Abstract
Despite numerous studies, a detailed description of the transthyretin (TTR) self-assembly mechanism and fibril structure in TTR amyloidoses remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry (HXMS) analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. The protofibrils only grow to an approximate final size of 2,900 kDa and a length of 70 nm and a comparative HXMS analysis of native and aggregated samples revealed a much higher average solvent exposure of TTR upon fibrillation. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state. Together, these data reveal that the fibrillar state interchanges with the solution state. Accordingly, we suggest that TTR fibrillation proceeds via addition of considerably unfolded monomers, and the continuous presence of amyloidogenic structures near the protofibril surface offers a plausible explanation for secondary nucleation. We argue that the presence of such dynamic structural equilibria must impact future therapeutic development strategies.
Highlights
Transthyretin (TTR) is a 55 kDa homotetrameric β -sheet-rich protein mainly produced in the liver, being responsible for the transport of the hormone thyroxine and vitamin A1,2
TTR was kept in demineralised water, and fibrillation was initiated by the addition of acetic acid and NaCl
transmission electron microscopy (TEM) micrographs reveal that already during the brief apparent lag-phase, small ill-defined aggregates were visualised (Fig. 2) and after 9 minutes of incubation, a few small protofibrils protruding between spherical aggregates were further visualised, corresponding to the very subtle increase in Thioflavin T (ThT) fluorescence observed
Summary
Transthyretin (TTR) is a 55 kDa homotetrameric β -sheet-rich protein mainly produced in the liver, being responsible for the transport of the hormone thyroxine and vitamin A1,2. In addition a tetramer (a dimer of dimers) has been proposed to be the building block of TTR amyloid fibrils, based on molecular packing observed in the crystal structure of a highly amyloidogenic variant of TTR23. Both types of models agree that a partial unfolding of the building block is essential for fibril formation[14,24,25,26,27] and that the process may proceed through fibrillogenic intermediate states, termed protofibrils. We suggest that continued focus on the considerably unfolded TTR monomer, which is highlighted for the first time in this study, is of key importance for further advancement in drug development against TTR amyloidoses
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