Toward the molecular mechanism(s) by which EGCG treatment remodels mature amyloid fibrils

Abstract

Protein misfolding and/or aggregation has been implicated as the cause of several human diseases, such as Alzheimer's and Parkinson's diseases and familial amyloid polyneuropathy. These maladies are referred to as amyloid diseases, named after the cross-β-sheet amyloid fibril aggregates or deposits common to these disorders. Epigallocatechin-3-gallate (EGCG), the principal polyphenol present in green tea, has been shown to be effective at preventing aggregation and is able to remodel amyloid fibrils comprising different amyloidogenic proteins, although the mechanistic underpinnings are unclear. Herein, we work toward an understanding of the molecular mechanism(s) by which EGCG remodels mature amyloid fibrils made up of Aβ(1-40), IAPP(8-24), or Sup35NM(7-16). We show that EGCG amyloid remodeling activity in vitro is dependent on auto-oxidation of the EGCG. Oxidized and unoxidized EGCG binds to amyloid fibrils, preventing the binding of thioflavin T. This engagement of the hydrophobic binding sites in Aβ(1-40), IAPP(8-24), or Sup35NM(Ac7-16) Y→F amyloid fibrils seems to be sufficient to explain the majority of the amyloid remodeling observed by EGCG treatment, although how EGCG oxidation drives remodeling remains unclear. Oxidized EGCG molecules react with free amines within the amyloid fibril through the formation of Schiff bases, cross-linking the fibrils, which may prevent dissociation and toxicity, but these aberrant post-translational modifications do not appear to be the major driving force for amyloid remodeling by EGCG treatment. These insights into the molecular mechanism of action of EGCG provide boundary conditions for exploring amyloid remodeling in more detail.

Figure 1

Effect of EGCG on ThT fluorescence of Aβ_1–40 amyloid fibrils. (A) Structure of EGCG. (B) Primary sequence of Aβ_1–40 peptide, lysines 16 and 28 are highlighted in blue. (C) Kinetics of Aβ_1–40 amyloid fibril remodeling monitored by ThT fluorescence incubated in the absence or presence of EGCG. (D) Experimental scheme of the centrifugation/wash protocol. (E) ThT fluorescence or (F) protein quantification by the Bradford assay (O.D.) of samples incubated for 0 or 24 h in the absence or presence of EGCG. The use of the centrifugation/wash protocol is denoted by (+). The buffer used for all assays was 50 mM phosphate buffer pH 7.4, 150 mM NaCl (25 °C). [Aβ_1–40 amyloid fibrils] = 65 µg/mL, [EGCG] = 30 µM. For ThT fluorescence, the ThT concentration used was 20 µM, Ex: 440 nm and Em: 485 nm.

Figure 2

Effect of EGCG on the morphology and seeding activity of Aβ_1–40. Aβ_1–40 (65 µg/mL) fibrils were incubated in the absence or presence of 30 µM EGCG for 24 h at 25 °C. The fibrils were processed using the centrifugation/wash protocol and one aliquot was used for AFM analysis (panels A and B) while the other aliquot was sonicated for 30 min in order to produce seeds used in the seeding experiment (panel C). AFM images of Aβ_1–40 fibrils incubated in the absence (A) or presence (B) of EGCG. The left images represent the amplitude image while the right images represent the height images. Inset: each image is 2 µm × 2 µm. (C) A monomeric solution of Aβ_1–40 peptide (65 µg/mL) was incubated in the absence or presence of 5% seeds in 50 mM phosphate buffer pH 7.4, 150 mM NaCl without agitation. The seeds were produced by sonication of Aβ_1–40 amyloid fibrils incubated in the absence or presence of EGCG (30 µM) for 24 h at 25 °C. The samples containing ThT (20 µM) were incubated at 25 °C in 96-well plate and every 10 min the fluorescence (excitation at 440 nm, emission at 485 nm) was monitored.

Figure 3

EGCG amyloid remodeling activity depends on auto-oxidation. (A) The stability of EGCG is enhanced in the presence of 1 µg/mL SOD1. (Inset A) The auto-oxidation of EGCG was monitored by RP-HPLC using a C18 column. (B) A kinetics experiment monitoring the ThT fluorescence using the centrifugation/wash protocol was performed with Aβ_1–40 amyloid fibrils in the presence of EGCG or EGCG + SOD1. Buffer alone was used as a control. (C) Filter retardation (FR) assay of aliquots of the samples described in Figure 3B stained by Ponceau or NBT, as indicated. For detailed experimental conditions see the legend of Figure 1.

Figure 4

The amyloid remodeling activity of EGCG does not require free amines. Acetic anhydride was used to acetylate any free amines in the Aβ_1–40 fibrils. The acetylated fibrils were incubated in the absence or presence of EGCG for 24 h and the ThT fluorescence using the centrifugation/wash protocol (A), and the FR assay stained with NBT or Ponceau (B) were monitored. Non-acetylated fibrils were used as control. (C) Acetylated and non-acetylated Aβ_1–40 amyloid fibrils were incubated in the absence or presence of 30 µM EGCG for 24 h. After the centrifugation/wash protocol, the pellets were resuspended in 8 M guanidine chloride (GndCl) and sonicated for 1 h. The samples were analyzed by RP-HPLC, monitoring the optical density at 220 nm (inset, non-acetylated fibril). The HPLC Aβ signal was calculated from the area under the peak.

Figure 5

The amyloid remodeling activity of EGCG on IAPP fibrils. IAPP_Ac8–24 (A) mature amyloid fibrils (65 mg/mL) were incubated in the absence or presence of 30 µM EGCG for 24 h at 25 °C. After the centrifugation/wash protocol, the ThT fluorescence (B), CD (D) and AFM images (E and F) were recorded. Inset of panels E and F: each image is 2 µm × 2 µm. An aliquot of these samples was boiled or not in 2% SDS and then applied to FR membrane that was stained by Ponceau (C).

Figure 6

The amyloid remodeling activity of EGCG is still partially functional in the presence of detergent micelles. (A) Experimental scheme of the centrifugation/wash protocol, highlighting the wash step performed with PBS or PBS containing Tween 20. IAPP_Ac8–24 mature amyloid fibrils (65 µg/mL) were incubated in the absence or presence of 30 µM EGCG for 24 h at 25 °C. After the centrifugation/wash protocol (A), the ThT fluorescence (B) was recorded. An aliquot of these samples (without boiling) was then applied to FR membrane that was stained by Ponceau (C) or NBT (D).

Figure 7

EGCG is incapable of remodeling Sup35NM_Ac7–16 amyloid fibrils. (A) Primary sequence of the peptides comprising Sup35NM_Ac7–16 (wt) and Sup35NM_Ac7–16 Y→F amyloid fibrils as well as the Grand Average of Hydropathicity index (GRAVY) of these peptides. (B) Sup35NM_Ac7–16 or Sup35NM_Ac7–16 Y→F mature amyloid fibrils (65 µg/mL) were incubated in the absence or presence of 30 µM EGCG for 24 h at 25 °C. After the centrifugation/wash protocol, the ThT fluorescence was recorded. (C and D) AFM images of Sup35NM_Ac7–16 Y→F mature amyloid fibrils incubated in the absence (C) or presence (D) of EGCG. Inset: each image is 2 µm × 2 µm.