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

<div><p>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 <i>in vitro</i> 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. </p> </div

CD spectra of 20 µM PrP(111-126) with varying Cu²⁺ concentrations (0, 0.3, 0.6, 0.9, 1.5, 1.8 mol. equiv) in PBS at 20 °C.

<p>(A) UV-CD and (B) visible-CD spectra of PrP(111-126) with 0 to 1.8 mole equivalent of Cu²⁺. (B, inset) intensity of 560 nm CD band vs. Cu²⁺ concentration. (C) CD spectra of PrP(111-126) with or without sonication for 120 seconds in the presence of 1 mole equivalent Cu²⁺.</p

Morphology of fibrillar assemblies of the PrP

<p>(111-126). AFM height images of (A) PrP(111-126) and (B) PrP(111-126) in the presence of Cu²⁺ ions. Height profiles of the fibrils for lines marked on the images: (C) PrP (111-126) and (D) PrP (111-126) with Cu²⁺. Scale bar represents 500nm.</p

Cell viability (MTT) assay.

<p>Monomeric and fibrillar form of PrP(111-126) and PrP(113-127) were treated to astrocyte cultures. (A) Cytotoxicity of monomers and fibrils at various concentrations of PrP(111-126) and at 25µM of PrP(113-127). (B) PrP(111-126) (20µM) in the presence of different concentrations of Cu²⁺and 25µM of PrP(113-127) with 25µM Cu²⁺.</p

Effect of microenvironment on the secondary structure of PrP

<p>(111-126). (A) CD spectra of 20µM PrP(111-126) in different TFE concentrations, 0% to 100% TFE, in PBS at 4 °C. (B) CD spectra of PrP(111-126) at pH 2, pH 5, pH 7, and pH 10.6 at 20°C. (C) CD spectrum of PrP(111-126) in 0.1%(CMC) of SDS, and in liposomes in deionized water at 20 °C. (D) surface area-pressure isotherm of PrP(111-126) at air-water interface, and (inset) CD spectrum of peptide film (20 layers) on quartz showing β-sheet formation.</p

Affects of exogenous Aβ_1–42 on the level of eNOS and autophagy in endothelial cells.

<p>Endothelial cells were treated with 5 µM and 10 µM Aβ_1–42 and incubated for 12 hrs. Protein levels of eNOS, nNOS, caveolin-1 and the autophagy marker LC3B were analyzed by Western blotting (A). (B), quantitation of protein levels of eNOS, nNOS and caveolin-1 normalized to β-actin control. Results are mean ± SD (n = 3–5). (C), quantitation of LC3B II/I ratio normalized to β-actin control. Results are mean ± SD (n = 3). **, P<0.01 compared to control.</p

Aβ_1–40 expression monitored by immunofluorescence microscopy.

<p>Endothelial cells were incubated with different concentrations of fibrils for 12 hrs and processed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058194#s2" target="_blank">materials and method</a>. Phase contrast (left panels) and fluorescence images (right panel) were taken showing the same field.</p

The morphological changes of Hep-1 cells incubated with Aβ_1–42 in the presence and absence of serum analyzed by scattering data.

<p>The effect of Aβ_1–42 concentration (A, D: 0 µM; B, E: 5 µM; C, F: 10 µM) in both forward and side scatter values is given in the dot plot.</p

The mean fluorescence values obtained from the flow cytometry histogram were plotted against incubated Aβ_1–42 concentration in the presence of serum (A) and under serum deprived condition (B).

<p>The mean fluorescence values obtained from the flow cytometry histogram were plotted against incubated Aβ_1–42 concentration in the presence of serum (A) and under serum deprived condition (B).</p

Detection of fibrillar Aβ_1–42 induced Aβ_1–40 production by flow cytometry.

<p>For the analysis of Aβ_1–40 synthesis in amyloidic condition, Hep-1 cells were treated with fibrillar Aβ_1–42 in the presence (A) or absence (B) of serum, stained with anti Aβ_1–40 antibodies (biotinylated) and streptavidin conjugated phycoerythrin. Histograms are shown for the control with no added amyloid (ctrl) and for cells incubated with various concentrations of Aβ_1–42.as indicated in the figure.</p