Fig 1: Dephosphorylation of RRM2 stimulates RRM2 binding with GSS to promote their corecruitment to the proteasome and subsequent activation of ferroptosis. a, b Reciprocal IP experiments of RRM2 and GSS in RRM2−/− HepG2 cells reconstituted with RRM2WT, RRM2T33A or RRM2T33E. The amount of proteins in the immunoprecipitates was normalized to that in whole-cell lysates (Input), and was graphed in the lower panel. c, d Reciprocal IP experiments for RRM2 and GSS in HepG2 cells with or without NU6102 (20 μM, 24 h) and SB203580 (10 μM, 24 h) treatment. The amount of protein in the immunoprecipitates was normalized to that in whole-cell lysates (Input), and was graphed in the lower panel. e Proximal protein ligation between endogenous GSS and the indicated exogenous RRM2-Myc, as measured by PLA in HepG2 cells expressing RRM2WT, RRM2T33A or RRM2T33E. Scale bar, 20 μm. The PLA signals were also calculated and graphed, and the data from the “Empty” group were arbitrarily set to 1. f RRM2WT, RRM2T33A or RRM2T33E was expressed in reconstituted RRM2−/− HepG2 cells. Immunoprecipitations were acquired with anti-PSMB5 antibodies and further analyzed by immunoblotting using anti-RRM2 and anti-GSS antibodies. GSS and RRM2 enrichment in the immunoprecipitates was calculated as the normalization to the levels in whole-cell lysates (input). g–i GSH (g), cell death (h) and 4-HNE (i) were measured in HepG2 and SMMC-7721 cells with or without RRM2 knockdown before they were further treated with Fer-1 (1 μM, 24 h), ZVAD-FMK (20 μM, 24 h), Nec-1 (20 μM, 24 h), or ectopically expressed GSS, RRM2T33A or RRM2WT in the presence or absence of NU6102 (20 μM, 24 h). The data are shown as the mean ± SD from three biological replicates (including IB). *P < 0.05, **P < 0.01 indicates statistical significance. The data from a–d and f–i were analyzed using one-way ANOVA
Fig 2: Diagnostic value of serum RRM2 in predicting liver cancer. a ROC curves for serum RRM2, AFP, and the combination of serum RRM2 and AFP for the discriminating patients with liver cancer from healthy individuals. The AUC value represents the combined effects of the sensitivity and specificity of single or combined biomarkers in the diagnosis of patients with liver cancer. All the data were obtained from three biological replicates. b Serum RRM2 is positively associated with tumor stage in liver cancer patients. The first quartile of serum RRM2 levels in liver cancer patients was no more than 300 pg/ml, the second quartile was from 300 to 1200 pg/ml, and the bottom quartile was more than 1200 pg/ml. Liver cancer patients were divided into three groups according to these quartiles. All the data were from three biological replicates. The associations between tumor stage and serum RRM2 in liver cancer patients were analyzed using the χ2 test. c Correlation between intracellular RRM2 and RRM2 in culture medium from liver cancer cell lines as analyzed by Spearman rank-correlation analysis. d RRM2 expression across multiple cancer types from the UALCAN database. TPM, transcriptions per million. e The model of the study. Under homeostasis, RRM2 is phosphorylated at the T33 site (can be dephosphorylated by NU6102) to sustain GSS and GSH levels and suppress potential ferroptosis. In the ferroptotic state, RRM2 is dephosphorylated and has increased affinity GSS, leading to the subsequent proteasomal degradation of both proteins. With such an effect, GSH eventually declines to facilitate ferroptosis. The data from a-c are shown from three biological replicates. Analysis of the receiver operator characteristics (ROC) and calculation of the area under the curve (AUC) was performed to find the diagnostic values of RRM2, AFP and the combination of RRM2 and AFP for the prediction of liver cancer. **P < 0.01 indicates statistical significance. Data from b were analyzed using the χ2 test. Data from c were analyzed by Spearman rank-correlation analysis. Data in d were analyzed using Student’s t test
Fig 3: Mechanism diagram of CA increasing ferroptosis sensitivity of liver cancer cells.Briefly, CA sensitizes ferroptosis by upregulating HERPUD1 in liver cancer cells. It was further analyzed that CA acts on MDM2 through HERPUD1, increases the ubiquitination of GSS, and then reduces the content of GSS to reduce the synthesis of GSH, thereby increasing the sensitivity of liver cancer cells to ferroptosis.
Fig 4: HERPUD1 affects the ubiquitination of GSS by acting on MDM2.A Half-life of GSS in Bel-7402 or Bel-7404 cells with or without HERPUD1 knocked out was detected by CHX chase experiments. The relative protein levels of GSS were normalized to those of GAPDH, and the “0 h” point was arbitrarily set to 100%. B Total ubiquitination of GSS was detected by co-IP experiments and measured by an anti-total ubiquitin antibody. C Bel-7402 and Bel-7404 cells were seeded at the indicated density in 10 cm petri dish and cultured for 24 h, then treated with: 10 μM erastin or 5 μM RSL3; 10 μM CA; pretreatment with 10 μM CA for 1 h followed by 10 μM erastin or 5 μM RSL3, in complete medium for 24 h. The drug-treated protein was then collected, co-IP was performed with anti-GSS antibody according to the previous steps, and measured by anti-total ubiquitin antibody. D UbiBrowser online software and the literature was used to identify GSS-related ubiquitinating enzymes. E Reciprocal IP experiments of GSS, MDM2 and SYVN1 in wild-type Bel-7402 and Bel-7404 cells. F Reciprocal IP experiments for detecting GSS, MDM2 and SYVN1 in Bel-7402 and Bel-7404 cells which HERPUD1 were overexpressed. G Endogenous MDM2 was immunoprecipitated by anti-MDM2 antibody and ubiquitination of MDM2 were measured by anti-ubiquitin antibody in Bel-7404 and Bel-7402 cells with or without HERPUD1 knocked out. Representative images of IB are shown in the figure. H Half-life of MDM2 was detected by CHX chase experiments in Bel-7402 and Bel-7404 cells with or without HERPUD1 knocked out. The relative protein levels of MDM2 were normalized to those of GAPDH, and the “0 h” point was arbitrarily set to 100%. Data were expressed as mean ± SD. Images of all the immunoblots are representative of three independent experiments.
Fig 5: RRM2 upregulates GSH by sustaining GSS. a The ferroptosis-related metabolic axis from glucose to GSH. b–g The levels of glycine (b), glutamate (c), cysteine (d), cystathionine (e), serine (f) and glucose (g) were measured in HepG2 and SMMC-7721 cells with or without ectopically expression or knocked down of RRM2. h, i mRNA levels of CBS, CTH, SHMT2, GSS and GPX4 were analyzed by qPCR in HepG2 (h) and SMMC-7721 cells (i) administered the indicated treatment. j, k Protein levels of CBS, CTH, SHMT2, GSS and GPX4 were analyzed by immunoblotting in HepG2 and SMMC-7721 cells (j). The level of RRM2 was normalized to that of GAPDH, and the normalized level of RRM2 in the untreated group was arbitrarily set to 100% (k). (l) GSH levels were measured in WT and GSS−/− HepG2 and SMMC-7721 cells with or without RRM2 overexpression or knockdown, as indicated. The data are shown as the mean ± SD from three biological replicates. **P < 0.01 indicates statistical significance. Data from b−i and k−l were analyzed using one-way ANOVA
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