Role of glycine-33 and methionine-35 in Alzheimer’s amyloid β-peptide 1–42-associated oxidative stress and neurotoxicity

AbstractRecent theoretical calculations predicted that Gly33 of one molecule of amyloid β-peptide (1–42) (Aβ(1–42)) is attacked by a putative sulfur-based free radical of methionine residue 35 of an adjacent peptide. This would lead to a carbon-centered free radical on Gly33 that would immediately bind oxygen to form a peroxyl free radical. Such peroxyl free radicals could contribute to the reported Aβ(1–42)-induced lipid peroxidation, protein oxidation, and neurotoxicity, all of which are prevented by the chain-breaking antioxidant vitamin E. In the theoretical calculations, it was shown that no other amino acid, only Gly, could undergo such a reaction. To test this prediction we studied the effects of substitution of Gly33 of Aβ(1–42) on protein oxidation and neurotoxicity of hippocampal neurons and free radical formation in synaptosomes and in solution. Gly33 of Aβ(1–42) was substituted by Val (Aβ(1–42G33V)). The substituted peptide showed almost no neuronal toxicity compared to the native Aβ(1–42) as well as significantly lowered levels of oxidized proteins. In addition, synaptosomes subjected to Aβ(1–42G33V) showed considerably lower dichlorofluorescein-dependent fluorescence – a measure of reactive oxygen species (ROS) – in comparison to native Aβ(1–42) treatment. The ability of the peptides to generate ROS was also evaluated by electron paramagnetic resonance (EPR) spin trapping methods using the ultrapure spin trap N-tert-butyl-α-phenylnitrone (PBN). While Aβ(1–42) gave a strong mixture of four- and six-line PBN-derived spectra, the intensity of the EPR signal generated by Aβ(1–42G33V) was far less. Finally, the ability of the peptides to form fibrils was evaluated by electron microscopy. Aβ(1–42G33V) does not form fibrils nearly as well as Aβ(1–42) after 48 h of incubation. The results suggest that Gly33 may be a possible site of free radical propagation processes that are initiated on Met35 of Aβ(1–42) and that contribute to the peptide’s toxicity in Alzheimer’s disease brain