Inhibiting transthyretin amyloid fibril formation via protein stabilization - PubMed (original) (raw)

Inhibiting transthyretin amyloid fibril formation via protein stabilization

G J Miroy et al. Proc Natl Acad Sci U S A. 1996.

Abstract

Transthyretin (TTR) amyloid fibril formation is observed systemically in familial amyloid polyneuropathy and senile systemic amyloidosis and appears to be the causative agent in these diseases. Herein, we demonstrate conclusively that thyroxine (10.8 microM) inhibits TTR fibril formation efficiently in vitro and does so by stabilizing the tetramer against dissociation and the subsequent conformational changes required for amyloid fibril formation. In addition, the nonnative ligand 2,4,6-triiodophenol, which binds to TTR with slightly increased affinity also inhibits TTR fibril formation by this mechanism. Sedimentation velocity experiments were employed to show that TTR undergoes dissociation (linked to a conformational change) to form the monomeric amyloidogenic intermediate, which self-assembles into amyloid in the absence, but not in the presence of thyroxine. These results demonstrate the feasibility of using small molecules to stabilize the native fold of a potentially amyloidogenic human protein, thus preventing the conformational changes, which appear to be the common link in several human amyloid diseases. This strategy and the compounds resulting from further development should prove useful for critically evaluating the amyloid hypothesis--i.e., the putative cause-and-effect relationship between TTR amyloid deposition and the onset of familial amyloid polyneuropathy and senile systemic amyloidosis.

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Figures

Figure 1

Figure 1

Schematic representation of the acid-mediated denaturation/amyloid fibril-forming pathway of TTR.

Figure 2

Figure 2

Ribbon diagram of tetrameric TTR illustrating the hourglass-shaped channel that runs through the center of the protein and serves as the binding site for T4 (3,3′-diiodothyroxine shown) molecules rendered as a space-filling structure (21). The four conserved water molecules are also shown in CPK (Corey–Pauling Space Filling Models) format.

Figure 3

Figure 3

Inhibition of TTR amyloid fibril formation by T4 and TIP. The extent of fibril formation in the presence and absence of inhibitor at pH 4.4 was probed at 72 h by an optical density measurement at 330 nm (hatched bars) and by a quantitative Congo red binding assay (solid bars). (A) Wild-type fibril formation. From left to right, fibril formation in the absence of T4 and in the presence of T4 alone (no TTR) and TTR fibril formation in the presence of the indicated equivalents of T4 (3 eq of T4 = 10.8 μM) are shown. Inhibition of (B) V-30-M and (C) L-55-P TTR by T4 at pH 5.0. (D) Inhibition of wild-type TTR by TIP. The labeling of the axes and the organization of the data in_B_–D is the same as that in_A_.

Figure 4

Figure 4

(A) Sedimentation velocity profile of wild-type TTR in the absence of T4 at pH 4.4. Solute distribution recorded at 235 nm (60,000 rpm). Scans for analysis were recorded every 3 min; for simplicity, only scans from every 24 min are shown. (B) Apparent sedimentation coefficient distribution (g*(s)t) as a function of sedimentation coefficient for wild-type TTR (0.2 mg/ml) in the absence of T4, indicating the formation of multiple species during the partial acid denaturation of TTR.

Figure 5

Figure 5

(A) Sedimentation velocity profile of wild-type TTR in the presence of T4 at pH 4.4. Solute distribution recorded at 235 nm (60,000 rpm). Scans for analysis were recorded every 3 min; for simplicity, only scans from every 15 min are shown. (B) Sedimentation equilibrium analysis of wild-type TTR at pH 4.4 in the presence of 10.8 μM. The solid line drawn through the data was obtained by fitting the absorbance vs. radial position_r_ to Eq. 2 for a homogeneous tetrameric species. The residual difference between the experimental data and the fitted data for each point is shown in the Inset.

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