Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting EnzymeReport as inadecuate




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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-Aβ12–16AAC 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-Aβ12–16AAC 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-Aβ12–16AAC 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-Aβ1–7C 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-Aβ1–7C and qf-Aβ12–16AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.



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

Source: http://plos.srce.hr/



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