Abstract
The islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone whose deposition as amyloid fibrils in the pancreatic islets is associated with type 2 diabetes. Previous studies have suggested that residue Asn-21 plays a critical role in the in vitro self-assembly of IAPP. Herein, we studied structure-self-assembly relationships focusing on position 21 to gain detailed insights into the molecular mechanisms of IAPP self-assembly and to probe the conformational nature of the toxic assemblies associated with β-cell death. Thioflavin T (ThT) fluorescence, CD spectroscopy, and transmission EM analysis revealed that the Asn-21 amide side chain is not required for IAPP nucleation and amyloid elongation, as N21A and N21G variants assembled into prototypical fibrils. In contrast, Asn-21 substitution with the conformationally constrained and turn-inducing residue Pro accelerated IAPP self-assembly. Successive substitutions with hydrophobic residues led to the formation of ThT-negative β-sheet-rich aggregates having high surface hydrophobicity. Cell-based assays revealed no direct correlation between the in vitro amyloidogenicity of these variants and their toxicity. In contrast, leakage of anionic lipid vesicles disclosed that membrane disruption is closely associated with cytotoxicity. We observed that the N21F variant self-assembles into worm-like aggregates, causing loss of lipid membrane structural integrity and inducing β-cell apoptosis. These results indicate that specific intra- and intermolecular interactions involving Asn-21 promote IAPP primary nucleation events by modulating the conformational conversion of the oligomeric intermediates into amyloid fibrils. Our study identifies position 21 as a hinge residue that modulates IAPP amyloidogenicity and cytotoxicity.
Highlights
The islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone whose deposition as amyloid fibrils in the pancreatic islets is associated with type 2 diabetes
Thioflavin T (ThT) fluorescence, CD spectroscopy, and transmission EM analysis revealed that the Asn-21 amide side chain is not required for IAPP nucleation and amyloid elongation, as N21A and N21G variants assembled into prototypical fibrils
The present study reveals that Asn-21 acts as a molecular hinge modulating IAPP amyloid formation and cytotoxicity
Summary
To gain mechanistic insights into IAPP self-assembly and toxicity to -cells, we prepared a library of analogs modified at Asn-21. The presence of an amide group on the residue 21 side chain is not a prerequisite for the formation of a ThT-positive cross--sheet structure, as the substitution N21A led to a slight acceleration of self-assembly. Introducing the turn-inducing residue Pro led to an acceleration of amyloid assembly, with N21P displaying a lag time of 4.2 Ϯ 0.8 h and a significant increase of ThT end-point fluorescence (Fig. 2B). The presence of a hydrophobic group (Phe, Leu) confined IAPP into a -sheet conformation immediately after solubilization These -sheet–rich species assembled under these conditions were ThT-negative (Fig. S3). The N21F analog did not form ThT-positive signal in the presence of lipid vesicles, a high percentage of membrane leakage was measured These results expose a certain association between cytotoxicity (Fig. 6) and membrane disruption (Fig. 7). We cannot rule out the possibility that the N21F wormlike aggregates disassemble and release soluble species, which induce cell death
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