Alternative Pathways of Human Islet Amyloid Polypeptide Aggregation Distinguished by 19F NMR-Detected Kinetics of Monomer Consumption

摘要

Amyloid formation, a complex process involving many intermediate states, is proposed to be the driving force for amyloid-related toxicity in common degenerative diseases. Unfortunately, the details of this process have been obscured by the limitations in the methods that can follow this reaction in real-time. We show that alternative pathways of aggregation can be distinguished by using ¹⁹F NMR to monitor monomer consumption along with complementary measurements of fibrillogenesis. The utility of this technique is demonstrated by tracking amyloid formation in the diabetes-related islet amyloid polypeptide (IAPP). Using this technique, we show IAPP fibrillizes without an appreciable build up of non-fibrillar intermediates, in contrast to the well-studied Aβ and α-synuclein proteins. To further develop the usage of ¹⁹F NMR, we have tracked the influence of the polyphenolic amyloid inhibitor epigallocatechin gallate (EGCG) on the aggregation pathway. Polyphenols have been shown to strongly inhibit amyloid formation in many systems. However, spectroscopic measurements of amyloid inhibition by these compounds can be severely compromised by background signals and competitive binding with extrinsic probes. Using ¹⁹F NMR, we show that thioflavin T strongly competes with EGCG for binding sites on IAPP fibers. By comparing the rates of monomer consumption and fiber formation, we are able to show that EGCG stabilizes non-fibrillar large aggregates during fibrillogenesis.