Development of a sensitive fluorescence detection system for high-throughput screening compounds which can promote the production of A

• Main Authors: Po-Ting Chen, 陳柏廷
• Other Authors: 王勝仕
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/55937707833165006038

碩士 === 國立臺灣大學 === 化學工程學研究所 === 97 === Amyloid-b (Ab), a physiological peptide, which is produced from amyloid precursor protein (APP) by sequential cleavages, and released into the extracellular spaces. The released Ab undergoes proteolytic degradation by multiple endopeptidases. The imbalance between the production and catabolism of Ab in the brain results in the accumulation of Ab leading to Alzheimer’s disease. Alzheimer’s disease is a neurodegenerative disease, and Ab in Alzheimer’s disease plays a pivotal role in the pathogenesis of the disease. Neprilysin (NEP) has been singled out as the most promising candidate in Ab cleaning in the therapeutics of Alzheimer’s disease. In this study, a quenched fluorogenic peptide substrate whose sequence was adopted from the first seven residues of the Ab peptide was synthesized to detect the activity of neprilysin. A fluorophore was attached to the C-terminal Cys residue but its fluorescence was quenched by the existence of a quencher linked to the N-terminus of the peptide. When this peptide substrate was degraded by an endopeptidase, the fluorescence lights up to serve as a sensitive detection system for endopeptidase. Our results showed that this assay system was extremely sensitive to NEP and insulin degrading enzyme (IDE) while inert or much less sensitive to other Ab-degrading enzymes. Moreover, based on our findings, a cell-based assay system was developed to high throughput screen chemicals which are able to enhance NEP and production in human SH-SY5Y cells. Key words：Amyloid-b, Ab-degrading enzymes, neprilysin