Role of C-terminal truncations of SAA protein for fibril formation and seeding in AA amyloidosis
Final Report Abstract
This project focused on systemic AA amyloidosis and the role of C-terminal truncated fragments of serum amyloid A (SAA) in the development of the disease. We used a battery of biochemical and biophysical techniques to systematically examine the properties of SAA fragments and their fibril formation ability along with their structure and seeding activity at molecular, cellular and organism level for the first time. Therefore, we recombinantly expressed and purified C-terminally truncated fragments of murine SAA1.1 (mSAA). Spectroscopic analysis of the peptides revealed that removal of only a few amino acids at the C-terminus of SAA leads to a significant loss of ordered secondary structure, suggesting that the C-terminus provides structural stability to the SAA fold. Examination of the solubility of the fragments showed the trend that shorter fragments exhibit a higher solubility. However, full-length mSAA protein and the mSAA(1-57) fragment the lowest solubility. This low solubility is reflected by the fibrillogenic potential. We found that all SAA fragments form fibrils in vitro at near physiological conditions and in the cell culture model of amyloid formation, whereby mSAA and mSAA(1-56) are associated with the shortest lag times and thus with the highest fibrillogenic potential. Structural analysis of in vitro formed fibrils with electron microscopy revealed that they do not differ in their morphology, which is mainly non-twisted. However, infrared spectroscopy showed that they possess differences in their secondary structure, suggesting variations in the structural organization of the different fragment fibrils. Examination of the seeding activity, that is the ability to accelerate fibril formation, of in vitro formed fragment fibrils in different assays (in vitro, in cell culture, in the AA mouse model) revealed remarkable results. While the fibrils show a strong seeding activity in vitro and in cell culture, they do not produce any seeding activity at all in the AA mouse model. In contrast, fibrils extracted from spleen tissue of amyloidogenic mice show a strong acceleration effect in the AA mouse model as well as in the other assays. Structural analysis of the fibrils extracted from amyloidogenic mice revealed a pronounced twisted morphology with crossovers, also indicating significant structural differences between in vitro and in vivo formed fibrils. These data are particularly striking because they show that simple presence of fibrils derived from SAA or a SAA fragment is not sufficient to generate seeding activity in vivo.
Publications
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Cellular mechanism of fibril formation from serum amyloid A1 protein. EMBO Rep., 2017, 18: 1352-1366
Claus S, Meinhardt K, Aumüller T, Puscalau-Girtu I, Linder J, Haupt C, Walther P, Syrovets T, Simmet T, & Fändrich M
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Common fibril structures imply systemically conserved protein misfolding pathways in vivo. Angew. Chemie Int. Ed. 2017, 56(26): 7510-7514
Annamalai K, Liberta F, Vielberg MT, Close W, Lilie H, Gührs KH, Schierhorn A, Koehler R, Schmidt A, Haupt C, Hegenbart U, Schönland S, Schmidt M, Groll M, Fändrich M
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Influence of C-terminal truncation of murine serum amyloid A on fibril structure. Sci. Rep. 2017, 7: 6170
Rennegarbe M, Lenter I, Schierhorn A & Haupt C
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Serum amyloid A forms stable oligomers that disrupt vesicles at lysosomal pH: Implications for the pathogenesis of reactive amyloidosis. Proc. Natl. Acad. Sci. USA 2017, 114(32):E6507-E6515
Jayaraman S, Gantz D, Haupt C & Gursky O
