Solvent Microenvironments and Copper Binding Alters the Conformation and Toxicity of a Prion Fragment

Abstract

The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable β-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited β-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu2+ ions inhibited the structural transition and fibril formation of the peptide in a cell free in vitro system. The fact that Cu2+ supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu2+ ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu2+ ions, owing to binding affinity of copper towards histidine moiety present in the peptide.

Neurometals in the Pathogenesis of Prion Diseases

“…We also found that Zn 2+ and Cu 2+ significantly inhibited PrP106–126 oligomerization using the thioflavin T (ThT) fluorescence assay, far-ultraviolet circular dichroism (CD) spectroscopy, and atomic force microscopy (AFM) imaging. Although Cu 2+ and Zn 2+ reportedly facilitate the aggregation of AβP [ 91 ], our results coincide with other studies, which indicate that PrP-induced conformational changes and toxicity are attenuated by Cu [ 92 , 93 ]. Moreover, Cu 2+ reportedly inhibits the aggregation of human islet amyloid peptide (amylin) [ 94 ], Thus, it is highly possible that Cu 2+ exhibits complex effects on the oligomerization of amyloidogenic proteins.…”

X-ray Absorption Spectroscopy Investigations of Copper(II) Coordination in the Human Amyloid β Peptide

“…Solution conditions previously have been found to have significant effects on peptide conformations. For example, the prion protein, PrP has been found to adopt different conformations, specifically with respect to the proportions of α-helical and β-sheet structures, in aqueous solutions, in surfactant solutions, and following sonication . Similarly, secondary structure and aggregation of Aβ peptides has been found to be influenced by solvent conditions.…”

“…For example, the prion protein, PrP has been found to adopt different conformations, specifically with respect to the proportions of α-helical and β-sheet structures, in aqueous solutions, in surfactant solutions, and following sonication. 109 Similarly, secondary structure and aggregation of Aβ peptides has been found to be influenced by solvent conditions. In early studies by Shen and Murphy, Aβ(1−39) was not found to have any detectable β-sheet content in neat dimethyl sulfoxide (DMSO); however, in 0.1% trifluoroacetate the peptide contained approximately 1 / 3 β-sheet, and approximately 2 / 3 β-sheet in 35% acetonitrile/0.1% trifluoroacetate, which is thought to be most similar to the fibrillar form of the peptide under physiological conditions.…”

The effect of a membrane-mimicking environment on the interactions of Cu2+with an amyloidogenic fragment of chicken prion protein

“…39 It is well known that the hydrophobic 112-125 region of hPrP undergoes random coil to α-helix transition in the presence of membrane like environments (surfactant or lipid micelles) and structuring solvents. [43][44][45][46][47] Similarly to what happens to other amyloidogenic proteins, like Aβ and αS, this structural rearrangement strongly impacts Cu 2+ binding modes in terms of both donor atoms and affinity. 47,48 As we recently demonstrated, the interaction of hPrP91-127 with anionic micelles facilitates simultaneous copper coordination to both His96 and His111, which on the other hand behaves as separate metal anchoring sites in the absence of micelle environment.…”