Development of Novel FRET Substrate for the Evaluation of β-Secretase (BACE1) and Its Application

Abstract

A neuropathological diagnosis of Alzheimer's disease (AD) relies on the detection of amyloid plaques, clumps of beta-amyloid made up of insoluble fibrils. Sequential cleavage by β-secretase (BACE) and γ-secretase produces the amyloid-β peptide fragment, which then aggregates into the clumps of amyloid plaques. As the β-secretase activity is specifically high in AD patient, the enzyme is regarded as the primary target of anti-AD reagent. All the conventional β-secretase inhibitors are substrate analogs, which means those inhibitors are competitive and reversible inhibitors. Therefore, a non-competitive enzyme inhibitor of β-secretase might be a perfect inhibitor for AD. To search for non-competitive enzyme inhibitor of β-secretase, we developed new method to evaluate non-competitive enzyme inhibitors easily by using flow injection analysis (FIA) in combination with immobilized enzyme. In general, FRET substrates linked by recognition sequence of β-secretase are used to evaluate the β-secretase activity. In this study, we constructed a novel FRET substrate which is made up of ECFP as the donor, and EYFP as the acceptor. These proteins are then linked with β-secretase recognition sequence with 8 amino acids. ECFP transfers its excitation energy to EYFP, causing EYFP to emit its characteristic fluorescence. By observing the wavelength using spectrophotometer, we can judge if the material or herbs have the ability to be the inhibitor of β-secretase. Therefore, both the FRET substrates and β-secretase we constructed can be produce with a large amount by using E. coli. And because the protein of FRET substrates and BACE1 can be obtained easily with the E. coli we constructed, therefore we will be able to search for the inhibitors of β-secretase by the FIA screening method with low cost.