Abstract
Glutathione peroxidase 4 (GPX4), as the only enzyme in mammals capable of reducing esterified phospholipid hydroperoxides within a cellular context, protects cells from ferroptosis. We identified a homozygous point mutation in the GPX4 gene, resulting in an R152H coding mutation, in three patients with Sedaghatian-type spondylometaphyseal dysplasia. Using structure-based analyses and cell models, including patient fibroblasts, of this variant, we found that the missense variant destabilized a critical loop, which disrupted the active site and caused a substantial loss of enzymatic function. We also found that the R152H variant of GPX4 is less susceptible to degradation, revealing the degradation mechanism of the GPX4 protein. Proof-of-concept therapeutic treatments, which overcome the impaired R152H GPX4 activity, including selenium supplementation, selective antioxidants and a deuterated polyunsaturated fatty acid were identified. In addition to revealing a general approach to investigating rare genetic diseases, we demonstrate the biochemical foundations of therapeutic strategies targeting GPX4.
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Data availability
Crystal structural coordinates were deposited in the RCSB, with accession codes PDB IDs: 7L8K, 7L8L, 7L8M, 7L8R, and 7L8Q. Publicly available datasets used in this study are: PDB IDs: 2OBI, 6HN3. Source data are provided with this paper.
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Acknowledgements
This study was supported by P01CA87497 (B.R.S.), R35CA209896 (B.R.S.) and R61NS109407 (B.R.S.), and the BMBF VIP+ program NEUROPROTEKT (03VP04260), the Ministry of Science and Higher Education of the Russian Federation (075-15-2019-1933) and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. GA 884754) (M.C.). We thank Q. Ran for calling to attention that three patients reported in this study shared the same homozygous variant. We are grateful to the patient with the R152H variant and the patient’s parent for providing their fibroblasts for this study. We thank the staff of the High-Throughput Crystallization Screening Center of the Hauptman-Woodward Medical Research Institute for screening of crystallization conditions and the staff of the Advanced Photon Source at Argonne National Laboratory for assistance with data collection. We also thank curegpx4.org for supporting the study and patients with GPX4 variants, and the roadmap effort for GPX4 disorders46.
Author information
Authors and Affiliations
Contributions
B.R.S. conceived and implemented the project after discussions with R.S. The planning and design of experiments was performed by H.L., T.S., R.S., M.C. and B.R.S. Computational modeling was conducted by H.L., as were biophysical assays, biochemical assays and cellular experiments. H.L. and X.X. conducted protein purification. F.F. crystallized the proteins and collected diffraction data to solve the crystal structures. T.S. and M.C. prepared GPX4-knockout Pfa1 and HT1080 cells. R.S., K.W. and J.F. conducted clinical observations of the patients. M.S.S. provided deuterium-reinforced linoleic acid. H.L. and B.R.S. wrote the manuscript with input from all authors.
Corresponding author
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Competing interests
B.R.S. is an inventor on patents and patent applications related to GPX4 and ferroptosis, and is a consultant to and cofounder of Inzen Therapeutics and Nevrox Limited, and is a member of the Scientific Advisory Board of Weatherwax Biotechnologies Corporation, and a consultant to Akin Gump Strauss Hauer & Feld LLP. M.C. is an inventor of ferroptosis-related patents and cofounder and shareholder of ROSCUE Therapeutics GmbH. J.F. participates in clinical trials with Biogen (Angelman’s syndrome) and J.F.’s spouse is Founder and Principal of Friedman Bioventure, which holds a variety of publicly traded and private biotechnology interests. M.S.S. is the Chief Scientific Officer of Retrotrope, Inc.
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Peer review information Nature Chemical Biology thanks Dohoon Kim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 In silico analysis of the impact of R152H mutation on GPX4 (GPX4U46C PDB: 2OBI) structure.
a, Structure of GPX4R152H was computationally modeled based on the crystal structure of GPX4U46C (PDB: 2OBI). As a control, structure of GPX4R152R was also computationally modeled using the same algorithm, which indeed represents WT GPX4 but excludes artifacts from computational process when compared with the modeled R152H protein structure. Technically the two modeled proteins are GPX4U46C-R152H and GPX4U46C. Protein surface are colored as below: hydrophobic (white), positive charge (red), and negative charge (blue). To visualize the pocket on top of R152 in the GPX4R152R structure, white dots were shown as indicator of space. Overlap of the R152H variant backbone with wild-type was performed in the panel on the right, where the major conformational change in the loop around His152 was colored (WT as in pale pink and R152H as in red). See Supplementary Note for rationale of using U46C GPX4. b, The alternation of surface mainly derived from an outstanding conformational change of the loop between Pro124 and Ala133, with which the side chains of Arg152 formed multiple hydrogen bonds in the wild-type, but not His152 in the mutant.
Extended Data Fig. 2 Molecular Dynamic (MD) simulation analysis of the impact of R152H mutation on GPX4.
a, RMSF of each residue in MD simulation based on the modeled GPX4 structure. Representative data from 5 times 100 ns trajectories were plotted. b, Distances between Cys46 and its catalytic partners Gln81/Trp136 were monitored in the MD simulation of GPX4R152H, as compared to GPX4R152R. Representative data from 5 times 100 ns trajectories were plotted.
Extended Data Fig. 3 Preparation of cell models of GPX4R152H.
a, HT-1080 transfected with pBP GFP-cGPX4WT, pBP GFP-cGPX4R152H, pBabepuro (pBP) empty vector, pBP GFP-cGPX4K48A, pBP GFP-cGPX4K48L and pBP GFP-cGPX4K125R-K127R were selected with puromycin and imaged with microscope. Triplicate experiments were repeated independently with similar results while the representative images were shown. The plotted scale bar is 400 µm. b, Total GPX4 enzymatic activity (endogenous apo-GPX4 and transfected exogenous GFP-tagged-GPX4) of control HT1080 (pBP, no expression of GFP-tagged-GPX4) and HT1080 cells overexpressing GFP-GPX4WT or GFP-GPX4R152H. Data are plotted as means ± SD of six replicate experiments. Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test was performed: n = 6, DF = 15 and P values were plotted. c, Western blot of control HT1080 (pBP) and HT1080 cells overexpressing GFP-GPX4WT or GFP-GPX4R152H using GPX4 and GAPDH antibodies. Expression levels of GFP-WT-GPX4 and GFP-R152H-GPX4 were quantified. Data are plotted as means ± SD, n = 4 biologically independent samples. Unpaired two-tailed t test was then performed and plotted: t = 0.3158, df=6, Pns = 0.7629. d, Western blot of Gpx4-knockout Pfa1 cells overexpressing exogenous human WT or R152H GPX4, Gpx4-knockout Pfa1 cells overexpressing exogenous murine WT or R152H mScarlet-tagged GPX4, and Gpx4-knockout HT1080 cells overexpressing exogenous murine WT or R152H mScarlet-tagged GPX4 using GPX4 and GAPDH antibodies. Expression levels of GPX4 were quantified. Data are plotted as means ± SD, n = 3 biologically independent samples. Ordinary two-way ANOVA followed by Sidak’s multiple comparisons test was performed and P values were plotted: n = 3, DF = 12. Full scan image is shown in the Supplementary Information.
Extended Data Fig. 4 Characterization of GPX4R152H in cells and in vitro.
a, HT1080 overexpressing exogenous WT or R152H GFP-GPX4 and a control line were tested for RSL3, ML162, and IKE sensitivity. Data are plotted as means ± SD, n = 3 biologically independent samples. b-c, SDS-PAGE gel of His-tagged GPX4U46C and GPX4U46C_R152H as stained by Coomasie Blue. Biologically independent duplicate experiments were performed and imaged. d-e, Distances between the catalytic triad in R152H variant were measured and labeled as compared to GPX4U46C (PDB:2OBI). f, Shift of Lys48 away from the active site in the GPX4R152H was plotted.
Extended Data Fig. 5 Characterization of Lysine 48 mutants of GPX4 in cells and in vitro.
a, Total GPX4 activity of HT1080 cells overexpressing GFP-GPX4WT, GFP-GPX4K48A, or GFP-GPX4K48L and a control line. Data are plotted as means ± SD of eleven biologically independent replicate experiments. Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test was performed and P values were plotted, n = 11, DF = 40. b, HT1080 overexpressing exogenous WT, K48A, or K48L GFP-GPX4 and a control line were tested for RSL3 sensitivity. Data are plotted as means ± SD of three biologically independent replicate experiments. c-d, SDS-PAGE gel of His-tagged GPX4U46C_K48A and GPX4U46C_K48L as stained by Coomasie Blue. Biologically independent duplicate (GPX4U46C_K48A) and quadruplicate (GPX4U46C_K48L) experiments were performed and imaged. e, The distances between catalytic residues Sec46 and Trp136 were recorded every 4.8 ps throughout MD simulations of GPX4WT (PDB: 6HN3), GPX4K48A, GPX4K48L, GPX4K48E, GPX4K48Q, and GPX4K48R. Representative data from three times 100 ns trajectories were plotted as means ± SD. Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test was performed: n = 20835, DF = 125004, all P**** < 1x10-20. f, Scheme illustrating the catalytic cycle of sulfur-containing variant of GPX4.
Extended Data Fig. 6 Characterization of Lysine 48 mutants of GPX4 in silico.
a, In silico docking of GSH to GPX4U46C, GPX4K48A, or GPX4K48L. Top covalent-docking pose of GSH on GPX4U46C (top left). 2D Ligand interaction diagram of GSH with GPX4U46C, GPX4K48A, or GPX4K48L in their individually top covalent-docking pose (top right and bottom panels). b, HT1080 overexpressing exogenous WT, K48A, or K48L GFP-GPX4 and a control line were tested for IKE sensitivity. Data are plotted as means ± SD of three biologically independent replicate experiments.
Extended Data Fig. 7 Investigation of GPX4 degradation mechanism after treatment with ferroptosis inducers.
a, HT1080 overexpressing exogenous WT or R152H GFP-GPX4 and a control line were tested for FIN56 sensitivity. Data are plotted as means ± SD, n = 3 biologically independent samples. b, Western blot of HT1080 OE GFP-GPX4WT and HT1080 OE GFP-GPX4R152H after treatment with ferroptosis inducers with GPX4 and GAPDH antibodies, with lanes arranged for cell line comparison. Triplicate experiments were repeated independently with similar results, which were shown in Extended Data Fig. 7c,e,f. c, Western blot of HT1080 OE GFP-GPX4WT and HT1080 OE GFP-GPX4R152H after treatment with ferroptosis inducers with GPX4 and GAPDH antibodies, with lanes arranged for ferroptosis inducer comparison. Triplicate experiments were repeated independently with similar results, which were shown in Extended Data Fig. 7b,e,f. d, The endogenous GPX4 in HT1080 OE GFP-R152H-GPX4 were tested for vulnerability to the degradation induced by RSL3, ML162, FIN56, and IKE. Data are plotted as means with range of two biologically independent experiments. The corresponding blots are shown in Extended Data Fig. 7b,c. e, Western blot of HT1080 OE GFP-GPX4WT after treatment with ferroptosis inducers with GPX4 and GAPDH antibodies. Triplicate experiments were repeated independently with similar results, which were shown in Extended Data Fig. 7b,c. f, Western blot of HT1080 OE GFP-GPX4R152H after treatment with ferroptosis inducers using GPX4 and GAPDH antibodies. Triplicate experiments were repeated independently with similar results, which were shown in Extended Data Fig. 7b,c.
Extended Data Fig. 8 Kinetic and mutagenesis study of GPX4 degradation mechanism after treatment with ferroptosis inducers.
a, HT1080 OE GFP-GPX4WT and HT1080 OE GFP-GPX4R152H were treated with 4 µM RSL3, 30 µg/ml cycloheximide, and 100 µM a-Tocopherol for 0, 2, 4, or 6 hours before Western Blot analysis of GPX4 and GADPH. Biologically independent duplicate experiments were performed and imaged. b, Western blot of HT1080 OE GPX4K125R_K127R after treatments with ferroptosis inducers using GPX4 and GAPDH antibodies. Duplicate experiments were performed and imaged. c, The endogenous GPX4 in HT1080 OE GFP-K125R-K127R-GPX4 were tested for vulnerability to the degradation induced by RSL3, ML162, FIN56, and IKE. Data are plotted as means with range of two biologically independent experiments. The corresponding blots are shown in Extended Data Fig. 8b.
Extended Data Fig. 9 Proof-of-concept treatments were tested on patient fibroblasts and Pfa1 cells, which were knocked out of endogenous GPX4 and transfected to overexpress human mScarlet-tagged GPX4WT (red) or GPX4R152H (blue).
a-b, Supplementations of methyl-seleno-cysteine and N-acetyl-cysteine were tested as proof-of-concept treatments on the patient and control fibroblasts. Data are plotted as means ± SD (n = 3 biologically independent samples). c, Viability was normalized to the corresponding DMSO control. Data are plotted as means ± SD (n = 3 biologically independent samples). See Supplementary Note for effects of α-tocopherol.
Extended Data Fig. 10 Proof-of-concept treatments were validated in Pfa1 cells, which were knocked out of endogenous GPX4 and transfected to overexpress murine mScarlet-tagged GPX4WT (red) or GPX4R152H (blue).
Viability was normalized to the corresponding DMSO control. Data are plotted as means ± SD (n = 3 biologically independent samples). See Supplementary Note for effects of α-tocopherol.
Supplementary information
Supplementary Information
Supplementary Tables 1–4 and Note.
41589_2021_915_MOESM3_ESM.mpeg
Supplementary Video 1 Representative movie for 100 ns MD simulation of WT GPX4. Structure of WT GPX4 is based on PDB structure 6HN3. R152 and active site residues C46, K48, Q81, and W136 are highlighted and shown as sticks.
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Supplementary Video 2 Representative movie for 100 ns MD simulation of GPX4R152H. Structure of R152H GPX4 is modeled based on PDB structure 6HN3. H152 and active site residues C46, K48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM5_ESM.mpeg
Supplementary Video 3 Representative movie for 100 ns MD simulation of GPX4U46C-R152R. Structure of GPX4U46C-R152R is modeled based on PDB structure 2OBI. Mutation site residue R152 and active site residues C46, Q81, and W136 are highlighted and shown as white sticks.
41589_2021_915_MOESM6_ESM.mpeg
Supplementary Video 4 Representative movie for 100 ns MD simulation of GPX4U46C-R152H. Structure of GPX4U46C-R152H is modeled based on PDB structure 2OBI. Mutation site residue H152 and active site residues C46, Q81, and W136 are highlighted and shown as green (H152) or white sticks.
41589_2021_915_MOESM7_ESM.mpeg
Supplementary Video 5 Representative movie for 100 ns water-free MD simulation of WT GPX4. Structure of WT GPX4 is based on PDB structure 6HN3. R152 and active site residues C46, K48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM8_ESM.mpeg
Supplementary Video 6 Representative movie for 100 ns water-free MD simulation of GPX4R152H. Structure of R152H GPX4 is modeled based on PDB structure 6HN3. H152 and active site residues C46, K48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM9_ESM.mpeg
Supplementary Video 7 Representative movie for 100 ns MD simulation of GPX4U46C with the camera zoomed in for a close-up of the active site. Structure of GPX4U46C is based on crystal structure reported in this work. Active site residues C46, K48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM10_ESM.mpeg
Supplementary Video 8 Representative movie for 100 ns MD simulation of GPX4U46C-K48A with the camera zoomed in for a close-up of the active site. Structure of GPX4U46C-K48A is based on crystal structure reported in this work. Active site residues C46, A48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM11_ESM.mpeg
Supplementary Video 9 Representative movie for 100 ns MD simulation of GPX4U46C-K48L with the camera zoomed in for a close-up of the active site. Structure of GPX4U46C-K48L is based on crystal structure reported in this work. Active site residues C46, L48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM12_ESM.mpeg
Supplementary Video 10 Representative movie for 100 ns MD simulation of WT GPX4 with the camera zoomed in for a close-up of the active site. Structure of WT GPX4 is based on PDB structure 6HN3. Active site residues U46, K48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM13_ESM.mpeg
Supplementary Video 11 Representative movie for 100 ns MD simulation of GPX4K48A with the camera zoomed in for a close-up of the active site. Structure of GPX4K48A is modeled based on PDB structure 6HN3. Active site residues U46, A48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM14_ESM.mpeg
Supplementary Video 12 Representative movie for 100 ns MD simulation of GPX4K48L with the camera zoomed in for a close-up of the active site. Structure of GPX4K48L is modeled based on PDB structure 6HN3. Active site residues U46, L48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM15_ESM.mpeg
Supplementary Video 13 Representative movie for 100 ns MD simulation of oxidized GPX4U46C (C46-SOOO-) with the camera zoomed in for a close-up of the active site. Structure of oxidized GPX4U46C is based on crystal structure reported in this work. Active site residues C46 (oxidized), K48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM16_ESM.mpeg
Supplementary Video 14 Representative movie for 100 ns MD simulation of oxidized GPX4U46C-K48A (C46-SOOO-) with the camera zoomed in for a close-up of the active site. Structure of oxidized GPX4U46C-K48A is modeled based on oxidized GPX4U46C-K48A crystal structure reported in this work. Active site residues C46 (oxidized), A48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM17_ESM.mpeg
Supplementary Video 15 Representative movie for 100 ns MD simulation of oxidized GPX4U46C-K48L (C46-SOOO-) with the camera zoomed in for a close-up of the active site. Structure of oxidized GPX4U46C-K48L is modeled based on oxidized GPX4U46C-K48L crystal structure reported in this work. Active site residues C46 (oxidized), L48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM18_ESM.mpeg
Supplementary Video 16 Representative movie for 100 ns MD simulation of GPX4K48E with the camera zoomed in for a close-up of the active site. Structure of GPX4K48E is modeled based on PDB structure 6HN3. Active site residues U46, E48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM19_ESM.mpeg
Supplementary Video 17 Representative movie for 100 ns MD simulation of GPX4K48Q with the camera zoomed in for a close-up of the active site. Structure of GPX4K48Q is modeled based on PDB structure 6HN3. Active site residues U46, Q48, Q81, and W136 are highlighted and shown as sticks.
41589_2021_915_MOESM20_ESM.mpeg
Supplementary Video 18 Representative movie for 100 ns MD simulation of GPX4K48R with the camera zoomed in for a close-up of the active site. Structure of GPX4K48R is modeled based on PDB structure 6HN3. Active site residues U46, R48, Q81, and W136 are highlighted and shown as sticks.
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Liu, H., Forouhar, F., Seibt, T. et al. Characterization of a patient-derived variant of GPX4 for precision therapy. Nat Chem Biol 18, 91–100 (2022). https://doi.org/10.1038/s41589-021-00915-2
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DOI: https://doi.org/10.1038/s41589-021-00915-2
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