Unraveling the Possible Mechanism Behind Leptomeningeal Amyloidosis Using as Model a Highly Unstable Transthyretin Tetramer

Abstract

Transthyretin (TTR) is a 127-residue -βsheet-rich protein homotetrameric that transports thyroxine in the blood and cerebrospinal fluid (CSF). Among all TTR variants, A25T is the most unstable tetramer. Its great instability induces TTR degradation in the endoplasmic reticulum of the hepatocytes, while thyroxine (T4, a natural ligand of TTR) leads to A25T secretion in the CSF by the choroid plexus. In the present study we aimed to determine the structure of A25T by X-ray crystallography in the apo form and in complex with T4. Also, by using high hydrostatic pressure, we have showed that the tetramers of A25T were less stable than the wt and L55P (the most aggressive variant of TTR ). Besides, A25T showed to be the most amyloidogenic variant thus far investigated, aggregating in conditions where wt and L55P remain mostly soluble. Using HPLC and native PAGE, we monitored acrylodan-labeled TTR aggregation in the human plasma. The aggregates formed displayed the typical amyloid structure. In the presence of monomers of T119M, a non-amyloidogenic variant, aggregation of A25T was remarkably reduced, pointing to the use of T119M monomers as a strategy to avoid TTR aggregation. The crystal structure of A25T, when compared to that of the wt protein, shaded light into the mechanism behind its increased amyloidogenicity: an expanded tetramer which is stabilized by a lower number of H-bonds and hydrophobic interactions. Interestingly, in the presence of T4 and lumiracoxib the structure of A25T was similar to that displayed by the wt protein. These data show that an expanded A25T tetramer with a decreased thermodynamic stability is prone to aggregate forming amyloid fibrils that trigger leptomeningeal amyloidosis. Support: CNPq and FAPERJ.