title: Imaging and Functional Analysis of γ-Secretase and Substrate in a Proteolipobead System with an Activity-Based Probe.
abstract: 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.
authors.affiliation: Department of Chemical Engineering and the Department of Biomedical Engineering, The City College of the City University of New York , 140th Street and Convent Avenue, New York, New York 10031, United States.
authors.forename: M Lane
authors.initials: ML
authors.lastname: Gilchrist
authors.affiliation: Chemistry Biology Program, Memorial Sloan-Kettering Cancer Center , 1275 York Avenue, New York, New York 10065, United States.
authors.forename: Kwangwook
authors.initials: K
authors.lastname: Ahn
authors.affiliation: Chemistry Biology Program, Memorial Sloan-Kettering Cancer Center , 1275 York Avenue, New York, New York 10065, United States.|Program of Pharmacology, Weill Graduate School of Medical Sciences of Cornell University , New York, New York 10021, United States.
authors.forename: Yue-Ming
authors.initials: YM
authors.lastname: Li
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D003902:Detergents; D008051:Lipid Bilayers; D015335:Molecular Probes; D011510:Proteolipids; C020485:proteoliposomes; D053829:Amyloid Precursor Protein Secretases