title: Alternative pathways of human islet amyloid polypeptide aggregation distinguished by (19)f nuclear magnetic resonance-detected kinetics of monomer consumption.
abstract: 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 (19)F nuclear magnetic resonance (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 buildup of nonfibrillar intermediates, in contrast to the well-studied Aβ and α-synuclein proteins. To further develop the usage of (19)F 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 (19)F 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 nonfibrillar large aggregates during fibrillogenesis.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Brender
authors.forename: Kevin
authors.initials: K
authors.lastname: Hartman
authors.forename: Ayyalusamy
authors.initials: A
authors.lastname: Ramamoorthy
authors.forename: E Neil G
authors.initials: EN
authors.lastname: Marsh
mesh_terms: D052160:Benzothiazoles; D002392:Catechin / Q000031:analogs & derivatives / Q000378:metabolism; D002942:Circular Dichroism; D005461:Fluorine; D006801:Humans; D058228:Islet Amyloid Polypeptide / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D058228:Islet Amyloid Polypeptide; D013844:Thiazoles; C009462:thioflavin T; D005461:Fluorine; D002392:Catechin; C045651:epigallocatechin gallate