Protease Resistance of ex vivo Amyloid Fibrils implies the proteolytic Selection of disease-associated Fibril Morphologies

Abstract

Several studies recently showed that ex vivo fibrils from patient or animal tissue were structurally different from in vitro formed fibrils from the same polypeptide chain. Analysis of serum amyloid A (SAA) and Aβ-derived amyloid fibrils additionally revealed that ex vivo fibrils were more protease stable than in vitro fibrils. These observations gave rise to the proteolytic selection hypothesis that suggested that disease-associated amyloid fibrils were selected inside the body by their ability to resist endogenous clearance mechanisms. We here show, for more than twenty different fibril samples, that ex vivo fibrils are more protease stable than in vitro fibrils. These data support the idea of a proteolytic selection of pathogenic amyloid fibril morphologies and help to explain why only few amino acid sequences lead to amyloid diseases, although many, if not all, polypeptide chains can form amyloid fibrils in vitro.

The AL Amyloid Fibril: Looking for a Link between Fibril Formation and Structure

“…In this context, a recent study hypothesized that all light chains have comparable intrinsic amyloidogenicity and that the interplay of protein sequence, environmental conditions and the presence and action of proteases determines whether or not a particular light chain is deposited as an amyloid fibril in a particular patient [38]. Another recent hypothesis is that disease-associated amyloid fibrils are selected in the body by their proteolytic stability, a mechanism termed proteolytic selection [39,40]. That is, protease-stable fibrils have a greater chance of escaping endogenous clearance mechanisms, proliferating and accumulating in a native cellular environment.…”

“…These studies showed that light chain-derived fibrils consist of an in-register parallel βsheet structure and that the polypeptide chain adopts a conformation with intermolecular contacts that is completely different from its native structure, suggesting that it undergoes dramatic structural rearrangements before fibril formation. However, it is crucial to point out that a growing number of studies show that in vitro fibrils differ in structure and stability from fibrils formed in the body [32,39,40,[58][59][60][61][62]. This suggests that it is important to focus on patient-derived fibrils when investigating the structural basis of a disease.…”

Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding

“…Seeding confers proteolytic stability to the daughter fibrils. Based on that proteolytic resistance constitutes a key difference between ex vivo amyloid fibrils and in vitro formed amyloid fibrils, including the in vitro and ex vivo fibrils from SAA1.1 protein 7,8,10,11 , we tested the susceptibility of the different fibril samples to proteolytic digestion. Subjecting samples of the seeded and unseeded in vitro fibrils, as well as of ex vivo AA amyloid fibrils to protease K digestion (Fig.…”

“…One example is the fibrils formed from SAA1.1, the fibril precursor protein in systemic AA amyloidosis 7 . Ex vivo amyloid fibrils from SAA1.1 or other proteins differ not only 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 .…”