A Cyclic Peptide Mimic of the β-Amyloid Binding Domain on Transthyretin

Abstract

Self-association of β-amyloid (Aβ) into oligomers and fibrils is associated with Alzheimer's disease (AD), motivating the search for compounds that bind to and inhibit Aβ oligomerization and/or neurotoxicity. Peptides are an attractive class of such compounds, with potential advantages over small molecules in affinity and specificity. Self-complementation and peptide library screening are two strategies that have been employed in the search for peptides that bind to Aβ. Alternatively, one could design Aβ-binding peptides based on knowledge of complementary binding proteins. One candidate protein, transthyretin (TTR), binds Aβ, inhibits aggregation, and reduces its toxicity. Previously, strand G of TTR was identified as part of a specific Aβ binding domain, and G16, a 16-mer peptide with a sequence that spans strands G and H of TTR, was synthesized and tested. Although both TTR and G16 bound to Aβ, they differed significantly in their effect on Aβ aggregation, and G16 was less effective than TTR at protecting neurons from Aβ toxicity. G16 lacks the β-strand/loop/β-strand structure of TTR's Aβ binding domain. To enforce proper residue alignment, we transplanted the G16 sequence onto a β-hairpin template. Two peptides with 18 and 22 amino acids were synthesized using an orthogonally protected glutamic acid derivative, and an N-to-C cyclization reaction was carried out to further restrict conformational flexibility. The cyclized 22-mer (but not the noncyclized 22-mer nor the 18-mer) strongly suppressed Aβ aggregation into fibrils, and protected neurons against Aβ toxicity. The imposition of structural constraints generated a much-improved peptidomimetic of the Aβ binding epitope on TTR.

Keywords: Alzheimer’s disease; Beta-amyloid; amyloid fibrils; cyclic peptide; peptidomimetics; transthyretin.

Figure 1

Cyclic peptide design strategy (a) Ribbon structure of transthyretin (PDB entry 1DVQ) monomer, showing strands G and H in blue. Sidechains from residues 102 to 125 are shown explicitly. Leu110, a critical residue for interaction, is shown in red. Sequence of strands G and H is shown, with the boxed region corresponding to the sequence of G16. (b) Design of cyclic peptides CG2 and CG3, mimicking the structure of strand G/loop/strand H using a dipeptide ^DPro-^LPro template (green box). Residues in red boxes are changed from the native sequence in order to facilitate cyclization.

Figure 2

Purification and characterization of β-hairpin peptides LG3 and CG3. (a) RP-HPLC analysis of crude peptide after on-resin cyclization and cleavage. Mass spectrometry analysis of the purified fractions confirmed the identity of peak 1 (LG3) and peak 2 (CG3). (b) CD spectra of G16, LG3, and CG3. *For this experiment, G16 was not capped.

Figure 3

(a) Gel electrophoresis analysis of photoinduced cross-linked (XL) and un-cross-linked (Non-XL) peptides. Peptide concentration was 36 µM, and peptides were visualized by silver staining. Non-cross-linked CG3 and LG3 were boiled and separated on the right two lanes for comparison. (b) TEM image of CG3, illustrating micelle-like aggregates of ~10–40 nm diameter. Vertical height of the image =200 nm.

Figure 4

Evidence of interaction between Aβ and peptides investigated by photoinduced cross-linking (PICUP). (a) Aβ was incubated without (−) or with peptides G16, CG3, LG3, CG2, LG2 and GF where Aβ concentration is in 10-fold excess. Samples were cross-linked prior to application to gels. Aβ was detected via Western blotting with 4G8. (b) Aβ at different aggregation state (freshly prepared or preaggregated) was incubated without (−) or with peptides G16 and CG3 prior to cross-linking and electrophoresis. Aβ was detected via Western blotting with 4G8.

Figure 5

Concentration dependence of the binding interaction between Aβ and CG3 investigated by photoinduced cross-linking (PICUP). Preaggregated Aβ (24 µM) was incubated without or with CG3 at varying concentrations. Samples were cross-linked prior to application to gels. Aβ was detected via Western blotting with 4G8. Density of the broad oligomer band at each concentration was measured using ImageJ and density of background was subtracted.

Figure 6

Evidence of interaction between Aβ and peptides investigated by proteolytic fragmentation assay. Aβ was incubated with or without G16, CG3, LG3, CG2 and GF for 24 h where peptide is in 3-fold excess. The relative rate of proteolytic fragmentation of Aβ was measured by addition of Proteinase K followed by dotting onto nitrocellulose membrane at different time points. Unfragmented species were detected by 6E10 and 4G8 antibodies.

Figure 7

Effect of peptides on Aβ aggregate growth kinetics measured by light scattering. The mean hydrodynamic diameter (top) and normalized scattering intensity (bottom) were measured for samples containing 140 µM Aβ without or with 14 µM CG3, LG3, CG2, G16 or GF. At the conditions of these experiments, concentrations of the peptides (in the absence of Aβ) were too low to contribute to the scattering signal.

Figure 8

Effect of peptides on Aβ aggregation measured by ThT fluorescence intensity. Samples containing 28 µM Aβ without or with 2.8 µM of the indicated peptide or protein were incubated at 37 °C for up to 48 h. After 1, 5, 24, and 48 h of incubation, samples were diluted 14-fold into a ThT-containing solution, and fluorescence emission intensity was measured immediately. Three replicates were averaged for each sample and the data for Aβ alone, Aβ+G16 and Aβ+CG3 represent the average of two independent experiments.

Figure 9

Effect of CG3 concentration and coincubation time on Aβ aggregation, measured by ThT fluorescence intensity. Samples containing 28 µM Aβ with the indicated concentration of CG3 were incubated at 37 °C for 1, 2, or 5 days. Samples were diluted 14-fold into a ThT-containing solution, fluorescence emission intensity was measured immediately and compared to a similarly prepared sample containing Aβ alone. Two to three measurements were averaged for each sample, and the data are the mean ± SD of two independent experiments. The curves are the nonlinear regression fit to the logistic equation but do not indicate any mechanistic interpretation.

Figure 10

TEM images, taken after 48 h incubation at 37 °C. (A and B) 28 µM Aβ, (C) 28 µM Aβ + 2.8 µM G16, and (D, E and F) 28 µM Aβ + 2.8 µM CG3. Scale bar for A and D is 500 nm and for B, C, E and F is 200 nm.

Figure 11

Effect of peptides on Aβ induced toxicity measured using the MTS assay with primary neuronal cultures. Dose-dependent protection afforded by (A) mTTR (B) G16, (C) CG3. In each graph, Aβ concentration was 10 µM and the molar concentration of the added protein or peptide is shown. (D) Comparison of efficacy of several peptides, all at 10 µM. *statistically different from vehicle (p < 0.05). #statistically different from Aβ (p < 0.05).