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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2022 Oct 24;119(44):e2214227119. doi: 10.1073/pnas.2214227119

HOIP modulates the stability of GPx4 by linear ubiquitination

Kangyun Dong a,b, Ran Wei a,b, Taijie Jin a,b, Mengmeng Zhang a, Jiali Shen a,b, Huaijiang Xiang a,b, Bing Shan a, Junying Yuan a,1, Ying Li a,1
PMCID: PMC9636971  PMID: 36279464

Significance

Defects in linear ubiquitination result in dysregulation of inflammation and cell death signaling pathways. However, Tnfr1 knockout fails to fully rescue the embryonic lethality of LUBAC deficiency, implicating additional key cellular substrates of LUBAC in promoting cell survival. Our study suggests a previously unrecognized role of LUBAC-mediated linear ubiquitination in regulating GPx4 stability and cellular redox status in control of cell death.

Keywords: GPx4, ferroptosis, TNF, LUBAC

Abstract

LUBAC-mediated linear ubiquitination plays a pivotal role in regulation of cell death and inflammatory pathways. Genetic deficiency in LUBAC components leads to severe immune dysfunction or embryonic lethality. LUBAC has been extensively studied for its role in mediating TNF signaling. However, Tnfr1 knockout is not able to fully rescue the embryonic lethality of LUBAC deficiency, suggesting that LUBAC may modify additional key cellular substrates in promoting cell survival. GPx4 is an important selenoprotein involved in regulating cellular redox homeostasis in defense against lipid peroxidation-mediated cell death known as ferroptosis. Here we demonstrate that LUBAC deficiency sensitizes to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation. LUBAC binds and stabilizes GPx4 by modulating its linear ubiquitination both in normal condition and under oxidative stress. Our findings identify GPx4 as a key substrate of LUBAC and a previously unrecognized role of LUBAC-mediated linear ubiquitination in regulating cellular redox status and cell death.

M1-linked ubiquitination of key cellular signaling proteins by linear ubiquitin chain assembly complex (LUBAC) performs important regulatory functions in the control of gene activation and prevention of cell death (1–4). LUBAC complex includes the catalytic subunit HOIP (HOIL-interacting protein), and regulatory subunits HOIL-1 (Heme-oxidized IRP2 ubiquitin ligase 1) and SHARPIN (Shank-associated RH domain-interacting protein) (5–7). The function of LUBAC in mediating TNFR1 signaling has been extensively characterized (3, 8, 9). The loss of HOIP or HOIL-1 sensitizes to TNF-mediated cell death, including both apoptosis and necroptosis (10–13). Deficiency in the LUBAC core components HOIP or HOIL-1 in mice results in embryonic lethality (10, 11). Genetic knockout mice of Hoip or Hoil-1 die around embryonic day (E) 10.5, which can be extended to E16.5 by double knockout of Tnfr1 in Hoip−/−Tnfr1−/− mice and Hoil-1−/−Tnfr1−/− mice (10, 11). Ripk3−/−Caspase-8−/−Hoil-1−/− MEFs (Mouse Embryonic Fibroblasts) are resistant to cell death induced by TNF and related cytokines; however, knockout of Ripk3 or Caspase-8 fails to rescue the embryonic lethality of Hoil-1−/− mice. Mlkl−/−Caspase-8−/−Hoil-1−/− mice and Mlkl−/−Caspase-8−/−Hoip−/− mice show a reduced embryonic lethality but nevertheless runted as adults (11, 14). These findings suggest that LUBAC may modify additional key cellular substrates in promoting cell survival.

Glutathione peroxidase 4 (GPx4, phospholipid hydroperoxide glutathione peroxidase PHGPx) is an important selenoprotein involved in regulating cellular redox homeostasis (15, 16). GPx4 performs an antioxidant function by directly reducing peroxidized phospholipids produced in cell membrane, including phospholipid hydroperoxides and cholesterol hydroperoxides (17, 18). However, we still know little about the mechanism that modulates GPx4 stability. GPx4 deficiency enhances cellular lipid peroxidation, which can lead to ferroptosis, an iron-dependent nonapoptotic cell death that can be elicited by chemical inhibitors of GPx4 or the cystine/glutamate antiporter system Xc− in cancer cells (19, 20). Knockout of Gpx4 induces lipid peroxidation-dependent, caspase-independent cell death in cells and embryonic lethality (17, 21). Inducible inactivation of GPx4 in cells and mice triggers lipid peroxidation and cell death (22, 23). Understanding the mechanism that regulates GPx4 may provide new insights into the cellular defense system against oxidative stress.

In this manuscript, we investigated the involvement of LUBAC in ferroptosis. We found that LUBAC deficiency sensitized cells to ferroptosis. We further characterized the mechanism by which LUBAC modulated the sensitivity to ferroptosis and found that LUBAC performed linear ubiquitination of GPx4 to enhance its stability and resistance to ferroptosis. Our study demonstrates the role and mechanism by which linear ubiquitination modulates the cellular redox status by controlling the stability of GPx4.

Results

LUBAC Regulates the Cellular Sensitivity to Ferroptosis.

We treated Hoip+/+ and Hoip−/− MEFs with Erastin and RSL3, two compounds that are known to induce ferroptosis (20, 24). We found that Hoip knockout sensitized to cell death induced by both Erastin and RSL3, which was inhibited upon the addition of Ferrostatin-1 (Fer-1) (Fig. 1 A and B). The protective effects of HOIP were also demonstrated when we evaluated the cell viabilities of Hoip+/+ and Hoip−/− MEFs treated with Erastin or RSL3 (Fig. 1 C and D and SI Appendix, Fig. S1 A–C). The sensitization of Hoip knockout to Erastin and RSL3 was both dose and time dependent (Fig. 1 C–F). The treatment of wild-type (WT) MEFs with Erastin (10 μM) and RSL3 (500 nM) for 8 h had minimum effect on survival, but could induce substantial cell death in Hoip−/− MEFs (Fig. 1 E and F). Remarkably, the increased sensitivity to ferroptosis was rescued by the expression of WT HOIP but not its inactive form (C879S), suggesting that regulation of ferroptosis sensitivity by LUBAC was dependent on its E3 ligase activity (Fig. 1 E and F). Small-molecule FIN56 can induce ferroptosis by promoting GPx4 degradation (25). HOIP deficiency also promoted FIN56-induced ferroptosis (SI Appendix, Fig. S1 C and D).

Fig. 1.

Fig. 1.

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LUBAC regulates the cellular sensitivity to ferroptosis. (A and B) Hoip+/+ and Hoip−/− MEFs were pretreated with or without Fer-1 (10 μM) and then Erastin for 8 h or RSL3 for 6 h as indicated. Samples were stained with PI (1 μg/mL) for dead cells and Hoechst 33342 (1 μg/mL) for total cells and then analyzed by Operetta CLS High-Content Analysis System. Six wells of cells were counted for each treatment condition. At least 1,000 cells in each well were counted. Mean ± SEM of n = 6. ****P < 0.0001. (C and D) Hoip+/+ and Hoip−/− MEFs were treated with RSL3 for 8 h and Erastin for 12 h as indicated. Cell viability was determined by the CellTiter-Glo assay. Mean ± SEM of n = 3. *P < 0.05, **P < 0.01. (E) Hoip−/− MEFs were reconstituted with expression vector of empty vector, HOIP WT, or HOIP C879S. Cells were then treated with Erastin (10 μM) for the indicated times. Cell viability was determined by the CellTiter-Glo assay. Mean ± SEM of n = 6. ****P < 0.0001. (F) Hoip−/− MEFs were reconstituted with expression vector of empty vector, HOIP WT, or HOIP C879S. Cells were then treated with RSL3 (500 nM) for the indicated times. Cell viability was determined by the CellTiter-Glo assay. Mean ± SEM of n = 4. **P < 0.01, ****P < 0.0001. (G–I) HT-22 cells were transfected with siRNA oligos targeting Cyld, Otulin, or NTC as control for 48 h. Cells were first pretreated with Nec-1s (10 μM), Fer-1 (10 μM), and zVAD (50 μM) and then treated with RSL3 (500 nM) for 6 h and Erastin (10 μM) for 8 h as indicated. Cell viability was determined by the CellTiter-Glo assay. Mean ± SEM of n = 3. n.s. no significance, **P < 0.01, ***P < 0.001. Cell lysates were analyzed by Western blotting with antibodies as indicated. DMSO, dimethyl sulfoxide.

Although loss of HOIP also sensitizes to TNFR1-mediated apoptosis and necroptosis (10), caspase inhibitor zVAD.fmk and RIPK1 inhibitor Nec-1s were unable to inhibit ferroptosis induced by Erastin and RSL3 in both Hoip+/+ and Hoip−/− MEFs (SI Appendix, Fig. S1 E and F), suggesting that LUBAC regulates cellular sensitivity to ferroptosis independent of its previous function in regulating apoptosis and necroptosis. SHARPIN and HOIL-1 are cofactors of LUBAC complex (5–7). Consistently, knockout of Sharpin or Hoil-1 also enhanced the sensitivity to ferroptosis in HT-22 cells (SI Appendix, Fig. S1G). Thus, LUBAC contributes to cellular resistance to ferroptosis.

CYLD (26) is a deubiquitinating enzyme (DUB) capable of removing M1-linked ubiquitin chains (27). We also tested whether CYLD was involved in regulating ferroptosis. Knockdown of CYLD reduced necroptosis as expected (28) (SI Appendix, Fig. S1H), suggesting siRNA oligos targeting CYLD worked well. Knockdown of CYLD by siRNAs did not influence ferroptosis induced by either Erastin or RSL3 in HT-22 cells (Fig. 1 G–I), suggesting that CYLD is not involved in regulating ferroptosis.

OTULIN is a DUB that positively regulates the activity of LUBAC (29–33). LUBAC activity is impaired in OTULIN-deficient cells. We tested the effect of OTULIN deficiency on ferroptosis. Knockdown of OTULIN sensitized to necroptosis as expected (34–37) (SI Appendix, Fig. S1H), confirming the functional knockdown by siRNA oligos targeting OTULIN. Knockdown of OTULIN also sensitized to cell death induced by both Erastin and RSL3 in HT-22 cells (Fig. 1 G–I). These results suggest a specific role of LUBAC activity in regulating ferroptosis.

Loss of LUBAC Promotes the Accumulation of Lipid Peroxides.

Ferroptosis is induced by the accumulation of lipid peroxides which can be inhibited by Fer-1 (20). The above data showed that Fer-1 inhibited ferroptosis induced by Erastin and RSL3 in Hoip+/+ and Hoip−/− MEFs (Fig. 1 A and B and SI Appendix, Fig. S1 A–C), suggesting that HOIP deficiency may sensitize to ferroptosis by promoting lipid peroxidation. Thus, we analyzed the status of lipid peroxidation in Hoip+/+ and Hoip−/− MEFs and found that HOIP deficiency promoted the accumulation of lipid peroxides induced by RSL3 and Erastin (Fig. 2A and SI Appendix, Fig. S2A). Consistently, restoring the expression of HOIP rescued the increased accumulation of lipid peroxides induced by RSL3 and Erastin (Fig. 2B and SI Appendix, Fig. S2B). Hence, LUBAC dysfunction results in promoting lipid peroxidation induced by Erastin and RSL3, and thus sensitizes cells to ferroptosis. These data suggest that LUBAC deficiency–sensitized ferroptosis is also lipid peroxidation dependent.

Fig. 2.

Fig. 2.

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LUBAC regulates the accumulation of lipid peroxides induced by Erastin. (A) Hoip+/+ and Hoip−/− MEFs were pretreated with Fer-1 (10 μM) and then treated with Erastin (10 μM) for the indicated times. Cells were then stained with 10 μM C11-BODIPY (Boron dipyrromethene fluoride) 581/591 in complete growth medium at 37 °C and analyzed by Guava easyCyte HT. (B) Hoip−/− MEFs reconstituted with expression vector of empty vector or Flag-HOIP were pretreated with Fer-1 (10 μM) and then treated with Erastin (10 μM) for the indicated times. Cells were then stained with 10 μM C11-BODIPY 581/591 in complete growth medium at 37 °C and analyzed by Guava easyCyte HT.

LUBAC Regulates GPx4 Stability to Modulate Ferroptosis.

We next explored the target of LUBAC in sensitizing to ferroptosis. Since RSL3 and FIN56 trigger ferroptosis by promoting GPx4 degradation (24, 25), and knockdown of GPx4 is sufficient to induce ferroptosis (22), we first considered GPx4 as the substrate mediated by LUBAC. Interestingly, we found that ferroptosis induced by knockdown of GPx4 was not affected by HOIP deficiency (Fig. 3 A and B). Furthermore, restoring the expression of HOIP also had no effect on ferroptosis in cells with GPx4 knockdown (SI Appendix, Fig. S3A). These results suggest that GPx4 is the candidate target of LUBAC in regulating ferroptosis.

Fig. 3.

Fig. 3.

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LUBAC regulates GPx4 stability to modulate ferroptosis. (A and B) Hoip+/+ and Hoip−/− MEFs were transfected with siRNA oligos targeting Gpx4 and NTC as control and treated with Fer-1 (10 μM), as indicated, for 48 h. Cell viability was determined by the CellTiter-Glo assay. Mean ± SEM of n = 6. ****P < 0.0001. Cell lysates were analyzed by Western blotting with antibodies as indicated. (C) Hoip+/+, Hoip−/− MEFs, and Hoip−/− MEFs reconstituted with expression vector of Flag-HOIP were treated with RSL3 (500 nM) for the indicated times. Cell lysates were analyzed by Western blotting with antibodies as indicated. (D) Hoip−/− MEFs reconstituted with expression vector of empty vector, HOIP WT, or HOIP C879S were treated with RSL3 (500 nM) for the indicated times. Cell lysates were analyzed by Western blotting with antibodies as indicated. (E and F) Hoip+/+ and Hoip−/− MEFs were treated with RSL3 (500 nM) for the indicated times. Cell lysates were analyzed by Western blotting with antibodies as indicated. (G) Hoip+/+ and Hoip−/− MEFs were treated with Na2SeO3 (1 μM) for the indicated times. Cell lysates were analyzed by Western blotting with antibodies as indicated. (H) Hoip+/+ and Hoip−/− MEFs were treated with Na2SeO3 (1 μM) followed by RSL3 (500 nM) for the indicated times. Cell viability was determined by the CellTiter-Glo assay. Mean ± SEM of n = 3. *P < 0.05, **P < 0.01, ****P < 0.0001. (I) Hoip+/+, Hoip−/− MEFs, and Hoip−/− MEFs reconstituted with expression vector of Flag-HOIP were treated with CHX (1 μg/mL) and RSL3 (500 nM) for 8 h. Cell lysates were analyzed by Western blotting with antibodies as indicated. (J) HEK293T cells were transfected with expression vectors of Flag-cGPx4 and HA-HOIP, as indicated, and with supplementation of Na2SeO3 (1 μM) for 24 h followed by CHX treatment (1 μg/mL), as indicated, for 8 h and RSL3 treatment (500 nM) for the indicated times before harvest. Cell lysates were analyzed by Western blotting with antibodies as indicated. (K) Hoip+/+ and Hoip−/− MEFs were treated with RSL3 (500 nM), PS-341 (200 nM), and E64D (10 μM), as indicated, for 6 h. Cell lysates were analyzed by Western blotting with antibodies as indicated.

LUBAC ubiquitinates several key components of TNF-RSC, such as TNFR1, TRADD, RIPK1, NEMO, and cFLIP, contributing to the stabilization of this protein complex and prevention of cell death (8, 12, 38). Moreover, LUBAC-mediated M1-linked ubiquitination is known to maintain protein stability of cFLIP (12). GPx4 levels are known to be reduced during ferroptosis (25). Interestingly, knockdown of OTULIN, but not that of CYLD, reduced the levels of GPx4 (Fig. 1I). OTULIN deficiency is known to reduce the stability of LUBAC (31, 33). Thus, we considered the possibility that M1-linked ubiquitination might also affect GPx4 stability. We found that the levels of GPx4 were lower in untreated control Hoip−/− MEFs compared to that of Hoip+/+ MEFs which can be restored upon complementation of HOIP (Fig. 3C). Moreover, the reduction in the levels of GPx4 was rescued by restoring the expression of WT HOIP but not by the inactive form (C879S) (Fig. 3 C and D and SI Appendix, Fig. S3B).

The protein stability of GPx4, which controls the lipid peroxidation and redox homeostasis, is regulated by its catalytic activity (24). We found that treatment of Hoip−/− MEFs with RSL3 and Erastin, which increases cellular reactive oxygen levels, accelerated the reduction of GPx4 compared to that of Hoip+/+ MEFs (Fig. 3 C–F and SI Appendix, Fig. S3 C and D). Conversely, the supplementation of sodium selenite (Na2SeO3) in culture medium, which was known to induce the protein levels of GPx4, can reduce the cellular reactive oxygen levels (39). We found that the ability of sodium selenite to increase the levels of GPx4 was impaired in Hoip−/− MEFs compared to that of Hoip+/+ MEFs (Fig. 3G). However, although supplementation of sodium selenite could increase the resistance against ferroptosis in both Hoip+/+ and Hoip−/− MEFs, the resistance of Hoip−/− MEFs in the presence of sodium selenite was still lower compared to that of Hoip+/+ MEFs (Fig. 3H).

Reduction in the abundance of GPx4 appeared to be specific, as the protein levels of other key regulators of ferroptosis, such as ACSL4 and SLC7A11, were not affected by HOIP deficiency (Fig. 3E and SI Appendix, Fig. S3C). The expression of FSP1 positively correlates with cellular resistance to ferroptosis (40, 41). Interestingly, the expression level of FSP1 was slightly higher in Hoip−/− MEFs, but HOIP deficiency still sensitized to ferroptosis, suggesting that the increased expression of FSP1 may be a compensatory resistance response against ferroptosis (Fig. 3E and SI Appendix, Fig. S3C). The protein levels of other selenoproteins, such as SELN and SELT, were also not affected by HOIP deficiency (Fig. 3F and SI Appendix, Fig. S3D).

Next, we determined whether HOIP deficiency sensitized GPx4 instability by affecting the transcription or protein synthesis. We found that HOIP deficiency had no effects on the messenger RNA levels of GPx4, whether or not ferroptosis was induced (SI Appendix, Fig. S3E). Furthermore, the reduction in the levels of GPx4 in Hoip−/− MEFs was further accelerated by the treatment of RSL3 together with protein synthesis inhibitor cycloheximide (CHX), compared to that of Hoip+/+ MEFs (Fig. 3I). These results suggest that HOIP deficiency may promote GPx4 degradation. In addition, under conditions where protein synthesis was inhibited by CHX, treatment with RSL3 induced the degradation of GPx4 protein, which was rescued by HOIP overexpression (Fig. 3J), suggesting that overexpression of HOIP stabilized GPx4 independent of protein synthesis. Furthermore, inhibition of proteasome by PS-341, but not lysosome inhibition by E64D, was able to rescue the decreased protein level of GPx4 induced by RSL3 in both Hoip+/+ and Hoip−/− MEFs, suggesting that treatment with RSL3 accelerates GPx4 degradation via proteasome pathway and that HOIP deficiency–accelerated GPx4 instability is dependent on protein degradation (Fig. 3K). On the other hand, the reduction in the levels of GPx4 in Hoip−/− MEFs treated with Erastin could be rescued by inhibitors of both proteasome and lysosome (SI Appendix, Fig. S3F), suggesting that HOIP deficiency can promote the degradation of GPx4 via both the proteasome and lysosome pathways under different conditions.

Taken together, these results suggest that LUBAC inhibits ferroptosis by repressing GPx4 degradation and HOIP deficiency–sensitized ferroptosis is still oxidative stress in nature.

LUBAC Mediates M1-Linked Ubiquitination of GPx4.

Since LUBAC deficiency sensitizes to lipid peroxidation–mediated ferroptosis, we next characterized the linear ubiquitination status during ferroptosis. We found that global M1-linked ubiquitination was dramatically increased when HT-22 cells were treated with RSL3 (Fig. 4A). In contrast, K63-linked ubiquitination was slightly increased in RSL3-treated cells, while K48-linked ubiquitination was slightly decreased (Fig. 4A). We also observed increased levels of global M1-linked ubiquitination in HT-1080 cells treated with either Erastin or RSL3 (SI Appendix, Fig. S4 A and B), indicating that ubiquitination, especially M1-linked ubiquitination, was activated during ferroptosis.

Fig. 4.

Fig. 4.

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LUBAC mediates M1-linked ubiquitination of GPx4. (A) HT-22 cells were treated with RSL3 (250 nM) for the indicated times. Cell lysates were analyzed by Western blotting with antibodies as indicated. (B) HT-1080 cells stably transfected with expression vector of 3×Flag-cGPx4 were treated with RSL3 (500 nM) for the indicated times and then lysed in 1% sodium dodecyl sulfate (SDS) lysis buffer followed by incubation at 100 °C for 30 min. Cell lysates were then subjected to immunoprecipitation via anti-Flag beads after 10-times dilution by Nonidet P-40 lysis buffer and analyzed by Western blotting with antibodies as indicated. (C) HT-1080 cells were treated with Erastin (10 μM) for the indicated times. Cell lysates were subjected to immunoprecipitation via the M1 antibody, which specifically recognizes M1-linked ubiquitination, and then analyzed by Western blotting with antibodies as indicated. (D) HEK293T cells were transfected with expression vectors of Flag-cGPx4 and Ub with HA-HOIP or HA-HOIP C885S, as indicated, with supplementation of Na2SeO3 (1 μM) for 24 h. Cell lysates were subjected to immunoprecipitation via anti-Flag beads and then analyzed by Western blotting with antibodies as indicated. (E) HEK293T cells were transfected with expression vectors of Flag-cGPx4 and Ub with HA-HOIP or HA-HOIP C885S, as indicated, with supplementation of Na2SeO3 (1 μM) for 24 h. Cells were lysed in 1% SDS lysis buffer and incubated at 100 °C for 30 min. Cell lysates were then subjected to immunoprecipitation via anti-Flag beads after 10-times dilution by Nonidet P-40 lysis buffer. All samples were then analyzed by Western blotting with antibodies as indicated. (F) Flag-cGPx4 overexpressed in HEK293T cells was immunoprecipitated by anti-Flag beads. The typical reaction system contained 1 μM His-UBA1, 2 μM Trx-UBE2L3, 100 μM Ub, 1 μM GST-HOIP (480 to 1072 aa), and 1 μM HOIL-1 UBL (53 to 135 aa) in the buffer with 50 mM Tris⋅HCl (pH7.5), 150 mM NaCl, 1 mM DTT, 10 mM ATP, and 10 mM MgCl2. Reaction mixtures were incubated with anti-Flag beads harboring Flag-cGPx4 at room temperature for the indicated times. All samples were then analyzed by Western blotting with antibodies as indicated.

The results described above suggest that LUBAC might negatively regulate GPx4 degradation during ferroptosis by mediating its M1-linked ubiquitination. We first demonstrated that GPx4 was ubiquitinated during ferroptosis (Fig. 4B). We also found that the M1-linked ubiquitination of GPx4 was stimulated at the early time points with Erastin and RSL3 (up to 2 h) but reduced at the late stage of ferroptosis at 4 h (Fig. 4C and SI Appendix, Fig. S4C).

Next, we checked whether GPx4 could be directly ubiquitinated by HOIP. When coexpressed with HOIP, but not the inactive mutant (C885S), GPx4 was modified by ubiquitin chains (Fig. 4D). To confirm that HOIP ubiquitinates GPx4 itself rather than its interactors, the samples were lysed and boiled to denature before Flag-cGPx4 was immunoprecipitated. The expression of HOIP, but not the inactive mutant (C885S), enhanced the ubiquitination of GPx4 (Fig. 4E). We also immunoprecipitated GPx4 and carried out in vitro ubiquitination assay by incubating with His-UBA1 (E1), Trx-UBE2L3 (E2), HOIL-1 UBL (53 to 135 aa), and GST-HOIP (480 to 1072 aa) for different times. GPx4 was ubiquitinated in vitro by HOIP complex (Fig. 4F), indicating that GPx4 was the substrate of LUBAC. Taken together, LUBAC targets GPx4 and catalyzes the formation of its M1-linked ubiquitin chains.

We next explored the potential ubiquitination sites of GPx4 induced by HOIP by mass spectrometry (SI Appendix, Fig. S4D). Mass spectrometry analysis identified multiple lysine residues on GPx4 that were ubiquitinated by LUBAC (SI Appendix, Fig. S4E). We generated HT-1080 cells stably expressing GPx4 with different lysine to arginine mutations and tested their effects on inhibiting ferroptosis. Further analysis showed that K47R, K117R, and K145R mutations reduced the ability of GPx4 to protect against ferroptosis induced by either RSL3 or Erastin (SI Appendix, Fig. S4 F–H), indicating K47, K117, and K145 as potential ubiquitination sites of GPx4 modified by LUBAC. In sum, LUBAC targets GPx4 and catalyzes the formation of its M1-linked ubiquitin chains to resist ferroptosis.

GPx4 Interacts with LUBAC in a Redox-Sensitive Manner.

Considering that LUBAC mediates M1-linked ubiquitination of GPx4, we first examined whether GPx4 might directly interact with LUBAC. Indeed, in cells cultured under normal conditions, we detected the interaction between endogenous GPx4 and HOIP as well as SHARPIN, which are present together in the LUBAC complex (6, 7) (Fig. 5A). We also detected the interaction between SHARPIN and GPx4 as well as that between HOIP and GPx4 in HEK293T cells, by coimmunoprecipitation (Fig. 5 B and C and SI Appendix, Fig. S5A). When ferroptosis was induced by RSL3, the interaction of GPx4 with HOIP and SHARPIN was maintained within a short period of time, but declined at longer time points (Fig. 5D and SI Appendix, Fig. S5 B and C). Thus, GPx4 may interact with LUBAC in a redox-sensitive manner.

Fig. 5.

Fig. 5.

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GPx4 recruits LUBAC to catalyze the formation of its M1-linked ubiquitin chains. (A) HT-1080 cells were lysed in Nonidet P-40 lysis buffer. Cell lysates were subjected to immunoprecipitation via GPx4 antibody or IgG as control and then analyzed by Western blotting with antibodies as indicated. (B) HEK293T cells were transfected with expression vectors of HA-HOIP and Flag-cGPx4, as indicated, with supplementation of Na2SeO3 (1 μM) for 24 h. Cell lysates were subjected to immunoprecipitation via anti-Flag beads and then analyzed by Western blotting with antibodies as indicated. (C) HEK293T cells were transfected with expression vectors of GFP-cGPx4 and Flag-SHARPIN, as indicated, with supplementation of Na2SeO3 (1 μM) for 24 h. Cell lysates were subjected to immunoprecipitation via anti-Flag beads and then analyzed by Western blotting with antibodies as indicated. (D) HT-1080 cells stably transfected with expression vector of 3×Flag-cGPx4 were treated with RSL3 (500 nM) for the indicated times. Cell lysates were then subjected to immunoprecipitation via anti-Flag beads and analyzed by Western blotting with antibodies as indicated. (E) HT-1080 cells were stably transfected with expression vector of 3xFlag-cGPx4 WT, U46C, or GFP as control. Cells were treated with RSL3 (500 nM) for the indicated times. Cell lysates were subjected to immunoprecipitation via anti-Flag beads and then analyzed by Western blotting with antibodies as indicated.

GPx4 is a selenoprotein containing the rare amino acid selenocysteine (Sec) in its active site, which is decoded by the UGA codon (42). Selenolate-based activity of GPx4 plays an indispensable role in prevention of ferroptosis; thus, mutating the active-site Sec (Sec-mutant) inactivates its activity in suppressing ferroptosis (43). Consistent with a redox sensitive interaction with LUBAC, U46C Sec-mutant GPx4 showed a reduced ability to recruit LUBAC, including both HOIP and SHARPIN compared to that of WT GPx4 (Fig. 5E). In addition, we found that HOIP preferred to interact with WT GPx4 compared with Sec-mutant GPx4, even when the expression levels of Sec-mutant GPx4 were higher (SI Appendix, Fig. S5D). Thus, the recruitment of LUBAC to GPx4 during ferroptosis may require its Sec site. Since the Sec in GPx4 is critical for its cellular antioxidant function (43), this result also suggests the redox regulation of its interaction with LUBAC.

Taken together, these results suggest that the interaction of GPx4 and LUBAC is regulated in a manner sensitive to the redox environment in cells.

Preexisting Ubiquitin Chains on GPx4 Enable LUBAC Recruitment.

The recruitment of LUBAC to signaling complexes has been found to be dependent upon the preexisting ubiquitin chains which interact with the NZF1 domain of HOIP or the NZF domain of SHARPIN or HOIL-1 (1, 5, 6, 44). Remarkably, the recruitment of LUBAC to GPx4 was changed in a manner consistent with its ubiquitination level during ferroptosis (Fig. 5 D and E), suggesting the recruitment of LUBAC may depend on the preexisting ubiquitin chains of GPx4. Furthermore, U46C GPx4, which showed a reduced ability to recruit HOIP and SHARPIN, also showed reduced levels of ubiquitination in general. These results suggest that the preexisting ubiquitination of GPx4 is sensitive to its redox catalytic status (Fig. 5E). Hence, we first checked whether the recruitment of LUBAC to GPx4 involved other ubiquitination events. We immunoprecipitated GPx4 complexes and incubated with GST-USP2, a DUB that can remove different types of ubiquitin chains (27). The result showed that the recruitment of HOIP to GPx4 was eliminated by incubation with purified GST-USP2 (Fig. 6A and SI Appendix, Fig. S5E). Additionally, the interaction between HOIP and GPx4 was also disrupted after in vitro deubiquitination assay mediated by GST-USP2 (SI Appendix, Fig. S5F). Since USP2 removes K48- and K63-linked ubiquitin chains (27), this result demonstrates the functional role of non-M1-linked ubiquitination in M1-linked ubiquitination of GPx4 and that GPx4 may recruit LUBAC via its preexisting ubiquitin chains during ferroptosis.

Fig. 6.

Fig. 6.

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Preexisting ubiquitin chains on GPx4 enable LUBAC recruitment. (A) HT-1080 cells stably transfected with expression vector of 3×Flag-cGPx4 were treated with RSL3 (500 nM) for 0.5 h. Samples were divided into two parts, and one part was incubated with purified GST-USP2 (258-605) (2 μM) at room temperature for 2 h. Cell lysates were then subjected to immunoprecipitation via anti-Flag beads. All samples were then analyzed by Western blotting with antibodies as indicated. (B) HT-1080 cells stably transfected with expression vector of 3×Flag-cGPx4 were treated with RSL3 (500 nM) for the indicated times. Cells were then subjected to immunoprecipitation via anti-Flag beads and analyzed by Western blotting with antibodies as indicated. (C) HEK293T cells were transfected with expression vectors of Flag-cGPx4 and His-Ub (K48), as indicated, with supplementation of Na2SeO3 (1 μM) for 24 h. Cells were treated with RSL3 (500 nM) for the indicated times. Cell lysates were then subjected to pull-down via Ni-NTA and analyzed by Western blotting with antibodies as indicated. (D) HEK293T cells were transfected with expression vectors of Flag-cGPx4 and His-Ub (K63), as indicated, with supplementation of Na2SeO3 (1 μM) for 24 h. Cells were treated with RSL3 (500 nM) for the indicated times. Cell lysates were then subjected to pull-down via Ni-NTA and analyzed by Western blotting with antibodies as indicated. (E) HEK293T cells were transfected with expression vectors of Flag-cGPx4 and His-Ub (K48), as indicated, with supplementation of Na2SeO3 (1 μM) for 24 h. Cells were pretreated with PS-341 (250 nM) for 6 h and then treated with RSL3 (500 nM) for 1 h. Cell lysates were then subjected to pull-down via Ni-NTA and analyzed by Western blotting with antibodies as indicated.

To explore the functional role of other types of ubiquitin chains on GPx4 during ferroptosis, we further verified K48- and K63-linked ubiquitination of GPx4 using antibodies specifically recognizing K48- and K63-linked ubiquitin chains (Fig. 6B). We also observed rapid stimulation of K48- and K63-linked ubiquitination of GPx4 induced by RSL3 using His-Ub-K48-only and His-Ub-K63-only, which gradually reduced over time (Fig. 6 C and D). Consistently, proteasome inhibition enhanced the signals of K48-linked ubiquitination (Fig. 6E). Hence, during ferroptosis, GPx4 is modified by multiple types of ubiquitin chains, which may provide the basis for the recruitment of LUBAC and, subsequently, M1-linked ubiquitination of GPx4 to antagonize ferroptosis at early time points.

Discussion

GPx4 is an important selenoprotein that is essential for the survival of cells and animals (17, 21). GPx4 deficiency leads to lipid peroxidation–dependent cell death, and thus suppression of phospholipid peroxidation is essential for cell survival in normal tissues in animals (16, 22). The role of LUBAC in modulating the TNF signaling pathway has been well established. However, the embryonic lethality of Hoip and Hoil-1 knockout mice can only be partially rescued by double knockout of Tnfr1 (10, 11). Thus, there should be additional key substrates of LUBAC that are important for cell survival. Here we provide evidence that supports the role of LUBAC in maintaining the cellular redox status by moderating the rate of lipid peroxidation. By performing M1-linked ubiquitination of GPx4, LUBAC maintains the stability of GPx4 in normal conditions and also when cells encounter oxidative stress. Our data suggest that GPx4 is modified by multiple types of ubiquitin chains during ferroptosis, which may provide an important basis for LUBAC to perform M1-linked ubiquitination of GPx4 and also explain why we had difficulty in demonstrating M1-linked ubiquitination of recombinant GPx4 by LUBAC in vitro. Consistently, M1-linked ubiquitination of IRAK1 by LUBAC also requires preexisting K63-linked ubiquitination (45). Since Gpx4 knockout mice die in early embryonic development around E8 in uterus (17, 21), the reduction of GPx4 levels may contribute to the embryonic lethality of Hoip and Hoil-1 knockout mice. In addition, it is interesting to note that the reduction in SHARPIN, a key regulatory component of the LUBAC complex, has been shown to promote oxidative stress-mediated inflammatory response in animal models of nonalcolic steatohepatitis (46). The reduced levels of LUBAC-mediated M1-linked ubiquitination of GPx4 may contribute to human inflammatory diseases that are characterized by increased oxidative stress. In addition, our study also demonstrates that GPx4 interacts with LUBAC in a manner sensitive to its selenolate status, suggesting that the selenolate state of GPx4 not only regulates the cellular redox state but also may affect its interacting partner, an interesting possibility which can be investigated in the future.

The loss of LUBAC-mediated M1-linked ubiquitination on GPx4 sensitizes cells to ferroptosis induced by compounds such as Erastin and RSL3. Our study suggests the loss of M1-linked ubiquitination of GPx4 mediated by LUBAC may sensitize cells to lipid peroxidation and ferroptosis through regulating GPx4 stability. Although we do not have the tool to determine the percentage of GPx4 that is subject to the M1 ubiquitination upon the activation of ferroptosis, the sensitized response of LUBAC-deficient cells to ferroptosis suggests the importance of this regulation. Reactive oxygen species generated by mammalian cells may function in signaling downstream of cytokines and transcription factors (47, 48). In particular, reactive oxygen intermediates have also been proposed to be involved in TNF signaling (49). Thus, regulation of GPx4 M1-linked ubiquitination by LUBAC may also be involved in the signaling process, including that of TNFR1. Our study expands the understanding about LUBAC biology and the mechanism by which LUBAC-mediated M1-linked ubiquitination supports cell survival.

Materials and Methods

Reagents and Antibodies.

The following commercial antibodies and reagents were used in this study: GPx4 (Abcam ab125066 and Santa Cruz sc-166570), SHARPIN (Proteintech 14626-1-AP), HOIL-1 (Santa Cruz sc-365523), CYLD (Santa Cruz sc-74435), OTULIN (Cell Signaling Technology 14127), β-actin (TransGen Biotech HC201), SLC7A11 (Cell Signaling Technology 98051), FSP1 (Proteintech 20886-1-AP), SELN (Santa Cruz sc-365824), SELT (Abcam ab176192), Flag (Cell Signaling Technology 2368), HA (SAB T501), Myc (Sigma-Aldrich m4493), M1-Ub (EMD Millipore MABS451), K63-Ub (EMD Millipore 05-1308), K48-Ub (EMD Millipore 05-1307), Ub (Dako Z0458), His (TransGen Biotech HT501), Anti-Flag affinity gel (Sigma-Aldrich A2220), Anti-Myc affinity gel (Biotool B23402), Anti-HA affinity gel (Biotool B23302), Ni-NTA His-Bind Resin (EMD Millipore 70666-4), RSL3 (TargetMol T3646), Erastin (TargetMol T1765), CHX (MCE HY-12320), E64D (MCE HY-100229), PS-341 (MCE HY-10227), Fer-1 (MCE HY-100579), and Na2SeO3 (Sigma-Aldrich 214485).

Statistics.

Data are expressed as the mean ± SEM. Error bars indicate the SEM. Pairwise comparisons between two groups were performed using the Student’s t test. Differences were considered statistically significant if *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001, or as not significant. At least three independent biological repeats were included in each data point. Each experiment was repeated at least three times.

Online Supplemental Material.

SI Appendix, Fig. S1 shows that deficiency in LUBAC components and OTULIN but not CYLD sensitized cells to ferroptosis. SI Appendix, Fig. S2 shows HOIP deficiency increased the accumulation of lipid peroxides induced by Erastin, which was rescued by restoring the expression of HOIP. SI Appendix, Fig. S3 shows HOIP deficiency promoted GPx4 degradation but not transcription. SI Appendix, Fig. S4 shows increased linear ubiquitination of GPx4 during ferroptosis and potential ubiquitination sites of GPx4 catalyzed by LUBAC. SI Appendix, Fig. S5 shows the interaction between GPx4 and HOIP.

Supplementary Material

Supplementary File

Acknowledgments

We thank Drs. Vishva Dixit and Kim Newton of Genentech for kindly providing M1, K48, and K63-ubiquitin chain-specific abs. We thank Dr. Henning Walczak for kindly providing Hoip+/+ and Hoip−/− MEF cells. We thank Dr. Lifeng Pan for kindly providing materials for in vitro ubiquitination assay, including GST-HOIP (480 to 1072 aa), HOIL-1 UBL (53 to 135 aa), His-UBA1, Trx-UBE2L3, and Ub. This work was supported by the China National Natural Science Foundation (Grants: 82188101, 21837004, 91849204, 92049303, and 32170755), China National Natural Science Youth Foundation (Grant: 31801163), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant: XDB39030200), the Shanghai Municipal Science and Technology Major Project (Grant: 2019SHZDZX02), and the Science and Technology Commission of Shanghai Municipality (Grant: 18JC1420500).

Footnotes

Reviewers: F.C., Zhejiang University; and M.C., Helmholtz Zentrum Munchen Deutsches Forschungszentrum fur Gesundheit und Umwelt.

The authors declare no competing interest.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2214227119/-/DCSupplemental.

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.

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Associated Data

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Supplementary Materials

Supplementary File
pnas.2214227119.sapp.pdf (1.2MB, pdf)

Data Availability Statement

All study data are included in the article and/or SI Appendix.

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