Structural characterisauon of the amyloidosenlc intermediate of nucleated pancreatic amyloid formation

Protein misfolding into a toxic conformation may underly a number of amyloidis-related diseases including CJD, AD, and familial amyloidosis disease. A common patholoc feature of these is the formation and deposition of cytotoxic amyloid fibrils. Also, a common molecular mechanism of fibril formation has been postulated for the involved proteins Pancreatic amyloid is formed by" the aggregation of IAPP and plays a major role in the pathology of NIDDM. Amyloid fibrils of hlAPP have been suggested to contain hlAPP in a highly pleated ant]parallel/J-sheet conformation. Using hIAPP fibrils as seeds, we show here tha.t hIAPP aggregation proceeds by nucleation-dependent polymerization. CD studies together with cross-linking demonstratpd that a conformational transition towards a dimeric, -sheet-rich amyloidogenic inlermediate precedes hIAPP nucleated self-assembly. ANS-binding fluorescence showed that this intermediate contains strongly exposed hydrophob]r patches. Furthermore, denaturation studies indicated a significant contributioxt of hydrophobic interactions in the 3-sheet formation and aggregation of hIAPP. These result.q indicate that preformed hIAPP fibrils act as a template to nu. cleate hlAPP association into soluble, hydrophobic and metastable dimeric sheet structnres which self-assemble to form insoluble aggregates. Our resxslts offer an insight into the mechanism of amyloid formation and may contribute to the design of inhibitors of amyloidogenesis and the treatment of amyloidosisrelated diseases.