Oligomerization Profile of Human Transthyretin Variants with Distinct Amyloidogenicity

Ana Frangolho,Rui M M Brito,Zaida L Almeida,Bruno E Correia,Daniela C Vaz

doi:10.3390/molecules25235698

Abstract

One of the molecular hallmarks of amyloidoses is ordered protein aggregation involving the initial formation of soluble protein oligomers that eventually grow into insoluble fibrils. The identification and characterization of molecular species critical for amyloid fibril formation and disease development have been the focus of intense analysis in the literature. Here, using photo-induced cross-linking of unmodified proteins (PICUP), we studied the early stages of oligomerization of human transthyretin (TTR), a plasma protein involved in amyloid diseases (ATTR amyloidosis) with multiple clinical manifestations. Upon comparison, the oligomerization processes of wild-type TTR (TTRwt) and several TTR variants (TTRV30M, TTRL55P, and TTRT119M) clearly show distinct oligomerization kinetics for the amyloidogenic variants but a similar oligomerization mechanism. The oligomerization kinetics of the TTR amyloidogenic variants under analysis showed a good correlation with their amyloidogenic potential, with the most amyloidogenic variants aggregating faster (TTRL55P > TTRV30M > TTRwt). Moreover, the early stage oligomerization mechanism for these variants involves stepwise addition of monomeric units to the growing oligomer. A completely different behavior was observed for the nonamyloidogenic TTRT119M variant, which does not form oligomers in the same acidic conditions and even for longer incubation times. Thorough characterization of the initial steps of TTR oligomerization is critical for better understanding the origin of ATTR cytotoxicity and developing novel therapeutic strategies for the treatment of ATTR amyloidosis.

Highlights

Transthyretin (TTR) is a homotetrameric protein found in plasma, cerebrospinal fluid, and the eye [1,2,3,4]
We investigated the early stages of TTR oligomerization using photo-induced cross-linking of unmodified proteins (PICUP) [32] and characterized the populations of oligomeric species in solution by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transmission electron microscopy (TEM)
The authors were able to validate the methodology by making use of different proteins and to demonstrate that PICUP is capable of distinguishing between monomeric and different oligomeric states, obtaining different distributions that varied from monomer and dimer [51] to tetramer [52] in the case of the oligomerization processes of wild-type TTR (TTRwt)

Summary

Introduction

Transthyretin (TTR) is a homotetrameric protein found in plasma, cerebrospinal fluid, and the eye [1,2,3,4]. Each TTR monomer is composed of a β-sandwich of two four-stranded β-sheets. TTR’s main known functions are thyroxine hormone (T4) transport and retinol transport in association with retinol-binding protein (RBP) [1,5]. TTR is known to have a neuroprotective role in several processes, such as decreasing brain amyloid-β deposition in mouse models [6,7]. TTR is implicated in several acquired and hereditary amyloid pathologies (ATTR, TTR amyloidosis) [1,8,9]. Whereas wild-type TTR (TTRwt) is associated with acquired amyloidosis

Methods

Results

Conclusion