Methionine oxidation perturbs the structural core of the prion protein and suggests a generic misfolding pathway.

Oxidative stress and misfolding of the prion protein (PrP(C)) are fundamental to prion diseases. We have therefore probed the effect of oxidation on the structure and stability of PrP(C). Urea unfolding studies indicate that H(2)O(2) oxidation reduces the thermodynamic stability of PrP(C) by as much as 9 kJ/mol. (1)H-(15)N NMR studies indicate methionine oxidation perturbs key hydrophobic residues on one face of helix-C as follows: Met-205, Val-209, and Met-212 together with residues Val-160 and Tyr-156. These hydrophobic residues pack together and form the structured core of the protein, stabilizing its ternary structure. Copper-catalyzed oxidation of PrP(C) causes a more significant alteration of the structure, generating a monomeric molten globule species that retains its native helical content. Further copper-catalyzed oxidation promotes extended ß-strand structures that lack a cooperative fold. This transition from the helical molten globule to ß-conformation has striking similarities to a misfolding intermediate generated at low pH. PrP may therefore share a generic misfolding pathway to amyloid fibers, irrespective of the conditions promoting misfolding. Our observations support the hypothesis that oxidation of PrP destabilizes the native fold of PrP(C), facilitating the transition to PrP(Sc). This study gives a structural and thermodynamic explanation for the high levels of oxidized methionine in scrapie isolates.

Evaluation of copper2+ affinities for the prion protein.

The prion protein (PrP) is a cell-surface Cu2+ binding glycoprotein which can bind six copper ions. The role of Cu2+ in PrP function, misfolding, and prion disease has generated much interest; however, the field has been hampered by a lack of consensus with regard to the affinity of Cu2+ for PrPC. Here we build on our understanding of the appearance of visible CD spectra for full-length PrP and fragments to determine the affinity of Cu2+ for four different binding modes, with dissociation constants ranging between 13 and 66 nM at pH 7.4.

Copper and the structural biology of the prion protein.

PrP (prion-related protein) is a cell-surface Cu(2+)-binding glycoprotein which, when misfolded, is responsible for a number of transmissible spongiform encephalopathies. The co-ordination geometry, stoichiometry and affinity of Cu(2+) for PrP are the subject of much debate. In the present paper, we review the recent progress we have made in these areas. As many as six Cu(2+) ions bind to PrP with submicromolar affinity. Initially, two Cu(2+) ions bind to full-length PrP in the amyloidogenic region, between the octarepeats and the structured domain, at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Competitive chelators have been used to determine the affinity of the first molar equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+)-binding sites support the suggestion that PrP could act as an antioxidant by binding potentially harmful Cu(2+) ions and sacrificially quenching of free radicals generated as a result of copper redox cycling. Finally, the effect of Cu(2+) on the prion structure and misassembly into oligomers and fibres is discussed.

Amyloid beta-Cu2+ complexes in both monomeric and fibrillar forms do not generate H2O2 catalytically but quench hydroxyl radicals.

Oxidative stress plays a key role in Alzheimer's disease (AD). In addition, the abnormally high Cu(2+) ion concentrations present in senile plaques has provoked a substantial interest in the relationship between the amyloid beta peptide (Abeta) found within plaques and redox-active copper ions. There have been a number of studies monitoring reactive oxygen species (ROS) generation by copper and ascorbate that suggest that Abeta acts as a prooxidant producing H2O2. However, others have indicated Abeta acts as an antioxidant, but to date most cell-free studies directly monitoring ROS have not supported this hypothesis. We therefore chose to look again at ROS generation by both monomeric and fibrillar forms of Abeta under aerobic conditions in the presence of Cu(2+) with/without the biological reductant ascorbate in a cell-free system. We used a variety of fluorescence and absorption based assays to monitor the production of ROS, as well as Cu(2+) reduction. In contrast to previous studies, we show here that Abeta does not generate any more ROS than controls of Cu(2+) and ascorbate. Abeta does not silence the redox activity of Cu(2+/+) via chelation, but rather hydroxyl radicals produced as a result of Fenton-Haber Weiss reactions of ascorbate and Cu(2+) rapidly react with Abeta; thus the potentially harmful radicals are quenched. In support of this, chemical modification of the Abeta peptide was examined using (1)H NMR, and specific oxidation sites within the peptide were identified at the histidine and methionine residues. Our studies add significant weight to a modified amyloid cascade hypothesis in which sporadic AD is the result of Abeta being upregulated as a response to oxidative stress. However, our results do not preclude the possibility that Abeta in an oligomeric form may concentrate the redox-active copper at neuronal membranes and so cause lipid peroxidation.

Prion protein does not redox-silence Cu2+, but is a sacrificial quencher of hydroxyl radicals.

Oxidative stress is believed to play a central role in the pathogenesis of prion diseases, a group of fatal neurodegenerative disorders associated with a conformational change in the prion protein (PrP(C)). The precise physiological function of PrP(C) remains uncertain; however, Cu(2+) binds to PrP(C) in vivo, suggesting a role for PrP(C) in copper homeostasis. Here we examine the oxidative processes associated with PrP(C) and Cu(2+). (1)H NMR was used to monitor chemical modifications of PrP fragments. Incubation of PrP fragments with ascorbate and CuCl(2) showed specific metal-catalyzed oxidation of histidine residues, His(96/111), and the methionine residues, Met(109/112). The octarepeat region protects His(96/111) and Met(109/112) from oxidation, suggesting that PrP(90-231) might be more prone to chemical modification. We show that Cu(2+/+) redox cycling is not 'silenced' by Cu(2+) binding to PrP, as indicated by H(2)O(2) production for full-length PrP. Surprisingly, although detection of Cu(+) indicates that the octarepeat region of PrP is capable of reducing Cu(2+) even in the absence of ascorbate, H(2)O(2) is not generated unless ascorbate is present. Full-length PrP and fragments cause a dramatic reduction in detectable hydroxyl radicals in an ascorbate/Cu(2+)/O(2) system; however, levels of H(2)O(2) production are unaffected. This suggests that PrP does not affect levels of hydroxyl radical production via Fentons cycling, but the radicals cause highly localized chemical modification of PrP(C).

Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer's disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1-28): insights from a range of complementary spectroscopic techniques.

There is now direct evidence that copper is bound to amyloid-beta peptide (Abeta) in senile plaque of Alzheimer's disease. Copper is also linked with the neurotoxicity of Abeta and free radical damage, and Cu(2+) chelators represent a possible therapy for Alzheimer's disease. We have therefore used a range of complementary spectroscopies to characterize the coordination of Cu(2+) to Abeta in solution. The mode of copper binding is highly pH-dependent. EPR spectroscopy indicates that both coppers have axial, Type II coordination geometry, square-planar or square-pyramidal, with nitrogen and oxygen ligands. Circular dichroism studies indicate that copper chelation causes a structural transition of Abeta. Competition studies with glycine and l-histidine indicate that copper binds to Abeta-(1-28) at pH 7.4 with an affinity of K(a) approximately 10(7) m(-1). (1)H NMR indicates that histidine residues are involved in Cu(2+) coordination but that Tyr(10) is not. Studies using analogues of Abeta-(1-28) in which each of the histidine residues have been replaced by alanine or in which the N terminus is acetylated suggest that the N terminus and His(13) are crucial for Cu(2+) binding and that His(6) and His(14) are also implicated. Evidence for the link between Alzheimer's disease and Cu(2+) is growing, and our studies have made a significant contribution to understanding the mode of Cu(2+) binding to Abeta in solution.