Activity‐Based Protein Profiling of Rhomboid Proteases in Liposomes
Activity‐Based Protein Profiling of Rhomboid Proteases in Liposomes
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DOI：
10.1002/cbic.201500213
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发表时间：
2015-07
期刊：
影响因子：
3.2
通讯作者：
Eliane V. Wolf;M. Seybold;R. Hadravová;K. Stříšovský;S. Verhelst
中科院分区：
文献类型：
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作者：
Eliane V. Wolf;M. Seybold;R. Hadravová;K. Stříšovský;S. Verhelst
虽然基于活性的蛋白质谱（ABPP）已被用于研究各种酶类，但其对膜内蛋白酶的应用仍处于起步阶段。膜内蛋白水解是激活处于休眠状态的膜内蛋白质的重要生化机制。菱形蛋白酶（膜内丝氨酸蛋白酶）嵌入膜的脂质双层中，并出现在所有系统发育域中。目前对菱形蛋白酶的研究主要集中在洗涤剂胶束环境中。在这里，我们报告的菱形在脂质体的重建。使用ABPP，我们已经能够检测到活跃的菱形在大型和巨型单层囊泡。我们已经发现菱形在胶束和脂质体中的抑制剂谱是相似的，从而验证了先前的抑制剂筛选。此外，脂质体上的荧光显微镜实验构成了在膜环境中菱形蛋白酶基于活性成像的第一步。
Although activity‐based protein profiling (ABPP) has been used to study a variety of enzyme classes, its application to intramembrane proteases is still in its infancy. Intramembrane proteolysis is an important biochemical mechanism for activating proteins residing within the membrane in a dormant state. Rhomboid proteases (intramembrane serine proteases) are embedded in the lipid bilayers of membranes and occur in all phylogenetic domains. The study of purified rhomboid proteases has mainly been performed in detergent micelle environments. Here we report on the reconstitution of rhomboids in liposomes. Using ABPP, we have been able to detect active rhomboids in large and giant unilamellar vesicles. We have found that the inhibitor profiles of rhomboids in micelles and liposomes are similar, thus validating previous inhibitor screenings. Moreover, fluorescence microscopy experiments on the liposomes constitute the first steps towards activity‐based imaging of rhomboid proteases in membrane environments.