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.
Highlights
The highly infectious and transmissible prion disease occurs due to the misfolding and aggregation of prion proteins, resulting in neurodegeneration [1,2,3,4]
When incubated at 37°C, the prion protein (PrP)(111-126) solution showed an increase in the β-sheet structure over time, characterized by a more intense negative band at 222 nm and positive band at 198 nm, with a cross over near 212 nm (Figure 1A)
Such transitions are well-known in other amyloid aggregates that are associated with protein conformational diseases [51,52,53,54,55]
Summary
The highly infectious and transmissible prion disease occurs due to the misfolding and aggregation of prion proteins, resulting in neurodegeneration [1,2,3,4]. To investigate which portions of the native protein sequence are involved in the conformational transition from PrPC to PrPSc and PrP amyloid, several groups have analyzed the secondary structures and fibrillogenic properties of synthetic PrP peptides [36]. Extensive work with these peptides has established that the glutamine-rich consecutive segment of PrP that spans residues 106-147 is important for deciphering the fibrillogenic properties of protein [37,38,39]. A detailed structural understanding of prion assemblies in various microenvironments and in the vicinity of metal ions is necessary to gather substantial information from amyloid toxicity
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