PRDX6 augments selenium utilization to limit iron toxicity and ferroptosis

Ferroptosis is a form of regulated cell death induced by iron-dependent accumulation of lipid hydroperoxides. Selenoprotein glutathione peroxidase 4 (GPX4) suppresses ferroptosis by detoxifying lipid hydroperoxides via a catalytic selenocysteine (Sec) residue. Sec, the genetically encoded 21st amino acid, is biosynthesized from a reactive selenium donor on its cognate tRNA[Ser]Sec. It is thought that intracellular selenium must be delivered ‘safely’ and ‘efficiently’ by a carrier protein owing to its high reactivity and very low concentrations. Here, we identified peroxiredoxin 6 (PRDX6) as a novel selenoprotein synthesis factor. Loss of PRDX6 decreases the expression of selenoproteins and induces ferroptosis via a reduction in GPX4. Mechanistically, PRDX6 increases the efficiency of intracellular selenium utilization by transferring selenium between proteins within the selenocysteyl-tRNA[Ser]Sec synthesis machinery, leading to efficient synthesis of selenocysteyl-tRNA[Ser]Sec. These findings highlight previously unidentified selenium metabolic systems and provide new insights into ferroptosis.

Ferroptosis is a form of regulated cell death induced by iron-dependent accumulation of lipid hydroperoxides.Selenoprotein glutathione peroxidase 4 (GPX4) suppresses ferroptosis by detoxifying lipid hydroperoxides via a catalytic selenocysteine (Sec) residue.Sec, the genetically encoded 21 st amino acid, is biosynthesized from a reactive selenium donor on its cognate tRNA [Ser]Sec .It is thought that intracellular selenium must be delivered 'safely' and 'efficiently' by a carrier protein owing to its high reactivity and very low concentrations.Here, we identified peroxiredoxin 6 (PRDX6) as a novel selenoprotein synthesis factor.Loss of PRDX6 decreases the expression of selenoproteins and induces ferroptosis via a reduction in GPX4.Mechanistically, PRDX6 increases the efficiency of intracellular selenium utilization by transferring selenium between proteins within the selenocysteyl-tRNA [Ser]Sec synthesis machinery, leading to efficient synthesis of selenocysteyl-tRNA [Ser]Sec .These findings highlight previously unidentified selenium metabolic systems and provide new insights into ferroptosis.
GPX4 is a selenoprotein in which the highly reactive 21 st amino acid Sec resides at the active site 4 .Sec is incorporated into the UGA codon when the Sec-insertion sequence (SECIS) element is present in the 3′-untranslated region of mRNAs [8][9][10] .Unlike other amino acids, selenocysteyl-transfer RNA for Sec (Sec-tRNA [Ser]Sec ) is biosynthesized from reactive selenium donor selenide on its cognate tRNA [Ser]Sec through intricate enzymatic reactions 8,11 .Selenide is phosphorylated by selenophosphate synthetase 2 (SEPHS2) [11][12][13] and subsequently incorporated into Sec-tRNA [Ser]Sec .Synthesis of selenoproteins is essential for life, as shown by studies describing embryonic lethality in mice lacking selenoprotein synthesis 14,15 .Despite its importance, however, the selenoprotein synthesis pathway remains unclear.In particular, highly reactive selenides are hypothesized to be delivered safely and efficiently by a carrier protein(s) to SEPHS2; however, they have not yet been identified 11,16 .
Here, we conducted a genome-wide iron-triggered ferroptosis screen and identified PRDX6 as a novel selenoprotein synthesis factor.Loss of PRDX6 greatly decreased the expression of selenoproteins, including GPX4, and triggered ferroptosis through a reduction in GPX4 levels.Mechanistically, PRDX6 functions as a selenide carrier, transferring selenide to SEPHS2 to facilitate efficient selenium utilization.

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https://doi.org/10.1038/s41594-024-01329-zCRISPR screen in which FBXL5 KO cells were infected with a GeCKOv2 library, followed by cultivation with or without FAC (Fig. 1c).Using a cutoff of P < 0.001, screening identified around 150 genes encoding iron-triggered ferroptosis suppressors or activators (Fig. 1d).As expected, one of the top hit genes encoding ferroptosis activators was IRP2; indeed, stabilization of IRP2 caused by loss of FBXL5 led to accumulation of redox-active iron in cells 24 (Fig. 1a).Other prominent hits included iron homeostasis regulators such as transferrin receptor 1 (TFRC), divalent metal transporter 1 (DMT1) and the V-ATPase proton pump complex, all of which are required for iron uptake 25 (Fig. 1d).We used the V-ATPase inhibitor bafilomycin A1 to confirm that organelle acidification is essential for iron uptake and iron-triggered ferroptosis (Extended Data Fig. 1d,e).More importantly, the high-confidence hit list included the known ferroptosis regulators acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) 26,27 (Fig. 1d), both of which are required for insertion of PUFAs into membrane phospholipids.Deletion of ACSL4 or LPCAT3 from FBXL5 KO cells restored cell viability (Fig. 1e and Extended Data Fig. 1f), further indicating that iron-triggered ferroptosis is also mediated by oxidation of PUFAs.In addition, we identified suppressor genes, the CoQ synthesis machinery and FSP1 (Fig. 1d) 6,7 .Deletion of diphosphate synthase subunit 2 (PDSS2), a component of the CoQ synthesis machinery, or FSP1 markedly increased the death of FBXL5 KO cells (Fig. 1f and Extended Data Fig. 1g); thus, lipid radical trapping by CoQH 2 is also involved in suppressing iron-triggered ferroptosis 6,7 .Interestingly, suppressor genes encoding mitochondrial electron transport chain complex I and II (CoQ oxidoreductase), but not complex III and IV, were enriched (Extended Data Fig. 1h), suggesting that CoQH 2 in mitochondria also suppresses ferroptosis.Therefore, our iron-triggered ferroptosis screen, which does not rely on conventional ferroptosis inducers such as RSL3 or erastin, is useful for identifying not only ferroptosis activators but also suppressors.
Increased selenium utilization suppresses ferroptosis and contributes to cancer progression 2,17 .We found that patients harboring cancers with high expression of PRDX6 have a poor prognosis.These findings provide insight into the previously unknown selenium metabolic system in cells as well as its potential as a target for cancer therapy.

Iron-induced ferroptosis screen
The prefix 'ferro' suggests that ferroptosis should be categorized as a form of iron toxicity; however, in most studies of ferroptosis, cell death is not induced by iron administration.Rather, it is induced by inhibiting erasers for lipid hydroperoxidation such as the GPX4 inhibitor RSL3 or the cystine transporter inhibitor erastin 3,18 .To re-analyze ferroptosis from the perspective of iron toxicity and to identify new regulators, we developed a ferroptosis induction system based on iron addition alone.The iron regulatory protein 2 (IRP2) and F-box and leucine-rich repeat protein 5 (FBXL5) system acts as a major regulator of cellular iron homeostasis by suppressing iron uptake and increasing iron storage by FBXL5-mediated degradation of IRP2 (refs.19-21).Therefore, to induce iron toxicity 22 , we knocked out FBXL5 from mouse embryonic fibroblasts (MEFs) using the CRISPR-Cas9 system (Fig. 1a).Treatment with ferric ammonium citrate (FAC) selectively killed FBXL5 knockout (KO) cells by increasing the cellular iron concentration, as detected by a fluorescent probe specific for Fe 2+ (Fig. 1b and Extended Data Fig. 1a,b) 23 .
Ferroptosis is thought to be triggered by hydroperoxidation of polyunsaturated fatty acids (PUFAs) in the cell membrane by redox-active iron 1 .BODIPY 581/591 C11 staining to detect hydroperoxidation of PUFAs revealed increased hydroperoxidation in iron-repleted FBXL5 KO cells (Extended Data Fig. 1c).Iron-triggered death of FBXL5 KO cells appeared to occur through ferroptosis because it was prevented by the ferroptosis inhibitor liproxstatin-1 (Fig. 1b).To identify genes involved in iron-triggered ferroptosis, we performed an unbiased genome-wide

Identification of PRDX6 as a regulator of GPX4 expression
Gene ontology analysis of the iron-triggered ferroptosis suppressors indicated that selenoprotein synthesis factors were the most enriched group (Extended Data Fig. 2a).The high-confidence suppressor list raised the possibility that loss of selenoprotein synthesis factors sensitizes cells to iron-triggered ferroptosis (Fig. 1d).Among selenoproteins, the master ferroptosis regulator GPX4 was ranked highly as a suppressor (Extended Data Fig. 2b).Therefore, we suspected that new GPX4 regulators might be included in our suppressor list (Extended Data Fig. 2c), and we performed a secondary screen using the abundance of GPX4 as a readout (Extended Data Fig. 2d).Unexpectedly, we found that deletion of PRDX6 greatly reduced expression of GPX4 (Extended Data Fig. 2d), which we confirmed using two different guide RNAs (gRNAs) (Fig. 2a).Surprisingly, loss of PRDX6 sensitized not only FBXL5 KO cells (Extended Data Fig. 3a) but also wild-type (WT) cells (Fig. 2b) to iron-triggered ferroptosis, whereas deletion of PRDX6 had no effect on iron homeostasis because FAC-induced stabilization of FBXL5 and degradation of IRP2 was similar to that in WT cells (Extended Data Fig. 3b) 19,20 .This finding ruled out the alternative possibility that loss of PRDX6 inhibits iron-induced ferroptosis by disrupting iron metabolism in cells.Increased iron-induced lipid hydroperoxidation in PRDX6 KO cells indicated that PRDX6 is an essential suppressor of iron-triggered ferroptosis (Extended Data Fig. 3c).In addition, we found that PRDX6 is also involved in suppressing ferroptosis mediated by imidazole ketone erastin 28 (IKE) and RSL3 (Fig. 2c,d).To address whether the decrease in GPX4 is responsible for ferroptosis sensitivity in PRDX6 KO cells, we expressed a partially active GPX4 mutant, in which Sec(U)46 is substituted by cysteine (GPX4 U/C) in PRDX6 KO cells (Extended Data Fig. 3d) 4 .Expression of the GPX4 U/C mutant rescued PRDX6 KO cells from iron-triggered ferroptosis (Fig. 2e), suggesting that PRDX6 suppresses cell death by maintaining expression of GPX4.PRDX6 belongs to the PRDXs family of antioxidant enzymes [29][30][31] .Among them, PRDX6 has a unique characteristic in that it contains a single conserved cysteine (C47) residue, whereas the other family members contain two 29 .PRDX6 exhibits three enzymatic activities: glutathione peroxidase (GPX) (C47) activity, phospholipase A2

Involvement of PRDX6 in selenoprotein synthesis
Next, we dissected the mechanism underlying the PRDX6-mediated increase in GPX4 expression.We found no obvious differences in the levels of GPX4 mRNA between PRDX6 KO and WT cells (Extended Data Fig. 4a), nor did proteasomal or lysosomal inhibitors restore GPX4 levels in PRDX6 KO cells (Extended Data Fig. 4b,c).Gene ontology analysis revealed enrichment of selenoprotein synthesis factors (Extended Data Fig. 2a).Moreover, we found that unbiased co-essentiality database tools 37 also indicated a strong functional connection between PRDX6 and genes involved in selenoprotein synthesis (Fig. 3a).Therefore, we reasoned that PRDX6 is involved in selenoprotein biosynthesis.Indeed, deletion of PRDX6 reduced expression of other selenoproteins, including selenoprotein N (SELN), GPX1 and SEPHS2 (Fig. 3b).The conserved Cys47 of PRDX6 is essential for expression of selenoproteins because selenoprotein expression was rescued by re-expression of PRDX6 WT, but not that of the C47S mutant, in PRDX6 KO cells (Fig. 3c).Moreover, loss of PRDX6 reduced the amount of selenoproteins in several human cancer cell lines (Extended Data Fig. 4d), indicating that the role of PRDX6 is conserved across many cancer types.
To confirm the involvement of PRDX6 in selenoprotein synthesis, we constructed cDNAs encoding model selenoproteins harboring an in-frame UGA codon at C70 or S175 of GFP (C70U and S175U), as well as the 3′UTR SECIS element of GPX4 (Fig. 3d).As expected, loss of phosphoseryl-tRNA [Ser]Sec kinase (PSTK) or SEPHS2, both of which are essential for synthesis of Sec-tRNA [Ser]Sec (ref.38), greatly reduced expression of both GFP C70U and S175U (Fig. 3d).Loss of PRDX6 also reduced expression of both model selenoproteins (Fig. 3d), whereas loss of PRDX6 did not affect expression of GFP WT (Extended Data Fig. 4e).These results clearly indicate that PRDX6 has a crucial role in Sec insertion into the UGA codon.

PRDX6 augments efficient utilization of selenium
Next, we explored the role of PRDX6 in selenoprotein synthesis.Both inorganic and organic forms of selenium can be used to synthesize Sec-tRNA [Ser]Sec (Extended Data Fig. 5a).Inorganic selenite is reduced by GSH and the thioredoxin system, whereas organic Sec is decomposed by selenocysteine lyase (SCLY), resulting in generation of the common active selenium donor selenide, which is important for Sec-tRNA [Ser]Sec synthesis (Extended Data Fig. 5a) 38 .Addition of excess selenite or selenocystine ((Sec) 2 ) to cultures of WT cells increased selenoprotein expression markedly, suggesting that the supply of selenium is limited under normal culture conditions (Fig. 4a).Although either selenium source failed to induce expression of selenoproteins in cells lacking PSTK or SEPHS2 (Fig. 4a), both of which are essential for Sec-tRNA [Ser]Sec synthesis 38 , we found that addition of excess selenium increased the amount of selenoproteins in PRDX6 KO cells, regardless of the selenium source (Fig. 4a).Addition of selenite or (Sec) 2 also protected MEFs lacking PRDX6 from iron-triggered and canonical ferroptosis (Fig. 4b,c and Extended Data Fig. 5b,c).Furthermore, addition of selenium increased selenoprotein expression and suppressed iron-triggered ferroptosis in HeLa cells lacking PRDX6 (Extended Data Fig. 5d,e).These results show that addition of excess selenium can compensate for loss of PRDX6, raising the possibility that selenium is not used efficiently by PRDX6 KO cells.Indeed, when selenite or (Sec) 2 was added to cultures in a dose-dependent or time-dependent manner, we found that the incremental increases in selenoprotein levels were much less efficient in PRDX6 KO than in WT cells (Fig. 4d,e and Extended Data Fig. 5f,g).PRDX6 is also involved in efficient utilization of selenium after treatment with SELENOP, a more physiological source of selenium 39 (Fig. 4f).Therefore, to more rigorously evaluate the hypothesis that PRDX6 is involved in the efficiency of selenium utilization, we adjusted the initial level of selenoproteins in WT and PRDX6 KO cells and then added selenium; this is because initial expression of selenoproteins was greatly reduced in PRDX6 KO cells (Fig. 3b).We found that expression of selenoproteins in WT cells cultivated in medium with a reduced concentration of fetal bovine serum (FBS) fell markedly; FBS is the sole source of selenium in this culture system (Extended Data Fig. 5h).Next, we added selenium to culture medium containing 1% FBS and found that selenoprotein synthesis was much more efficient in WT cells than in KO cells (Extended Data Fig. 5h).Furthermore, even when PRDX6 KO cells were pretreated with selenium and then fed additional selenium, selenoprotein synthesis was lower in PRDX6 KO cells than in WT cells (Extended Data Fig. 5i).These results strongly support our hypothesis that PRDX6 is involved in efficient utilization of selenium for Sec-tRNA [Ser]Sec synthesis.Next, to evaluate the amount of Sec-tRNA [Ser]Sec in cells, we established a highly sensitive in vitro reconstructive system for selenoprotein synthesis by modifying a Sec UGA readthrough luciferase reporter assay 40 .Given that wheat germ lacks the Sec incorporation machinery 41 , eukaryotic elongation factor Sec-tRNA [Ser]Sec -specific (eEFSec) and SECIS binding protein 2 (SBP2) were added to the wheat germ lysate together with a Sec-tRNA [Ser]Sec source to establish the reconstitution system (Fig. 4g).Addition of aminoacyl-tRNAs (aa-tRNAs) purified from WT or SEPHS2 KO cells (the former, but not the latter, contains Sec-tRNA [Ser]Sec ) revealed that aa-tRNAs from WT cells increased selenoprotein synthesis in the presence of eEFSec and SBP2, as evaluated by measuring luciferase luminescence (Fig. 4h).Moreover, aa-tRNAs from WT cells cultivated with (Sec) 2 further enhanced selenoprotein synthesis.However, aa-tRNAs purified from MEFs lacking SEPHS2 did not show increased synthesis, even when KO cells were cultivated with (Sec) 2 (Fig. 4h), confirming the validity of the system for evaluating the amount of Sec-tRNA [Ser]Sec in aa-tRNA sources.Next, we purified aa-tRNAs from WT or PRDX6 KO cells cultured with or without (Sec) 2 (Fig. 4i).Purified aa-tRNAs from PRDX6 KO cells cultivated in the absence of (Sec) 2 failed to increase selenoprotein synthesis, and (Sec) 2 treatment increased it only slightly.These results clearly indicate that PRDX6 is involved in the efficient use of selenium for Sec-tRNA [Ser]Sec synthesis.

PRDX6 augments selenium utilization as a selenide carrier
Next, we explored the mechanism(s) by which PRDX6 increases the efficiency of selenium utilization for Sec-tRNA [Ser]Sec synthesis.First, we overexpressed enzymes involved in Sec-tRNA [Ser]Sec synthesis in PRDX6 KO cells (Extended Data Fig. 5a) and found that overexpression (with the exception of SEPHS2 and PRDX6) failed to restore GPX4 expression (Fig. 5a).Thus, SEPHS2, a selenoprotein that phosphorylates selenide for Sec-tRNA [Ser]Sec synthesis 11 , appears to have a critical role in GPX4 expression in PRDX6 KO cells.Previous studies have shown that a SEPHS2 mutant in which Sec is replaced by cysteine (SEPHS2 U/C) rescued the SEPHS2-deficient phenotype 42,43 (Extended Data Fig. 6a).Therefore, we overexpressed SEPHS2 U/C or WT in PRDX6 KO cells and found that SEPHS2 WT, but not the U/C mutant, restored GPX4 expression markedly (Extended Data Fig. 6b).However, we found that overexpression of SEPHS2 WT in PRDX6 KO failed to fully restore expression of other selenoproteins such as SELN and GPX1 (Extended Data Fig. 6b).We additionally found that overexpression of SEPHS2 WT also restored expression of model selenoproteins (Extended Data Fig. 6c) and protected cells from iron-triggered and canonical ferroptosis (Extended Data Fig. 6d-f).These results raise the possibility that loss of PRDX6 suppresses the expression of selenoproteins through a reduction of SEPHS2, not by facilitating utilization of selenium.To rule out this possibility, we expressed SEPHS2 WT at endogenous levels in PRDX6 KO cells because the amount of SEPHS2 in overexpressing cells was much higher than that in cells expressing endogenous SEPHS2 (Fig. 5a).We found that the level of endogenous SEPHS2 WT failed to reverse loss of selenoprotein expression in PRDX6 KO cells (Fig. 5b) and did not suppress iron-triggered and canonical ferroptosis (Extended Data Fig. 6d-f).In addition, when PRDX6 KO cells were pretreated with selenite or (Sec) 2 to adjust SEPHS2 expression in KO cells to levels comparable to those in WT cells before the time course experiment, we observed that WT cells utilized selenium more efficiently than PRDX6 KO cells (Extended Data Fig. 5i).These results show clearly that PRDX6 is necessary for effective utilization of selenium to generate Sec-tRNA [Ser]Sec in cells expressing endogenous levels of SEPHS2.Importantly, we also found that Sec-tRNA [Ser]Sec levels were still lower in PRDX6 KO cells overexpressing SEPHS2 than in WT cells (Fig. 5c), suggesting that overexpression of SEPHS2 cannot compensate fully for the reduced efficiency of selenium utilization owing to loss of PRDX6.Collectively, the data suggest that excess selenium as well as overexpression of SEPHS2 WT, but not the U/C mutant, compensates for the loss of PRDX6, albeit not very effectively.Thus, these results imply that SEPHS2 itself can acquire selenium through the Sec residue of SEPHS2; however, PRDX6 greatly facilitates the utilization of selenium by SEPHS2.
Owing to its high reactivity, it was thought that selenide must associate with an unidentified carrier protein through a cysteine residue for efficient transfer to SEPHS2 (refs.11,16).Addition of excess (Sec) 2 was toxic to cells; however, we found that PRDX6, in which C47 is critical, suppresses (Sec) 2 toxicity (Extended Data Fig. 6g).Considering that C47 of PRDX6 also has a crucial role in the efficient synthesis of selenoproteins (Figs.2f and 3c), it is highly likely that PRDX6 is a potential selenide carrier.A previous report showed that rhodanese forms perselenide (-S-SeH) at cysteine residues in the presence of selenite and GSH 44 .Therefore, we incubated recombinant PRDX6 WT or C47S proteins with selenite and GSH, followed by the detection of PRDX6-bound selenium by inductively coupled plasma mass spectrometry (ICP-MS) (Extended Data Fig. 6h).We found that PRDX6 WT binds to selenium more effectively than the C47S mutant.Next, we performed mass spectrometry analyses to further characterize the PRDX6-selenium intermediate.The results showed that C47 of PRDX6 forms a perselenide bond when reacting with GSH and selenite, as well as with SCLY and Sec (Extended Data Fig. 7).Moreover, we found that about 10% of PRDX6 C47 formed a perselenide bond in a 5 min reaction with selenite and GSH, or with SCLY and Sec (Fig. 5d,e).Selenide carrier proteins should receive selenide from SCLY and then transfer it to SEPHS2 for phosphorylation (Extended Data Fig. 5a).Proximity ligation assays (PLAs) revealed that endogenous PRDX6 bound to both SCLY and SEPHS2 effectively (Fig. 5f and Extended Data Fig. 8a).To confirm binding of PRDX6 to SCLY and SEPHS2, we expressed HA-TurboID-PRDX6 or HA-TurboID-PRDX1 (a paralog of PRDX6) in WT or PRDX6 KO MEFs.
As shown in Fig. 5g and Extended Data Fig. 8b,c, PRDX6 bound specifically to SEPHS2 and SCLY.
Selenide is delivered to and phosphorylated by SEPHS2 to generate selenophosphate for Sec-tRNA [Ser]Sec synthesis 11 .Given that selenophosphate is an unstable product 45 , we evaluated the production of AMP, a by-product of selenophosphate synthesis, to evaluate the acceleration of selenophosphate synthesis by PRDX6.We used a recombinant SEPHS2 U/C protein that retains enzyme activity for selenophosphate synthesis 42,46 instead of SEPHS2 WT because expression of SEPHS2 WT is extremely low (Fig. 5a) and it is difficult to purify.In the selenophosphate synthesis reaction in which SCLY-mediated decomposition of Sec is used as a selenium source, addition of PRDX6 WT efficiently increased the synthesis of selenophosphate, whereas C47S did not (Fig. 5h).When PRDX6 WT or C47S was preincubated with selenide to generate the PRDX6-selenide complex as a selenium source, PRDX6 WT accelerated SEPHS2 U/C-mediated production of AMP (Fig. 5i).Collectively, these data suggest that PRDX6 facilitates Sec-tRNA [Ser]Sec synthesis by functioning as a selenide carrier protein (Fig. 5j).

PRDX6 and cancer
Most selenoproteins have a role in maintaining redox balance by reducing oxidative insults, and high expression of selenoproteins is associated with malignancy grade 8,43 .Consistent with the relationship between selenoproteins and cancer, database analyses revealed that expression of PRDX6 is higher in cancerous tissues than in normal tissues 47 (Extended Data Fig. 9a) and that high expression of PRDX6 correlates with a poor prognosis for various cancers 48 (Extended Data Fig. 9b).A previous report showed that intractable pancreatic cancer cells are sensitive to ferroptosis 49 .Database analysis also revealed that expression of PRDX6 is high in pancreatic cancer and that high expression correlates with a poor prognosis (Extended Data Fig. 9).We found that loss of PRDX6 from two pancreatic cancer cell lines suppressed growth, a phenomenon reversed by the addition of selenium or liproxstatin-1 (Extended Data Fig. 10a-d).To consolidate our findings, we obtained several cancer cells and evaluated their sensitivity to iron-triggered ferroptosis (Extended Data Fig. 10e,f).The results showed that MYC-N-amplified neuroblastoma cells (SK-N-DZ and NB-1) were sensitive to iron-induced ferroptosis (Extended Data Fig. 10e,f).We also found that loss of PRDX6 from SK-N-DZ cells suppressed their growth markedly (Extended Data Fig. 10g,h).A previous study reported that MYC-N increases intracellular iron by upregulating the expression of TFRC, a protein required for iron uptake 50 .In addition, cancer cells are generally addicted to iron, which drives proliferation.Thus, inhibiting PRDX6 may be an effective strategy for sensitizing cancer cells specifically to iron-triggered ferroptosis.

Discussion
Here, we developed a ferroptosis induction system that can kill cells upon addition of iron to the culture medium (Fig. 1c,d).We used this system to conduct a genome-wide CRISPR screen and identified PRDX6 as a selenoprotein synthesis factor.Although PRDX6 has long been considered an antioxidant enzyme 29,31 , we show here that it exerts antioxidant effects indirectly by facilitating the expression of selenoproteins.
Although organic and inorganic selenium are metabolized to selenide, which is then used for selenoprotein synthesis 38 , the metabolic pathway has not yet been shown conclusively.Our results indicate that PRDX6 is an unidentified selenide carrier protein that facilitates the efficient utilization of selenide by SEPHS2 (Figs. 4 and 5).Among the PRDX family, which comprises six proteins (PRDX1-PRDX6), the two-cysteine proteins PRDX1-PRDX5 harbor a disulfide

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https://doi.org/10.1038/s41594-024-01329-zbond between the two conserved cysteine residues, which is reduced by the thioredoxin system.The one-cysteine protein PRDX6 is unique in that it is thought to be reduced by GSH and to have GPX activity 29,30 .However, we showed conclusively in this study that PRDX6 does not possess GPX activity (Fig. 2g and Extended Data Fig. 3g).Instead, we found that a C47S mutant of PRDX6 profoundly reduces the amount of selenoproteins, including GPX1 and GPX4 (Fig. 3c), both of which are well-known proteins that exhibit GPX activity.Given that the GPX function of PRDX6 has been evaluated mainly using PRDX6 knockdown or KO cells [51][52][53] , the reduced GPX activity in PRDX6-deficient cells might be caused by a reduction in GPX1 and GPX4.Consistent with our results (Fig. 2g and Extended Data Fig. 3g), other reports used purified proteins to show that PRDX6 has no GPX activity [34][35][36] .Therefore, we conclude that PRDX6 is not an antioxidant enzyme like PRDX1-PRDX5 but it does function as a selenide carrier protein.This was confirmed by our finding that PRDX1, an antioxidant enzyme that is considered to be a paralog of PRDX6, did not restore GPX4 expression in PRDX6 KO MEFs (Extended Data Fig. 8b).
Both inorganic and organic selenium function as sources for SEPHS2 to generate selenoproteins 38 .Although the mechanism underlying delivery of selenium to SPEHS2 remains unknown, we clearly showed that PRDX6 facilitates the synthesis of selenoproteins (Fig. 4d-f and Extended Data Fig. 5f-i) and forms a perselenide bond at the C47 of both selenium sources (Fig. 5d,e and Extended Data Fig. 7).We also found that PRDX6 binds to both SEPHS2 and SCLY; the latter is an enzyme that releases selenide from Sec.Thus, PRDX6 appears to have a crucial role in delivering selenide from organic selenium by accepting selenide from SCLY and delivering it to SEPHS2.Although the mechanisms underlying the metabolism of inorganic selenium and its delivery to SEPHS2 remain unknown, our results strongly suggest that PRDX6 also has a role in the delivery of selenide to SEPHS2, even from inorganic selenium.Therefore, the identification of PRDX6 as a selenium carrier protein opens new avenues of research into intracellular selenium dynamics.
It is noteworthy that overexpression of SEPHS2 WT substantially restored the expression of selenoproteins in PRDX6 KO cells (Fig. 5a and Extended Data Fig. 6b,c).These observations imply that SEPHS2 can bind directly to selenide to produce selenophosphate in the absence of PRDX6, albeit much less efficiently; indeed, endogenous levels of SEPHS2 failed to restore selenoprotein expression.Given that the SEPHS2 U/C mutant could not restore expression of selenoproteins in PRDX6 KO cells (Extended Data Fig. 6b,c), the Sec residue in SEPHS2 seems to facilitate utilization of selenide, although the precise molecular mechanism remains unsolved.The Sec residue and C47 of PRDX6 have a low pK a and high reactivity 29,54 ; therefore, we suspect that highly reactive residues may be important for the reaction with selenide in cells.
We also observed that addition of excess selenium (both organic and inorganic forms) restored the amount of selenoproteins in PRDX6 KO cells (Fig. 4a-c).Therefore, the ability of SEPHS2, which reacts directly with selenide to induce the synthesis of some selenoproteins, may underlie the finding of trace amounts of selenoproteins in PRDX6 KO cells.These observations appear to be consistent with differences in the phenotypes of PRDX6 KO mice and mice lacking selenoprotein synthesis 14,15,55,56 .Two lines of PRDX6 KO mice have been reported 55,56 .Both are viable and fertile, and neither shows an overt phenotype, although they are hyper-sensitive to various oxidative stresses.By contrast, mice lacking selenoprotein synthesis are embryonic lethal 14,15 .The observation that loss of PRDX6 does not completely attenuate selenoprotein synthesis may be advantageous with respect to the development of PRDX6 inhibitors as anti-cancer drugs.Effective inducers of ferroptosis are believed to be a promising strategy for anti-cancer therapy, and GPX4 is regarded as a suitable target 3 .However, inhibition of GPX4 may have severe side effects because GPX4 KO mice are embryonic lethal 57 .Taken together, the present results suggest that PRDX6 may be an alternative target for anti-cancer drugs, with fewer side effects.

Generation of FBXL5 KO MEFs
To generate FBXL5 KO MEFs, a NEPA21 electroporator (NEPAGENE) was used to electroporate MEFs with the pX459 plasmid containing an sgRNA sequence specific for mFBXL5.Cells were then treated with puromycin for 2 days.Following selection, the cells were seeded at a low density and isolated colonies were picked.To identify FBXL5 KO cells, expression of FBXL5 was analyzed by immunoblotting.

Lentivirus production and generation of CRISPR-Cas9-mediated KO cell lines
Lentivirus was produced by co-transfecting HEK293T cells with the LentiCRISPRv2 (puro, bsr or hygro)-containing guide sequences, the psPAX2 packaging plasmid and the VSV-G envelope plasmid using PEI MAX (Polysciences) transfection reagent.On the following day, the medium was replaced with fresh medium.After 2 days of culture, lentivirus-containing supernatant was collected and used to infect target cells overnight in the presence of 10 μg ml −1 polybrene (Merck).Infected cells were selected with puromycin, blasticidin or hygromycin.

Retroviral expression
pMXs-IP, pMXs-neo or pMXs-IRES-Bsr containing appropriate inserts were transfected into PLATE packaging cells or GP2-293 cells along with the pVSV-G plasmid.The resultant viruses were used to infect target cells in the presence of 10 μg ml −1 polybrene.Stably transduced cells were selected using puromycin, G-418 or blasticidin.

Cell lysis and western blotting
Cells were washed with PBS and lysed with lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, and protease inhibitor cocktail (Sigma-Aldrich)).The lysates were clarified by centrifugation at 20,400g for 20 min at 4 °C.Protein concentrations were determined using the Bradford assay (Nacalai Tesque), and equal amounts of protein were mixed with SDS sample buffer.Samples were heated for 5 min at 95 °C, separated by SDS-PAGE and transferred onto PVDF membranes (Millipore).After blocking in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% (w/v) nonfat dry milk, the membrane was incubated with the appropriate primary antibodies followed by appropriate secondary antibodies.The membranes were visualized using enhanced chemiluminescence and analyzed on a LAS4000mini or LAS3000 instrument (GE Healthcare). https://doi.org/10.1038/s41594-024-01329-z

Cell viability assay
Cells were plated in 96-well plates (in triplicate and at a density of 2,500-10,000 cells per well) in the presence or absence of FAC, RSL3 or IKE.After culture for 24-48 h, 10 μl of Cell Counting Kit-8 (DOJINDO) was added for 1-2 h.Absorbance at 450 nm was measured using a SpectraMax M5 microplate reader (Molecular Devices).Cell viability, monitored continuously using the iCELLigence or xCELLigence system (ACEA Bioscience), was expressed as an impedance-based cell index.GPX4 KO cells (4,500 cells per well) were plated onto an E-Plate L8 PET (ACEA Bioscience), and pancreatic ductal adenocarcinoma cells (3,000 cells per well) and SK-N-DZ cells (6,000 cells per well) were plated on an E-Plate 16 PET (ACEA Bioscience).SK-N-DZ cells were cultured in DMEM supplemented with 10% FBS, 1× non-essential amino acids and 0.25 mM l-glutamine (Fujifilm).The cell index was monitored continuously.Recording data were analyzed by RTCA software lite v.2.2.1.

Cell staining using BODIPY 581/591 C11, and iron staining
For BODIPY 581/591 C11 (Invitrogen) staining, cells were plated in a six-well plate and treated with FAC.After 1.5-24 h, the medium was removed and the cells were labeled with DMEM containing 10 μM BODIPY 581/591 C11 at 37 °C for 30 min.Cells were then washed three times with PBS and detached from the plate using trypsin.Initial cell population gating (FSC-Area versus FSC-height) was used to ensure doublet exclusion, and green fluorescence was measured by using FACS Canto II (BD Biosciences) and FACS Diva software v.6.1.2(Becton Dickinson).Data were analyzed using FlowJo software (v.9.9.6).For iron staining, cells were plated in a 35-mm glass bottom dish and treated with 25 μg ml −1 FAC for 4 h.Cells were then washed three times with HBSS and labeled at 37 °C for 30 min with 1 μM FerroOrange (DOJINDO) followed by visualization under a Fv1000 confocal microscope (Olympus).

Genome-wide CRISPR screening
FBXL5 KO MEFs were infected with the mouse GeCKO v.2 library 59 at a multiplicity of infection of 0.3 and selected with puromycin for 1 week post infection.The cells were then treated with 100 μg ml −1 FAC for 48 h and cultured for a further 24 h in fresh medium.The cells were lysed in NTE buffer (15 mM Tris-HCl (pH 7.5), 150 mM NaCl and 1 mM EDTA), and genomic DNA from non-treated and FAC-treated cells was prepared by phenol-chloroform extraction and isopropanol precipitation.sgRNA sequences were amplified from genomic DNA by PCR using Herculase II Fusion DNA polymerase.The resultant amplicons were gel-extracted and subjected to DNA sequencing on a Novaseq 6000 (Illumina) sequencer.Sequence data were analyzed using the MAGeCK pipeline.

PLA
The PLA was conducted using the Proximity Ligation Kit (Sigma-Aldrich).A549 cells, either empty or expressing Myc-SEPHS2 or Myc-SCLY, were cultured on micro cover glass slips in a 6-well plate.Cells were washed with PBS and fixed for 20 min with 4% formaldehyde in PBS at room temperature (25-27 °C).Cells were then washed twice with PBS and permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min.Cells were washed twice with PBS and then incubated at 37 °C for 1 h with Duolink blocking solution.Cells were then incubated at room temperature for 1 h with primary antibodies targeting PRDX6 (rabbit polyclonal; 1:100) and Myc (mouse monoclonal; 1:200).Cells were washed twice for 5 min with Duolink wash buffer A at room temperature and then incubated with a secondary antibody (anti-mouse minus and anti-rabbit plus) at 37 °C for 1 h.Cells were washed twice at room temperature for 5 min with Duolink wash buffer A and then incubated with ligase solution at 37 °C for 30 min.Next, the cells were washed twice with Duolink wash buffer A and incubated with polymerase in amplification buffer at 37 °C for 100 min.Cells were washed twice at room temperature for 10 min with Duolink wash buffer B, followed by 0.01% Duolink wash buffer B for 1 min.Finally, the cover glasses were mounted with Duolink PLA mounting medium with DAPI.Protein-protein interactions were visualized under an Fv1000 confocal microscope (Olympus).PLA foci were counted by ImageJ (v.2.3.0).

TurboID pulldown
Cells were treated (or not) for 30 min with 50 μM biotin, washed with PBS and lysed with lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100 and 1 mM PMSF).The lysates were clarified by centrifugation at 20,400g for 20 min at 4 °C.Protein concentrations were determined using the Bradford assay, and equal amounts of protein and Streptavidin Sepharose (Cytiva) were incubated overnight at 4 °C by rotation.The beads were washed three times with lysis buffer and one time with PBS.Proteins were eluted from beads using SDS sample buffer supplemented with 2 mM biotin and then analyzed by western blotting.
Expression of His-TEV-hSCLY and hSEPHS2-His was induced by the addition of 0.2 mM IPTG, and culture was continued overnight at 15 °C.Cells were collected by centrifugation and frozen rapidly.Subsequently, the cells were resuspended in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM 2-mercaptoethanol, 2 mM PMSF and a protease inhibitor cocktail and then lysed by sonication at 4 °C.Insoluble material was removed by centrifugation at 23,700g for 20 min at 4 °C.Proteins were purified using Ni-NTA beads.The eluted samples were desalted in 20 mM Tris-HCl (pH 7.5) and 1 mM DTT buffer on a PD10 column.
Expression of His-TEV-eEFSec and His-TEV-SBP2 was induced by the addition of 0.3 mM IPTG, and culture was continued at 30 °C for 4 h.Cells were collected by centrifugation and frozen rapidly.Subsequently, the cells were resuspended in buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole, 10% glycerol, 10 mM 2-mercaptoethanol, 1 mM PMSF and a protease inhibitor cocktail and then lysed by sonication at 4 °C.Insoluble material was removed by centrifugation at 23,700g for 20 min at 4 °C.Proteins were purified using Ni-NTA beads.Eluted samples were desalted on a PD10 column.eEFsec was stored in buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl and 1 mM DTT, and SBP2 was stored in buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol and 1 mM DTT.

GPX assay
GPX activity was measured using a GPX4 Inhibitor Screening Assay Kit (Cayman Chemical).First, 0.5 μg of GPX4 protein (included in the kit) or 5 μg of PRDX6 protein was dissolved in 50 μl of GPX4 assay buffer (included in the kit).Then, 20 μl of the GSH/GSH reductase mix (included in the kit) was added and mixed.Next, 20 μl of the NADPH solution (included in the kit) was added and mixed.Finally, 10 μl of cumene hydroperoxide (included in the kit), hydrogen peroxide (final concentration, 500 μM) or tert-butyl hydroperoxide (final concentration, 500 μM) was added and mixed, and absorbance https://doi.org/10.1038/s41594-024-01329-z at 340 nm was measured using SpectraMax M5 microplate reader (Molecular Devices).

Isolation of aa-tRNAs
aa-tRNAs were isolated from WT, PRDX6 KO and SEPHS2 KO MEF cells.In brief, cells were treated (or not) with 50 nM (Sec) 2 for 105 min, followed by addition of 20 μg ml −1 cycloheximide for 15 min.Cells were then washed three times in PBS and detached using trypsin.After centrifugation at 200g, 3.2 ml of DPEC water, 0.8 ml of 5× T buffer (50 mM NaOAc, 3.25 M NaCl, 50 mM MgCl 2 and 5 mM EDTA) and 2 ml of acid-phenol pH 4.2 (Nippon gene) were added to the cell pellets in that order.The solution was mixed briefly and centrifuged at 12,000g for 5 min at 4 °C.The aqueous phase was transferred to another tube and re-extracted with 2 ml of acid-phenol (pH 4.2).RNA was precipitated with 2.5 volumes of 100% ethanol, washed twice with 75% ethanol and air-dried for 5 min.The aa-tRNA pellet was resuspended in 5 mM NaOAc buffer.
Luciferase activity was measured in a Lumat Luminometer (Berthold).

ICP-MS
The reaction (100 μl) contained 40 μM sodium selenite, 160 μM GSH and 40 μM (or not) recombinant PRDX6 WT or C47S in degassed buffer containing 1 mM EDTA and 50 mM Tris-HCl (pH 7.0).Reactions were incubated at 25 °C for 5 min.Any selenium that did not bind to PRDX6 protein was removed using a NAP-5 column.The protein solution was then concentrated with an Amicon 3K concentrator.A small aliquot (50 μl) of the protein solution was put into a glass test tube, and the proteins were decomposed by addition of 0.1 ml of 60% HNO 3 followed by heating at 170°C for 2 h.The digested samples were diluted with Milli-Q water, and the concentration of selenium was measured by ICP-MS (Agilent 8800 ICP-MS/MS; Agilent Technologies).The O 2 mass-shift mode was used to monitor the signal intensity of 80 Se 16 O, with a dwell time of 100 ms.Selenium concentrations were measured from a standard calibration curve.
Reactions were incubated at 25 °C for 5 min.Then, PRDX6 was alkylated with 10 mM iodoacetamide in 0.02% ProteaseMAX surfactant at 37 °C for 10 min, followed by digestion with 27 mg ml −1 Trypsin Gold at 37 °C for 3 h.Products were analyzed using liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry (LC-ESI-Q-TOF MS/MS).LC-ESI-Q-TOF analysis was performed using a 6545XT AdvanceBio LC-Q-TOF apparatus (Agilent Technologies) connected to the Agilent HPLC system.Analysis of the modifications to the active center cysteine (Cys47) was performed using Agilent MassHunter BioConfirm software v.10.0.The modification levels in DFTPVCTTELGR peptide, which includes the active center cysteine residue, were detected by monitoring at m/z 698.3341 (for CysS-AM (AM, iodoacetamide adduct)), m/z 738.2942 (for CysSSe-AM) and m/z 862.3172 (for CysSSe-SG).The efficiency of trypsin-mediated protein digestion was normalized using the FHDFLGDSWGILFSHPR peptide (m/z 678.0041).The level of selenium modification in the fragment containing the active center cysteine was assessed by determining the relative ratio between the intensity of peptide-CysSSe-AM and that of peptide-CysS-AM.Samples of peptide-CysS-AM subjected to trypsin digestion followed by reduction with TCEP and subsequent alkylation with iodoacetamide were used to measure peptides corresponding to peptide-CysSSe-AM.

Statical analysis and reproducibility
Data are presented as the mean ± s.d. or mean ± s.e.m.GraphPad Prism9 v.9.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Boxplot data of differential gene expression in tumor and normal tissues were obtained from TNMplot (https://tnmplot.com/analysis).All data are available in the article and the supplementary information, and from the corresponding authors upon reasonable request.Source data are provided with this paper.

Fig. 1 |
Fig. 1 | Genome-wide CRISPR screening for iron-triggered ferroptosis.a, WT or FBXL5 KO MEFs were treated with FAC (100 μg ml −1 ) for the indicated periods, and cell lysates were analyzed by immunoblotting.Data are representative of three independent experiments.b, Viability of WT or FBXL5 KO MEFs treated for 48 h with FAC (100 μg ml −1 ) in the presence of liproxstatin-1 (0, 1, 3 or 5 μM).c, Schematic showing the CRISPR-Cas9 screening strategy.d, Volcano plots showing the regulatory genes involved in iron-triggered ferroptosis.The P value

Fig. 3 |
Fig. 3 | PRDX6 is involved in selenoprotein synthesis.a, Co-essentiality network analysis of PRDX6 using FIREWORKS.b, Immunoblot analysis of lysates from cells expressing sgEmpty, sgControl or sgRNAs targeting PRDX6.Data are representative of three independent experiments.c, Immunoblot analysis of lysates from control or PRDX6 KO cells stably expressing the PRDX6 WT or C47S mutant.Data are representative of three independent experiments.

Fig. 4 |
Fig.4| PRDX6 augments efficient selenium utilization.a, Immunoblot analysis of lysates from control or the indicated KO cells cultured for 32 h in the presence of sodium selenite (1 μM) or (Sec) 2 (1 μM).Data are representative of two independent experiments.b,c, WT or PRDX6 KO cells were pretreated for 2 days with (Sec) 2 (100 nM) or sodium selenite (100 nM).Then, cells were treated for 48 h with FAC (50, 100 μg ml −1 ) in the presence of (Sec) 2 (100 nM) (b) or sodium selenite (100 nM) (c), and viability was measured.Viability data are presented as the mean ± s.d. of three biological replicates.d-f, Time course of selenoprotein expression by WT or PRDX6 KO MEFs in the presence of 50 nM (Sec) 2 (d), 100 nM sodium selenite (e) or 0.5 μg ml −1 SELENOP (f).Data are representative of two (in f) or three (in d and e) independent experiments.g, Schematic showing the in vitro reconstruction system used to evaluate the amount of Sec-tRNA[Ser]Sec .h,i, aa-tRNAs, which were purified from the indicated cells treated (or not) for 105 min with 50 nM (Sec) 2 , followed by treatment for 15 min with cycloheximide, were added to wheat germ extract in the presence or absence of eEFSec and SBP2.Data of aa-tRNAs from WT or SEPHS2 KO cells (h), and WT or PRDX6 KO cells (i) are shown.Data are presented as the mean ± s.d. of three independent experiments.**P < 0.01 (WT ± eEFSec and SBP2, P = 0.0037; PRDX6 WT versus KO, P = 0.0031); ****P < 0.0001; two-way ANOVA.

Fig. 5 |
Fig. 5 | PRDX6 is a selenide carrier protein that enables efficient selenium utilization.a, Immunoblot analysis of lysates from control or PRDX6 KO cells stably expressing enzymes involved in Sec-tRNA [Ser]Sec synthesis.Data are representative of three independent experiments.b, Immunoblot analysis of lysates from control or PRDX6 KO cells stably expressing SEPHS2 WT at endogenous levels.Data are representative of three independent experiments.c, aa-tRNAs, purified from the indicated cells treated with cycloheximide for 15 min, were added to wheat germ extract in the presence of eEFSec and SBP2.Data are expressed as the mean ± s.d. of three independent experiments.****P < 0.0001; *P < 0.05 (SEPHS2 U/C versus SEPHS2 WT O.E., P = 0.0213; SEPHS2 WT O.E. versus SEPHS2 WT, P = 0.0422); NS (P = 0.6212); one-way ANOVA.d,e, Percentage of C47 modification as calculated by mass spectrometry analysis.PRDX6 was incubated with GSH and sodium selenite (d), or with Sec and SCLY (e) for 5 min before mass spectrometry analyses.Data are presented as the mean ± s.d of three biological replicates.f, The physical association between PRDX6 and SEPHS2 was detected in a PLA.Scale bars, 20 μm.Data are representative of three independent experiments.Quantification of the PLA dots is also shown.Data are presented as the mean ± s.d.(n = 42 cells (empty-PRDX6), n = 67 cells (SEPHS2-PRDX6) examined), ****P < 0.0001; unpaired two-sided t-test.g, MEFs expressing HA-TurboID-empty, -PRDX1 or -PRDX6 were cultured for 30 min with DMSO or 50 μM biotin.Cell lysates and pulldown samples were analyzed by immunoblotting.Data are representative of two independent experiments.h,i, PRDX6-mediated enhancement of selenophosphate-mediated synthesis of SEPHS2 was evaluated by measuring the AMP product.Data are presented as the mean ± s.d. of three independent experiments in which selenocysteine and SCLY were used as the selenium source; *P < 0.05 (P = 0.0423); **P < 0.01 (P = 0.004); one-way ANOVA (h) or selenium bound to PRDX6 was used as the selenium source; **P < 0.01 (P = 0.0018); ***P < 0.001 (P = 0.0001); one-way ANOVA (i).j, Schematic showing the role of PRDX6 as a selenide carrier.

Extended Data Fig. 1 |
Characterization iron-induced cell death in FBXL5 KO cells.a, Phase-contrast images of cells after treatment with FAC (100 μg ml -1 ) for 48 h.Scale bars, 200 μm.Data are representative of three independent experiments.b, Fluorescence microscopy images of cells stained with a FerroOrange probe after treatment for 4 h with FAC (25 μg ml-1  ).Scale bars, 20 μm.Data are representative of three independent experiments.c, Flow cytometry analysis of lipid hydroperoxidation levels in WT or FBXL5 KO cells by C11-BODIPY staining after treatment with FAC (100 μg ml -1 l) for 24 h.Data are representative of two independent experiments.d, WT MEFs were pretreated for 3 h with bafilomycin A1 (100 nM), followed by FAC (100 μg ml -1 ) for the indicated times.Cell lysates were analyzed by immunoblotting.Data are representative of two independent experiments.e, Viability of FBXL5 KO MEFs treated for 24 h with FAC (100 μg ml -1 ) in the presence of bafilomycin A1 (0, 10, 100 nM).Data are presented as the mean ± s.d. of three biological replicates.f, g, Immunoblot analysis of lysates from the indicated cells.Data (f) are representative of two independent experiments.Expression check of (g) is a single experiment.h, Volcano plots of iron-triggered ferroptosis screen, focusing on the mitochondrial electron transport chain (complex I and II: left, complex III and IV: right).The p value and fold change were generated by the MAGeCK test.ExtendedData 2 | Validation of hit genes, and identification of PRDX6 as a GPX4 regulator.a, Hit suppressor genes [p-value cut off (p < 0.001)] were examined by gene ontology analysis.The p value was generated by the David gene ontology test.b, Negative score ranking of selenoproteins from the screening list.c, MAGeCK rank of suppressor genes.The p value and fold change were generated by the MAGeCK test.d, Immunoblot analysis of lysates from cells expressing sgRNAs targeting the indicated genes.Data are representative of two independent experiments.Extended Data Fig. 3 | PRDX6 regulates ferroptosis by maintaining expression of GPX4.a, Viability of FBXL5 KO cells or FBXL5/PRDX6 double knockout cells treated for 24 h with FAC (50 or 100 μg ml -1 ), and immunoblot analysis of lysates from the indicated cells.Viability data are presented as the mean ± s.d. of three biological replicates.b, WT or PRDX6 KO MEFs were treated with FAC (25 μg ml -1 ) for the indicated times, and cell lysates were analyzed by immunoblotting.Data are representative of two independent experiments.c, Flow cytometry analysis of lipid hydroperoxidation levels in WT and PRDX6 KO cells, as assessed by C11-BODIPY staining after treatment with FAC (50 μg ml -1 ) for 90 min.Data are representative of three independent experiments.d, Immunoblot analysis of lysates from control of PRDX6 KO cells expressing the GPX4 U/C mutant.Data are representative of two independent experiments.e, f, Viability of PRDX6 KO cells stably expressing the indicated PRDX6 mutants after incubation for 24 h in the presence of RSL3 (0.3 μM) (e) or IKE (0.3 μM) (f).Viability data are presented as the mean ± s.d. of three biological replicates.g, Measurement of GPX activity using recombinant proteins.Hydrogen peroxide (left) or tert-butyl hydroperoxide (right) were used as a substrate.Data are presented as the mean ± s.e.m of three independent experiments.h, Flow cytometry analysis of lipid hydroperoxidation after C11-BODIPY staining of WT or GPX4 KO cells stably expressing PRDX6 after removal of liiproxstatin-1 for 24 h.Data are representative of two independent experiments.i, Immunoblot analysis of lysates from the indicated cells.Data are representative of two independent experiments.Extended Data Fig. 4 | PRDX6 regulates expression of selenoproteins.a, Quantification of GPX4 mRNA levels by RT-qPCR.Data are expressed as the mean ± s.d of three biological replicates.ns; not significant; P (sgControl vs. sgPRDX6 #1 = 0.9523; sgControl vs. sgPRDX6 #2 = 0.9607); one-way ANOVA.b, c, Immunoblot analysis of lysates from control or PRDX6 KO cells incubated for 4 or 8 h in the presence of E64d/pep (10 μg ml -1 ) (b) or MG-132 (10 μM) (c).d, Immunoblot analysis of lysates from control or PRDX6 KO cells derived from the indicated human cancer cell lines (Hela, A549, H226, HepG2, NB-1).e, Immunoblot analysis of lysates from control cells or cells with KO of the indicated genes and stably expressing Myc-GFP WT.Data (b-e) are representative of two independent experiments.Extended Data Fig. 5 | PRDX6 regulates selenium utilization efficiency.a, Schematic showing selenium metabolism and the selenoprotein synthesis pathway.b, c, WT or PRDX6 KO cells were pretreated for 2 days with (Sec) 2 (100 nM) or sodium selenite (100 nM).Then, cells were treated for 24 h with RSL3 (0.3 or 0.6 μM) (b) or IKE (0.3 or 0.4 μM) (c) in the presence of 100 nM (Sec) 2 or 100 nM sodium selenite.d, Immunoblot analysis of lysates from control or PRDX6 KO Hela cells cultured for 48 h in the presence of sodium selenite (100 nM) or (Sec) 2 (100 nM).e, WT or PRDX6 KO Hela cells were pretreated with (Sec) 2 (100 nM) or sodium selenite (100 nM) for 2 days.Then, cells were treated for 48 h with FAC (100 or 200 μg ml -1 ) in the presence of (Sec) 2 (100 nM) or sodium selenite (100 nM), and cell viability was measured.f, g, Immunoblot analysis of lysates from control or PRDX6 KO cells cultured for 32 h in the presence of the indicated concentrations of (Sec) 2 (f) or sodium selenite (g).h, WT or PRDX6 KO cells were incubated for 24 h with 1% FBS in the presence of liproxstatin-1.Then, cells were treated with 50 nM (Sec) 2 or 100 nM sodium selenite for the indicated times.Cell lysates were analyzed by immunoblotting.i, PRDX6 KO cells were preincubated for 36 h with 10 nM (Sec) 2 or 40 nM sodium selenite.Then, WT or PRDX6 KO cells pretreated with selenium were exposed to 50 nM (Sec) 2 or 200 nM sodium selenite for the indicated periods and the lysates were analyzed by immunoblotting.Data are representative of two (d, h, i) or three (f, g) independent experiments.Viability data (b, c, e) are presented as the mean ± s.d. of three biological replicates.Extended Data Fig. 6 | Overexpression of SEPHS2 WT rescues the PRDX6 KO phenotype.a, Immunoblot analysis of lysates from control or SEPHS2 KO cells stably expressing the SEPHS2 U/C mutant.b, Immunoblot analysis of lysates from control or PRDX6 KO cells stably expressing the SEPHS2 WT or U/C mutant.c, Immunoblot analysis of lysates from cells stably expressing Myc-GFP C70U or S175U in control or PRDX6 KO cells stably expressing the SEPHS2 WT or U/ C mutant.Data (a-c) are representative of two independent experiments.d-f, Viability of the indicated cells in the presence of FAC (50 or 100 μg ml -1 ) for 48 h (d), or RSL3 (0.3 or 0.6 μM) (e) or IKE (0.3 or 0.45 μM) (f) for 24 h.Viability data are presented as the mean ± s.d. of three biological replicates.g, Viability of the indicated cells treated for 48 h with (Sec) 2 (10 μM) and liproxstatin-1 (3 μM).Viability data are presented as the mean ± s.d. of three biological replicates.*P (PRDX6 WT vs. C47S = 0.0243; WT vs. sgSEPHS2 = 0.0238) < 0.05; one-way ANOVA.h, Coomassie-stained SDS-PAGE gel showing the recombinant PRDX6 WT or C47S mutant used for ICP-MS analysis.ICP-MS analysis of the amount of selenium bound to recombinant PRDX6 WT or C47S.Data are presented as the mean ± s.d. of three independent experiments.*P (=0.0141) < 0.05, ***P (=0.0003) < 0.001; one-way ANOVA.Extended Data Fig. 8 | Interaction between PRDX6 and SEPHS2 or SCLY.a, The physical association between PRDX6 and SCLY was detected in a PLA assay.Scale bars, 20 μm.Data are representative of three independent experiments.Quantification of the PLA dots is also shown.Data are presented as the mean ± s.d.[n = 52 cells (Empty-PRDX6), n = 50 cells (SCLY-PRDX6) examined], ****P < 0.0001; unpaired two-sided t-test.b, c PRDX6 KO MEFs expressing HA-TurboID-empty, -PRDX1, or -PRDX6 were cultured for 30 min with DMSO or 50 μM biotin.Cell lysates and pulldown samples were analyzed by immunoblotting.The association between PRDX6 and SEPHS2 (b) or between PRDX6 and SCLY (c) was analyzed.Data are representative of two (c) or three (b) independent experiments.Extended Data Fig. 9 | Expression of PRDX6 is associated with a poor prognosis for several cancers.a, Expression profile of PRDX6 in several types of normal and tumor tissue.Data were obtained from TNMplot (https://tnmplot.com/analysis/), which is a web server that stores data regarding expression of normal and cancer-related genes.Those with higher values in tumor tissue compared to normal tissue and with significant differences (Mann-Whitney U test) are marked in red (P < 0.01).b, Kaplan-Meier survival curves comparing cancer samples showing high PRDX6 expression with samples showing low PRDX6 expression (curves were constructed using Kaplan-Meier plotter).Extended Data Fig. 10 | Expression of PRDX6 is important for survival of cancer cell lines.a, Immunoblot analysis of lysates from WT or PRDX6 KO Panc-1 cells.b, Continuous monitoring of the viability of WT or PRDX6 KO Panc-1 cells in the presence or absence of sodium selenite (100 nM), (Sec) 2 (100 nM), or liproxstatin-1 (1 μM).c, Immunoblot analysis of lysates from WT or PRDX6 KO MIA PaCa-2 cells.d, Continuous monitoring of the viability of WT or PRDX6 KO MIA PaCa-2 cells in the presence or absence of sodium selenite (100 nM), (Sec) 2 (100 nM), or liproxstatin-1 (1 μM).e, Immunoblot analysis of lysates from indicated cell lines.f, Viability of indicated cell lines in the presence of FAC (50 or 100 μg ml -1 ) for 48 h.Viability data are presented as the mean ± s.d. of three biological replicates.g, Immunoblot analysis of lysates from WT or PRDX6 KO SK-N-DZ cells.h, Continuous monitoring of the viability of WT or PRDX6 KO SK-N-DZ cells in the presence or absence of sodium selenite (100 nM), (Sec) 2 (100 nM), or liproxstatin-1 (1 μM).Viability data (b, d, h) are presented as the mean ± s.e.m of three biological replicates.Data (a, c, e, g) are representative of two independent experiments.
4.0 was used to calculate P values using Student's t-test, one-way ANOVA or two-way ANOVA, followed by Tukey's multiple comparison test.The P values are presented in the figure legends.All experiments were reproduced at least twice, each with similar results (the expression checks shown in Extended Data Figs.1g and 3a were single experiments).