM Aβ, (b) 140 μM Aβ + 12 μM G16, and (c) 12 μM G16. Scale bar is 200 nm in all images.

Figure 7

Thioflavin T fluorescence intensity. Samples were prepared with Aβ alone (30 μM) or with the indicated peptide or protein (2.5 μM), and then analyzed immediately or incubated for 20 h prior to analysis. Samples were diluted 6.5-fold into ThT-containing solution, and fluorescence emission intensity was measured immediately. Background intensity was measured with ThT-containing solution and subtracted. *Differs from Aβ alone (p < 0.05).

Figure 8

(top) Toxicity was measured in primary neuronal cultures using the MTS assay. G16 or Gsc was added at the indicated concentration and incubated with Aβ (10 μM) for 1 h before addition to neuronal cultures. (bottom) TUNEL staining was used to confirm the results of the MTS assay. Aβ alone (10 μM) was toxic; 5 μM G16 prevented Aβ toxicity.

Figure 9

EM of G16 taken after 9 day incubation at 37 °C and 12 μM.