Transthyretin Binding Heterogeneity and Anti-amyloidogenic Activity of Natural Polyphenols and Their Metabolites

Paola Florio,Claudia Folli,Michele Cianci,Daniele Del Rio,Giuseppe Zanotti,Rodolfo Berni

doi:10.1074/jbc.m115.690172

Abstract

Transthyretin (TTR) is an amyloidogenic protein, the amyloidogenic potential of which is enhanced by a number of specific point mutations. The ability to inhibit TTR fibrillogenesis is known for several classes of compounds, including natural polyphenols, which protect the native state of TTR by specifically interacting with its thyroxine binding sites. Comparative analyses of the interaction and of the ability to protect the TTR native state for polyphenols, both stilbenoids and flavonoids, and some of their main metabolites have been carried out. A main finding of this investigation was the highly preferential binding of resveratrol and thyroxine, both characterized by negative binding cooperativity, to distinct sites in TTR, consistent with the data of x-ray analysis of TTR in complex with both ligands. Although revealing the ability of the two thyroxine binding sites of TTR to discriminate between different ligands, this feature has allowed us to evaluate the interactions of polyphenols with both resveratrol and thyroxine preferential binding sites, by using resveratrol and radiolabeled T4 as probes. Among flavonoids, genistein and apigenin were able to effectively displace resveratrol from its preferential binding site, whereas genistein also showed the ability to interact, albeit weakly, with the preferential thyroxine binding site. Several glucuronidated polyphenol metabolites did not exhibit significant competition for resveratrol and thyroxine preferential binding sites and lacked the ability to stabilize TTR. However, resveratrol-3-O-sulfate was able to significantly protect the protein native state. A rationale for the in vitro properties found for polyphenol metabolites was provided by x-ray analysis of their complexes with TTR.

Highlights

Amyloidoses are relevant human diseases that are characterized by the extracellular deposition of normally soluble proteins
Human transthyretin (TTR)3 represents a relevant amyloidogenic protein whose amyloidogenic potential is enhanced by a large number of specific point mutations
Polyphenol-Transthyretin Interactions and Heterogeneity each other through several interactions mediated by the TTR ligand, which leads to a drastic stabilization of the native TTR tetramer (2, 14 –16)

Summary

Experimental Procedures

Materials—L-Thyroxine (T4), genistein, apigenin, and transresveratrol, were from Sigma-Aldrich; resveratrol-3-O-sulfate, resveratrol-4Ј-O-glucuronide, and resveratrol-3-O-glucuronide were from Bertin Pharma; and genistein-7-O-glucuronide and daidzein-7-O-glucuronide were from Extrasynthese. Recombinant WT TTR (3 ␮M) was incubated with a trace amount (ϳ0.3 nM) of radiolabeled T4 or with 3 ␮M nonradiolabeled T4 in the presence of ϳ0.3 nM radiolabeled T4, in the absence or in the presence of increasing concentrations of polyphenols, in 50 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.4, for 1 h at room temperature. 7.2 ␮M WT TTR was preincubated with 3 molar equivalents of polyphenols or with DMSO or 3 molar equivalents of diflunisal as negative and positive controls, respectively, at neutral pH (10 mM sodium phosphate buffer, pH 7, 100 mM KCl, 1 mM EDTA) for 3 h at room temperature, prior to incubation at acidic pH upon addition of an equal volume of 100 mM sodium acetate, 100 mM KCl, 1 mM EDTA, pH 4.2 (final pH 4.3), at 37 °C to promote fibrillogenesis. The ligand orientation was determined by inspecting the electron density map, calculated withFobs Ϫ Fcalccoefficients, and calculated with phases from the model, deprived of the ligand

Results

TTR monomer

Discussion

Data collection Source