Glutaredoxin-2 is required to control proton leak through uncoupling protein-3

Abstract

Glutathionylation has emerged as a key modification required for controlling protein function in response to changes in cell redox status. Recently, we showed that the glutathionylation state of uncoupling protein-3 (UCP3) modulates the leak of protons back into the mitochondrial matrix, thus controlling reactive oxygen species production. However, whether or not UCP3 glutathionylation is mediated enzymatically has remained unknown because previous work relied on the use of pharmacological agents, such as diamide, to alter the UCP3 glutathionylation state. Here, we demonstrate that glutaredoxin-2 (Grx2), a matrix oxidoreductase, is required to glutathionylate and inhibit UCP3. Analysis of bioenergetics in skeletal muscle mitochondria revealed that knock-out of Grx2 (Grx2(-/-)) increased proton leak in a UCP3-dependent manner. These effects were reversed using diamide, a glutathionylation catalyst. Importantly, the increased leak did not compromise coupled respiration. Knockdown of Grx2 augmented proton leak-dependent respiration in primary myotubes from wild type mice, an effect that was absent in UCP3(-/-) cells. These results confirm that Grx2 deactivates UCP3 by glutathionylation. To our knowledge, this is the first enzyme identified to regulate UCP3 by glutathionylation and is the first study on the role of Grx2 in the regulation of energy metabolism.

FIGURE 1.

Impact of Grx2^−/− on whole body energetics and mouse physiology. a, mouse weight. Student's t test, n = 4, mean ± S.E. b, effect of Grx2^−/− on tissue and organ weights. Student's t test, n = 4, mean ± S.E. BAT, interscapular brown adipose tissue; GWAT, gonadal white adipose tissue. c, hematoxylin and eosin staining of gastrocnemius (×40 objective) and liver (×20 objective) cross-sections. d, measurement of whole body energetics by indirect calorimetry. n = 6. e, impact of Grx2^−/− on respiratory exchange ratio (RER) and mouse activity in WT and Grx2^−/− mice during light and dark phases. Student's t test, n = 6, mean ± S.E. f, daily food intake. Measurements were taken every 3–4 days and corrected for spillage. n = 5, mean ± S.E.

FIGURE 2.

Grx2^−/− alters 2GSH/GSSG redox potential and the glutathionylated proteome but does not induce oxidative damage. a, measurement of the absolute levels of GSH and GSSG in liver and muscle mitochondria from WT and Grx2^−/− mice. 2GSH/GSSG was calculated from the absolute GSH and GSSG levels. E_h was calculated using the Nernst equation as described under “Materials and Methods.” ** indicates p ≤ 0.01. Student's t test, n = 4, mean ± S.E. b, serum levels of GSH and GSSG in WT and Grx2^−/− mice. n = 4, mean ± S.E., Student's t test. c, measurement of HNE-His adducts in liver and muscle mitochondria from WT and Grx2^−/− mice. n = 4, mean ± S.E., Student's t test. d, assessment of the glutathionylated proteome in liver and muscle mitochondria. Gels were run under nonreducing conditions to preserve PSSG adducts. Samples prepared in 2% (v/v) β-mercaptoethanol served as the control. Blots were quantified using ImageJ software. Student's t test, n = 5, mean ± S.E. e, immunodetection of glutaredoxin-2, Grx2; thioredoxin-2, Trx2; and uncoupling protein-3, UCP3. SDH, succinate dehydrogenase.

FIGURE 3.

Effect of Grx2^−/− on complex I activity in liver and muscle mitochondria. Reactions were performed on digitonized mitochondria in the absence (a) or presence (b) of 2 mm DTT. Student's t test, n = 4, mean ± S.E.

FIGURE 4.

Levels of TCA cycle metabolites and ATP in liver (a and c) and muscle (b and d) mitochondria, as assessed by HPLC. Student's t test, n = 5, mean ± S.E.

FIGURE 5.

Impact of Grx2^−/− on bioenergetics of mitochondria from liver (a) and skeletal muscle (b). Bioenergetic determinations were performed with the Seahorse XF24 Extracellular Flux Analyzer. Mitochondria (10 μg) were attached to the surface of Seahorse TC plates and then treated sequentially with ADP (0.1 mm), oligomycin (oligo; 2.5 μg/ml) and FCCP (8 μm) to test state 3, state 4, and maximal respiration, respectively. % contribution of respiration to ATP production was calculated by dividing state 4 respiration (proton leak-dependent) by state 3 respiration. Student's t test, n = 4, mean ± S.E. Pyr, pyruvate; Mal, malate.

FIGURE 6.

Effect of H₂O₂ and diamide on mitochondrial proton leak. a, impact of successive H₂O₂ and diamide treatments on proton leak in mitochondria isolated from muscle of WT and Grx2^−/− mice. One-way analysis of variance with Fisher's protected least significant difference post hoc test, n = 4, mean ± S.E. * and # correspond to comparisons with WT or Grx2^−/− (oligomycin (oligo) conditions). b, impact of successive H₂O₂ (Per) and diamide (Dia) treatments on proton leak in mitochondria isolated from muscle of WT and UCP3^−/− mice. One-way analysis of variance with Fisher's protected least significant difference post hoc test, n = 4, mean ± S.E. * and # correspond with comparisons to WT or UCP3^−/− (oligomycin conditions). c, Grx2 is required to glutathionylate UCP3. Mitochondria were treated with BioGEE, and proteins were immunoprecipitated and then tested for UCP3 by immunoblot. d, visualization of UCP3 glutathionylation status by immunofluorescent microscopy. Gastrocnemius muscle sections were fixed and treated with antibodies directed against UCP3 (green) and BioGEE (red). Co-localization of both stains provides a yellow fluorescence. Preincubation in NEM or DTT were used as controls. Scale bar, 10 μm.

FIGURE 7.

Grx2 is required to modulate proton leak through UCP3 in intact skeletal muscle cells. Primary myotubes from WT or UCP3^−/− mice were transduced with shGrx2 lentivirus to knock down Grx2. Scrambled shRNA served as a control. a, oxygen consumption trace showing the bioenergetic responses of WT and UCP3^−/− primary myotubes transduced with scrambled or shGrx2 lentiviral particles. O, oligomycin; D, diamide; F, FCCP; A, antimycin A. b, Grx2 knockdown increases proton leak in primary WT but not UCP3^−/− myotubes. Student's t test, n = 4, mean ± S.E. c, immunoblot for UCP3 and Grx2 in WT and UCP3^−/− myotubes knocked down for Grx2. SDH, succinate dehydrogenase.