Suire Caitlin N, Brizuela Monica K, Leissring Malcolm A
Institute for Memory Impairments and Neurological Disorders, University of California, Irvine (UCI MIND), Irvine, CA 92697, USA.
Department of Neurobiology and Behavior, University of California, Irvine, CA 92697, USA.
Methods Protoc. 2020 Nov 24;3(4):81. doi: 10.3390/mps3040081.
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.
胰淀素是一种胰腺肽激素，它调节葡萄糖稳态，但在2型糖尿病中也会聚集形成胰岛淀粉样蛋白。鉴于其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重新产生了兴趣。在这里，我们描述了三种新型胰淀素降解检测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动物胰淀素（荧光素-啮齿动物胰淀素-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（基于荧光共振能量转移的胰淀素，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更适合高通量筛选（HTS）的基于荧光偏振（FP）的方案，而FRAM底物具有允许连续监测蛋白水解活性的优点。所有三种检测方法都能产生高度定量的数据，并且对二甲基亚砜具有抗性，基于FRAM和FP的FrAB检测方法非常适合HTS应用。
