Stability and fibril formation properties of human and fish transthyretin, and of theEscherichia colitransthyretin‐related protein

Abstract

Human transthyretin (hTTR) is one of several proteins known to cause amyloid disease. Conformational changes in its native structure result in aggregation of the protein, leading to insoluble amyloid fibrils. The transthyretin (TTR)‐related proteins comprise a protein family of 5‐hydroxyisourate hydrolases with structural similarity to TTR. In this study, we tested the amyloidogenic properties, if any, of sea bream TTR (sbTTR) and Escherichia coli transthyretin‐related protein (ecTRP), which share 52% and 30% sequence identity, respectively, with hTTR. We obtained filamentous structures from all three proteins under various conditions, but, interestingly, different structures displayed different tinctorial properties. hTTR and sbTTR formed thin, curved fibrils at low pH (pH 2–3) that bound thioflavin‐T (thioflavin‐T‐positive) but did not stain with Congo Red (CR) (CR‐negative). Aggregates formed at the slightly higher pH of 4.0–5.5 had different morphology, displaying predominantly amorphous structures. CR‐positive material of hTTR was found in this material, in agreement with previous results. ecTRP remained soluble at pH 2–12 at ambient temperatures. By raising of the temperature, fibril formation could be induced at neutral pH in all three proteins. Most of these temperature‐induced fibrils were thicker and straighter than the in vitro fibrils seen at low pH. In other words, the temperature‐induced fibrils were more similar to fibrils seen in vivo. The melting temperature of ecTRP was 66.7 °C. This is approximately 30 °C lower than the melting temperatures of sbTTR and hTTR. Information from the crystal structures was used to identify possible explanations for the reduced thermostability of ecTRP.

Binding site asymmetry in human transthyretin: insights from a joint neutron and X-ray crystallographic analysis using perdeuterated protein

“…Despite its ability to form fibrils, transthyretin is a very stable protein. It withstands temperature changes from 277 to 353 K and pH variation between 5.5 and 12.0 without significant damage (Lundberg et al, 2009). Studies concerning its evolutionary development show that the central residues in the binding channel have not changed in over 400 million years (Prapunpoj & Leelawatwattana, 2009).…”

“…There are numerous theories concerning the fibril formation of transthyretin. One of the most popular is the hypothesis that a complete degradation to the monomeric species has to occur prior to aggregation (Lundberg et al, 2009;Wiseman et al, 2005). Other publications suggest a mechanism via the dimeric state of the protein (Serag et al, 2001).…”

“…The fibrils so formed are usually of poor quality and do not (as visualized by electron microscopy) resemble fibrils from natural sources. Furthermore, TTR disassembles into a dimer when exposed to 5% SDS, and the monomeric form is only obtained after boiling the sample (Lundberg et al, 2009). A probe for the existence of a monomeric species in vivo is the measurement of subunit exchange under physiological conditions.…”

Mechanisms of Transthyretin Inhibition of IAPP Amyloid Formation

“…This result is also corroborated by us and is presented in Supplementary files Figure S1. TTR, in contrast, results in an increased aggregation rate as a function of low pH [48,84]. Given that IAPP amyloid formation likely is an intracellular event (occurring within the IAPP containing granules of the β-cells), it is of interest to investigate the inhibitory effect of TTR also at a low pH.…”

Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption

“…However, the native tetrameric form of TTRwt has high conformational stability [ 47 , 48 , 49 ], and the amyloid formation process in these conditions is lengthy and thus is unsuitable to be used in screening protocols. After a careful analysis of the available literature [ 21 , 50 , 51 ], three aggregate and fibril formation protocols were selected for comparison: acidification at pH 2.0 and pH 4.4, and heating at pH 7.4. Furthermore, the effect of changing the pH from 2.0 or 4.4 to physiological conditions (pH 7.4), after aggregation, was also studied.…”