The Transthyretin/Oleuropein Aglycone Complex: A New Tool against TTR Amyloidosis.

Francesco Bemporad,Monica Bucciantini,Massimo Stefani,Matteo Ramazzotti,Manuela Leri

doi:10.3390/ph15030277

Abstract

The release of monomers from the homotetrameric protein transthyretin (TTR) is the first event of a cascade, eventually leading to sporadic or familial TTR amyloidoses. Thus, ligands able to stabilize TTR and inhibit monomer release are subject of intense scrutiny as potential treatments against these pathologies. Here, we investigated the interaction between TTR and a non-glycated derivative of the main olive polyphenol, oleuropein (OleA), known to interfere with TTR aggregation. We coupled fluorescence studies with molecular docking to investigate the OleA/TTR interaction using wild-type TTR, a monomeric variant, and the L55P cardiotoxic mutant. We characterized a fluorescence band emitted by OleA upon formation of the OleA/TTR complex. Exploiting this signal, we found that a poorly specific non-stoichiometric interaction occurs on the surface of the protein and a more specific stabilizing interaction takes place in the ligand binding pocket of TTR, exhibiting a KD of 3.23 ± 0.32 µM, with two distinct binding sites. OleA interacts with TTR in different modes, stabilizing it and preventing its dissociation into monomers, with subsequent misfolding. This result paves the way to the possible use of OleA to prevent degenerative diseases associated with TTR misfolding.

Highlights

IntroductionTTR is a homotetramer, where the four monomers are assembled with a 2:2:2 symmetry resulting in a dimer of dimers
Transthyretin (TTR), known as prealbumin, is a 55 KDa circulating protein produced by liver and, to a lesser extent, by choroid plexus, with a well-established role in the transport of thyroxine (T4) and retinol in the plasma and the cerebrospinal fluid [1].Structurally, TTR is a homotetramer, where the four monomers are assembled with a 2:2:2 symmetry resulting in a dimer of dimers
We found that the presence of oleuropein aglycone (OleA) during non-native aggregation under acid conditions of wt-TTR and L55P-TTR induced an increase in Trp quenching; this finding suggests that the stabilization by OleA of meta-stable misfolded intermediates of TTR relies on the reduction in their surface hydrophobicity

Summary

Introduction

TTR is a homotetramer, where the four monomers are assembled with a 2:2:2 symmetry resulting in a dimer of dimers. Monomer interaction is stabilized through H-bonds involving the two-edge H and F β-strands; the back-to-back association of the two dimers is stabilized by a limited number of contacts, giving rise to two symmetrical binding sites for T4, at the dimer–dimer interface [2]. Human TTR possesses a relatively high intrinsic propensity to β-aggregation [3], and such a propensity is strongly enhanced in a set of genetic TTR variants as a result of a destabilization of the native TTR structure [4]. Over 130 TTR gene mutations have been associated with pathological phenotypes, with autosomal dominant inheritance [5,6,7,8].

Methods

Results

Conclusion