Abstract
Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection.
Highlights
Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure
Using the same buffer and solution conditions as in the formation of unseeded in vitro fibrils, we analyzed the effect of 0.01 mg/mL ex vivo fibrils on the kinetics of fibril formation and on the structure of the resulting fibrils
We followed the formation of fibrils in real time based on thioflavin T (ThT) fluorescence (Fig. 2a), which depends on the binding of ThT to amyloid fibrils[23]
Summary
Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. Ex vivo amyloid fibrils from SAA1.1 or other proteins differ by their structure from in vitro formed fibrils, they are more protease stable[7,8,10]. These observations gave rise to the proteolytic selection hypothesis which assumes that disease-associated amyloid fibrils were selected within the body due to their ability to escape the endogenous proteolysis machinery[7,11]. The ordered conformation of in vitro fibrils is formed by residues Gly[1] to Ala[37]
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