Identification of Transthyretin Domains Involved with Binding to Beta-Amyloid: In Search of Nature's Anti-Alzheimer's ‘Drug'

Alzheimer's disease (AD) is the most common age-associated neurodegenerative disease. Characteristic features include extracellular senile plaques, intraneuronal neurofibrillary tangles, and extensive neuronal cell death. The major proteinaceous component of the plaques is beta-amyloid (Ab). Ab spontaneously self-assembles through a multi-step process into soluble oligomers and fibrillar aggregates. Numerous studies have established that Ab aggregation is causally linked to neurodegeneration; most researchers currently believe that it is the soluble oligomers that are most toxic to neurons.

Transthyretin (TTR) is a homotetrameric transport protein present in both blood and cerebrospinal fluid (CSF). Several studies have demonstrated that TTR binds to Ab, and alters its aggregation. Interestingly, TTR itself is prone to aggregation and is linked to other age-related amyloid disorders. Recent experiments with transgenic AD mice suggest that TTR may protect neurons against Ab-induced damage, raising the intriguing possibility that TTR is a natural anti-AD 'drug'.

The aim of this study is to identify the specific regions of TTR involved with binding to Ab. By doing so, we hope to be able to design mimetics that could replace TTR's natural protective activity that is lost with age or disease. We have used crosslinking and tryptic cleavage coupled with mass spectrometry, along with immobilized peptide arrays, to isolate two regions of TTR, the G strand and the E-strand-E/F loop, as likely domains involved with TTR-Ab association. We have also shown that TTR's natural ligands, thyroxine and retinol-binding protein, which bind to TTR at these identified regions, compete with Ab for binding to TTR.  We synthesized peptides corresponding to the putative binding regions of TTR and found that these peptides specifically inhibit Ab binding to TTR, but in a nonlinear fashion.  We are using limited alanine-scanning mutagenesis to identify specific residues on TTR involved with binding to Ab. This study is complicated by the fact that the quaternary structure and stability of TTR also strongly influences TTR-Ab association. Finally, we plan to examine the effect of active peptides and TTR mutants on Ab oligomerization and toxicity.