The role of copper(II) in the aggregation of human amylin.

Alessandro Sinopoli,Jeremy J Titman,Giuseppe Grasso,Angela Flagiello,Danilo Milardi,Giuseppe Pappalardo,Antonio Magrì,Matteo Pappalardo,Giuseppe Caruso,Vincenzo Giuseppe Nicoletti,Pietro Pucci

doi:10.1039/c4mt00130c

Abstract

Amylin is a 37-residue peptide hormone produced by the islet β-cells of pancreas and the formation of amylin aggregates is strongly associated with β-cell degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Aβ and α-synuclein and there is evidence that amylin self-assembly is also largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure, which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non-fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported.

Highlights

Conformational disease is a general term often used to identify a number of disorders which are characterized by aggregation and deposition of specific proteins
It was interesting that, apart from the expected inhibitory role of copper(II) on amylin degradation by Insulin-degrading enzyme (IDE),[34,36,45] the enzyme was no longer able to cut the peptide in the middle part of its sequence, indicating that conformational changes driven by copper(II) binding to this segment of peptide chain renders the latter unavailable for enzymatic hydrolysis
Copper induces a conformational change onto the h-amylin molecule which shifts the equilibrium toward a more structured form, the lack of β-sheet structures as seen by Circular Dichroism (CD) indicates the absence of an induced fibrillar form of the protein

Summary

Introduction

Conformational disease is a general term often used to identify a number of disorders which are characterized by aggregation and deposition of specific proteins. A common feature of all amyloid deposits is the association in situ with a number of cofactors such as membrane lipids, other proteins, glycosaminoglycans and metals.[11] In the case of amylin, many modulators of its aggregating properties have been identified.[12,13,14,15,16,17,18,19,20,21] In this scenario, the role that some metal ions have in the aggregation of human[22,23,24] or rat[25] amylin and its fragments has been investigated in recent years. The latter seems to be involved in the proteolytic processing of many other different substrates,[37,38] which often function as modulators of its activity[39,40] and degradation of amylin by IDE has already been reported in the literature.[41,42] the cleavage sites of IDE on amylin seem to depend on the particular conditions used, as different peptide fragments are reported in different published works.[41,42] In this scenario, considering the link that might exist between copper(II)

Methods

Results

Conclusion