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

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. SIMILAR CONTENT BEING VIEWED BY OTHERS CELLULAR DEGRADATION SYSTEMS IN FERROPTOSIS Article 18 January 2021 CRL2-KLHDC3 E3 UBIQUITIN LIGASE COMPLEX

SUPPRESSES FERROPTOSIS THROUGH PROMOTING P14ARF DEGRADATION Article 06 November 2021 THE MARCHF6 E3 UBIQUITIN LIGASE ACTS AS AN NADPH SENSOR FOR THE REGULATION OF FERROPTOSIS Article 08

August 2022 INTRODUCTION Regulated cell death is initiated by intracellular and extracellular perturbations that trigger tightly orchestrated molecular programs [1]. Ferroptosis is a form of

non-apoptotic cell death mediated by iron-dependent lipid peroxidation and loss of plasma membrane integrity [2, 3]. Recent studies have implicated ferroptosis in several pathologies, such

as neurodegeneration and cancer [4]. Execution of ferroptosis is tightly linked to lipid and glutathione metabolism [5] and several chemical compounds have been shown to induce ferroptosis.

Glutathione peroxidase 4 (GPX4) is a critical enzyme protecting from lipid reactive oxygen species (ROS) formation and, thus, from ferroptosis by most prominently using glutathione for its

antioxidative activity [6]. While depletion of the reduced glutathione pool predisposes cells to ferroptosis, the compound RSL3 directly inhibits GPX4 [2, 7, 8]. Thus, oxidative stress is

sensed and mitigated by GPX4 and its inactivation leads to lipid peroxidation and cell death in cells and mouse models [8]. Interestingly, ferroptosis is linked to adaptive changes in

protein homeostasis, as ferroptosis initiation is associated with diminished proteasomal activity and restoration of proteasomal activity protects cells from ferroptotic cell death [9]. The

ubiquitin-proteasome system (UPS) manages the degradation of unwanted, obsolete, or damaged proteins [10]. The UPS controls almost all cellular processes by precision proteolysis, enabled by

specific ubiquitin ligases [11]. Attachment of ubiquitin to these proteins is the prerequisite for the ATP-dependent degradation by the proteasome. The 26S proteasome is a multi-subunit

protease complex, which consists of the barrel-shaped 20S proteolytic core particle capped with one or two 19S regulatory particles for binding to the polyubiquitylated proteins [12]. While

it is accepted that the UPS is a critical pillar of cellular health, the roles of UPS remodeling and the nature of these changes in ferroptosis remain unclear. We and others have shown that

the transcription factor Nuclear factor erythroid-2, like-1 (NFE2L1, also known as TCF11 or NRF1) regulates ferroptosis [9, 13]. NFE2L1 upregulates the expression of genes encoding

proteasome subunit genes [14, 15], thus restoring proteasomal activity and protecting from ferroptosis. NFE2L1 mediates the adaptive component of proteasomal activity and is subject to

complex posttranslational regulation: NFE2L1 is a cap’n’collar transcription factor tethered in the endoplasmic reticulum membrane. In a simplified model, NFE2L1 undergoes deglycosylation by

N-glycanase 1 (NGLY1, also known as PNGase) and proteolytic cleavage by the protease DNA damage-inducible 1 homolog 2 (DDI2) [16, 17]. However, the resulting fragment containing the

bZIP-DNA-binding domain-containing is virtually absent in most cells, as it is being continuously ubiquitylated and degraded by the proteasome [18, 19]. Upon treatment with chemical

proteasome inhibitors or if the levels of ubiquitylated proteins exceed the capacity of the available proteasomes, NFE2L1 escapes degradation and restores proteasomal activity [14]. However,

the impact of DDI2-mediated activation of NFE2L1 on UPS in ferroptosis remains unknown. Here, we determine the impact of ferroptosis on UPS and global ubiquitylation using an unbiased MS

approach. Furthermore, we demonstrate a critical role of DDI2-mediated activation of the NFE2L1 pathway in calibrating the UPS for ferroptosis protection. METHODS CELL CULTURE, REVERSE

TRANSFECTION, AND TREATMENTS Human EA.hy926 cells and mouse WT1 cells were used for cellular experiments. EA.hy926 DDI2 KO cells were created previously [19]. Cells were cultured in DMEM

Glutamax (Thermo), supplemented with 10% v/v fetal bovine serum (FBS, Sigma) and 1% v/v Penicillin-Streptomycin (10.000 U/ml, Thermo). Cells were incubated at 37 °C, 5% v/v CO2 and were

passaged two to three times a week. For experiments 300,000, 100,000, and 25,000 cells were seeded at 6, 24, and 96-well cell culture plates, respectively. Primary GPX4 mutant fibroblasts

(derived from a patient with a homozygous mutation c.647 G > A in exon 6 of GPX4) and control cells were kindly provided by Sanath K. Ramesh (curegpx4.org). These cells were incubated in

DMEM GlutaMax supplemented with 10% v/v FBS and 1% v/v PS, supplemented with 10 µM ferrostatin-1 (SelleckChem). Reverse transfection with SMARTpool siRNA (Dharmacon) was performed using 30 

nM of siRNA in RNAiMAX (Thermo). Cells were incubated for 48 h after transfection, and then treatments were performed. For overexpression experiments, 250 ng of each plasmid construct (Empty

vector, DDI2 WT, DDI D252, NFE2L1 WT, and NFE2L1-8ND) was added to TransIT-X2® Transfection Reagent (Mirus) and after 10 min incubation, cells were added to the medium. For treatments,

different concentrations of RSL3 (SelleckChem), FIN56 (SelleckChem) and ferrostatin-1 (SelleckChem) were used as indicated. Inhibition of proteasome and DDI2 was performed using bortezomib

(SelleckChem) and nelfinavir mesylate (Sigma), respectively. Cell viability was assessed by AquaBluer (MultiTarget Pharmaceuticals), by changing the medium of cells to 1:100 of AquaBluer in

phenol red-free DMEM GlutaMax, after 20 h of treatment. Cell plates were incubated for 4 h at 37 °C incubator and using a Spark Reader (Tecan) fluorescence was measured at emission of 590 nm

with an excitation of 540 nm. PROTEIN EXTRACTION AND IMMUNOBLOTTING Cells were lysed in RIPA buffer (150 mM NaCl (Merck), 5 mM EDTA (Merck), 50 mM Tris pH 8 (Merck), 1% v/v IGEPAL CA-630

(Sigma), 0.5% w/v sodium deoxycholate (Sigma Aldrich), 0.1% w/v SDS (Roth),1 mM protease inhibitors (Sigma)) for 3 min in TissueLyser II (30 Hz; Qiagen). Cell lysates were centrifuged for 15

 min (4 °C, 21,000 _g_) and the concentration of proteins in supernatant was determined by Pierce BCA Protein Assay (Thermo) according to the manual. Proteins were denatured for 5 min at 95 

°C in Bolt LDS Sample buffer with 5% v/v 2-mercaptoethanol (Sigma). 10-30 µg of proteins were loaded in Bolt 4-12% Bis-Tris gels (Thermo) followed by transferring onto a 0.2 mm PVDF membrane

(Bio-Rad) at 25 V, 1.3 A for 7 min. Membranes were blocked in TBS-T (200 mM Tris (Merck), 1.36 mM NaCl (Merck), 0.1% Tween-20 (Sigma)) containing 5% w/v milk powder for 1 h at room

temperature after staining in Ponceau-S (Sigma Aldrich). Incubation by primary antibodies (Supplementary Table 1) was performed overnight at 4 °C followed by washing membranes three times

for 10 min with TBS-T and incubation with secondary antibodies for 1 h at room temperature. SuperSignal West Pico PLUS (Thermo) was used for developing blots in a ChemiDoc MP imager

(Bio-Rad). All uncropped blots can be found in Supplementary Figs. S1-S3. PROTEASOME ACTIVITY Cells lysis was performed in lysis buffer (40 mM Tris pH 7.2 (Merck), 50 nM NaCl (Merck), 5 mM

MgCl2(6H2O) (Merck), 10% v/v glycerol (Sigma), 2 mM ATP (Sigma), 2 mM 2-mercaptoethanol (Sigma). Proteasome Activity Fluorometric Assay II kit (UBPBio, J41110) was used to measure proteasome

activity. BCA Protein Assay (Bio-Rad) was used to normalize the results to protein levels. NATIVE PAGE Cells were lysed in lysis buffer (50 mM Tris/HCl pH 7.5, 2 mM DTT, 10% v/v glycerol, 5

 mM MgCl2, 0.05% v/v Digitonin, 2 mM ATP) containing phosphatase inhibitor (PhosphoStop, Roche Diagnostics) as described previously [20]. Samples were incubated on ice for 20 min and

centrifuged twice. Concentration of proteins was determined with Bio-Rad Protein Assay Kit II. 15 µg of protein were loaded in NuPAGE 3-8% Tris-Acetate gels (Thermo) and run at constant

voltage of 150 V for 4 h. Gels were kept for 30 min at 37 °C in activity buffer (1 mM MgCl2, 50 mM Tris, 1 mM DTT) with 0.05 mM substrate Suc-Leu-Leu-Val-Tyr-AMC (Bachem). ChemiDoc MP

(Bio-Rad) was used to measure the fluorescent signal. Afterwards, to prepare samples for blotting gel was incubated in a solubilization buffer (2% w/v SDS, 1.5% v/v 2-Mercaptoethanol, 66 mM

Na2CO3) for 15 min. Samples were transferred to a PVDF membrane at 40 mA through tank transfer. The membrane was kept for 1 h in the ROTI-block (Roth) and overnight in primary antibody

(1:1000). The day after, the membrane was incubated for 3 h in the secondary antibody (1:10,000) at room temperature and developed as described above. NFE2L1 REPORTER ASSAY HEK293a cells

stably expressing short half-life firefly luciferase driven by upstream activator sequence (UAS) promoter and a chimeric NFE2L1 in which the DNA-binding domain was replaced by the

UAS-targeting Gal4 DNA-binding domain [21]. The assay measures nuclear translocation and its transactivation by binding of NFE2L1-UAS to a luciferase promoter [21]. 30,000 cells were seeded

in 96-well plates and after treatment with RSL3, cells were lysed, and luciferase emission was measured using Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s

instructions. GENE EXPRESSION ANALYSIS RNA extraction was performed using Nucleospin RNA kit (Macherey Nagel), based on manufacturer’s instruction, and the concentration of RNAs were

measured with a NanoDrop spectrophotometer (Implen). To prepare complementary DNA (cDNA), 500 ng RNA were added to 2 µL of MaximaTM H Master Mix 5x (Thermo Fischer) and the total volume was

adjusted to 10 µL with H2O. The cDNA was diluted 1:40 in H2O, and 4 µL of cDNA, 5 µL of PowerUpTM SYBR Green Master Mix (Thermo), and 1 µL OF 5 µM primer stock (Supplementary Table 2) were

used to measure Relative gene expression. Cycle thresholds (Ct) of gene of interest were measured using a Quant-Studio 5 RealTime PCR system (Thermo). Relative gene expression was normalized

to TATA-box binding protein (_TBP_) levels by the ddCt-method. PROTEIN DIGESTION AND PURIFICATION FOR MS Protein digestion was performed as described previously [22,23,24]. Cells were lysed

in SDC buffer (1% v/v SDC in 100 mM Tris-HCl, pH 8.5) followed by boiling for 5 min at 95 °C while shaking at 1000 rpm. Protein concentrations of lysates were determined after 15 min of

sonication (Bioruptor, Diagenode, cycles of 30 s) using the Pierce BCA Protein Assay (Thermo). CAA and TCEP (final concentrations: 40 mM and 10 mM respectively) were added to 5 mg protein.

After 10 min incubation of samples at 45 °C in the dark shaking at 1000 rpm, Trypsin (1:50 w/w) and LysC (1:50 w/w) were added. Samples then were kept overnight at 37 °C while shaking at

1000 rpm for digestion. For proteome analysis, sample aliquots (~15 µg) were desalted in SDB-RPS StageTips (Empore). Briefly, samples were diluted with 1% TFA in isopropanol to a final

volume of 200 µl, loaded onto StageTips, and sequentially washed with 200 µl of 1% v/v TFA in isopropanol and 200 µl 0.2% v/v TFA/2% v/v ACN. Peptides were eluted with freshly prepared 60 µl

of 1.25% v/v ammonium hydroxide (NH4OH)/80% v/v ACN and dried using a SpeedVac centrifuge (Eppendorf). Dried peptides were resuspended in 6 µL buffer A (2% v/v ACN/0.1% v/v TFA). Di-Gly

enrichment samples were diluted with 1% v/v TFA in isopropanol (1:5). For peptide cleanup, SDB-RPS cartridges (Strata™-X-C, 200 mg/6 ml, Phenomenex Inc.) were equilibrated with 8 bed volumes

(BV) of 30% v/v MeOH/1% v/v TFA and washed with 8 BV of 0.2% v/v TFA. Samples were loaded by gravity flow and sequentially washed twice with 8 BV 1% TFA in isopropanol and once with 8 BV

0.2% v/v TFA/2% v/v ACN. Peptides were eluted with 2 × 4 BV 1.25% v/v NH4OH/80% v/v ACN and diluted with ddH2O to a final of 35% v/v ACN. Samples were dried via a SpeedVac Centrifuge

overnight. DIGLY PEPTIDE ENRICHMENT We used the PTMScan® Ubiquitin Remnant Motif (Cell Signaling). The peptides were resuspended in 500 µL immunoaffinity purification (IAP) buffer and

sonicated (Bioruptor) for 15 min. BCA Protein Assay (Thermo) was used to determine protein concentration. The antibody-coupled beads were cross-linked as previously described [22, 24]. One

vial of antibody coupled beads was washed with cold cross-linking buffer (2000 _g_, 1 min). Subsequently the beads were incubated in 1 mL cross-linking buffer at room temperature for 30 min

under gentle agitation. The reaction was stopped by washing twice with 1 mL cold quenching buffer (200 mM ethanolamine, pH 8.0) and incubating for 2 h in quenching buffer at room temperature

under gentle agitation. Cross-linked beads were washed three times with 1 mL cold IAP buffer and directly used for peptide enrichment. For DiGly enrichment, 3 mg of peptide is used with 1/8

of a vial of cross-linked antibody beads. Peptides are added to the cross-linked beads and the volume is adjusted to 1 mL with IAP buffer and incubated for 2 h at 4 °C under gentle

agitation. The beads are washed twice with cold IAP buffer and twice ddH2O via centrifugation. The enriched peptides were eluted by adding 200 µL 0.2% v/v TFA onto the beads, incubating 5 

min at 1400 rpm and centrifuging for 1 min at 100 _g_. The supernatant was then transferred to SDB-RPS StageTips and the peptides washed, eluted and dried as previously described for total

proteome samples. LC-MS/MS PROTEOME AND UBIQUITOME MEASUREMENTS Liquid chromatography of total proteome and ubiquitome samples was performed on an EASYnLCTM 1200 (Thermo) with a constant

flow rate of 10 µL/min and a binary buffer system consisting of buffer A (0.1% v/v formic acid) and buffer B (80% v/v acetonitrile, 0.1% v/v formic acid) at 60 °C. The column was in-house

made, 50 cm long with 75 µm inner diameter and packed with C18 ReproSil (Dr. Maisch GmbH, 1.9 µm). The elution gradient for the ubiquitome started at 5% buffer B, increasing to 25% after 73 

min, 50% after 105 min and 95% after 110 min. The gradient for the proteome started at 5% buffer B and increased to 20% after 30 min, further increased at a rate of 1% per minute to 29%,

following up to 45% after 45 min and to 95% after 50 min. The MS was performed on a Exploris480 with injection of 500 ng peptide (Thermo). Fragmented ions were analyzed in Data Independent

Acquisition (DIA) mode with 66 isolation windows of variable sizes. The scan range was 300–1650 m/z with a scan time of 120 min, an Orbitrap resolution of 120,000 and a maximum injection

time of 54 ms. MS2 scans were performed with a higher-energy collisional dissociation (HCD) of 30% at a resolution of 15,000 and a maximum injection time of 22 ms. The MS measurement of the

proteome was performed equivalently, yet including HighField Asymmetric Waveform Ion Mobility (FAIMS) with a correction voltage of −50 V and a scan time of 60 min. The injection time for the

full scan was 45 s and the MS2 injection time was set to 22 s. PROTEOME AND UBIQUITOME DATA ANALYSIS DIA raw files were processed using Spectronaut (13.12.200217.43655) in directDIA mode.

The FASTA files used for the search were: Uniprot Homo sapiens (29.03.2022) with 20609 entries, Uniprot Homo sapiens isoforms (29.03.2022) with 77157 entries and MaxQuant Contaminants for

filtering: 245 entries. Analysis was performed via Perseus (version 1.6.2.3). For the ubiquitome samples the output was converted with the plugin “Peptide Collapse” (version 1.4.2). Values

were log2-transformed and missing values were replaced by imputation from normal distribution with a width of 0.3 and downshift 1.8 separately for each sample (For full data please see

Supplementary Table 3). Ubiquitome was normalized to total proteome. Comparison between conditions of proteome and ubiquitome was performed via Student’s T-test in R 4.2.2 (_P_ value cutoff

0.5). STATISTICS All data were analyzed with Microsoft Excel, GraphPad Prism, and R (4.2.2). Data were visualized in GraphPad Prism and shown as mean ± standard deviation (SD) with plotting

individual data distribution of technical replicates. Except for the proteomics, every experiment was replicated twice. Sample size was not based on power calculations. 1-way ANOVA with

Dunnett’s Post-hoc Test was used when comparing three or more groups and one variable, and 2-way ANOVA followed by Tukey’s Test was used for comparing two groups with two variables.

_P_-values lower than 0.05 were considered significant and are as such indicated in the graphs with an asterisk between groups. RESULTS RSL3-INDUCED FERROPTOSIS DIMINISHES PROTEASOMAL

ACTIVITY AND ACTIVATES NFE2L1 It remains largely unclear how the UPS is involved in the execution of ferroptosis. For two specific reasons this is important to understand: First, it is

unclear if the regulation of UPS is a ferroptosis-specific event or simply a consequence of proteasome dysfunction. Second, as critical regulators of ferroptosis such as GPX4 have been shown

to be regulated by UPS [9], understanding global and site-specific ubiquitylation will potentially aid the discovery of novel key players in ferroptosis. Treatment of EA.hy926 cells with

the ferroptosis inducer RSL3 led to higher levels of ubiquitin (Fig. 1a and Supplementary Fig. S4). Interestingly, the effect of RSL3 on ubiquitin levels was somewhat comparable if not

higher to treatment of cells with the established chemical proteasome inhibitor bortezomib (BTZ) (Fig. 1a). We next asked if this effect also triggered the activation of NFE2L1, which an

important part of UPS defense against inhibition of the proteasome. Indeed, treatment with RSL3 led to higher protein levels of the cleaved fragment of NFE2L1 (ca. 95 kDa) and lower levels

of the full-length protein in a time- and dose-dependent fashion (Fig. 1b). This increase in cleaved fragment was associated with higher levels of NFE2L1 in the nucleus (Fig. 1c). Based on

native PAGE analysis and measuring degradation of fluorogenic peptides, RSL3 diminished proteasomal activity (Fig. 1d, e). Hence, we tested the possibility that RSL3 may have off-target

effects directly on proteasomal activity. At concentrations well-above what is used in our cell assays there was no direct inhibition of proteasomal activity in cell lysates, unlike what is

seen with regular proteasome inhibitors. (Fig. 1f). This diminishes the possibility that the RSL3-induced decrease in proteasome activity and increase in ubiquitin levels are consequences of

off-target pharmacological inhibition of the proteasome by direct binding of RSL3. REMODELING OF THE UBIQUITOME IN FERROPTOSIS IS DISTINCT FROM PROTEASOME INHIBITION To better understand

the calibration of UPS during ferroptosis, unequivocally determine the levels of ubiquitin, and to chart the specific lysine residues modified with ubiquitin, we performed a mass

spectrometry-based analysis of the proteome and ubiquitylated proteins using the so-called Di-Gly method of immunoprecipitating ubiquitin remnant motifs that are produced by trypsin

digestion [24]. We identified 7063 peptides in the proteome and 3974 peptides in the ubiquitome. These analyses confirmed the increase in ubiquitin levels upon RSL3 treatment (Fig. 1g). To

delineate and compare how RSL3 and BTZ treatments impact the ubiquitome, we performed principal component analysis and found that the three groups cluster significantly apart from each other

(Fig. 1h). This indicates that the nature of ubiquitome remodeling induced by RSL3 is distinct from common proteasome inhibition, e.g. by BTZ. Next, to better understand the mechanisms

involved in the execution of ferroptosis, we analyzed the cellular pathways implicated in the remodeling of the ubiquitome. Here, we found that gene ontology (GO) terms linked to regulation

of protein catabolic process and establishment of protein localization to organelle were among the differentially regulated pathways (Fig. 1i and Supplementary Table 3). In summary, this set

of data highlights the remodeling of the ubiquitome, implicates the regulation of cell death signaling pathways by the UPS, and shows the activation of the proteostatic bounce-back response

of NFE2L1 upon impairment of proteasomal activity upon RSL3-induced ferroptosis. DDI2 FACILITATES THE RSL3-INDUCED PROTEOLYTIC ACTIVATION OF NFE2L1 As we observed an RSL3-induced increase

in ubiquitin levels and NFE2L1 activation, we addressed the importance of the protease DDI2 for NFE2L1 cleavage under these conditions. We performed the next experiments in a cell line, in

which DDI2 was deleted by CRISPR/Cas9 technology. Similar to BTZ treatment, RSL3 led to NFE2L1 activation in the parental cell line. However, in the DDI2 KO cells, neither BTZ nor RSL3

treatment led to changes in NFE2L1 protein levels (Fig. 2a), indicating that the activation of NFE2L1 during ferroptosis is critically dependent on DDI2. This effect on NFE2L1 was also

reproduced using FIN56 for ferroptosis induction (Supplementary Fig. S4). To rule out that this is a cell line-dependent false negative effect, we re-expressed DDI2 in KO cells, and this

restored BTZ-induced activation of NFE2L1 (Fig. 2b). Furthermore, reconstitution of DDI2 KO cells with a construct carrying proteolytically inactive DDI2 (DDI2-D252) did not restore the

activation of NFE2L1 compared to re-expression of the WT protein (Fig. 2b). Silencing DDI2 expression by transfection of siRNA also showed lower levels of the cleaved form of NFE2L1 in cells

treated with BTZ and RSL3 (Fig. 2c). Next, we used the established chemical inhibitor of DDI2 nelfinavir (NFV), which is clinically used for the treatment of patients with HIV infections

[25]. Both BTZ and RSL3-induced activation of NFE2L1 was lower in cells treated with NFV compared to control treated cells (Fig. 2d). This was independent of _NFE2L1_ gene expression, which

was unchanged (Fig. 2e). We used the chemical inhibition strategy in a nuclear translocation reporter assay, which is a surrogate for NFE2L1 transcriptional activity [21]. NFV treatment led

to lower reporter activity when cells were stimulated with RSL3, indicating that the nuclear translocation of NFE2L1 during ferroptosis is enabled by DDI2 (Fig. 2f). In summary, these

experiments show that DDI2 facilitates the RSL3-induced proteolytic activation and nuclear translocation of NFE2L1. DDI2 IS REQUIRED FOR THE UPS BOUNCE BACK RESPONSE DURING FERROPTOSIS To

analyze the impact of the DDI2-NFE2L1 switch for maintaining proper proteostasis during ferroptosis, we analyzed ubiquitylation levels of DDI2 KO compared to WT cells. Immunoblot of BTZ

showed an accumulation of ubiquitylated proteins of high molecular weights in DDI2 KO cells (Fig. 3a). Interestingly, this effect on ubiquitin levels was also observed when the cells were

treated with RSL3, highlighting the necessity of DDI2 for maintaining proper proteostasis during ferroptosis (Fig. 3a). In-gel measurement of proteasome activity further showed the

importance of DDI2 in regulating baseline proteasome activity, as cells treated with NFV showed both lower 30S activity and expression of proteasome subunits in the native PAGE (Fig. 3b). To

check if proteasome activity in the context of ferroptosis was also affected by inhibiting DDI2 we used fluorometric peptides and measured the proteasome activity, which revealed that the

trypsin-like activity was lower upon loss of DDI2 (Fig. 3c). This shows that DDI2 is required for sustaining proteasomal activity during ferroptosis. While GO analysis indicated that

establishment of protein localization to organelle is among the most impacted pathways (Fig. 3d and Supplementary Table 3), Volcano plot visualization and principal component analysis of the

ubiquitome data further proved higher ubiquitylation status in DDI2 KO cells compared to parental WT cells (Fig. 3e and Supplementary Table 3). In summary, the activity of DDI2 is required

to activate the NFE2L1-bounce back mechanism of proteasome function in response to ferroptosis initiation to maintain proper proteostasis. PROTEOLYTIC ACTIVATION OF NFE2L1 BY DDI2 PROTECTS

FROM FERROPTOTIC CELL DEATH As the main outcome of ferroptosis induction is cell death, we determined the viability of cells upon RSL3 treatment in the presence or absence of the established

ferroptosis inhibitor ferrostatin (Fig. 4a). Moreover, we checked if targeting the activity DDI2 with NFV sensitized cells to ferroptosis and potentiated RSL3-induced cell death. NFV

treatment increased the cytotoxicity of RSL3, which was rescued by ferrostatin (Fig. 4a). We also proved that the appearance of cleaved NFE2L1 protein was dependent on ferroptosis per se as

ferrostatin abolished the effect (Fig. 4b). In line with the impact on the NFE2L1-ubiquitin-proteasome system, DDI2 KO cells were more susceptible to ferroptotic death compared to the WT

cells (Fig. 4c). We also checked the effect of silencing DDI2 using siRNA on the viability of primary fibroblasts from a patient with Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD),

carrying mutant GPX4 compared to healthy control cells. In this distinct genetic ferroptosis cell system, both _NFE2L1_ and _DDI2_ silencing lowered the viability of cells when ferrostatin

was removed. (Fig. 4d). As previously described, following the translocation of NFE2L1 to the cytosolic face of ER membrane, NGLY1 removes _N_-linked glycans from NFE2L1, which is a distinct

and important posttranslational step for NFE2L1 activation during ferroptosis [13]. As others have suggested that deglycosylation of NFE2L1 by NGLY1 takes place prior to its cleavage by

DDI2 (Fig. 4e), we determined if the NFE2L1-8ND mutant, which contains mutations in the glycosylation sites and is considered constitutively active [13], was processed in the absence of DDI2

under ferroptotic conditions. We transfected WT NFE2L1 and mutant NFE2L1-8ND in DDI2 KO cells. While control cells showed the BTZ- and RSL3-induced cleaved form of NFE2L1, immunoblot of

DDI2 KO cells showed no cleaved form of NFE2L1 compared with control cells and this effect was not overcome by using the 8ND mutant form of NFE2L1 (Fig. 4e). Also, to validate if NFE2L1-8ND

protected cells from ferroptosis, we performed a cell viability assay, but found no differences between wild-type and mutant forms of NFE2L1 upon RSL3 treatment (Fig. 4f). These experiments

highlight the critical role of DDI2 for NFE2L1 activation and subsequent protection from ferroptosis. Finally, we reproduced the key results in WT1 cells, which is are more sensitive to RSL3

compared to the EA.hy926 cells (Supplementary Fig. S5). DISCUSSION The execution of ferroptosis and molecular approaches to manipulate its outcomes is an emerging field of research with

important implications for metabolism, cancer, and neurodegenerative diseases. While the UPS is clearly involved in ferroptosis [9], little was known about the nature and relevance of this

observation. Here, we unequivocally demonstrate the RSL3-induced recalibration of UPS and activation of the proteasome bounce-back mechanism of NFE2L1 by DDI2 in two independent cell lines,

human and mouse, with distinct sensitivity to ferroptosis. In the absence of DDI2, cells accumulate hyperubiquitylated proteins, reflecting a maladaptive regulation of the UPS. Consequently,

DDI2 is required to proteolytically activate NFE2L1 and protect from RSL3-induced and genetically driven ferroptotic cell death. Upon induction of ferroptosis, cells are required to

increase proteostatic defense mechanisms, possibly caused by lipid ROS-induced perturbations before the eventual death of the cell. Usually, the proteasome defuses and recycles damaged or

obsolete proteins by ubiquitin-dependent and independent degradation. Perhaps, one key finding of our study is that RSL3-induced ferroptosis compromises proteasome activity. However, the

nature of this insult was distinct from chemical proteasome inhibition by itself, arguing for more than impact on the ubiquitome. In other words, the recalibration of UPS and ubiquitome

during ferroptosis is not only caused by proteasome inhibition. Interestingly, among the ubiquitylated proteins increased upon RSL3 treatment we found many UPS components, including those

constituting proteasomes itself. Future work will have to investigate the relevance of these modifications and delineate the posttranslational regulation of proteasome activity during

ferroptosis. In our global ubiquitome analysis, we found several known players that either suppress or promote ferroptosis. Previous studies investigating deubiquitylating enzymes have found

a role for ubiquitylation and degradation of GPX4, Nedd4 or VDAC2/3 [26, 27] but the relevance for these mechanisms is still unclear. We also find GPX4 and enzymes of glutathione production

to be hyperubiquitylated upon RSL3 treatment and in the absence of DDI2, suggesting that there is a specific impact on reduced proteasome function on key pathways of ferroptosis. On a more

general note, our results show lack or dysfunction of DDI2 lead to a global increase in ubiquitylation and reduced survival of the cells when ferroptosis is induced. While

hyperubiquitylation increases proteotoxic stress levels, it is possible that the turnover of certain key regulators is disturbed and contributes to the observed ferroptosis sensitivity of

DDI2 KO cells. However, as our means of inducing ferroptosis are largely GPX4-dependent, GPX4 is likely not required for the execution of this response. We have previously shown that also

other distinct means of inducing ferroptosis such as erastin or FIN56 exhibit changes in proteasomal activity [9]. Thus, reduced proteasome function is a hallmark of ferroptosis, and there

is overlap between BTZ and RSL3-induced hyperubiquitylation. The role of DDI2 in the complex series of events leading to NFE2L1 activation should be viewed as required and permissive for the

bounce-back response. While reconstituting DDI2 KO cells with WT DDI2 rescued RSL3-induced NFE2L1 activation, a protease-deficient mutant did not. However, proteolytic processing is not the

only posttranslational modification required for the activation of NFE2L1, as NGLY1 plays a critical role deglycosylation of NFE2L1 and modulates susceptibility of cells to ferroptosis

[13]. In our hands, overexpression of deglycosylated NFE2L1 did not rescue the sensitivity of DDI2 KO cells, which underlines the importance of the DDI2-mediated proteolytic cleavage step

for the activation of NFE2L1 during ferroptosis. Our data using NFV for inhibiting DDI2 indicate that this could be a potential add-on treatment for targeting ferroptosis in cancer therapy,

similar to what has been rationalized for proteasome inhibitors. Interfering with proteasome function and preventing NFE2L1 activation at the same time might further sensitize cancer cells

to ferroptosis. Finally, NFE2L1 is also ubiquitylated by the E3 ubiquitin ligase Hrd1 at the ER for ER-associated protein degradation [28], by β-TrCP in the nucleus for removal of NFE2L1

[28], or by UBE4A for cleavage by DDI2 [29], but the details of these regulatory steps for ferroptosis remain unexplored. It will be important to further dissect the posttranslational

regulation of NFE2L1 during ferroptosis, as this might reveal more angles to therapeutically exploit the calibration of the UPS for manipulating ferroptosis outcomes. DATA AVAILABILITY All

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Download references ACKNOWLEDGEMENTS We thank Silvia Weidner and Thomas Pitsch for excellent technical assistance. We are grateful to Joel Guerra and Leonardo Matta for help with the

experiments. We thank all lab members for discussions and the enjoyable atmosphere. We thank Scott Dixon for providing us with the NFE2L1-8ND construct. We acknowledge BioRender.com for help

with the figures. We apologize to colleagues whose work we could not cite due to space limitations. FUNDING AO was supported by the Integrated Research Training Group of the Deutsche

Forschungsgemeinschaft (DFG) CRC1123. SK was supported by the Ludwig-Maximilians-University Medical Faculty program FöFoLe for MD students. SM and EK was supported by DFG RTG2719 PRO. DTH

and NK were supported by DFG _Emmy-Noether Program_ (KR5166/1-1). AB was supported by DFG (SFB1123-B10 & SPP2306 BA4925/2-1), the Deutsches Zentrum für Herz-Kreislauf-Forschung (DZHK),

and the European Research Council (ERC) Starting Grant PROTEOFIT. Open Access funding enabled and organized by Projekt DEAL. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Institute for

Cardiovascular Prevention (IPEK), Faculty of Medicine, Ludwig-Maximilians-University, Munich, Germany Anahita Ofoghi, Stefan Kotschi, Imke L. Lemmer, Nienke Willemsen, Batoul Bayer, Anna S.

Jung & Alexander Bartelt * Institute for Diabetes and Cancer (IDC), Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany Imke L. Lemmer, Anna S.

Jung & Alexander Bartelt * Institute for Diabetes and Obesity (IDO), Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany Daniel T. Haas & 

Natalie Krahmer * Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald, Germany Sophie Möller, Stefanie Haberecht-Müller & Elke Krüger *

German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Munich, Germany Alexander Bartelt * Department of Molecular Metabolism & Sabri Ülker Center for

Metabolic Research, Harvard T.H. Chan School of Public Health, Boston, USA Alexander Bartelt Authors * Anahita Ofoghi View author publications You can also search for this author inPubMed 

Google Scholar * Stefan Kotschi View author publications You can also search for this author inPubMed Google Scholar * Imke L. Lemmer View author publications You can also search for this

author inPubMed Google Scholar * Daniel T. Haas View author publications You can also search for this author inPubMed Google Scholar * Nienke Willemsen View author publications You can also

search for this author inPubMed Google Scholar * Batoul Bayer View author publications You can also search for this author inPubMed Google Scholar * Anna S. Jung View author publications You

can also search for this author inPubMed Google Scholar * Sophie Möller View author publications You can also search for this author inPubMed Google Scholar * Stefanie Haberecht-Müller View

author publications You can also search for this author inPubMed Google Scholar * Elke Krüger View author publications You can also search for this author inPubMed Google Scholar * Natalie

Krahmer View author publications You can also search for this author inPubMed Google Scholar * Alexander Bartelt View author publications You can also search for this author inPubMed Google

Scholar CONTRIBUTIONS AO designed and performed experiments, analyzed data, and wrote the manuscript. SK, ILL, DH, ASJ, and NK performed and analyzed proteomics. NW and BB helped with cell

analysis. SM, SH-M, and EK generated cell models. AB designed experiments, analyzed data, and wrote the manuscript. All authors read and commented on the manuscript. CORRESPONDING AUTHOR

Correspondence to Alexander Bartelt. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ETHICS APPROVAL All methods were performed in accordance with the

relevant guidelines and regulations. Human cell lines were generated previously as described in the methods section of this manuscript. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer

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ARTICLE CITE THIS ARTICLE Ofoghi, A., Kotschi, S., Lemmer, I.L. _et al._ Activating the NFE2L1-ubiquitin-proteasome system by DDI2 protects from ferroptosis. _Cell Death Differ_ 32, 480–487

(2025). https://doi.org/10.1038/s41418-024-01398-z Download citation * Received: 05 July 2023 * Revised: 30 September 2024 * Accepted: 02 October 2024 * Published: 09 October 2024 * Issue

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