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.     

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.     

Uncoupling proteins 2 and 3 interact with members of the 14.3.3 family.

Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms theta, beta, zeta were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the gamma isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 theta could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria.     

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.     

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.     

The conserved regulation of mitochondrial uncoupling proteins: From unicellular eukaryotes to mammals

Uncoupling proteins (UCPs) belong to the mitochondrial anion carrier protein family and mediate regulated proton leak across the inner mitochondrial membrane. Free fatty acids, aldehydes such as hydroxynonenal, and retinoids activate UCPs. However, there are some controversies about the effective action of retinoids and aldehydes alone; thus, only free fatty acids are commonly accepted positive effectors of UCPs. Purine nucleotides such as GTP inhibit UCP-mediated mitochondrial proton leak. In turn, membranous coenzyme Q may play a role as a redox state-dependent metabolic sensor that modulates the complete activation/inhibition of UCPs. Such regulation has been observed for UCPs in microorganisms, plant and animal UCP1 homologues, and UCP1 in mammalian brown adipose tissue. The origin of UCPs is still under debate, but UCP homologues have been identified in all systematic groups of eukaryotes. Despite the differing levels of amino acid/DNA sequence similarities, functional studies in unicellular and multicellular organisms, from amoebae to mammals, suggest that the mechanistic regulation of UCP activity is evolutionarily well conserved. This review focuses on the regulatory feedback loops of UCPs involving free fatty acids, aldehydes, retinoids, purine nucleotides, and coenzyme Q (particularly its reduction level), which may derive from the early stages of evolution as UCP first emerged.     

Role of intrahelical arginine residues in functional properties of uncoupling protein (UCP1)

The functional role of the four intrahelical arginines in uncoupling protein (UCP1) from brown adipose tissue were studied in mutants where they were replaced by noncharged residues. Wild-type and mutant UCP1 were expressed in Saccharomyces cerevisiae. As measured in isolated UCP1, nucleotide binding was largely lost in mutants of R83, R182, and R276 occurring in three repeated domains and common to mitochondrial carrier family, whereas mutation of the UCP typical R91 shows normal binding capacity but > 20-fold lower binding affinity and a near loss of pH dependency of binding. In reconstituted UCP1, fatty acid dependent H(+) transport is retained in all four mutants, but inhibition by nucleotide changes according to the binding ability of UCP1. Cl(-) transport is inhibited only by mutations of arginines in the first domain (R83 and R91). Also in isolated mitochondria H(+) transport and respiration with all four mutants is similar to wt, and inhibition by GDP is found only in R91T. The three "regular" arginines are suggested to influence the nucleotide binding site indirectly via a charge network and the "extra" R91 directly via an ion bond with the previously characterised pH sensor E190. The mutants were also used to assess intrahelical control of UCP1. In the yeast cells expressing UCP1, the aerobic growth could be reduced by fatty acid addition only with the nucleotide insensitive mutants. This demonstrates an intracellular control of UCP1 by nucleotides and fatty acids.     

Structural organization and mutational analysis of the human uncoupling protein-2 (hUCP2) gene

Uncoupling proteins (UCPs) are mitochondrial membrane transporters which are involved in dissipating the proton electrochemical gradient thereby releasing stored energy as heat. This implies a major role of UCPs in energy metabolism and thermogenesis which when deregulated are key risk factors for the development of obesity and other eating disorders. From the three different human UCPs identified so far by gene cloning both UCP2 and UCP3 were mapped in close proximity (75-150 kb) to regions of human chromosome 11 (11q13) that have been linked to obesity and hyperinsulinaemia. At the amino acid level hUCP2 has about 55% identity to hUCP1 while hUCP3 is 71% identical to hUCP2. In this study we have deduced the genomic structure of the human UCP2 gene by PCR and direct sequence analysis. The hUCP2 gene spans over 8.7 kb distributed on 8 exons. The localization of the exon/intron boundaries within the coding region matches precisely that of the hUCP1 gene and is almost conserved in the recently discovered hUCP3 gene as well. The high degree of homology at the nucleotide level and the conservation of the exon /intron boundaries among the three UCP genes suggests that they may have evolved from a common ancestor or are the result from gene duplication events. Mutational analysis of the hUCP2 gene in a cohort of 172 children (aged 7 - 13) of Caucasian origin revealed a polymorphism in exon 4 (C to T transition at position 164 of the cDNA resulting in the substitution of an alanine by a valine at codon 55) and an insertion polymorphism in exon 8. The insertion polymorphism consists of a 45 bp repeat located 150 bp downstream of the stop codon in the 3'-UTR. The allele frequencies were 0.63 and 0.37 for the alanine and valine encoded alleles, respectively, and 0.71 versus 0.29 for the insertion polymorphism. The allele frequencies of both polymorphisms were not significantly elevated in a subgroup of 25 children characterized by low Resting Metabolic Rates (RMR). So far a direct correlation of the observed genotype with (RMR) and Body Mass Index (BMI) was not evident. Expression studies of the wild type and mutant forms of UCP2 should clarify the functional consequences these polymorphisms may have on energy metabolism and body weight regulation.     

Expression,Folding, and Proton Transport Activity of Human Uncoupling Protein-1 (UCP1) in Lipid Membranes: EVIDENCE FOR ASSOCIATED FUNCTIONAL FORMS*

Uncoupling protein-1 (UCP1) is abundantly expressed in the mitochondrial inner membrane of brown adipose tissues and has an important role in heat generation, mediated by its proton transport function. The structure and function of UCP1 are not fully understood, partially due to the difficulty in obtaining native-like folded proteins in vitro. In this study, using the auto-induction method, we have successfully expressed UCP1 in Escherichia coli membranes in high yield. Overexpressed UCP1 in bacterial membranes was extracted using mild detergents and reconstituted into phospholipid bilayers for biochemical studies. UCP1 was folded in octyl glucoside, as indicated by its high helical content and binding to ATP, a known UCP1 proton transport inhibitor. Reconstituted UCP1 in phospholipid vesicles also exhibited highly helical structures and proton transport that is activated by fatty acids and inhibited by purine nucleotides. Self-associated functional forms of UCP1 in lipid membranes were observed for the first time. The self-assembly of UCP1 into tetramers was unambiguously characterized by circular dichroism and fluorescence spectroscopy, analytical ultracentrifugation, and semi-native gel electrophoresis. In addition, the mitochondrial lipid cardiolipin stabilized the structure of associated UCP1 and enhanced the proton transport activity of the protein. The existence of the functional oligomeric states of UCP1 in the lipid membranes has important implications for understanding the structure and proton transport mechanism of this protein in brown adipose tissues as well as structure-function relationships of other mammalian UCPs in other tissues.     

Fatty acid activation of the uncoupling proteins requires the presence of the central matrix loop from UCP1

Noradrenaline signals the initiation of brown fat thermogenesis and the fatty acids liberated by the hormone-stimulated lipolysis act as second messengers to activate the uncoupling protein UCP1. UCP1 is a mitochondrial transporter that catalyses the re-entry of protons to the mitochondrial matrix thus allowing a regulated discharge of the proton gradient. The high affinity of UCP1 for fatty acids is a distinct feature of this uncoupling protein. The uncoupling proteins belong to a protein superfamily formed by the mitochondrial metabolite carriers. Members of this family present a tripartite structure where a domain containing two transmembrane helices, linked by a long hydrophilic loop, is repeated three times. Using protein chimeras, where the repeats had been swapped between UCP1 and UCP3, it has been shown that the central third of UCP1 is necessary and sufficient for the response of the protein to fatty acids. We have extended those studies and in the present report we have generated protein chimeras where different regions of the second repeat of UCP1 have been sequentially replaced with their UCP2 counterparts. The resulting chimeras present a progressive degradation of the characteristic bioenergetic properties of UCP1. We demonstrate that the presence of the second matrix loop is necessary for the high affinity activation of UCP1 by fatty acids.     

Links between fatty acids and expression of UCP2 and UCP3 mRNAs

Physiological and pathological states that are associated with elevated plasma fatty acids (FAs) increase uncoupling protein 2 (UCP2) mRNA in white adipose tissue and UCP3 mRNA in skeletal muscle and heart. A direct effect of unsaturated fatty acids from all classes has been shown in various cultured cells. There is evidence that FAs could induce expression of UCPs by acting as ligands for peroxisome proliferator-activated receptors, influencing the function of sterol responsive element binding protein or activating 5'-AMP-activated protein kinase. Oleic acid has been shown to stimulate the activity of the promoter regions of UCP2 and UCP3 genes and the FA responsive regions are beginning to be characterised.     

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.     

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.     

Canine mitochondrial uncoupling proteins: structure and mRNA expression of three isoforms in adult beagles

Uncoupling proteins (UCPs) are members of the mitochondrial transporter family that dissipate the proton gradient as heat more than via ATP synthesis. In the present study, nucleotide and amino acid sequences of UCPs 1, 2 and 3 of a dog were determined, and their mRNA expression in various peripheral tissues was examined. The sequences were highly (76-97%) homologous to those of other species. Although lower homologies (60-74%) were found when compared among the three canine UCPs, their deduced amino acid sequences had some common domains, such as three mitochondrial carrier protein motifs, six transmembrane alpha-helix domains, and putative purine nucleotide binding domains. By Northern blot analyses, UCP1 mRNA was not detected in any tissues examined. UCP2 mRNA was expressed in most tissues, particularly abundantly in adipose tissue, spleen and lung. Two sizes of UCP3 mRNA were found exclusively in heart and skeletal muscle. These results suggest that canine UCPs have uncoupling activity, and are involved in the regulation of metabolic heat production and/or energy expenditure, as do those of other species.     

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.     

Characterization of the porcine uncoupling proteins 2 and 3 (UCP2 & UCP3) and their localization to chromosome 9 p by somatic cell hybrids

Uncoupling proteins (UCPs) are mitochondrial membrane transporters that are involved in thermogenesis. Heat is generated by dissipation of the proton gradient at the inner mitochondrial membrane, without coupling to any other energy consuming process. A cDNA library from porcine white adipose tissue was screened for clones encoding porcine uncoupling proteins 2 and 3. Ten independent clones were identified and both strands of selected clones were sequenced. Comparison of the sequences with their human homologues revealed an identity of about 87% at the nucleotide level and over 90% at the level of the putative amino acid sequence. Using the INRA hybrid panel, the porcine UCP2 and UCP3 genes were mapped to SSC 9 p21-p24. This localization is consistent with the assignment of human UCP2 and UCP3 to HSA 11q13.     

Uncoupling proteins UCP2 and UCP3 from mitochondria of liver and skeletal muscle of ground squirrel Spermophilus undulatus do not transport pyruvate in contrast to UCP1 from brown adipose tissue

Physiological role of mitochondrial uncoupling proteins UCP2 and UCP3, homologous to UCP1 from brown adipose tissue, is unclear. It was proposed recently that UCP2 and UCP3 are metabolic triggers that switch oxidation of glucose to oxidation of fatty acids, exporting pyruvate from mitochondria. In the present study we tried to verify this hypothesis using ground squirrels (Spermophilus undulatus), since expression of all UCPs in different tissues increases during winter season, and UCP1 is abundant in brown fat. We confirmed the possibility of nonspecific transport of pyruvate through UCP1 in brown fat mitochondria and tried to identify similar transport in liver and skeletal muscle mitochondria where UCP2 and UCP3 are expressed. Transport of pyruvate mediated by UCP1 in mitochondria of brown fat was observed using valinomycin-induced swelling of non-respiring mitochondria in 55 mM potassium pyruvate and was inhibited by GDP. In contrast, mitochondria of liver and skeletal muscles in similar conditions did not exhibit electrogenic transport of pyruvate anions that could be related to functioning of UCP2 and UCP3. At the same time, functioning of pyruvate carrier was detected in these mitochondria by nigericin-induced passive swelling or valinomycin-induced active swelling in potassium pyruvate that was inhibited by α-CHC, a specific inhibitor of the pyruvate carrier. Thus, our results suggest that in contrast to UCP1 of brown fat, UCP2 and UCP3 from intact liver and skeletal muscle mitochondria of winter active ground squirrels are unable to carry out pyruvate transport.