Transthyretin variants with improved inhibition of β-amyloid aggregation

Abstract

Aggregation of β-amyloid (Aβ) is widely believed to cause neuronal dysfunction in Alzheimer's disease. Transthyretin (TTR) binds to Aβ and inhibits its aggregation and neurotoxicity. TTR is a homotetrameric protein, with each monomer containing a short α-helix and two anti-parallel β-sheets. Dimers pack into tetramers to form a hydrophobic cavity. Here we report the discovery of a TTR mutant, N98A, that was more effective at inhibiting Aβ aggregation than wild-type (WT) TTR, although N98A and WT bound Aβ equally. The N98A mutation is located on a flexible loop distant from the putative Aβ-binding sites and does not alter secondary and tertiary structures nor prevent correct assembly into tetramers. Under non-physiological conditions, N98A tetramers were kinetically and thermodynamically less stable than WT, suggesting a difference in the tetramer folded structure. In vivo, the lone cysteine in TTR is frequently modified by S-cysteinylation or S-sulfonation. Like the N98A mutation, S-cysteinylation of TTR modestly decreased tetramer stability and increased TTR's effectiveness at inhibiting Aβ aggregation. Collectively, these data indicate that a subtle change in TTR tetramer structure measurably increases TTR's ability to inhibit Aβ aggregation.

Keywords: amyloid; protein aggregation; transthyretin.

Fig. 1

Ribbon structure of human TTR. (A) Monomer, (B) dimer and (C) tetramer with S23 (blue), S100 (green) and N98 (red) highlighted. Generated from PDB entry 1DVQ. Each monomer has an ‘inner sheet’ of strands D, A, G and H, and an ‘outer sheet’ of strands C, B, E and F as well as a lone α-helix between the E and F strands.

Fig. 2

ThT analysis of TTR-mediated inhibition of Aβ aggregation. TTR (WT and mutants, 3.6 μM) was incubated with Aβ (28 μM) at 37°C for 2, 24 or 48 h. Fibrils were detected using ThT. Data shown are mean ± SD. TTR alone (WT or mutants) does not have any ThT fluorescence signal (not shown).

Fig. 3

DLS analysis of TTR-mediated inhibition of Aβ aggregation. The increase in the mean hydrodynamic diameter of aggregates of Aβ (28 μM) alone (filled squares), with WT TTR (3.6 μM, empty circles) or with N98A (filled triangles) at 37°C was measured by DLS.

Fig. 4

NTA of Aβ aggregate growth. Size distribution of Aβ (28 μM) incubated with WT (3.6 μM; black) or with N98A (3.6 μM; gray) for (A) 20 min and (B) 120 min.

Fig. 5

TEM images of Aβ with TTR mutants. Aβ incubated alone (A) or with WT (B) or N98A (C) at 37°C for 24 h. All scale bars represent 500 nm in length.

Fig. 6

ANS fluorescence of TTR alanine mutants. WT (filled circles), S23A (diamonds), S100A (plus symbols) and N98A (filled triangles). TTR concentration was 1 μM. ANS (29 μM) was excited at 370 nm, and emission spectra were collected.

Fig. 7

Thermodynamic stability of TTR mutants measured via urea denaturation. The ratio of Trp fluorescence intensities, a measure of the degree of unfolding, for (A) WT (filled circles), S23A (diamonds) and N98A (filled triangles) at 0.9 μM. (B) Concentration dependence of urea denaturation for WT at 0.9 µM (filled circles), WT at 9 µM (empty circles), N98A at 0.9 µM (filled triangles) and N98A at 9 µM (empty triangles). Curves are fits of the data to an apparent two-state model (Hurshman Babbes et al., 2008).

Fig. 8

ANS fluorescence of oxidized TTR. WT (filled squares), S-TTR (empty circles) and C-TTR (empty triangles). TTR concentration was 1 μM. ANS (29 μM) was excited at 370 nm.

Fig. 9

Effect of oxidation of TTR on inhibition of Aβ aggregation. TTR (3.6 μM) was incubated with Aβ (28 μM) at 37°C for 24 h. ThT was used to assess the extent of fibril formation.

Fig. 10

TEM images of Aβ with oxidized TTR. Aβ incubated alone (A) or with TTR (B), S-TTR (C) or C-TTR (D) at 37°C for 3 days. All scale bars represent 500 nm in length.