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 Polyneurop athy (FAP) are unknown, as well as the factors determining the develop ment of this pathology. To get some insights into this process, we hav e first tested a fluorimetric assay with thioflavine T, as a quantitat ive method for transthyretin (TTR) amyloid estimation, using amyloid i solated from post-mortem tissues of a FAP patient. Then production of amyloid fibrils from soluble TTR was achieved by acidification and opt imized for protein concentration and pH. The effect of different ions such as metal and sulphate ions in the process of amyloid formation fr om wild type TTR was compared using a kinetic assay. Under the conditi ons 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 s usceptible to amyloid formation as compared to the wild type protein; on the contrary, the Met119 variant is more resistant than the other T TR proteins towards precipitation into amyloid. These results are in a greement with the pathological conditions associated with these mutati ons. This type of assay has a wide application for testing the influen ce of other factors, such as therapeutical agents, on amyloid formatio n.