The atypical E3 ligase HOIL-1 safeguards the ribosome during cellular stress.

Figures

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  Fig. 1. HOIL-1 loss exacerbates cardiac remodelling and hypertrophy under pressure overload. a, A schematic of the experimental design assessing cardiac hypertrophy in WT (n = 8, 3 males and 5 females) and HOIL-1ΔRBR (n = 10, 3 males and 7 females) mice following TAC surgery. b, The peak aortic flow velocity assessed by echocardiography 1 week pre- and post-TAC. c, The LV diameter assessed by echocardiography. d, The LV volume assessed by echocardiography. e, H&E-stained male heart sections 9 weeks post-TAC. Scale bar, 30 µm. f, A violin plot of cardiomyocyte cross-sectional area and width quantified from H&E images (n = 3 male mice and n = 3 female mice per genotype). The black line represents the median. g, Periodic acid–Schiff-stained male heart sections 9 weeks post-TAC. Arrowheads indicate glycogen aggregates. Scale bar, 35 µm. h, The quantification of glycogen aggregates using Periodic acid–Schiff-stained heart sections. Control mice were age-matched and not subjected to TAC. i, Ejection fractions assessed by echocardiography. j, The lung weight to body weight ratio assessing pulmonary oedema 9 weeks post-TAC (WT n = 3 male mice and n = 5 female mice, HOIL-1ΔRBR n = 3 male mice and…

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  Fig. 2. HOIL-1 mitigates nutrient stress. a, Cell death in AC16 cells after 48 h glucose starvation (n = 3 biological replicates). b, Cell death in 786-O cells after 48 h glucose starvation (n = 3 biological replicates). c, Cell death in MEFs after 48 h glucose starvation (n = 4 biological replicates). d, Cell death in AC16 cells after 48 h amino acid starvation (n = 4 biological replicates). e, Cell death in 786-O cells after 24 h amino acid starvation (n = 3 biological replicates). f, Cell death in MEFs after 48 h amino acid starvation (n = 4 biological replicates). g, Cell death in AC16 cells treated with cell death inhibitors (Q-VD 5 µM, necrostatin-1 30 µM, ferrostatin-1 2 µM and digoxin 1 µg ml−1) concurrent with 48 h glucose starvation (n = 3 biological replicates). h, MitoSOX Red mean fluorescence intensity (MFI) assessed by flow cytometry in AC16 cells grown in full media or after 24 h glucose starvation (n = 3 biological replicates). i, Representative image of MitoSOX Red staining after TEMPOL. No scale bar available given acquisition settings. j, Cell death in AC16 cells treated with TEMPOL (50 µM) concurrent with 48 h glucose starvation (n = 3 biological replicates). Data are…

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  Fig. 3. The nutrient stress metabolome reveals glucose starvation causes disulfidptosis mediated by cystine influx via xCT. a, A schematic of the experimental design examining the metabolome in AC16 cells in full medium or after 3 h or 24 h glucose starvation. b, PLS-DA of the metabolomics dataset. c, A volcano plot of targeted metabolites in parent and HOIL-1ΔRBR AC16 cells after 24 h glucose starvation. Significant when −log(P value) ≥1.3. d, SLC7A11 mRNA levels (normalized to TBP) in AC16 cells after 24 h glucose starvation. Data are represented as the fold change over full medium (n = 3 biological replicates). e, Immunoblot analysis of xCT levels in AC16 cells during glucose starvation. f, A schematic of xCT-dependent nutrient and metabolite flux. g, Cell death in AC16 cells treated with erastin (2 µM), sulfasalazine (500 µM) or DMSO vehicle concurrent with 48 h glucose starvation (n = 4 biological replicates). h, Cell death in xCTKO AC16 cells after 48 h glucose starvation (n = 3 biological replicates). Representative immunoblot of xCT KO. i, The measurement of NADP+ and NADPH ratios during glucose starvation in AC16 cells (n = 3 biological replicates). j, Cell death in AC16 cells…

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  Fig. 4. ATF4 orchestrates disulfidptosis. a, Immunoblot analysis of AC16 cells after glucose starvation for the indicated timepoints. b, ATF4 and NRF2 target genes in AC16 cells measured by RT–qPCR. Data are presented as the fold change over full medium (n = 3 biological replicates). c, Cell death in AC16 cells transfected with ATF4, NRF2 or non-targeting control (NTC) siRNA 48 h before glucose starvation for 48 h (n = 3 biological replicates). d, Immunoblot analysis of AC16 cells transfected with ATF4, NRF2 or NTC siRNA 48 h before glucose starvation for 24 h. e, Immunoblot analysis of 786-O cells after 24 h glucose starvation. Two different HOIL-1ΔRBR clones are shown. f, Cell death in 786-O cells transfected with ATF4 or NTC siRNA 48 h before glucose starvation for 48 h (n = 4 biological replicates). g, Cell death in 786-O cells treated with erastin (2 µM), sulfasalazine (500 µM) or DMSO vehicle concurrent with 48 h glucose starvation (n = 4 biological replicates). h, Immunoblot analysis of AC16 cells after DMSO vehicle, CCCP (5 µM, 25 µM) or glucose starvation for 24 h. i, Immunoblot analysis of AC16 cells after amino acid or glucose starvation for the indicated timepoints. j, A…

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  Fig. 5. Loss of the HOIL-1 RBR domain remodels the ribosome ubiquitin landscape and renders glucose starvation ribotoxic. a, A schematic of the experimental design. b, A volcano plot showing differentially ubiquitinated peptides after 8 h glucose starvation. Significant at −log(FDR) ≥2s.d. ≤−2s.d. c, Gene Ontology enrichment analysis of proteins with reduced ubiquitin-modified peptides in HOIL-1ΔRBR cells relative to parent AC16 cells after 8 h glucose starvation. d, A schematic of RSR signalling. e, An immunoblot analysis of AC16 cells after DMSO vehicle, TNF (10 ng ml−1), anisomycin (5 µM) or glucose starvation for the indicated timepoints. ZAKα phosphorylation was assessed using a Phos-Tag acrylamide gel. f, An immunoblot analysis of AC16 cells after glucose starvation for the indicated timepoints. g, An immunoblot analysis of AC16 cells transfected with ZAK or NTC siRNA 48 h before sodium arsenite (10 µM) or glucose starvation for the indicated timepoints. h, Cell death in AC16 cells transfected with ZAK or NTC siRNA before glucose starvation for 48 h (n = 4 biological replicates). i, Cell death in 786-O cells transfected with ZAK or NTC siRNA before glucose starvation for 48 h (n =…

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  Fig. 6. HOIL-1 resolves translational stress by promoting quality control-associated ubiquitination and limiting ribosome collisions. a, Cell death in AC16 cells transfected with ZAK or NTC siRNA 48 h before anisomycin (5 µM) treatment for 24 h (n = 4 biological replicates). b, Cell death in AC16 cells treated with the indicated ribotoxic stressors for 24 h (n = 3 biological replicates). c,e,g, Immunoblot analysis of RPS10 and RPS20 ubiquitination during anisomycin treatment in AC16 cells (c), 786-O cells (e) or MEFs (g). d,f,h, Quantification of RPS10 and RPS20 ubiquitination by immunoblot densitometry analysis from three independent experiments in AC16 cells (d), 786-O cells (f) or MEFs (h). Data were calculated as the percentage of the maximum RPS-ubiquitin (Ub)/total RPS within the experiment and represented as the mean ± s.e.m. i, Polysome profiles from RNase A-digested lysates of AC16 cells treated with anisomycin (5 µM) for 1 h or left untreated. j, Polysome profiles from RNase A-digested lysates of AC16 cells starved of glucose for 24 h or fed full medium. Data are presented as mean ± s.e.m. from one representative experiment. P values were determined by two-way ANOVA followed by…

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  Fig. 7. Mutant HOIL-1 binds PABPC1 and excludes MKRN1, triggering ribotoxic stress during glucose starvation. a, A schematic of the experimental design examining the ΔRBR HOIL-1 proximitome in AC16 cells in full medium or after 6 h glucose starvation. b, A volcano plot showing log2FCs of proteins detected in ΔRBR HOIL-1-MiniTurbo streptavidin pulldown samples over GFP-MiniTurbo samples after 6 h glucose starvation. Significant when −log10(P value) >1.3 and log2FC >0.6. c, Immunoblot analysis following endogenous HOIL-1 or IgG immunoprecipitation from 786-O lysates. d, Immunoblot analysis of HEK293T cells transfected with the indicated plasmids, followed by denaturing lysis and GFP-Trap immunoprecipitation. e, Immunoblot analysis following endogenous PABPC1 or IgG immunoprecipitation from AC16 lysates. The asterisk denotes the IgG heavy chain and the arrowheads denote full-length HOIL-1. f, The quantification of MKRN1–PABPC1 binding under glucose starvation by immunoblot densitometry analysis from three independent experiments, as in e. The ratio of MKRN1:PABPC1 in parental AC16 lysates in full medium was set to 1.0. g, Immunoblot analysis of AC16 cells stably expressing PABPC1-GFP…

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  Fig. 8. HOIL-1, PABPC1 and MKRN1 suppress ZAKα-driven disulfidptosis during glucose starvation. a, The proposed model. Cells in full glucose medium are in homeostasis. During glucose starvation in WT cells, HOIL-1 binds and ubiquitinates PABPC1, which resolves stress and stalled ribosomes by facilitating quality control via MKRN1. In HOIL-1KO cells, MKRN1 retains PABPC1 binding and quality control is unaffected. HOIL-1ΔRBR acts as a dominant-negative by binding to PABPC1 and excluding MKRN1 from the complex. Unresolved stalled ribosomes in HOIL-1ΔRBR cells activate ZAKα, which promotes p38 phosphorylation and ATF4 translation, leading to disulfidptosis.

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  Extended Data Fig. 1. HOIL-1 loss does not influence cardiac fibrosis or cardiomyocyte apoptosis under pressure overload. a, Hematoxylin and eosin (H&E)-stained sections from untreated male mice. Scale bar = 30 µm. b, Violin plot of cardiomyocyte cross-sectional area and width quantified from H&E images (n = 3 WT male mice, n = 3 HOIL-1ΔRBR male mice, n = 4 WT female mice, n = 2 HOIL-1ΔRBR female mice). Black line is the median. c, Masson’s Trichrome-stained male heart sections 9 weeks post-TAC. Arrowheads indicate interstitial collagen. Scale bar = 45 µm. d, Cardiac fibrosis quantified from Masson’s Trichrome images (n = 3 male mice, n = 3 female mice per genotype). e, TUNEL-stained heart sections 9 weeks post-TAC. Arrowheads indicate TUNEL+ nuclei. Scale bar = 60 µm. f, Proportion of TUNEL+ nuclei quantified from TUNEL images (n = 3 male mice, n = 3 female mice per genotype). Data are presented as the mean ± s.e.m (d, f). P values were determined by unpaired two-tailed Student’s t-test (b, d, f). Source numerical data are available in Source Data. Source data

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  Extended Data Fig. 2. Loss of the HOIL-1 RBR impairs glycogen breakdown. a, Model system. b, Immunoblot analysis of AC16 cells. Arrowhead indicates ΔRBR HOIL-1. Asterisks indicate non-specific bands. c, Genomic DNA sequencing from AC16 cells confirming deletion and premature stop codon in HOIL-1ΔRBR cells. Red text is the gRNA target sequence. d, Fraction of cellular glycogen remaining after 1 h glucose starvation in AC16 cells. Pooled data from three independent experiments. Each data point is the mean of 3 biological replicates. Data are presented as mean ± s.d. e, Glucose-1-phosphate levels in AC16 cells after glucose starvation measured by LC-MS (n = 6 biological replicates). Data are presented as mean ± s.e.m. f, Immunoblot analysis of HOIL-1ΔRBR 786-O cells. g, Immunoblot analysis of HOIL-1ΔRING1 MEFs. P values were determined by unpaired two-tailed Student’s t-test (d) or two-way ANOVA followed by Dunnett’s post-hoc comparison (e). Source numerical data and uncropped blots are available in Source Data. Source data

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  Extended Data Fig. 3. Glucose starvation causes noncanonical cell death. a, Schematic. Glycogen phosphorylase inhibitor (GPI) reduces glycogen breakdown. b, Cell death in AC16 cells pre-treated with GPI for 1 h followed by 48 h glucose starvation (n = 3 biological replicates). c, Cell death in AC16 cells transfected with GYS1 or non-targeting control (NTC) siRNA prior to glucose starvation for 48 h (n = 3 biological replicates). d, Cell death in HOIPKO AC16 cells after 48 h glucose starvation (n = 4 biological replicates). e, Cell death in AC16 cells after TNF (20 ng/ml) + CHX (6 µg/ml) treatment for 24 h or glucose starvation for 48 h in combination with HOIPIN-8 (30 µM) (n = 2 biological replicates). f, Cell death in AC16 cells transfected with OTULIN or NTC siRNA prior to glucose starvation for 48 h (n = 3 biological replicates). g, Cell death in AC16 cells treated with DMSO vehicle, Torin-1 (250 nM), MHY1485 (20 µM), or Compound C (7 µM) concurrent with 48 h glucose starvation (n = 3 biological replicates). h, Immunoblot analysis for AMPK and mTOR-related proteins in AC16 cells following glucose starvation for the indicated time-points. Data are presented as mean ± s.e.m from one representative…

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  Extended Data Fig. 4. NADPH collapse does not cause glucose starvation-induced cell death. a, Heatmap of metabolite levels after 24 h glucose starvation represented as log2(FC) of parental AC16 cells. b, Pathway analysis of metabolites enriched in HOIL-1ΔRBR cells after 24 h glucose starvation. c, Cell death in AC16 cells after DMSO vehicle, uridine (25 mM), hydroxyurea (0.2 mM, 2.0 mM), brequinar (0.1 µM, 1.0 µM), or oxythiamine (0.5 mM, 5 mM) treatment concurrent with 48 h glucose starvation (n = 3 biological replicates). d, Cell death in AC16 cells stably overexpressing (OE) xCT treated with erastin (2 µM) concurrent with 48 h glucose starvation (n = 3 biological replicates for all except parent-xCTOE + erastin, n = 2 biological replicates). e, Cell death in AC16 grown in L-cystine and glucose-free media supplemented with L-cystine (200 µM) or sulfasalazine (500 µM) for 48 h (n = 4 biological replicates). f, Schematic of NADP + and/NADPH flux. g, Measurement of NADP+ and NADPH ratio during glucose starvation in xCTKO AC16 cells (n = 3 biological replicates). h, Measurement of NADP+ and NADPH ratio in AC16 cells treated with G6PD inhibitor (G6PDi-1, 50 µM) during glucose starvation (n = 3 biological…