Epigallocatechin-3-gallate remodels apolipoprotein A-I amyloid fibrils into soluble oligomers in the presence of heparin

Abstract

Amyloid deposits of WT apolipoprotein A-I (apoA-I), the main protein component of high-density lipoprotein, accumulate in atherosclerotic plaques where they may contribute to coronary artery disease by increasing plaque burden and instability. Using CD analysis, solid-state NMR spectroscopy, and transmission EM, we report here a surprising cooperative effect of heparin and the green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG), a known inhibitor and modulator of amyloid formation, on apoA-I fibrils. We found that heparin, a proxy for glycosaminoglycan (GAG) polysaccharides that co-localize ubiquitously with amyloid in vivo, accelerates the rate of apoA-I formation from monomeric protein and associates with insoluble fibrils. Mature, insoluble apoA-I fibrils bound EGCG (KD = 30 ± 3 μm; Bmax = 40 ± 3 μm), but EGCG did not alter the kinetics of apoA-I amyloid assembly from monomer in the presence or absence of heparin. EGCG selectively increased the mobility of specific backbone and side-chain sites of apoA-I fibrils formed in the absence of heparin, but the fibrils largely retained their original morphology and remained insoluble. By contrast, fibrils formed in the presence of heparin were mobilized extensively by the addition of equimolar EGCG, and the fibrils were remodeled into soluble 20-nm-diameter oligomers with a largely α-helical structure that were nontoxic to human umbilical artery endothelial cells. These results argue for a protective effect of EGCG on apoA-I amyloid associated with atherosclerosis and suggest that EGCG-induced remodeling of amyloid may be tightly regulated by GAGs and other amyloid co-factors in vivo, depending on EGCG bioavailability.

CryoEM reveals how the small molecule EGCG binds to Alzheimer’s brain-derived tau fibrils and initiates fibril disaggregation

“…An alternative explanation that EGCG prevents fibrils from adhering to the EM grid is unlikely because the disappearance of fibrils becomes gradually pronounced over time in spite of all other variables remaining unchanged, and also because others report that EGCG disaggregates fibrils of other amyloids, including α-synuclein, amyloid β, and apolipoprotein A-I. 12,15 The effect of EGCG treatment on AD-tau could differ from recombinant tau fibrils since recombinant fibrils of tau form different polymorphs. To test whether EGCG disaggregates brain-purified AD-tau fibrils, we examined negatively stained EM grids of purified AD-tau at various times following incubation with EGCG.…”

“…Epigallocatechin gallate (EGCG), a small molecule natural product in green tea, inhibits aggregation of numerous amyloid proteins and, despite their tremendous stability, disaggregates fibrils of some amyloids into smaller units. [11][12][13][14][15] Evidence suggesting that EGCG disaggregates fibrils of tau includes studies in primary rat neurons showing that EGCG enhances clearance of phosphorylated tau, 16 although the reported effects of EGCG are somewhat pleiotropic and range from the inhibition aggregation to altered post-translational modification. 17,18 Here, we sought to illuminate the effects direct binding of EGCG to AD-tau by single-particle cryoEM structure determination.…”

“…Remarkably, the small molecule natural product EGCG is reported to inhibit at least 14 different amyloids, in part by disaggregating amyloid fibrils, although also by blocking aggregation of protein monomers. [11][12][13]15,29 Despite potent in vitro anti-amyloid activity, EGCG is not a proven clinical therapeutic. The lack of clinical efficacy of EGCG stems from poor drug-like properties, which includes limited bioavailability and lack of specificity.…”

The Environment Is a Key Factor in Determining the Anti-Amyloid Efficacy of EGCG

“…Epigallocatechin-3-gallate (EGCG), the main polyphenol found in green tea, has been reported to effectively inhibit the aggregation of a number of amyloidogenic peptides and proteins, including amyloid-β (related to AD) [32,33], α-synuclein (related to PD) [33][34][35][36], islet amyloid polypeptide (related to type-II diabetes) [37,38], huntingtin exon 1 (related to Huntington's disease) [39], tau (related to AD and tauopathies) [40], superoxide dismutase (related to amyotrophic lateral sclerosis) [41], prion proteins (related to prion diseases) [42], and others. In addition, it has been shown that EGCG can induce remodeling and/or dissociation of pre-existing aggregate species [33,34,36,43,44]. Taken together, EGCG appears to be a "universal" inhibitor of amyloid fibril formation, suggesting that this molecule could be used as a therapeutic agent for the prevention and treatment of amyloid-related disorders.…”

Circular Dichroism Spectroscopy Identifies the β-Adrenoceptor Agonist Salbutamol As a Direct Inhibitor of Tau Filament Formation in Vitro

“…5,7,8 In this context, much research has been carried out into the inhibition of amyloidosis by natural aromatic compounds, [16][17][18][19][20][21][22][23][24] including polyphenols from dietary substances that recognise the generic amyloid cross-β motif and alter the aggregation kinetics or restructure many amyloidogenic proteins into non-toxic species in vitro. [22][23][24][25][26] For example, the phenolic rings of polyphenol compounds interfere with the stacking of aromatic residues and hydroxyl groups, thereby destabilising the amyloid core and increasing its solubility. [27][28][29] A common first step toward identifying potential inhibitors is in vitro screening of compounds that reduce tau or Aβ self-assembly kinetics and yield.…”