'In vitro' amyloid fibril formation from transthyretin: the influence of ions and the amyloidogenicity of TTR variants.

The mechanisms of amyloid formation in Familial Amyloidotic Polyneuropathy (FAP) are unknown, as well as the factors determining the development of this pathology. To get some insights into this process, we have first tested a fluorimetric assay with thioflavine T, as a quantitative method for transthyretin (TTR) amyloid estimation, using amyloid isolated from post-mortem tissues of a FAP patient. Then production of amyloid fibrils from soluble TTR was achieved by acidification and optimized for protein concentration and pH. The effect of different ions such as metal and sulphate ions in the process of amyloid formation from wild type TTR was compared using a kinetic assay. Under the conditions tested sulphate diminishes the amount of amyloid formed from wild type TTR and in addition appears to promote aggregation of preexisting amyloid fibrils. The relative amyloidogenicity of three TTR variants, TTR Met30, TTR Pro55 and TTR Met119 respectively, was evaluated using a pH dependent assay. It was shown that the Pro55 variant is highly susceptible to amyloid formation as compared to the wild type protein; on the contrary, the Met119 variant is more resistant than the other TTR proteins towards precipitation into amyloid. These results are in agreement with the pathological conditions associated with these mutations. This type of assay has a wide application for testing the influence of other factors, such as therapeutical agents, on amyloid formation.