(−)-Epigallocatechin-3-Gallate (EGCG) Maintains κ-Casein in Its Pre-Fibrillar State without Redirecting Its Aggregation Pathway

Abstract

The polyphenol (-)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-beta, alphasynuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer's, Parkinson's and Huntington's diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered offfolding pathway that results in the formation of non-toxic amorphous aggregates. whether this anti-fibril activity is specific to these disease-related target proteins or ismore generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMkappa-CN (reduced and carboxymethylated kappa-casein) and thereby protect pheochromocytoma-12 cells from RCMkappa-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation byRCMkappa-CN [the IC50 for 50 uM RCMkappa-CN is 1 uM]. Biophysical studies reveal that EGCG prevents RCMkappa-CN fibril formation by stabilising RCMkappa-CN in its nativelike state rather than by redirecting its aggregation to the disordered, amorphous aggregation pathway. Thus, while it appears that EGCG is a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves this inhibition is specific to the target fibril-forming polypeptide. It is proposed that EGCG is directed to the amyloidogenic sheet-turn-sheet motif of monomeric RCMkappa-CN with high affinity by strong non-specific hydrophobic associations. Additional non-covalent pi-pi stacking interactions between the polyphenolic and aromatic residues common to the amyloidogenic sequence are also implicated. The polyphenol (−)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-β, α-synuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer's, Parkinson's and Huntington's diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered off-folding pathway that results in the formation of non-toxic amorphous aggregates. Whether this anti-fibril activity is specific to these disease-related target proteins or is more generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMκ-CN (reduced and carboxymethylated κ-casein) and thereby protect pheochromocytoma-12 cells from RCMκ-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation by RCMκ-CN [the IC 50 for 50 μM RCMκ-CN is 13 ± 1 μM]. Biophys...

How Epigallocatechin Gallate Can Inhibit α-Synuclein Oligomer Toxicity in Vitro

“…1A) are also found in numerous other fibril inhibitors (14, 15, 18 -20). EGCG is not specific toward ␣SN and has been shown to inhibit the fibrillation of numerous proteins (21)(22)(23)(24)(25)(26)(27). At equimolar concentrations, EGCG preferentially binds the C terminus of ␣SN (Asp-119, Ser-129, Glu-130, and Asp-135) (13).…”

Natural Compounds May Open New Routes to Treatment of Amyloid Diseases

“…Carboxymethylated casein forms fibrils with β-sheet structure that bind to the amyloid dye thioflavin T; it has, therefore, been used as a generic model system for amyloid formation [97]. EGCG inhibits the fibril formation of casein through its binding to a β-sheet-turnsheet motif of the protein [98]. The protein remains in solution as a monomer after EGCG binding.…”

“…The protein remains in solution as a monomer after EGCG binding. The casein model, however, is atypical for amyloidogenic proteins in that the β-sheet-turn-sheet motif structure, which is characteristic of many amyloid fibrils [99], is already present in the monomeric form of the protein [98]. In contrast, the Aβ peptide, as well as most other proteins, forms these structures during aggregation into oligomeric and protofibrillar intermediates of the amyloid cascade [100].…”

Carboxymethylated-κ-casein: A convenient tool for the identification of polyphenolic inhibitors of amyloid fibril formation

“…Like Ab fibrils, amyloid fibrils formed from RCMj-CN are cytotoxic. 10 Fibril formation by RCM-j-CN can be easily monitored using an in situ ThT fluorescence binding assay similar to that used in assays that study the fibrilisation of Ab. 11 The mechanism of such fibril formation by RCM-j-CN has recently been studied in detail, 12 and this protein has shown itself to be a convenient model with which to study generic aspects of fibril formation.…”

“…Very recently we showed that EGCG also efficiently inhibits fibril formation by RCM-j-CN and protects pheochromocytoma-12 cells from RCM-j-CN amyloid-induced toxicity. 10 Given the difficulties typically encountered with the screening of small molecules against synthetic Ab, it was decided that RCM-j-CN be investigated for its potential to serve as a convenient initial screen for small molecule Ab anti-fibrilisation activity. Herein are described the results of an investigation of the ability of a set of common polyphenolics and their derivatives to inhibit fibril formation by RCM-j-CN, as measured by the in situ ThT fluorescence binding assay.…”