A novel mechano‐enzymatic cleavage mechanism underlies transthyretin amyloidogenesis

Abstract

The mechanisms underlying transthyretin-related amyloidosis in vivo remain unclear. The abundance of the 49–127 transthyretin fragment in ex vivo deposits suggests that a proteolytic cleavage has a crucial role in destabilizing the tetramer and releasing the highly amyloidogenic 49–127 truncated protomer. Here, we investigate the mechanism of cleavage and release of the 49–127 fragment from the prototypic S52P variant, and we show that the proteolysis/fibrillogenesis pathway is common to several amyloidogenic variants of transthyretin and requires the action of biomechanical forces provided by the shear stress of physiological fluid flow. Crucially, the non-amyloidogenic and protective T119M variant is neither cleaved nor generates fibrils under these conditions. We propose that a mechano-enzymatic mechanism mediates transthyretin amyloid fibrillogenesis in vivo. This may be particularly important in the heart where shear stress is greatest; indeed, the 49–127 transthyretin fragment is particularly abundant in cardiac amyloid. Finally, we show that existing transthyretin stabilizers, including tafamidis, inhibit proteolysis-mediated transthyretin fibrillogenesis with different efficiency in different variants; however, inhibition is complete only when both binding sites are occupied.

Inhibition of the mechano-enzymatic amyloidogenesis of transthyretin: role of ligand affinity, binding cooperativity and occupancy of the inner channel

“…( C ) Aggregation of 18 µM V122I TTR was quantified as spectrophotometric turbidity at 400 nm normalized to 100% for aggregation of the protein in the absence of ligands. We know from previous work that TTR aggregation in this system is in the form of authentic amyloid fibrils 3, 4 . All data shown represent mean (SD) of three independent experiments, and *** represents P < 0.001. …”

Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation

“…Mutations directly or indirectly affecting the stability of the AC/BD dimer-dimer interface tend to promote exacerbated protein aggregation and severe disease phenotypes, highlighting the crucial role played by the native quaternary structure of TTR at preventing protein deposition. It is worth to point out here that an alternative mechano-enzymatic cleavage mechanism has been recently proposed to underlie transthyretin amyloidogenesis in vivo 43. According to this new view, the combined effect of proteolysis and shearing would result in the release of a highly amyloidogenic 49–127 truncated protomer, usually found in ex vivo deposits.…”

“…Thus, reinforcing the contacts at the quaternary dimer-dimer interface appears as a requirement to impose kinetic stability on the entire TTR tetramer. It is worth to emphasize here that T119M TTR is also totally resistant to proteolytic cleavage under shearing43.…”

“…According to this new view, the combined effect of proteolysis and shearing would result in the release of a highly amyloidogenic 49–127 truncated protomer, usually found in ex vivo deposits. Destabilized TTR variants would be more susceptible to proteolysis than TTR WT43.…”

Peptide probes detect misfolded transthyretin oligomers in plasma of hereditary amyloidosis patients

“…Our data suggest that TTR is cleaved prior to deposition and after rate-limiting dissociation of the native tetramer, as tafamidis treatment stops the aberrant endoproteolysis presumably by kinetically stabilizing the native tetramer. Aberrant endoproteolysis of the monomer could change the structure of the TTR aggregates that are formed, leading to unique proteotoxicity mechanisms and potentially explaining why there is early onset versus late onset V30M FAP, or why FAP in males typically progresses faster than in women (51, 52). …”