Mechanisms of nucleotide-mediated inhibition of mitochondrial uncoupling proteins

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 proteinnucleotide 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.

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 millimolar concentrations of ATP in mitochondria. The goal of this project was a quantitative characterization of the UCP-PN interactions using (i) electrophysiological measurements of the transmembrane current and (ii) high-resolution atomic force microscopy (AFM). We used the well-defined model of bilayer membranes for detailed studies of PN binding and inhibition kinetics in the presence of different (tri-, di- and monophosphate) purine nucleotides. The critical evaluation of the results from both methods revealed that despite the high homology the inhibition mechanisms of UCP1 and UCP in the presence of PN are different. We updated the existing mechanism of UCP1 inhibition and for the first time proposed a mechanism of UCP3 inhibition by PN. We depicted phosphate as a novel and significant inhibitor of UCPs, acting independently from PNs. To perform direct measurements of UCP-PN bond lifetime we developed a new method which combined recognition and force spectroscopy to track and to measure the binding forces of low amounts of protein. Our results 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.