High-avidity binding drives nucleation of amyloidogenic transthyretin monomer
Li Gao, Xinfang Xie, Pan Liu, Jing Jin
Li Gao, Xinfang Xie, Pan Liu, Jing Jin
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Research Article Aging Cardiology

High-avidity binding drives nucleation of amyloidogenic transthyretin monomer

Abstract

Amyloidosis involves stepwise growth of fibrils assembled from soluble precursors. Transthyretin (TTR) naturally folds into a stable tetramer, whereas conditions and mutations that foster aberrant monomer formations facilitate TTR oligomeric aggregation and subsequent fibril extension. We investigated the early assembly of oligomers by WT TTR compared with its V30M and V122I variants. We monitored time-dependent redistribution among monomer, dimer, tetramer, and oligomer contents in the presence and absence of multimeric TTR seeds. The seeds were artificially constructed recombinant multimers that contained 20–40 TTR subunits via engineered biotin-streptavidin (SA) interactions. As expected, these multimer seeds rapidly nucleated TTR monomers into larger complexes, while having less effect on dimers and tetramers. In vivo, SA-induced multimers formed TTR-like deposits in the heart and the kidney following i.v. injection in mice. While all 3 variants prominently deposited glomerulus in the kidney, only V30M resulted in extensive deposition in the heart. The cardiac TTR deposits varied in size and shape and were localized in the intermyofibrillar space along the capillaries. These results are consistent with the notion of monomeric TTR engaging in high-avidity interactions with tissue amyloids. Our multimeric induction approach provides a model for studying the initiation of TTR deposition in the heart.

Authors

Li Gao, Xinfang Xie, Pan Liu, Jing Jin

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Figure 4

Rapid coaggregation of TTRWT monomers by artificially induced SA-TTR complexes.

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Rapid coaggregation of TTRWT monomers by artificially induced SA-TTR com...
(A) TTRWT was incubated in the presence or absence of SA-induced TTRV30M seeds (right and left panels, respectively; preparation of induced TTRV30M seeds is shown in Supplemental Figure 1) in 10:1 w/w ratio for a total of 180 minutes. Aliquots were taken from the reaction at indicated time points and subsequently analyzed by SEC. The seeds-alone sample was separately analyzed by SEC. Monomeric (M) dimeric (D), tetrameric (T), and oligomeric (O) contents are indicated. Insets below show the M peaks in the time series. The arrow shows the range of O increases on top of the seed amount. Additional results of TTRWT, TTRV30M, and TTRV122I with the seeds are in Supplemental Figures 3 and 4. (B) Monomeric content of TTRWT before induction was purified by SEC and then subjected to conditions with or without the TTRV30M seeds (top-left and bottom-left panels, respectively). Aliquots taken from 0, 1 (immediately after the spiking-in of seeds), 10, and 60 minutes were analyzed by SEC. There was the contrasting difference between the presence or absence of the seeds. Without the seeds, there was a gradual shift of monomer contents to newly formed dimers and tetramers. With the seeds, there was a rapid increase of high molecular weight of TTR oligomers (arrow indicates the change in levels). Within minutes, the multimeric seeds depleted most monomers in forming high molecular weight protein complexes. In contrast to TTRWT monomers, dimeric and tetrameric fractions did not react to seed-induced aggregation. All experiments were repeated 3 times.