Investigation of Molecular Mechanism of Transthyretin Oligomerization Associated with ATTR Amyloidosis

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Transthyretin amyloidosis (ATTR) has classically been diagnosed predominantly post-observation of amyloid fibril deposition on soft tissues within the body, however, recent studies have shown that oligomers which arise during the aggregation process are much more cytotoxic than their amyloid fibril counterparts.2,6,7 Additionally, it has also been found that cleaved C-terminal fragments, within the peptide range of T49-E127, circulate in-vivo with these oligomers and amyloids, providing further insight into the vast complexity of amyloidogenic species circulating within patients.5,11 Previous mechanistic studies of transthyretin misfolding and aggregation have shed light on the capability of the native protein to first dissociate into its constituent monomers, which are then capable of self-associating into dimeric species that subsequently form hexamers and eventually large cytotoxic oligomers.2,6,9 Whilst searching for the specific amino acid sequences involved in amyloidosis within ATTR, it was found that mutations E92P, in the A91-T96 peptide chain, or T119W, in the T119-N124 peptide chain, were both able to completely inhibit aggregation.23 This study seeks to examine the specific molecular mechanism behind transthyretin dimerization in misfolding pathways by introducing the E92P and T119W mutations directly into the H-H’ and F-F’ ß-strand of the native dimeric interface of TTR. \\n Structural characterization profiles of non-native TTR species which arise during the aggregation process of wild-type TTR (WT-TTR), as well as those of the highly amyloidogenic mutations V30M and L55P were propagated under acidic and kinetically favorable conditions to induce aggregation while maintaining an observable timeline. The WT and mutant forms of TTR underwent similar aggregation processes, involving large concentrations of tetramers first dissociating into monomers, these misfolded monomers then form misfolded dimers, and at the peak of dimer formation high molecular weight oligomers begin to form. These observations indicate the importance of the misfolded monomer’s ability to form into misfolded dimers with regards to the eventual formation of oligomeric species. In order to examine if the native dimeric interface sites, along the H-H’ and F-F’ ß-strand bonds, are integral parts of WT-TTR and L55P-TTR’s ability to aggregate into misfolded dimeric species, proline substitutions were introduced into the E92 peptide within their F-strands as well as tryptophan substitutions into the T119 peptide within their H-strands. These two bulky peptide substitutions showed clear inhibition of non-native oligomeric species under acidic conditions as well as in samples which had been proteolytically cleaved at the L48-T49 peptide bond using trypsin digestion. As these mutations may be having a stabilizing effect on the native tetramer during the dissociation phase, rather than the intended inhibitory effect during the aggregation phase, leading to the observed decrease in oligomerization, a monomeric variant of TTR (m-TTR) was also studied in similar fashion to the WT-TTR and L55P-TTR trials. Similar to the previous wild-type and L55P studies, the inhibited m-TTR samples proved successful in eliminating dimerization. These results indicate that oligomerization of TTR is dependent upon the ability of its dissociated misfolded monomers to form into misfolded dimers, and by introducing large sidechains into locations within the dimeric interface, it is possible to eliminate the ability of these misfolded monomers to aggregate into larger amyloidogenic species.