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.     

Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3

Evidence for the physiological functions of UCP2 and UCP3 is critically reviewed. They do not mediate adaptive thermogenesis, but they may be significantly thermogenic under specific pharmacological conditions. There is strong evidence that the mild regulated uncoupling they cause attenuates mitochondrial ROS production, protects against cellular damage, and diminishes insulin secretion. Evidence that they export fatty acids physiologically is weak. UCP2 and UCP3 are important potential targets for treatment of aging, degenerative diseases, diabetes, and perhaps obesity.     

Reconstitution of novel mitochondrial uncoupling proteins UCP2 and UCP3

Reconstitution of novel mitochondrial uncoupling proteins, human UCP2 and UCP3, expressed in yeast, was performed to characterize fatty acid (FA)-induced H⁺ efflux in the resulted proteoliposomes. We now demonstrate for the first time that representatives of physiologically abundant long chain FAs, saturated or unsaturated, activate H⁺ translocation in UCP2- and UCP3-proteoliposomes. Efficiency of lauric, palmitic or linoleic acid was roughly the same, but oleic acid induced faster H⁺ uniport. We have confirmed that ATP and GTP inhibit such FA-induced H⁺ uniport mediated by UCP2 and UCP3. Coenzyme Q₁₀ did not further significantly activate the observed H⁺ efflux. In conclusion, careful instant reconstitution yields intact functional recombinant proteins, UCP2 and UCP3, the activity of which is comparable with UCP1.     

The reactions catalysed by the mitochondrial uncoupling proteins UCP2 and UCP3

The mitochondrial uncoupling proteins UCP2 and UCP3 may be important in attenuating mitochondrial production of reactive oxygen species, in insulin signalling (UCP2), and perhaps in thermogenesis and other processes. To understand their physiological roles, it is necessary to know what reactions they are able to catalyse. We critically examine the evidence for proton transport and anion transport by UCP2 and UCP3. 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. However, they do not catalyse proton leak in the absence of such acute activation. They can also catalyse export of fatty acid and other anions, although the relationship of anion transport to proton transport remains controversial.     

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.     

Alkylsulfonates activate the uncoupling protein UCP1: implications for the transport mechanism

Fatty acids activate the uncoupling protein UCP1 by a still controversial mechanism. Two models have been put forward where the fatty acid operates as either substrate ("fatty acid cycling hypothesis") or prosthetic group ("proton buffering model"). Two sets of experiments that should help to discriminate between the two hypothetical mechanisms are presented. We show that undecanosulfonate activates UCP1 in respiring mitochondria under conditions identical to those required for the activation by fatty acids. Since alkylsulfonates cannot cross the lipid bilayer, these experiments rule out the fatty acid cycling hypothesis as the mechanism of uncoupling. We also demonstrate that without added nucleotides and upon careful removal of endogenous fatty acids, brown adipose tissue (BAT) mitochondria from cold-adapted hamsters respire at the full uncoupled rate. Addition of nucleotides lower the respiratory rate tenfold. The high activity observed in the absence of the two regulatory ligands is an indication that UCP1 displays an intrinsic proton conductance that is fatty acid-independent. We propose that the fatty acid uncoupling mediated by other members of the mitochondrial transporter family probably involves a carrier to pore transition and therefore has little in common with the activation of UCP1.     

Molecular identification of uncoupling proteins (UCP2 and UCP3) and absence of UCP1 in the marsupial Tasmanian bettong,Bettongia gaimardi

This study has identified the expression of uncoupling proteins in a marsupial using molecular techniques. The Tasmanian bettong, Bettongia gaimardi, increases non-shivering thermogenesis (NST) in response to cold exposure and norepinephrine, although previous studies have been unable to demonstrate the presence of brown adipose tissue or uncoupling protein 1 (UCP1). This study used molecular techniques to confirm the absence of UCP1 as well as ascertain if this species expresses UCP2 and/or UCP3. Tissue samples from four B. gaimardi were taken prior to and post-cold exposure at 4-5 degrees C for 2 weeks. The tissues were then examined for UCP1, UCP2 and UCP3 expression using Western blotting. UCP2 and UCP3 were amplified through RT-PCR and subsequently sequenced to confirm molecular identity. Our work confirms that B. gaimardi does not express UCP1 and that this species expresses both uncoupling proteins 2 and 3. The sequencing of the amplified B. gaimardi UCP2 and UCP3 cDNAs have revealed a 74% homology with rat UCP2 cDNA, and 65% homology with rat UCP3 cDNA. Although this work has not yet characterised the functional properties of these proteins in the marsupial, it does suggest a possible mechanism to explain the existence of NST in B. gaimardi.     

The human uncoupling proteins 5 and 6 (UCP5/SLC25A14 and UCP6/SLC25A30) transport sulfur oxyanions,phosphate and dicarboxylates

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family. In this work, two members of this family, UCP5 (BMCP1, brain mitochondrial carrier protein 1 encoded by SLC25A14) and UCP6 (KMCP1, kidney mitochondrial carrier protein 1 encoded by SLC25A30) have been thoroughly characterized biochemically. They were overexpressed in bacteria, purified and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that UCP5 and UCP6 transport inorganic anions (sulfate, sulfite, thiosulfate and phosphate) and, to a lesser extent, a variety of dicarboxylates (e.g. malonate, malate and citramalate) and, even more so, aspartate and (only UCP5) glutamate and tricarboxylates. Both carriers catalyzed a fast counter-exchange transport and a very low uniport of substrates. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors at various degrees. The transport affinities of UCP5 and UCP6 were higher for sulfate and thiosulfate than for any other substrate, whereas the specific activity of UCP5 was much higher than that of UCP6. It is proposed that a main physiological role of UCP5 and UCP6 is to catalyze the export of sulfite and thiosulfate (the H₂S degradation products) from the mitochondria, thereby modulating the level of the important signal molecule H₂S.     

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.     

Toward understanding the molecular mechanism of a geminivirus C4 protein

Geminiviruses are ssDNA plant viruses that cause significant agricultural losses worldwide. The viruses do not encode a polymerase protein and must reprogram differentiated host cells to re-enter the S-phase of the cell cycle for the virus to gain access to the host-replication machinery for propagation. To date, 3 Beet curly top virus (BCTV) encoded proteins have been shown to restore DNA replication competency: the replication-initiator protein (Rep), the C2 protein, and the C4 protein. Ectopic expression of the BCTV C4 protein leads to a severe developmental phenotype characterized by extensive hyperplasia. We recently demonstrated that C4 interacts with 7 of the 10 members of the Arabidopsis thaliana SHAGGY-like protein kinase gene family and characterized the interactions of C4 and C4 mutants with AtSKs. Herein, we propose a model of how C4 functions.     

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 and phylogenesis--UCP4 as the ancestral uncoupling protein

We searched for the previously defined uncoupling protein (UCP) signatures [Jezek, P. and Urbánková, E. (2000) IUBMB Life 49, 63-70] in genomes of Drosophila melanogaster, Caenorhabditis elegans, Dictyostelium discoideum, and Arabidopsis thaliana. We identified four UCPs in Drosophila and one in Caenorhabditis or Dictyostelium as close relatives of human UCP4 (BMCP), but distant from UCP1, UCP2, UCP3, and two plant UCPs of Arabidopsis. But the third Arabidopsis UCP is the closest UCP4 relative. This suggests that UCP4 represents the ancestral UCP from which other mammalian and plant UCPs diverged. Speculations on UCP4 participation in apoptosis are thus supported by its early phylogenetic occurrence.     

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 mechanism and redox regulation of mitochondrial uncoupling protein 1 (UCP1)

Brown adipose tissue (BAT) and brown in white (brite) adipose tissue, termed also beige adipose tissue, are major sites of mammalian nonshivering thermogenesis. Mitochondrial uncoupling protein 1 (UCP1), specific for these tissues, is the key factor for heat production. Recent molecular aspects of UCP1 structure provide support for the fatty acid cycling model of coupling, i.e. when UCP1 expels fatty acid anions in a uniport mode from the matrix, while uncoupling. Protonophoretic function is ensured by return of the protonated fatty acid to the matrix independent of UCP1. This mechanism is advantageous for mitochondrial uncoupling and compatible with heat production in a pro-thermogenic environment, such as BAT. It must still be verified whether posttranslational modification of UCP1, such as sulfenylation of Cys253, linked to redox activity, promotes UCP1 activity. BAT biogenesis and UCP1 expression, has also been linked to the pro-oxidant state of mitochondria, further endorsing a redox signalling link promoting an establishment of pro-thermogenic state. We discuss circumstances under which promotion of superoxide formation exceeds its attenuation by uncoupling in mitochondria and throughout point out areas of future research into UCP1 function.     

Toward an understanding of structure and function of ion channels

The second half of the 1980s is certain to be considered a turning point in the study of ion channels. Within the last few years, monumental advances in the application of molecular biology, single-channel recording, and direct molecular characterization have been brought to bear on the problem of relating the molecular structure of the ion channel proteins to their function in the cell membrane. Structure-function relationships can now be studied at a level of detail that was unimagined a decade ago. Recently, advances made with the techniques of molecular biology appear to have dominated the literature in this field; however, innovative strategies of structural characterization and electrical measurements of functioning channels in native and artificial membranes continue to break new ground. This paper is a selective review of current progress in understanding structure-function relationships in ion channels. The relative usefulness of determining amino acid sequences of channel proteins together with the resulting deductions about 3-dimensional structure and function will be evaluated with respect to the potential importance of studying the channel molecules more directly by biochemical, immunological, and electrophysiological methods. A full understanding of the details of channel structure and its relationship to function may be realized in the near future as a result of the interdisciplinary application of biophysical, biochemical, and molecular biological techniques.