Final Report Summary - FIBRILLATION (The structure-based design of a blocker of formation of amyloid fibers of the segment AADTWE in the mutant D38A of the protein transthyretin, which causes familial amyloidosis.)
The tetrameric thyroxine-transport protein transthyretin (TTR) forms amyloid fibrils upon dissociation and monomer unfolding. The aggregation of transthyretin has been reported as the cause of life-threatening diseases such as senile systemic amyloidosis, familial amyloidotic polyneuropathy and familial amyloidotic cardiomyopathy. The standard treatment of familial cases of TTR amyloidosis has been liver transplantation. Recent research has identified compounds, such as tafamidis and diflunisal, that bind to the thyroxine-binding site, thereby stabilizing the native tetramer and hindering aggregation. Here we propose a different approach to inhibiting aggregation: binding of non-natural peptides to TTR strands F and H that are necessary for protein aggregation.
Combining mutational and structural studies of TTR variants and isolated peptides, we uncovered the involvement of the strands F and H in TTR aggregation (Figures 1, 2 and 3). This finding is consistent with NMR relaxation dispersion studies that showed that monomeric TTR undergoes conformational fluctuations of the beta-strand H, that are propagated along the entire beta-sheet, and the strand F, at physiological pH (Jiang et al., 2013). We speculate that the S85P substitutions, near strand F, constrain this flexibility to prevent aggregation, while substitutions E92P or V94P, located in strand F, would hinder self-recognition and further aggregation. We anticipate that the thorough study of the crystal structures of our engineered TTR variants will add new insights into the conformational understanding of amyloidogenicity of TTR.
To validate our identification of the fibril-forming segments, we designed analog peptides that bind to the F and H strands, but which do not permit additional monomers to attach, thus preventing amyloid formation (Figure 4). These inhibitors in fact slowed fibril formation, validating our identification of the fibril-forming segments (Figure 5). We propose the specific binding of small peptides as an additional approach. The Eisenberg lab has previously performed structure-based design of peptides or compounds to disrupt fibril development and/or growth (Sievers et al., 2011, Jiang et al., 2013). For the design of TTR aggregation blockers, N-methyl residues were incorporated into the peptide sequence to increase effectiveness (Figure 5B). This is advantageous, because non-natural amino acids can increase peptide stability by reducing proteolytic degradation (Cruz et al., 2004, Eldridge et al., 2009). Our peptide inhibitors have a synergistic effect suggesting that they have separate binding sites (Figure 5B). In addition, the mechanism of inhibition of aggregation does not involve protein stabilization, and the blockers do not compete with T4 for the hydrophobic pocket (Figure 5F).
In summary, our data suggest the mechanism of inhibition of TTR aggregation by peptide blockers as represented in Figure 6. Upon dissociation, the binding of the peptide inhibitors to their identical segments within the TTR monomer hinder the consequent unfolding, thus favoring dimerization and tetramer assembly. The peptide inhibitors bind to the monomer through self-recognition only when the binding site is exposed (Figure 6A). Thus, the hydrophobic pocket of the tetramer remains accessible for stabilizers such as tafamidis or diflunisal (Castano et al., 2012, Bulawa et al., 2012). The combination of our inhibitors with stabilizing compounds might be an effective therapeutic approach.
Combining mutational and structural studies of TTR variants and isolated peptides, we uncovered the involvement of the strands F and H in TTR aggregation (Figures 1, 2 and 3). This finding is consistent with NMR relaxation dispersion studies that showed that monomeric TTR undergoes conformational fluctuations of the beta-strand H, that are propagated along the entire beta-sheet, and the strand F, at physiological pH (Jiang et al., 2013). We speculate that the S85P substitutions, near strand F, constrain this flexibility to prevent aggregation, while substitutions E92P or V94P, located in strand F, would hinder self-recognition and further aggregation. We anticipate that the thorough study of the crystal structures of our engineered TTR variants will add new insights into the conformational understanding of amyloidogenicity of TTR.
To validate our identification of the fibril-forming segments, we designed analog peptides that bind to the F and H strands, but which do not permit additional monomers to attach, thus preventing amyloid formation (Figure 4). These inhibitors in fact slowed fibril formation, validating our identification of the fibril-forming segments (Figure 5). We propose the specific binding of small peptides as an additional approach. The Eisenberg lab has previously performed structure-based design of peptides or compounds to disrupt fibril development and/or growth (Sievers et al., 2011, Jiang et al., 2013). For the design of TTR aggregation blockers, N-methyl residues were incorporated into the peptide sequence to increase effectiveness (Figure 5B). This is advantageous, because non-natural amino acids can increase peptide stability by reducing proteolytic degradation (Cruz et al., 2004, Eldridge et al., 2009). Our peptide inhibitors have a synergistic effect suggesting that they have separate binding sites (Figure 5B). In addition, the mechanism of inhibition of aggregation does not involve protein stabilization, and the blockers do not compete with T4 for the hydrophobic pocket (Figure 5F).
In summary, our data suggest the mechanism of inhibition of TTR aggregation by peptide blockers as represented in Figure 6. Upon dissociation, the binding of the peptide inhibitors to their identical segments within the TTR monomer hinder the consequent unfolding, thus favoring dimerization and tetramer assembly. The peptide inhibitors bind to the monomer through self-recognition only when the binding site is exposed (Figure 6A). Thus, the hydrophobic pocket of the tetramer remains accessible for stabilizers such as tafamidis or diflunisal (Castano et al., 2012, Bulawa et al., 2012). The combination of our inhibitors with stabilizing compounds might be an effective therapeutic approach.
