Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin.
Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin.
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DOI：
10.3390/mps3040081
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发表时间：
2020-11-24
影响因子：
2.4
通讯作者：
Leissring MA
中科院分区：
文献类型：
--
作者：
Suire CN;Brizuela MK;Leissring MA
胰淀素是一种胰肽激素，可调节葡萄糖稳态，但在 2 型糖尿病中也会聚集形成胰岛淀粉样蛋白。鉴于糊精在健康和疾病中的作用，人们对胰岛素降解酶 (IDE) 和其他蛋白酶对胰淀素的蛋白水解降解产生了新的兴趣。在这里，我们描述了三种新的胰淀素降解测定方法的开发和详细表征，其中两种基于荧光素化和生物素化形式的啮齿类胰淀素（荧光素-啮齿类胰淀素-生物素，FrAB），可用于任何胰淀素蛋白酶，另一种基于内部淬灭的荧光底物（基于 FRET 的胰淀素，FRAM），它对 IDE 更具特异性。基于 FrAB 的底物可用于易于实施的基于荧光的方案或基于荧光偏振 (FP) 的方案，更适合高通量筛选 (HTS)，而 FRAM 底物具有允许连续监测蛋白水解活性的优点。所有三种检测均可产生高度定量的数据，并且耐 DMSO，并且 FRAM 和基于 FP 的 FrAB 检测非常适合 HTS 应用。
Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. The FrAB-based substrate can be used in a readily implemented fluorescence-based protocol or in a fluorescence polarization (FP)-based protocol that is more amenable to high-throughput screening (HTS), whereas the FRAM substrate has the advantage of permitting continuous monitoring of proteolytic activity. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications.