Structural analysis of SAA-amyloid fibrils by cryo-EM and NMR
Biochemistry
Final Report Abstract
We were able to reconstruct several AA amyloid fibril structures at high resolution of cryo-EM data and model the resolved electron density maps. The ordered part of the density of the mouse fibril corresponds to residues 1–69 of mSAA1.1, the ordered part of the human fibril to hSAA1.1 residues 2–55. In two morphologies of ex vivo AA amyloid fibrils from mouse and one morphology from human were reconstructed, that differ only in the amount of protofilaments but the structure of a single protofilament is nearly identical. Comparing fibrils from mouse with the human fibril the overall structure is different but the first 21 amino acids show a remarkable similar beta arch fold. Also, our structure of the human fibril shows that the first amino acid Arg1 needs to be cleaved off beforehand to be able to from the human fibril morphology. Additionally, in vitro mSAA1-derived amyloid fibrils morphologies with and without seeds were reconstructed and modeled. The in vitro fibrils without seed that show different structures compared with ex vivo fibril structures from mouse. If seeds were added from murine ex vivo fibrils we could demonstrates not only that it is possible to replicate the ex vivo fibril structure, but by using full length mSAA1 we can assemble the ex vivo fibril structure without truncating SAA1 peptide. This allows to investigate the kinetics and the mechanism of ex vivo-like fibril proliferation with detailed biophysical studies by NMR. Subjecting samples of the seeded and unseeded in vitro fibrils, as well as of ex vivo AA amyloid fibrils to protease K digestion we find ex vivo fibrils to be substantially more protease resistant than the unseeded in vitro fibrils. Seeded in vitro fibrils were also stable to proteolytic digestion and persisted for more than 2 h under the conditions of our experiment, similar to the ex vivo fibrils. Hence, proteolytic resistance arises, at least partly, from the fold of the fibril protein. But as they are not as stable as ex vivo fibrils, it points to additional in vivo factors that increase the stability against proteases even more. For example, it is possible that molecules attached onto the surface of ex vivo fibrils that act as protective layer. These data suggest that pathogenic amyloid fibrils may originate from proteolytic selection, allowing specific fibril morphologies to proliferate and to cause damage to the surrounding tissue. Overall the presented work demonstrate that systemic AA amyloidosis is an excellent model system for studying phenomena that are relevant for a broad range of protein misfolding diseases and understanding mechanisms of fibril formation. Most of the aim and sometimes more could be reached, except the mutational testing could not be done in time.
Publications
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Cryo-EM fibril structures from systemic AA amyloidosis reveal the species complementarity of pathological amyloids. Nature Communications 2019 10(1), 1–10
Liberta, F., Loerch, S., Rennegarbe, M., Schierhorn, A., Westermark, P., Westermark, G. T., Hazenberg, B. P. C. C., Grigorieff, N., Fändrich, M., Schmidt, M.
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AA amyloid fibrils from diseased tissue are structurally different from in vitro formed SAA fibrils. Nature Communications 2021, 12(1)
Bansal, A., Schmidt, M., Rennegarbe, M., Haupt, C., Liberta, F., Stecher, S., Puscalau- Girtu, I., Biedermann, A., Fändrich, M.
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Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding. Nature Communications 2022,13(1)
Heerde, T., Rennegarbe, M., Biedermann, A., Savran, D., Pfeiffer, P. B., Hitzenberger, M., Baur, J., Puscalau-Girtu, I., Zacharias, M., Schwierz, N., Haupt, C., Schmidt, M., Fändrich, M.
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SAA fibrils involved in AA amyloidosis are similar in bulk and by single particle reconstitution: A MAS solid-state NMR study. J. Struct. Biol. X 2022, 6, e100069 (2022)
Sundaria, A., Liberta, F., Savran, D., Sarkar, R., Rodina, N., Peters, C., Schwierz, N., Haupt, C., Schmidt, M. & Reif, B.
