The preaggregated state of an amyloidogenic protein: Hydrostatic pressure converts native transthyretin into the amyloidogenic state

Abstract

Protein misfolding and aggregation cause several diseases, by mechanisms that are poorly understood. The formation of amyloid aggregates is the hallmark of most of these diseases. Here, the properties and formation of amyloidogenic intermediates of transthyretin (TTR) were investigated by the use of hydrostatic pressure and spectroscopic techniques. Native TTR tetramers (T 4) were denatured by high pressure into a conformation that exposes tryptophan residues to the aqueous environment. This conformation was able to bind the hydrophobic probe bis-(8-anilinonaphthalene-1-sulfonate), indicating persistence of elements of secondary and tertiary structure. Lowering the temperature facilitated the pressure-induced denaturation of TTR, which suggests an important role of entropy in stabilizing the native protein. Gel filtration chromatography showed that after a cycle of compression-decompression at 1°C, the main species present was a tetramer, with a small population of monomers. This tetramer, designated T 4*, had a non-native conformation: it bound more bis-(8-anilinonaphthalene-1-sulfonate) than native T4, was less stable under pressure, and on decompression formed aggregates under mild acidic conditions (pH 5-5.6). Our data show that hydrostatic pressure converts native tetramers of TTR into an altered state that shares properties with a previously described amyloidogenic intermediate, and it may be an intermediate that lies on the aggregation pathway. This ''preaggregated'' state, which we call T 4*, provides insight into the question of how a correctly folded protein may degenerate into the aggregation pathway in amyloidogenic diseases.

Hydration and Packing are Crucial to Amyloidogenesis as Revealed by Pressure Studies on Transthyretin Variants that Either Protect or Worsen Amyloid Disease

“…22 It can be observed in Figure 6 that wt TTR dissociates at lower pressures in the second cycle of compression, indicating that T 4 p is less stable than the native T 4 TTR (Figure 6, circles). At 1 8C, the T 4 p species does not aggregate.…”

“…The second cycle of compression of the wt protein is closer to the compression curve of L55P, while the wt decompression curve overlaps with the L55P compression curve. It is possible that the conformation of T 4 p observed for the wt TTR, which has a large hydrophobic segment exposed to the solvent as measured by bis-ANS binding, 22 could resemble the native, amyloidogenic conformation of L55P that undergoes aggregation at much milder pH values. Figure 6(B) shows the p 1/2 values at 1 8C for all the mutants studied thus far, as well as the values found for the first and second compression curves, and for the decompression curve of the wt protein.…”

“…This altered tetramer is called T 4 p and is thought to represent a preaggregate state of TTR. 22 More recently, Niraula and co-workers described a decrease in thermodynamic stability of V30M in relation to the wt protein at neutral pH. 33 In the present study, we compare the stability against pressure of wt TTR to variants (T119M, L55P and V30M).…”

“…22 To investigate whether pressure could induce fibril formation in the variants of TTR, the proteins were incubated at pH 5.6 under 3000 bar (1 bar ¼ 10 5 Pa) for 60 minutes at 37 8C. This incubation time is enough to shift the tryptophan emission completely to the red.…”

“…22 -24 The acid-induced denaturation experiments have suggested a monomer as precursor for fibrillogenesis, while recent evidence obtained by the use of high hydrostatic pressure (HHP) pointed to the possible participation of an altered tetramer in equilibrium with monomers as the raw material for fibril formation in senile systemic amyloidosis. 22 Recently, the crystal structure of the highly amyloidogenic triple mutant G53S/E54D/L55S at 2.3 Å resolution revealed that it is a tetramer. 25 Other evidence for the possible participation of an oligomeric species (either a dimer or a multiple of it) as the building block for TTR aggregation comes from the studies performed by Serag and co-workers, where cysteine substitution mutants were employed.…”