(386e) Subtle Defects In Transthyretin Quaternary Structure Prevent Its Inhibitory Action On Beta-Amyloid Aggregation

Deposition of beta-amyloid (Abeta) fibrils is an early event in the neurodegenerative processes associated with Alzheimer's disease. According to the ?amyloid cascade? hypothesis, Abeta aggregation and its subsequent deposition as fibrils, is the underlying cause of neurotoxicity. In transgenic mice that express large quantities of the human Abeta precursor protein and generate excess Abeta, surprisingly little neuronal death is observed. Microarray analysis and histochemical studies indicated that these transgenic mice synthesize the soluble homotetrameric protein transthyretin at levels several-fold above normal. These results lead to the hypothesis that increased TTR synthesis is a protective response, and that TTR inhibits Abeta toxicity. We tested the hypothesis that TTR inhibits Abeta toxicity by directly affecting its aggregation. To test this hypothesis, we used several biophysical tools to examine Abeta aggregation kinetics in the presence of TTR purified from human plasma (pTTR). At substoichiometric ratios, pTTR drastically decreased the rate of aggregation without affecting the fraction of Abeta in the aggregate pool. Detailed analysis of the data using a mathematical model demonstrated that the decrease in aggregation rate was due to both a decrease in the rate of elongation relative to the rate of initiation of filaments and a decrease in lateral association of filaments to fibrils. Tryptophan quenching data indicated that transthyretin binds weakly to Abeta. Taken together, the data support a hypothesis wherein pTTR preferentially binds to aggregated rather than monomeric Abeta and arrests further growth of the aggregates. Next we produced recombinant TTR (rTTR) in E. coli using an intein-based system, and purified the protein from inclusion bodies using chitin bead adsorption. rTTR was identical to pTTR by mass spectroscopy, circular dichroism, tryptophan fluorescence, size exclusion chromatography, static light scattering, and native and denatured gel electrophoresis. We concluded from these analyses that rTTR was correctly folded and assembled. Surprisingly, rTTR failed to inhibit Abeta aggregation and showed no binding to Abeta by tryptophan quenching. Dynamic light scattering and gel electrophoresis revealed that rTTR tetramers were less stable than pTTR under acidic conditions. By ANS fluorescence we observed greater solvent exposure of hydrophobic sites in rTTR than in pTTR. Chemical denaturation of pTTR followed by refolding produced tetramers with reduced acid stability. We speculate that the diverse folding environments of pTTR (in vivo) and rTTR (in vitro) lead to subtle differences in quaternary structure that have profound effects on TTR-Abeta association and TTR inhibition of Abeta aggregation.