| Mitochondrial role of Apoptosis-Inducing Factor (AIF): Oxidative Phosphorylation and Reactive Oxygen Species. | |
Abstract/OtherAbstract
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The apoptotic function of Apoptosis-inducing factor (AIF) is well documented in the literature, but its physiological role in the mitochondrion is less certain. Using a small interfering RNA (siRNA) strategy, we studied whether modulation of AIF expression in cultured cells influenced the production of reactive oxygen species (ROS). We found that siAIF-transfected cells had reduced AIF protein levels and this was paralleled by a significant increase in ROS. We tested the generality of this response by using two different human cell lines, the hepatoma cell line Hep3B and cervix carcinoma line HeLa, and also by employing a mouse ES AIF-KO cell line. The increased ROS were mitochondrial in origin as a similar silencing strategy in cells devoid of a functioning mitochondrial electron transport chain (ETC) did not result in a ROS-increase. The augmented ROS levels were sufficient to activate Hypoxia-inducible factor 1α (HIF-1α), a ROS-sensitive transcription factor, and this effect could be reversed using antioxidants, both the broad-range general antioxidant (N-acetyl cysteine) and a specific mitochondrial-targeted antioxidant (MitoQ), proving the implication of ROS in the HIF-1α stabilization. We also studied another two redox-sensitive transcription factor and thus observed up-regulation in the expression of Nuclear factor (erythroidderived 2)-like 2 (Nrf2), however without major changes in Nuclear factor-kappa B (NF-κB) levels. Examination of the cellular oxygen consumption rate revealed that AIFdepleted cells had a major impairment of respiration, at Complex I in the ETC. Western blot analysis also showed a loss of Complex I 39 and 20 kDa subunits. Studies using the antioxidants mentioned above, revealed that the respiratory competence could be regained in AIF-silenced cells. However, neither of the antioxidant treatments we used could recover Complex I assembly. Studies of the energetic state of siAIF cells showed that despite a 30% decrease in the overall intact cell respiration, these cells maintain normal basal levels of ATP, presumably due to a higher glycolytic capacity and a lower proliferation rate. Moreover, we analyzed the expression of another redox-active protein, thioredoxin, by Western blot and found that the mitochondrial isoform, Trx2, was significantly decreased when AIF was silenced. Preliminary co-immunoprecipitation analyses and proteomic studies failed to show any direct correlation between AIF and Trx2 at the protein level. Our results lead us to the conclusion that the defect in respiration in siAIF cells is downstream of Complex I protein loss and is presumably due to ROS-mediated damage to the ETC. This suggests an integral mitochondrial function of AIF, as a redox modifier and chaperone-like molecule, necessary for Complex I assembly. Additional studies are required to define the detailed mechanism of the AIF enzymatic activity in the mitochondrion and to establish its binding partners. __________________________________________________________________________________________________ |
Authors
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Apostolova, Nadezda |
Contributors
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Esplugues Mota, Juan Vte., James McCreath, Kenneth, Cervera Zamora, Ana Mª |
Publication Detail
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Publisher : Universitat de València Type : text Format : application/pdf |
Date Detail
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2008-03-12 |
Subject
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FARMACOLOGIA |
Coverage
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- |
Relation
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- |
Source
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http://www.tesisenxarxa.net |
Copyright Information
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Copyright information available at source archive |
Other Details
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Languages : eng |
Export Citation
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