(570b) Competition between Ligands: How Retinol-Binding Protein and Beta-Amyloid Compete for Binding to Transthyretin

Transthyretin (TTR) is a soluble transport protein that circulates in blood and cerebrospinal fluid. One of its primary functions is the transport of retinol (vitamin A), via coordination with retinol-binding protein (RBP), to target cells. Once internalized, retinol and its oxidative products regulate transcription of a large number of genes. Retinol must also be delivered across the blood-brain barrier, where it plays essential roles in maintaining neuronal health. The mechanism by which retinol is taken up by cells or crosses the blood-brain barrier is still under investigation; both receptor-mediated and receptor-independent mechanisms have been proposed. TTR is a homotetrameric protein; each monomeric subunit contains two four-stranded β-sheets and a lone α-helix. RBP binds to multiple monomeric subunits in the tetramer, with contacts primarily involving TTR’s α-helix. Although two RBP can bind per TTR, under biological conditions the binding stoichiometry is 1:1.

Recently it has been discovered that TTR may serve a second role, as an inhibitor of the Alzheimer-related beta-amyloid (Aβ). Neuronal dysfunction in Alzheimer’s disease is generally believed to be caused by aggregation of Aβ. Multiple lines of evidence demonstrate that TTR binds to Aβ, thereby inhibiting Aβ aggregation and neurotoxicity. We have shown that TTR contains two Aβ binding domains: the solvent exposed α-helix and a β-strand that is in an interior hydrophobic cavity in the folded TTR tetramer. Our data indicate that Aβ binding to the α-helix triggers destabilization of TTR tetramers, thus opening up the interior binding sites on TTR for greater Aβ binding. This hypothesis is supported by strong evidence that TTR monomers (with exposed ‘interior’ sites) are significantly more effective than tetramers at binding Aβ and inhibiting its aggregation.

Importantly, the RBP binding site on TTR overlaps with the Aβ binding domain on TTR’s α-helix. Thus, we wondered to what extent RBP and Aβ compete with each other for binding to TTR, or whether RBP interferes with TTR’s inhibitory role against Aβ. We report that holo-RBP (hRBP, retinol complexed with RBP) significantly hindered TTR’s protection against Aβ aggregation. This effect was characterized by thioflavin T fluorescence, light scattering, and nanoparticle tracking analysis. Next we determined that RBP does not directly compete with Aβ for binding to TTR. This is likely because the RBP-TTR binding stoichiometry is effectively 1:1, leaving unbound α-helix sites available for Aβ binding. The question remains, then, as to why RBP prevents TTR from inhibiting Aβ aggregation when it does not prevent Aβ binding. A plausible explanation arises from the recognition that RBP stabilizes TTR tetramer against dissociation into monomer subunits. Specifically, the tetramer stability enhancement by hRBP counteracts Aβ-induced tetramer dissociation, thus preventing access of Aβ to the interior binding sites on TTR. Fluorescence experiments, currently underway, are designed to provide direct evidence supporting this hypothesis. Our data demonstrate that RBP is competitive with Aβ in preventing TTR from carrying out its biological function of inhibiting Aβ aggregation. We are currently exploring whether the opposite is also true: does Aβ compete with RBP to prevent TTR from assisting with retinol delivery? Since there is evidence that Alzheimer’s patients suffer retinol deficiencies, this is potentially a previously unrecognized mechanism by which Aβ interferes with neuronal health.