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. 2021 Jan 5;33(1):174-189.e7.
doi: 10.1016/j.cmet.2020.12.007. Epub 2020 Dec 22.

Non-canonical Glutamate-Cysteine Ligase Activity Protects against Ferroptosis

Yun Pyo Kang  1 , Andrea Mockabee-Macias  1 , Chang Jiang  1 , Aimee Falzone  1 , Nicolas Prieto-Farigua  1 , Everett Stone  2 , Isaac S Harris  3 , Gina M DeNicola  4
Affiliations

Non-canonical Glutamate-Cysteine Ligase Activity Protects against Ferroptosis

Yun Pyo Kang et al. Cell Metab. .

Abstract

Cysteine is required for maintaining cellular redox homeostasis in both normal and transformed cells. Deprivation of cysteine induces the iron-dependent form of cell death known as ferroptosis; however, the metabolic consequences of cysteine starvation beyond impairment of glutathione synthesis are poorly characterized. Here, we find that cystine starvation of non-small-cell lung cancer cell lines induces an unexpected accumulation of γ-glutamyl-peptides, which are produced due to a non-canonical activity of glutamate-cysteine ligase catalytic subunit (GCLC). This activity is enriched in cell lines with high levels of NRF2, a key transcriptional regulator of GCLC, but is also inducible in healthy murine tissues following cysteine limitation. γ-glutamyl-peptide synthesis limits the accumulation of glutamate, thereby protecting against ferroptosis. These results indicate that GCLC has a glutathione-independent, non-canonical role in the protection against ferroptosis by maintaining glutamate homeostasis under cystine starvation.

Keywords: GCLC; NRF2; cysteine; cystine; ferroptosis; glutamate; γ-glutamyl.

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Conflict of interest statement

Declaration of Interests I.S.H. is a consultant for Ono Pharma USA. E.S. is an inventor of intellectual property related cyst(e)inase, and has an equity interest in Aeglea Biotherapeutics, a company pursuing the commercial development of cyst(e)inase. These companies had no role in funding or the design of this study.

Figures

Figure 1.
Figure 1.. Transsulfuration cannot support NSCLC cysteine pools.
(A) Measurement of NSCLC cell death under cystine starved (0 mM) or replete (200 mM) conditions treated with Vehicle (0.1% DMSO) or Ferrostatin-1 (Fer-1, 10 μM) (N=4). Cell death was determined by Incucyte analysis of Sytox Green staining over 73 hrs, followed by normalization to cell density. Area under the curve (AUC) calculations are presented here. Full curves can be found in Figure S1D. (B) Schematic depiction of [13C3]-serine tracing into cysteine (M+3, 3 carbons labeled) and glutathione (M+2, 2 carbons labeled from glycine; M+3, 3 carbons labeled from cysteine). (C-F) Quantitation of [13C3]-serine tracing into (C) serine, (D) glycine, (E) cysteine, and (F) glutathione (GSH) following culture under cystine starved (−) or replete (+) conditions for 4 hrs (N=3). For A and C-F, data are shown as mean ± SD. N is number of biological replicates. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. For A, a one-way ANOVA with Bonferroni’s multiple comparison test was used for statistical analyses. An unpaired two-tailed t test was used for the statistical comparisons between non-labeled M+0 fraction in E and M+2 labeling fractions in F.
Figure 2.
Figure 2.. Cystine starvation induces glutamate-derived γ-glutamyl-peptide accumulation.
(A) Scatter plot comparison of non-targeted metabolomics features in A549 cells cultured under cystine replete (+Cys2) and starved conditions (-Cys2) for 4 hrs (left). The mean intensity of median-normalized LC-MS peaks of each group (N=3) are plotted on the axes, and each dot represents an individual LC-MS peak. The LC-MS peaks that highly accumulated under cystine starvation (red dots) were further identified and annotated (right). (B-C) A549 cell 13C5, 15N2-Gln tracing into (B) Gln, Glu, GSH, and (C) γ-Glu-peptides following culture in cystine replete or starved conditions for 4 hrs (N=3). (D) Correlation between ferroptotic cell death (AUC, from Figure 1A) and the levels of γ-Glu-peptides across 13 NSCLC cell lines. The γ-Glu-peptides were analyzed following cystine starvation for 12 hrs and normalized to the mean value of H1581 cells under cystine replete conditions (N=13). For B-D, data are shown as mean ± SD. N is number of biological replicates. For D, Pearson correlation test was used for statistical analysis.
Figure 3.
Figure 3.. GCLC mediates γ-glutamyl-peptide synthesis in cell culture.
(A) Correlation of γ-Glu-dipeptides (data from Figure 2D) with GCLC expression in NSCLC cell lines. The GCLC protein expression of each cell line was normalized to the amount of HSP90 protein. The western blot can be found in Figure 5A. (B) Representative immunoblots of parental A549 cells, GCLC KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GCLC (+GCLCRes), and GSS KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GSS (+GSSRes). β-actin was used for the loading control. (C-D) Intracellular GSH levels (C) and γ-Glu-peptides levels (D) in the cells from (B) under cystine replete or starved conditions for 2.5 hrs (N=3). The data were normalized to the mean value of parental A549 cells under cystine replete conditions. (E) Schematic depicting the non-canonical, γ-Glu-peptide synthesis activity of GCLC. For C and D, data are presented as mean ± SD. n.d., not detected. N is number of biological replicates. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001; ##P<0.01, ###P<0.001, ####P<0.0001. For C, a one-way ANOVA with Bonferroni’s multiple comparison test was used for statistical analyses for the comparison of Parental, GCLCKOA/B + GFP, and GSSKO A/B + GFP. For the comparison between GCLC KO or GSS KO group (GCLCKO A/B + GFP or GSSKO A/B + GFP) and their GCLC or GSS reconstituted group (GCLC KOA/B + GCLCRes or GSSKO A/B + GSSKOA/B + GSSRes), an unpaired two-tailed t test was used. For D, an unpaired two-tailed t test was used for the comparison with parental cells under cystine replete conditions [+(Cys)2].
Figure 4.
Figure 4.. GCLC mediates γ-glutamyl-peptide synthesis in vivo.
(A) Schematic depicting the Cyst(e)inase and BSO treatment schedule for the depletion of extracellular cyst(e)ine and inhibition of Gclc. Cyst(e)inase (75 mg/kg) or vehicle (PSB) were administered, followed by treatment with BSO (100 mmol/kg) or vehicle (saline) 24 hours later. Tissues were collected after 4 hrs. (B) Evaluation of Gclc mRNA expression after tamoxifen (Tam)-inducible Gclc deletion in the adult mouse. Gclcf/f (control, Gclc functional) and Gclc−/− (Gclc knockout). The mRNA levels are normalized to the mean value of Gclcf/f mouse tissues (N=4). (C-E) Analysis of serum cystine (C), GSSG (D), and γ-Glu-peptide levels (E) in mice treated with Cyst(e)inase/BSO. (F-H) Analysis of liver cysteine (F), GSH (G), and γ-Glu-peptide levels (H) in the mice from (C-E). The metabolite levels are normalized to the mean value of PBS/saline treated mice (N=5). (I-J) Analysis of serum GSSG (I) and γ-Glu-peptide levels (J) in Gclcf/f and Gclc−/− mice. (K-L) Analysis of and liver GSH (K) and γ-Glu-peptide levels (L) in the mice from (I-J). The metabolite levels are normalized to the mean value of Gclcf/f mice (N=4). For B-L, data are presented as mean ± SD. N is number of biological replicates. n.d., not detected. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. For B and I-L, an unpaired two-tailed t test was used for the statistical comparisons. For C-H, a one-way ANOVA with Bonferroni’s multiple comparison test was used for statistical analyses.
Figure 5.
Figure 5.. NRF2 promotes γ-glutamyl-peptide synthesis via GCLC.
(A) Representative immunoblot of NRF2, GCLC, GSS, and GCLM expression in NRF2LOW and NRF2HIGH (KEAP1 mutant: A549, HCC15, H460, H2172, H1792, and H1944) NSCLC cell lines. HSP90 is used as loading control. (B) Comparison of γ-Glu-dipeptide levels in KEAP1WT and KEAPMUT NSCLC cell lines. Individual cell line data are found in Figure 5SA, and also correspond to the data in Figure 2D. The γ-Glu-peptides were analyzed following culture in cysteine replete and starved conditions for 12 hrs and normalized to the mean value of H1581 cells under cystine replete conditions. (C) Representative immunoblot of NRF2, GCLC, and GCLM expression in NRF2 KO A549 cells transduced with empty vector (EV) or NRF2. β-actin is used as loading control. (D-E) Analysis of intracellular γ-Glu-dipeptides (D) and GSH levels (E) in the cells from (C) under cystine replete or starved conditions in the presence and absence of 100 μM BSO for 12 hrs. (F) Representative immunoblot of NRF2, GCLC, and GCLM expression in Calu3 cells pre-treated with 100 nM of KI-696 or vehicle (Veh, 0.1% DMSO) for 48 hrs. b-actin is used as loading control. (G-H) Analysis of intracellular γ-Glu-dipeptides (G) and GSH levels (H) in the cells from (F) under cystine replete or starved conditions in the presence and absence of 100 μM BSO for 12 hrs. For B, D, E, G, and H, data are presented as mean ± SD. N is number of biological replicates. n.d., not detected. **P<0.01, ***P<0.001, and ****P<0.0001. For B, D, E, G, and H, a one-way ANOVA with Bonferroni’s multiple comparison test was used for statistical analyses.
Figure 6.
Figure 6.. Dipeptide synthesis protects KEAP1 mutant cells from ferroptosis.
(A) Correlation between cell death under cystine starvation (AUC, from Figure 1A) and GCLC expression in NSCLC cells (N=13). GCLC expression is normalized to HSP90 expression and can be found in Figure 5A. (B) Evaluation of the influence γ-Glu-dipeptide treatment on the death of GCLC KO A549 cells under cystine starvation. Cells were treated with γ-Glud-ipeptides at the indicated concentration or Fer-1 (10 μM) as a positive control. Cell death was monitored with Sytox Green every 2 hrs for 49 hrs, normalized to cell density, and then AUCs were calculated (N=3). (C) Evaluation of the death of NRF2HIGH (N=7) and NRF2LOW (N=10) cell lines under cysteine replete and starved conditions in the presence and absence of 100 μM BSO. Each dot represents the mean AUC of each NSCLC cell line. Cell death was monitored with Sytox Green and individual cell line data is found in Figure S6B. (D) Evaluation of the death of NRF2 KO A549 cells transduced with empty vector (EV) or NRF2 under cystine replete and starved conditions in the presence and absence of 100 μM BSO (N=3). Cell death was monitored by Sytox Green every 2 hours for 65 hours, followed by AUC calculation. (E) Evaluation of the death of Calu3 cells pre-treated with 100 nM of KI-696 or vehicle (Veh, 0.1% DMSO) for 48 hrs, followed by culture under cystine replete and starved conditions in the presence and absence of 100 μM BSO (N=3). Cell death was monitored with Sytox Green every 2 hours for 47 hours, followed by AUC calculation. (F) Evaluation of the death of parental A549 cells, GCLC KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GCLC (+GCLCRes), and GSS KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GSS (+GSSRes) following cystine starvation for the indicated time points (N=3). (G) Evaluation of the death of GCLC KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GCLC (+GCLCRes), and GSS KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GSS (+GSSRes) treated with of Erastin (5 μM) for the indicated time points (N=3). (H) Evaluation of the death of the cells from (G) under the cystine starved conditions in the presence and absence of 100 μM BSO. (N=3). (I) Evaluation of the death of H1299Cas9 cells infected with sgRNAs (sgCon, sgGCLC or sgGSS), followed by reconstitution with GFP (+GFP), sgRNA-resistant GCLC (+GCLCRes), or sgRNA-resistant GSS (+GSSRes) under cystine starved conditions (N=4). For F-H, results are representative of 2 independent GCLC and GSS KO clones. For F-I, cell death was monitored by Cytotox Red (CR) every 2 hrs (I), 3 hrs (F, G) or 4 hrs (H). For B-I, data are presented as mean ± SD. N is number of biological replicates. *P<0.05, ***P<0.001, and ****P<0.0001. For A, a Pearson correlation analysis was used. For B-E, a one-way ANOVA with Bonferroni’s multiple comparison test was used for statistical analyses.
Figure 7.
Figure 7.. Dipeptide synthesis scavenges glutamate.
(A) Correlation between the intracellular concentrations of γ-Glu-peptide substrate amino acids (Glu, Gly, Ala, Val, Thr, Leu) following cystine starvation for 12 hours and cystine starvation AUCs (from Figure 1A) (N=13). Individual cell line Glu concentrations can be found in Figure 7D. (B) Correlation between xCT protein expression and the Glu export rate under cystine replete and starved conditions for 12 hrs in NSCLC cells. xCT expression is normalized to HSP90 expression and can be found in Figure S7A (N=13). (C) Correlation between GCLC protein expression and intracellular Glu levels following NSCLC culture in cysteine replete and starved conditions for 12 hours. GCLC expression is normalized to HSP90 expression and can be found in Figure 5A (N=13). (D) (Left) Analysis of intracellular Glu concentrations in NSCLC cell lines cultured under cystine replete or starved conditions for 12 hrs (N=3). (Right) Comparison of Glu concentrations between KEAP1 WT (N=7) and KEAP1 MUT NSCLC (N=6) cells. (E) Analysis of relative intracellular Glu levels in the NRF2 KO A549 cells transduced with empty vector (EV) or NRF2 cultured under cystine replete or starved conditions in the presence and absence of 100 μM BSO for 12 hrs (N=3). (F) Analysis of relative intracellular Glu levels in the Calu3 cells pre-treated with 100 nM of KI-696 or vehicle (Veh, 0.1% DMSO) for 48 hrs, followed by culture in cystine replete or starved conditions in the presence and absence of 100 μM BSO for 12 hrs (N=3). (G) Analysis of relative intracellular Glu levels of the parental A549 cells, GCLC KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GCLC (+GCLCRes), and GSS KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GSS (+GSSRes) cultured under cystine replete or starved conditions for 2.5 hrs (N=3). (H) (Left) Relative Glu levels in mouse liver treated with Cyst(e)inase or vehicle (PBS) for 24 hrs followed by treatment with BSO or vehicle (saline) for 4 hrs (for all exp group, N=5), and (Right) Relative Glu levels in the liver of Gclcf/f and Gclc−/− mice (for all exp group, N=4). (I) Analysis of the death of NRF2LOW (N=10) and NRF2HIGH (N=7) cells under cystine starved conditions treated with GluEE (5 mM) or AOA (0.5 mM), or Gln starvation for 49 hrs. Cell death was analyzed with Sytox Green and the mean AUC of each cell lines can be found in Figure S7I. (J) Analysis of intracellular A549 ROS levels with CellROX green following culture under cystine replete and starved conditions in the presence and absence of AOA (0.5 mM) or Gln starvation for 14 hrs (N=3). (K) Analysis of intracellular A549 ROS levels with CellROX green following culture under cystine replete and starved conditions in the presence and absence of GluEE (5 mM) for 8 hrs (N=3). (L) Schematic depiction of the GSH-independent, non-canonical role of GCLC in the protection against ferroptosis by maintaining glutamate homeostasis under cystine starvation. For D-K, data are presented as mean ± SD. N is number of biological replicates. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. For A-C, a Pearson correlation analysis was used. For D (left), G, H (right), and I, Unpaired two-tailed t tests were used for the statistical comparisons. For G, * is for the comparison with parental cells under cystine replete conditions [+(Cys)2] and # is for the comparison of parental group under cystine starved conditions [−(Cys)2]. For D (right), E, F, H (left), J, and K, one-way ANOVA with Bonferroni’s multiple comparison tests were used for statistical analyses.
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