Effect of methionine-35 oxidation on the aggregation of amyloid-ß peptide.

Aggregation of Aß peptides into amyloid plaques is considered to trigger the Alzheimer's disease (AD), however the mechanism behind the AD onset has remained elusive. It is assumed that the insoluble Aß aggregates enhance oxidative stress (OS) by generating free radicals with the assistance of bound copper ions. The aim of our study was to establish the role of Met35 residue in the oxidation and peptide aggregation processes. Met35 can be readily oxidized by H2O2. The fibrillization of Aß with Met35 oxidized to sulfoxide was three times slower compared to that of the regular peptide. The fibrils of regular and oxidized peptides looked similar under transmission electron microscopy. The relatively small inhibitory effect of methionine oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. The peptide oxidation pattern was more complex when copper ions were present: addition of one oxygen atom was still the fastest process, however, it was accompanied by multiple unspecific modifications of peptide residues. Addition of copper ions to the Aß with oxidized Met35 in the presence of H2O2, resulted a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in the peptide molecule do not depend on the oxidation state of Met35 residue. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aß-Cu(II) complex.

Label-free high-throughput screening assay for inhibitors of Alzheimer's amyloid-ß peptide aggregation based on MALDI MS.

Aggregation of amyloid-ß (Aß) peptides is causatively linked to Alzheimer's disease (AD); thus, suppression of this process by small molecule inhibitors is a widely accepted therapeutic and preventive strategy for AD. Screening of the inhibitors of Aß aggregation deserves much attention; however, despite intensive efforts, there are only a few high-throughput screening methods available, all of them having drawbacks related to the application of external fluorescent probes or artificial Aß derivatives. We have developed a label-free MALDI MS-based screening test for inhibitors of Aß42 fibrillization that exhibits high sensitivity, speed, and automation possibilities suitable for high-throughput screening. The test was evaluated by transmission electron microscopy and compared with a fluorimetric thioflavin-based assay, where interference of a number of tested compounds with thioflavin T binding and/or fluorescence caused false-positive results. The MALDI MS-based method can significantly speed up in vitro screening of compound libraries for inhibitors of Aß42 fibrillization.