Presence of intrinsically disordered proteins can inhibit the nucleation phase of amyloid fibril formation of Aβ(1-42) in amino acid sequence independent manner

Abstract

The molecular shield effect was studied for intrinsically disordered proteins (IDPs) that do not adopt compact and stable protein folds. IDPs are found among many stress-responsive gene products and cryoprotective- and drought-protective proteins. We recently reported that some fragments of human genome-derived IDPs are cryoprotective for cellular enzymes, despite a lack of relevant amino acid sequence motifs. This sequence-independent IDP function may reflect their molecular shield effect. This study examined the inhibitory activity of IDPs against fibril formation in an amyloid beta peptide (Aβ(1-42)) model system. Four of five human genome-derived IDPs (size range 20 to 44 amino acids) showed concentration-dependent inhibition of amyloid formation (IC₅₀ range between 60 and 130 μM against 20 μM Aβ(1-42)). The IC₅₀ value was two orders of magnitude lower than that of polyethylene-glycol and dextran, used as neutral hydrophilic polymer controls. Nuclear magnetic resonance with ¹⁵ N-labeled Aβ(1-42) revealed no relevant molecular interactions between Aβ(1-42) and IDPs. The inhibitory activities were abolished by adding external amyloid-formation seeds. Therefore, IDPs seemed to act only at the amyloid nucleation phase but not at the elongation phase. These results suggest that IDPs (0.1 mM or less) have a molecular shield effect that prevents aggregation of susceptible molecules.

Figure 1

The effect of human genome–derived IDPs and other crowding agents on Aβ(1–42) amyloid fibril formation probed using thioflavin T (ThT). (A) Aβ(1–42) peptide (20 µM) was incubated for 72 h at 37 ℃ in the absence or presence of 0.01–20 mM IDP (C1) (blue), or PEG 6000 (brown), dextran 6 (yellow). The formation of Aβ(1–42) fibrils was monitored by ThT fluorescence and normalized to 1.0 with the intensity of the Aβ(1–42) alone. (B) 20 µM Aβ(1–42) peptide was incubated for 72 h at 37 ℃ in the presence of 10–160 µM IDPs. IDP-B4 (purple), C1 (blue), E1 (orange), FK20 (green), and D10 (red). Error bars, s.d. from three independent experiments.

Figure 2

Estimation of apparent IC₅₀ value of IDPs against Aβ(1–42) fibril formation. (A) B4, (B) C1, (C) E1, (D) FK20, (E) D10. Error bars, s.d. from three independent experiments.

Figure 3

Observation of Aβ(1–42)-derived amyloid fibrils by negatively stained transmission electron microscopy with or without IDPs. TEM images of 10 µM Aβ(1–42) were obtained after 24 h of incubation either alone (A) or in the presence of 40 µM IDP-C1 (B) or 10 µM IDP-D10 (C). Scale bars indicate 200 nm.

Figure 4

Absence of relevant interaction of IDPs with Aβ(1–42), assessed by solution nuclear magnetic resonance (NMR). (A, C, E) Overlay of the 2D ¹H–¹⁵N HSQC spectra of Aβ(1–42) with various concentration of IDP-C1 at 15 ℃, pH 7.4. Aβ(1–42) (20 µM) with of 0 µM (cyan), 10 µM (red), 20 µM (yellow), 40 µM (green), and 80 µM (blue) of C1 (A), D10 (C), or FK20 (E). (B, D, F) Normalized chemical shift perturbation Δδ derived from ¹H–¹⁵N HSQC spectra of Aβ(1–42) with 80 µM C1 (B), D10 (D), or FK20 (F) were plotted against residue numbers of Aβ(1–42).

Figure 5

Reversal of IDP inhibition of fibril formation of Aβ(1–42) by addition of external amyloid seed. (A) Aβ(1–42) peptide (20 µM) was incubated for 72 h at 37 ℃ with 5% of external Aβ(1–42) amyloid seed in the presence of 80 µM C1 (blue), 80 µM FK20 (green), and 20 µM D10 (red). Thioflavin T (ThT) fluorescence intensity of the Aβ(1–42) alone with 5% seed was set to 1.0. (B) Comparison of relative ThT intensity from panel (A) at 72 h. (C) Aβ(1–42) (20 µM) peptide and 20 µM IDP-D10 was co-incubated for 72 h at 37 ℃ with various amounts of the external Aβ(1–42) amyloid seed. The seed amounts were 0.1% seed (black), 0.5% seed (orange), 1% seed (purple), 2.5% seed (yellow), and 5% seed (red), respectively. (D) Comparison of relative ThT intensity from panel (C) at 72 h. Error bars, s.d. from three independent experiments.

Figure 6

Schematic representation of molecular shield effect of human genome-derived IDPs against amyloid fibril formation of Aβ(1–42). (A) Aβ(1–42) monomer is considered to be an equilibrium between two conformations, the fibrillization-ready conformation (short arrow) and relaxed conformation (filled circle). Only when several fibrillization-ready Aβ(1–42) monomers encountered, the nucleus was formed (short pile of arrows). (B) The case of Aβ(1–42) alone. The nuclei are formed at the early nucleation phase, then the fibrils (long pile of arrow) grow by addition of fibrillization-ready monomers at the elongation phase. (C) The case of Aβ(1–42) and IDPs (except of D10). IDPs may not interfere equilibrium between fibrillization-ready and relaxed conformations of the monomer. IDPs may interfere the formation of the nucleus, thereby inhibiting fibril formation. (D) The case of Aβ(1–42) and IDP-D10. D10 probably binds to the nucleus and inhibits fibrillization.