Mechanismus der Nukleotide-vermittelten Inhibierung mitochondrialer Entkopplerproteine

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.

Zentrum für Biologische Wissenschaften