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J Biol Chem. 2017 May 26;292(21):8811-8822. doi: 10.1074/jbc.M116.761718. Epub 2017 Apr 7.

Introducing inducible fluorescent split cholesterol oxidase to mammalian cells.

Author information

1
From the Department of Biochemistry and Developmental Biology and.
2
Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki 00290, Finland.
3
Minerva Foundation Institute for Medical Research, Helsinki 00290, Finland, and.
4
Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki 00290, Finland, elina.ikonen@helsinki.fi.
5
From the Department of Biochemistry and Developmental Biology and vladislav.verkhusha@einstein.yu.edu.
6
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.

Abstract

Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned from Chromobacterium sp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments. The addition of rapamycin reconstituted a fluorescent enzyme, termed split GFP-COase, the fluorescence level of which correlated with its oxidation activity. A rapid decrease of cellular cholesterol induced by intracellular expression of the split GFP-COase promoted the dissociation of a cholesterol biosensor D4H from the plasma membrane. The process was reversible as upon rapamycin removal, the split GFP-COase fluorescence was lost, and cellular cholesterol levels returned to normal. These data demonstrate that the split GFP-COase provides a novel tool to manipulate cholesterol in mammalian cells.

KEYWORDS:

cholesterol; cholesterol metabolism; cholesterol regulation; cholesterol-binding protein; enzyme

PMID:
28391244
PMCID:
PMC5448107
DOI:
10.1074/jbc.M116.761718
[Indexed for MEDLINE]
Free PMC Article

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