Authors: Yun Pyo Kang1, Andrea Mockabee-Macias1 2 1,* · 2020. 5. 29. · Cysteine inadequacy can induce an iron-dependent form of cell death known as ferroptosis (Dixon et al., 2012)

Title: Non-canonical glutamate-cysteine ligase activity protects against ferroptosis

Authors: Yun Pyo Kang1, Andrea Mockabee-Macias1, Chang Jiang1, Isaac S. Harris2, and Gina

M. DeNicola1,*.

Affiliation: 1 Department of Cancer Physiology, H. Lee. Moffitt Cancer Center, Tampa, Florida, USA 2 University of Rochester Medical Center, Rochester, New York, USA

*For correspondence: [email protected]

Keywords: cystine, cysteine, ferroptosis, GCLC, glutamate, γ-glutamyl

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 uncharacterized. Here, we find that cystine starvation promotes ferroptosis not

only through the inhibition of glutathione (GSH) synthesis, but also through the accumulation of

glutamate. Surprisingly, we find that glutamate-cysteine ligase catalytic subunit (GCLC) prevents

glutamate accumulation through the generation of alternative γ-glutamyl peptides. Further,

inhibition of GCLC accelerates ferroptosis under cystine starvation in a GSH-independent

manner. These results indicate that GCLC has an additional, non-canonical role in the protection

against ferroptosis to maintain glutamate homeostasis under cystine starvation.

.CC-BY 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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https://doi.org/10.1101/2020.05.29.123802

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Introduction

Amino acids can play critical biosynthetic functions beyond their use for protein synthesis. A

notable example is the thiol-containing amino acid cysteine. Cysteine-derived molecules are

crucial for multiple cellular processes as a consequence of their sulfur moiety, which facilitates

diverse functions, including enzyme catalysis, energy transfer, and redox metabolism (Furuyama

and Sassa, 2000; Martinez-Reyes et al., 2016; Rouault, 2012; Solmonson and DeBerardinis,

2018; Vyas et al., 2016). Cysteine is a rate-limiting substrate for the synthesis of glutathione

(GSH) (Stipanuk et al., 2006), the most abundant intracellular antioxidant (Winterbourn and

Hampton, 2008). GSH is a tripeptide consisting of the amino acids cysteine, glutamate and

glycine. The synthesis of GSH occurs in two steps (Anderson, 1998). First, glutamate and

cysteine are ligated by GCLC, producing the dipeptide γ-glutamyl cysteine (γ-Glu-Cys). Next,

glycine is added to γ-Glu-Cys, producing the tripeptide GSH (γ-Glu-Cys-Gly). The antioxidant

activity of GSH is a consequence of its function as a cofactor to multiple antioxidant proteins,

including glutaredoxins (GRXs), GSH peroxidases (GPXs), and GSH S-transferases, thereby

removing reactive oxygen species (ROS) (Harris and DeNicola, 2020).

Because of both its reactive thiol moiety and its essential function in redox homeostasis, cysteine

levels are tightly regulated. While cysteine excess is prevented by overflow into the taurine

pathway (Stipanuk et al., 2009), cysteine demand is met by inducible regulation of cystine import.

Following oxidative stress, the expression of the cystine/glutamate exchange transporter xCT is

induced, (Habib et al., 2015) thereby facilitating the uptake of cystine and its reduction to cysteine.

In some tissues, most notably the liver, cysteine is also synthesized from homocysteine and

serine via the transsulfuration pathway (Beatty and Reed, 1980; Rao et al., 1990; Reed and

Orrenius, 1977). Given the important roles of cysteine, many cancers overexpress xCT (Ji et al.,

2018; Takeuchi et al., 2013; Timmerman et al., 2013), which is positively regulated by oncogenic

RAS (Lim et al., 2019) and NRF2 (Sasaki et al., 2002), and negatively regulated by the tumor

suppressor p53 (Jiang et al., 2015). Pharmacological targeting of cystine uptake can effectively

induce cancer cell death (Cramer et al., 2017; Dixon et al., 2012; Zhang et al., 2019), and cystine

starvation can impair growth in multiple in-vivo cancer models (Cramer et al., 2017; Zhang et al.,

2019).



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Cysteine inadequacy can induce an iron-dependent form of cell death known as ferroptosis

(Dixon et al., 2012). Ferroptosis is triggered by the reaction of polyunsaturated fatty acids (PUFA)

in membrane lipids with peroxyl radicals produced from iron (Fe2+) and ROS (Cao and Dixon,

2016; Yang et al., 2014), thereby inducing lipid peroxidation. Consistently, processes that

promote ferroptosis include increased ferritin uptake (Gao et al., 2015), ferritin degradation

(Mancias et al., 2014), synthesis of PUFA containing lipids (Dixon et al., 2015; Doll et al., 2017),

and mitochondrial ROS production (Gao et al., 2015; Gao et al., 2019). However, while cysteine

is directly linked to GSH synthesis, which can influence the levels of ROS and lipid peroxides

via GPX4 (Yang et al., 2014), cysteine availability can also influence the levels of cofactors and

metabolites within cells beyond its use for GSH synthesis. Importantly, the metabolic

consequences of cystine starvation are poorly understood.

To understand the metabolic consequences of the cystine starvation, we performed quantitative

stable isotope labeled metabolite tracing in non-small cell lung cancer (NSCLC) cells. We found

that glutamate accumulated due to impaired GSH synthesis, and promoted ferroptosis. Further,

we identified that under cysteine-deprived conditions, GCLC used other small, non-charged

amino acids in place of cysteine to generate γ-glutamyl peptides. This promiscuous activity

prevented glutamate accumulation to protect against ferroptosis. γ-glutamyl peptide synthesis

by GCLC was also evident in mouse tissues.

Results

Cystine starvation impairs GSH synthesis prior to the onset of ferroptosis

To evaluate the consequence of cystine starvation in NSCLC cells, we starved a panel of cell

lines of extracellular cystine and first monitored viability over time. Cystine starvation induced

the death of most cell lines between 24-48 hrs, with the exception of H460 and H1944 cells,

which were more resistant (Figure 1A). Cell death was confirmed to be ferroptosis due to both

the ability of the ferroptosis inhibitor Ferrostatin-1 (Fer-1) and iron chelator DFO (Dixon et al.,

2012) to rescue cell death (Figure 1A) and the morphological changes characteristic of

ferroptosis (Figure S1A). Next, we examined the metabolic consequences of cystine starvation.

To understand the immediate consequences of cystine starvation, we starved cells for 4 hrs,

which caused the rapid depletion of intracellular cysteine to almost undetectable levels in all cell



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lines (Figure 1B). To determine whether cystine starvation influenced cellular processes, we first

examined glutathione (GSH) synthesis. Cysteine is a rate limiting metabolite of GSH synthesis

(Stipanuk et al., 2006) and GSH also plays an important role in ferroptosis via ROS metabolism

(Dixon et al., 2012) and substrate of GPX4 (Conrad and Friedmann Angeli, 2015). Therefore, to

evaluate the effect of extracellular cystine starvation on GSH synthesis, we conducted

quantitative 13C3-serine tracing. 13C3-serine can be metabolized to 13C2-glycine (M+2) and 13C3-

cysteine (M+3), which are subsequently incorporated into GSH (M+2 and M+3, respectively,

Figure 1C). After 4 hours labeling, most of the serine fraction and half of the glycine fraction were

labeled (Figure S1B and S1C). Importantly, while the amount of newly synthesized glycine was

equivalent or increased in the cell lines following cystine starvation, the amount of M+2 glycine

incorporated into GSH was dramatically depleted. Minimal M+3 labeling was detected. In

addition, total GSH levels were lower, consistent with an inhibition of GSH synthesis. These

results indicate that the extracellular cystine starvation rapidly depletes intracellular cysteine

availability for GSH synthesis, which precedes the induction of ferroptosis.

Inhibition of GSH synthesis promotes glutamate accumulation

GSH synthesis consumes glutamate and glycine in addition to cysteine. We observed that

inhibition of GSH synthesis was associated with an accumulation of intracellular glycine and

glutamate in multiple NSCLC cell lines following cystine starvation (Figure 2A). In addition,

glutamate export is obligatory for cystine import and glutamate accumulation may also be

influenced by reduced cystine/glutamate exchange. Consistently, we observed that cystine

starvation decreased glutamate exportation (Figure 2A and D). Because glutamate was

previously shown to contribute ferroptosis (Gao et al., 2015), we examined whether glutamate

plays a causal role in cystine-starvation induced ferroptosis in NSCLC cells. We treated cells

with 5 mM glutamate diethyl ester (GlutEE), a concentration we confirmed increases intracellular

glutamate to similar levels as cystine starvation in A549 cells (Figure S2A and 2A). We found

that GlutEE treatment accelerated ferroptosis in multiple NSCLC cells (Figure 2B), while

glutamine starvation, which depleted intracellular glutamate (Figure S2D), or treatment with the

transaminase inhibitor AOA could rescue ferroptosis (Figure 2C). Interestingly, the effects of

AOA could be overridden by treatment with dimethyl-alpha-ketoglurate (DMαKG), suggesting

αKG or its downstream metabolite mediates the effects of glutamate. Glutamate was previously



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shown to promote ferroptosis via the TCA cycle and ROS generated from the oxidative

phosphorylation (Gao et al., 2015; Gao et al., 2019). Consistently, we found that GlutEE

promoted ROS accumulation under cysteine starved conditions (Figure S2B). Therefore, these

results demonstrate that inhibition of GSH synthesis by cystine starvation not only depletes GSH,

but also induces the accumulation of glutamate to promote ferroptosis.

GCLC prevents ferroptosis independent of GSH production

Multiple studies have demonstrated a potent, synergistic effect of GSH synthesis inhibition with

BSO with limitation of cystine uptake or availability (Cramer et al., 2017; Harris et al., 2015).

Consistently, we observe that BSO treatment promotes ferroptosis under cystine starvation in

most NSCLC cell lines (Figure 3A). However, we find that cystine starvation rapidly inhibits

intracellular GSH synthesis (Figure 1B). Importantly, while BSO treatment depleted GSH in

cystine replete cells as expected, it did not change GSH levels in cystine starved cells (Figure

3B). Thus, the accelerated cell death induced by BSO under cystine starvation may not be

explained by the depletion of GSH. Therefore, we examined whether GCLC could play a role in

cystine-starvation induced ferroptosis independent of GSH synthesis. To evaluate this question,

we generated GCLC and GSS KO A549 clones (Figure 3C). Importantly, both clones were

defective in GSH synthesis as evidenced by significantly reduced intracellular GSH levels

effectively compared to parental cells (Figure 3D). Importantly, the GCLC KO clones

demonstrated accelerated ferroptosis induction under cystine starvation compared to parental

cells, which could be rescued by GCLC cDNA, while the GSS KO clones did not (Figure 3E).

Further, BSO treatment induced ferroptosis in the GSSKO clone (Figure 3F), despite the

absence of GSH (Figure 3D), further confirming the GSH-independent role of GCLC in

ferroptosis protection. Finally, GCLC KO, but not GSS KO, accelerated ferroptosis induction

under cystine starvation following acute deletion in H1299 cells (Figure S3A-C), suggesting this

is not a consequence of adaptation in single cell clones. Together, these data indicate that GCLC

can prevent cystine starvation-induced ferroptosis of NSCLC cells independent of GSH

production (Figure 3G).

Cystine starvation induces GCLC-dependent γ-glutamyl peptide accumulation



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To determine the mechanism of GSH-independent protection against ferroptosis by GCLC, we

conducted non-targeted metabolomics. Interestingly, we discovered a cluster of LC-MS peaks

which were highly depleted by BSO treatment following cystine starvation in A549 cells (Figure

4A). Further, these peaks were the same ones that were the most highly accumulated by

extracellular cystine starvation (Figure 4A). These unknown LC-MS peaks were identified as γ-

glutamyl-di or tri-peptides, which all contain a glutamate-derived moiety (Figure 4A). Authentic

standards for γ-glutamyl threonine (γ-Glu-Thr) and γ-glutamyl-alanyl-glycine (γ-Glu-Ala-Gly)

were not available, thus we further validated their identity via stable isotope labeled metabolite

tracing. The 13C5, 15N2-glutamine tracing result indicated that both γ-Glu-Thr and γ-Glu-Ala-Gly

were derived from glutamate (Figure 4D). Further, 2, 3, 3-2H3-serine tracing showed that γ-Glu-

Ala-Gly was derived from the glycine (Figure S4A). We extended these observations to other

NSCLC cell lines and found that cystine starvation consistently promoted the accumulation of γ-

glutamyl peptides, which was inhibited by treatment with BSO (Figure 4B). These results suggest

that cystine starvation promotes the accumulation of γ-glutamyl peptides by the GSH synthesis

pathway.

γ-glutamyl peptide synthesis by GCLC scavenges glutamate to protect against

ferroptosis

The tripeptide γ-glutamyl-2-aminobutyryl-glycine (γ-Glu-2AB-Gly) is known to be generated by

GCLC and GSS (Huang et al., 1988; Oppenheimer et al., 1979) in a similar manner to GSH by

substituting 2-aminobutyrate for cysteine (Figure S4B). Consequently, the accumulation of γ-

Glu-2AB-Gly under cysteine starvation can be explained by cysteine unavailability for GCLC

(Figure 4B and S4B). In contrast, γ-glutamyl-dipeptides are reported to be derived from GSH by

γ-glutamyl transferase (GGT) extracellularly (Figure S4B) (Hanigan and Pitot, 1985). However, 13C5, 15N2-glutamine tracing demonstrated that while the newly labeled GSH fraction was very

small, as expected, glutamate and γ-glutamyl-dipeptides were newly labeled to ~ 50% in cystine

starved A549 cells (Figure 4C and D), suggesting that the γ-glutamyl dipeptides were

synthesized from glutamate but not from GSH (Figure S4B). Because γ-glutamyl-valine is

synthesized by the Saccharomyces cerevisiae glutamate-cysteine ligase (Sofyanovich et al.,

2019), and γ-glutamyl dipeptide synthesis by mouse liver extracts was recently shown to be

GCLC-dependent (Kobayashi et al., 2020), we hypothesized that the γ-glutamyl dipeptides were



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directly generated by GCLC rather than GSH metabolism by GGT. To evaluate this, we

evaluated the γ-glutamyl dipeptide levels in the GCLC and GSS KO clones. Importantly, the γ-

glutamyl dipeptides that were accumulated following cystine starvation in parental cells were

dramatically depleted only in the GCLC KO clones (Figure 5A). Interestingly, the γ-glutamyl

dipeptide levels were generally higher in GSS KO clones than parental lines, which can be

explained by the feedback inhibition of GCLC by GSH, and more weakly by γ-Glu-2AB-Gly

(Richman and Meister, 1975) (Figure 5A). In addition, both γ-Glu-2AB-Gly and γ-Glu-Ala-Gly

tripeptides were depleted by both GCLC and GSS KO compared to parental cells, as GSS

activity is required for the ligation of glycine. Consistent alterations of γ-glutamyl peptides were

observed in GCLC and GSS KO H1299 cells, which were rescued by GCLC or GSS restoration

(Figure S5A). These results indicate that γ-glutamyl dipeptides are directly generated by GCLC

under cystine starved condition (Figure 5B).

As we found that GCLC inhibition with BSO treatment or genetic KO could accelerate ferroptosis

under cystine starvation (Figure 3A and E), we examined whether GCLC-mediated γ-glutamyl

dipeptide synthesis plays a causal role in this process. Because glutamate accumulation

promoted ferroptosis (Figure 2), we evaluated the ability of γ-glutamyl dipeptides to serve as a

glutamate sink. Both inhibition of GCLC with BSO treatment and GCLC KO increased

intracellular glutamate levels under cystine starvation, while GSS KO was actually protective

(Figure 5C and D). We also observed an accumulation of glutamate in GCLC KO, but not GSS

KO H1299 cells under cystine starvation (Figure S5B). Importantly, the γ-glutamyl dipeptides

themselves did not play a protective role against ferroptosis as their supplementation did not

rescue cystine starvation-induced ferroptosis of GCLC KO clones (Figure S5C). Finally, cystine

starvation-induced ferroptosis of GCLC KO cells was rescued by both glutamine starvation and

AOA treatment (Figure S5D). Together, these results demonstrate that GCLC has a non-

canonical role in ferroptosis to balance the glutamate pool to protect against ferroptosis under

cystine starvation (Figure S5E).

GCLC regulates glutamate homeostasis in vivo

Finally, we examined whether GCLC mediates the synthesis of γ-glutamyl peptides in vivo under

normal physiological conditions. To this end, systemic Gclc deletion was induced in an adult



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mouse (Figure 6A) and we examined the effect in the liver, kidney and serum. Efficacy of Gclc

deletion was evident by the depletion of glutathione by 75-90% in these tissues (Figures 6B-D).

While glutathione was present in the reduced (GSH) form in tissues, the serum had

predominantly the oxidized form (GSSG), which may either be due to the oxidizing extracellular

conditions or oxidation during sample preparation. Further, we found that Gclc KO liver, kidney,

and serum were also depleted of γ-glutamyl-peptides, including both the dipeptides and

tripeptides (Figures 6B-D). In addition, deletion of Gclc increased glutamate levels in the liver

and serum, but not the kidney (Figures 6B-D). Overall, these results indicate that GCLC plays a

causal role in the homeostatic control of glutamate and γ-glutamyl peptide metabolism in vivo

(Figure 6E).

Discussion

The findings reported herein demonstrate that g-glutamyl peptide synthesis by GCLC provides

GSH-independent protection from ferroptosis following cystine starvation. While cystine

starvation-induced ferroptosis has commonly been attributed to the depletion of cellular GSH,

we show that cystine starvation induces complex metabolic changes within cells. Our work does

not exclude the importance of GSH in the protection against ferroptosis. GSH is a major

intracellular antioxidant and substrate for GPX4 for lipid hydroperoxide detoxification (Dixon and

Stockwell, 2019; Yang et al., 2014). However, our work demonstrates that inhibition of GSH

synthesis causes a metabolic imbalance and accumulation of the amino acids glycine and

glutamate, which plays a causal role in ferroptosis induction. Our findings are consistent with

prior reports demonstrating that glutamate contributes to ferroptosis via ROS generation in the

mitochondria (Gao et al., 2015; Gao et al., 2019). Previous studies have found that combined

inhibition of cystine uptake with glutathione synthesis can synergistically inhibit the viability of

cells and tumors (Cramer et al., 2017; Harris et al., 2015). Our work has important implications

for the interpretation of studies using BSO to inhibit GCLC. While many of those results may be

attributed to GSH depletion, the contribution of GCLC to g-glutamyl peptide synthesis and

glutamate scavenging may also play a very important role, particularly in the context of xCT

inhibition, where cells cannot export glutamate. Our findings warrant the development of potent

GSS inhibitors for the study of ferroptosis to distinguish these mechanisms. These inhibitors



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would also be valuable for therapeutic combinations with ferroptosis inducers, although they may

increase glutamate scavenging, which may affect cellular responses.

Our in vivo results provide direct genetic evidence to support the GCLC-mediated g-glutamyl

peptide production that was recently been reported in mouse liver extracts (Kobayashi et al.,

2020), where glutamate could be ligated with other amino acids in a reaction inhibited by BSO.

The promiscuity of GCLC toward amino acids other than cysteine is not a unique feature of this

enzyme, and has been shown for many other metabolic enzymes. For example, serine palmitoyl

transferase will also metabolize alanine or glycine when serine is limiting (Penno et al., 2010)

and glutamate-aspartate aminotransferase will also metabolize cysteine sulfinic acid (Weinstein

et al., 1988). In the case of GCLC, this feature may have been selected for during evolution, as

the S. cerevisiae homolog (Gsh1p) also has the ability to at least use valine (Sofyanovich et al.,

2019). Additional work is needed to determine which other amino acids are accepted by S.

cerevisiae Gsh1p. For the human enzyme, small, non-charged amino acids that are structurally

similar to cysteine can be used based on their appearance in g-glutamyl peptides, although the

full spectrum of amino acids has not been tested in a direct enzymatic assay.

Our findings may extend beyond conditions of cysteine deficiency. Systemic deletion of mouse

Gclc revealed that Gclc plays a role in the regulation of glutamate and g-glutamyl peptides levels

in normal tissue. Notably, glutamate accumulation was only observed in the liver but not the

kidney. Liver plays a critical role in GSH synthesis to supply the rest of the organism, which may

consume significantly more glutamate in liver than kidney (Ookhtens and Kaplowitz, 1998).

Similarly, cancer cells synthesize a significant amount of GSH (Balendiran et al., 2004; Huang

et al., 2001; Soini et al., 2001; Sun et al., 2019; Tatebe et al., 2002) and use glutamate for cystine

export (Ji et al., 2018; Shin et al., 2017; Takeuchi et al., 2013; Timmerman et al., 2013), which

may explain the robust accumulation of glutamate following cystine starvation in our NSCLC

cells. It is important to note that, in contrast to cell culture, depletion of g-glutamyl peptides in

Gclc KO tissue may be a consequence of both canonical, extracellular GGT mediated g-glutamyl

dipeptide production and the intracellular GCLC-mediated pathway. However, the accumulation

of glutamate and the depletion of g-glutamyl tripeptides, which require the activity of GSS,

strongly suggests that these peptides are being produced intracellularly. Supportively, the



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activity of GGT is negligible in the mouse liver compared to kidney (Kobayashi et al., 2020). It is

not known whether g-glutamyl peptides have additional functions in tissues beyond serving as a

reservoir for glutamate, and potentially other amino acids. g-glutamyl peptides levels have been

shown to be increased under conditions of liver injury, including drug-induced injury, hepatitis

infection, liver cirrhosis, and hepatocellular carcinoma (Soga et al., 2011). Additional work is

needed to understand the role of g-glutamyl peptide synthesis in these diseases.

We also find that GSS may regulate glycine homeostasis by producing g-glutamyl tripeptides,

including g-Glu-2AB-Gly and g-Glu-Ala-Gly. Future work is needed to both understand whether

GSS can use other amino acids besides glycine and determine the full spectrum of g-glutamyl

tripeptides produced by GSS. Further, we find that GSS deficiency actually enhances g-glutamyl

dipeptide synthesis, which can be explained by loss of feedback inhibition of GCLC by GSH.

These findings raise interesting implications for the metabolic phenotypes of patients with inborn

errors of glutathione metabolism. Although extremely rare, mutations in GCLC and GSS result

in hemolytic anemia. Interestingly, GSS mutant patients also present with 5-oxo-prolinuria, which

is not observed in GCLC deficiency (Ristoff and Larsson, 2007). While this 5-oxo-prolinuria has

been attributed to the accumulation of g-glutamylcysteine and its metabolism to 5-oxo-proline by

g-glutamylcyclotransferase (GGCT) (Ristoff and Larsson, 2007), our work suggests that other g-

glutamyl-amino acids are likely produced in this situation to contribute to 5-oxo-prolinuria. This

is likely to depend on the availability of cysteine, which would likely become limiting if the

feedback inhibition of GCLC is lost due to an inability to synthesize GSH.



https://doi.org/10.1101/2020.05.29.123802


Key Resources REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies GCLC (Mouse polyclonal Ab) Santa Cruz Biotechnology Cat#: sc-390811

Lot#: 1917 RRID: AB-2736837

GSS (Mouse polyclonal Ab) Novus Biologicals Cat#: NBP2-03351 Lot#: A01 RRID: N/A

HSP90 (Rabbit polyclonal Ab) Cell Signaling Technology Cat#: 4874S Lot# 5 RRID: 2121214

Chemicals, Peptides, and Recombinant Proteins Sytox Green Thermo Fisher Scientific Cat#: S7020 DMSO VWR Scientific Inc Cat#: 97063-136 0.4% PFA in PBS Thermo Fisher Scientific Cat#: J19943-K2 Tamoxifen Sigma-Aldrich Cat#: T5648-5G Arginine Sigma-Aldrich Cat#:A6969-25G Aspartate MP Biomedicals Cat#:219463380 Asparagine Sigma-Aldrich Cat#:A4159-25G Glutamate Sigma-Aldrich Cat#:G8415-100G Glutamine VWR Cat#:VWRL0131-

0100 Glycine VWR Cat#:BP381-1 Histidine Sigma-Aldrich Cat#:H5659-25G Hydroxy-L-proline VWR Cat#:TCH0296-5G Isoleucine VWR Cat#:AAJ63045-14 Leucine Sigma-Aldrich Cat#:L8912-25G Lysine Sigma-Aldrich Cat#:L8662-25G Methionine Sigma-Aldrich Cat#:M5308-25G Phenylalanine Sigma-Aldrich Cat#:P5482-25G Proline Sigma-Aldrich Cat#:P5607-25G Threonine VWR Cat#:97064-026 Tryptophan Sigma-Aldrich Cat#:T8941-25G Tyrosine Sigma-Aldrich Cat#:T1145-25G Valine Sigma-Aldrich Cat#:V0513-25G Glucose Sigma-Aldrich Cat#:G7021-100G [2H5]-GSH Santa Cruz Biotechnology Cat#: sc-489493 [13C3, 15N]-cysteine Cambridge Isotope

Laboratories Cat#: CNLM-3871-H-0.25



https://doi.org/10.1101/2020.05.29.123802


[2, 3, 3-2H3]-serine Cambridge Isotope Laboratories

Cat#: DLM-582- 0.1

[13C3]-serine Cambridge Isotope Laboratories

Cat#: CLM-1574-H-0.1

[13C5, 15N2]-glutamine Cambridge Isotope Laboratories

Cat#: CNLM-1275-H-0.1

METABOLOMICS AMINO ACID MIX STANDARD

Cambridge Isotope Laboratories

Cat#: MSK-A2-1.2

γ-glutamyl-alanine Santa Cruz Biotechnology Cat#: sc-300878 γ-glutamyl-glycine Bachem Cat#: 4003498.0001 γ-glutamyl-leucine Bachem Cat#: 4005004.0001 γ-glutamyl-valine Bachem Cat#: 4003707.0250 MeOH (HPLC grade) Sigma Aldrich Cat#: 34860-1 L-R H2O (HPLC grade) Fisher Chemical Cat#: W5-1 Acetonitrile (HPLC grade) Honeywell Cat#: 34967 N-ethylmaleimide (NEM) Alfa Aesar Cat#: 40526-06 Blasticidin Invivogen Cat#: ant-bl-1 Puromycin Invivogen Cat#: ant-pr-1 Hygromycin Invivogen Cat#: ant-hg-1 Glutamate diethyl ester (GlutEE) TCI Chemicals Cat#: G0179-5G Dimethyl alpha-ketoglutarate (DMαKG) Sigma Aldrich 349631-5G Cystine Sigma Aldrich Cat#: C6727-25G Ferrostatin-1 (Fer-1) Cayman Chemical

Company Cat#: 17729

Deferoxamine (DFO) Sigma Aldrich Cat#: D9533-1G AOA Santa Cruz Biotechnology Cat#: sc-207410 SHIN-1 Dr. Rabinowitz lab

Department of Chemistry and Lewis-Sigler Institute for Integrative Genomics (Princeton University)

N/A

L-Buthionine-(S,R)-Sulfoximine (BSO) Cayman Chemical Company or Sigma Aldrich

Cat#: 14484 or Cat#: B2515-500MG

2755 Glutamate Standard YSI Cat#: 027055 Critical Commercial Assays

CellRox green Fisher Scientific Cat#: C10444 Experimental Models: Cell Lines



https://doi.org/10.1101/2020.05.29.123802


PC9 Dr John Minna, Hamon Cancer Center Collection (University of Texas-Southwestern Medical Center)

RRID: CVCL_B260

H810 Dr John Minna, Hamon Cancer Center Collection (University of Texas-Southwestern Medical Center)

RRID: CVCL_1590


RRID: CVCL_1537

Calu3 Dr John Minna, Hamon Cancer Center Collection (University of Texas-Southwestern Medical Center)

RRID: CVCL_0609


RRID: CVCL_1479


RRID: CVCL_1511


RRID: CVCL_1524


RRID: CVCL_1550



https://doi.org/10.1101/2020.05.29.123802



RRID: CVCL_1495


RRID: CVCL_1508


RRID: CVCL_0459

HCC15 Dr John Minna, Hamon Cancer Center Collection (University of Texas-Southwestern Medical Center)

RRID: CVCL_2057

H2009 ATCC Cat#: CRL-5911 RRID: CVCL_1514



H441 ATCC Cat#: HTB-174 RRID: CVCL_1512

A549 ATCC Cat#: CCL-185 RRID:CVCL_1561

Lenti-X 293T Clontech Cat#: 632180 RRID: N/A

Experimental Models: Organisms/Strains Gclcf/f (Chen et al., 2007) R26-CreERT2 (Ventura et al., 2007)

Oligonucleotides



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Guide RNA for lentiCRISPR-V2 GCLC. Forward: 5’-caccgTAGATGTGCAGGAACTGG-3’ Reverse: 5’-aaacCCAGTTCCTGCACATCTAc-3

(Harris et al., 2019) N/A

Guide RNA for lentiCRISPR-V2 GSS. Forward: 5’-caccgGGTCTCTGGACCAAGACCGA-3’ Reverse: 5’-aaacTCGGTCTTGGTCCAGAGACc-3’

This study N/A

PCR primer for pLenti-hygromycin-GCLC. Forward: 5’-cgactctagaggatccatggggctgctgtcc-3’ Reverse: 5’-gaggttgattgtcgacctagttggatgagtcagttttacttcc-3’

This study N/A

PCR primer for pLenti-hygromycin-GSS. Forward: 5’-cgactctagaggatccatggccaccaactgg-3’ Reverse: 5’-gaggttgattgtcgactcacacagggtatgggttgtc-3’

This study N/A

Site directed mutagenesis primer for pLenti-hygromycin-GCLCRes. Forward: 5’- CCTGCACATCTACCACG -3’ Reverse: 5’- AACTGGAAGATCCCGTGCCG -3’

This study N/A

Site directed mutagenesis primer for pLenti-hygromycin-GSSRes. Forward: 5’- AAGACCGAAGACTGTTTGTGG -3’, Reverse: 5’- GGTCCAGAGACCCCTTTT-3’

This study N/A

Recombinant DNA lentiCas9-Blast Addgene Cat#: 52962 lentiCRISPR-V2 Addgene Cat#: 52961 lentiCRISPR-V2 GCLC; Using BsmBI restriction site, primers were annealed and cloned to progenitor of lentiCRISPR-V2.

This study N/A



https://doi.org/10.1101/2020.05.29.123802


lentiCRISPR-V2 GSS; Using BsmBI restriction site, primers were annealed and cloned to progenitor of lentiCRISPR-V2

This study N/A

MGC Human GCLC Sequence-Verified cDNA (pCMV-SPORT6-GCLC)

Dharmacon Cat#: MHS6278-202759380

MGC Human GSS Sequence-Verified cDNA (pOTB7-GSS)

Dharmacon Cat#: MHS6278-202830404

pLenti-hygro-GFP Addgene Cat#: 17446 pLenti-hygro-GCLC resistant to sgGCLC (pLGH-GCLCRes); The GFP of pLenti-hygro-GFP was excised and replaced with human GCLC cDNA using MGC Human GCLC Sequence-Verified cDNA (pCMV-SPORT6-GCLC) as a PCR template. The pLent-hygro-GCLC resistant to sgGCLC was further generated by the site-directed mutagenesis.

This study N/A

pLenti-hygro-GSS resistant to sgGSS (pLGH-GSSRes); The GFP of pLenti-hygro-GFP was excised and replaced with human GSS cDNA using MGC Human GSS Sequence-Verified cDNA (pOTB7-GSS) as a PCR template. The pLent-hygro-GSS resistant to sgGSS was further generated by the site-directed mutagenesis.

This study N/A

pCMV-dR8.2 dvpr Addgene Cat#: 8455 pCMV-VSV-G Addgene Cat#: 8454

Software and Algorithms EL Maven https://resources.elucidata.

io/elmaven Version 0.6. 1 or 0.10.0

GraphPrism https://www.graphpad.com/scientific-software/prism/

Version 8

IncuCyte S3 Essen BioScience IncuCyte S3 2018B Other

RPMI 1640 Medium Modified w/o L-Glutamine, w/o Amino acids, Glucose (Powder)

US biological Cat# R9010-01



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cysteine/cystine/glutamine/methionine free RPMI

MP Biomedicals Cat# 091646454

Dialyzed FBS (dFBS) Sigma Aldrich Cat# F0392 Methods

Animal Experiments

All animal studies were performed according to protocols approved by the Institutional Animal

Care and Use Committee, the Standing Committee on Animals at Harvard University. Gclcf/f

(Chen et al., 2007) and R26-CreERT2 (Ventura et al., 2007) mice were bred to generate Gclcf/f;

R26-CreERT2 mice (C57B6 background). Gclcf/f (Gclc WT) and Gclcf/f; R26-CreERT2 (Gclc KO)

adult mice (aged 11-17 weeks old) were treated with tamoxifen (20 mg/ml; dissolved in corn oil)

via daily intraperitoneal injection for 5 consecutive days at 160 mg/kg (tamoxifen/mouse body

mass). Mice were humanely euthanized (isoflurane inhalation followed by cervical dislocation)

14-16 days later and necropsies were performed. Tissues (liver, kidney) were removed and

serum was isolated; both were snap frozen on dry ice and stored at -80℃ prior to analysis.

Lentivirus generation

Lentiviruses were generated by overnight PEI transfection of 90% confluent Lenti-X 293T cells

(Clonetech) with target lentiviral plasmid, and packaging plasmids pCMV-dR8.2 dvpr and pCMV-

VSV-G in DMEM (10% FBS). The next day, the medium was changed to fresh DMEM (10%

FBS). After 24 hrs, the first batch of virus contained medium was collected and filtered by 0.45

μm PES filter. The second batch of virus contained medium was collected as following above,

further combined to the first batch and stored at -80°C until virus infection.

Lentiviral infection of NSCLC cells.

To increase of gene deletion efficiency in a polyclonal population, H1299 cells with stable

Streptococcus pyogenes Cas9 expression (H1299Cas9) were established by lentiviral

transduction and blasticidin selection (3 μg/mL) for 5 days, using lentiCas9-Blast (Greenfeld et

al., 2015). To generate GCLC or GSS KO cells, H1299Cas9 and A549 cells were infected with the

empty pLenti-CRISPR-V2 or pLenti-CRISPR-V2 encoding sgGCLC or sgGSS with 2 μg/mL of

polybrene, followed by puromycin selection (1 μg/mL) for 4 days. To select single clones for



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GCLC or GSS KO, A549 cells were further diluted to 0.5 cells/well in 96 well dishes in RPMI

supplemented with 1 μM of Fer-1. Cells were grown for 2 weeks, followed by expansion of clones.

GCLC or GSS KO was verified by loss of GCLC or GSS protein via immunoblotting.

Subsequently, cells were reconstituted with sgRNA-resistant cDNAs by infection with pLenti-

hygro-GFP, pLenti-hygro-GCLC resistant to sgGCLC (pLGH-GCLCRes), or pLenti-hygro-GSS

resistant to sgGSS (pLGH-GSSRes) followed by hygromycin selection (300 μg/mL) for 5 days.

Dead cell measurement with Incucyte

Cells were plated in black walled 96 well pates at a density of 10,000 cells/well in 100 μL final

volume. The next day, the medium was changed to 100 μL of experimental medium containing

20 nM of Sytox Green as follows: For the extracellular cystine starvation, cysteine/cystine,

methionine and glutamine free RPMI (MP Biomedicals) was supplemented with 100 μM

methionine and 10% dialyzed FBS (dFBS) containing 2 mM glutamine and/or 200 μM cystine as

indicated. In addition, 10 μM of Ferrostatin-1 (Fer-1), 100 μM of DFO, 5 mM of GlutEE, 0.5 or 5

mM of AOA, or 100 μM of BSO was further supplemented as indicated. The number of dead

cells and cell confluence were measured by IncuCyte S3 live-cell analysis system (Essen

BioScience, Ann Arbor, MI, USA) in a humidified tissue culture incubator at 37°C with 5% CO2.

Data were acquired by 10X objective lens in phase contrast and green fluorescence (excited

wavelength: 460 nm, emission wavelength: 524 nm, acquisition time: 400 ms) channel. Images

were acquired from each well at 3 hr intervals. Image and data processing were performed with

IncuCyte S3 software (Essen BioScience, Ann Arbor, MI, USA). Dead cell number was

normalized to cell confluence [Number of Sytox Green positive cells/mm2/cell confluent (%) of

total image].

Crystal Violet based cell viability assay.

Cells were plated in 96-well plates at a density of 10,000-20,000 cells/well in 100 μL final volume.

The next day, the medium was changed to 100 μL medium with different experimental conditions

as indicated. At the indicated time points, cells were fixed with 4% Paraformaldehyde, stained

with crystal violet, washed and dried. The crystal violet was dissolved in 10% acetic acid and the

absorbance was measured by 600 nm wavelength. The relative cell number was normalized to

control cells of each experimental set.



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Sample preparation for the liver tissue targeted metabolomics.

The frozen liver tissue samples were pulverized using a pre-chilled BioPulverizer (59012MS,

BioSpec), weighed frozen, and then placed on dry ice. The tissue metabolites were extracted in

80% MeOH at a final tissue concentration of 50mg/mL for 24 hrs at −80°C. After centrifugation

(17000 g, 20 min, 4°C), the supernatant was analyzed by LC-HRMS.

Sample preparation for the quantitative intracellular 13C3-serine tracing and cysteine

quantification.

NSCLC cells were plated in 6 well dishes and pre-conditioned in RPMI medium containing dFBS

(10%) overnight. For serine tracing, RPMI 1640 Medium without glucose and amino acids (MP

Biomedicals) was prepared from powder according the manufacturer’s instructions and

supplemented with glucose and amino acids to meet the RPMI formulation with the exception of

serine and cystine. The following day, the cells were quickly washed with 1 mL of warm

serine/cystine-free RPMI medium, followed by feeding with 13C3-serine containing medium

(serine/cystine-free RPMI + 10% dFBS + 1% Pen/Strep + 300 μM 13C3-Serine) lacking or

supplemented with 200 μM cystine. After 4 hrs, the medium was aspirated and the cells were

quickly washed with ice cold PBS. As described in previous study (Kang et al., 2019), the cellular

metabolites were extracted and derivatized with 0.5 mL of ice-cold extraction solvent (80%

MeOH:20% H2O containing 25 mM NEM and 10 mM ammonium formate, pH 7.0). The

concentration of internal standards in the extraction solvent were as follows: 4.18 μM [2H5]-GSH-

NEM, 2.49 μM [13C3, 15N]-serine and 2.48 uM [13C2, 15N]-glycine from METABOLOMICS AMINO

ACID MIX STANDARD (Cambridge Isotope Laboratories). For cysteine quantification, the

extraction solvent contained 10 μM [13C2, 15N]-cysteine-NEM. [2H5]-GSH-NEM and [13C2, 15N]-

cysteine-NEM were pre-prepared by reaction of 50 mM NEM (10 mM ammonium formate, pH

7.0) for 30 min as previously described (Kang et al., 2019). After incubation on ice for 30 min,

the NEM-derivatized metabolite extracts were cleared by centrifugation and the supernatant was

analyzed by LC-HRMS at the positive mode. Cell volume and number were determined using a

Scepter 2.0 cell counter (Millipore) and used to calculate the intracellular metabolite

concentrations.



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Sample preparation for intracellular 2, 3, 3- 2H3-serine tracing.

A549 cells were prepared as described for 13C3-serine tracing but medium containing 300 μM [2,

3, 3-2H3-serine]-was used. Further, 0.5 μM of SHIN-1 (Ducker et al., 2017) was included in the

cystine starved condition. After 12 hrs, the medium was aspirated, and cells were quickly washed

with ice cold PBS, and cellular metabolites were extracted with 1 mL of 80% MeOH (−80°C, 15

min). After scraping, the metabolite extract was transferred into an Eppendorf tube and cleared

by centrifugation (17000 g, 20 min, 4°C), followed by LC-HRMS analysis at the negative mode.

Sample preparation for the intracellular non-targeted metabolomics, and Glycine and

Glutamate quantification.

NSCLC cells were plated in 6 well dishes so they were 70% confluent at extraction and pre-

conditioned in RPMI medium containing dFBS (10%) overnight. The following day, the medium

was aspirated and the cells were quickly washed with 1 mL of RPMI (10% dFBS, 1% P/S),

followed by feeding with conditioning medium as indicated. 1 mL of the medium supernatant was

collected to assay glutamate as indicated. For the non-targeted metabolomics approach,

medium was aspirated and cells were quickly washed with ice cold PBS, followed by extraction

of cellular metabolites with 0.5 mL of 80% MeOH (−80°C, 15 min). For intracellular glutamate

and glycine quantification, the extraction solvent also contained 2.49 uM [13C5, 15N]-glutamate

and 2.48 uM [13C2, 15N]-glycine from METABOLOMICS AMINO ACID MIX STANDARD

(Cambridge Isotope Laboratories). After scraping, the metabolite extract was transferred into an

Eppendorf tube and cleared by centrifugation (17000 g, 20 min, 4°C), followed by LC-HRMS

analysis in the negative or positive mode. For glutamate and glycine quantification, cell volume

and number were further determined using a Scepter 2.0 cell counter (Millipore).

Quantitation of extracellular glutamate exportation

The extracellular medium collected above was transferred to the 96 well plates and the medium

glutamate levels was measured by YSI 2900 (Yellow springs, OH, USA) using 2755 glutamate

standard (5 mM) The extracellular glutamate secretion rate (nmol/µL of cell volume/hrs)

determined from cell volume measurements above.

LC-MS analysis.



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The LC-MS condition was identical with previously established method (Kang et al., 2019). For

the chromatographic metabolite separation, the Vanquish UPLC systems coupled to a Q

Exactive HF (QE-HF) mass spectrometer equipped with HESI (Thermo Fisher Scientific,

Waltham, MA). The column was a SeQuant ZIC-pHILIC LC column, 5 µm, 150 × 4.6 mm

(MilliporeSigma, Burlington, MA) with a SeQuant ZIC-pHILIC guard column, 20 × 4.6 mm

(MilliporeSigma, Burlington, MA). Mobile phase A was 10 mM (NH4)2CO3 and 0.05% NH4OH in

H2O while mobile phase B was 100% ACN. The column chamber temperature was set to 30°C.

The mobile phases were eluted as following gradient condition. 0-13min: 80% to 20% of mobile

phase B, 13-15min: 20% of mobile phase B. The ESI ionization mode was positive or negative.

The MS scan range (m/z) was set to 60-900. The mass resolution was 120,000 and the AGC

target was 3 × 106. The capillary voltage and capillary temperature were set to 3.5 KV and 320°C,

respectively. The 5 µL of sample was loaded. For targeted metabolomics, the LC-MS peaks

were manually identified and integrated by EL-Maven (Version 0. 6. 1) by matching with a

previously established in-house library (Kang et al., 2019). The peak area of target metabolites

was further normalized by the median value of identified metabolite peak areas or the peak area

of stable isotope labeled internal standards for further quantification as previously described

(Bennett et al., 2008). For the non-targeted metabolomics approach, the LC-MS peaks were

automatically extracted and aligned using the Automated Feature Detection function of EL-

Maven. After the normalization with median value of the intensities of LC-MS peaks, the

statistical analysis was conducted. The γ-glutamyl peptides peaks were putatively identified by

matching m/z value with online HMDB database (http://www.hmdb.ca), and further confirmed by

matching with m/z value and retention time of authentic standards. The standards of γ-glutamyl

threonine and γ-glutamyl-alanyl-glycine were not available and were instead validated by stable

isotope labeled metabolite tracing (Figure S4A and B), as described in result.

ROS measurement by CellRox green

NSCLC cells were plated at 70,000 cells/well to 24 well dishes and pre-conditioned in RPMI

medium containing dFBS (10%) overnight. The following day, the medium was aspirated and

the cells were quickly washed with the warm PBS followed by feeding with the indicated medium

conditions. The CellRox green was added to the cells at a final concentration of 5 μM for the

final 30 min of the experiment. Cells were washed with PBS, detached with trypsin, and



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transferred to Eppendorf tubes. After spin down (10sec, 17,000g), the supernatant was aspirated

and the pellet was re-suspended in 500 μL of PBS and filtered into FACS tubes. The mean

fluorescence intensity (MFI) of Green fluorescence from the 4,000 discrete events was

determined by AccuriTM C6 Flow cytometry (BD biosciences, An Arbor, Mi, USA). The MFI value

was obtained by the AccuriTM C6 software (BD biosciences, An Arbor, Mi, USA) and further

normalized by the control of experimental set.

Immunoblotting

Cell lysates were prepared in RIPA buffer (20 mM Tris-HCl, pH7.5; 150 mM NaCl, 1 mM EDTA,

1 mM EGTA, 1% NP-40, 1% sodium deoxycholate) containing protease inhibitors. After protein

quantification using Biorad DC assay, the samples were mixed with reducing buffer (v/v, 5:1)

containing 2-mercaptoethanol. The proteins were separated by SDS-PAGE using NuPAGE [4-

12% Bis-Tris gels (Invitrogen)] and transferred to 0.45 um Nitrocellulose membrane (GE

Healthcare). The membrane was blocked by 5% non-fat milk in TBST for 15 min, and the primary

antibodies were incubated overnight in blocking buffer as follows: GCLC - 1/1000 dilution with

5% non-fat milk in TBST; GSS - 1/2000 dilution with 5% non-fat milk in TBST; HSP90 - 1/5000

dilution with 5% non-fat milk in TBST. After wash membrane 3 times using TBST for 10 min of

each, the 10,000 times diluted secondary antibody in 5% non-fat milk in TBST (goat anti-rabbit

or goat anti-mouse, Jackson ImmunoResearch) were attached to membrane for 1 hr. After wash

the membrane in TBST for 3 times for 10 min of each, the enhanced chemo-luminescence signal

was measured by exposing to the x-ray film followed by fixing and developing.

Statistical analysis

Statistical analyses were conducted with Graph Pad Prism 8. For the comparison of two groups,

two-tailed Student’s t-test was used. For the comparison of more than 3 experimental groups,

one-way ANOVA was used with Bonferroni’s multiple comparison test.

Lead Contact

Further information and requests for resources and reagents should be directed to and will be

fulfilled by the Lead Contact, Dr. Gina M. DeNicola ([email protected])



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Acknowledgement

We would like to thank Dr. Joshua D. Rabinowitz for SHIN-1, Dr. Vince Luca, Dr. David

Gonzalez-Perez and Elliot Medina for flow cytometry assistance, Dr. Min Liu for LC-MS

assistance, Chen Tingan for Incucyte assistance, and all members of the DeNicola laboratory

for their very helpful discussions. This work was supported by grants from the NIH/NCI (R37-

CA230042) to G.M.D, the Ludwig Center at Harvard to I.S.H., the AACR-Takeda Oncology Lung

Cancer Research Fellowship (19-40-38-KANG) to Y.P.K., a National Pancreas Foundation grant

to C.J. This work was also supported by the Analytic Microscopy and the

Proteomics/Metabolomics Cores, which are funded in part by Moffitt’s Cancer Center Support

Grant (NCI, P30-CA076292), and grants from the Moffitt Foundation, and a Florida Bankhead-

Coley grant (06BS-02–9614) to the Proteomics/Metabolomics core.

Author Contributions

Y.P.K and G.M.D. designed the study and interpreted experimental results. Y.P.K performed all

metabolomics experiments, Y.P.K performed western blotting and cell viability experiments with

assistance from A.M-M., Y.P.K. generated KO cell lines with assistance from C.J., I.S.H.

generated Gclc knockout mice and collected tissues, Y.P.K and G.M.D wrote the manuscript

and all authors commented on it.

Declaration of Interests

The authors declare no competing financial interests. I.S.H. is a consultant for Ono Pharma USA,

who had no role in funding or design of this study.



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Figure legends: Figure 1. Cysteine starvation induces ferroptosis and impairs GSH synthesis. (A) Time

course Measurement of NSCLC cell death under cystine starved (0µM) or replete (200µM)

conditions treated with Vehicle (0.1% DMSO), Ferrostatin-1 (Fer-1, 10 μM) or DFO (100 μM).

(N=4). Cell death was determined by Sytox Green staining and normalized to cell density. (B)

Quantitation of intracellular cysteine (N=3) and (C) quantitative [13C3]-serine tracing into

glutathione following media change to cystine starved (-) or replete (+) conditions for 4 hrs. (N=3)

Data shown as mean ± SEM. N is number of biological replicates. **P<0.01, ***P<0.001, and

****P<0.0001. Unpaired two-tailed t test between M+2 labeling fractions was used for the

statistical comparisons in (B).

Figure 2. Inhibition of GSH synthesis promotes ferroptosis via glutamate accumulation.

(A) Quantitation of intracellular glutamate (Glut, upper) and glycine (Gly, middle), and exportation

rate of glutamate (bottom) in NSCLC cell lines under cystine replete or starved conditions for 12

hrs. (N=3). (B) Measurement of NSCLC cell death under cystine starved or replete conditions

treated with Vehicle, Fer-1 (10 uM), and/or glutamate diethyl ester (GlutEE, 5 mM) as indicated

(N=4 except H1299, cystine 0 μM + GlutEE group: N=3). (C) Measurement of NSCLC cell death

under cystine starved or replete conditions treated with Vehicle or AOA (0.5 mM), or without

media glutamine (-Gln) (N=4). Vehicle-treated cystine replete and starved data are from (B). For

A-C, data are shown as mean ± SEM (A, B, and C). n.s., not significant; *P<0.05, **P<0.01,

***P<0.001, and ****P<0.0001. N is number of biological replicates. An unpaired two-tailed t test

was used for statistical analysis in (A). (D) Schematic depiction of glutamate accumulation-

mediated ferroptosis promotion under cystine starved conditions.

Figure 3. GCLC prevents ferroptosis independent of GSH production. (A) Measurement of

NSCLC cell death under cystine starved or replete conditions treated with Vehicle (0.1% DMSO)

or BSO (100 μM). (N=4). Vehicle-treated cystine replete and starved data are from Fig. 1A. (B)

Intracellular glutathione levels under cystine starved or replete conditions treated with Vehicle

(0.1% DMSO) or BSO (100 μM) for 13 hrs. (N=3). Data was normalized by the mean value of

vehicle-treated cystine replete conditions. (C) Representative immunoblots of GCLC, GSS, and

HSP90 (loading control) from A549 GCLC KO clones (GCLCKOA and GCLCKOB), GSS KO



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clones (GSSKOA and GSSKOB), and parental A549 cells. (D) Intracellular GSH levels of A549

GCLC KO clones, GSS KO clones, and parental cells under cystine replete or starved conditions

for 3 hrs (N=3). Data was normalized by mean value of parental cells under cystine replete

conditions. (E) Relative cell number 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) under cystine starved condition treated with vehicle

or Fer-1 (10 μM) as indicated for 16 hrs (N=3). Data are normalized to the mean value of vehicle-

treated cystine replete conditions. (F) Relative cell number of A549 GSSKO cells under cystine

starved conditions treated with vehicle (0.1% DMSO), Fer-1 (10 μM), or DFO (100 μM) as

indicated for 16 hrs (N=3). Data are normalized to the mean value of vehicle-treated cystine

replete conditions. For A, B, D-F, data are shown as mean ± SEM. N is number of biological

replicates. n.s., not significant; **P<0.01, ****P<0.0001, and ####P<0.0001. For B, D, E, F, a one-

way ANOVA with Bonferroni’s multiple comparison test was used for statistical analyses. (G)

Schematic depicting the glutathione synthesis-independent role of GCLC to prevent ferroptosis

under cystine starved conditions.

Figure 4. Cystine starvation promotes γ-Glut-dipeptide production by GCLC. (A) Scatter

plot comparison of non-targeted metabolomics features in A549 cells (Left) between control and

BSO treated groups under cystine starved conditions and (middle) between cystine replete and

starved conditions. 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 highly altered

LC-MS peaks (red dots) were further identified and annotated (right). (B) The intensity of γ-

glutamyl di- and tripeptides in 4 NSCLC cells following cystine replete or starved condition and

treatment with vehicle or without BSO (100 μM) for 13 hrs (N=3). Data are normalized to the

median value of all LC-MS features in each sample. 13C5, 15N2-Gln tracing of A549 cells into (C)

Gln, Glut, GSH, and (D) γ-Glut-peptides following cystine starvation for 4 hrs (N=3). For B-D,

data are shown as mean ± SEM. N is number of biological replicates. n.d., not detected;

****P<0.0001. For B, a one-way ANOVA with Bonferroni’s multiple comparison test was used.

Figure 5. γ-glutamyl peptide synthesis by GCLC scavenges glutamate to protect against

ferroptosis. (A) Intracellular γ-glutamyl peptide levels in parental A549s and GCLC and GSS



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KO clones following cystine replete or starved conditions for 3 hrs (N=3). Data was normalized

by the mean value of parental lines under cystine replete conditions. (B) Schematic depicting

the non-canonical, γ-glutamyl dipeptide synthesis function of GCLC under cystine starvation.

GCLC consumes glutamate to produce alternative γ-glutamyl amino acids. (C) Intracellular

glutamate levels following cystine replete or starved conditions and treatment with vehicle or

BSO (100 μM) for 13 hrs (N=3). Data were normalized to the mean value of vehicle-treated

cystine replete conditions. (D) Intracellular glutamate levels in parental A549 cells and GCLC

and GSS KO clones following cystine replete or starved condition for 3 hrs (N=3). Data are

normalized to the mean value of cysteine-replete parental lines. For A, C, and D, data are shown

as mean ± SEM. N is number of biological replicates. n.d., not detected; n.s., not significant;

*P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. For A, C, and D, a one-way ANOVA with

Bonferroni’s multiple comparison test was used.

Figure 6. GCLC regulates glutamate homeostasis in vivo. (A) Tamoxifen (Tam)-inducible

deletion in the adult mouse. Gclcf/f (control, Gclc functional) and Gclc-/- (Gclc knockout).

Measurement of GSH, γ-Glu-peptides, and Glut levels in (B) liver and (C) kidney of Gclcf/f and

Gclc-/- mice. Measurement of GSSG, γ-Glu-peptides, and Glut levels of (D) serum in Gclcf/f and

Gclc-/- mice. For B-D, Data were normalized to the mean value of Gclcf/f group. For B-D, data

are shown as mean ± SEM. N is number of biological replicates. n.s., not significant; *P<0.05,

**P<0.01, ***P<0.001, and ****P<0.0001. For B-D, Unpaired two-tailed t test was used for the

statistical comparisons. (E) Schematic depiction of GCLC-mediated metabolic alterations in

mouse tissue.

Supplementary Figure legends: Figure S1. Cysteine starvation induces ferroptosis and impairs GSH synthesis. (Related

to Figure 1) (A) Representative image of sytox green (SG) stained A549 cells under cystine

starvation conditions for 1 or 49 hours treated with Vehicle (0.1% DMSO), Fer-1 (10 μM), or DFO

(100 μM). Images show the same well position at the two different time points (1 or 49 hrs). A

representative sytox green positive (SG+) cell is indicated with a white arrow. (B) Quantitative

[13C3]-Serine labeling of (B) serine (Ser) and (C) glycine (Gly) following media change to cystine



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starved (-) or replete (+) conditions for 4 hrs. (N=3 biological replicates). Data shown as mean

± SEM.

Figure S2. Inhibition of GSH synthesis promotes ferroptosis via glutamate accumulation.

(Related to Figure 2) (A) Quantitative measurement of intracellular glutamate levels in A549

cells cultured with the indicated concentration of GlutEE (N=3). (B) Measurement of intracellular

ROS levels under cystine starved or replete condition treated with vehicle or GlutEE (5 mM) for

4 hrs (N=3). Data was normalized to the mean value of cells under cystine replete conditions

without GlutEE. (C) Measurement of cell number under cystine starved or replete condition with

vehicle, GlutEE (5 mM), dimethyl alpha-ketoglutarate (DMαKG, 5 mM), and/or AOA (0.5 mM) as

indicated for 17 hrs (N=3). Data was normalized to the mean value of vehicle-treated cells under

cystine replete conditions. (D) Measurement of intracellular glutamate levels under cystine

starved or replete conditions cultured with or without media glutamine for 3 hrs (N=3). For A-D,

data are shown as mean ± SEM. N is number of biological replicates. n.s., not significant;

*P<0.05, ***P<0.001, and ****P<0.0001. For A and C, an unpaired two-tailed t test was used for

statistical analyses. For B and D, a one-way ANOVA with Bonferroni’s multiple comparison test

was used.

Figure S3. GCLC prevents ferroptosis independent of GSH production. (Related to Figure

3). (A) Representative immunoblots of GCLC and GSS from H1299Cas9 cells transfected with

LentiCrisprV2 with control sgRNA (sgCon) or sgRNA targeting GCLC (sgGCLC) and GSS

(sgGSS). Ponceau staining was used for the loading control. (B) Intracellular GSH levels of

H1299Cas9 infected with sgCon, sgGCLC or sgGSS reconstituted with GFP (+GFP), sgRNA-

resistant GCLC (+GCLCRes), or sgRNA-resistant GSS (+GSSRes) as indicated under cystine

replete or starved condition for 3 hrs (N=3). Data was normalized to the mean value of sgCon

with GFP cells under cystine replete conditions. (C) Relative cell number of H1299Cas9 infected

with sgCon, sgGCLC or sgGSS reconstituted with GFP, GCLCRes, or GSSRes as indicated under

cystine starved condition treated with vehicle (0.1% DMSO), Fer-1 (10 μM), DFO (100 μM) for

13 hrs (N=3). Data was normalized to the mean value of vehicle-treated cystine replete

conditions. Data shown as mean ± SEM (B and C). N is number of biological replicates.



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***P<0.001, ****P<0.0001. One-way ANOVA with Bonferroni’s multiple comparison test was

used to determined statistical significance for B and C.

Figure S4. Cystine starvation promotes γ-Glut-dipeptide production by GCLC. (Related to

Figure 4). (A) 2, 3, 3-2H3-Ser tracing of A549 cells under cystine replete or starved condition in

the presence and absence of SHIN-1 (0.5 uM) as indicated for 12 hrs (N=3). (B) Schematic

depiction of the mechanism of γ-glutamyl dipeptides synthesis under cystine replete and starved

conditions. While γ-glutamyl transferase (GGT) can generate of γ-glutamyl dipeptides from

glutathione, GCLC can generate γ-glutamyl dipeptides from glutamate and amino acids under

cystine starved conditions.

Figure 5S. γ-glutamyl peptide synthesis by GCLC scavenges glutamate to protect against

ferroptosis (Related to Figure 5). (A-B) Measurement of intracellular (A) γ-glutamyl-peptide

and (B) Glut levels of H1299Cas9 infected with sgCon, sgGCLC or sgGSS reconstituted with GFP

(+GFP), sgRNA-resistant GCLC (+GCLCRes), or sgRNA-resistant GSS (+GSSRes) as indicated

under cystine replete or starved conditions for 3 hrs (N=3). Data was normalized to the mean

value of sgCon +GFP cells under cystine replete conditions. (C) Analysis of cell number of GCLC

KO A549 clones following cystine replete or starved conditions and treatment with vehicle, γ-

glutamyl dipeptides (γ-Glu-Ala, γ-Glu-Leu, γ-Glu-Gly, and γ-Glu-Val; 2 mM), Fer-1 (10 uM), or

DFO (100 uM) as indicated for 14 hrs (N=3). The data were normalized to the mean value of

cystine replete, vehicle treated conditions for each clone. (D) Analysis of cell number of GCLC

KO A549 cells following cystine replete or starved conditions and treatment with vehicle,

glutamine starvation (0, 0.02, 0.2 mM), AOA (5 mM), Fer-1 (10 uM), or DFO (100 uM) as

indicated for 12 hrs (N=3). The data were normalized to the mean value of cystine replete,

vehicle treated conditions for each clone. For A-D, data are shown as mean ± SEM. N is

number of biological replicates. n.s., not significant; *P<0.05, ***P<0.001, ****P<0.0001. For A-

D, one-way ANOVA with Bonferroni’s multiple comparison test was used for statistical analyses.

(E) Schematic depiction of GSH-independent GCLC function that prevents ferroptosis under

cystine starvation via depletion of glutamate.



https://doi.org/10.1101/2020.05.29.123802


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Figure 1.

+ - + - + - + - + - + - + - + - + - + -0

5

10

15

GSH

(mM

)

M+0

(Cys)2:

A549Calu

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93H20

09

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******** ****

****

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(Cys)2

(Cys)2

Gly

Cys

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ExtracellularIntracellular

Glu

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CthHcys

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GSS

0 20 40 600

500

1000

A549

(Cys)2 200 µM(Cys)2 200 µM + DFO(Cys)2 200 µM + Fer1(Cys)2 0 µM(Cys)2 0 µM + DFO(Cys)2 0 µM + Fer1

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Figure 2

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Figure 3

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Figure 4

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- Metabolite annotation of featured LC-MS peaks (!)

* Identifed by matching with m/z and retention time of authentic standardƒ Identified by matching with m/z and 13C,15N or 2H labeled metabolite tracing

203.0674 5.24 γ-Glutamyl-glycine (γ-Glu-Gly)*217.0830 4.93 γ-Glutamyl-alanine (γ-Glu-Ala)*245.1142 4.02 γ-Glutamyl-valine (γ-Glu-Val)*247.0936 5.00 γ-Glutamyl-threonine (γ-Glu-Thr)ƒ

259.1299 3.28 γ-Glutamyl-leucine (γ-Glu-Leu)*274.1044 4.86 γ-Glutamyl-alanyl-glycine (γ-Glu-Ala-Gly)ƒ

288.1201 4.29 γ-Glutamyl-2-aminobutyryl-glycine (γ-Glu-2AB-Gly)*

Metabolite (abbreviation)m/z RT(min)

C

γ-Glu-

Gly

γ-Glu-

Ala

γ-Glu-

Val

γ-Glu-

Thr

γ-Glu-

Leu

γ-Glu-

Ala-Gly

γ-Glu-

2AB-G

ly0

2×105

4×105

6×105

Peak

Are

a

M+0M+5M+6

Gln GluGSH

0

5×107

1×108

Peak

Are

a M+0M+5M+6M+7

- (Cys)2

D

- (Cys)2



https://doi.org/10.1101/2020.05.29.123802


Figure 5

A

C D

- + - + - + - + - + - + - + - +0.00.51.01.52.0

Glu

tR

elat

ive

Inte

nsity

+ - + - + - + -BSO:

(Cys)2:

A549 H1299 H1975 HCC15

* * **** ***

**

p=0.057**** **

***

γ-Glu-Leu γ-Glu-Thr γ-Glu-Ala γ-Glu-Val γ-Glu-Gly γ-Glu-Ala-Gly γ-Glu-2AB-Gly0.1

1

10

100

Rel

ativ

e In

tens

ity

(Cys)2: + + - + - + - + - + - + -Parental GCLCKOA GCLCKOB GSSKOA GSSKOB

****

-

************

****

************

****

********

**

*******

***p=0.083

********

********

****

n.s.n.s.

**** **** ******** ****

************

****************

γ-Glu-dipeptides γ-Glu-tripeptides

Par

enta

lG

CLC

KO

AG

CLC

KO

BG

SS

KO

AG

SS

KO

BP

aren

tal

GC

LCK

OA

GC

LCK

OB

GS

SK

OA

GS

SK

OB

0

1

2

3

Glu

tR

elat

ive

Inte

nsity

+ (Cys)2 - (Cys)2****

******

Cys

g-GluCys

Glut

Gly

AAs

g-Glu-AAs

g-GluCysGly(GSH)

GCLC

GSS

- (Cys)2

GGT

Canonical

Non-Canonical

Cys

g-GluCys

Glut

Gly

AAs

g-GluAAs

g-GluCysGly(GSH)

GCLC

GSS

+ (Cys)2

GGT

Canonical

Non-Canonical

B



https://doi.org/10.1101/2020.05.29.123802


Figure 6

GSH0.0

0.5

1.0R

elat

ive

Inte

nsity

Gclcf/f

Gclc-/-

****

γ-Glu-

Gly

γ-Glu-

Val

γ-Glu-

Leu

γ-Glu-

Ala-Gly

γ-Glu-

2AB-G

ly0.0

0.5

1.0

1.5

Rel

ativ

e In

tens

ity

Gclcf/f

Gclc-/-

**

P=0.

080

**

***

Glut0

1

2

3

Rel

ativ

e In

tens

ity

Gclcf/f

Gclc-/-

***

Liver

γ-Glu-

Gly

γ-Glu-

Val

γ-Glu-

Leu

γ-Glu-

Ala-Gly

γ-Glu-

2AB-G

ly0

1

2

3

Rel

ativ

e In

tens

ity

Gclcf/f

Gclc-/-

* *

n.s.

**

p=0.0636

GSH0.0

0.5

1.0

1.5

Rel

ativ

e In

tens

ity

Gclcf/f

Gclc-/-

**

Glut0.0

0.5

1.0

1.5

Rel

ativ

e In

tens

ity

Gclcf/f

Gclc-/-

n.s.

γ-Glu-

Gly

γ-Glu-

Ala

γ-Glu-

Val

γ-Glu-

Thr

γ-Glu-

Leu

γ-Glu-

Ala-Gly

γ-Glu-

2AB-G

ly0.0

0.5

1.0

1.5

Rel

ativ

e In

tens

ity

Gclcf/f

Gclc-/-

n.s.

******

****** ***

**

Serum

GSSG0.0

0.5

1.0

Rel

ativ

e In

tens

ity

Gclcf/f

Gclc-/-

*

Glut0

1

2

3

Rel

ativ

e In

tens

ity

Gclcf/f

Gclc-/-

p=0.082

A B C

DE

Kidney

Gclcf/f

Cys

GSH

Glut

Gly

Glut

GCLC

GSS

!-Glut-AAs

AAs


Intracellular

Extracellular + AA!-Glut-AAsGSH

GGT

Gclc-/-

Cys

GSH

Glut

Gly

Glut

GCLC

GSS

!-Glut-AAs

AAs(Ox)GSH

+ AA!-Glut-AAsGSH

GGT

GSSG GSSG

Gclcf/f

;CreERT2Gclcf/f

+ Tam

Gclc-/-Gclcf/f

+ Tam

Figure 1S

+ - + - + - + - + - + - + - + - + - + -0

1

2

3

4

Ser (

mM

)

M+0M+3

A549

Calu3

H1299

H1581

H1792

H1944

H1975

H1993

H2009

H2347

(Cys)2: + - + - + - + - + - + - + - + - + - + -0

2

4

6

Gly

(mM

)M+0M+2

A549

Calu3

H1299

H1581

H1792

H1944

H1975

H1993

H2009

H2347

(Cys)2:

A

B C

1 hr

49 hr

- Fer-1 DFOA549, + 20 nM of Sytox Green (SG)

SG+

Figure 2S

0 50

2

4

6

8

10

Glu

t (m

M)

GlutEE (mM):

***

+ - + -0.0

0.5

1.0

1.5

Glu

tR

elat

ive

inte

nsity

(Cys)2:Gln:

+ -

*** ***

****

+ + - -0

1

2

3

RO

S R

elat

ive

inte

nsity

**

***

GlutEE: + +(Cys)2:

A B

C D

- + - - + - - + - - + -0.0

0.5

1.0

Rel

ativ

e C

ell N

umbe

r (Cys)20 µM(Cys)2200 µM

5 mM GlutEE:

0.5 mM AOA: - +

n.s.

5 mM DMαKG:

****

- - + - - + - - + - - +

Figure 3S

A B

- + - + - + - + - +0.0

0.5

1.0

Rel

ativ

e C

ell N

umbe

r

(Cys)2 0 µMH1299Cas9

sgCon

+ GFP

sgGCLC

Fer-1:

sgGSS

+ GCLC Res

+ GFP+ GSS Res

+ GFP

**** ***

********

GCLCGSS

sgCon

sgGCLC

sgGSS

H1299

Ponceau

H1299Cas9

sgCon

+GFP

sgGCLC

+GFP

sgGCLC

+GCLC

Res

sgGSS+G

FP

sgGSS+G

SS Res

sgCon

+GFP

sgGCLC

+GFP

sgGCLC

+GCLC

Res

sgGSS+G

FP

sgGSS+G

SS Res

0.0

0.5

1.0

GSH

Rel

ativ

e In

tens

ity

+ (Cys)2 - (Cys)2

H1299Cas9

**** ********

**** ****

************

C

Figure 4S

A

+ - -0

1×105

2×105

Peak

Are

a

γ-Glut-Ala-Gly

m+0m+1

(Cys)2:SHIN-1: - - +

*###* *

g-Glu-Ala-Gly

Glut

2H-Gly

Ala

g-Glu-Ala

GCLC

GSS

2, 3, 3-2H3-Ser

SHMT1/2

1 2 3

1 2

SHIN-11 2

+ - -0.0

5.0×106

1.0×107

1.5×107

2.0×107

Peak

Are

a

Glym+0m+1

(Cys)2:SHIN-1: - - +

*###*** ****#

B

GSH

Cys

g-Glu-Cys

g-Glu-2AB-Gly

Cys-Gly

g-Glu-AAs

Glut

Gly

2AB

gGlu-2AB

g-Glu-Cys-Gly(GSH)

GCLC

GSS

g-Glu-AAs

GGTIntracellular

AAs

Extracellular

+ (Cys)2

GSH

Cys

g-Glu-Cys

g-Glu-2AB-Gly

Cys-Gly

g-Glu-AAs

Glut

Gly

2AB

g-Glu-2AB

g-Glu-Cys-Gly(GSH)

GCLC

GSS

g-Glu-AAs

GGTIntracellular

AAs

Extracellular

- (Cys)2

?

Figure 5S

-γ-

Glu

Ala

2m

Mγ-

Glu

Leu

2mM

γ-G

luG

ly 2

mM

γ-G

luVa

l 2m

MFe

r 10µ

MD

FO 1

00µM

-γ-

Glu

Ala

2m

Mγ-

Glu

Leu

2mM

γ-G

luG

ly 2

mM

γ-G

luVa

l 2m

MFe

r 10µ

MD

FO 1

00µM

-γ-

Glu

Ala

2m

Mγ-

Glu

Leu

2mM

γ-G

luG

ly 2

mM

γ-G

luVa

l 2m

MFe

r 10µ

MD

FO 1

00µM

-γ-

Glu

Ala

2m

Mγ-

Glu

Leu

2mM

γ-G

luG

ly 2

mM

γ-G

luVa

l 2m

MFe

r 10µ

MD

FO 1

00µM

0.0

0.5

1.0

Rel

ativ

e C

ell N

umbe

r

GCLCKOA GCLCKOB

**** ****

+ (Cys)2 - (Cys)2 + (Cys)2 - (Cys)2

A B

20.

20.

02 0 2 2 2 20.

20.

02 0 2 2 2

0.0

0.5

1.0

Rel

ativ

e C

ell N

umbe

r

GCLCKOB

Gln (mM):

AOA:Fer-1:DFO:

++

+

+ (Cys)2

---- - ----- - -

---- --

++

+

---- - ----- - -

---- --

- (Cys)2

n.s.

***

n.s.

*

********

D

(Cys)2

(Cys)2

Gly

Cys

g-GluCys

GSH

Extracellular

Intracellular

Glu

Glu

GCLC

GSS

g-GluAAs

AAs

Ferroptosis

+ (Cys)2

xCT

(Cys)2

(Cys)2

Gly

Cys

g-GluCys

GSH

Glu

Glu

GCLC

GSS

g-GluAAs

AAs

Ferroptosis

Ferroptosis

- (Cys)2

xCTFerroptosis

sgCon

+GFP

sgGCLC

+GFP

sgGCLC

+GCLC

Res

sgGSS+G

FP

sgGSS+G

SS Res

sgCon

+GFP

sgGCLC

+GFP

sgGCLC

+GCLC

Res

sgGSS+G

FP

sgGSS+G

SS Res

0.0

0.5

1.0

1.5

Glu

tR

elat

ive

Inte

nsity

**

******

H1299Cas9

+ (Cys)2 - (Cys)2

C

E

0

1

2

H1299Cas9

+ (Cys)2 - (Cys)2

sgCon+GFP

sgGCLC+GFP

sgGCLC+GCLCRes

sgGSS+GFP

sgGSS+GSSRes

γ-G

lu-G

ly

n.s.

n.s.

*****

***

0

5

10

15

γ-G

lu-A

la

**

****

********

****

0

2

4

γ-G

lu-V

al

****

***

********

***

0

5

10

γ-G

lu-T

hr

********

**** ********

0

2

4

6

8

γ-G

lu-L

eu

****

****

******** ****

0

20

40

60

80

γ-G

lu-A

la-G

ly

n.s.

****

p=0.22 p=0.12

p=0.14

0

2

4

6

8

γ-G

lu-2

AB-G

ly

****

********

***n.s.

Rel

ativ

e in

tens

ity

γ-Glu-dipeptides

γ-Glu-tripeptides

Documents

Authors: Yun Pyo Kang1, Andrea Mockabee-Macias1 2 1,* · 2020. 5. 29. · Cysteine inadequacy can induce an iron-dependent form of cell death known as ferroptosis (Dixon et al., 2012)