Histidine protonation as a possible mechanism for human transthyretin amyloidosis: modelled against wallaby transthyretin
Creators: name: D'SOUZA, Damian
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310101
subject: Analytical biochemistry
classificationCode: 310112
subject: Structural biology (incl. macromolecular modelling)

description: Transthyretin (TTR) is an essential component in the transport of thyroid hormones and retinol. TTR is a tetrameric protein that can undergo misfolding and ß-sheet fibrillar assembly causing disease (amyloidosis) wherever deposited. Several mutations within the TTR peptide sequence have been identified that instigate TTR tetramer dissociation and misfolding preceding disease states, however wild-type TTR can also undergo amyloidosis. Histidine (His) residues have a side-chain pKa of ~ 6.0, susceptible to physiological pH changes that alter their bonding capacities, thus facilitating misfolding similar to the effects of mutations within the protein sequence. Previous preliminary data had suggested that wallaby TTR was more stable than human TTR despite a high sequence identity between the two proteins.  The overall aim of this thesis was to investigate differences in the stability of human and wallaby TTRs, under the hypothesis that these differences were due to the position of His residues within each tetramer. 
There was no known structure for wallaby TTR prior to this thesis. A molecular model of the predicted 3D structure of wallaby TTR was generated and superimposed on a known crystal structure of human TTR (PDB: 3U2I) with a peptide backbone Cα RMSD of ~ 0.45. Each human TTR monomer contains four His (His31, His56, His88, and His90) that are susceptible to protonation. Wallaby TTR contains His56 and His88 conserved in its TTR sequence, however does not contain His31 or His90. This supported a hypothesis that the position and protonation of His31 and His90 in human TTR, absent in wallaby TTR (Lys31 and Tyr90), make human TTR less stable. 
Further comparisons of human TTR stability to wallaby TTR stability were carried out to investigate why human TTR is less stable than wallaby TTR. Human and wallaby TTRs were expressed in E.coli to determine if the recombinant human TTR formed amyloid similar to reports in the literature, and to compare similarities or differences in amyloid capabilities to  wallaby TTR. Human and wallaby TTR underwent amyloidosis at varying intensities when incubated in pHs 4.5-7.5, with increased amyloidosis evident with the presence of denser fibrils formed on decreasing pH (increasing acidity) shown using congo red (CR), transmission electron microscopy TEM and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR). These were the first studies to show the potential for TTR amyloid formation in marsupials.
Dynamic light scattering (DLS) showed that after ageing the human and wallaby TTRs, the percentage of native tetramer (~ 10 nm) in most cases decreased while the presence of smaller molecules (~ 0.1-1 nm) and larger aggregates/oligomers (&gt; 100 nm) increased similar to the predicted amyloidosis pathway of tetramer dissociation, monomer misfolding and aggregation in to amyloid fibrils. The percentage of tetramer dissociation to fibril oligomerisation occured more significantly in human TTR compared to wallaby TTR. ThioflavinT (ThT) fluorescence made it clear that the human TTR were less stable than the wallaby TTR at physiological pHs (7.5-6.5) as there was an increase in ThT fluorescence (indicating fibril/amyloid formation) in human TTR within 24 hours of incubation but no increase in ThT fluorescence in the wallaby TTR samples. These results indicated tetramer dissociation kinetics were sufficient for human TTR to progress through to amyloid faster than wallaby TTR.
While studying the structural features that result in human TTR forming amyloid faster than wallaby TTR, the ability of a potential peptide inhibitor 2-[(3,5-Dichlorophenyl) amino]benzoic acid (MW 282.12) (originally synthesised and termed Victorian College of Pharmacy compound 6, VCP6) to prevent TTR amyloidosis and to further understand potential drug designs in the prevention of TTR amyloid formation, was used. VCP6s is an analogue of thyroxine (3',5',3,5 -tetraiodo-L-thyronine, T4) that inhibits human TTR amyloidosis at pH 4.4. The potential to inhibit human and/or wallaby TTR amyloidosis through stabilization of the tetramer via the T4- binding pocket, was tested at physiological pHs 7.5 - 6.5. VCP6 had an inhibitory effect on human and wallaby TTR aggregation to amyloid fibril formation through binding to the T4 pocket, stabilising both tetramers. VCP6 had a reduced effect on human TTR fibril formation at pH 6.5, while having 50-100 % of an inhibitory effect on wallaby TTR fibril formation at pH 6.5. This finding supported the hypothesis that His31 and His90 (not present in wallaby TTR), may facilitate human TTR tetramer dissociation and amyloid formation through changes in protonation states.
The predicted wallaby TTR structure was modelled around a previously solved human TTR crystal structure and does not necessarily reflect the actual conformation of wallaby TTR. For this reason, an X-ray crystal structure was sought for wallaby TTR to better address the hypothesis that the occurrence and position of His residues in human TTR, not present in wallaby TTR, makes human TTR less stable. An X-ray crystal structure for wallaby TTR was solved to ~ 2.5 Å using molecular replacement against human TTR. An overall RMSD of 0.97 indicated a highly conserved overall structure between the solved structure of wallaby TTR and a known crystal structure of human TTR (PDB: 3U2I). The structural homology was strong with an almost perfect overlap of their peptide backbones, secondary, tertiary and quaternary features. The difference in His56 orientation between human and wallaby TTR, as well as the lack of His31 and His90 in wallaby TTR (present in human TTR), provides an insight towards understanding the increased amyloidosis potential of human TTR compared with wallaby TTR.