Fig 2: Regulation of the Txn system and its effect on ERK and AKT phosphorylation in primary culture of mutant Kras pancreatic cellsPrimary pancreatic cells were derived from EL-Kras and KC mice and treated with the TxnR inhibitor auranofin. (A,B) Regulation of ERK and AKT phosphorylation by auranofin in (A) EL-Kras and (B) KC mice. (C,D) Auranofin induced changes in Prdx1 expression in (C) EL-Kras and (D) KC mice in non-reducing westerns (EL-Kras n=4, KC n=4). (Statistical analysis shown in Supplementary Figures 10 and 11. Supporting Txn and Srxn data shown in Supplementary Figures 11 and 12.)

Fig 3: Regulation of Kras signaling by Peroxiredoxin-1- In our model of Kras associated disruption of the Txn system, Kras activated signaling results in decreased Txn and Srxn expressionWe propose that (A) decreased Txn and Srxn expression result in elevated levels of oxidized Prdx1 in the cell and decreased inhibition of ERK signaling by reduced Prdx1Cys-SH. Upon overoxidation and/or oligomerization, Prdx1's function changes and becomes tumorigenic by enhancing pERK related signaling. Prdx1Oligo in this model represents higher molecular weight Prdx1 bands (p100, p250+). The effect of Prdx1 on AKT signaling may also decrease as a result of Prdx1's diminished redox function and its inability to reduce the active site of PTEN. (B) The redox cycle of Prdx1 and the Txn system. Reduction of Prdx1Cys-SOH and Prdx1 disulfide (Prdx1DS) requires Txn to return Prdx1 to its reduced, Prdx1Cys-SH state. Upon further oxidation to Prdx1Cys-SO2- or when Txn expression is inhibited (i.e. Kras suppressed Txn expression or auranofin treatment), Srxn is required to reduce Prdx1. Auranofin inhibits the function of TxnR, which reduces oxidized Txn.

Fig 4: ßHB upregulates Trx1. (A) Dosage effect of ßHB on Trx1 expression. Cardiomyocytes were treated with the indicated concentrations of ßHB for 16 h. n = 6–8 (Trx1) and 5–7 (Prdx1). (B) Time-dependent effect of 1 mM ßHB on Trx1 in cardiomyocytes. n = 5–7. (C) ßHB upregulates Trx1 in cardiac fibroblasts. Cardiac fibroblasts were treated with 3 mM ßHB for 16 h. n = 6. (D) Ketogenic diet consumption upregulates Trx1 in the heart in vivo. n = 4. Western blot analyses with indicated antibodies were performed. * p < 0.05 (A–D).

Fig 5: Nrf2 promotes the accumulation of intracellular cysteine and sulfur-containing metabolites.(a) Schematic of the murine Keap1R554Q allele. The Keap1R554Q allele was created by inserting a loxP-flanked wild-type Keap1 cDNA containing exons 3–5 upstream of a R554Q mutation in the endogenous exon 4 of the Keap1 gene. Prior to recombination, wild-type Keap1 protein is expressed due to splicing into the cDNA containing exons 3–5, and is referred to as Keap1WT. Following Cre-mediated excision of the loxP-flanked cargo, Keap1R554Q is expressed at physiological levels and is referred to as Keap1 R554Q. PA, poly A signal. (b) Western blot analysis of Nrf2, Nqo1, and ß-Actin levels following Nrf2 stabilization in wild-type (WT) vs. homozygous Keap1R554Q/R554Q (R554Q) MEFs. (c) LC-HRMS metabolomics profiling of Keap1R554Q/R554Q MEFs compared to Keap1WT/WT MEFs. GSH, glutathione. CSA, cysteine sulfinic acid. HTAU, hypotaurine. CTH, cystathionine. NAC, N-acetyl cysteine. SAH, S-adenosyl homocysteine. aKG, a-ketoglutarate. PYR, pyruvate. N = 3, representative of 2 individual MEF lines. (d–i) Quantitation of cystine ([CYS]2, d), cysteine (CYS, e), glutathione (GSH, f), cysteine sulfinic acid (CSA, g), hypotaurine (HTAU, h) and taurine (TAU, i) levels in Keap1R554Q/R554Q MEFs compared to Keap1WT/WT MEFs. Cysteine and glutathione were derivatized with N-ethylmaleamide (NEM) to prevent oxidation during extraction here and for all quantification experiments. TXN, thioredoxin. TXN-ox, oxidized thioredoxin. GSH-ox, oxidized glutathione. GLU, glutamate. GLY, glycine. N = 3.10.7554/eLife.45572.005Figure 1—source data 1.Nrf2 promotes the accumulation of intracellular cysteine and sulfur-containing metabolites.