Produção Nacional
Revisado por pares
Hydroperoxy Fatty Acid Cycling Mediated by Mitochondrial Uncoupling Protein UCP2
2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês
10.1074/jbc.m405339200
ISSN1083-351X
AutoresMartin Jabůrek, Sayuri Miyamoto, Paolo Di Mascio, Keith Garlid, Petr Ježek,
Tópico(s)Biochemical effects in animals
ResumoFunctional 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. 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. Mitochondria produce a substantial amount of superoxide anion (O2⋅¯) (1Loschen G. Flohe L. Chance B FEBS Lett. 1971; 18: 261-264Crossref PubMed Scopus (449) Google Scholar, 2Muller F.L. Liu Y. Van Remmen H. J. Biol. Chem. 2004; (August 17, 10.1074/jbc.M407715200)Google Scholar, 3Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7531) Google Scholar, 4Halliwell B. Gutteridge J.M.C. 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Neurochem. 2002; 80: 780-787Crossref PubMed Scopus (974) Google Scholar) at Complex I (by a yet unresolved mechanism, O2⋅¯ is released to the matrix) (2Muller F.L. Liu Y. Van Remmen H. J. Biol. Chem. 2004; (August 17, 10.1074/jbc.M407715200)Google Scholar) and via autooxidation of the ubisemiquinone anion radical at Complex III (1Loschen G. Flohe L. Chance B FEBS Lett. 1971; 18: 261-264Crossref PubMed Scopus (449) Google Scholar, 2Muller F.L. Liu Y. Van Remmen H. J. Biol. Chem. 2004; (August 17, 10.1074/jbc.M407715200)Google Scholar, 3Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7531) Google Scholar, 4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, 2002: 936Google Scholar, 5Cadenas E. Davies K.J.A. Free Radic. Biol. Med. 2000; 29: 222-230Crossref PubMed Scopus (2386) Google Scholar, 6Raha S. Robinson B.H. Trends Biochem. Sci. 2000; 25: 502-507Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 7Skulachev V.P. Biochim. Biophys. Acta. 1998; 1363: 100-124Crossref PubMed Scopus (818) Google Scholar, 8Korshunov S.S. Skulachev V.P. Starkov A.A. FEBS Lett. 1997; 416: 15-18Crossref PubMed Scopus (1401) Google Scholar, 9Korshunov S.S. Korkina O.V. Ruuge E.K. Skulachev V.P. Starkov A.A. FEBS Lett. 1998; 435: 215-218Crossref PubMed Scopus (174) Google Scholar, 10Kowaltowski A.J. Costa A.D.T. Vercesi A.E. FEBS Lett. 1998; 425: 213-216Crossref PubMed Scopus (146) Google Scholar, 11Liu Y. Fiskum G. Schubert D. J. Neurochem. 2002; 80: 780-787Crossref PubMed Scopus (974) Google Scholar), where O2⋅¯ could be released into both sides of the membrane (2Muller F.L. Liu Y. Van Remmen H. J. Biol. Chem. 2004; (August 17, 10.1074/jbc.M407715200)Google Scholar). A spectrum of radical and non-radical compounds is produced from O2⋅¯, which are commonly called reactive oxygen species (ROS). 1The abbreviations used are: ROS, reactive oxygen species; FA(s), fatty acid(s); FAOH, hydroxy-fatty acid; FAOOH or FAOOH-COOH, fatty acid hydroperoxides; HNE, 4-hydroxy-2-nonenal; LA, linoleic acid; LAOOH, linoleic acid hydroperoxides; PLA2, phospholipase A2; PUFAs, polyunsaturated FAs; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium; TES, N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid; UCP2, "ubiquitous" uncoupling protein; UCP, uncoupling protein. These include radicals (O2⋅¯, hydroperoxyl (HO2·), hydroxyl (·OH), peroxyl (RO2·), alkoxyl (RO·)) and nonradical species such as H2O2 and singlet oxygen (3Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7531) Google Scholar, 4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, 2002: 936Google Scholar). The list is extended by peroxynitrite, which is formed by the reaction of O2⋅¯ with nitric oxide, ·NO (12Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar, 13Rubbo H. Radi R. Trujillo M. Telleri R. Kalyanaraman B. Barnes S. Kirk M. Freeman B.A. J. Biol. Chem. 1994; 269: 26066-26075Abstract Full Text PDF PubMed Google Scholar). Most of the O2⋅¯ is converted to H2O2 by matrix manganese-superoxide dismutase (14Melov S. Ann. N. Y. Acad. Sci. 2002; 959: 219-225Crossref Scopus (56) Google Scholar) and by cytosolic CuZn-superoxide dismutase, which exists also in the mitochondrial intramembrane space (2Muller F.L. Liu Y. Van Remmen H. J. Biol. Chem. 2004; (August 17, 10.1074/jbc.M407715200)Google Scholar). H2O2, which has been recognized as an important signaling molecule (15Sen C.K. Packer L. FASEB J. 1996; 10: 709-720Crossref PubMed Scopus (1777) Google Scholar, 16Xie Z. Kometiani P. Liu J. Li J. Shapiro J.I. Askari A. J. Biol. 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At pH 6.8, ∼1% of O2⋅¯ is protonated to highly reactive species, hydroperoxyl radical HO2· (pKa 4.8) (4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, 2002: 936Google Scholar). H2O2 may be converted into highly reactive ·OH by a reaction with transition metals, namely with Fe2+ (Fenton reaction) (3Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7531) Google Scholar, 4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, 2002: 936Google Scholar). The ·OH is an extremely reactive oxidizing radical that will react to most biomolecules at diffusion-controlled rates. The hydroxyl radical can also oxidize nitrite to nitrogen dioxide (·NO2) and react with bicarbonate yielding the very reactive carbonate radical anion under physiological conditions (4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, 2002: 936Google Scholar). All of these highly reactive radicals (HO2·, ·OH, and CO3⋅¯) attack proteins and DNA, but, in addition, a substantial lipoperoxidation must occur in vivo, because polyunsaturated hydrocarbon chains are very susceptible to allelic hydrogen abstraction (3Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7531) Google Scholar, 4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, 2002: 936Google Scholar, 18Spiteller G. Ann. N. Y. Acad. Sci. 2002; 959: 30-44Crossref PubMed Scopus (37) Google Scholar, 19Aikens J. Dix T.A. J. Biol. Chem. 1991; 266: 15091-15098Abstract Full Text PDF PubMed Google Scholar, 20Miyamoto S. Dupas C. Murota K. Terao J. Lipids. 2003; 38: 641-649Crossref PubMed Scopus (31) Google Scholar). Once carbon-centered lipid radicals (RC·R) are generated, they react with O2 giving peroxyl radicals (ROO·), which react for example with the neighbor polyunsaturated hydrocarbon side chain yielding hydroperoxides (ROOH) and other RC·R. The reaction is thus propagated by many cycles (18Spiteller G. Ann. N. Y. Acad. Sci. 2002; 959: 30-44Crossref PubMed Scopus (37) Google Scholar). Fatty acid hydroperoxides (FAOOH) can be cleaved off from the phospholipids by phospholipase A2 (PLA2) (20Miyamoto S. Dupas C. Murota K. Terao J. Lipids. 2003; 38: 641-649Crossref PubMed Scopus (31) Google Scholar), e.g. by its mitochondrial Ca2+-dependent isoform induced by superoxide (21Guidarelli A. Cantoni O. Biochem. J. 2002; 366: 307-314Crossref PubMed Google Scholar) or by the Ca2+-independent isoform. FAOOH are transient, non-radical but reactive species, which are eventually degraded by glutathione peroxidase or phospholipid hydroperoxide glutathione peroxidase to the corresponding hydroxy fatty acids (FAOH). FAOOH may also decompose to toxic epoxy acids and α,β,γ,δ-unsaturated aldehydes (18Spiteller G. Ann. N. Y. Acad. Sci. 2002; 959: 30-44Crossref PubMed Scopus (37) Google Scholar). It has been found that even a slight increase of H+ backflux into the matrix may substantially suppress mitochondrial ROS formation (7Skulachev V.P. Biochim. Biophys. Acta. 1998; 1363: 100-124Crossref PubMed Scopus (818) Google Scholar, 8Korshunov S.S. Skulachev V.P. Starkov A.A. FEBS Lett. 1997; 416: 15-18Crossref PubMed Scopus (1401) Google Scholar, 9Korshunov S.S. Korkina O.V. Ruuge E.K. Skulachev V.P. Starkov A.A. FEBS Lett. 1998; 435: 215-218Crossref PubMed Scopus (174) Google Scholar, 10Kowaltowski A.J. Costa A.D.T. Vercesi A.E. FEBS Lett. 1998; 425: 213-216Crossref PubMed Scopus (146) Google Scholar, 11Liu Y. Fiskum G. Schubert D. J. Neurochem. 2002; 80: 780-787Crossref PubMed Scopus (974) Google Scholar). The physiological H+ backflow during ATP synthesis also suppresses ROS formation (7Skulachev V.P. Biochim. Biophys. Acta. 1998; 1363: 100-124Crossref PubMed Scopus (818) Google Scholar, 8Korshunov S.S. Skulachev V.P. Starkov A.A. FEBS Lett. 1997; 416: 15-18Crossref PubMed Scopus (1401) Google Scholar, 9Korshunov S.S. Korkina O.V. Ruuge E.K. Skulachev V.P. Starkov A.A. FEBS Lett. 1998; 435: 215-218Crossref PubMed Scopus (174) Google Scholar); therefore most ROS are produced in the non-phosphorylating state. This effect of uncoupling or ATP synthesis is due to increased respiration and hence concomitantly shortened lifetime of UQ⋅¯ and the lowered oxygen tension in the microenvironment which results in reduced rate of O2⋅¯ formation (7Skulachev V.P. Biochim. Biophys. 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The strong acidic character of the former (38Garlid K.D. Orosz D.E. Modrianský M. Vassanelli S. Ježek P. J. Biol. Chem. 1996; 271: 2615-2620Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 43Jabůrek M. Vařecha M. Ježek P. Garlid K.D. J. Biol. Chem. 2001; 276: 31897-31905Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and a "U-shape" of the latter compounds in the membrane (48Ježek P. Modrianský M. Garlid K.D. FEBS Lett. 1997; 408: 161-165Crossref PubMed Scopus (71) Google Scholar) prevent their flip-flop with protons across the bilayer. We examined interactions of linoleic acid hydroperoxides (LAOOH) (50Lima E.S. Di Mascio P. Rubbo H. Abdalla D.S. Biochemistry. 2002; 41: 10717-10722Crossref PubMed Scopus (93) Google Scholar, 51Miyamoto S. Martinez G.R. Medeiros M.H.G. Di Mascio P. J. Am. Chem. Soc. 2003; 125: 6172-6179Crossref PubMed Scopus (165) Google Scholar) with liposomal membranes and with reconstituted UCP2. We found that LAOOH does rapidly flip-flop across the membrane, resulting in the delivery of protons to the interior. Moreover, LAOOH supports uncoupling in liposomes containing UCP2, and it does so with a higher affinity than was observed for linoleic acid itself. We concluded that LAOOH is an efficient cycling substrate of UCP2. The finding that FAOOH supports UCP-mediated uncoupling suggests an alternative explanation for the reported activation of UCP2 by externally generated superoxide observed exclusively in the presence of FAs (33Echtay K.S. Roussel D. St-Pierre J. Jekabson M.B. Cadenas S. Stuart J.A. Harper J.A. Roebuck S.J. Morrison A. Pickering S. Clapham J.C. Brand M.D. Nature. 2002; 415: 96-99Crossref PubMed Scopus (1150) Google Scholar). Our suggested mechanism may serve physiologically as a feedback regulation accelerating the suppression of mitochondrial ROS production. Most of the chemicals were purchased from Sigma. LAOOH (Fig. 1) was synthesized in the São Paulo laboratory as described previously (50Lima E.S. Di Mascio P. Rubbo H. Abdalla D.S. Biochemistry. 2002; 41: 10717-10722Crossref PubMed Scopus (93) Google Scholar, 51Miyamoto S. Martinez G.R. Medeiros M.H.G. Di Mascio P. J. Am. Chem. Soc. 2003; 125: 6172-6179Crossref PubMed Scopus (165) Google Scholar). Hydroxylapatite, Bio-Gel HTP, and Bio-Beads SM2 were from Bio-Rad. Octylpentaoxyethylene was from Bachem Feinchemikalien, Bubendorf, Switzerland. Other materials for reconstitution were from the same sources as described elsewhere (32Žácková M. Škobisová E. Urbánková E. Ježek P. J. Biol. Chem. 2003; 278: 20761-20769Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 38Garlid K.D. Orosz D.E. Modrianský M. Vassanelli S. Ježek P. J. Biol. Chem. 1996; 271: 2615-2620Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 39Ježek P. Modrianský M. Garlid K.D. FEBS Lett. 1997; 408: 166-170Crossref PubMed Scopus (68) Google Scholar, 43Jabůrek M. Vařecha M. Ježek P. Garlid K.D. J. Biol. Chem. 2001; 276: 31897-31905Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 45Jabůrek M. Vařecha M. Gimeno R.E. Dembski M. Ježek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 46Jabůrek M. Garlid K.D. J. Biol. Chem. 2003; 278: 25825-25831Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). All other chemicals were of a reagent grade. Escherichia coli Expression of UCP2—Bacterial strains BL21 (Novagen) containing plasmids pET21a with inserted cDNA, coding for human UCP2, were donated by Dr. R. E. Gimeno and Louis A. Tartaglia (Millennium Pharmaceuticals, Inc., Cambridge, MA). Cell cultures were grown, and the expression of UCP2, induced by isopropyl 1-thio-β-d-galactopyranoside and including the inclusion bodies preparation, was performed as previously described (45Jabůrek M. Vařecha M. Gimeno R.E. Dembski M. Ježek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Protein content was assayed by the Amido Black method (52Kaplan R.S. Pedersen P.L. Anal. Biochem. 1985; 150: 97-104Crossref PubMed Scopus (187) Google Scholar). Extraction of UCP2 from Inclusion Bodies—A pelleted aliquot of inclusion bodies (∼2 mg of protein) was washed and solubilized with Sarkosyl as described previously (46Jabůrek M. Garlid K.D. J. Biol. Chem. 2003; 278: 25825-25831Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The resulting supernatant was diluted rapidly in 6 ml of 10% glycerol, 1% Triton X-114, and 1 mm ATP. This mixture was supplemented with 0.8 g of Dowex 11A8 and incubated for 2 h at 4 °C with constant mixing. The extract was passed through an additional Dowex 11A8 column (2.5 ml). The collected protein was supplemented with 0.1 mm ATP and 20 mg of soybean asolectin/mg of starting protein. The resulted extract was incubated overnight at 4 °C and concentrated using the Ultrafree-15 centrifugal filter device (Millipore). Aliquots of ∼0.6 mg of protein were stored at –80 °C. Purification and Reconstitution of E. coli-expressed UCP2—The extracted protein was dialyzed twice for 3 h and once overnight at a 1:100 dilution against the internal medium (tetraethylammonium salts of 30 mm TES, 80 mm SO4, 2 mm EDTA, pH 7.2). The dialyzed protein was centrifuged at 14,000 × g for 10 min to remove any precipitate, and the supernatant was passed through a hydroxylapatite (Bio-Gel HTP) column (46Jabůrek M. Garlid K.D. J. Biol. Chem. 2003; 278: 25825-25831Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The recombinant UCP2 was reconstituted into liposomes as described recently (46Jabůrek M. Garlid K.D. J. Biol. Chem. 2003; 278: 25825-25831Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Reconstitution involved supplementing the extract by additional lipids (solubilized in octylpentaoxyethylene and adjusted to total 40 mg of egg yolk lecithin, type XI-E, supplemented with 5% cardiolipin), detergent removal on Bio-Beads SM2, and external probe removal on Sephadex G50–300. The protein content of proteoliposomes was estimated by the Amido Black method (52Kaplan R.S. Pedersen P.L. Anal. Biochem. 1985; 150: 97-104Crossref PubMed Scopus (187) Google Scholar). Usually, a lipid-to-protein ratio of about 200 or higher was obtained. Fluorescent Monitoring of K+and H+Fluxes in Proteoliposomes—Ion fluxes in proteoliposomes were measured using a RF5301 PC fluorometer (Shimadzu, Japan), equipped with polarization filters (Polaroid) in cross-orientation, to decrease interference by scattered light. Proton fluxes, i.e. changes of intraliposomal H+ concentration, were monitored by SPQ fluorescence changes (2 mm SPQ) because of quenching by the TES anion (38Garlid K.D. Orosz D.E. Modrianský M. Vassanelli S. Ježek P. J. Biol. Chem. 1996; 271: 2615-2620Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 39Ježek P. Modrianský M. Garlid K.D. FEBS Lett. 1997; 408: 166-170Crossref PubMed Scopus (68) Google Scholar, 43Jabůrek M. Vařecha M. Ježek P. Garlid K.D. J. Biol. Chem. 2001; 276: 31897-31905Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 45Jabůrek M. Vařecha M. Gimeno R.E. Dembski M. Ježek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scho
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