Protein L-isoaspartyl O-methyltransferase inhibits amyloid β fibrillogenesis in vitro

Fibrillar aggregates of beta-amyloid peptide (A beta) are major constituents of the senile plaques found in the brains of patients suffering from Alzheimer's disease (AD). Previous studies have shown that spontaneous isomerization or racemization of aspartyl residues in A beta peptides leads to conformational changes in the secondary structure and increased aggregative ability of the peptides. Protein L-isoaspartyl O-methyltransferase (PIMT, EC 2.1.1.77) is a repairing enzyme converting L-isoaspartyl/D-aspartyl residues in damaged proteins to normal L-aspartyl residues. In this study it was investigated, whether PIMT is able to modulate A beta fibrillogenesis in vitro by methylation of isoaspartyl residue using purified 5A beta and PIMT. A Thioflavin-T (Th-T) binding assay conducted after aging A beta in vitro (37 degrees C, pH 7.4 in PBS) revealed that PIMT inhibited the increase of fluorescence caused by amyloid fibrillogenesis. Western blot analysis revealed that high molecular A beta aggregates (>200 kDa) only occurred during A beta incubation, while they were reduced in response to incubation with PIMT and AdoMet. Additionally, circular dichroism (CD) showed that the beta-sheet structure was increased in A beta peptides in a time-dependent fashion, while PIMT suppressed the beta-sheet transition after 24 h. Finally, transmission electron microscopy (TEM) revealed that PIMT reduced the size of the A beta aggregates and induced a different pathway, leading to the formation of amorphous structures. Taken together, these findings indicate that isoaspartyl methylation leads to partial blockade of fibrillogenesis of A beta by inhibiting the beta transition in the A beta peptide.