Martin Jabůrek

Transport Function and Regulation of Mitochondrial Uncoupling Proteins 2 and 3

Uncoupling protein 1 (UCP1) dissipates energy and generates heat by catalyzing back-flux of protons into the mitochondrial matrix, probably by a fatty acid cycling mechanism. If the newly discovered UCP2 and UCP3 function similarly, they will enhance peripheral energy expenditure and are potential molecular targets for the treatment of obesity. We expressed UCP2 and UCP3 inEscherichia coli and reconstituted the detergent-extracted proteins into liposomes. Ion flux studies show that purified UCP2 and UCP3 behave identically to UCP1. They catalyze electrophoretic flux of protons and alkylsulfonates, and proton flux exhibits an obligatory requirement for fatty acids. Proton flux is inhibited by purine nucleotides but with much lower affinity than observed with UCP1. These findings are consistent with the hypothesis that UCP2 and UCP3 behave as uncoupling proteins in the cell.

The mechanism of proton transport mediated by mitochondrial uncoupling proteins

The effort to understand the mechanism of uncoupling by UCP has devolved into two models – the fatty acid protonophore model and the proton buffering model. Evidence for each hypothesis is summarized and evaluated. We also evaluate the obligatory requirement for fatty acids in UCP1‐mediated uncoupling and the question of fatty acid affinity for UCP1. The structural bases of UCP transport function and nucleotide inhibition are discussed in light of recent mutagenesis studies and in relationship to the sequences of newly discovered UCPs.

How do uncoupling proteins uncouple

According to the proton buffering model, introduced by Klingenberg, UCP1 conducts protons through a hydrophilic pathway lined with fatty acid head groups that buffer the protons as they move across the membrane. According to the fatty acid protonophore model, introduced by Garlid, UCPs do not conduct protons at all. Rather, like all members of this gene family, they are anion carriers. A variety of anions are transported, but the physiological substrates are fatty acid (FA) anions. Because the carboxylate head group is translocated by UCP, and because the protonated FA rapidly diffuses across the membrane, this mechanism permits FA to behave as regulated cycling protonophores. Favoring the latter mechanism is the fact that the head group of long-chain alkylsulfonates, strong acid analogues of FA, is also translocated by UCP.

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 (Km values 97 μm for LAOOH and 275 μm 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 Km values being 90 and 350 μm, 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.

Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers

Uncoupling proteins 1 (UCP1) and 2 (UCP2) belong to the family of mitochondrial anion transporters and share 59% sequence identity with each other. Whereas UCP1 was shown to be responsible for the rapid production of heat in brown adipose tissue, the primary function and transport properties of ubiquitously expressed UCP2 are controversially discussed. Here, for the first time, the activation pattern of the recombinant human UCP2 in comparison to the recom‐binant human UCP1 are studied using a well‐defined system of planar lipid bilayers. It is shown that despite apparently different physiological functions, hUCP2 exhibited its protonophoric function similar to hUCP1—exclusively in the presence of long‐chain fatty acids (FA). The calculated hUCP2 transport rate of 4.5 s−1 is the same order of magnitude, as shown previously for UCP1. It leads to the conclusion that the differences in the activity of both proteins in living mitochondria are based exclusively on their different expression level. Both proteins are activated much more effectively by polyunsaturated than by saturated FA. The proton and total membrane conductances increased in the range palmitic < oleic < eicosatrie‐noic < linoleic < retinoic < arachidonic acids. The higher uncoupling protein (UCP)—dependent conductance in the presence of polyunsaturated FA is explained on the basis of the FA cycling hypothesis.—Beck, V., Jabůrek, M., Demina, T., Rupprecht, A., Porter, R. K., Ježek, P., Pohl, E. E. Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers. FASEB J. 21, 1137–1144 (2007)

The nucleotide regulatory sites on the mitochondrial KATP channel face the cytosol.

The mitochondrial KATP channel (mitoKATP) is richly endowed with regulatory sites for metabolites and drugs, but the topological location of these sites is unknown. Thus, it is not known whether ATP, GTP and acyl CoA esters regulate mitoKATP from the matrix or cytosolic side of the inner membrane, nor whether they all act from the same side. The experiments reported in this paper provide an unambiguous answer to these questions. Electrophysiological experiments in bilayer membranes containing purified mitoKATP showed that current is blocked asymmetrically by ATP. K+ flux experiments using proteoliposomes containing purified mitoKATP showed that mitoKATP is unipolar with respect to regulation by Mg2+, ATP, GTP, and palmitoyl CoA and that all of these ligands react on the same pole of the protein. This demonstration was made possible by the new finding that mitoKATP is 85-90% oriented inward or outward in liposomes, depending on the presence or absence of Mg2+ in the reconstitution buffer. K+ flux experiments in respiring rat liver mitochondria showed that mitoKATP was inhibited by palmitoyl CoA and activated by GTP when these agents were added to the external medium. Given that the inner membrane is impermeant to these ligands and that mitoKATP is unipolar with respect to nucleotide regulation, it follows that the regulatory sites on mitoKATP face the cytosol.

Antioxidant activity by a synergy of redox-sensitive mitochondrial phospholipase A2 and uncoupling protein-2 in lung and spleen.

Mitochondrial uncoupling protein-2 (UCP2) has been suggested to participate in the attenuation of the reactive oxygen species production, but the mechanism of action and the physiological significance of UCP2 activity remain controversial. Here we tested the hypothesis that UCP2 provides feedback downregulation of oxidative stress in vivo via synergy with an H2O2-activated mitochondrial calcium-independent phospholipase A2 (mt-iPLA2). Tert-butylhydroperoxide or H2O2 induced free fatty acid release from mitochondrial membranes as detected by gas chromatography/mass spectrometry, which was inhibited by r-bromoenol lactone (r-BEL) but not by its stereoisomer s-BEL, suggesting participation of mt-iPLA2γ isoform. Tert-butylhydroperoxide or H2O2 also induced increase in respiration and decrease in mitochondrial membrane potential in lung and spleen mitochondria from control but not UCP2-knockout mice. These data suggest that mt-iPLA2γ-dependent release of free fatty acids promotes UCP2-dependent uncoupling. Upon such uncoupling, mitochondrial superoxide formation decreased instantly also in the s-BEL presence, but not when mt-iPLA2 was blocked by R-BEL and not in mitochondria from UCP2-knockout mice. Mt-iPLA2γ was alternatively activated by H2O2 produced probably in conjunction with the electron-transferring flavoprotein:ubiquinone oxidoreductase (ETFQOR), acting in fatty acid β-oxidation. Palmitoyl-d,l-carnitine addition to mouse lung mitochondria, respiring with succinate plus rotenone, caused a respiration increase that was sensitive to r-BEL and insensitive to s-BEL. We thus demonstrate for the first time that UCP2, functional due to fatty acids released by redox-activated mt-iPLA2γ, suppresses mitochondrial superoxide production by its uncoupling action. In conclusion, H2O2-activated mt-iPLA2γ and UCP2 act in concert to protect against oxidative stress.

Distribution of mitochondrial nucleoids upon mitochondrial network fragmentation and network reintegration in HEPG2 cells

Mitochondrial DNA (mtDNA) is organized in nucleoids in complex with accessory proteins, proteins of mtDNA replication and gene expression machinery. A robust mtDNA genome is represented by hundreds to thousands of nucleoids in cell mitochondrion. Detailed information is lacking about the dynamics of nucleoid distribution within the mitochondrial network upon physiological and pathological events. Therefore, we used confocal microscopy to study mitochondrial nucleoid redistribution upon mitochondrial fission and following reintegration of the mitochondrial network. Fission was induced by oxidative stress at respiration inhibition by rotenone or upon elimination of the protonmotive force by uncoupling or upon canceling its electrical component, ΔΨ(m), by valinomycin; and by silencing of mitofusin MFN2. Agent withdrawal resulted in concomitant mitochondrial network reintegration. We found two major principal morphological states: (i) a tubular state of the mitochondrial network with equidistant nucleoid spacing, 1.10±0.2 nucleoids per μm, and (ii) a fragmented state of solitary spheroid objects in which several nucleoids were clustered. We rarely observed singular mitochondrial fragments with a single nucleoid inside and very seldom we observed empty fragments. Reintegration of fragments into the mitochondrial network re-established the tubular state with equidistant nucleoid spacing. The two major morphological states coexisted at intermediate stages. These observations suggest that both mitochondrial network fission and reconnection of the disintegrated network are nucleoid-centric, i.e., fission and new mitochondrial tubule formation are initiated around nucleoids. Analyses of combinations of these morphological icons thus provide a basis for a future mitochondrial morphology diagnostics.

CHANNEL CHARACTER OF UNCOUPLING PROTEIN-MEDIATED TRANSPORT

Mitochondrial uncoupling proteins (UCPs) are pure anion uniporters, which mediate fatty acid (FA) uniport leading to FA cycling. Protonated FAs then flip‐flop back across the lipid bilayer. An existence of pure proton channel in UCPs is excluded by the equivalent flux‐voltage dependencies for uniport of FAs and halide anions, which are best described by the Eyring barrier variant with a single energy well in the middle of two peaks. Experiments with FAs unable to flip and alkylsulfonates also support this view. Phylogenetically, UCPs took advantage of the common FA‐uncoupling function of SLC25 family carriers and dropped their solute transport function.

Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA

Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5′-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor® 488 or 647 dye, we obtained the fluorescent “L-ND5 probe” containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5′-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5′-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.