Analysis of the Minimal Amyloid-forming Fragment of the Islet Amyloid Polypeptide

Ronit Azriel,Ehud Gazit

doi:10.1074/jbc.m102883200

Abstract

The development of type II diabetes was shown to be associated with the formation of amyloid fibrils consisted of the islet amyloid polypeptide (IAPP or amylin). Recently, a short functional hexapeptide fragment of IAPP (NH(2)-NFGAIL-COOH) was found to form fibrils that are very similar to those formed by the full-length polypeptide. To better understand the specific role of the residues that compose the fragment, we performed a systematic alanine scan of the IAPP "basic amyloidogenic units." Turbidity assay experiments demonstrated that the wild-type peptide and the Asn(1) --> Ala and Gly(3) --> Ala peptides had the highest rate of aggregate formation, whereas the Phe(2) --> Ala peptide did not form any detectable aggregates. Dynamic light-scattering experiments demonstrated that all peptides except the Phe(2) --> Ala form large multimeric structures. Electron microscopy and Congo red staining confirmed that the structures formed by the various peptides are indeed amyloid fibrils. Taken together, the results of our study provide clear experimental evidence for the key role of phenylalanine residue in amyloid formation by IAPP. In contrast, glycine, a residue that was suggested to facilitate amyloid formation in other systems, has only a minor role, if any, in this case. Our results are discussed in the context of the remarkable occurrence of aromatic residues in short functional fragments and potent inhibitors of amyloid-related polypeptides. We hypothesize that pi-pi interactions may play a significant role in the molecular recognition and self-assembly processes that lead to amyloid formation.

Highlights

The development of type II diabetes was shown to be associated with the formation of amyloid fibrils consisted of the islet amyloid polypeptide (IAPP or amylin)
The minimal amyloid-forming fragment of IAPP provides a unique case of an extremely short peptide fragment that contains all OF the structural information needed to mediate the molecular recognition and self-assembly processes that lead to amyloid formation
The results presented in this article clearly indicate that a specific pattern of molecular recognition, rather than nonspecific hydrophobic interactions, directs this process of self-assembly in the case of IAPP fibrils formation

Summary

EXPERIMENTAL PROCEDURES

Peptides Synthesis—Peptide synthesis was performed by PeptidoGenic Research & Co. Inc. (Livermore, CA). Kinetic Aggregation Assay—Freshly prepared stock solutions were prepared by dissolving lyophilized form of the peptides in Me2SO at a concentration of 100 mM. Prepared stock solution with a concentration of 10 mM peptide in Me2SO was diluted in to 10 mM Tris buffer, pH 7.2 (which was filtered through a 0.2-␮m filter) to a final concentration of 100 ␮M peptide and 1% Me2SO. Congo Red Staining and Birefringence—A suspension of peptide fibrils in 10 mM Tris buffer, pH 7.2 (aged for 4 days), was allowed to dry on a glass microscope slide. Staining was performed by the addition of a solution of 1 mM CR in 10 mM Tris buffer, pH 7.2, for about 1 min followed by rinsing with double-distilled water to remove excess CR and drying. Electron Microscopy—A solution of peptides (2 mM) in 10 mM Tris buffer, pH 7.2, was incubated overnight at room temperature. Samples were viewed in a JEOL 1200EX electron microscope operating at 80 kV

RESULTS

Short active sequencea

PHGGGWGQ PQGGYQQYNc

DISCUSSION

PHENYLALANINE RESIDUE IN AMYLOID FORMATION Ronit Azriel and Ehud Gazit