The preaggregated state of an amyloidogenic protein: Hydrostatic pressure converts native transthyretin into the amyloidogenic state

摘要

Protein misfolding and aggregation cause several diseases, by mechanisms that are poorly understood. The formation of amyloid aggregates is the hallmark of most of these diseases. Here, the properties and formation of amyloidogenic intermediates of trans- thyretin (TTR) were investigated by the use of hydrostatic pressure and spectroscopic techniques. Native TTR tetramers (T_4) were denatured by high pressure into a con formation that exposes tryptophan residues to the aqueous environment. This conforma- tion was able to bind the hydrophobic probe bis-(8-anilinonaph- thalene-1 -sulfonate), indicating persistence of elements of second- ary and tertiary structure. Lowering the temperature facilitated the pressure-induced denaturation of TTR, which suggests an impor- tant role of entropy in stabilizing the native protein. Gel filtration chromatography showed that after a cycle of compression-decom- pression at 1℃. the main species present was a tetramer, with a small population of monomers. This tetramer. designated T_4~*, had a non-native conformation: it bound more bis-(8-anilinonaphtha- lene-1-sulfonate) than native T_4, was less stable under pressure. and on decompression formed aggregates under mild acidic con- ditions (pH 5--5.6). Our data show that hydrostatic pressure con- verts native tetramers of TTR into an altered state that shares properties with a previously described amyloidogenic intermedi- ate, and it may be an intermediate that lies on the aggregation pathway. This "preaggregated" state, which we call T_4~*, provides insight into the question of how a correctly folded protein may degenerate into the aggregation pathway in amyloidogenic diseases.