Coenzyme A protects against ferroptosis via CoAlation of thioredoxin reductase 2

Abstract

The Cystine-xCT transporter-Glutathione (GSH)-GPX4 axis is the canonical pathway to protect against ferroptosis. While not required for ferroptosis-inducing compounds (FINs) targeting GPX4, FINs targeting the xCT transporter require mitochondria and its lipid peroxidation to trigger ferroptosis. However, the mechanism underlying the difference between these FINs is still unknown. Given that cysteine is also required for coenzyme A (CoA) biosynthesis, here we show that CoA supplementation specifically prevents ferroptosis induced by xCT inhibitors but not GPX4 inhibitors. We find that, auranofin, a thioredoxin reductase inhibitor, abolishes the protective effect of CoA. We also find that CoA availability determines the enzymatic activity of thioredoxin reductase, but not thioredoxin. Importantly, the mitochondrial thioredoxin system, but not the cytosolic thioredoxin system, determines CoA-mediated ferroptosis inhibition. Our data show that the CoA regulates the in vitro enzymatic activity of mitochondrial thioredoxin reductase (TXNRD2) by covalently modifying the thiol group of cysteine (CoAlation) on Cys-483. Replacing Cys-483 with alanine on TXNRD2 abolishes its in vitro enzymatic activity and ability to protect cells from ferroptosis. Targeting xCT to limit cysteine import and, therefore, CoA biosynthesis reduced CoAlation on TXNRD2, an effect that was rescued by CoA supplementation. Furthermore, the fibroblasts from patients with disrupted CoA metabolism demonstrate increased mitochondrial lipid peroxidation. In organotypic brain slice cultures, inhibition of CoA biosynthesis leads to an oxidized thioredoxin system, mitochondrial lipid peroxidation, and loss in cell viability, which were all rescued by ferrostatin-1. These findings identify CoA-mediated post-translation modification to regulate the thioredoxin system as an alternative ferroptosis protection pathway with potential clinical relevance for patients with disrupted CoA metabolism.

Keywords: CoAlation; TXNRD2; ferroptosis; mitochondria; thioredoxin.

Figure 1. CoA is a class 1 FIN-specific ferroptosis inhibitor

(A-B) CoA supplementation increased the levels of intracellular CoA (A) and acetyl-CoA (B) as quantified by LC-MS/MS analysis. HT-1080 cells were treated with H2O, CoA (30 μM, 100 μM) for 18 hours for LC-MS/MS analysis. (C) CoA inhibited erastin-induced ferroptosis. HT-1080 cells were treated with increasing doses of erastin, either alone or in combination with CoA (100 μM) or deferoxamine (DFO, 80 μM), ferrostatin-1 (Fer-1, 10 μM), liproxstatin-1 (lipro, 2 μM), Trolox (100 μM). The cell viability was quantified by Cell-Titer Glo assay. (D-E) Erastin-induced lipid peroxidation (2 μM, 18 hours) in HT-1080 cells was inhibited by CoA treatment as determined by C11-BODIPY staining (D) and the quantification of % lipid peroxidation positive cells (E). (F-G) CoA (100 μM) inhibited erastin (2.5 μM, 20 hours)-induced membrane rupture in HT-1080 cells as observed by CellTox Green under fluorescence microscope (F) and quantified by a plate reader (G). (H-I) CoA (100 μM) failed to inhibit class 2 FIN-induced ferroptosis in HT-1080 cells including ML162 (20 hours) (H) and RSL3 (20 hours) (I). (J) CoA (100 μM) failed to inhibit class 3 FIN (FIN56)-induced ferroptosis in HT-1080 cells.

Figure 2. CoA regulates mitochondrial thioredoxin system

(A) Auranofin (0.5 μM) abolished CoA (100 μM)-mediated ferroptosis protection of HT-1080 cells caused by indicated levels of erastin for 20 hours as quantified by Cell-Titer Glo assay. (B) Erastin (1.25 μM, 16 hours) significantly repressed the thioredoxin reductase activity in HT-1080 cell lysates, which can be restored by CoA (100 μM) supplementation. (C) The knockdown of TXN2, but not TXN1, mitigated CoA-mediated ferroptosis protection. (D) The TXNRD2-TXN2 interaction was abolished by erastin, which can be restored by CoA supplement. HT-1080 cells overexpressing TXNRD2 and TXN2 were treated with erastin (2.5 μM, 18 hours) or in combination with CoA (100 μM) and were lysed with NEM for co-immunoprecipitation. (E) Erastin-induced decrease in the monomers (reduced and active forms) of PRDX3 in HT-1080 cells was rescued by CoA supplementation as determined by Western blots. (F) Quantification of the monomer/dimer ratio of PRDX3 upon erastin treatment with or without CoA supplementation. (G) COASY knockdown in HT-1080 cells by two independent COASY-targeting siRNAs reduced the monomer (reduced and active forms) of PRDX3 in Western blots. (H) Quantification of the monomer/dimer ratio of PRDX3 upon the knockdown of COASY. (I) COASY knockdown by COASY shRNA sensitized HT-1080 cells to BSO treatment, which was rescued by CoA (100 μM), 4’-phosphopantetheine (4’-PPT, 100 μM), ferrostatin-1 (Fer-1, 10 μM). (J) CoA inhibited ferroptosis induced by the combination of BSO and PANKi. HT-1080 cells were treated with PANKi (2.5 μM) and an increasing dose of BSO in combination with ferrostatin-1 (Fer-1, 10 μM), liproxstatin-1 (lipro, 2 μM), Trolox (100 μM), or CoA (100 μM). The cell viability was quantified by Cell-Titer Glo assay. (K-L) PANKi, but not BSO, increased mitochondrial lipid peroxidation (PANKi 2.5 μM, BSO 1 mM, 18 hours) in HT-1080 cells as determined by the sensor of mitochondrial lipid peroxidation (mitoPerOx) staining (K) and the quantification of % mitochondrial lipid peroxidation positive cells (L). (M) COASY knockdown in HT-1080 cells triggered mitochondrial lipid peroxidation, which was rescued by CoA treatment, as quantification by % mitochondrial lipid peroxidation (mitoPerOX) positive cells.

Figure 3. CoAlation of Cys-483 on TXNRD2 protein regulates the thioredoxin reductase activity.

(A) CoA supplement increased the CoAlation of TXNRD2 and its interaction with TXN2. HT-1080 cells overexpressing TXNRD2 and TXN2 were supplemented with CoA (100 μM, 18 hours), and the TXNRD2-TXN2 interaction was evaluated by co-immunoprecipitation. (B) The specificity of the CoAlation antibody was verified by DTT, which abolishs the disulfide bond. V5-tag purified TXNRD2 was treated with oxidized CoA or combined with DTT, resolved on non-reducing PAGE, and blotted for Western blots. (C) CoAlation increased thioredoxin reductase activity of TXNRD2. The enzymatic activities of thioredoxin reductase of purified TXNRD2 protein with or without CoAlation or in combination with TXNRD inhibitor (Auranofin). The graph also included the background (bk) and positive control of thioredoxin reductase (TrxR(+)). (D) Tandem mass spectrum of CoA-modified peptide “C*GASYAQVMR” within TXNRD2. (E) Extracted Ion chromatograms (EIC) of the 2+ precursor ion corresponding to “C*GASYAQVMR in the DMSO control non-CoA sample and CoA treated sample demonstrating the lack of modified peptide in the DMSO control. (F) Replacing Cys-483 with Alanine abolished most of the CoAlation on the TXNRD2 protein. Purified wild-type or C483A mutant TXNRD2 proteins were resolved on non-reducing PAGE and Western blots for protein CoAlation. (G) Replacing Cys-483 with Alanine in TXNRD2 (C483A) protein abolished thioredoxin reductase activity. bk, background. (H) Erastin reduced the CoAlation on TXNRD2, which was restored by CoA treatment. HT-1080 cells overexpressing TXNRD2 were treated with erastin (2 μM) or in combination with CoA (100 μM) for 16 hours. TXNRD2 proteins were purified by V5 tag and Western blots for CoAlation. (I) Cys-483 on TXNRD2 determines its function against ferroptosis induced by the combination of PANKi and BSO. HT-1080 cells were transduced with lentiviral sgRNA against TXNRD2 to knock out endogenous TXNRD2 expression and overexpressed with TXNRD2 wild-type or C483A mutant with synonymous mutation to avoid targeting by TXNRD2 sgRNA. These cell lines were treated with the combination of BSO (300 μM) and various concentrations of PANKi for Cell-Titer Glo assay.

Figure 4. Disruption of CoA biosynthesis in PKAN fibroblasts and OBSC leads to mitochondrial lipid peroxidation.

(A) PANK1 knockdown sensitized HT-1080 cells to BSO treatment. HT-1080 cells with shRNA targeting PANK1, PANK2, or PANK3 were treated with different doses of BSO for three days for Cell-Titer Glo assay. (B) PANK1, PANK2, and COASY knockdown by shRNA in HT-1080 cells showed an increase in mitochondrial lipid peroxidation. (C-D) PKAN, when compared with healthy, fibroblasts showed elevated mitochondrial lipid peroxidation. Four pairs of fibroblasts from PKAN patients and unaffected individuals were stained with the sensor of mitochondrial lipid peroxidation (mitoPerOx) (C) and the quantification of % mitochondrial lipid peroxidation positive cells (D). (E-F) The cell death triggered by inhibiting CoA using PANKi in OBSC was rescued by ferrostatin-1. OBSC transfected with YFP were treated with PANKi (2.5 μM) or in combination with ferrostatin-1 (Fer-1, 2 μM) for one day, and the neuron numbers were accessed by fluorescence microscopy (E) and quantified in (F). (G-H) The elevated mitochondrial lipid peroxidation by PANKi was rescued by ferrostatin-1. After 1 day of treatment with PANKi (2.5 μM) and Fer-1 (2 μM), OBSC was disassociated and stained with mitoPerOx (G) for quantification (H). (I-J) PANKi treatment of brain slices repressed the levels of reduced and active 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 homogenized to blot for mitochondrial PRDX3 (I) and quantification (J).