Mitochondrial UCPs: new insights into regulation and impact

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.     

Redox State of Endogenous Coenzyme Q Modulates the Inhibition of Linoleic Acid-Induced Uncoupling by Guanosine Triphosphate in Isolated Skeletal Muscle Mitochondria

The skeletal muscle mitochondria contain two isoforms of uncoupling protein, UCP2 and mainly UCP3, which had been shown to be activated by free fatty acids and inhibited by purine nucleotides in reconstituted systems. On the contrary in isolated mitochondria, the protonophoretic action of muscle UCPs had failed to be demonstrated in the absence of superoxide production. We showed here for the first time that muscle UCPs were activated in state 3 respiration by linoleic acid and dissipated energy from oxidative phosphorylation by decreasing the ADP/O ratio. The efficiency of UCPs in mitochondrial uncoupling increased when the state 3 respiratory rate decreased. The inhibition of the linoleic acid-induced uncoupling by a purine nucleotide (GTP), was not observed in state 4 respiration, in uninhibited state 3 respiration, as well as in state 3 respiration inhibited by complex III inhibitors. On the contrary, the progressive inhibition of state 3 respiration by n -butyl malonate, which inhibits the uptake of succinate, led to a full inhibitory effect of GTP. Therefore, as the inhibitory effect of GTP was observed only when the reduced state of coenzyme Q was decreased, we propose that the coenzyme Q redox state could be a metabolic sensor that modulates the purine nucleotide inhibition of FFA-activated UCPs in muscle mitochondria.     

棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白活性及质子漏的影响

本文旨在通过观察棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白(uncoupling proteins,UCPs)活性的影响及脑线粒体质子漏与膜电位的改变,探讨UCPs在介导游离脂肪酸对低氧时线粒体氧化磷酸化功能改变中的作用.将SpragueDawley大鼠随机分为对照组、急性低氧组和慢性低氧组.低氧大鼠于低压舱内模拟海拔5 000 m高原23 h/d作低氧暴露,分别连续低氧3 d和30 d.用差速密度梯度离心法提取脑线粒体,[3H-GTP法测定UCPs含量与活性,TPMP 电极与Clark氧电极结合法测量线粒体质子漏,罗丹明123荧光法测定线粒体膜电位.结果显示,低氧使脑线粒体内UCPs含量与活性升高、质子漏增加、线粒体膜电位降低;同时,低氧暴露降低脑线粒体对棕榈酸的反应性,UCPs活性的改变率低于对照组,且线粒体UCPs含量、质子漏、膜电位变化率亦出现相同趋势.线粒体质子漏与反映UCPs活性的Kd值呈线性负相关(P<0.01 r=-0.906),与反映UCPs含量的Bmax呈线性正相关(P<0.01,r=0.856),与膜电位呈线性负相关(P<0.01,r=-0.880).以上结果提示,低氧导致的脑线粒体质子漏增加及膜电位降低与线粒体内UCPs活性升高有关,同时低氧暴露能降低脑线粒体对棕榈酸的反应性,提示在高原低氧环境下,游离脂肪酸升高在维持线粒体能量代谢中起着自身保护和调节机制.     

Reconstitution of recombinant uncoupling proteins: UCP1, -2, and -3 have similar affinities for ATP and are unaffected by coenzyme Q10

The successful development of recombinant expression and reconstitution protocols has enabled a detailed study of the transport properties and regulation of the uncoupling proteins (UCP). We optimized conditions of isolation and refolding of bacterially expressed uncoupling proteins and reexamined the transport properties and regulation of bacterially expressed UCP1, -2, and -3 reconstituted in liposomes. We show for the first time that ATP inhibits UCP1, -2, and -3 with similar affinities. The Ki values for ATP inhibition were 50 microm (UCP1), 70 microm (UCP2), and 120 microm (UCP3) at pH 7.2. These affinities for ATP are similar to those obtained with native UCP1 isolated from brown adipose tissue mitochondria (Ki = 65 microm at pH 7.2). The Vmax values for proton transport were also similar among the UCPs, ranging from 8 to 20 micromol.min(-1).mg(-1), depending on experimental conditions. We also examined the effect of coenzyme Q on fatty acid-catalyzed proton flux in liposomes containing recombinant UCP1, -2, and -3. We found that coenzyme Q had no effect on the fatty acid-dependent proton transport catalyzed by any of the UCPs nor did it affect nucleotide regulation of the UCPs. We conclude that coenzyme Q is not a cofactor of UCP-mediated proton transport.     

Respiration under control of uncoupling proteins: Clinical perspective

The term 'uncoupling protein' was originally used for the mitochondrial membrane protein UCP1, which is uniquely present in mitochondria of brown adipocytes, thermogenic cells that regulate body temperature in small rodents, hibernators and mammalian newborns. In these cells, UCP1 acts as a proton carrier activated by free fatty acids and creates a shunt between complexes of the respiratory chain and ATP-synthase resulting in a futile proton cycling and dissipation of oxidation energy as heat. Recent identification of new homologues to UCP1 expressed in brown and white adipose tissue, muscle, brain and other tissues together with the hypothesis that these novel uncoupling proteins (UCPs) may regulate thermogenesis and/or fatty acid metabolism and furthermore may protect against free radical oxygen species production have generated considerable optimism for rapid advances in the identification of new targets for pharmacological management of complex pathological syndromes such as obesity, type 2 diabetes or chronic inflammatory diseases. However, since the physiological and biochemical roles of the novel UCPs are not yet clear, the main challenge today consists first of all in providing mechanistic explanation for their functions in cellular physiology. This lively awaited information may be the basis for potential pharmacological targeting of the UCPs in future.     

Mitochondrial uncoupling proteins: regulation and physiological role

Uncoupling proteins (UCPs), members of mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. UCPs can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis. A physiological function of the first described UCP, UCP1 or termogenin, present in mitochondria of mammalian brown adipose tissues is well established. UCP1 plays a role in nonshivering thermogenesis in mammals. The widespread presence of UCPs in eukaryotes, in non-thermogenic tissues of animals, plants and in unicellular organisms implies that these proteins may elicit other functions than thermogenesis. However, the physiological functions of UCP1 homologues are still under debate. They can regulate energy metabolism through modulation of the electrochemical proton gradient and production of ROS. Functional activation of UCPs is proposed to decrease ROS production. Moreover, products of lipid peroxidation can activate UCPs and promote feedback down-regulation of mitochondrial ROS production.     

Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.

Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C-terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1-198) and hUCP2 (amino acids 211-309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1-210) and mUCP1 (amino acids 199-307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide-binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide-binding site is present in the C-terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N-terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C-terminal domain on nucleotide binding, they also demonstrate that interactions with the N-terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold-induced thermogenesis.     

Evidence for Involvement of Uncoupling Proteins in Cerebral Mitochondrial Oxidative Phosphorylation Deficiency of Rats Exposed to 5,000 m High Altitude

The present study aimed to investigate the change of proton leak and discuss the role of cerebral uncoupling proteins (UCPs) and its regulatory molecules non-esterified fatty acid (NEFA) in high altitude mitochondrial oxidative phosphorylation deficiency. The model group animals were exposed to acute high altitude hypoxia, and the mitochondrial respiration, protein leak, UCPs abundance/activity and cerebral NEFA concentration were measured. We found that in the model group, cerebral mitochondrial oxidative phosphorylation was severely impaired with decreased ST3 respiration rate and ATP pool. Proton leak kinetics curves demonstrated an increase in proton leak; GTP binding assay pointed out that total cerebral UCPs activity significantly increased; Q-PCR and western blot showed upregulated expression of UCP4 and UCP5. Moreover, cerebral NEFA concentration increased. In conclusion, UCPs mediated proton leak is closely related to cerebral mitochondria oxidative phosphorylation deficiency during acute high altitude hypoxia and NEFA is involved in this signaling pathway.     

A sequence related to a DNA recognition element is essential for the inhibition by nucleotides of proton transport through the mitochondrial uncoupling protein.

The uncoupling protein (UCP) is uniquely expressed in brown adipose tissue, which is a thermogenic organ of mammals. The UCP uncouples mitochondrial respiration from ATP production by introducing a proton conducting pathway through the mitochondrial inner membrane. The activity of the UCP is regulated: nucleotide binding to the UCP inhibits proton conductance whereas free fatty acids increase it. The similarities between the UCP, the ADP/ATP carrier and the DNA recognition element found in the DNA binding domain of the estrogen receptor suggested that these proteins could share common features in their respective interactions with free nucleotides or DNA, and thus defined a putative 'nucleotide recognition element' in the UCP. This article provides demonstration of the validity of this hypothesis. The putative nucleotide recognition element corresponding to the amino acids 261-269 of the UCP was gradually destroyed, and these mutant proteins were expressed in yeast. Flow cytometry, measuring the mitochondrial membrane potential in vivo, showed increased uncoupling activities of these mutant proteins, and was corroborated with studies with isolated mitochondria. The deletion of the three amino acids Phe267, Lys268 and Gly269, resulted in a mutant where proton leak could be activated by fatty acids but not inhibited by nucleotides.     

Mitochondrial uncoupling proteins in unicellular eukaryotes

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH₂) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species.     

Fatty Acids Change the Conformation of Uncoupling Protein 1 (UCP1)

UCP1 catalyzes proton leak across the mitochondrial inner membrane to disengage substrate oxidation from ATP production. It is well established that UCP1 is activated by fatty acids and inhibited by purine nucleotides, but precisely how this regulation occurs remains unsettled. Although fatty acids can competitively overcome nucleotide inhibition in functional assays, fatty acids have little effect on purine nucleotide binding. Here, we present the first demonstration that fatty acids induce a conformational change in UCP1. Palmitate dramatically changed the binding kinetics of 2′/3′-O-(N-methylanthraniloyl)-GDP, a fluorescently labeled nucleotide analog, for UCP1. Furthermore, palmitate accelerated the rate of enzymatic proteolysis of UCP1. The altered kinetics of both processes indicate that fatty acids change the conformation of UCP1, reconciling the apparent discrepancy between existing functional and ligand binding data. Our results provide a framework for how fatty acids and nucleotides compete to regulate the activity of UCP1.     

4-hydroxy-2-nonenal and uncoupling proteins: an approach for regulation of mitochondrial ROS production

One factor that has the potential to regulate reactive oxygen species (ROS) generation is the mild uncoupling of oxidative phosphorylation, i.e. proton (H(+)) leak across the mitochondrial inner membrane. Proton leak has been shown to attenuate ROS generation, whereas ROS and their derivatives (such as superoxide and hydroxynonenal) have been shown to induce H(+) leak through uncoupling proteins (UCPs). This suggests the existence of a feedback loop between ROS and H(+) leak mediated through UCPs. Although the physiological functions of the new UCPs, such as UCP2 and UCP3, are still not established, extensive data support the idea that these mitochondrial carrier proteins are involved in the control of ROS generation. The molecular basis of both ROS generation and hydroxynonenal-induced uncoupling through UCPs is reviewed and the consequences of their interaction for protection against excessive ROS production at the expense of energy production is discussed.     

Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate

Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by purine nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.     

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system

Mitochondrial inner membrane uncoupling proteins (UCPs) facilitate transmembrane (TM) proton flux and consequently reduce the membrane potential and ATP production. It has been proposed that the three neuronal human UCPs (UCP2, UCP4 and UCP5) in the central nervous system (CNS) play significant roles in reducing cellular oxidative stress. However, the structure and ion transport mechanism of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1 in bacterial membranes and applied this system to obtain highly pure neuronal UCPs in high yields. In the present study, we report on the structure and function of the three neuronal UCP homologues. Reconstituted neuronal UCPs were dominantly helical in lipid membranes and transported protons in the presence of physiologically-relevant fatty acid (FA) activators. Under similar conditions, all neuronal UCPs also exhibited chloride transport activities that were partially inhibited by FAs. CD, fluorescence and MS measurements and semi-native gel electrophoresis collectively suggest that the reconstituted proteins self-associate in the lipid membranes. Based on SDS titration experiments and other evidence, a general molecular model for the monomeric, dimeric and tetrameric functional forms of UCPs in lipid membranes is proposed. In addition to their shared structural and ion transport features, neuronal UCPs differ in their conformations and proton transport activities (and possibly mechanism) in the presence of different FA activators. The differences in FA-activated UCP-mediated proton transport could serve as an essential factor in understanding and differentiating the physiological roles of UCP homologues in the CNS.     

Evolution of mitochondrial uncoupling proteins: novel invertebrate UCP homologues suggest early evolutionary divergence of the UCP family

Current hypothesis about the evolution of uncoupling proteins (UCPs) proposed by suggests that UCP4 is the earliest form of UCP ancestral to all other UCP orthologues. However, this hypothesis is difficult to reconcile with a narrow tissue distribution of UCP4 (which is a brain-specific isoform), suggesting highly specialized rather than anfcestral function for this protein. We searched for UCP2, UCP3, and UCP5 homologues in invertebrate genomes using amplification with degenerate primers designed against UCP2-specific conserved sequences and/or BLASTP search with stringent ad hoc criteria to distinguish between homologues and orthologues of different UCPs. Our study identified invertebrate UCP homologues similar to UCP2 and 3 (which we termed UCP6) and an invertebrate homologue of UCP5. Phylogenetic analysis indicates that there are at least three clades of UCPs in invertebrates, which are closely related to vertebrate UCP1-3, UCP4, and UCP5, respectively, and shows early evolutionary divergence of UCPs, which pre-dates the divergence of protostomes and deuterostomes. It also suggests that the newly identified UCP6 proteins from invertebrates are ancestral to the vertebrate UCP1, UCP2, and UCP3, and that divergence of these three vertebrate orthologues occurred late in evolution of the vertebrates. This study refutes the hypothesis of Hanak and Jezek (2001) that UCP4 is an ancestral form for all UCPs, and shows early evolutionary diversification of this protein family, which corresponds to their proposed functional diversity in regulation of proton leak, antioxidant defense and apoptosis.     

Mitochondrial uncoupling proteins--facts and fantasies

Instead of a comprehensive review, we describe the basic undisputed facts and a modest contribution of our group to the fascinating area of the research on mitochondrial uncoupling proteins. After defining the terms uncoupling, leak, protein-mediated uncoupling, we discuss the assumption that due to their low abundance the novel mitochondrial uncoupling proteins (UCP2 to UCP5) can provide only a mild uncoupling, i.e. can decrease the proton motive force by several mV only. Contrary to this, the highly thermogenic role of UCP1 in brown adipose tissue is not given only by its high content (approximately 5 % of mitochondrial proteins) but also by the low ATP synthase content and high capacity respiratory chain. Fatty acid cycling mechanism as a plausible explanation for the protonophoretic function of all UCPs and some other mitochondrial carriers is described together with the experiments supporting it. The phylogenesis of all UCPs, estimated UCP2 content in several tissues, and details of UCP2 activation are described on the basis of our experiments. Functional activation of UCP2 is proposed to decrease reactive oxygen species (ROS) production. Moreover, reaction products of lipoperoxidation such as cleaved hydroperoxy-fatty acids and hydroxy-fatty acid can activate UCP2 and promote feedback down-regulation of mitochondrial ROS production.     

Toward understanding the mechanism of ion transport activity of neuronal uncoupling proteins UCP2, UCP4, and UCP5

Neuronal uncoupling proteins (UCP2, UCP4, and UCP5) have crucial roles in the function and protection of the central nervous system (CNS). Extensive biochemical studies of UCP2 have provided ample evidence of its participation in proton and anion transport. To date, functional studies of UCP4 and UCP5 are scarce. In this study, we show for the first time that, despite a low level of amino acid sequence identity with the previously characterized UCPs (UCP1-UCP3), UCP4 and UCP5 share their functional properties. Recombinantly expressed in Escherichia coli, UCP2, UCP4, and UCP5 were isolated and reconstituted into liposome systems, where their conformations and ion (proton and chloride) transport properties were examined. All three neuronal UCPs are able to transport protons across lipid membranes with characteristics similar to those of the archetypal protein UCP1, which is activated by fatty acids and inhibited by purine nucleotides. Neuronal UCPs also exhibit transmembrane chloride transport activity. Circular dichroism spectroscopy shows that these three transporters exist in different conformations. In addition, their structures and functions are differentially modulated by the mitochondrial lipid cardiolipin. In total, this study supports the existence of general conformational and ion transport features in neuronal UCPs. On the other hand, it also emphasizes the subtle structural and functional differences between UCPs that could distinguish their physiological roles. Differentiation between structure-function relationships of neuronal UCPs is essential for understanding their physiological functions in the CNS.