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Malic enzyme 2 maintains protein stability of mutant p53 through 2-hydroxyglutarate

Nature Metabolism volume 4, pages 225–238 (2022)Cite this article

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Abstract

Many types of cancer feature TP53 mutations with oncogenic properties. However, whether the oncogenic activity of mutant p53 is affected by the cellular metabolic state is unknown. Here we show that cancer-associated mutant p53 protein is stabilized by 2-hydroxyglutarate generated by malic enzyme 2. Mechanistically, malic enzyme 2 promotes the production of 2-hydroxyglutarate by adjusting glutaminolysis, as well as through a reaction that requires pyruvate and NADPH. Malic enzyme 2 depletion decreases cellular 2-hydroxyglutarate levels in vitro and in vivo, whereas elevated malic enzyme 2 expression increases 2-hydroxyglutarate production. We further show that 2-hydroxyglutarate binds directly to mutant p53, which reduces Mdm2-mediated mutant p53 ubiquitination and degradation. 2-Hydroxyglutarate supplementation is sufficient for maintaining mutant p53 protein stability in malic enzyme 2-depleted cells, and restores tumour growth of malic enzyme 2-ablated cells, but not of cells that lack mutant p53. Our findings reveal the previously unrecognized versatility of malic enzyme 2 catalytic functions, and uncover a role for mutant p53 in sensing cellular 2-hydroxyglutarate levels, which contribute to the stabilization of mutant p53 and tumour growth.

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Fig. 1: ME2 regulates mutp53 stability in a Mdm2-dependent manner.
Fig. 2: ME2 modulates cellular 2-HG abundance to control mutp53 degradation.
Fig. 3: ME2 promotes 2-HG production via distinct mechanisms.
Fig. 4: 2-HG binds to mutp53 and abrogates its ubiquitination by Mdm2.
Fig. 5: Mutp53 sensing 2-HG contributes to ME2-mediated tumour growth.

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Data availability

The source and relevant information for all publicly available RNA sequence or gene mutation data are indicated in the corresponding figure legends or Methods. Source data are provided with this paper. Data that support other findings of this study are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank X. Yang, K.E. Wellen, C.B. Thompson, B. Vogelstein, W.S. El-Deiry and L. Yu for reagents and/or helpful comments. We thank X. Lan for helping with gene expression and mutation analysis in human cancer patients. We thank all of the Jiang laboratory members for technical assistance and/or discussion. We thank X. Wang, L. Xu and X. Liu for helping with the liquid chromatography tandem mass spectrometry experiments. We are grateful to N. Wang for helping with the protein structural analysis. This work was supported by the National Natural Science Foundation of China (82125030 and 81930082), The National Key R&D Program of China (2019YFA0801701) and Tsinghua-Peking Center for Life Sciences to P.J. and by CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-1-I2M-016), National Key Research and Development Program of China (2019YFA0802600), CAMS Basic Research Fund (2019-RC-HL-007) and State Key Laboratory Special Fund (2060204) and to WD.

Author information

Author notes

  1. These authors contributed equally: Mengjia Zhao, Pengbo Yao, Youxiang Mao, Jinjun Wu.

Authors and Affiliations

  1. School of Life Science, Tsinghua University, and Tsinghua-Peking Center for Life Sciences, Beijing, China

    Mengjia Zhao, Pengbo Yao, Youxiang Mao, Jinjun Wu, Chenhui Geng, Jie Cheng & Peng Jiang

  2. State Key Laboratory of Medical Molecular Biology, Department of Cell Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China

    Mengjia Zhao & Wenjing Du

  3. Center of Pharmaceutical Technology, Tsinghua University, Beijing, China

    Weihua Wang

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Contributions

M.Z., P.Y., Y.M. and J.W. performed all the experiments except those mentioned below. W.W. helped with some of the mass spectrometry analysis. C.G. and J.C. performed the bioinformatics analysis. M.Z., Y.M., P.Y. and P.J. designed the experiments and analysed the data. W.D. generated the ME2 knockout mice and supervised the project. P.J. conceived and supervised the project, and wrote the manuscript.

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Correspondence to Wenjing Du or Peng Jiang.

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Nature Metabolism thanks Stefan Kempa and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editors: Christoph Schmitt; George Caputa.

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Extended data

Extended Data Fig. 1 ME2, a non-target gene of mutant p53, affects mutant p53 transcriptional activity.

a, Related to Fig. 1a. SF188 cells were transfected with control siRNA (siCtrl) or siRNA against ME1, ME2, and/or p53 for 48 hours, and RNA were extracted for real-time PCR analysis of target knockdown efficiency and expression of p53 (n = 3 biological independent samples). b, Reverse sequencing analysis of mutation(s) in TP53 gene in SF188 cells. Blue shading indicates the mutation position. A GGA-to-GAA mutation has been detected in TP53 gene in these cells. c and d, SF188 cells transduced with lentivirus expressing sgRNA targeting p53 or sgRNA only (Ctrl) were transfected with control siRNA (siCtrl) or siRNA against ME1, or ME2 for 48 hours, and RNA were extracted for real-time PCR analysis of expression of indicated genes (n = 3 biological independent samples). e and f, HCT116 cells and U2OS cells were transfected with Ctrl or p53 siRNA (sip53) for 48 hours. mRNA and protein expression were determined by quantitative RT-PCR (e) and western blotting (f) respectively. n = 4 biological independent wells. g and h, U251MG cells, SF188 cells and MDA-MB-231 cells were transfected with Ctrl or p53 siRNA for 48 hours. mRNA and protein expression were determined by quantitative RT-PCR (g) and western blotting (h) respectively. n = 4 independent wells. i-k, HEK293 cells and p53-null H1299 cells were transiently transfected with different mutant p53 cDNAs individually or vector control plasmid (-) as indicated for 48 hours. mRNA and protein expression were determined by quantitative RT-PCR (i, j) and western blotting (k) respectively. n = 4 independent wells. Data in a, c, d, e, g, i, j are means ± SD, P values were determined by unpaired two-tailed Student’s t-tests. All western blot data in this study are representative of three independent experiments unless otherwise noted.

Source data

Extended Data Fig. 2 ME2 controls mutant p53 stability.

a, mRNA levels of p53R280K in MDA-MB-231 cells treated with Ctrl or ME2 siRNA (siME2) for 48 hours (n = 3 biological independent samples). b, and d, Sequencing analysis of mutations in TP53 gene in MDA-MB-231 cells (b) and U251MG cells (d). Blue shadings indicate the mutation positions. An AGA-to-AAA mutation in TP53 gene has been detected in MDA-MB-231 cells, and a CGT-to-CAT in TP53 gene in U251MG cells. c, Western blot analysis of p53 expression in MDA-MB-231 cells transfected with Ctrl, ME1, or ME2 siRNA for 48 hours. e, U251MG cells transfected with Ctrl or ME2 siRNA for 48 hours were treated with 200 µg/mL CHX for another indicated time points in the presence or absence of 10 µM MG132. Protein expression was analyzed by western blot (n = 3 biological replicates). Relative p53/actin ratios are shown. f, U251MG cells transfected with Ctrl, ME2 and/or Mdm2 siRNA as indicated for 48 hours were treated with 200 µg/mL CHX for another indicated time points. Protein expression was analyzed by western blot. Relative p53/actin ratios are shown. Data in a, e and f are mean ± SD, P values were determined by unpaired two-tailed Student’s t-tests.

Source data

Extended Data Fig. 3 2-HG accumulation augments protein levels, not mRNA transcription, of mutant p53.

a, Lysates from SF188 cells were subjected to immunoprecipitation using indicated antibodies followed by immunoblotting analysis. b, Colon tissue lysates from three pairs of ME2-/- and ME2+/+ mice were analyzed for the expression of ME2 by western blot. c and d, Western blot of SF188 cells (c) and MDA-MB-231 cells (d) treated with 8 mM 2-HG or left untreated (0) for 24 hours. e, Western blot of SF188 cells treated with 5 mM 2-HG or 5 mM α-KG as indicated. f, p53 mRNA levels in SF188 cells and MDA-MB-231 cells cultured in medium containing 0 or 5 mM 2-HG for 24 hours. (n = 3 biological independent samples). g and h, SF188 cells (g) and H1299 cells (h) transfected with luciferase constructs containing p53 promoter, p53 3’UTR region or vector control were treated with 5 mM 2-HG for 36 hours. Renilla vector pGL3 Basic-CMV was used as a transfection internal control. The relative luciferase activity was normalized to the co-transfected Renila activity. (n = 3 biological independent samples). i and l, Western blot of MDA-MB-231 cells (i) and p53+/+ HCT116 cells (l) cultured in medium containing 0 or 8 mM 2-HG for 36 hours prior to 200 μg/mL CHX treatment for indicated time points. Relative p53/actin ratios are shown. j, Western blot of SF188 cells treated with 8 mM 2-HG, 8 mM α-KG or left untreated for 36 hours followed by 200 μg/mL CHX treatment for indicated time points. Relative p53/actin ratios are shown. k, Western blot of Flag-p53R248W-expressing HEK293 cells treated with 8 mM 2-HG, or left untreated for 36 hours followed by 100 μg/mL CHX treatment for indicated time points. Relative p53/actin ratios are shown. m, Western blot analysis of p53G266E expression in SF188 cells stably expressing Flag-IDH1, Flag-IDH1R132H or vector control. n, Western blot of SF188 cells cultured in standard medium or in acidic medium (pH6.0) for 24 hours followed by 200 μg/mL CHX treatment for indicated time points. Relative p53/actin ratios are shown. Data in f, g, h are means ± SD, P values were determined by unpaired two-tailed Student’s t-tests.

Source data

Extended Data Fig. 4 Regulation of 2-HG production by ME2 and effect of pyruvate on mutp53 stability in SF188 cells.

a-e, The structure analysis of 2-HG. (a) The scheme of 2-HG in LC-MS/MS. (b) Relative levels of 2-HG in SF188 cells transfected with Ctrl or ME2 siRNA for 48 hours were determined by Thermo Fisher UHPLC Q-Exactive MS. (n = 3 biological independent samples) (c) 2-HG standard MS1 Spectra by Thermo Fisher UHPLC Q-Exactive MS. (d-e) 2-HG MS1 Spectra by Thermo Fisher UHPLC Q-Exactive MS in SF188 cells treated with Ctrl (d) or ME2 siRNA (e) for 48 hours. f, NADPH and position-specific labelled pyruvate (pyruvate 13C-labelled at the C1, C2, C3, C2 and C3, or all-carbon positions) were incubated with purified ME2 or vector control proteins (Ctrl) for 60 min at 37°C as indicated. 2-HG production was determined by LC-MS analysis and the incorporation of 13C atoms into 2-HG are denoted as m + n, where n is the number of 13C atoms. The insert shows Coomassie blue-stained SDS-PAGE gel of ME2 protein. n = 3 biological independent samples. g, Relative p53G266E protein expression in SF188 cells treated with CHX in the presence or absence of 8 mM pyruvate as indicated. h, SF188 cells treated with control Ctrl or ME2 siRNA for 48 hours in the presence or absence of 8 mM pyruvate were collected after 4 hours of 10 μM MG132 treatment and subjected to immunoprecipitation using anti-p53 antibody. Immunoprecipitates and input were analyzed by western blot. i, Relative 2-HG levels in SF188 cells treated with Ctrl or ME2 siRNA, and/or 8 mM pyruvate as indicated for 48 hours. (n = 3 biological independent samples). j, SF188 cells transfected with Ctrl or ME2 siRNA were cultured in medium containing 8 mM pyruvate, 2-HG or α-KG as indicated for 48 hours. p53G266E expression was measured by western blot analysis. Data in b, f, i are means ± SD, P values were determined by unpaired two-tailed Student’s t-tests. All immunoprecipitation and western blot experiments were repeated three times.

Source data

Extended Data Fig. 5 Effect of 2-HG on Mdm2-mediated ubiquitination of mutant p53.

a-c, In vitro p53 ubiquitination assay was performed using purified HA-p53R282W (a), HA-p53R273H (b) and HA-p53 (c) incubated with purified Flag-Mdm2, E1, E2 and/or ubiquitin (Ub) in the presence or absence of 5 mM 2-HG for 60 min. Mixtures were immunoprecipitated with anti-HA agarose, and analyzed by western blot. d, Purified ME2 was incubated with pyruvate or NADPH for 60 min at room temperature (RT) before incubating with purified HA-p53G266E, Flag-Mdm2, E1, E2 and/or ubiquitin (Ub) for another 60 min. Mixtures were then immunoprecipitated with anti-HA agarose and analyzed by western blot. e, Lysates from SF188 cells transfected with Ctrl or ME2 siRNA for 48 hours were collected after 6 hours of 10 μM MG132 treatment, and immunoprecipated with anti-p53 antibody. Immunoprecipitates and input were analyzed by western blot using anti-p53 (HRP) and anti-Ub antibodies respectively. f, MDA-MB-231 cells transfected with Ctrl or ME2 siRNA for 48 hours in the presence or absence of 5 mM 2-HG were collected after 6 hours of 10 μM MG132 treatment and subjected to immunoprecipitation using anti-p53 antibody. Immunoprecipitates and input were analyzed by western blot using anti-p53 (HRP) and anti-Ub antibodies respectively. g and h, also related to Fig. 4e, Recombinant Flag-tagged p53 mutants (p53G266E, p53R175H, p53R280K and p53R282W) and p53 were analyzed on SDS-PAGE followed by coomassie staining of the gels. i and j, Surface plasmon resonance (BIAcore) measurement of the interaction between 2-HG and purified p53R175H (i), p53R282W(i), or p53(j). The Graphs of equilibrium response units (RU) and compound concentrations are shown. The estimated Kd of p53R175H and 2-HG interaction is about 37 nM, and around 66 nM for that of p53R282W and 2-HG interaction. All immunoprecipitation and western blot experiments were repeated independently three times.

Source data

Extended Data Fig. 6 2-HG binds to the C-terminus of mutant p53, and R306A mutation abolishes the binding of p53G266E with 2-HG.

a, Recombinant GST-tagged p53G266E and p53 were analyzed on SDS-PAGE followed by Coomassie staining of the gels. b, ITC binding assay of p53 with 2-HG, and data were fitted to a two sets of site binding models. c and d, ITC binding assay of p53G266E with α-KG (c) or citrate (d), and data were fitted to a two sets of site binding models. e and f, Direct binding of 2-HG to purified p53R175H (e), not p53 (f) was measured by DARTS assays. Purified proteins were incubated with the indicated doses of 2-HG respectively for 1 hour on ice and 0.5 hour at RT followed by pronase digestion. g-i, Direct binding of 2-HG to p53 mutants in p53-/- HCT116 cells stably expressing different p53 mutant proteins individually as indicated was measured by DARTS assays. Cells were incubated with the various doses of 2-HG for 1 hour on ice and 0.5 hour at RT, followed by pronase digestion (see Methods for details). j-n, Surface plasmon resonance (BIAcore) measurement of the interaction between 2-HG and purified truncated mutant p53 proteins as indicated. The Graphs of equilibrium response units (RU) and compound concentrations are shown (k-n). The estimated Kd are indicated. Recombinant Flag-tagged proteins were analyzed on SDS-PAGE followed by Coomassie staining of the gels (j). o-r, Surface plasmon resonance (BIAcore) measurement of the interaction between 2-HG and purified point-mutated p53G266E proteins as indicated. The Graphs of equilibrium response units (RU) and compound concentrations are shown (p-r). The estimated Kd are indicated. Recombinant Flag-tagged proteins were analyzed on SDS-PAGE followed by Coomassie staining of the gels (o). s, The predicted structure of p53 binding with 2-HG through AutoDock Vina. t, Intermolecular interactions between 2-HG and its binding pocket. The amino acid of p53 is from 130 to 315. Experiments in “a-r” were repeated in triple.

Source data

Extended Data Fig. 7 2-HG suppresses Mdm2-mediated p53G266E ubiquitination, and doesn’t impact on Mdm2 auto-ubiquitination.

a and b, HEK293 cells transfected with Flag-p53G266E (a) or Flag-p53R248W (b), together with HA-Ub and/or HA-Mdm2 in the presence or absence of 8 mM 2-HG were treated with MG132 for 6 hours. Cell lysates were prepared and then immunoprecipitated with anti-Flag antibody. Input and bound proteins were analyzed by western blot using indicated antibodies. c, HEK293 cells transfected with Flag-p53G266E, HA-Ub and/or HA-Mdm2 in the presence or absence of 8 mM α-KG were treated with MG132 for 6 hours. Cell lysates were prepared and then immunoprecipitated with anti-Flag antibody. Input and bound proteins were analyzed by western blot using indicated antibodies. d, HEK293 cells transfected with either Flag-p53 or Flag-p53G266E, together with HA-Ub and/or HA-Mdm2 in the presence or absence of 2-HG were treated with MG132 for 6 hours. Cell were collected and lyzed for immunoprecipitation using anti-Flag antibody. Input and bound proteins were analyzed by western blot using indicated antibodies. e, HEK293 cells transfected with HA-Ub and/or Flag-Mdm2 as indicated were treated with or without 5 mM 2-HG. After 6 hours of MG132 treatment, cells were lysed and subjected to immunoprecipitation assay using anti-Flag antibody. Immunoprecipitates and input were analyzed by western blot. f, Bio Layer Interferometry (BLI) analysis of 2-HG and Mdm2 interactions. Sensograms were fitted with a global two-site binding model. Soluble Flag-Mdm2 protein was immobilized to streptavidin-coated biosensors, and the binding of 2-HG and Mdm2 was assessed at the indicated concentrations (no binding was detected). The insert shows Coomassie blue-stained SDS-PAGE gel of Mdm2 protein. All the experiments were repeated in triple.

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Extended Data Fig. 8 2-HG disrupts interaction between Mdm2 and mutant p53, and correlation of ME2 expression with cellular 2-HG abundance and survival of cancer patients.

a-b and d-g, Purified Flag-Mdm2 proteins were incubated with HA-p53G266E (a, d, f) or HA-p53R175H (b, e, g) proteins bound on HA-beads in the presence of increasing amounts of 2-HG, α-KG or pyruvate for 1 hour. Immunoprecipitates were analyzed by western blot using indicated antibodies. c and h, Purified Flag-Mdm2 proteins were incubated with HA-tagged p53R248W (c) or p53 (h) proteins bound on HA-beads in the presence of increasing amounts of 2-HG for 1 hour. Immunoprecipitates were analyzed by western blot using indicated antibodies. i, Absolute amounts of 2-HG in SF188 cells transfected with Ctrl or ME2 siRNA. The dissociation constant Kd of p53G266E for 2-HG is indicated. j and k, Absolute amounts of 2-HG in MDA-MB-231 cells (j) and U251MG cells (k) transfected with Ctrl or ME2 siRNA for 48 hours. Western blot confirms ME2 knockdown efficiency and shows expression of p53. l, Western blot of SF188 cells treated with increasing amounts of Ocytl-2-HG for 24 hours. m, Expression of ME2 in normal brain tissues and gliomas of different stages was determined by immunohistochemistry (proteinatlas.org). Magnified regions of ME2 staining are provided. n, Kaplan–Meier survival curves of patients with glioma based on ME2 expression. Data were generated from French dataset via ‘R2: Genomics Analysis and Visualization Platform. In the cohort (n = 273), a z-score threshold of 2.0 was used to identify tumours with high levels of ME2 expression; this included 6.97% (n = 19) of tumours. o, Related to Fig. 5b. Kaplan-Meier survival curves of patients with liver cancer based on ME2 expression and IDH1/2 mutations. In this TCGA liver hepatocellular carcinoma cohort (TCGA LIHC, n = 371), IDH1/2 mutations were observed in 3% of patient tumors (n = 11), and TP53 mutations in 30% samples (n = 107). The top 1/4 and bottom 1/4 of samples were selected based on the expression levels of ME2 to test the correlation between ME2 levels and overall survival in patients with or without IDH1/2 alterations. Data in i, j, k are means ± S.D, unpaired two-tailed Student’s t-tests.

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Extended Data Fig. 9 2-HG supplementation restores anchorage-independent growth of p53 mutant tumour cells depletion of ME2 in soft agar.

a-c, p53-/- HCT116 cells stably expressing p53R273H (a), p53R175H (b) or p53R282W(c) were transfected with Ctrl, ME2 or p53 siRNA for 24 hours. Equal number of the cells were then cultured in soft agar medium for 2 weeks in the presence or absence of 8 mM 2-HG. Numbers of colonies with a diameter greater than 20 µm were quantified. In a, n = 6 (for each vehicle treatment) or 3 (for each 2-HG treatment) biological independent wells. In b and c, n = 3 biological independent wells. Protein expression was measured by western blot. Representative images of colonies stained with crystal violet at day 14 are shown. d, Colony formation assay of MDA-MB-231 cells treated with Ctrl, ME2 or p53 siRNA in the presence or absence of 8 mM 2-HG. Numbers of colonies with a diameter greater than 20 µm were quantified. n = 3 biological independent wells. Representative images of colonies stained with crystal violet at day 14. p53 expression was measured by western blot. e, Colony formation assay of HCT116 cells treated with control, ME2 or p53 siRNA in the presence or absence of 8 mM 2-HG. Numbers of colonies with a diameter greater than 20 µm were quantified. n = 4 biological independent wells. Representative images of colonies stained with crystal violet at day 14. p53 expression was measured by western blot. Data in a-e are means ± SD, P values were determined by unpaired two-tailed Student’s t-tests.

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Extended Data Fig. 10 Effect of 2-HG on ME2-mediated tumour growth, and TP53 mutation correlates with ME2 mRNA expression, and mutations of D2HGDH, L2HGDH and IDH1.

a, Schematic summarizing the dual characteristics of ME2 enzymatic functions, and a role for 2-HG in preventing mutant p53 degradation by Mdm2. Whether ME2 produces pyruvate or 2-HG may dependent on cellular demands of these metabolites, and viability of sufficient NADPH. b, Results from fisher’s exact test showed the group with higher expression level of ME2 was significantly higher TP53 mutation frequency. Please see Methods for details. c, Correlation analysis of mutant p53 and ME2 expression in in human cancer patients from The Cancer Genome Atlas (TCGA) of pan cancer. The color shades of the circles represent the expression of mutant p53 and the blue line represents the expression of ME2. d-f, Correlation of TP53 mutation frequency and mutation status of IDH1(d), D2HGDH (e) or L2HGDH (f) in human cancer patients from The Cancer Genome Atlas (TCGA) of pan cancer was analyzed via cBioPortal for Cancer Genomics. Data in b-f are means ± SD. P values were determined by Fisher’s exact test.

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Zhao, M., Yao, P., Mao, Y. et al. Malic enzyme 2 maintains protein stability of mutant p53 through 2-hydroxyglutarate. Nat Metab 4, 225–238 (2022). https://doi.org/10.1038/s42255-022-00532-w

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