Identification of beta-amyloid-binding sites on transthyretin

Abstract

Transthyretin (TTR) binds to the Alzheimer-related peptide beta-amyloid (Aβ), and may protect against Aβ-induced neurotoxicity. In this work, the specific domains on TTR involved with binding to Aβ were probed. An array was constructed of peptides derived from overlapping sequences from TTR. Strong binding of Aβ to TIAALLSPYSYS (residues 106-117) was detected, corresponding to strand G on the inner β-sheet of TTR. Aβ bound weakly to four contiguous peptides spanning residues 59-83, which includes strand E through the E/F helix and loop. To further pinpoint specific residues on TTR involved with Aβ binding, nine alanine mutants were generated: I68A, I73A, K76A, L82A, I84A, S85A, L17A, T106A and L110A. Aβ binding was significantly inhibited only in L82A and L110A, indicating that Aβ binding to TTR is mediated through these bulky hydrophobic leucines. Aβ binding to L17A and S85A was significantly higher than to wild-type TTR. Enhancement of binding in L17A is postulated to arise from reduced steric restriction to the interior L110 site, since these two residues are adjacent in the native protein. The S85A mutation caused a reduction in TTR tetramer stability; increased Aβ binding is postulated to be a direct consequence of the reduced quaternary stability.

Mechanisms of Transthyretin Inhibition of β-Amyloid AggregationIn Vitro

“…Our estimation of the K D differs substantially from previously estimated binding parameters of K S at 2300 M Ϫ1 (Liu and Murphy, 2006) by tryptophan fluorescence quenching and K D of 28 nM using competition binding (Costa et al, 2008b). Our NMR data showed that the A␤ binding site on TTR involves amino acids in and around the T 4 binding site of the tetramer, partially confirming and extending the recently reported involvement of TTR residues L17, L110, and L82 (but S85) (Du et al, 2012;Yang et al, 2013).…”

“…Experiments showing reduced inhibition of A␤ aggregation when the T 4 site is occupied by small molecules confirm its involvement in A␤ binding. These results are consistent with, but more definitive than, previous data from cross-linking experiments, alanine scanning mutagenesis, and peptide inhibition of TTR-A␤ interaction, which suggested major roles for residues L110 in strand G and L82 in the EF helix/loop in binding A␤ (Du et al, 2012;Yang et al, 2013). Our data suggest that L82, rather than serving as an A␤ oligomer sensor (Yang et al, 2013), may influence the orientation of the side chain of W79, which usually points to the T 4 binding site.…”

“…TTR-A␤ complexes can be coimmunoprecipitated from APP23 cortical lysates and from similar preparations of some human AD brains (Li et al, 2011). Studies in which A␤ or A␤ are preincubated with TTR have demonstrated TTR inhibition of A␤-induced cytotoxicity in a variety of assay systems (Mazur-Kolecka et al, 1995;Giunta et al, 2005;Costa et al, 2008b;Li et al, 2011).…”

Cross-interactions between the Alzheimer Disease Amyloid-β Peptide and Other Amyloid Proteins: A Further Aspect of the Amyloid Cascade Hypothesis

“…TTR appears to be a major A␤-sequestering protein in human CSF, inhibiting and even reverting the formation of amyloid fibrils, as well as reducing their toxicity in vitro (58,59). TTR forms a complex with A␤ monomers/dimers, with stronger binding affinity observed for the more structurally flexible S85A TTR mutant (60), reinforcing the idea that less structured forms of amyloid proteins may be more likely to engage in cross-amyloid interactions.…”

Designed Trpzip-3 β-Hairpin Inhibits Amyloid Formation in Two Different Amyloid Systems

“…Interestingly a recent account suggested that β-amyloid binds to TTR; however, it appears that the interaction occurs after tetramer dissociation. 14 We are interested in the development of broad-spectrum inhibitors of amyloid formation. The trpzip peptides are among the smallest β-hairpin peptides known to fold spontaneously without the use of disulfide-bonds or metal ions.…”