Disruption of the CD Loop by Enzymatic Cleavage Promotesthe Formation of Toxic Transthyretin Oligomers through a Common TransthyretinMisfolding Pathway
2020
Amyloid
formation of full-length TTR involves dissociation of the
native tetramers into misfolded monomers that self-assemble into amyloid.
In addition to the full-length TTR, C-terminal fragments including
residues 49–127 were also observed in vivo, implying the presence
of additional misfolding pathways. It was previously proposed that
a proteolytic cleavage might lead to the formation of the C-terminal
fragment TTR amyloid. Here, we report mechanistic studies of misfolding
and aggregation of a TTR variant (G53A) in the absence and presence
of a serine protease. A proteolytic cleavage of G53A in the CD loop
(K48 and T49) with agitation promoted TTR misfolding and aggregation,
suggesting that the proteolytic cleavage may lead to the aggregation
of the C-terminal fragment (residues 49–127). To gain more
detailed insights into TTR
misfolding promoted by proteolytic cleavage, we investigated structural
changes in G53A TTR in the presence and absence of trypsin. Our combined
biophysical analyses revealed that the proteolytic cleavage accelerated
the formation of spherical small oligomers, which exhibited cytotoxic
activities. However, the truncated TTR appeared to maintain native-like
structures, rather than the C-terminal fragment (residues 49–127)
being released and unfolded from the native state. In addition,
our solid-state nuclear magnetic resonance and Fourier transform infrared
structural studies showed that the two aggregates derived from the
full-length and cleaved TTR exhibited nearly identical molecular structural
features, suggesting that the proteolytic cleavage in the CD loop
destabilizes the native tetrameric structure and accelerates oligomer
formation through a common TTR misfolding and aggregation mechanism
rather than through a distinct molecular mechanism.
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