Origin of mitochondrial proton leak: comparative investigation of Adenine Nucleotide, Translocase, Phosphate and Aspartat/Glutamate Carriers
Zusammenfassung der Projektergebnisse
The goal of the project was (i) to investigate the specific proton leak in mitochondria mediated by uncoupling proteins, adenine nucleotide translocase (ANT), phosphate (PIC) or oxoglutarate carriers (OGC), and (ii) to reveal which factors may influence proton leak, for example, membrane lipid composition, fatty acids (FA) and/or transmembrane potential. Other research groups published pertinent data subsequent to the start of our present project, which indicated for the first time the presence of phosphoinositolphosphate derivatives (PIP) in mitochondria. We thus shifted the priority of the investigations to (ii) of the project. The involvement of PIPs is a completely new and exciting mechanism that explains the specific protein-mediated proton leak in stem cells. For the investigation of PIPs in artificial bilayers, we used uncoupling protein 2, and not the originally planned proteins, because UCP2 was shown to be abundant in stem cells and was previously characterized by our and other groups. We have demonstrated that PIPs substantially potentiated UCP-mediated membrane conductance, Gm, even in micromolar concentrations in the presence of fatty acids. We hypothesized that Gm enhancement due to the build-up of PIP substrates in tyrosine phosphatase (PTPMT1)-depleted mitochondria critically alters mitochondrial substrate utilization within the stem cell, leading to bioenergetic stress and differentiation block. Interestingly, the magnitude of the effect did not correlate with the PIP charge. This means that the molecular mechanism of the UCP-PIP interaction remained unclear and will be evaluated in a new project. We have furthermore demonstrated that FA alone mediated an essential proton leak, although it was much smaller than the one generated by UCPs in the presence of FAs. Both correlated with the FA saturation degree and chain length. We have shown that Gm is very sensitive to the transmembrane potential and is at its maximum at pH equal to the FA’s pK, similar to the specific proton leak mediated by UCPs. One important additional aspect was that we quantified UCP amounts in different tissues for the first time, using recombinant proteins produced in this project. This was crucial for establishing a functional link between UCPs-mediated proton leak and their function in different tissues. The latter has implications for the treatment of obesity (UCP1), neurodegenerative diseases (UCP4), immunological processes and cancer (UCP2).
Projektbezogene Publikationen (Auswahl)
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(2009). Comparative analysis of uncoupling protein 4 distribution in various tissues under physiological conditions and during development. Biochim. Biophys. Acta 1788, 2309-2319
Smorodchenko, A., Rupprecht, A., Sarilova, I., Ninnemann, O., Brauer, A. U., Franke, K., Schumacher, S., Techritz, S., Nitsch, R., Schuelke, M., and Pohl, E.E.
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(2010). Role of the transmembrane potential in the membrane proton leak. Biophys. J. 98, 1503-1511
Rupprecht, A., Sokolenko, E. A., Beck, V., Ninnemann, O., Jaburek, M., Trimbuch, T., Klishin, S. S., Jezek, P., Skulachev, V. P., and Pohl, E.E.
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(2011). Linking of sensor molecules with amino groups to amino-functionalized AFM tips. Bioconjugate Chem. 22, 1239–1248
Wildling, L., Unterauer, B., Zhu, R., Rupprecht, A., Haselgruebler, T., Rankl, C., Ebner, A., Vater, D., Pollheimer, P., Pohl, E.E., Hinterdorfer, P., Gruber, H. J.
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(2011). Role of mitochondrial uncoupling protein 4 in rat inner ear. Mol. Cell. Neurosci. 47(4), 244-53
Smorodchenko, A., Rupprecht, A., Fuchs, J., Gross, J., Pohl, E.E.
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(2012) Quantification of Uncoupling protein 2 reveals its main expression in immune cells and selective up-regulation during T-cell proliferation. PLoS ONE 7(8): e41406
Rupprecht, A., Bräuer, A.U., Smorodchenko, A., Goyn, J., Hilse K.E., Shabalina I.G., Infante-Duarte, C., Pohl, E.E.
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(2013) A differentiation checkpoint in hematopoietic stem cells activated by bioenergetic stress. Cell Stem Cell 12, 62–74
Yu, W.M, Liu, X., Shen, J.H., Jovanovic, O., Pohl, E.E., Gerson, S.L., Finkel, T., Broxmeyer, H.E., Qu, C.K.
