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Selective Targeting of a Redox-active Ubiquinone to Mitochondria within Cells

ANTIOXIDANT AND ANTIAPOPTOTIC PROPERTIES*
Open AccessPublished:February 16, 2001DOI:https://doi.org/10.1074/jbc.M009093200
      With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells. Here we report on the development of a novel antioxidant that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of cell death to be inferred. This antioxidant, named mitoQ, is a ubiquinone derivative targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the ubiquinone moiety inserted into the lipid bilayer and was reduced by the respiratory chain. The ubiquinol derivative thus formed was an effective antioxidant that prevented lipid peroxidation and protected mitochondria from oxidative damage. After detoxifying a reactive oxygen species, the ubiquinol moiety was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. In cell culture studies, the mitochondrially localized antioxidant protected mammalian cells from hydrogen peroxide-induced apoptosis but not from apoptosis induced by staurosporine or tumor necrosis factor-α. This was compared with untargeted ubiquinone analogs, which were ineffective in preventing apoptosis. These results suggest that mitochondrial oxidative stress may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis factor-α. We have shown that selectively manipulating mitochondrial antioxidant status with targeted and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death. This approach will have further applications in investigating mitochondrial dysfunction in a range of experimental models.
      IR
      infrared
      AMC
      aminomethylcoumarin
      DMEM
      Dulbecco's modified Eagle's medium
      FCCP
      carbonyl cyanide p-trifluoromethoxyphenylhydrazone
      LDH
      lactate dehydrogenase
      MDA
      malondialdehyde
      mitoquinol
      10-(6′-ubiquinolyl)decyltriphenylphosphonium
      mitoquinone
      10-(6′-ubiquinonyl)decyltriphenylphosphonium
      mitoQ
      mixture of mitoquinol and mitoquinone
      Q1
      ubiquinone-1
      Q2
      ubiquinone-2
      TBARS
      thiobarbituric acid-reactive species
      TPMP
      methyltriphenylphosphonium cation
      PBS
      phosphate-buffered saline
      MOPS
      4-morpholinepropanesulfonic acid
      The mitochondrial respiratory chain is a major source of superoxide and, therefore, mitochondria accumulate oxidative damage more rapidly than the rest of the cell, contributing to mitochondrial dysfunction and cell death in degenerative diseases and in aging (
      • Wallace D.C.
      ,
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      ,
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      ,
      • Beckman K.B.
      • Ames B.N.
      ,
      • Michikawa Y.
      • Mazzucchelli F.
      • Bresolin N.
      • Scarlato G.
      • Attardi G.
      ). Mitochondria are also central to activating apoptosis and oxidative damage can lead to cell death, however, the significance of mitochondrial oxidative damage for cell death is unclear (
      • Polyak K.
      • Xia Y.
      • Zweier J.L.
      • Kinzler K.W.
      • Vogelstein B.
      ,
      • Kroemer G.
      • Dallaporta B.
      • Resche-Rignon M.
      ,
      • Hampton M.B.
      • Orenius S.
      ). One approach to this problem is to selectively target antioxidants to mitochondria (
      • Murphy M.P.
      ,
      • Murphy M.P.
      • Smith R.A.J.
      ,
      • Matthews R.T.
      • Yang L.
      • Browne S.
      • Bail M.
      • Beal M.F.
      ). This should allow the relative importance of mitochondrial and cytoplasmic oxidative stress for cell death to be distinguished, and also enable the contribution of mitochondrial damage to aging, diabetes, and cancer to be investigated in cell and animal models.
      Derivatives of ubiquinol are promising antioxidants to target to mitochondria (
      • Matthews R.T.
      • Yang L.
      • Browne S.
      • Bail M.
      • Beal M.F.
      ,
      • Lass A.
      • Forster M.J.
      • Sohal R.S.
      ). In mammals ubiquinone comprises a 2,3-dimethoxy-5-methylbenzoquinone core with a hydrophobic 45- to 50-carbon chain at the 6 position (
      • Crane F.L.
      • Barr R.
      ,
      • Crane F.L.
      ). Mitochondrial ubiquinone is a respiratory chain component buried within the lipid core of the inner membrane where it accepts two electrons from complexes I or II becoming reduced to ubiquinol, which then donates electrons to complex III (
      • Crane F.L.
      ). The ubiquinone pool in vivo is largely reduced and ubiquinol is an effective antioxidant, as well as being a mobile electron carrier (
      • Lass A.
      • Sohal R.S.
      ,
      • Kagan V.E.
      • Serbinova E.A.
      • Stoyanovsky D.A.
      • Khwaja S.
      • Packer L.
      ,
      • Maguire J.J.
      • Wilson D.S.
      • Packer L.
      ,
      • Ernster L.
      • Forsmark P.
      • Nordenbrand K.
      ). Ubiquinol acts as an antioxidant by donating a hydrogen atom from one of its hydroxyl groups to a lipid peroxyl radical, thereby decreasing lipid peroxidation within the mitochondrial inner membrane (
      • Ernster L.
      • Forsmark P.
      • Nordenbrand K.
      ,
      • Takada M.
      • Ikenoya S.
      • Yuzuriha T.
      • Katayama K.
      ,
      • Ingold K.U.
      • Bowry V.W.
      • Stocker R.
      • Walling C.
      ). The ubisemiquinone radicals thus formed disproportionate to ubiquinone and ubiquinol (
      • Land E.J.
      • Swallow A.J.
      ), or react with oxygen to form superoxide and ubiquinone thereby transferring the radical to the aqueous phase for detoxification by superoxide dismutase and peroxidases (
      • Maguire J.J.
      • Wilson D.S.
      • Packer L.
      ,
      • Ingold K.U.
      • Bowry V.W.
      • Stocker R.
      • Walling C.
      ). The respiratory chain then recycles ubiquinone back to ubiquinol to restore its antioxidant function. Vitamin E is another important antioxidant within the mitochondrial inner membrane, and the tocopheroxyl radical thus formed is regenerated to active vitamin E by reaction with ubiquinol or ubisemiquinone (
      • Lass A.
      • Sohal R.S.
      ,
      • Maguire J.J.
      • Wilson D.S.
      • Packer L.
      ,
      • Ingold K.U.
      • Bowry V.W.
      • Stocker R.
      • Walling C.
      ,
      • Stoyanovsky D.A.
      • Osipov A.N.
      • Quinn P.J.
      • Kagan V.E.
      ,
      • Mukai K.
      • Kikuchi S.
      • Urano S.
      ). Therefore, in vivo ubiquinol probably acts as an antioxidant by direct reaction with peroxyl radicals and by regenerating vitamin E (
      • Kagan V.E.
      • Serbinova E.A.
      • Stoyanovsky D.A.
      • Khwaja S.
      • Packer L.
      ,
      • Maguire J.J.
      • Wilson D.S.
      • Packer L.
      ,
      • Ingold K.U.
      • Bowry V.W.
      • Stocker R.
      • Walling C.
      ).
      The low solubility of ubiquinone in water makes it difficult to usein vitro, and animals must be fed ubiquinone-enriched diets for several weeks to increase levels in subsequently isolated mitochondria (
      • Matthews R.T.
      • Yang L.
      • Browne S.
      • Bail M.
      • Beal M.F.
      ,
      • Crane F.L.
      ). Therefore, to manipulate mitochondrial ubiquinone content in vitro we synthesized a ubiquinone analog selectively targeted to mitochondria by addition of a lipophilic triphenylphosphonium cation. Such lipophilic cations easily permeate lipid bilayers and accumulate in mitochondria within cells, driven by the large mitochondrial membrane potential (
      • Murphy M.P.
      ,
      • Murphy M.P.
      • Smith R.A.J.
      ,
      • Liberman E.A.
      • Topali V.P.
      • Tsofina L.M.
      • Jasaitis A.A.
      • Skulachev V.P.
      ). Here we report on the antioxidant and antiapoptotic properties of this mitochondrially targeted ubiquinone derivative.

      Acknowledgments

      We thank Prof. Cathy Clarke, UCLA and Prof. Ian Dawes, University of New South Wales, Sydney for supplying yeast strains.

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