Avian UCP: The Killjoy in the Evolution of the Mitochondrial Uncoupling Proteins

The understanding of mitochondrial functioning is of prime importance since it combines the production of energy as adenosine triphosphate (ATP) with an efficient chain of redox reactions, but also with the unavoidable production of reactive oxygen species (ROS) involved in aging. Mitochondrial respiration may be uncoupled from ATP synthesis by a proton leak induced by the thermogenic uncoupling protein 1 (UCP1). Mild uncoupling activity, as proposed for UCP2, UCP3, and avian UCP could theoretically control ROS production, but the nature of their transport activities is far from being definitively understood. The recent discovery of a UCP1 gene in fish has balanced the evolutionary view of uncoupling protein history. The thermogenic proton transport of mammalian UCP1 seems now to be a late evolutionary characteristic and the hypothesis that ancestral UCPs may carry other substrates is tempting. Using in silico genome analyses among taxa and a biochemical approach, we present a detailed phylogenetic analysis of UCPs and investigate whether avian UCP is a good candidate for pleiotropic mitochondrial activities, knowing that only one UCP has been characterized in the avian genome, unlike all other vertebrates. We show, here, that the avian class seems to be the only vertebrate lineage lacking two of the UCP1/2/3 homologues present in fish and mammals. We suggest, based on phylogenetic evidence and synteny of the UCP genes, that birds have lost UCP1 and UCP2. The phylogeny also supports the history of two rounds of duplication during vertebrate evolution. The avian uncoupling protein then represents a unique opportunity to explore how UCPs' activities are controlled, but also to understand why birds exhibit such a particular relationship between high metabolism and slow rate of aging.     

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.     

Mitochondrial uncoupling proteins--what is their physiological role?

The physiological functions of the mitochondrial uncoupling proteins (UCP2 and UCP3) are still under debate. There is, however, ample evidence to indicate that, in contrast to UCP1, they are not crucial for nonshivering thermogenesis and do not catalyze the basal proton conductance of mitochondria. Rather, there is good evidence that they increase mitochondrial proton conductance when activated by superoxide, reactive oxygen species derivatives such as hydroxynonenal, and other alkenals or their analogues. This review critically examines the evidence of the different proposed mechanisms for UCPs functions, namely (a) to export fatty acid anions from mitochondria, (b) to regulate insulin secretion in pancreatic beta-cells, and (c) to cause mild uncoupling and so diminish mitochondrial superoxide production, hence protecting against oxidative damage. Beside, available scientific data on UCP4 and UCP5/BMCP1 will be reviewed. However, their physiological function has not yet been established.     

UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged

The relationship between UCP2 and UCP3 expression and mitochondrial proton conductance of rat skeletal muscle was examined. Rats were starved for 24 h and the levels of UCP2 and UCP3 mRNA and UCP3 protein were determined by Northern and Western blots. Proton conductance was measured by titrating mitochondrial respiration rate and membrane potential with malonate. Starvation increased UCP2 and UCP3 mRNA levels more than 5-fold and 4-fold, respectively, and UCP3 protein levels by 2-fold. However, proton conductance remained unchanged. These results suggest either that Northern and Western blots do not reflect the levels of active protein or that these UCPs do not catalyse the basal proton conductance in skeletal muscle mitochondria.     

Alkylsulfonates as probes of uncoupling protein transport mechanism. Ion pair transport demonstrates that direct H(+) translocation by UCP1 is not necessary for uncoupling

The mechanism of fatty acid-dependent uncoupling by mitochondrial uncoupling proteins (UCP) is still in debate. We have hypothesized that the anionic fatty acid head group is translocated by UCP, and the proton is transported electroneutrally in the bilayer by flip-flop of the protonated fatty acid. Alkylsulfonates are useful as probes of the UCP transport mechanism. They are analogues of fatty acids, and they are transported by UCP1, UCP2, and UCP3. We show that undecanesulfonate and laurate are mutually competitive inhibitors, supporting the hypothesis that fatty acid anion is transported by UCP1. Alkylsulfonates cannot be protonated because of their low pK(a), consequently, they cannot catalyze electroneutral proton transport in the bilayer and cannot support uncoupling by UCP. We report for the first time that propranolol forms permeant ion pairs with the alkylsulfonates, thereby removing this restriction. Because a proton is transported with the neutral ion pair, the sulfonate is able to deliver protons across the bilayer, behaving as if it were a fatty acid. When ion pair transport is combined with UCP1, we now observe electrophoretic proton transport and uncoupling of brown adipose tissue mitochondria. These experiments confirm that the proton transport of UCP-mediated uncoupling takes place in the lipid bilayer and not via UCP itself. Thus, UCP1, like other members of its gene family, translocates anions and does not translocate protons.     

Molecular evolution of UCP1 and the evolutionary history of mammalian non-shivering thermogenesis

Background  

Uncoupling protein 1 (UCP1) is a mitochondrial anion carrier, expressed in brown adipose tissue (BAT) of Eutherians. UCP1 is responsible for uncoupling mitochondrial proton transport from the production of ATP, thereby dissipating heat; it is essential for non-shivering thermogenesis (NST) in mammalian BAT. UCP1 orthologs have been identified in non-Eutherian mammals, fish and amphibians. Yet, UCP1 has a unique function in Eutherians in that it is necessary in the production of heat (NST). As such, this study aims to determine the evolutionary mode of UCP1 in Eutherians, where there is clear evidence of UCP1-dependent NST in BAT.     

UCP2, UCP3, avUCP, what do they do when proton transport is not stimulated? Possible relevance to pyruvate and glutamine metabolism

Uncoupling proteins (UCPs) are specialized members of the mitochondrial transporter family. They allow passive proton transport through the mitochondrial inner membrane. This activity leads to uncoupling of mitochondrial respiration and to energy waste, which is well documented with UCP1 in brown adipose tissue. The uncoupling activity of the new UCPs (discovered after 1997), such as UCP2 and UCP3 in mammals or avUCP in birds, is more difficult to characterize. However, extensive data support the idea that the new UCPs are involved in the control of reactive oxygen species (ROS) generation. This fits with the hypothesis that mild uncoupling caused by the UCPs prevents ROS production. Activators and inhibitors regulate the proton transport activity of the UCPs. In the absence of activators of proton transport, the UCP allows the permeation of other ions. We suggest that this activity has physiological significance and, for example, UCP3 expressed in glycolytic muscle fibres may be a passive pyruvate transporter ensuring equilibrium between glycolysis and oxidative phosphorylation. Induction of UCP2 expression by glutamine strengthens the proposal that new UCPs could act to determine the choice of mitochondrial substrate. This would obviously have an impact on mitochondrial bioenergetics and ROS production.     

Hydroperoxy fatty acid cycling mediated by mitochondrial uncoupling protein UCP2

Functional activation of mitochondrial uncoupling protein-2 (UCP2) is proposed to decrease reactive oxygen species production. Skulachev and Goglia (Skulachev, V. P., and Goglia, F. (2003) FASEB J. 17, 1585-1591) hypothesized that hydroperoxy fatty acid anions are translocated by UCPs but cannot flip-flop across the membrane. We found that the second aspect is otherwise; the addition of synthesized linoleic acid hydroperoxides (LAOOH, a mix of four isomers) caused a fast flip-flop-dependent acidification of liposomes, comparable with the linoleic acid (LA)-dependent acidification. Using Escherichia coli-expressed UCP2 reconstituted into liposomes we found that LAOOH induced purine nucleotide-sensitive H(+) uniport in UCP2-proteoliposomes with higher affinity than LA (K(m) values 97 microM for LAOOH and 275 microM for LA). In UCP2-proteoliposomes LAOOH also induced purine nucleotide-sensitive K(+) influx balanced by anionic charge transfer, indicating that LAOOH was also transported as an anion with higher affinity than linoleate anion, the K(m) values being 90 and 350 microM, respectively. These data suggest that hydroperoxy fatty acids are transported via UCP2 by a fatty acid cycling mechanism. This may alternatively explain the observed activation of UCP2 by the externally generated superoxide. The ability of LAOOH to induce UCP2-mediated H(+) uniport points to the essential role of superoxide reaction products, such as hydroperoxyl radical, hydroxyl radical, or peroxynitrite, initiating lipoperoxidation, the released products of which support the UCP2-mediated uncoupling and promote the feedback down-regulation of mitochondrial reactive oxygen species production.     

Uncoupling protein 3 protects aconitase against inactivation in isolated skeletal muscle mitochondria

Mitochondrial uncoupling proteins only catalyse proton transport when they are activated. Activators include superoxide and reactive alkenals, suggesting new physiological functions for UCP2 and UCP3: their activation by superoxide when protonmotive force is high causes mild uncoupling, which lowers protonmotive force and attenuates superoxide generation by the electron transport chain. This feedback loop acts to prevent excessive mitochondrial superoxide production. Superoxide inactivates aconitase in the mitochondrial matrix, so aconitase activity provides a sensitive measure of the effects of UCPs on matrix superoxide. We find that inhibition of UCP3 in isolated skeletal muscle mitochondria by GDP decreases aconitase activity by 25% after 20 min incubation. The GDP effect is absent in skeletal muscle mitochondria from UCP3 knockout mice, showing that it is mediated by UCP3. Protection of aconitase by UCP3 in the absence of nucleotides does not require added fatty acids. The purine nucleoside diphosphates and triphosphates cause aconitase inactivation, but the monophosphates and CDP do not, consistent with the known nucleotide specificity of UCP3. The IC₅₀ for GDP is about 100 μM. These findings support the proposal that UCP3 attenuates endogenous radical production by the mitochondrial electron transport chain at high protonmotive force.     

Uncoupling protein 3 protects aconitase against inactivation in isolated skeletal muscle mitochondria

Mitochondrial uncoupling proteins only catalyse proton transport when they are activated. Activators include superoxide and reactive alkenals, suggesting new physiological functions for UCP2 and UCP3: their activation by superoxide when protonmotive force is high causes mild uncoupling, which lowers protonmotive force and attenuates superoxide generation by the electron transport chain. This feedback loop acts to prevent excessive mitochondrial superoxide production. Superoxide inactivates aconitase in the mitochondrial matrix, so aconitase activity provides a sensitive measure of the effects of UCPs on matrix superoxide. We find that inhibition of UCP3 in isolated skeletal muscle mitochondria by GDP decreases aconitase activity by 25% after 20 min incubation. The GDP effect is absent in skeletal muscle mitochondria from UCP3 knockout mice, showing that it is mediated by UCP3. Protection of aconitase by UCP3 in the absence of nucleotides does not require added fatty acids. The purine nucleoside diphosphates and triphosphates cause aconitase inactivation, but the monophosphates and CDP do not, consistent with the known nucleotide specificity of UCP3. The IC(50) for GDP is about 100 microM. These findings support the proposal that UCP3 attenuates endogenous radical production by the mitochondrial electron transport chain at high protonmotive force.     

Glutathionylation acts as a control switch for uncoupling proteins UCP2 and UCP3

The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactive oxygen species and glutathionylation were found to activate and deactivate UCP3-dependent increases in non-phosphorylating respiration. We identified both Cys(25) and Cys(259) as the major glutathionylation sites on UCP3. Additional experiments in thymocytes from wild-type and UCP2 null mice demonstrated that glutathionylation similarly diminishes non-phosphorylating respiration. Our results illustrate that UCP2- and UCP3-mediated state 4 respiration is controlled by reversible glutathionylation. Altogether, these findings advance our understanding of the roles UCP2 and UCP3 play in modulating metabolic efficiency, cell signaling, and oxidative stress processes.     

Loss of UCP2 Attenuates Mitochondrial Dysfunction without Altering ROS Production and Uncoupling Activity

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.     

Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria

Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.     

Hyperoxia-mediated oxidative stress increases expression of UCP3 mRNA and protein in skeletal muscle

The uncoupling protein-3 (UCP3) is a mitochondrial protein expressed mainly in skeletal muscle. Among several hypotheses for its physiological function, UCP3 has been proposed to prevent excessive production of reactive oxygen species. In the present study, we evaluated the effect of an oxidative stress induced by hyperoxia on UCP3 expression in mouse skeletal muscle and C2C12 myotubes. We found that the hyperoxia-mediated oxidative stress was associated with a 5-fold and 3-fold increase of UCP3 mRNA and protein levels, respectively, in mouse muscle. Hyperoxia also enhanced reactive oxygen species production and UCP3 mRNA expression in C2C12 myotubes. Our findings support the view that both in vivo and in vitro UCP3 may modulate reactive oxygen species production in response to an oxidative stress.     

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.     

Mitochondrial uncoupling,ROS generation and cardioprotection

Mitochondrial oxidative phosphorylation is incompletely coupled, since protons translocated to the intermembrane space by specific respiratory complexes of the electron transport chain can return to the mitochondrial matrix independently of the ATP synthase —a process known as proton leak— generating heat instead of ATP. Proton leak across the inner mitochondrial membrane increases the respiration rate and decreases the electrochemical proton gradient (Δp), and is an important mechanism for energy dissipation that accounts for up to 25% of the basal metabolic rate. It is well established that mitochondrial superoxide production is steeply dependent on Δp in isolated mitochondria and, correspondingly, mitochondrial uncoupling has been identified as a cytoprotective strategy under conditions of oxidative stress, including diabetes, drug-resistance in tumor cells, ischemia-reperfusion (IR) injury or aging. Mitochondrial uncoupling proteins (UCPs) are able to lower the efficiency of oxidative phosphorylation and are involved in the control of mitochondrial reactive oxygen species (ROS) production. There is strong evidence that UCP2 and UCP3, the UCP1 homologues expressed in the heart, protect against mitochondrial oxidative damage by reducing the production of ROS. This review first analyzes the relationship between mitochondrial proton leak and ROS generation, and then focuses on the cardioprotective role of chemical uncoupling and uncoupling mediated by UCPs. This includes their protective effects against cardiac IR, a condition known to increase ROS production, and their roles in modulating cardiovascular risk factors such as obesity, diabetes and atherosclerosis.     

Uncoupling protein 1 affects the yeast mitoproteome and oxygen free radical production

Uncoupling protein 1 (UCP1) is a mitochondrial inner membrane protein that dissipates the proton electrochemical gradient built up by the respiratory chain. Its activity is stimulated by free fatty acids and inhibited by purine nucleotides. Here we investigated how active and regulated recombinant UCP1 expressed in yeast at approximately 1 and approximately 10 microg/mg of total mitochondrial proteins induced changes in the mitochondrial proteome and in oxygen free radical production. Using two-dimensional differential in-gel electrophoresis (2D-DIGE), we found that most of the proteins involved in the response to ectopically expressed UCP1 are related to energy metabolism. We also quantified the cellular H(2)O(2) release in the absence or in the presence of UCP1. Our results suggest that UCP1 has a dual influence on free radical generation. On one side, FFA-activated UCP1 was able to decrease the superoxide anion production, demonstrating that a decrease in the generation of reactive oxygen species is an obligatory outcome of UCP1 activity even in a heterologous context. On the other side, an increase in UCP1 content was concomitant with an increase in the basal release of superoxide anion by mitochondria as a side consequence of the overall increase in oxidative metabolism.     

The mitochondrial uncoupling-protein homologues

Uncoupling protein(UCP)1 is an integral membrane protein that is located in the mitochondrial inner membrane of brown adipocytes. Its physiological role is to mediate a regulated, thermogenic proton leak. UCP2 and UCP3 are recently identified UCP1 homologues. They also mediate regulated proton leak, and might function to control the production of superoxide and other downstream reactive oxygen species. However, their role in normal physiology remains unknown. Recent studies have shown that UCP2 has an important part in the pathogenesis of type-2 diabetes. The obscure roles of the UCP homologues in normal physiology, together with their emerging role in pathophysiology, provide exciting potential for further investigation.     

Physiological regulation of the transport activity in the uncoupling proteins UCP1 and UCP2

Brown fat is a thermogenic organ that allows newborns and small mammals to maintain a stable body temperature when exposed to cold. The heat generation capacity is based on the uncoupling of respiration from ATP synthesis mediated by the uncoupling protein UCP1. The first studies on the properties of these mitochondria revealed that fatty acid removal was an absolute prerequisite for respiratory control. Thus fatty acids, that are substrate for oxidation, were proposed as regulators of respiration. However, their ability to uncouple all types of mitochondria and the demonstration that several mitochondrial carriers catalyze the translocation of the fatty acid anion have made them unlikely candidates for a specific role in brown fat. Nevertheless, data strongly argue for a physiological function. First, fatty acids mimic the noradrenaline effects on adipocytes. Second, there exists a precise correlation between fatty acid sensitivity and the levels of UCP1. Finally, fatty acids increase the conductance by facilitating proton translocation, a mechanism that is distinct from the fatty acid uncoupling mediated by other mitochondrial carriers. The regulation of UCP1 and UCP2 by retinoids and the lack of effects of fatty acids on UCP2 or UCP3 are starting to set differences among the new uncoupling proteins.     

线粒体解偶联蛋白UCP2的研究进展

本文综述了线粒体解偶联蛋白2（uncoupling protein2,UCP2）研究方面的进展。UCP2定位于线粒体内膜上,通过消散线粒体内膜的质子梯度调节线粒体的功能,包括线粒体内膜电位、ATP合成、呼吸链ROS产生、线粒体钙库的存储和释放等。目前,UCP2的质子漏机理并不清楚,但体内实验表明UCP2活性可被过氧化物激活。特别是近年来UCP2调控胰岛素分泌方面的研究取得了重要进展。