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Biochim Biophys Acta. 2012 Sep;1817(9):1660-70. doi: 10.1016/j.bbabio.2012.05.014. Epub 2012 Jun 4.

Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artefacts and reveals no contribution to basal proton leak.

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1
Institute for Diabetes and Obesity, Helmholtz Zentrum Munich, Neuherberg, Germany. martin.jastroch@helmholtz-muenchen.de

Abstract

Mechanistic studies on uncoupling proteins (UCPs) not only are important to identify their cellular function but also are pivotal to identify potential drug targets to manipulate mitochondrial energy transduction. So far, functional and comparative studies of uncoupling proteins in their native environment are hampered by different mitochondrial, cellular and genetic backgrounds. Artificial systems such as yeast ectopically expressing UCPs or liposomes with reconstituted UCPs were employed to address crucial mechanistic questions but these systems also produced inconsistencies with results from native mitochondria. We here introduce a novel mammalian cell culture system (Human Embryonic Kidney 293 - HEK293) to study UCP1 function. Stably transfected HEK293 cell lines were derived that contain mouse UCP1 at concentrations comparable to tissue mitochondria. In this cell-based test system UCP1 displays native functional behaviour as it can be activated with fatty acids (palmitate) and inhibited with purine nucleotides guanosine-diphosphate (GDP). The catalytic centre activity of the UCP1 homodimer in HEK293 is comparable to activities in brown adipose tissue supporting functionality of UCP1. Importantly, at higher protein levels than in yeast mitochondria, UCP1 in HEK293 cell mitochondria is fully inhibitable and does not contribute to basal proton conductance, thereby emphasizing the requirement of UCP1 activation for therapeutic purposes. These findings and resulting analysis on UCP1 characteristics demonstrate that the mammalian HEK293 cell system is suitable for mechanistic and comparative functional studies on UCPs and provides a non-confounding mitochondrial, cellular and genetic background.

PMID:
22676960
DOI:
10.1016/j.bbabio.2012.05.014
[Indexed for MEDLINE]
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