Conformational transitions of islet amyloid polypeptide (IAPP) in amyloid formation in vitro

Amyloid aggregates have been recognized to be a pathological hallmark of se veral fatal diseases, including Alzheimer's disease, the prion-related dise ases, and type II diabetes. Pancreatic amyloidosis is characterized by the deposition of amyloid consisting of islet amyloid polypeptide (IAPP). We fo llowed the steps preceding IAPP insolubilization and amyloid formation in v itro using a variety of biochemical methods, including a filtration assay, far and near-UV circular dichroism (CD) spectropolarimetry, 1-anilino-8-nap hthalenesulfonic acid (ANS) binding, and atomic force (AFM) and electron (E M) microscopy. IAPP insolubilization and amyloid formation followed kinetic s that were consistent with the nucleation-dependent polymerization mechani sm. Nucleation of IAPP amyloid formation with traces of preformed fibrils i nduced a rapid conformational transition into P-sheets that subsequently ag gregated into insoluble amyloid fibrils. Transition proceeded via a molten globule-like conformeric state with large contents of secondary structure, fluctuating tertiary and quaternary aromatic interactions, and strongly sol vent-exposed hydrophobic patches. In the temperature denaturation pathway a t 5 mu M peptide, we found that this state was mostly populated at about 45 degrees C, and either aggregated rapidly into amyloid by prolonged exposur e to this temperature, or melted into denaturated but still structured IAPP , when heated further to 65 degrees C. The state at 45 degrees C was also f ound to be populated at 4.25 M GdnHCl at 25 degrees C during GdnHCl-induced equilibrium denaturation, and was stable in solution for several hours bef ore aggregating into amyloid fibrils. Our studies suggested that this amylo idogenic state was a self-associated form of an aggregation-prone, partiall y folded state of IAPP. We propose that this partially folded population an d its self-associated forms are in a concentration-dependent equilibrium wi th a non-amyloidogenic IAPP conformer and may act as early, soluble precurs ors of P-sheet and amyloid formation. Our findings on the molecular mechani sm of IAPP amyloid formation in vitro should assist in gaining insight into the pathogenesis and inhibition of pancreatic amyloidosis and other amyloi d-related diseases. (C) 1999 Academic Press.