(7g) Identification of Transthyretin Domains Involved with Binding to Beta-Amyloid: In Search of Nature's Anti-Alzheimer's ‘Drug'

Du, J., University of Wisconsin-Madison

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

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