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Mechanism of nucleotide-mediated inhibition of mitochondrial uncoupling proteins

Abstract
A tight proton transport regulation in the inner mitochondrial membrane is crucial for physiological
processes such as ATP synthesis and heat production as demonstrated for mitochondrial uncoupling
protein 1, UCP1, or regulation of the reactive oxygen species (ROS) as proposed for uncoupling protein 2,
UCP2. Specific regulation of proton transport is thus becoming increasingly important in the therapy of
obesity and of inflammatory, neurodegenerative and ischemic diseases. We and other research groups
have shown previously that UCP1- and UCP2-mediated proton transport is inhibited by purine nucleotides
(PN). However, the molecular mechanism of inhibition is not understood. Moreover, the unresolved
mystery is how UCP operates in vivo despite the permanent presence of high (millimolar) concentrations
of ATP in mitochondria.
The goal of this project is a quantitative characterization of the UCP-PN interactions using (i)
electrophysiological measurements of the transmembrane current and (ii) high-resolution atomic force
microscopy (AFM). Two modes of AFM - topographical and recognition mode (TREC) - will be applied.
The attachment of PN to cantilevers enables identification of selective protein–nucleotide binding
simultaneously to target protein imaging. The data will be compared with electrophysiological
measurements characterizing both PN binding and inhibition. The well-defined model of bilayer
membranes will be employed for the first time for detailed studies of PN binding and inhibition kinetics as
a function of pH, osmolarity and transmembrane potential. Different (tri-, di- and monophosphate) purine
nucleotides will be comparatively investigated. The critical evaluation of the results from both methods in
the light of the recently published NMR structure of UCP2 will provide new insights into the structure of
the uncoupling proteins and mechanism of the protein-nucleotide interaction. The latter will pave the way
for new pharmacological approaches against the diseases mentioned above.
Statistik Austria science classification
106006         Biophysics
301110         Physiology
301114         Cell biology
301303         Medical biochemistry
Lemma
Inhibierung von Entkopplerproteinen
Project leader
Pohl Elena
Duration
01.02.13-31.01.17
Type of Research
Basic research
Vetmed Research Units
Institute of Physiology, Pathohysiology and Biophysics, Unit of Physiology and Biophysics
Funded by
FWF - Fonds zur Förderung der wissenschaftlichen Forschung, Sensengasse 1, 1090 Wien, Austria

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8 Publications

Moschinger, M; Hilse, KE; Rupprecht, A; Zeitz, U; Erben, RG; Rülicke, T; Pohl, EE (2019): Age-related sex differences in the expression of important disease-linked mitochondrial proteins in mice. Biol Sex Differ. 2019; 10(1):56
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Kreiter, J; Rupprecht, A; Zimmermann, L; Moschinger, M; Rokitskaya, TI; Antonenko, YN; Gille, L; Fedorova, M; Pohl, EE (2019): Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins. Biophys J. 2019; 117(10):1845-1857
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Pohl, EE; Rupprecht, A; Macher, G; Hilse, KE (2019): Important Trends in UCP3 Investigation. Front Physiol. 2019; 10:470
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Hilse, KE; Rupprecht, A; Egerbacher, M; Bardakji, S; Zimmermann, L; Wulczyn, AEMS; Pohl, EE (2018): The Expression of Uncoupling Protein 3 Coincides With the Fatty Acid Oxidation Type of Metabolism in Adult Murine Heart. Front Physiol. 2018; 9:747
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Pashkovskaya, AA; Vazdar, M; Zimmermann, L; Jovanovic, O; Pohl, P; Pohl, EE (2018): Mechanism of Long-Chain Free Fatty Acid Protonation at the Membrane-Water Interface. Biophys J. 2018; 114(9):2142-2151
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Macher, G; Koehler, M; Rupprecht, A; Kreiter, J; Hinterdorfer, P; Pohl, EE (2018): Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate. Biochim Biophys Acta. 2018; 1860(3):664-672
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Hilse, KE; Kalinovich, AV; Rupprecht, A; Smorodchenko, A; Zeitz, U; Staniek, K; Erben, RG; Pohl, EE (2016): The expression of UCP3 directly correlates to UCP1 abundance in brown adipose tissue. Biochim Biophys Acta. 2016; 1857(1):72-78
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Zhu, R; Rupprecht, A; Ebner, A; Haselgrübler, T; Gruber, HJ; Hinterdorfer, P; Pohl, EE (2013): Mapping the nucleotide binding site of uncoupling protein 1 using atomic force microscopy. J Am Chem Soc. 2013; 135(9):3640-3646
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