Veterinary and Biomedical Sciences, Department of


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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 12, pp. 8365–8379, March 22, 2013. DOI 10.1074/jbc.M112.442905


© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Used by permission.


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.

Supplementary files attached below.

jbc.M112.442905-1.docx (13 kB)
Supplemental Table 1 (.docx, 14 KB) - Circulating metabolite levels.

jbc.M112.442905-2.pptx (170 kB)
Supplemental Figure 1 (.pptx, 174 KB) - Protein levels of antioxidant enzymes and ANT.

jbc.M112.442905-3.pptx (343 kB)
Supplemental Figure 2 (.pptx, 352 KB) - Validation of Seahorse protocol for measurement of mitochondrial bioenergetics.

jbc.M112.442905-4.pptx (166 kB)
Supplemental Figure 3 (.pptx, 171 KB) - Seahorse trace for analysis of bioenergetics of liver and muscle mitochondria from wild-type and Grx2-/- mice.

jbc.M112.442905-5.pptx (236 kB)
Supplemental Figure 4 (.pptx, 242 KB) - Mitochondrial energetics of liver and muscle mitochondria from wild-type and Grx2-/- mice using succinate as energy substrate.

jbc.M112.442905-6.pptx (139 kB)
Supplemental Figure 5 (.pptx, 143 KB) - Contribution of ANT to proton leak in liver and muscle mitochondria from wild-type and Grx2-/- mice.