Imaging and Functional Analysis of γ-Secretase and Substrate in a Proteolipobead System with an Activity-Based Probe

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• 作者：M. Lane Gilchrist ; Kwangwook Ahn ; Yue-Ming Li
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：January 19, 2016
• 年：2016
• 卷：88
• 期：2
• 页码：1303-1311
• 全文大小：484K
• ISSN：1520-6882

文摘

Investigation of intramembranal protease catalysis demands the generation of intact biomembrane assemblies with structural integrity and lateral mobility. Here, we report the development of a microsphere supported-biomembrane platform enabling characterization of γ-secretase and substrate within proteolipobead assemblies via microscopy and flow cytometry. The active enzyme loading levels were tracked using an activity-based probe, with the biomembranes delineated by carbocyanine lipid reporters. Proteolipobeads formed from HeLa proteoliposomes gave rise to homogeneous distributions of active γ-secretase within supported biomembranes with native-like fluidity. The substrate loading into supported biomembranes was detergent-dependent, as evidenced by even colocalization of substrate and lipid tracers in confocal 3D imaging of individual proteolipobeads. Moreover, the loading level was tunable with bulk substrate concentration. γ-Secretase substrate cleavage and its inhibition within γ-secretase proteolipobeads were observed. This platform offers a means to visualize enzyme and substrate loading, activity, and inhibition in a controllable biomembrane microenvironment.