Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption.

Elisabete Ferreira,Rui M M Brito,Marta Silva E Sousa,Zaida L Almeida,Pedro F Cruz,Paula Veríssimo

doi:10.3390/ijms23010391

Elisabete Ferreira, Rui M M Brito + Show 4 more

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https://doi.org/10.3390/ijms23010391

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Abstract

Several degenerative amyloid diseases, with no fully effective treatment, affect millions of people worldwide. These pathologies—amyloidoses—are known to be associated with the formation of ordered protein aggregates and highly stable and insoluble amyloid fibrils, which are deposited in multiple tissues and organs. The disruption of preformed amyloid aggregates and fibrils is one possible therapeutic strategy against amyloidosis; however, only a few compounds have been identified as possible fibril disruptors in vivo to date. To properly identify chemical compounds as potential fibril disruptors, a reliable, fast, and economic screening protocol must be developed. For this purpose, three amyloid fibril formation protocols using transthyretin (TTR), a plasma protein involved in several amyloidoses, were studied using thioflavin-T fluorescence assays, circular dichroism (CD), turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM), in order to characterize and select the most appropriate fibril formation protocol. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was successfully used to study the interaction of doxycycline, a known amyloid fibril disruptor, with preformed wild-type TTR (TTRwt) aggregates and fibrils. DLS and TEM were also used to characterize the effect of doxycycline on TTRwt amyloid species disaggregation. A comparison of the TTR amyloid morphology formed in different experimental conditions is also presented.

Highlights

Considerable attention is given to a group of protein misfolding diseases known as amyloidosis [1,2,3,4]
In order to summarize the three aggregation protocols of TTRwt used in this study, Table 1 shows the main requirements for these protocols in terms of experimental conditions, and Table 2 summarizes the main characteristics of the aggregates and fibrils formed during aggregation and the result of pH changes on these amyloid structures
No treatments are available to effectively address all the clinical manifestations associated to ATTR

Summary

Introduction

Considerable attention is given to a group of protein misfolding diseases known as amyloidosis [1,2,3,4]. There are approximately 50 of these pathologies, involving different precursor proteins and associated with the formation of extracellular amyloid fibrils or intracellular inclusions with amyloid-like characteristics [3,5]. Amyloidoses can affect different organs, namely the heart, liver, kidneys, nervous system, spleen, skin, and gastrointestinal tract, among others. Severe amyloidosis can lead to life-threatening organ failure and death. Amyloid-β peptide, prion protein, α-synuclein, and transthyretin (TTR) are examples of the more than 40 different human peptides and proteins identified in amyloid deposits that are at the origin of the known amyloidoses [3]

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