Mitochondrial thioltransferase (glutaredoxin 2) has GSH-dependent and thioredoxin reductase-dependent peroxidase activities in vitro and in lens epithelial cellsстатья
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Дата последнего поиска статьи во внешних источниках: 10 августа 2018 г.
Аннотация:Thioltransferase (or Grx) belongs to the oxidoreductase family and is known to regulate redox homeostasis in cells. Mitochondrial Grx2 is a recent discovery, but its function is largely unknown. In this study we investigate Grx2 function by examining its potential peroxidase activity using lens epithelial cells (LEC). cDNA for human and mouse Grx2 was cloned into pET21d(+) vector and used to produce respective recombinant Grx2 for kinetic studies. cDNA for human Grx2 was transfected into human LEC and used for in vivo studies. Both human and mouse Grx2 showed glutathione (GSH)-dependent and thioredoxin reductase (TR)-dependent peroxidase activity. The catalytic efficiency of human and mouse Grx2 was lower than that of glutathione peroxidases (2.5 and 0.8×104 s–1M–1, respectively), but comparable with TR-dependent peroxiredoxins (16.5 and 2.7×104 s–1M–1, respectively). TR-dependent peroxidase activity increased 2-fold in the transfected cells and was completely abolished by addition of anti-Grx2 antibody (Ab). Flow cytometry (FACS) analysis and confocal microscopy revealed that cells preloaded with pure Grx2 detoxified peroxides more efficiently. Grx2 over-expression protected cells against H2O2-mediated disruption of mitochondrial transmembrane potential. These results suggest that Grx2 has a novel function as a peroxidase, accepting electrons both from GSH and TR. This unique property may play a role in protecting the mitochondria from oxidative damage.—Fernando, M. R., Lechner, J. M., Löfgren, S., Gladyshev, V. N., Lou, M. F. Mitochondrial thioltransferase (glutaredoxin 2) has GSH-dependent and thioredoxin reductase-dependent peroxidase activities in vitro and in lens epithelial cells.
mitochondria in mammalian cells are the main site of aerobic energy metabolism. Most of the oxygen utilized by the cell is consumed by the oxidative phosphorylation that operates within the mitochondria. Less than 1% of the total oxygen is converted to reactive oxygen species (ROS), including superoxide anions (1⤻ 2⤻ 3)⤻ . Superoxide anions thus generated are converted to hydrogen peroxide (H2O2) by the mitochondrial enzyme manganese superoxide dismutase (SOD) (MnSOD). Even though H2O2 is a relatively stable oxidant, it can be converted to the highly reactive hydroxyl radical by a metal ion via the Fenton reaction (3)⤻ . Hydrogen peroxide and free radicals generated in this way within the mitochondria may damage the reactive moieties of biologically important macromolecules such as proteins, lipids, and DNA (4⤻ , 5)⤻ . This damage may eventually challenge the integrity of the organelle. ROS have been implicated in mediating mitochondrial permeability transition, which is an initial event in the process of cell death induced by Ca2+ and inorganic phosphate. This mitochondrial permeability transition can be prevented by antioxidants (6)⤻ . Mitochondria have several oxidation defense systems, including enzymes such as glutathione peroxidase (Gpx), MnSOD, thioredoxin-2 (Trx2), and thioredoxin reductase (TR3). The mitochondria is also defended by antioxidants such as glutathione (GSH), vitamin E, and ascorbate.
Mammalian mitochondrial thioltransferase (glutaredoxin 2 or Grx2) is a recently (7⤻ , 8)⤻ identified 18 kDa protein belonging to the thiol/disulfide exchange oxidoreductase family. This new protein is 34% identical to the previously known cytosolic thioltransferase (also known as Grx1) and exhibits GSH-dependent hydroxyethyl disulfide reducing activity in vitro (7⤻ , 8)⤻ . Grx2 can reduce S-glutathionylated proteins with high affinity, accepting electrons from either GSH or thioredoxin reductase (9)⤻ . It has also been shown that silencing of Grx2 by RNA-mediated short interfering method increases the sensitivity of HeLa cells toward the anticancer drugs doxorubicin and phenylarsine oxide (10)⤻ . Recent findings show that Grx2 plays a role in attenuating apoptosis by preventing cytochrome c release in Grx2 overexpressed HeLa cells (11)⤻ . Human Grx2 has been characterized as an iron-sulfur protein, which may play an important role as redox sensor in mitochondria (12)⤻ . Using mouse and human recombinant Grx2 and overexpressed human Grx2 in mammalian cells, the current study provides evidence that mammalian Grx2 possesses a novel GSH- and TR-dependent peroxidase activity.