Abstract
Characterization of small oligomers formed at an early stage of amyloid formation is critical to understanding molecular mechanism of pathogenic aggregation process. Here we identified and characterized cytotoxic oligomeric intermediates populated during transthyretin (TTR) aggregation process. Under the amyloid-forming conditions, TTR initially forms a dimer through interactions between outer strands. The dimers are then associated to form a hexamer with a spherical shape, which serves as a building block to self-assemble into cytotoxic oligomers. Notably, wild-type (WT) TTR tends to form linear oligomers, while a TTR variant (G53A) prefers forming annular oligomers with pore-like structures. Structural analyses of the amyloidogenic intermediates using circular dichroism (CD) and solid-state NMR reveal that the dimer and oligomers have a significant degree of native-like β-sheet structures (35–38%), but with more disordered regions (~60%) than those of native TTR. The TTR variant oligomers are also less structured than WT oligomers. The partially folded nature of the oligomeric intermediates might be a common structural property of cytotoxic oligomers. The higher flexibility of the dimer and oligomers may also compensate for the entropic loss due to the oligomerization of the monomers.
Highlights
Protein misfolding and amyloid formation is implicated in various debilitating human diseases including Alzheimer’s diseases and amyloidoses[1,2,3]
TTR misfolding and amyloid formation is associated with numerous amyloidoses including senile systemic amyloidosis, familial amyloidotic polyneuropathy, and familial amyloid cardiomyopathy[25,26,27]
In order to detect small oligomeric species that may form at an early stage of aggregation, we incubated wild-type (WT) TTR under the amyloidogenic pH of 4.4 at low temperature (4 °C)
Summary
Protein misfolding and amyloid formation is implicated in various debilitating human diseases including Alzheimer’s diseases and amyloidoses[1,2,3]. The protein aggregation process involves conformational changes from native polypeptides to aggregation-prone intermediates that self-assemble into β-structured amyloid. Identification and characterization of various cytotoxic species populated during the complicated aggregation process would be of great importance in developing therapeutic strategies for the protein misfolding disorders. We report a detailed molecular mechanism of transthyretin (TTR) oligomerization process through characterization of small intermediate states of WT and a mutant form of TTR (G53A). The aggregation-prone monomers effectively self-assemble into insoluble amyloid via downhill mechanism[32], and isolation of oligomeric intermediate states for biochemical characterization has been a daunting task. We isolated small oligomeric species populated during TTR oligomerization process and investigated structural features of the cytotoxic www.nature.com/scientificreports/. Our combined analyses of the oligomeric species provided valuable insights into aggregation pathways to the formation of two distinct cytotoxic oligomers and into the structural features of the oligomeric species
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