Functional Characterization of Mitochondrial Uncoupling Protein 2 by EPR Studies

Uncoupling protein1 (UCP1) in brown adipose tissue was discovered earlier as the main uncoupling source of respiration. We describe the basic facts and a modest contribution of our group to the area of research on mitochondrial uncoupling proteins. After defining the terms uncoupling, leak, proton-mediated uncoupling, we discuss the assumption that due to its low abundance, uncoupling protein 2 (UCP2) can provide only mild uncoupling, i.e. can decrease the proton motive force by several mV only. A fatty acid cycling mechanism is described as a plausible explanation for the protonophoretic function of all uncoupling proteins together with our experiments supporting it. A speculation for the phylogenesis of all uncoupling proteins can be deduced by estimated UCP2 content in several tissues, and details of its activation are explained on the basis of our experiments. In the present study a solubilization and refolding method for UCP2 from inclusion bodies was developed and characterized. As it was known and also demonstrated from previous experiments on UCP1 that fatty acids are substrates, we used the same procedure to study the function of UCP2. Utilizing spin-labelled fatty acids (SLFA) for our experiments we demonstrated the binding of fatty acids to UCP2, and the competition of other natural fatty acids like oleic acid, palmitic acid, arachidonic acid and eicosatrienoic acid to the preformed complex emphasizes the presence of a fatty acid binding site for mitochondrial UCP2. The findings were observed by EPR spectroscopy where the highly immobilized spectra with presence of spin-labelled fatty acid eventually end up as free spin label spectra with a particular concentration of the natural fatty acid added to the UCP2 bound with spin-labelled fatty acid. This fits in significantly with the earlier findings of UCP1 and also leads to assumption of functional explanation about the physiological relevance between the uncoupling proteins functions. The present study, in which representative and sensitive parameters for EPR spectroscopy were established, at the same time describes the concentration effects of fatty acids upon the protein bound with spin-labelled fatty acids which are much of importance in comparison to physiological levels, being in the micromolar range (µM) as compared with milli molar (mM) as for UCP1 previously. In appropriate examples, different fatty acids are used and compared with competitors like alkylsulfonates also emphasizing the function of the protein. And the studies with the effect of nucleotides inhibition demonstrate that there exists a putative binding site for fatty acids. Much significance lies in demonstration with the spin-labelled-ATP studies where competition of ATP to the protein bound to spin-labelled ATP explains about the inhibition effect of nucleotides on the UCP2. So the present study applies different methods for the functional characterization of UCP2. The studies of natural fatty acids and alkylsulfonates with UCP2 bound to spin-labelled fatty acid, and study of nucleotide inhibition on UCP2 are closely related and give the much awaited answer to the question of functional similarities between UCP1 and UCP2. This supports the discussion of many groups which predict the functional similarity between these two proteins based upon sequence homology. Also many attempts have been reported in literature to explain the physiological functional relevance where by this present study can also be added to as we now suppose from the present conclusions of our experiments.