Role of glycine-33 and methionine-35 in Alzheimer's amyloid beta-peptide 1-42-associated oxidative stress and neurotoxicity.
Recent theoretical calculations predicted that Gly33 of one molecule of amyloid beta-peptide (1-42) (Abeta(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 Abeta(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 Abeta(1-42) on protein oxidation and neurotoxicity of hippocampal neurons and free radical formation in synaptosomes and in solution. Gly33 of Abeta(1-42) was substituted by Val (Abeta(1-42G33V)). The substituted peptide showed almost no neuronal toxicity compared to the native Abeta(1-42) as well as significantly lowered levels of oxidized proteins. In addition, synaptosomes subjected to Abeta(1-42G33V) showed considerably lower dichlorofluorescein-dependent fluorescence - a measure of reactive oxygen species (ROS) - in comparison to native Abeta(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-alpha-phenylnitrone (PBN). While Abeta(1-42) gave a strong mixture of four- and six-line PBN-derived spectra, the intensity of the EPR signal generated by Abeta(1-42G33V) was far less. Finally, the ability of the peptides to form fibrils was evaluated by electron microscopy. Abeta(1-42G33V) does not form fibrils nearly as well as Abeta(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 Abeta(1-42) and that contribute to the peptide's toxicity in Alzheimer's disease brain.
Kanski
Jaroslaw
J
Department of Chemistry, University of Kentucky, Lexington 40506-0055, USA.
Varadarajan
Sridhar
S
Aksenova
Marina
M
Butterfield
D Allan
DA
D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000502:physiology / Q000633:toxicity*; D000818:Animals; D002478:Cells, Cultured; D004578:Electron Spin Resonance Spectroscopy; D005452:Fluoresceins; D005609:Free Radicals / Q000032:analysis; D005998:Glycine / Q000737:chemistry*; D006624:Hippocampus / Q000196:embryology; D008715:Methionine / Q000737:chemistry*; D008854:Microscopy, Electron; D009154:Mutation; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000502:physiology / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism; D019097:Spin Trapping; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D014633:Valine / Q000737:chemistry
D016229:Amyloid beta-Peptides; D005452:Fluoresceins; D005609:Free Radicals; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; C075222:amyloid beta-protein (1-42); C029569:diacetyldichlorofluorescein; D008715:Methionine; D014633:Valine; D005998:Glycine