Design of peptide substrate for sensitively and specifically detecting Two Aβ-degrading enzymes: Neprilysin and angiotensin-converting enzyme

Po Ting Chen, Chao Long Chen, Lilian Tsai Wei Lin, Chun Hsien Lo, Chaur Jong Hu, Rita P Y Chen, Steven S S Wang

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2 Citations (Scopus)

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.

Original languageEnglish
Article numbere0153360
JournalPLoS One
Volume11
Issue number4
DOIs
Publication statusPublished - Apr 1 2016

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ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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