Examining the Effects of Modulation of Peptide Substrate Sequence on the Enzyme Specificity in the Sensitive fluoresence Assay

• Main Authors: Chao-Long Chen, 陳昭隆
• Other Authors: Sheng-Shih Wang
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/46574455475591752141

碩士 === 國立臺灣大學 === 化學工程學研究所 === 99 === Abstract Amyloid-b (Ab) is a physiological peptide, which is produced from amyloid precursor protein (APP) by sequential cleavages, and then 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. Reduction of Ab production is considered a feasible way in the therapeutics for Alzheimer’s disease. The enzymes that are capable of hydrolyzing Ab include neprilysin (NEP), insulin degrading enzyme (IDE), endothelin converting enzyme (ECE-1), angiotensin-converting enzyme (ACE), plasmin and matrix metalloproteinase (MMPs). In this study, we synthesized a quenched fluorogenic peptide substrate whose seven-residue sequence was adopted from the twelfth to eighteenth residues of Ab peptide with a cystenine residue (Cys) added at its C-terminal. The fluorescence emission contributed by the fluorophore attached to the C-terminal Cys residue was quenched by the presence of the quencher linked to the N-terminus of the peptide. The synthetic peptide substrate mentioned above was named as qf-Ab(12-18)C. The fluorescence was emitted when this peptide substrate was degraded by enzymes. We observed in our study that four enzymes exhibit activity toward our synthetic peptide substrate qf-Ab(12-18)C, including NEP, ACE, IDE, and ECE-1. Our results showed that the former two have higher activities, whereas the latter two have lower activities, suggesting that qf-Ab(12-18)C lacks a good enzyme specificity. Next, we made an attempt to modify the sequence of the peptide substrate, changing the last two residues from luecine and valine to two alanine. Following the same method mentioned previously, a new peptide substrate was obtained and called qf-Ab(12-16)AAC. Only two enzymes, including NEP and ACE, were found to be effective in hydrolyzing the new substrate qf-Ab(12-16)AAC. It is evident that the new peptide substrate qf-Ab(12-16)AAC possesses a superior enzyme specificity over qf-Ab(12-18)C. Moreover, the cell-based assay was performed in our study. Our previous investigation showed that qf-Ab(1-7)C can be hydrolyzed by only two enzymes, NEP and IDE. However, we found in this work that NEP and ACE are able to hydrolyze qf-Ab(12-16)AAC. We believe the combination of these two different peptide substrates allows us to develop a fluorescence-based platform for detecting the chemicals which can enhance or reduce the activity of important Ab-degarding enzymes. Key words：Amyloid-b, Ab-degrading enzymes, neprilysin, angiotensin-converting enzyme, insulin degrading enzyme, fluorescence resonance energy transfer (FRET).