Meanwhile Westermark and collaborators have elegantly characterized the constituents of TTR amyloid fibrils extracted from cardiac amyloid deposits and adipose tissue, identifying two clearly distinct categories ( 8, 9). The protease responsible has not yet been identified, but the highly specific cleavage suggests that it could be a trypsin-like serine protease. In addition to full-length TTR monomers, Lundgren’s group identified the residue 49-127 fragment, generated by proteolytic cleavage of the peptide bond Lys48–Thr49 ( 7), as the main fragment present in ex vivo TTR amyloid fibrils. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the β-turn linking strands C and D caused by loss of the wild-type hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants. The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation.
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