Design of Peptide Substrate for Sensitively and Specifically Detecting Two A?-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme.

ABSTRACT: Upregulation of neprilysin (NEP) to reduce A? accumulation in the brain is a promising strategy for the prevention of Alzheimer's disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-A?(12-16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-A?(12-16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 ?M. Moreover, qf-A?(12-16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other A?-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-A?(1-7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-A?(1-7)C and qf-A?(12-16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.

SUBMITTER: Chen PT 

PROVIDER: S-EPMC4838334 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature