Activating the NFE2L1-ubiquitin-proteasome system by DDI2 protects from ferroptosis

Abstract

Ferroptosis is an iron-dependent, non-apoptotic form of cell death initiated by oxidative stress and lipid peroxidation. Recent evidence has linked ferroptosis to the action of the transcription factor Nuclear factor erythroid-2 derived,-like-1 (NFE2L1). NFE2L1 regulates proteasome abundance in an adaptive fashion, maintaining protein quality control to secure cellular homeostasis, but the regulation of NFE2L1 during ferroptosis and the role of the ubiquitin-proteasome system (UPS) herein are still unclear. In the present study, using an unbiased proteomic approach charting the specific ubiquitylation sites, we show that induction of ferroptosis leads to recalibration of the UPS. RSL3-induced ferroptosis inhibits proteasome activity and leads to global hyperubiquitylation, which is linked to NFE2L1 activation. As NFE2L1 resides in the endoplasmic reticulum tethered to the membrane, it undergoes complex posttranslational modification steps to become active and induce the expression of proteasome subunit genes. We show that proteolytic cleavage of NFE2L1 by the aspartyl protease DNA-damage inducible 1 homolog 2 (DDI2) is a critical step for the ferroptosis-induced feed-back loop of proteasome function. Cells lacking DDI2 cannot activate NFE2L1 in response to RSL3 and show global hyperubiquitylation. Genetic or chemical induction of ferroptosis in cells with a disrupted DDI2-NFE2L1 pathway diminishes proteasomal activity and promotes cell death. Also, treating cells with the clinical drug nelfinavir, which inhibits DDI2, sensitized cells to ferroptosis. In conclusion, our results provide new insight into the importance of the UPS in ferroptosis and highlight the role of the DDI2-NFE2L1 as a potential therapeutic target. Manipulating DDI2-NFE2L1 activity through chemical inhibition might help sensitizing cells to ferroptosis, thus enhancing existing cancer therapies.

Fig. 1. RSL3-induced calibration of the UPS is distinct from proteasome inhibition.

a Immunoblot of ubiquitin from EA.hy926 cells treated with 5 µM RSL3 and 100 nM bortezomib (BTZ) for 9 h and 3 h, respectively. b Immunoblot of NFE2L1 in EA.hy926 cells treated with indicated time points and concentrations of RSL3 (FL: full-length form ca. 120 kD; CL: cleaved ca. 95 kD). c Immunoblot of NFE2L1 in nuclear and cytoplasmic fractions isolated from EA.hy926 cells treated as indicated for 3 h. d Native page of EA.hy926 cells treated with 5 µM RSL3 and 100 nM BTZ for 6 h with in-gel activity and immunoblot of the ɑ1-7 (20S) subunits. e Proteasomal activity in EA.hy926 cells treated with 5 µM RSL3 for 3 h. f Proteasomal activity assay in EA.hy926 cells with the extracts being incubated with indicated concentrations and time points of RSL3 and proteasome inhibitor MG132. g Volcano Plot of the ubiquitome of EA.hy926 cells treated for 9 h with 5 µM RSL3 P_adj < 0.05 indicated in red. (n = 4 replicates). h Principal component analysis (PCA) of the ubiquitome of EA.hy926 cells treated with 5 µM RSL3 and 100 nM BTZ (n = 4 replicates). i Top 10 enriched gene ontology (GO) terms in the ubiquitome of EA.hy926 cells treated for 9 h with 5 µM RSL3.

Fig. 2. DDI2 is required for RSL3-induced proteolytic processing of NFE2L1.

a Immunoblot of NFE2L1 and DDI2 in EA.hy926 wild-type (Parental WT) and DDI2 knockout (KO) cells treated with 5 µM RSL3 and 100 nM bortezomib (BTZ) for 9 h and 3 h, respectively. b Immunoblot of NFE2L1 in DDI2 KO cells transfected with empty vector, wild-type and D252 protease-dead DDI2 carrying plasmids followed by 3 h treatment with 100 nM BTZ (FL: full-length form ca. 120 kDa; CL: cleaved ca. 95 kDa). c Immunoblot of NFE2L1 in WT EA.hy926 cells treated with DDI2 siRNA for 48 h, and then 9 h of RSL3 or 3 h of 100 nM BTZ. d Immunoblot of NFE2L1 in WT EA.hy926 cells after treatment with 50 µM of protease DDI2 inhibitor nelfinavir (NFV) for 4 h, 5 µM RSL3 for 9 h, and 100 nM BTZ for 3 h. e Relative gene expression of NFE2L1 in cells treated with 10 µM of NFV and 5 µM RSL3 for 9 h. f NFE2L1 luciferase nuclear translocation reporter assay in HEK293a cells after 24 h treatment with 5 µM of NFV and 20 h with 5 µM RSL3.

Fig. 3. DDI2 is required for the UPS bounce-back response during ferroptosis.

a Immunoblot of ubiquitin in EA.hy926 cells treated with 5 µM RSL3 and 100 nM bortezomib (BTZ) for 9 h and 3 h, respectively. b Native PAGE of EA.hy926 cells treated with 10 µM NFV for 20 h and immunoblot of the ɑ1-7 (20S) subunits. c Proteasomal activity of EA.hy926 cells treated with 5 µM NFV for 9 h and 3 h of 5 µM RSL3. d Top 10 enriched gene ontology (GO) terms in the ubiquitome of DDI2 (knockout) KO compared to wild-type (WT) parental cells. e Volcano Plot of the DDI2 KO cells compared to WT cells P_adj < 0.05 indicated in red. (n = 3 replicates). f Principal component analysis (PCA) of the ubiquitome of EaHy926 WT vs. DDI2 KO cells.

Fig. 4. Loss of DDI2 sensitizes cells to chemical and genetic ferroptosis.

a Cell viability in EA.hy926 cells treated with 5 µM NFV and 10 µM ferrostatin for 20 h. b Immunoblot of NFE2L1 in wild-type (Parental WT) and DDI2 knockout (KO) EA.hy926 cells treated 9 h with 10 µM Ferrostatin (Fer-1) and indicated concentrations of RSL3, and 3 h with 100 nM bortezomib (BTZ). c Cell viability of DDI2 KO versus WT EA.hy926 cells after 20 h of treatment with 5 µM RSL3. d Cell viability of SSMD cells (GPX4 mut) and healthy controls (GPX4 WT) after siRNA-mediated silencing of NFE2L1 and DDI2. e Immunoblot of 95 kD cleaved form of NFE2L1 in WT and DDI2 KO EA.hy926 cells transfected with WT NFE2L1 and NFE2L1-8ND mutant plasmids followed by 9 h treatment with 5 µM RSL3 and 100 nM BTZ. f Cell viability of DDI2 KO cells transfected with WT NFE2L1 and NFE2L1-8ND mutant plasmids followed by 9 h treatment with 5 µM RSL3.