Imaging and functional analysis of γ-secretase and substrate in a proteolipobead system with an activity-based probe Journal Article
| Authors: | Gilchrist, M. L.; Ahn, K.; Li, Y. M. |
| Article 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. © 2015 American Chemical Society. |
| Keywords: | enzyme inhibition; probes; enzymes; substrates; microenvironments; colocalization; proteoliposomes; activity-based probe; bulk substrates; homogeneous distribution; lateral mobility; substrate loading; loading |
| Journal Title: | Analytical Chemistry |
| Volume: | 88 |
| Issue: | 2 |
| ISSN: | 0003-2700 |
| Publisher: | American Chemical Society |
| Date Published: | 2016-01-19 |
| Start Page: | 1303 |
| End Page: | 1311 |
| Language: | English |
| DOI: | 10.1021/acs.analchem.5b03762 |
| PROVIDER: | scopus |
| PUBMED: | 26699370 |
| PMCID: | PMC4911041 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 3 March 2016 -- Source: Scopus |
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