EMBO/soda-nel huggingface dataset:
Cytosolic GDH1 degradation restricts protein synthesis to sustain tumor cell survival following amino acid deprivation (2021)
Figure 1

Figure 1. GDH1 promotes protein synthesis by maintaining ribosomal protein (RP) expression and inversely correlates with the CRC patient prognosis.

(A) 786-0 or 769-P cells stably expressing shNT or shGDH1 were treated with or without aa starvation for the indicated times, and then cell viability was assessed.

(B) 786-0 or 769-P cells stably expressing shNT or shGDH1 were reconstituted with or without various amounts of rGDH1 WT (3*WT indicates 3-fold expression WT) and then treated with aa starvation for the indicated time. Cell viability was subsequently assessed.

(C-E) RNA-seq analyses were performed in 786-0 cells stably expressing shNT or shGDH1 treated with or without aa starvation for 12 h. Gene ontology cellular components (C) and KEGG enrichment (D) analysis of differentially expressed genes was performed using shNT- and shGDH1-expressing 786-0 cells. The enrichment of differentially expressed ribosomal protein-coding genes was measured by GSEA (E).

(F) Heatmap of all of the ribosomal protein-coding genes in shNT and shGDH1 cells.

(G) Coomassie brilliant blue (CBB) staining and protein synthesis assays using the Cayman kit (601100) were performed in cells with or without GDH1. Western blotting (WB) was performed with the indicated antibodies. β-Actin was used as a loading control(left). The Click-iT HPG kit (C10428) is also used to detect newly synthesized proteins in cells with or without GDH1 (right).

(H) Boxplots of GDH1 expression between primary CRC tumors and adjacent normal tissues in the TCGA-KIRC cohort. In the boxplot, medians, 0.25/0.75 quantile, and min/max were represented by the central lines, the box limits, and the whiskers, respectively. (N: normal, n = 72; T: tumor, n = 534.) Unpaired t-test was used with ***p < 0.001.

(I) IHC staining of GDH1 in primary KIRC tumors and adjacent normal tissues collected by us was performed (left). Quantitative analysis of the GDH1 protein levels was performed using unpaired t-test (right). ***p < 0.001. Scale bars: 50 μm.

(J) Boxplots of GDH1 expression in four stages in the TCGA-KIRC cohort. In the boxplot, medians, 0.25/0.75 quantile, and min/max were represented by the central lines, the box limits, and the whiskers, respectively.(I: n = 267, II: n = 57, III: n = 123, IV: n = 84). One way ANOVA test was used with **p < 0.01.

(K) Overall survival of patients in different tumor stages in the TCGA-KIRC cohort. The R package "survminer"-based maximally selected log-rank statistic was used to determine the best cutoff. The log-rank test was used to determine the p value.

Data information: (A, B and G) The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t-test. (**p < 0.01; ***p < 0.001.) See also Expanded View Figure 1.
Cytosolic GDH1 degradation restricts protein synthesis to sustain tumor cell survival following amino acid deprivation (2021)
Figure 2

Figure 2. The starvation of aa induces GDH1 translocation and reduces protein stability.

(A) 786-0 cells were subjected to aa starvation for the indicated times. The protein level of GDH1 was determined by WB using the indicated antibody.

(B) The mRNA level of GDH1 in the cells in (A) was determined by qPCR using specific primers against GDH1 mRNA.

(C) 786-0 cells were treated with or without aa starvation for 6 hours and then treated with CHX (1 μM) for the indicated times. The protein level of GDH1 was determined by western blotting (left), and the relative GDH1 protein levels were calculated (right). CHX: cycloheximide. Unpaired t-test was used.

(D) Immunofluorescence (IF) assay was performed to determine the subcellular location of GDH1 with or without aa starvation. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.

(E) Cytoplasm-mitochondrial fractionation assay was performed to determine the subcellular location of GDH1 with or without aa starvation. The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.

(F) The total GDH1 protein in 786-0 and 769-P cells with or without aa starvation for 12 hours was measured by WB.

(G) Immunofluorescence (IF) assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.

(H) Cytoplasm-mitochondrial fractionation assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.

(I) 786-0 cells with FLAG-tagged WT or dMSP GDH1 were treated with CHX (1 μM) for the indicated times. The protein level of FLAG-tagged WT or dMSP GDH1 was determined by western blotting (left). The relative WT or dMSP GDH1 protein level was calculated (right). Unpaired two-tailed t-test was used.

Data information: (B-C and I) The data are represented as mean ± SEM of three independent experiments. The one-way ANOVA test (B) and unpaired t-test were used (C and I). (N.S.: not significant; ***p < 0.001). See also Expanded View Figure 2.
Cytosolic GDH1 degradation restricts protein synthesis to sustain tumor cell survival following amino acid deprivation (2021)
Figure 3

Figure 3. Ubiquitination of a cluster of lysines impairs GDH1 protein stability under aa starvation.

(A) 786-0 or 769-P cells were pretreated with or without aa starvation for 6 hours, and then MG132 (10 μM) or CQ (1 μM) was added to the cell culture medium for 12 hours. The protein level of GDH1 was determined by WB with the indicated antibody.

(B) In 786-0 cells, FLAG-tagged GDH1 was co-transfected with or without HA-tagged ubiquitin for 48 hours, and then the cells were treated with or without aa starvation for 6 hours. Immunoprecipitation was performed using an antibody against FLAG followed by western blotting using the indicated antibodies.

(C) In 786-0 cells, FLAG-tagged GDH1 was co-transfected with HA-tagged ubiquitin for 48 hours, and then the cells were treated with or without aa starvation for 6 hours. Immunoprecipitation (IP) was performed using an antibody against FLAG, and the ubiquitination of FLAG-GDH1 was identified by LC-MS/MS.

(D) FLAG-tagged GDH1 (including WT and mutants with different lysine mutations to arginine) was co-transfected with HA-tagged ubiquitin for 48 hours, and then cells were subjected to aa starvation for 12 hours before collecting cells. WB and qPCR separately tested the levels of GDH1 protein and mRNA, respectively.

(E) FLAG-tagged GDH1 (including WT and 3KR mutant) and HA-tagged ubiquitin-expressing 786-0 cells were pretreated with aa starvation for 12 hours before treatment with MG132 (10 μM) for 12 hours. Immunoprecipitation was performed using an antibody against FLAG followed by WB using the indicated antibodies. 3KR mutant: lysine 183, 191, 200 to arginine 183, 191, 200.

(F) FLAG-tagged GDH1 (including WT and 3KR mutant) was transfected into HEK293T cells and enriched using an antibody against FLAG. GDH1 enzymatic activity was quantified, and the quantity of WT and 3KR GDH1 was evaluated by CBB staining. Unpaired two-tailed t-test was used.

(G) Restored expression of wild-type or 3KR mutant in 786-0 cells lacking GDH1 followed by pretreatment of the cells with or without aa starvation for 24 hours. Cells were collected, and total RNA was extracted for further qPCR using primers against the mRNA of RP genes.

(H) 786-0 or 769-P cells stably expressing shGDH1 were reconstituted with rGDH1 WT or 3KR mutant and then treated with or without aa starvation for the indicated time. Cell viability was determined. Unpaired two-tailed t-test was used.

Data information: (D, F and H) The data are represented as the mean ± SEM of three independent experiments. The one-way ANOVA test (D) and unpaired t-test were used (F and H). (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). See also Expanded View Figure 3.
Cytosolic GDH1 degradation restricts protein synthesis to sustain tumor cell survival following amino acid deprivation (2021)
Figure 4

Figure 4. RNF213 is required for GDH1 ubiquitination under aa starvation.

(A) 786-0 cells were transfected with individual siRNAs that targeted 335 E3 ligases (three siRNAs per gene; indicated as 'siGene' on the x axis) for 48 hours and treated with aa starvation for 12 hours in duplicates. Next, the intensity of GDH1-GFP in each well was recorded using Opera Phenix (Perkin Elmer). The data are presented as a volcano plot. The siRNAs with P < 0.0001 (two-tailed Student's t-test) and a GFP intensity ratio (normalized to the non-targeting siRNA) >16 were considered to be effective siRNAs (red). E3 ligases targeted by more than two effective siRNAs were selected as candidates. siE3s, E3 ligase siRNAs; siNC, non-targeting control siRNA.

(B) 786-0 cells were transfected with siRNA targeting RNF213, CHFR or TRIM27 for 48 hours and then were subjected to aa starvation for 12 hours. WB was performed using the indicated antibodies.

(C-D) 786-0 cells were treated with MG132 (10 μM) under aa starvation. Antibodies against GDH1 (C) or RNF213 (D) were used to enrich protein complexes in cells with or without GDH1. IgG was used as a control. WB was performed using the indicated antibodies.

(E) 786-0 cells were treated with MG132 (10 μM) under aa starvation. The cells were transfected with siNC or siRNF213 for 48 hours, and then an antibody against GDH1 was used to enrich the GDH1-associated complex. IgG was used as a control. WB was performed using the indicated antibodies.

(F) 786-0 cells were transfected with siNC or siRNF213 for 24 hours and then were transfected with or without His-ubiquitin for 36 hours. The indicated cells were treated with or without MG132 (10 μM) under aa starvation. Beads against His were used to enrich His-ubiquitin-associated proteins, which were eluted by PBS containing 250 mM imidazole. WB was performed using the indicated antibodies.

(G) Restored expression of WT or 3KR GDH1 in 786-0 cells lacking GDH1 followed by transfection with or without His-ubiquitin for 36 hours. The indicated cells were treated with MG132 (10 μM) under aa starvation. Beads against His were used to enrich His-ubiquitin-associated proteins in the indicated cells. WB was performed using the indicated antibodies.

(H) 786-0 or 769-P cells (WT or stably expressing shGDH1) were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for the indicated times. Cell viability was determined. Unpaired t-test was used with ***p < 0.001.

(I) IHCs of KIRC clinical samples were performed using antibodies against GDH1 or RNF213 (upper panel). The correlated IHC signal between GDH1 and RNF213 in KIRC samples was calculated (lower panel). The cor.test was used to determine the p-value. Scale bars: 50 μm.
Cytosolic GDH1 degradation restricts protein synthesis to sustain tumor cell survival following amino acid deprivation (2021)
Figure 5

Figure 5. Increased cellular αKG sensitizes tumor cells to aa starvation.

(A-C) 786-0 cells expressing WT or enzymatically dead (ED) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours. Western blotting was performed using the indicated antibodies (A). The intracellular level of αKG was measured using an αKG determination kit and normalized to 786-0 cells expressing WT GDH1 transfected with siNC under normal culture conditions. Unpaired t-test was used (B). In the above cells in (A), qPCR was used to measure the mRNA of the indicated RP genes (C).

(D-E) 786-0 cells expressing WT or ED GDH1 were supplemented with or without 500 μM methylated αKG for 36 hours, and then cells were collected to extract total mRNA. The mRNA levels of the indicated RP genes were tested using qPCR (D), and WB was performed using the indicated antibodies (E).

(F) Protein synthesis assays using the Cayman kit and Coomassie brilliant blue (CBB) staining were performed in the indicated cells. WB was performed using the indicated antibodies (left), and the relative intensity of protein synthesis was quantified (right). Unpaired t-test was used.

(G) 786-0 cells expressing WT or ED GDH1 were treated with or without aa starvation for the indicated times. Cell viability was determined. Unpaired two-tailed t-test was used.

(H-I) 786-0 or 769-P cells were supplemented with methylated αKG (500 μM), 2HG (500 μM) or αKG (500 μM) +2HG (500 μM) for 36 hours under aa starvation conditions. Cell viability was determined (H), and the mRNA levels of the indicated RP genes were tested using qPCR (I).

(J) KIRC clinical samples were collected for αKG measurement, and we assessed overall survival (left panel) with log-rank test according to the dividing groups of high and low intracellular levels of αKG with the best separation (right panel). Unpaired t-test was used in the right panel with ***p < 0.001.

Data information: (B and F-H) The data are represented as the means ± SEM of three independent experiments (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). See also Appendix Figure S1.
Cytosolic GDH1 degradation restricts protein synthesis to sustain tumor cell survival following amino acid deprivation (2021)
Figure 6

Figure 6. KDM activity regulated by αKG is required to demethylate H3K9me3 and H3K27me3 on the promoter of RP genes.

(A) 786-0 cells expressing shGDH1 with restored expression of WT or ED GDH1 were supplemented with or without 500 μM methylated αKG for 48 hours, and then the cells were collected to extract total protein. WB was performed using the indicated antibodies.

(B) In 786-0 cells expressing shGDH1 with restored expression of WT or ED GDH1, the ratio (ED/WT) of H3K9me3, H3K27me3 and H3K36me3 targeting RP genes was calculated. The outer circle in the chord diagram represents the ratio of ribosomal protein-coding genes targeted by each histone lysine methylation compared with the control. The inner circles represent the sum of the indicated ratios. The IgG was used as a control.

(C) The relative enrichment of KDMs targeting ribosomal protein-coding genes in 786-0 cells. The outer circle represents the enrichment value of ribosomal protein-coding genes by each KDM. The inner circles represent the sum of the indicated values. The IgG was used as a control.

(D) 786-0 cells were transfected with siRNAs targeting KDM4A or KDM6A for 56 hours, and the cells were fixed with 1% formaldehyde. ChIP assays were performed using antibodies against H3K9me3 or H3K27me3. qPCR tested the enrichment of the above histone H3 lysine modifications on the promoters of the indicated RP genes.

(E) 786-0 cells were transfected with siRNAs targeting KDM4A or KDM6A and then were supplemented with or without methylated αKG (500 μM) for 48 hours. The cells were collected to extract total RNA, and qPCR was used to test the mRNA level of the indicated RP genes.

(F) Isothermal titration calorimetry (ITC) was used to test the binding affinity of αKG (1 mM) to recombinant KDM4A (0.05 mM) or KDM6A (0.05 mM) (left), and then statistical analysis was performed (right). The data are represented as the means ± SEM of three independent experiments. Unpaired t-test was used (N.S.: not significant).

(G) An in vitro demethylation assay was performed with recombinant KDM4A (0.05 mM) or KDM6A (0.05 mM), and the indicated histone H3 lysine methylation peptides under 50-μM αKG conditions (no αKG was used as a blank control). Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in the reaction system.

(H) 786-0 cells were supplemented with or without 2HG (500 μM) for 48 hours, and the cells were fixed with 1% formaldehyde. ChIP assays were performed using antibodies against H3K9me3 or H3K27me3. qPCR was used to test the enrichment of the above histone H3 lysine modifications on the promoters of the indicated RP genes.

See also Expanded View Figure 5.
Cytosolic GDH1 degradation restricts protein synthesis to sustain tumor cell survival following amino acid deprivation (2021)
Figure 7

Figure 7. Loss of GDH1 or its enzymatic activity or its protein stability promotes solid tumor formation, and GDH1 expression is negatively associated with KIRC progression.

(A) 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 were subjected to aa starvation for 24 hours. PI/Annexin V double staining of the indicated cells was performed, and the number of apoptotic cells in each group was calculated. The data are represented as the mean ± SEM of three independent experiments. Unpaired t-test was used with **p < 0.01.

(B-G) Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The solid tumor formed by the indicated 786-0 cells is shown (B). The tumor growth rate (C), tumor weight (D) and αKG levels in tumors (E) were recorded and analyzed. The tumors were collected to extract total RNA, and qPCR was used to test the mRNA level of the indicated RPL/S genes (F). The staining of Ki67 (G, upper panel) and TUNEL (G, lower panel) using tumors from (B) are shown and analyzed (G, right panel). We used 6 mice in each group. Scale bars: 50 μm.

(H) Schematic model of the αKG-mediated regulation of RP genes. We uncovered the mechanism by which KIRC cells respond to aa starvation and regulate their protein synthesis to save limited nutrients. Specifically, in KIRC cells under aa starvation, GDH1 is transferred from the mitochondria to the cytoplasm and degraded by the ubiquitin degradation pathway mediated by the E3 ligase RNF213. The degradation of GDH1 directly leads to decreased intracellular αKG, causing the weakened activity of KDM. H3K9me3 and H3K27me3 were demethylated by KDM4 and KDM6, particularly KDM4A and KDM6A, which occupied the promoter of RP genes and cooperatively downregulated the expression of the RP gene during aa starvation. The tumor cells can then preserve the remaining amino acids for survival. Notably, because RP genes are regulated by operons in prokaryotes, we revealed that the expression of most RP genes in renal cell carcinoma cells is mediated by the metabolite αKG.

Data information: (C-E and G) All statistical analyses were conducted using unpaired t-test (n = 6). (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). See also Appendix Figure S2.

Legends for expanded view figures
Lactate induces metabolic and epigenetic reprogramming of pro-inflammatory Th17 cells (2022)
Figure 4

Figure 4. Lactate induces histone lactylation in T cells.

A Western blots of acid-extracted histones from activated CD4+ T cells showing global histone lactylation and H3K18 lactylation levels in the presence and absence of glucose at 24 hours after stimulation of cells. One of three similar experiments is shown.

B Schematic diagram of glycolysis and inhibitory compounds used for inhibition of lactate production (E and F). While 2-Deoxy-D-glucose (2-DG) competitively inhibits the glycolysis, dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), which results in enhanced activity of pyruvate dehydrogenase (PDH) and elevated pyruvate oxidation.

C, D Western blots of acid-extracted histones from activated CD4+ T cells in the presence of glycolysis inhibitor 2-DG and PDK inhibitor DCA, respectively. Cells were analysed for global lactylation and H3K18 lactylation 24 hours after activation of TCR with anti-CD3 (5 µg/ml).

E Immunoblotting of acid-extracted lactylated histones from anti-CD3-activated CD4+ T cells in the presence of extracellular lactate (25 mM) at 24 hours of cell culture. One representative out of three experiments is shown. For all immunoblotting experiments, ex vivo purified, non-activated CD4+ T cells were used as control lymphocytes.

F ChIP-sequncing showing H3K18 lactylation signals in control Th17 cells and lactate-treated Th17 lymphocytes (25 mM). Biological duplicates are shown.

G KEGG-pathway analysis (biological processes) of H3K18la-specific genes in lactate-treated Th17 cells. Statistical significance was determined using DAVID software.

EV Figure legends
Listeria monocytogenes ActA-mediated escape from autophagic recognition (2009)
figf1

(a) MDCK/pGFP–LC3 cells infected with a wild-type or ΔactA2 strain of L. monocytogenes were stained with an anti-Listeria antibody (red) and rhodamine phalloidin (stains F-actin; light blue). Scale bars, 10 μm. Arrowheads indicate GFP–LC3-positive bacteria. (b) High magnification of framed images in a, and graphs show intensity of the fluoresence signal along the arrow. (c) Quantification of the number of GFP–LC3-positive bacteria shown in a. Data are mean ± s.e.m. At least 500 bacteria were counted in each experiment (n = 7 and 6 experiments at 2 and 4 h, respectively). (d) LC3 levels in infected MDCK cells. MDCK cells were infected with wild-type or ΔactA2 bacteria for the indicated times. EBSS (Earle's balanced salt solution, a starvation medium) and rapamycin (100 μg ml−1) were used as controls. (e–h) Intracellular survival of wild-type or ΔactA2 bacteria in MDCK cells (e; n = 8), MDCK cells treated with 10 mM 3-MA (f; n = 4), atg5+/+ MEF cells (g; n = 8 and 7 for wild-type and ΔactA2, respectively) or atg5−/− MEF cells (h; n = 4 and 3 for wild-type and ΔactA2, respectively). Data are presented as the number of colony-forming units (CFU) relative to that at 30 min after infection, and are mean ± s.e.m. WT, wild type. A full scan of d is shown in Supplementary Information, Fig. S7.