tailieunhanh - Báo cáo Y học: Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells

The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms, UCP3, is abun-dantly expressed in skeletal muscle, the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels inyeast systems,where it leads to theuncouplingof cell respiration, suggesting that UCP3may indeed be capable of dissipating the mitochondrial proton gradient. | Eur. J. Biochem. 269 1373-1381 2002 FEBS 2002 Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells Danilo Guerini1 Elisabetta Prati1 Urvi Desai2 Hans Peter Nick1 Rolf Flammer1 Stephan Griininger1 Frederic Cumin1 Machael Kaleko2 Sheila Connelly2 and Michele Chiesi1 1Metabolic and Cardiovascular Diseases Novartis Pharmaceuticals Ltd Basel Switzerland 2Genetic Therapy Inc. Gaithersburg MD USA The uncoupling proteins UCPs are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms UCP3 is abundantly expressed in skeletal muscle the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels in yeast systems where it leads to the uncoupling of cell respiration suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient. This effect however was recently shown to be a consequence of the high level of expression and incorrect folding of the protein and not to its intrinsic uncoupling activity. In the present study we investigated the properties of UCP3 overexpressed in a relevant mammalian host system such as the rat myoblast L6 cell line. UCP3 was expressed in relatively low levels 1 Ltg-mg 1 membrane protein with the help of an adenovirus vector. Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90 of the cells and that its staining pattern was characteristic for mitochondrial localization. The oxygen consumption of L6 cells under nonphosphorylating conditions increased concomitantly with the levels of UCP3 expression. However uncoupling was associated with an inhibition of the maximal respiratory capacity of mitochondria and was not affected by purine nucleotides and free fatty acids. Moreover recombinant UCP3 was resistant to Triton X-100 extraction under conditions that fully solubilize membrane bound proteins. Thus UCP3 can be uniformly .