(2020). Two polyphenols with diverse mechanisms towards amyloidosis: differential modulation of the fibrillation pathway of human lysozyme by curcumin and EGCG. Journal of Biomolecular Structure and Dynamics. Ahead of Print.
Abstract The effect of resveratrol, a polyphenol in red wine, on the amyloid fibril formation of human lysozyme (HuL) was investigated, towards elucidating the mechanism of resveratrol action and probing its role as a possible modulator of lysozyme aggregation and toxicity. By using a number of biophysical tools, resveratrol was observed to alter the fibrillization kinetics of HuL and inhibit its fibrillization by binding with weak to moderate affinity to the conformations populated at the early stages of the pathway with concomitant stabilization of these initial conformations. The marginal decrease in the lifetime of HuL in the presence of resveratrol by time‐resolved fluorescence measurements indicated the involvement of a static quenching mechanism in the interaction between HuL and resveratrol. Docking studies predicted the binding of resveratrol to aggregation‐prone regions in HuL, and structure and activity analyses demonstrated the retention of much of the α‐helical structure and activity of HuL in the presence of resveratrol. Resveratrol modulated the fibrillization pathway towards less‐hydrophobic, less‐toxic, off‐pathway aggregates. These results demonstrate that binding of resveratrol to HuL could protect against the formation of pathogenic, cytotoxic aggregates formed in amyloidogenic disorders, such as systemic amyloidosis; thus suggesting its potential as a plausible therapeutic agent against lysozyme amyloidosis.
The aggregation of the protein alpha synuclein (α-Syn), a known contributor in Parkinson's disease (PD) pathogenesis is triggered by transition metal ions through occupational exposure and disrupted metal ion homeostasis. Naturally occurring small molecules such as polyphenols have emerged as promising inhibitors of α-Syn fibrillation and toxicity and could be potential therapeutic agents against PD. Here, using an array of biophysical tools combined with cellular assays, we demonstrate that the novel polyphenolic compound scutellarin efficiently inhibits the uninduced and metal-induced fibrillation of α-Syn by acting at the nucleation stage and stabilizes a partially folded intermediate of α-Syn to form SDS-resistant, higher-order oligomers (∼680 kDa) and also disaggregates preformed fibrils of α-Syn into similar type of higher-order oligomers. ANS binding assay, fluorescence lifetime measurements and cell-toxicity experiments reveal scutellarin-generated oligomers as compact, low hydrophobicity structures with modulated surface properties and significantly reduced cytotoxicity than the fibrillation intermediates of α-Syn control. Fluorescence spectroscopy and isothermal titration calorimetry establish the binding between scutellarin and α-Syn to be non-covalent in nature and of moderate affinity (Ka ∼ 105 M-1). Molecular docking approaches suggest binding of scutellarin to the residues present in the NAC region and C-terminus of monomeric α-Syn and the C-terminal residues of fibrillar α-Syn, demonstrating inhibition of fibrillation upon binding to these residues and possible stabilization of the autoinhibitory conformation of α-Syn. These findings reveal interesting insights into the mechanism of scutellarin action and establish it as an efficient modulator of uninduced as well as metal-induced α-Syn fibrillation and toxicity.
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