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

Fibrillar aggregates of β-amyloid peptide (Aβ) 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 Aβ 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 Aβ fibrillogenesis in vitro by methylation of isoaspartyl residue using purified 5Aβ and PIMT. A Thioflavin-T (Th-T) binding assay conducted after aging Aβ in vitro (37 °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 Aβ aggregates (>200 kDa) only occurred during Aβ incubation, while they were reduced in response to incubation with PIMT and AdoMet. Additionally, circular dichroism (CD) showed that the β-sheet structure was increased in Aβ peptides in a time-dependent fashion, while PIMT suppressed the β-sheet transition after 24 h. Finally, transmission electron microscopy (TEM) revealed that PIMT reduced the size of the Aβ 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 Aβ by inhibiting the β transition in the Aβ peptide.