Abstract
The mitochondria are the major intracellular source of reactive oxygen species (ROS), which are generated during cellular respiration. The role of peroxiredoxin (Prx) III, a 2-Cys Prx family member, in the scavenging of mitochondrial H(2)O(2) has recently been emphasized. While eliminating H(2)O(2), Prx can become overoxidized and inactivated by modifying the active cysteine into cysteine sulfinic acid (Cys-SO(2)H). When 2-Cys Prxs are inactivated in vitro, sulfiredoxin (Srx) reduces the cysteine sulfinic acid to cysteines. However, whereas Srx is localized in the cytoplasm, Prx III is present exclusively in the mitochondria. Although Srx reduces sulfinic Prx III in vitro, it remains unclear whether the reduction of Prx III in cells is actually mediated by Srx. Our gain- and loss-of-function experiments show that Srx is responsible for reducing not only sulfinic cytosolic Prxs (I and II) but also sulfinic mitochondrial Prx III. We further demonstrate that Srx translocates from the cytosol to mitochondria in response to oxidative stress. Overexpression of mitochondrion-targeted Srx promotes the regeneration of sulfinic Prx III and results in cellular resistance to apoptosis, with enhanced elimination of mitochondrial H(2)O(2) and decreased rates of mitochondrial membrane potential collapse. These results indicate that Srx plays a crucial role in the reactivation of sulfinic mitochondrial Prx III and that its mitochondrial translocation is critical in maintaining the balance between mitochondrial H(2)O(2) production and elimination.
Highlights
Peroxiredoxins (Prxs)2 are a family of enzymes that catalyze the reduction of hydrogen peroxide (H2O2) and hydroperox
We previously developed a method to monitor the Srx could translocate to the mitochondria in response to H2O2. reduction of sulfinic 2-Cys Prx enzymes by immunoblot analy- To examine this possibility, we investigated the intracellular sis with antibodies that recog- localization of Srx under basal conditions and after oxidative nize both sulfinic and sulfonic forms of these proteins [25]. stress, using subcellular fractionation (Fig. 2A) and confocal
Effects of Mitochondria-targeted Srx Overexpression on Rotenone-induced Apoptotic Phenotypes—We looked at the effects of ectopic mitochondrion-targeted Srx (mitoSrx) expression on steady-state level of sulfinic Prx III in cells treated with rotenone, which inhibits NADH conversion to NAD through respiratory Complex I (Fig. 4A)
Summary
Materials—Dulbecco’s minimum essential cell culture medium, and F-12 Nutrient Mixture (Ham’s F-12) medium were obtained from WelGENE Inc. (Daegu, Republic of Korea). pcDNA3 vector, Alexa-488-conjugated goat antibodies to mouse IgG, Alexa-594-conjugated goat antibodies to rabbit IgG, MitoTracker Red CMXRos, tetramethylrhodamine ethyl ester (TMRE), and 5,6-chloromethyl-2Ј,7Ј-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) were purchased from Invitrogen. pFLAG-CMV2 vector, FLAG M2 monoclonal, and anti-FLAG polyclonal antibodies were obtained from Sigma-Aldrich. FLAG-tagged Srx was detected with FLAG M2 monoclonal antibodies (10 g/ml) and Alexa-488-conjugated goat antibodies raised against mouse IgG (5 g/ml). Endogenous Prx III was detected with polyclonal antibodies (10 g/ml) and Alexa-594-conjugated goat antibodies raised against rabbit IgG (5 g/ml). To evaluate mitochondrial membrane potential changes (⌬⌿m), cells (2 ϫ 105) were incubated with 100 nM of TMRE for 20 min at 37 °C, and the shifts in red fluorescence emissions of TMRE were measured. Consistent with the results obtained by one-dimensional immunoblot analysis, the ure intracellular ROS, detached cells were loaded with 5 M reduction of sulfinic forms of Prx III was promoted by Srx over-.
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