Coenzyme A protects against ferroptosis via CoAlation of mitochondrial thioredoxin reductase
Chao-Chieh Lin, Yi-Tzu Lin, Ssu-Yu Chen, Yasaman Setayeshpour, Yubin Chen, Denise E. Dunn, Taylor Nguyen, Alexander A. Mestre, Adrija Banerjee, Lalitha Guruprasad, Erik J. Soderblom, Guo-Fang Zhang, Chen-Yong Lin, Valeriy Filonenko, Suh Young Jeong, Scott R. Floyd, Susan J. Hayflick, Ivan Gout, Jen-Tsan Chi
Chao-Chieh Lin, Yi-Tzu Lin, Ssu-Yu Chen, Yasaman Setayeshpour, Yubin Chen, Denise E. Dunn, Taylor Nguyen, Alexander A. Mestre, Adrija Banerjee, Lalitha Guruprasad, Erik J. Soderblom, Guo-Fang Zhang, Chen-Yong Lin, Valeriy Filonenko, Suh Young Jeong, Scott R. Floyd, Susan J. Hayflick, Ivan Gout, Jen-Tsan Chi
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Research Article Cell biology Metabolism

Coenzyme A protects against ferroptosis via CoAlation of mitochondrial thioredoxin reductase

Abstract

The cystine-xCT transporter/glutathione/GPX4 axis is the canonical pathway protecting cells from ferroptosis. Whereas GPX4-targeting ferroptosis-inducing compounds (FINs) act independently of mitochondria, xCT-targeting FINs require mitochondrial lipid peroxidation, though the mechanism remains unclear. Because cysteine is also a precursor for coenzyme A (CoA) biosynthesis, here, we demonstrated that CoA supplementation selectively prevented ferroptosis triggered by xCT inhibition by regulating the mitochondrial thioredoxin system. Our data showed that CoA regulated the in vitro enzymatic activity of mitochondrial thioredoxin reductase-2 (TXNRD2) by covalently modifying the thiol group of cysteine (CoAlation) on Cys-483. Replacing Cys-483 with alanine on TXNRD2 abolished its enzymatic activity and ability to protect cells against ferroptosis. Targeting xCT to limit cysteine import and, therefore, CoA biosynthesis reduced CoAlation on TXNRD2. Furthermore, the fibroblasts from patients with disrupted CoA metabolism had increased mitochondrial lipid peroxidation. In organotypic brain slice cultures, inhibition of CoA biosynthesis led to an oxidized thioredoxin system, increased mitochondrial lipid peroxidation, and loss of cell viability, which were all rescued by ferrostatin-1. These findings identified CoA-mediated posttranslational modification to regulate the thioredoxin system as an alternative ferroptosis protection pathway with potential clinical relevance for patients with disrupted CoA metabolism.

Authors

Chao-Chieh Lin, Yi-Tzu Lin, Ssu-Yu Chen, Yasaman Setayeshpour, Yubin Chen, Denise E. Dunn, Taylor Nguyen, Alexander A. Mestre, Adrija Banerjee, Lalitha Guruprasad, Erik J. Soderblom, Guo-Fang Zhang, Chen-Yong Lin, Valeriy Filonenko, Suh Young Jeong, Scott R. Floyd, Susan J. Hayflick, Ivan Gout, Jen-Tsan Chi

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Figure 6

Disruption of CoA biosynthesis in PKAN fibroblasts and OBSC leads to mitochondrial lipid peroxidation.

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Disruption of CoA biosynthesis in PKAN fibroblasts and OBSC leads to mit...
(A) PANK1 knockdown sensitized HT-1080 cells to BSO treatment. HT-1080 cells with shRNA targeting PANK1, PANK2, or PANK3 were treated with BSO for 3 days for Cell-Titer Glo assay. Data were analyzed by 2-way ANOVA and Šídák’s multiple comparisons; n = 3 independent biological replicates. (B) PANK1, PANK2, and COASY knockdown in HT-1080 cells showed an increase in mitochondrial lipid peroxidation. (C and D) Primary fibroblasts from patients with PKAN and age- and sex-matched unaffected individuals were stained with mitoPerOx, a probe that detects mitochondrial lipid peroxides. Representative images of mitoPerOx staining in (C) demonstrate a visibly higher signal intensity in PKAN fibroblasts. Quantification of the percentage of mitoPerOx+ cells is presented in (D), confirming a significant increase in mitochondrial lipid peroxidation in fibroblasts from patients with PKAN. Data were analyzed by unpaired t test. (E and F) The cell death triggered by inhibiting CoA using PANKi in OBSC was rescued by Fer-1. OBSCs transfected with YFP were treated with PANKi (2.5 μM) or in combination with Fer-1 (2 μM, 24 hours). Neuronal viability was assessed by fluorescence microscopy: the number of YFP+ neurons remaining after treatment was quantified by fluorescence microscopy as a measure of cell survival (E and F). Representative images of YFP+ neurons are shown in (E), and quantification is presented in (F). (G and H) The elevated mitochondrial lipid peroxidation by PANKi was rescued by Fer-1. After 1 day of treatment with PANKi (2.5 μM) and Fer-1 (2 μM), OBSC was stained with mitoPerOx (G) for quantification (H). (I and J) PANKi treatment of brain tissue sections repressed the levels of reduced form of PRDX3, which was rescued by Fer-1. OBSC treated with PANKi (2.5 μM) or in combination with Fer-1 (2 μM) were blotted with PRDX3 (I) and quantification (J). (B, F, H, and J) Data were analyzed by 1-way ANOVA and Tukey’s multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are mean ± SEM.