Trans-Suppression of Misfolding in an Amyloid Disease

See allHide authors and affiliations

Science  28 Sep 2001:
Vol. 293, Issue 5539, pp. 2459-2462
DOI: 10.1126/science.1062245


The transthyretin (TTR) amyloid diseases, representative of numerous misfolding disorders, are of considerable interest because there are mutations that cause or suppress disease. The Val30 → Met30 (V30M) TTR mutation is the most prevalent cause of familial amyloid polyneuropathy in heterozygotes, whereas a Thr119 → Met119(T119M) mutation on the second TTR allele protects V30M carriers from disease. Here, we show that the incorporation of one or more T119M TTR subunits into a predominantly V30M tetramer strongly stabilized the mixed tetramer against dissociation. Dissociation is required for amyloid formation, so these findings provide a molecular explanation for intragenictrans-suppression of amyloidosis. The data also suggest a potential therapeutic strategy, provide insight into tissue-specific deposition and amyloid composition, and support the validity of the amyloid hypothesis in human disease.

The amyloidoses are a large group of protein misfolding diseases (1–3). The 80 transthyretin (TTR) amyloid diseases are representative of those where a full-length protein composes the fibrils. The TTR familial amyloid polyneuropathy (FAP) mutations (e.g., V30M) make TTR more susceptible to dissociation and the conformational changes that enable amyloid deposition and pathology. Compound heterozygotes having V30M and T119M TTR mutations on different alleles have few, if any, manifestations of FAP (4, 5), suggesting that the incorporation of a stabilizing subunit in an oligomeric protein such as TTR can protect against misfolding and disease. Previous studies document the increased stability of T119M-containing TTR and the decreased stability of the V30M-containing TTR, relative to the wild type (WT) (6–8).

Here, we evaluate the amyloidogenicity and stability of individual tetramers with defined V30M and/or T119M subunit composition to explain the T119M trans-suppression that protects compound heterozygotes from disease. The V30M/T119M TTR hybrid tetramers were synthesized by double transformation and coexpression inE. coli. An anionic tandem FLAG tag (FT2) sequence (DYKDDDDK)2(9) was added to the NH2-terminus of T119M TTR to facilitate ion exchange separation of tetramers having different numbers of T119M subunits (nondenaturing conditions; Fig. 1A). The greater the number of FT2-T119M subunits in the TTR tetramer, the longer the retention time. Reverse-phase high-pressure liquid chromatography (RP-HPLC) (Fig. 1B) (denaturing conditions) and SDS–polyacrylamide gel electrophoresis (10) analysis of individual peaks collected from a preparative anion exchange separation revealed that tetramers1, 2, 3, 4, and5 contained zero, one, two, three, and four FT2-T119M subunits, respectively, with the balance being V30M. Tetramers 2 to 5 dissociate very slowly at 37°C (no exchange after 5 days), allowing studies on tetramers1 through 5 to be carried out without complications from postseparation subunit exchange (11). Appending the FT2 sequence to the NH2-terminus of WT TTR does not alter its stability, rate of subunit self-exchange, or pH-dependent amyloidogenicity (11). Isolated tetramers composed of WT and/or T119M, and WT and/or V30M subunits, were prepared analogously.

Figure 1

Coexpression, isolation, and analysis of TTR tetramers composed of V30M and/or FT2-T119M subunits. Escherichia coli [BL21-Gold (DE3), Stratagene] was transformed with two plasmids, pET29a containing the genes for FT2-T119M TTR (11) and kanamycin (kan) resistance, as well as pMMHα that includes the genes for V30M-TTR and ampicillin (amp) resistance. Selection for doubly transformed BL21-Gold (DE3) was performed by growth in LB-media containing kan and amp. Expression and purification of the hybrid tetramers was performed as described (10). (A) Preparative anion exchange chromatography of the hybrid tetramers reveals that they can be separated. The elution time of tetramers1 through 5 increased in proportion to the number of FT2-T119M subunits composing the tetramer, from 0 to 4 subunits, respectively. (B) Verification of tetramer subunit composition was performed by RP-HPLC/electrospray ionization mass spectrometry (ESI-MS) under acidic conditions (1% trifluoroacetic acid) using a CH3CN elution gradient that denatures the hybrid tetramers into unfolded subunits, the elution time being shorter for the FT2-T119M (peak A) relative to V30M subunits (peak B) due to their increased polarity.

Because partial acid denaturation (endocytic pathway) has been implicated in fibril formation for several amyloid proteins (1), we evaluated the pH-dependent solubility of tetramers1 to 5. The V30M homotetramer 1 and tetramer 2 precipitated over a pH range of 4.8 to 4.0. However, tetramer 2 incorporating a single FT2-T119M subunit exhibited significantly reduced deposition (10). The inclusion of two or more suppressor subunits rendered tetramers 3, 4, and5 soluble from pH 7 to 3. The amyloidogenicity of hybrid tetramers 2, 3, and 4 was evaluated at pH 4.4 (the pH maximum of V30M fibril formation from homotetramer1) by both turbidity and thioflavin T binding. Substituting V30M subunits with FT2-T119M subunits in the TTR tetramer caused a stoichiometry-dependent inhibition of fibril formation (Fig. 2A). The inclusion of one suppressor subunit (tetramer 2) reduced amyloidogenicity by 50%, whereas the presence of two or more suppressor subunits (e.g., tetramer3, 4, or 5) reduced amyloidogenicity by more than 90% at physiological TTR concentrations (at 37°C). Comparing tetramers 1 through 5 from compound heterozygotes to V30M/FT2-WT tetramer 1 and tetramers 6 to 9 (found in heterozygous FAP patients) revealed that the WT subunits are poor amyloid fibril suppressors (Fig. 2, A and B). WT TTR amyloidogenesis (pH 4.4) was inhibited by 45 and 80% by the incorporation of one and two FT2-T119M subunits, respectively (tetramers 11and 12; Fig. 2C), suggesting generality.

Figure 2

Amyloidogenicity of tetramers with defined subunit composition. The percent amyloidogenicity of (A) tetramers 1 to 5 composed of 0, 1, 2, 3, and 4 FT2-T119M subunits and (B) tetramer 1 and tetramers 6 to9 composed of 0, 1, 2, 3, and 4 FT2-WT subunits, respectively (the remainder being V30M) were evaluated at pH 4.4 after 72 hours as described (10) (amyloidogenicity of tetramer 1 was set to 100%). (C) Percent amyloidogenicity of tetramers 10 to 13 and tetramer 5 composed of 0, 1, 2, 3, and 4 FT2-T119M subunits, respectively, the remainder being WT (amyloidogenicity of tetramer 10 was set to 100%). Amyloid quantification was performed by measuring turbidity at 400 nm (open bars) and ThT fluorescence at 482 nm (filled bars).

The influence of suppressor subunit stoichiometry on tetramer stability was evaluated by the guanidinium thiocyanate (GdmSCN)–mediated transition from folded tetramer to unfolded monomers (Fig. 3A). GdmSCN denaturation exhibits hysteresis due to anion stabilization of the tetramer—ideal for this application, because it prevents reequilibration of hybrid tetramer subunit stoichiometry during measurements (12). Stability differences can be detected by comparing denaturant curve midpoints (Δc 1/2), whereas changes in the free energy of unfolding (ΔΔG NU) can be estimated by the linear extrapolation method (imperfect due to hysteresis) (13). Tetramer stability increased in a nonlinear fashion with T119M subunit stoichiometry, and the Δc 1/2 between homotetramers 1 and5 was 0.36 M, with ΔΔG NU ≈ 1.8 kcal/mol (10). The trans-suppression effect is similar in magnitude to the cis-global suppressor mutations that prevent P22 tailspike (14) and P53 tumor suppressor (15) misfolding. Furthermore, subunit exchange experiments show that homotetrameric T119M TTR self-exchanges subunits >100-fold slower than both WT and V30M TTR, likely owing to the differential stabilization of the tetramer relative to the transition state for dissociation (Fig. 3C). Thus, the T119M stabilization of the tetramer in combination with the high kinetic barriers for dissociation rationalize the mechanistic basis for intragenic trans-suppression. In comparison, increasing the WT stoichiometry relative to V30M revealed a modest stability increase [Δc 1/2 = 0.10 M; ΔΔG NU ≈ 0.5 kcal/mol (10)] and similar subunit self-exchange rates (Fig. 3, B and C). Tetramer 1 and tetramers 6 to9 also exhibited very similar amyloidogenicity at pH 4.4 (Fig. 2B), suggesting similar stability toward acid denaturation, consistent with epidemiology results showing no major differences in FAP pathology or age of onset when comparing V30M homozygotes to heterozygotes (16–19).

Figure 3

Stability of hybrid tetramers and TTR homotetramer subunit exchange kinetics. (A) Guanidinium thiocyanate (GdmSCN) denaturation curves of tetramers1 to 5. Symbol definition: tetramer 1(•); tetramer 2 (▵); tetramer 3 (□); tetramer 4 (▪); and tetramer 5 (▴). (B) GdmSCN denaturation curves of tetramer 1and tetramers 6 to 9. Symbol definition: tetramer 1 (•); tetramer 6 (▵); tetramer 7 (□); tetramer 8 (▪); and tetramer9 (▴). The unfolding data was collected and analyzed as described (28). (C) Homotetramer subunit self-exchange time courses. Subunit exchange (4°C) was initiated by mixing equimolar concentrations (1.8 μM) of unlabeled and FT2-labeled homotetramers of otherwise identical sequence. Plotted is the appearance of a tetramer composed of two FT2-labeled and two unlabeled subunits. Symbols: WT (•), V30M (□), and T119M (▴). V30M and WT show very similar subunit self-exchange kinetics, however, T119M is >100-fold slower. The detailed experimental procedure for these types of experiments has been reported (11).

Mixing equimolar amounts of FT2-T119M homotetramers (or T119M homotetramers) with V30M homotetramers under conditions precluding exchange had no effect on V30M fibril formation (Fig. 4, A and B), suggesting that the stabilizing effect of T119M requires subunit exchange. Hybrid tetramers formed by a modest extent of subunit exchange (Fig. 4C) inhibited amyloidosis by 20% (20), whereas a near-statistical mixture of tetramers created by reconstitution of unfolded V30M and T119M monomers (20) inhibited amyloid formation by 70% (Fig. 4E). The extent of subunit mixing determined the extent of amyloid inhibition observed (Fig. 4, A through E).

Figure 4

Fibril formation as a function of the extent of hybrid tetramer formation. Chromatograms from analytical anion exchange chromatography quantifying the amount of subunit exchange are shown on the left and the extent of fibril formation represented by bar graphs (turbidity at 400 nm) on the right. (A) The extent of amyloidogenicity from the V30M homotetramer 1 [0.2 mg/ml (pH 4.4); 37 °C; 72 hours; set to 100%]. Entry (B) displays the amyloidogenicity arising from mixing homotetramers 1 and5 (open bar) and 1 (filled bar) with an untagged T119M homotetramer immediately before triggering fibril formation by lowering the pH. No suppression of V30M amyloidogenicity occurs, due to lack of subunit exchange. (C) When tetramer 1and the T119M homotetramer (filled bar) or tetramers 1 and5 (open bar) are preincubated at 4°C for 18 days (20) some exchange occurs, consistent with the modest suppression of amyloidogenicity. (D) The unfolding/refolding protocol (26) provides assembly-competent monomeric T119M suppressor subunits which exchange with subunits in V30M homotetramer1 after 24 hours (4°C). The amyloidogenicity of the tetramers resulting from exchange between native V30M and monomeric T119M (filled bar) or FT2-T119M (open bar) is shown. (E) Amyloid formation from co-reconstituted TTR [V30M and T119M (filled bar) or FT2-T119M (open bar)] as described in (20), affording a near-statistical distribution of tetramers (1:4:6:4:1), exhibited a 70% inhibition of amyloid formation.

Little is known about the distribution of tetramers 1to 5 in compound heterozygotes because of our inability to separate them under nondenaturing conditions. Expression levels and kinetics of folding, degradation, and subunit exchange as well as thermodynamic differences among tetramers 1 to 5contribute to the observed distribution. The propensity of V30M and T119M subunits to form hybrid tetramers is high, as demonstrated by the distribution revealed by coexpression of these proteins in E. coli (Fig. 1A; favoring T119M-rich tetramers) and the near-statistical distribution of tetramers arising from in vitro reconstitution of equimolar unfolded subunits (Fig. 4E) (10). T119M suppression requiring hybrid tetramer formation appears similar in vitro and in vivo, on the basis of suppression of amyloidogenicity and the benign disease phenotype, respectively (4, 5). We presume that the reason pathology is observed at all in compound heterozygotes is due to the low concentration of amyloidogenic tetramer 1 and modestly amyloidogenic tetramer2 which can form fibrils, unlike tetramers 3,4, and 5. To explain the tissue-specific deposition of V30M TTR and the bias toward fibrils rich in V30M subunits (21–23), we propose that a given tissue imposes a denaturation stress of fixed magnitude that can only utilize the less stable tetramers as amyloid precursors (e.g., 1 and to a lesser extent 2) (24). Both heterozygotes (aggressive disease phenotype) and compound heterozygotes have comparable amounts of V30M TTR subunits, yet the latter exhibit a benign disease phenotype (4, 5). Thus, it is very unlikely that the V30M TTR subunit causes the disease by a mechanism associated with its normal fold. The dramatic reduction in amyloidogenicity (in vitro) of tetramers mimicking those found in compound heterozygotes supports the amyloid hypothesis origin of FAP.

V30M FAP is now treated by liver transplantation, replacing V30M TTR with WT TTR, resulting in the clearance of amyloid and a dramatic health improvement—further supporting the amyloid hypothesis (25). The predictions made by the data outlined above, if confirmed in vivo, suggest that a gene therapy approach for the synthesis of the T119M trans-suppressor subunits in the liver is likely to inhibit TTR amyloid formation. Administration of T119M subunits that could be incorporated into TTR tetramers after secretion represents an alternative strategy (Fig. 4D) (26).

  • * To whom correspondence should be addressed. E-mail: jkelly{at}


View Abstract

Navigate This Article