Abstract

β-amyloid (Aβ) aggregates involved in Alzheimer's disease (AD) are resistant to proteases but could be destabilized by small peptides designed to target specific hydrophobic regions of Aβ that take part in aggregate assembly. Since thrombin and AD are intricately connected, and elastase modulates thrombin activity, elastase-digested thrombin peptides were verified for intervention in the Aβ-aggregation pathway. Intact or elastase-digested thrombin destabilized Aβ fibril, as demonstrated by thioflavin T assay. Peptides were synthesized employing thrombin as a template, of which, a hexapeptide (T3) showed maximum destabilization at 1µm. ExPASy peptide cutter software coupled with mass spectrometric analysis confirmed the generation of T3 peptide from elastase-digested thrombin. TEM micrographs revealed that 30-day incubation of preformed Aβ fibrils or monomers with T3 resulted in destabilization or inhibition, respectively, leading mostly to particles of 1.74±0.17nm, which roughly corresponded to Aβ monomer. Surface plasmon resonance employing CM5 chip coupled with Aβ40 mouse monoclonal antibody showed a drop in response when T3 was incubated with Aβ fibrils between 2 and 8h. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and confocal microscopy demonstrated the ability of T3 to rescue neuroblastoma cells from Aβ oligomer-induced cytotoxic damage. Although no [Aβ-T3] adduct could be detected by mass spectrometry, an initial interaction appeared to facilitate the process of destabilization/inhibition of aggregation. T3 was comparable to standard β-sheet breaker peptides, LPFFD and KLVFF in terms of Aβ aggregate destabilization. High hydrophobicity values coupled with recognition and breaking elements make T3 a potential candidate for future therapeutic applications.

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