Reductive Modification of a Methionine Residue in the Amyloid-beta Peptide

Amyloid-b peptide (Ab) is a 39-43 amino acid peptide strongly involved in the development of Alzheimers disease. In the plasma, the major form is AbACHTUNGTRENUNG(1-40), which aggregates to form neuritic plaques and neurofibrillary tangles in the diseased brain.[1] Special attention has been paid to the role of methionine residue 35 (Met35), which has been shown to play a critical role in the oxidative-stress and neurotoxicity properties exhibited by Ab. This was demonstrated by replacing Met35 with other amino acids.[2, 3] The molecular basis of this stress involves oxidation to the methionine radical cation and subsequent formation of methionine sulfoxide and/or electron transfer to other residues.[4, 5] However, a clear picture of the chemical degradation paths and their influence on the disease aetiology is far from being achieved. In the context of free-radical damage, the concept of reductive stress has received much less attention than that of oxidative stress.[6] The reductive attack of HC atoms on methionine is known to cause degradation, with formation of a-aminobutyric acid and CH3SH.[7] The sulfur-centered radical intermediates involved in such degradation have been recently connected with a type of tandem protein-lipid damage, based on the reaction of thiyl radicals with unsaturated fatty acids.[8] We were interested in applying a chemical biology approach to focus on radical damage of Ab, by replacement of Met35 with a-aminobutyric acid in the peptide sequence. The replacement of methionine by other amino acids inAb is known to occur by genetic mutation, as in the case of valine, which causes the disease to appear at very early ages.[9] In this paper we describe the reaction ofHC atoms with the 40-residueAbACHTUNGTRENUNG(1-40) or its reversed sequenceAbACHTUNGTRENUNG(40- 1), the detection of tandem protein-lipid damage, and the isolation and identification of the peptide modified at the Met35 residue. To begin with, the reaction of HC atoms with the methionine moiety was monitored by using trans-lipids as markers.[8, 10] Liposomes were prepared from 1-palmitoyl-2- oleoyl phosphatidylcholine (POPC), in the form of large unilamellar vesicles (LUVETs) with a 100 nm diameter; these vesicles were obtained by membrane extrusion with LiposoFast.[ 10, 11] AbACHTUNGTRENUNG(1-40) or AbACHTUNGTRENUNG(40-1) was added to this suspension. A small amount of tert-butanol (0.20m), which did not exceed 1% of the total volume, was also added. The oleate (cis-9-octadecenoic acid) content of POPC was kept at 8.7 mm, whereas three peptide concentrations (150, 300, 450 mm), corresponding to lipid/peptide ratios of 58:1, 58:2, and 58:3, respectively, were used. In at least two of these conditions (58:1 and 58:2 ratios), the membrane association of the peptide could be neglected, according to recent data.[12] The mixtures were saturated with N2Oprior to g irradiation at a dose rate of 12.0 Gymin1. After irradiation, lipid isolation, and derivatization to the corresponding fatty acid methyl esters,[13] GC analysis was used to determine the cis/trans ratio.[11]