Conference Agenda

Uncoupling protein 1 (UCP1) is a mitochondrial carrier protein that catalyses proton leak in brown adipose tissue mitochondria to release energy as heat for thermoregulation, and may help combat metabolic disease if targeted for activation in humans. The protein is activated by free fatty acids and inhibited by cytosolic purine nucleotides via unresolved molecular processes. Importantly, we have recently resolved the first high resolution cryo-electron microscopy structure of human UCP1, with bound GTP, putting in place a valuable structural context [1]. UCP1 is monomeric with a typical carrier fold of SLC25 members, binding three cardiolipin molecules. Our analysis reveals in detail how purine nucleotides interact in the central cavity of UCP1 in a pH-dependent manner to lock the protein in a proton impermeable state. Notably, recent ATP-bound, nucleotide-free and dinitrophenol-bound structures, reported by others [2] retain a similar proton impermeable arrangement, showing that the activation mechanism has not been resolved. Our evaluation of structural and biochemical data suggests that UCP1 activation by fatty acids utilises dynamic state changes within a common carrier transport process to facilitate proton leak.

[1] S.A. Jones, P. Gogoi, J.J. Ruprecht, M.S. King, Y. Lee, T. Zögg, E. Pardon , D. Chand, S. Steimle, D.M. Copeman, C.A. Cotrim, J. Steyaert, P.G. Crichton, V. Moiseenkova-Bell V, E.R.S. Kunji. Structural basis of purine nucleotide inhibition of human uncoupling protein 1. Sci Adv. 9 (2023) eadh4251.

[2] Y. Kang, L. Chen. Structural basis for the binding of DNP and purine nucleotides onto UCP1. Nature 620 (2023) 226-231.

UCP1 was the first so-called uncoupling protein to be identified and is probably the only physiologically functional uncoupling protein. The acute regulation of UCP1 activity remains an important molecular and physiological issue. UCP1 activity is generally believed to be inhibited physiologically by purine nucleotides and activated by fatty acids released through lipolysis. However, at least experimentally, other substances – not normally considered as conventional fatty acids – such as retinoic acid, PFOA, TTNBP and ibuprofen, have also been shown to be able to function as UCP1 activators. Recently, in detailed patch-clamp studies of mitoplasts, traditional uncouplers, such as FCCP and DNP, have been suggested to mediate (some of) their uncoupling effects through activation of members of the mitochondrial carrier family, such as ANT and UCP1. We have examined whether these patch-clamp observations are transferable to studies of UCP1 activity in classical bioenergetic experiments of the control of thermogenesis (oxygen consumption) in isolated brown-fat mitochondria. We found that a direct involvement of UCP1 in the uncoupling action of FCCP and DNP could not be observed under these circumstances; these results thus underscore that different experimental systems may contribute complementarily to the understanding of UCP1 action. – For translational studies, it is important to examine whether human and mouse UCP1 are similarly regulated and whether the uncoupling activity is fully confined to the protein itself or whether the specific environment of the brown adipose tissue is necessary to allow the protein to display full activity; this was examined through ectopic UCP1 expression. Physiologically, the issue still remains as to whether UCP1 is the only protein able to mediate both classical nonshivering thermogenesis and diet-induced thermogenesis, in mice and in humans.

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat metabolic syndrome. Thermogenic tissues such as brown and beige fats develop highly specialized mitochondria for heat production. Mitochondria of other tissues, which primarily produce ATP, also convert up to 25% of the total mitochondrial energy production into heat and can, therefore, have a considerable impact on the physiology of the whole body. Mitochondrial thermogenesis is not only essential for maintaining the body temperature, but also prevents diet-induced obesity and reduces the production of reactive oxygen species (ROS) to protect cells from oxidative damage. Since mitochondrial thermogenesis is a key regulator of cellular metabolism, a mechanistic understanding of this fundamental process will help in the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction. Importantly, the precise molecular mechanisms that control acute activation of thermogenesis in mitochondria are poorly defined. This lack of information is largely due to a dearth of methods for the direct measurement of uncoupling proteins. The recent development of patch-clamp methodology applied to mitochondria enabled, for the first time, the direct study of the phenomenon at the origin of mitochondrial thermogenesis, H+ leak through the IMM, and the first biophysical characterization of mitochondrial transporters responsible for it, the uncoupling protein 1 (UCP1), specific of brown and beige fats, and the ADP/ATP transporter (AAC) for all other tissues. This unique approach will provide new insights into the mechanisms that control H+ leak and mitochondrial thermogenesis in general and how they can be targeted to combat metabolic syndrome.

Several proteins of the SLC25 family (ATP/ADP carrier, oxoglutarate carrier, phosphate carrier, dicarboxylate carrier) transport protons across the inner mitochondrial membrane in the presence of uncouplers similar to UCP1, a key player in the non-shivering thermogenesis. The significance and the molecular mechanism are unknown. I will discuss putative mechanisms that describe proteins as either proton carriers that function in the presence of long-chain fatty acids (FA), or FA anion transporters (fatty acid cycling mechanism). A four-step mechanism for the “sliding” of the FA anion from the matrix to the mitochondrial intermembrane space as a part of the FA cycling mechanism will be presented. Understanding proton transport in mitochondria could lead to these proteins being used as drug targets for the treatment of obesity, cardiovascular and neurodegenerative diseases.

Uncoupling proteins 2 and 3 (UCP2 and UCP3) are the closest homologs of UCP1, the canonical uncoupler responsible for non-shivering thermogenesis in mammals. UCP2 and UCP3 share approximately 70% of their protein sequences. When expressed in bacteria and reconstituted into liposomes, these proteins transport nearly identical substrates such as aspartate, malate, oxaloacetate, phosphate, and sulfate. However, they differ in their transport mode and kinetic constants. UCP3 functions strictly as an exchanger, whereas UCP2 can transport substrates unidirectionally. The substrate affinities of UCP3 for most transported substrates are similar to those of UCP2, except for aspartate, which is 7-8 times higher. Swelling experiments conducted on mitochondria from yeast expressing UCP2 or UCP3 demonstrated that UCP2 catalyzes a proton-coupled symport of phosphate, whereas UCP3 exchanges aspartate for malate plus a proton. This latter exchange reaction has also been confirmed by complementation studies carried out in a yeast cell model lacking the aspartate-malate shuttle and grown on oleate. In the same yeast model, UCP2 was able to catalyze only the exchange of aspartate for phosphate plus a proton. Both proteins were unable to catalyze malate/oxaloacetate reaction. The complementation experiments also elucidated the directionality of the exchange reactions, revealing that aspartate is the substrate exported out of mitochondria. Although both proteins catalyze the export of aspartate from mitochondria, that of UCP2 leads to a reduction of four-carbon metabolites in the matrix which, lowers the oxidation of acetyl-CoA-producing carbon sources such as glucose or fatty acids, and increases that of glutamine. In pancreatic ductal adenocarcinoma, UCP2 catalyzes the export of glutamine-derived aspartate out of mitochondria, which cells use to produce NADPH in the cytosol, crucial for controlling ROS levels and proliferating. On the other hand, although an aspartate/malate exchange activity has been suggested in skeletal muscle for managing amino acid metabolism, further studies are necessary to dissect the physiological function of UCP3 in this tissue.