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PLoS One. 2016 Apr 20;11(4):e0153360. doi: 10.1371/journal.pone.0153360. eCollection 2016.

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

Author information

1
Institute of Biochemical Sciences, National Taiwan University, Taipei, 10617, Taiwan.
2
Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.
3
Department of Chemical Engineering, National Taiwan University, Taipei, 10617, Taiwan.
4
Department of Neurology, Shuang-Ho Hospital, Taipei Medical University, Taipei, 110, Taiwan.

Abstract

Upregulation of neprilysin (NEP) to reduce Aβ 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-Aβ(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-Aβ(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 μM. Moreover, qf-Aβ(12-16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(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-Aβ(1-7)C and qf-Aβ(12-16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.

PMID:
27096746
PMCID:
PMC4838334
DOI:
10.1371/journal.pone.0153360
[Indexed for MEDLINE]
Free PMC Article

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