(750d) Monomers and Multimers: Role of Quaternary Structure in Regulating Transthyretin-Beta-Amyloid Association

Transthyretin (TTR) is a circulating transport protein, present in both blood and cerebrospinal fluid. The protein is natively assembled into a homotetramer ? a dimer of dimers. Each 14 kDa monomer folds into a β-pleat sandwhichof two four-stranded β-sheets. Dimers are formed through extensive hydrogen bonding between the F and H stands of each monomer. Tetramers are assembled via hydrophobically-driven association of two dimers in a face-to-face manner, held together by loops which project from the edge of the dimer. Normally TTR tetramers are quite stable at neutral pH, but will dissociate to monomers at slightly acidic pH. Subtle conformational rearrangement of monomers will lead spontaneously to aggregation into large amyloid fibrils. TTR amyloid deposits are linked to senile systemic amyloidosis, a common disease of aging. Alzheimer's disease is another age-associated disorder that is characterized by amyloid deposition. β-amyloid (Aβ) is the main protein component of the amyloid plaques associated with this neurodegenerative disease. Interestingly, results from several recent studies provide support for the hypothesis that these two amyloidogenic proteins interact, and that this interaction is biologically relevant. For example, upregulation of TTR expression in Tg2576 mice was linked to protection from toxic effects of Aβ deposition [Stein, T.D. and Johnson, J.A. (2002) J. Neurosci. 22: 7380-7388]. These observations motivated our detailed study of TTR- Aβ association. Specifically, we examined the role of TTR's quaternary structure in modulating its association with Aβ. We produced recombinant wtTTR as well as two mutants: one mutation (T119M) stabilizes tetramers against dissociation whereas the other (F87M/L110M) produces a stable monomeric TTR that does not assemble into tetramers. Using on enzyme-linked immunoassays, we show that Aβ monomers bind more strongly to TTR monomers than to TTR tetramers, and that the binding interaction likely involves the central region (residues 17-24) of Aβ and the TTR residues at the monomer-monomer interface. The data further suggest that TTR tetramers interact weakly with Aβ, most likely with aggregates. Light scattering and electron microscopy studies demonstrate that the outcome of the TTR-Aβ interaction strongly depends on TTR quaternary structure. While TTR tetramers may modestly enhance aggregation, TTR monomers decidedly arrest Aβ aggregate growth. To further identify the specific interacting regions of TTR and Aβ, we crosslinked monomeric TTR and Aβ using a mixture of the isotope-labeled (d0 and d4) cross-linker BS3 (bis[sulfosuccinimidyl]suberate). Nano-HPLC/nano-ESI-LTQ mass spectrometry was used to analyze the peptide mixture resulted from tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes. MS/MS analysis points out Lys-9 residue near the AB loop of TTR to be cross-linked with Lys-28 of Aβ. Given the fact that the principle source of TTR dimer-dimer contacts is the AB loop and this loop is also the only region where all four monomers come together, we conclude that TTR tetramer/monomer stability plays an important role in its ability to interact with Aβ. Several factors, including thyroxine binding, oxidation, or pH, dramatically alter the stability of TTR tetramers. Since TTR appears to be an important natural modulator of Aβ aggregation and toxicity, then subtle changes in the TTR tetramer-monomer equilibrium could have major biological consequences. These results suggest new avenues for AD prevention by bolstering TTR's natural protective effects.