Identification of the interfacial regions in misfolded transthyretin oligomers.

Misfolding and aggregation of transthyretin (TTR) is associated with numerous ATTR amyloidosis. TTR aggregates extracted from ATTR patients consist of not only full-length TTR, but also N-terminally truncated TTR fragments that can be produced by proteolytic cleavage, suggesting the presence of multiple misfolding pathways. Here, we report mechanistic studies of an early stage of TTR aggregation to probe the oligomerization process for the full-length as well as N-terminally truncated TTR. Our kinetic analyses using size exclusion chromatography revealed that amyloidogenic monomers dissociated from wild-type (WT) as well as pathogenic variants (V30M and L55P) form misfolded dimers, which self-assemble into oligomers, precursors of fibril formation. Dimeric interfaces in the full-length misfolded oligomers were investigated by examining the effect of single-point mutations on the two ß-strands (F and H). The single-point mutations on the two ß-strands (E92P on strand F and T119W on strand H) inhibited the dimerization of misfolded monomers, while the TTR variants can still form native dimers through the same F and H strands. These results suggest that the two strands are involved in intermolecular associations for both native and misfolded dimers, but detailed intermolecular interactions are different in the two forms of dimers. In the presence of a proteolytic enzyme, TTR aggregation is greatly accelerated. The two mutations on the two ß-strands, however, inhibited TTR aggregation even in the presence of a proteolytic enzyme, trypsin. These results suggest that the two ß-strands (F and H) play a critical role in aggregation of the N-terminally truncated TTR as well.

Toxic Misfolded Transthyretin Oligomers with Different Molecular Conformations Formed through Distinct Oligomerization Pathways.

Protein aggregation is initiated by structural changes from native polypeptides to cytotoxic oligomers, which form cross-ß structured amyloid. Identification and characterization of oligomeric intermediates are critically important for understanding not only the molecular mechanism of aggregation but also the cytotoxic nature of amyloid oligomers. Preparation of misfolded oligomers for structural characterization is, however, challenging because of their transient, heterogeneous nature. Here, we report two distinct misfolded transthyretin (TTR) oligomers formed through different oligomerization pathways. A pathogenic TTR variant with a strong aggregation propensity (L55P) was used to prepare misfolded oligomers at physiological pH. Our mechanistic studies showed that the full-length TTR initially forms small oligomers, which self-assemble into short protofibrils at later stages. Enzymatic cleavage of the CD loop was also used to induce the formation of N-terminally truncated oligomers, which was detected in ex vivo cardiac TTR aggregates extracted from the tissues of patients. Structural characterization of the oligomers using solid-state nuclear magnetic resonance and circular dichroism revealed that the two TTR misfolded oligomers have distinct molecular conformations. In addition, the proteolytically cleaved TTR oligomers exhibit a higher surface hydrophobicity, suggesting the presence of distinct oligomerization pathways for TTR oligomer formation. Cytotoxicity assays also revealed that the cytotoxicity of cleaved oligomers is stronger than that of the full-length TTR oligomers, indicating that hydrophobicity might be an important property of toxic oligomers. These comparative biophysical analyses suggest that the toxic cleaved TTR oligomers formed through a different misfoling pathway may adopt distinct structural features that produce higher surface hydrophobicity, leading to the stronger cytotoxic activities.