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用于灵敏且特异性检测两种β-淀粉样蛋白降解酶(中性内肽酶和血管紧张素转换酶)的肽底物设计

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

作者信息

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

机构信息

Institute of Biochemical Sciences, National Taiwan University, Taipei, 10617, Taiwan.

Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.

出版信息

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

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.

摘要

上调中性内肽酶(NEP)以减少大脑中β淀粉样蛋白(Aβ)的积累是预防阿尔茨海默病(AD)的一种有前景的策略。本报告描述了一种淬灭荧光肽底物qf-Aβ(12 - 16)AAC(序列为VHHQKAAC)的设计与合成,其荧光团Alexa - 350连接到C端半胱氨酸的侧链,淬灭剂Dabcyl连接到N端。该肽在切割后发出强烈荧光。我们的结果表明,qf-Aβ(12 - 16)AAC对NEP比先前报道的肽底物更敏感,因此在肽浓度为2 μM时,低至0.03 nM的NEP浓度都能被检测到。此外,qf-Aβ(12 - 16)AAC对NEP和血管紧张素转换酶(ACE)都具有优异的酶特异性,但对其他Aβ降解酶无活性。该肽与先前报道的对NEP和胰岛素降解酶(IDE)敏感的肽底物qf-Aβ(1 - 7)C联合使用,可用于高通量筛选仅上调NEP的化合物。以qf-Aβ(1 - 7)C和qf-Aβ(12 - 16)AAC为底物的基于细胞的活性测定实验结果证实,在神经母细胞瘤细胞中,生长抑素处理最有可能上调IDE,而不是NEP。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ae16/4838334/8f6593d820fb/pone.0153360.g001.jpg

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