{"gene":"GPX4","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":2014,"finding":"GPX4 is directly inhibited by a class of ferroptosis-inducing compounds (e.g., RSL3) identified via chemoproteomics; GPX4 overexpression suppresses and knockdown promotes ferroptotic cell death induced by 12 different ferroptosis inducers, establishing GPX4 as an essential regulator of ferroptosis.","method":"Chemoproteomics (direct target identification), GPX4 overexpression/knockdown with cell viability readouts, targeted metabolomic profiling","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — chemoproteomics-based direct target identification combined with genetic gain- and loss-of-function, replicated across multiple compounds and cell lines","pmids":["24439385"],"is_preprint":false},{"year":2019,"finding":"GPX4 functions as the glutathione-dependent lipid hydroperoxidase that converts lipid hydroperoxides into non-toxic lipid alcohols, thereby suppressing ferroptosis; FSP1 acts in a parallel non-mitochondrial CoQ antioxidant pathway independent of GPX4.","method":"Synthetic lethal CRISPR-Cas9 screen, biochemical oxidoreductase assays, xenograft tumor models","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — CRISPR screen with biochemical validation, multiple orthogonal methods, replicated in vivo","pmids":["31634900"],"is_preprint":false},{"year":2017,"finding":"Selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation; cells expressing a cysteine variant of GPX4 (Sec→Cys) are highly sensitive to peroxide-induced ferroptosis, demonstrating that selenolate-based catalysis is specifically required for neuronal survival and prevention of fatal epileptic seizures in vivo.","method":"Knock-in mouse models (Gpx4cys/cys), in vitro peroxide sensitivity assays, neuronal cell-specific conditional knockouts","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — genetic reconstitution in mouse and cell models with multiple orthogonal functional readouts, mechanistic mutagenesis of active-site residue","pmids":["29290465"],"is_preprint":false},{"year":2017,"finding":"Drug-tolerant persister cancer cells in a high mesenchymal therapy-resistant state acquire dependency on GPX4 for survival; loss of GPX4 function causes selective ferroptotic death of persister cells in vitro and prevents tumor relapse in mice.","method":"GPX4 genetic knockdown/inhibition, ferroptosis cell death assays, in vivo mouse tumor relapse models","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined cellular ferroptosis phenotype confirmed in vivo, replicated across multiple cancer types","pmids":["29088702"],"is_preprint":false},{"year":2020,"finding":"GPX4 deficiency enhances cellular lipid peroxidation, which leads to carbonylation of STING at C88 and inhibits STING trafficking from the ER to the Golgi complex, thereby specifically attenuating the cGAS-STING innate immune pathway; GPX4-maintained redox homeostasis is required for STING activation.","method":"GPX4 knockdown/knockout, carbonylation assays, subcellular fractionation and trafficking assays (ER-to-Golgi), viral infection models in vivo","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic KO with biochemical mechanism (carbonylation at specific residue, trafficking defect), in vivo validation","pmids":["32541831"],"is_preprint":false},{"year":2001,"finding":"PHGPx (GPX4) acts as a protein thiol peroxidase in sperm, using its selenocysteine to oxidize specific capsule protein thiols via a peroxide-dependent mechanism, leading to cross-linking of mitochondrial capsule proteins and accounting for the selenium dependency of spermatogenesis.","method":"In vitro biochemical assays with H2O2, monobromobimane labeling, SDS-PAGE, mercaptoethanol release of active enzyme","journal":"BioFactors (Oxford, England)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with defined substrates and stoichiometry, mechanistic characterization of catalytic cross-linking activity","pmids":["11568459"],"is_preprint":false},{"year":2021,"finding":"GPX4 is the only enzyme capable of reducing esterified phospholipid hydroperoxides within a cellular context; a patient-derived R152H mutation destabilizes a critical loop adjacent to the active site, causing substantial loss of enzymatic function while paradoxically reducing GPX4 susceptibility to degradation.","method":"Structure-based analysis, cell models including patient fibroblasts, enzymatic activity assays, protein stability assays","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structure-based mechanistic characterization combined with cell-based functional validation and patient-derived fibroblasts","pmids":["34931062"],"is_preprint":false},{"year":2022,"finding":"An allosteric site on GPX4 (distinct from the active site) is involved in native regeneration of GPX4 under low glutathione conditions; covalent binding of inhibitors to this site causes conformational change, inhibition of activity, and subsequent cellular GPX4 protein degradation. Co-crystal structures of six inhibitors bound in this site were determined.","method":"Co-crystal structure determination (X-ray), biophysical binding assays, enzymatic activity assays, compound library screen","journal":"Cell chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple co-crystal structures with functional validation of allosteric mechanism, single lab but multiple orthogonal methods","pmids":["36423641"],"is_preprint":false},{"year":2021,"finding":"NMR characterization of GPX4 reveals an electrostatic (cationic patch) mechanism for membrane binding; mutagenesis identifies specific cationic patch residues required for membrane and cardiolipin headgroup binding, and a novel lipid binding site adjacent to the catalytic site. The cationic patch also mediates DNA binding, explaining the nuclear isoform's ability to target DNA-bound protamines.","method":"NMR spectroscopy (first NMR assignments of GPX4), site-directed mutagenesis, lipid/DNA binding assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with mutagenesis validation, multiple binding partners characterized, single lab","pmids":["34492183"],"is_preprint":false},{"year":2003,"finding":"Testis-specific nuclear form of GPX4 (PHGPx/snGPx) is generated by transcription initiation from an alternative promoter (not by alternative splicing) located in intron 1, producing an arginine-rich N-terminus responsible for nuclear localization and chromatin binding, with expression restricted to late stages of spermatogenesis.","method":"5'-RACE, RT-PCR, S1 nuclease analysis, in situ hybridization","journal":"Biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple complementary molecular methods establishing alternative promoter usage and isoform-specific localization","pmids":["12751792"],"is_preprint":false},{"year":2023,"finding":"Copper promotes GPX4 autophagic degradation and ferroptosis by directly binding to GPX4 cysteines C107 and C148, inducing GPX4 ubiquitination and aggregation; TAX1BP1 then acts as the autophagic receptor mediating GPX4 degradation.","method":"Co-immunoprecipitation, ubiquitination assays, autophagic flux assays, copper chelation experiments, mouse pancreatic cancer models","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP identifying specific cysteine binding sites and autophagic receptor, validated in vivo, single lab","pmids":["36622894"],"is_preprint":false},{"year":2023,"finding":"Creatine kinase B (CKB), when phosphorylated at T133 by AKT downstream of IGF1R signaling, acts as a non-canonical protein kinase to phosphorylate GPX4 at S104; this phosphorylation prevents HSC70 binding to GPX4 and blocks chaperone-mediated autophagy (CMA)-dependent GPX4 degradation, thereby suppressing ferroptosis.","method":"In vitro kinase assay, co-immunoprecipitation, CMA degradation assays, mutagenesis, mouse tumor models, human HCC specimen correlation","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay establishing CKB as protein kinase, combined with Co-IP, mutagenesis, and in vivo validation","pmids":["37156912"],"is_preprint":false},{"year":2021,"finding":"Legumain facilitates chaperone-mediated autophagy (CMA) of GPX4 in acute kidney injury: legumain directly interacts with HSC70, HSP90, and GPX4 (shown by immunoprecipitation), and legumain deficiency prevents CMA-dependent GPX4 degradation and tubular ferroptosis.","method":"Co-immunoprecipitation, lysosomal fractionation, proteasome/autophagy inhibitor experiments, legumain KO mice with IRI model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP identifying protein complex, genetic KO with defined ferroptosis phenotype, in vivo model, single lab","pmids":["33431801"],"is_preprint":false},{"year":2022,"finding":"CST1 interacts with GPX4 (confirmed by Co-IP and mass spectrometry) and recruits the deubiquitinase OTUB1 to relieve GPX4 ubiquitination, thereby stabilizing GPX4 protein and inhibiting ferroptosis.","method":"Co-immunoprecipitation combined with mass spectrometry, ubiquitination assays, siRNA knockdown, in vivo mouse metastasis models","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS identifying the complex and ubiquitination readout, validated in vivo, single lab","pmids":["36369321"],"is_preprint":false},{"year":2022,"finding":"LUBAC (via its catalytic subunit HOIP) binds GPX4 and stabilizes it through linear (M1-linked) ubiquitination both under basal conditions and oxidative stress; LUBAC deficiency promotes GPX4 degradation and sensitizes cells to ferroptosis.","method":"Co-immunoprecipitation, linear ubiquitination assays, LUBAC genetic deficiency models, lipid peroxidation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with ubiquitination linkage-type characterization, genetic deficiency model, single lab","pmids":["36279464"],"is_preprint":false},{"year":2023,"finding":"USP8 (ubiquitin-specific protease 8) directly interacts with and deubiquitinates GPX4, leading to GPX4 protein stabilization; USP8 inhibition destabilizes GPX4 and sensitizes cancer cells to ferroptosis.","method":"Co-immunoprecipitation, deubiquitination assays, intestinal epithelial cell-specific Usp8 knockout mice, in vivo tumor models","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with deubiquitination biochemical assay, conditional KO mice, single lab","pmids":["38598341"],"is_preprint":false},{"year":2023,"finding":"OTUD5 acts as a deubiquitinase that binds GPX4, stabilizes it, and confers ferroptosis resistance; during ischemia-reperfusion, mTORC1-mediated autophagy degrades OTUD5, causing GPX4 decay and subsequent ferroptosis in renal tubular cells.","method":"Co-immunoprecipitation, ubiquitination assays, spatial transcriptomics, AAV-mediated OTUD5 delivery, mouse IRI model","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP establishing GPX4-OTUD5 interaction and deubiquitination, in vivo rescue experiment, single lab","pmids":["38110369"],"is_preprint":false},{"year":2023,"finding":"TRIM26 directly interacts with GPX4 via its RING domain and catalyzes K63-linked ubiquitination of GPX4 at K107 and K117, switching polyubiquitination from K48 to K63 linkage and thereby enhancing GPX4 protein stability; PLK1-mediated S127 phosphorylation of TRIM26 enhances this interaction.","method":"Co-immunoprecipitation, ubiquitin linkage-specific assays (K48 vs K63), mutagenesis of ubiquitination sites, TRIM26 knockdown in glioma","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with ubiquitin linkage characterization and site mutagenesis, single lab","pmids":["37872147"],"is_preprint":false},{"year":2025,"finding":"GPX4 is S-palmitoylated on cysteine 66 by the acyltransferase ZDHHC20, which increases GPX4 protein stability; APT2 acts as the depalmitoylase of GPX4. Disrupting GPX4 palmitoylation sensitizes cancer cells to ferroptosis in vitro and in vivo.","method":"Palmitoylation assays (acyl-RAC), mutagenesis (C66), ZDHHC20/APT2 knockdown/inhibition, xenograft and orthotopic tumor models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — site-specific PTM identification with mutagenesis, writer/eraser identified biochemically, validated in multiple in vivo models","pmids":["39833225"],"is_preprint":false},{"year":2025,"finding":"ZDHHC8 palmitoylates GPX4 at Cys75, stabilizing GPX4 and suppressing ferroptosis; pharmacological inhibition of ZDHHC8 with PF-670462 promotes ZDHHC8 degradation, reduces GPX4 palmitoylation, and enhances ferroptosis sensitivity in tumor cells.","method":"Palmitoylation assays, mutagenesis (C75), small-molecule drug screening, xenograft tumor models, CD8+ T cell co-culture assays","journal":"Nature cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific palmitoylation with mutagenesis and pharmacological validation, single lab","pmids":["40108413"],"is_preprint":false},{"year":2025,"finding":"PRMT5 catalyzes symmetric dimethylation of GPX4 at arginine 152 (R152), which prevents Cullin1-FBW7 E3 ligase binding to GPX4 and blocks ubiquitination-mediated GPX4 degradation, thereby prolonging GPX4 half-life and suppressing ferroptosis.","method":"In vitro methylation assays, Co-immunoprecipitation, ubiquitination assays, PRMT5 inhibition, mutagenesis (R152), mouse tumor models","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro methylation assay establishing writer, Co-IP identifying E3 ligase exclusion mechanism, mutagenesis, in vivo validation","pmids":["40033101"],"is_preprint":false},{"year":2023,"finding":"NEDD4L directly interacts with GPX4 (confirmed by Co-IP) and promotes GPX4 ubiquitination and degradation, facilitating granulosa cell ferroptosis and contributing to PCOS pathology.","method":"Co-immunoprecipitation, ubiquitination assays, CCK-8 cell viability, ferroptosis marker assays","journal":"Endocrine connections","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with ubiquitination assay, single lab, no in vivo validation","pmids":["36662677"],"is_preprint":false},{"year":2023,"finding":"STUB1 is identified as an E3 ubiquitin ligase of GPX4, promoting GPX4 ubiquitination at site K191 and its degradation, thereby inducing ferroptosis in gastrointestinal stromal tumors.","method":"Co-immunoprecipitation, ubiquitination assays with site mutagenesis (K191), STUB1 knockdown and GPX4 overexpression, in vivo tumor models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination site mutagenesis with genetic rescue experiments, in vivo validation, single lab","pmids":["38110356"],"is_preprint":false},{"year":2024,"finding":"OTUD5 deubiquitylates and stabilizes GPX4; p53 suppresses OTUD5 transcription, thereby promoting GPX4 ubiquitination and degradation and inducing ferroptosis in gastric cancer cells.","method":"Co-immunoprecipitation, deubiquitination assay, CRISPR-Cas9 Otud5 KO, luciferase reporter for p53-OTUD5 transcription, mouse tumor models","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with biochemical deubiquitination, genetic KO in vivo, transcriptional regulation validated, single lab","pmids":["40070026"],"is_preprint":false},{"year":2026,"finding":"FUNDC1 interacts with GPX4 via its 96–133 amino acid domain (shown by Co-IP), facilitating GPX4 recruitment from cytoplasm into mitochondria via the TOM/TIM import complex; within mitochondria, GPX4 is degraded by mitophagy, triggering hepatocyte ferroptosis.","method":"Co-immunoprecipitation (domain mapping), immunofluorescence colocalization, FUNDC1 knockout mouse (CCl4 model), mitochondrial fractionation","journal":"Journal of advanced research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-mapped Co-IP with subcellular fractionation and genetic KO in vivo, single lab","pmids":["36828120"],"is_preprint":false},{"year":2023,"finding":"STAT3 binds to the GPX4 promoter region and promotes its transcription (confirmed by ChIP and dual-luciferase reporter assay); thiostrepton blocks GPX4 expression by inhibiting STAT3, thereby inducing ferroptosis in pancreatic cancer cells.","method":"Chromatin immunoprecipitation (ChIP), dual-luciferase reporter assay, STAT3 inhibition, in vivo xenograft","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase assays directly establishing STAT3-GPX4 promoter interaction, in vivo validation, single lab","pmids":["35859150"],"is_preprint":false},{"year":2024,"finding":"Itaconate (produced by Irg1/aconitate decarboxylase 1) directly alkylates GPX4 at cysteine 66, allosterically enhancing GPX4 enzymatic activity, thereby protecting neurons from ferroptosis after intracerebral hemorrhage.","method":"Bioorthogonal itaconate-alkyne (ITalk) probe, GPX4 activity assay using phosphatidylcholine hydroperoxide, Irg1 transgenic/KO mouse models, in vitro cortical neuron ferroptosis assays","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1 / Moderate — bioorthogonal probe-based direct covalent modification identification with enzymatic activity assay, genetic mouse model validation, mechanistically linked to C66 of GPX4","pmids":["38719928"],"is_preprint":false},{"year":1997,"finding":"PHGPx (GPX4) shares an active site catalytic triad (selenocysteine, glutamine, tryptophan) with tetrameric GPXs; its unique ability to accommodate large lipophilic substrates (phospholipid hydroperoxides) is defined by structural and kinetic data. PHGPx also exhibits broad donor-substrate specificity (not restricted to GSH) and binds specific mitochondrial proteins via protein-protein interaction, suggesting a role in oxidizing specific protein thiols.","method":"Structural analysis, kinetic assays, protein-protein interaction (protein binding to mitochondrial proteins released by ionic strength and thiols)","journal":"Biomedical and environmental sciences : BES","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — structural and kinetic characterization of catalytic mechanism, single review-based summary but citing original structural/kinetic data","pmids":["9315326"],"is_preprint":false},{"year":2003,"finding":"Overexpression of mitochondrial PHGPx (mPHGPx) in neonatal rat cardiac myocytes reduces lipid peroxidation (malondialdehyde/HNE), decreases cytochrome c release from mitochondria, and preserves electron transport chain complex IV function following simulated ischemia/reoxygenation, demonstrating a direct mitochondrial protective role.","method":"Adenoviral overexpression of mPHGPx and cPHGPx, MDA/HNE quantification, cytochrome c release assay, complex IV activity assay, simulated I/R in neonatal cardiomyocytes","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct overexpression with multiple biochemical readouts including mitochondrial function, single lab","pmids":["14583338"],"is_preprint":false},{"year":2021,"finding":"GPX4 is required for follicular helper T (TFH) cell survival; selective deletion of GPX4 in T cells abrogates TFH cells and germinal center responses in immunized mice, establishing the selenium-GPX4-ferroptosis axis as a critical regulator of TFH homeostasis.","method":"T cell-specific GPX4 conditional knockout mice, immunization experiments, flow cytometry for TFH and germinal center B cells, selenium supplementation experiments","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific conditional KO with defined cellular phenotype (TFH depletion, germinal center loss), in vivo immunization model, replicated with selenium intervention","pmids":["34413521"],"is_preprint":false},{"year":2022,"finding":"GPX4 deficiency in macrophages and lung tissue results in ferroptotic cell death during Mycobacterium tuberculosis infection; Gpx4-deficient mice show increased lung necrosis and bacterial burden while Gpx4-overexpressing transgenic mice show decreased necrosis, establishing the GPX4/GSH axis as a host-resistance determinant in TB.","method":"Gpx4-deficient and Gpx4-transgenic mouse models, Mtb infection, ferrostatin-1 rescue in macrophages, lipid peroxidation assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — both loss- and gain-of-function mouse genetic models with defined infectious disease phenotypes, pharmacological rescue with lipid peroxidation inhibitor","pmids":["36069923"],"is_preprint":false},{"year":2022,"finding":"Adipocyte-specific GPX4 knockout spontaneously causes adipocyte hypertrophy, lipid peroxidation, inflammatory cytokine expression (TNF-α, IL-1β, IL-6, CXCL1), macrophage infiltration, glucose intolerance, and hepatic insulin resistance independent of ferroptosis-associated cell death.","method":"Conditional adipocyte-specific Gpx4 knockout (Adipoq-Cre and Fabp4-Cre), metabolic phenotyping (GTT, ITT), lipid peroxidation assays, cytokine measurement, TUNEL staining","journal":"International journal of obesity (2005)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — two independent conditional KO mouse models with metabolic phenotyping and orthogonal biochemical readouts, single lab","pmids":["35031697"],"is_preprint":false},{"year":2024,"finding":"PKA phosphorylates and promotes degradation of ALKBH5 (an m6A demethylase), thereby increasing m6A modification and stability of GPX4 mRNA; ALKBH5 deletion maintains GPX4 m6A modification and suppresses ferroptotic cell death, establishing a PKA-ALKBH5-GPX4 m6A regulatory axis.","method":"PKA kinase assays, ALKBH5 phosphorylation and degradation assays, m6A quantification of GPX4 mRNA, ALKBH5 knockout, ferroptosis cell death assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase assay with downstream m6A mRNA modification readout and genetic validation, single lab","pmids":["39901038"],"is_preprint":false},{"year":2023,"finding":"RSL3 and ML162 (commonly used GPX4 inhibitors) completely lack capacity to inhibit recombinant selenoprotein GPX4 enzymatic activity in vitro; instead, they are efficient inhibitors of TXNRD1. This negative result suggests prior mechanistic studies attributing ferroptosis induction by these compounds solely to GPX4 inhibition need re-evaluation.","method":"In vitro enzymatic activity assays with recombinant GPX4 and TXNRD1, cell death assays with ferrostatin-1 rescue","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct in vitro enzymatic assay with recombinant proteins, single lab; important negative result regarding direct GPX4 inhibition by RSL3/ML162","pmids":["37087975"],"is_preprint":false},{"year":2025,"finding":"TRAF6 ubiquitinates GPX4, promoting its recognition by the autophagic receptor p62 and selective autophagic degradation; a GPX4-targeted AUTAC exploiting this pathway induces GPX4 degradation and ferroptosis in breast cancer.","method":"Co-immunoprecipitation, ubiquitination assays, selective autophagy flux assays, AUTAC compound design, patient-derived organoids, in vivo tumor models","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with ubiquitination and autophagic receptor identification, validated with TPD approach in organoids and in vivo, single lab","pmids":["40394165"],"is_preprint":false},{"year":2001,"finding":"PHGPx (GPX4) was cloned as an anti-apoptotic and growth-promoting gene in Burkitt lymphoma cells, where cystine availability limiting glutathione biosynthesis determines cell susceptibility to oxidative stress-induced apoptosis, and PHGPx overexpression provides protection.","method":"Expression cloning in BL cells, overexpression rescue of low-density/low-serum-induced apoptosis","journal":"BioFactors (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional cloning with defined cellular phenotypic rescue, single lab","pmids":["11568455"],"is_preprint":false},{"year":1997,"finding":"Co-transfection of both PHGPx cDNA and selenophosphate synthase (SelD) cDNA into endothelial cells produces significantly higher PHGPx activity than either alone, especially under selenium-limiting conditions, demonstrating that selenocysteine incorporation machinery (SelD) is rate-limiting for GPX4 expression and function.","method":"Stable transfection of endothelial cells (ECV304), PHGPx activity assays, hydroperoxide reduction assays, H2O2 cytotoxicity assays","journal":"Biomedical and environmental sciences : BES","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional co-transfection experiment with biochemical and cytotoxicity readouts, single lab","pmids":["9315307"],"is_preprint":false}],"current_model":"GPX4 is a selenocysteine-containing lipid hydroperoxidase that reduces phospholipid hydroperoxides to non-toxic lipid alcohols using glutathione as the reducing substrate, thereby preventing iron-dependent lipid peroxidation and ferroptosis; its activity, stability, and subcellular localization are regulated at multiple levels including selenocysteine-based catalysis (superior to Cys for resisting overoxidation), alternative promoter-driven isoform expression (cytosolic, mitochondrial, nuclear), and an extensive post-translational modification network encompassing K48/K63/M1-linked ubiquitination by E3 ligases (STUB1, NEDD4L, CUL1-FBW7) and stabilization by deubiquitinases (USP8, OTUD5, OTUB1), S-palmitoylation at C66/C75 by ZDHHC20/ZDHHC8 (reversed by APT2), arginine-152 methylation by PRMT5, and serine-104 phosphorylation by CKB; direct covalent modification at C66 by itaconate allosterically activates GPX4; copper-induced ubiquitination at C107/C148 and chaperone-mediated autophagy (legumain/HSC70) or selective autophagy (TAX1BP1, TRAF6-p62) drive its degradation; transcriptional control by STAT3, KLF11, and epigenetic mechanisms (m6A via METTL3/ALKBH5/FTO, DNMT-mediated promoter methylation) modulates GPX4 levels; functionally, GPX4 is essential for TFH cell survival, innate immune STING activation (by preventing lipid peroxidation-induced STING carbonylation at C88), and host resistance to Mycobacterium tuberculosis, and in adipocytes it suppresses metabolic inflammation independently of overt ferroptosis."},"narrative":{"mechanistic_narrative":"GPX4 is a selenocysteine-dependent glutathione peroxidase that constitutes the central cellular defense against iron-dependent lipid peroxidation, and its loss precipitates ferroptotic cell death [PMID:24439385, PMID:31634900]. It is uniquely capable of reducing esterified phospholipid hydroperoxides within membranes to non-toxic lipid alcohols, a reaction no other enzyme performs in a cellular context [PMID:31634900, PMID:34931062]; an electrostatic cationic patch mediates membrane and cardiolipin binding adjacent to the catalytic site [PMID:34492183], and the selenolate active-site catalysis is specifically required to resist irreversible overoxidation, distinguishing GPX4 from a cysteine variant and being essential for neuronal survival in vivo [PMID:29290465]. This antioxidant function makes GPX4 a survival dependency in therapy-resistant high-mesenchymal persister cancer cells [PMID:29088702], and a patient-derived R152H active-site loop mutation that abolishes activity links GPX4 dysfunction to human disease [PMID:34931062]. Beyond its canonical hydroperoxidase role, GPX4 acts as a protein-thiol peroxidase that cross-links mitochondrial capsule proteins during spermatogenesis [PMID:11568459], and alternative-promoter usage generates cytosolic, mitochondrial, and arginine-rich nuclear isoforms with distinct localizations [PMID:12751792, PMID:14583338]. GPX4 abundance is set by a dense post-translational network: stabilizing palmitoylation at C66/C75 (ZDHHC20/ZDHHC8, reversed by APT2) [PMID:39833225, PMID:40108413], R152 symmetric dimethylation by PRMT5 that excludes the Cullin1-FBW7 ligase [PMID:40033101], S104 phosphorylation by CKB that blocks chaperone-mediated autophagy [PMID:37156912], stabilizing deubiquitination (USP8, OTUD5, OTUB1 recruited by CST1) and linear ubiquitination by LUBAC [PMID:36369321, PMID:36279464, PMID:38598341, PMID:38110369], opposed by degradative ubiquitination (STUB1 at K191, NEDD4L, copper-induced modification of C107/C148) and selective/chaperone-mediated autophagy via TAX1BP1, p62/TRAF6, legumain-HSC70, and FUNDC1-mediated mitochondrial import [PMID:36622894, PMID:38110356, PMID:33431801, PMID:36828120, PMID:40394165], with transcriptional and m6A control by STAT3 and the PKA-ALKBH5 axis [PMID:35859150, PMID:39901038]. Functionally, GPX4-maintained redox homeostasis sustains follicular helper T cell and germinal center responses [PMID:34413521], enables cGAS-STING innate immunity by preventing lipid-peroxidation-driven STING carbonylation at C88 [PMID:32541831], confers host resistance to Mycobacterium tuberculosis [PMID:36069923], and in adipocytes suppresses metabolic inflammation independently of overt ferroptotic death [PMID:35031697]. Direct covalent modification of C66 by itaconate allosterically activates the enzyme to protect neurons [PMID:38719928].","teleology":[{"year":1997,"claim":"Established the catalytic basis for GPX4's distinctive substrate range, answering how a glutathione peroxidase could act on bulky lipid hydroperoxides and protein thiols.","evidence":"Structural and kinetic analysis with protein-binding assays defining a Sec/Gln/Trp catalytic triad and broad donor specificity","pmids":["9315326"],"confidence":"Medium","gaps":["Did not resolve the membrane-binding mode","Protein-thiol substrates not identified at residue level"]},{"year":1997,"claim":"Showed that selenocysteine incorporation machinery, not the GPX4 coding sequence alone, limits functional enzyme production, framing selenium availability as a control point.","evidence":"Co-transfection of PHGPx with selenophosphate synthase (SelD) in endothelial cells with activity and cytotoxicity readouts","pmids":["9315307"],"confidence":"Medium","gaps":["Single cell type","Did not address endogenous regulation of Sec machinery"]},{"year":2001,"claim":"Demonstrated a protein-thiol peroxidase moonlighting activity, explaining the selenium dependency of spermatogenesis beyond simple antioxidant function.","evidence":"In vitro biochemistry with H2O2, thiol labeling and SDS-PAGE of mitochondrial capsule protein cross-linking","pmids":["11568459"],"confidence":"High","gaps":["In vitro only","Identity of all physiological thiol substrates incomplete"]},{"year":2001,"claim":"Connected GPX4 expression to cell survival under cystine/glutathione limitation, an early anti-apoptotic/growth-promoting role.","evidence":"Expression cloning in Burkitt lymphoma cells with overexpression rescue of oxidative apoptosis","pmids":["11568455"],"confidence":"Medium","gaps":["Predates ferroptosis framework","Mechanism of protection not at molecular resolution"]},{"year":2003,"claim":"Defined the molecular origin of the nuclear GPX4 isoform, answering how subcellular targeting diversity arises without alternative splicing.","evidence":"5'-RACE, RT-PCR, S1 nuclease and in situ hybridization identifying an intron-1 alternative promoter generating an Arg-rich N-terminus","pmids":["12751792"],"confidence":"High","gaps":["Regulation of alternative promoter not defined","Function of nuclear isoform beyond chromatin binding unclear"]},{"year":2003,"claim":"Provided direct evidence that mitochondrial GPX4 protects organelle function, linking isoform localization to physiological cardioprotection.","evidence":"Adenoviral mPHGPx/cPHGPx overexpression in cardiomyocytes with MDA/HNE, cytochrome c release and complex IV assays in I/R","pmids":["14583338"],"confidence":"Medium","gaps":["Overexpression only","Endogenous mitochondrial GPX4 contribution not isolated"]},{"year":2014,"claim":"Identified GPX4 as the essential regulator of ferroptosis, defining the cell death modality it suppresses.","evidence":"Chemoproteomic target identification of RSL3 plus GPX4 gain/loss-of-function across multiple inducers and cell lines","pmids":["24439385"],"confidence":"High","gaps":["Direct enzymatic inhibition by RSL3 later questioned","Endogenous lipid substrate spectrum not fully mapped"]},{"year":2017,"claim":"Explained why GPX4 uses selenocysteine rather than cysteine, establishing resistance to overoxidation as the catalytic rationale for an essential enzyme.","evidence":"Gpx4 Sec-to-Cys knock-in mice and conditional neuronal knockouts with peroxide sensitivity assays","pmids":["29290465"],"confidence":"High","gaps":["Mechanism of selenolate regeneration in cells not fully defined"]},{"year":2017,"claim":"Revealed GPX4 as a selective vulnerability of drug-tolerant persister cancer cells, giving therapeutic relevance to the ferroptosis dependency.","evidence":"GPX4 inhibition/knockdown in mesenchymal persister cells with ferroptosis assays and in vivo relapse models","pmids":["29088702"],"confidence":"High","gaps":["Why the mesenchymal state imposes GPX4 dependency not fully mechanistic"]},{"year":2019,"claim":"Defined GPX4 as the glutathione-dependent lipid hydroperoxidase and positioned FSP1 as a parallel, GPX4-independent antioxidant arm of ferroptosis defense.","evidence":"Synthetic-lethal CRISPR screen, biochemical oxidoreductase assays and xenografts","pmids":["31634900"],"confidence":"High","gaps":["Crosstalk thresholds between GPX4 and FSP1 pathways not quantified"]},{"year":2020,"claim":"Linked GPX4 redox control to innate immunity, showing lipid peroxidation carbonylates STING and blocks its trafficking.","evidence":"GPX4 knockdown/knockout with STING C88 carbonylation, ER-to-Golgi trafficking assays and viral infection models","pmids":["32541831"],"confidence":"High","gaps":["Direct lipid species mediating STING carbonylation not pinned down"]},{"year":2021,"claim":"Resolved the structural basis of membrane and DNA engagement, explaining how GPX4 reaches membrane lipid substrates and how the nuclear isoform targets chromatin.","evidence":"First NMR assignments of GPX4 with mutagenesis of a cationic patch and lipid/DNA binding assays","pmids":["34492183"],"confidence":"High","gaps":["Membrane orientation during catalysis not directly visualized"]},{"year":2021,"claim":"Established GPX4 as required for follicular helper T cell survival, extending the selenium-GPX4-ferroptosis axis to adaptive immunity.","evidence":"T cell-specific Gpx4 conditional knockout with immunization, flow cytometry and selenium supplementation","pmids":["34413521"],"confidence":"High","gaps":["Why TFH cells are selectively vulnerable not fully explained"]},{"year":2021,"claim":"Initiated the degradation arm of GPX4 regulation, showing chaperone-mediated autophagy controls GPX4 turnover in injury.","evidence":"Co-IP of legumain with HSC70/HSP90/GPX4, lysosomal fractionation and legumain-KO IRI model","pmids":["33431801"],"confidence":"Medium","gaps":["Single lab Co-IP","Direct CMA targeting motif on GPX4 not defined"]},{"year":2021,"claim":"Established GPX4 as the only enzyme reducing esterified phospholipid hydroperoxides in cells and connected an active-site-loop mutation to human disease.","evidence":"Structure-based analysis, patient fibroblasts and enzymatic/stability assays of R152H","pmids":["34931062"],"confidence":"High","gaps":["Full clinical spectrum of GPX4 mutations not enumerated here"]},{"year":2022,"claim":"Identified an allosteric regeneration site distinct from the active site, defining a druggable conformational control point.","evidence":"Six co-crystal structures of inhibitors with biophysical and activity assays","pmids":["36423641"],"confidence":"High","gaps":["Endogenous ligand of the allosteric site unknown"]},{"year":2022,"claim":"Began assembling the ubiquitin-stabilization network by showing LUBAC adds protective linear ubiquitin chains to GPX4.","evidence":"Co-IP, M1-linkage ubiquitination assays and LUBAC-deficient ferroptosis sensitivity","pmids":["36279464"],"confidence":"Medium","gaps":["Single lab","Site of M1 attachment not mapped"]},{"year":2022,"claim":"Showed deubiquitinase recruitment stabilizes GPX4, identifying CST1-OTUB1 as a protective module.","evidence":"Co-IP/MS, ubiquitination assays and in vivo metastasis models","pmids":["36369321"],"confidence":"Medium","gaps":["Single lab","Generality across tissues untested"]},{"year":2022,"claim":"Extended GPX4's protective role to infection, defining the GPX4/GSH axis as a tuberculosis host-resistance determinant.","evidence":"Gpx4-deficient and -transgenic mice in Mtb infection with ferrostatin-1 rescue","pmids":["36069923"],"confidence":"High","gaps":["Cell-intrinsic vs tissue-level contributions not fully separated"]},{"year":2022,"claim":"Revealed a ferroptosis-independent metabolic function of GPX4 in adipocytes restraining inflammation.","evidence":"Two adipocyte-specific Gpx4 KO lines with metabolic phenotyping, cytokine and TUNEL assays","pmids":["35031697"],"confidence":"High","gaps":["Lipid mediators driving inflammation not identified","Single lab"]},{"year":2023,"claim":"Defined copper as a direct trigger of GPX4 autophagic degradation, coupling metal stress to ferroptosis.","evidence":"Co-IP, ubiquitination/aggregation assays mapping Cu binding to C107/C148 and TAX1BP1 receptor in pancreatic cancer models","pmids":["36622894"],"confidence":"Medium","gaps":["Single lab","Stoichiometry of copper binding undefined"]},{"year":2023,"claim":"Identified CKB as a non-canonical kinase that phosphorylates GPX4 to block its CMA degradation, linking growth-factor signaling to ferroptosis resistance.","evidence":"In vitro kinase assay, Co-IP, S104 mutagenesis, CMA assays and HCC tumor models","pmids":["37156912"],"confidence":"High","gaps":["Phosphatase reversing S104 not identified"]},{"year":2023,"claim":"Showed USP8 directly deubiquitinates and stabilizes GPX4, adding a DUB to the protective network.","evidence":"Co-IP, deubiquitination assays and intestinal-epithelial Usp8 conditional KO mice","pmids":["38598341"],"confidence":"Medium","gaps":["Single lab","Linkage type removed not specified"]},{"year":2023,"claim":"Identified OTUD5 as a stabilizing DUB whose autophagic loss drives GPX4 decay during ischemia-reperfusion.","evidence":"Co-IP, ubiquitination assays, spatial transcriptomics and AAV-OTUD5 rescue in IRI","pmids":["38110369"],"confidence":"Medium","gaps":["Single lab","Ubiquitin site on GPX4 not mapped"]},{"year":2023,"claim":"Revealed a ubiquitin-linkage switch in which TRIM26 builds stabilizing K63 chains, illustrating linkage-type control of GPX4 fate.","evidence":"Co-IP, K48/K63 linkage assays and K107/K117 mutagenesis in glioma","pmids":["37872147"],"confidence":"Medium","gaps":["Single lab","PLK1-TRIM26 axis generality untested"]},{"year":2023,"claim":"Identified STUB1 as a degradative E3 ligase ubiquitinating GPX4 at K191, defining a pro-ferroptotic destabilizer.","evidence":"Co-IP, K191 mutagenesis, genetic rescue and in vivo GIST tumor models","pmids":["38110356"],"confidence":"Medium","gaps":["Single lab","Conditions selecting STUB1 over stabilizers unclear"]},{"year":2023,"claim":"Established direct transcriptional control of GPX4 by STAT3, defining a targetable input to GPX4 abundance.","evidence":"ChIP, dual-luciferase reporter, STAT3 inhibition and xenografts in pancreatic cancer","pmids":["35859150"],"confidence":"Medium","gaps":["Single lab","Other transcription factors at the locus not co-assessed"]},{"year":2023,"claim":"Added NEDD4L as a degradative E3 ligase of GPX4 in a granulosa-cell ferroptosis context.","evidence":"Co-IP and ubiquitination assays with viability and ferroptosis markers","pmids":["36662677"],"confidence":"Low","gaps":["Single Co-IP without in vivo validation","Ubiquitination site not mapped"]},{"year":2024,"claim":"Showed itaconate directly alkylates C66 to allosterically activate GPX4, defining an endogenous metabolite as a positive regulator.","evidence":"Bioorthogonal ITalk probe, phosphatidylcholine-hydroperoxide activity assay and Irg1 mouse models in ICH","pmids":["38719928"],"confidence":"High","gaps":["Structural basis of allosteric activation not solved"]},{"year":2024,"claim":"Defined an m6A layer of control, with PKA-driven ALKBH5 degradation increasing GPX4 mRNA stability.","evidence":"PKA kinase assays, ALKBH5 degradation, GPX4 mRNA m6A quantification and ALKBH5 KO","pmids":["39901038"],"confidence":"Medium","gaps":["Single lab","m6A reader for GPX4 transcript not identified"]},{"year":2024,"claim":"Linked tumor suppressor p53 to GPX4 turnover via transcriptional repression of the DUB OTUD5.","evidence":"Co-IP, deubiquitination assay, Otud5 CRISPR KO, p53-OTUD5 luciferase reporter and tumor models","pmids":["40070026"],"confidence":"Medium","gaps":["Single lab","Relation to other p53-ferroptosis routes unresolved"]},{"year":2025,"claim":"Identified S-palmitoylation at C66 by ZDHHC20 (reversed by APT2) as a stability switch for GPX4.","evidence":"Acyl-RAC, C66 mutagenesis, ZDHHC20/APT2 perturbation and multiple in vivo tumor models","pmids":["39833225"],"confidence":"High","gaps":["Interplay between C66 palmitoylation and C66 itaconate alkylation not reconciled"]},{"year":2025,"claim":"Added ZDHHC8-mediated C75 palmitoylation as a second stabilizing acylation and a pharmacological target.","evidence":"Palmitoylation assays, C75 mutagenesis, PF-670462 and xenograft/CD8 T cell co-culture","pmids":["40108413"],"confidence":"Medium","gaps":["Single lab","Relative contribution of C66 vs C75 palmitoylation unclear"]},{"year":2025,"claim":"Showed PRMT5-mediated R152 methylation stabilizes GPX4 by excluding the Cullin1-FBW7 ligase, adding arginine methylation to the PTM network.","evidence":"In vitro methylation, Co-IP, R152 mutagenesis, ubiquitination assays and tumor models","pmids":["40033101"],"confidence":"High","gaps":["Demethylase for R152 not identified","Relationship of R152 methylation to the R152H disease mutation not addressed"]},{"year":2025,"claim":"Defined TRAF6-p62 selective autophagy as a route for GPX4 degradation and validated it with a degrader strategy.","evidence":"Co-IP, ubiquitination/autophagy flux assays, AUTAC design, organoids and tumor models","pmids":["40394165"],"confidence":"Medium","gaps":["Single lab","Ubiquitin site engaged by p62 not mapped"]},{"year":2026,"claim":"Showed FUNDC1 recruits GPX4 into mitochondria for mitophagic degradation, coupling subcellular import to GPX4 turnover.","evidence":"Domain-mapped Co-IP, colocalization, mitochondrial fractionation and FUNDC1-KO liver injury model","pmids":["36828120"],"confidence":"Medium","gaps":["Single lab","How import competes with cytosolic GPX4 function unclear"]},{"year":null,"claim":"Whether widely used inhibitors directly inhibit GPX4 enzymatic activity remains unsettled, complicating interpretation of decades of pharmacological ferroptosis data.","evidence":"In vitro assays showing RSL3 and ML162 fail to inhibit recombinant selenoprotein GPX4 and instead inhibit TXNRD1","pmids":[],"confidence":"Medium","gaps":["Need reconciliation of cellular target engagement with recombinant-enzyme data","Identity of true cellular targets of these compounds incompletely resolved","Whether allosteric/covalent inhibition occurs only on membrane-bound or modified GPX4 untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,1,5,6,27]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[1,6,26]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[8]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[8,9]},{"term_id":"GO:0016209","term_label":"antioxidant activity","supporting_discovery_ids":[0,1,28]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[9,24]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[24,28]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,9]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4,29,30]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[11,18,20,22,34]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[10,12,34,24]}],"complexes":[],"partners":["STING","CKB","PRMT5","ZDHHC20","USP8","OTUD5","STUB1","TRAF6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P36969","full_name":"Phospholipid hydroperoxide glutathione peroxidase GPX4","aliases":["Glutathione peroxidase 4","GPx-4","GSHPx-4"],"length_aa":197,"mass_kda":22.2,"function":"Essential antioxidant peroxidase that directly reduces phospholipid hydroperoxide even if they are incorporated in membranes and lipoproteins (PubMed:40281343). Can also reduce cholesterol hydroperoxide and thymine hydroperoxide (By similarity). Plays a key role in protecting cells from oxidative damage by preventing membrane lipid peroxidation (PubMed:40281343). Required to prevent cells from ferroptosis, a non-apoptotic cell death resulting from an iron-dependent accumulation of lipid reactive oxygen species (PubMed:24439385, PubMed:40281343). The presence of selenocysteine (Sec) versus Cys at the active site is essential for life: it provides resistance to overoxidation and prevents cells against ferroptosis (By similarity). The presence of Sec at the active site is also essential for the survival of a specific type of parvalbumin-positive interneurons, thereby preventing against fatal epileptic seizures (By similarity). May be required to protect cells from the toxicity of ingested lipid hydroperoxides (By similarity). Required for normal sperm development and male fertility (By similarity). Essential for maturation and survival of photoreceptor cells (By similarity). Plays a role in a primary T-cell response to viral and parasitic infection by protecting T-cells from ferroptosis and by supporting T-cell expansion (By similarity). Plays a role of glutathione peroxidase in platelets in the arachidonic acid metabolism (PubMed:11115402). Reduces hydroperoxy ester lipids formed by a 15-lipoxygenase that may play a role as down-regulator of the cellular 15-lipoxygenase pathway (By similarity). Can reduce fatty acid-derived hydroperoxides (PubMed:11115402, PubMed:36608588). Can also reduce small soluble hydroperoxides such as H2O2, cumene hydroperoxide and tert-butyl hydroperoxide (PubMed:17630701, PubMed:36608588)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P36969/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GPX4","classification":"Not Classified","n_dependent_lines":748,"n_total_lines":1208,"dependency_fraction":0.6192052980132451},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/GPX4","total_profiled":1310},"omim":[{"mim_id":"620198","title":"THYROID HORMONE METABOLISM, ABNORMAL, 3; THMA3","url":"https://www.omim.org/entry/620198"},{"mim_id":"605159","title":"APOPTOSIS-INDUCING FACTOR, MITOCHONDRIA-ASSOCIATED, 2; AIFM2","url":"https://www.omim.org/entry/605159"},{"mim_id":"604851","title":"G-RICH RNA SEQUENCE-BINDING FACTOR 1; GRSF1","url":"https://www.omim.org/entry/604851"},{"mim_id":"250220","title":"SPONDYLOMETAPHYSEAL DYSPLASIA, SEDAGHATIAN TYPE; SMDS","url":"https://www.omim.org/entry/250220"},{"mim_id":"189909","title":"ZINC FINGER E BOX-BINDING HOMEOBOX 1; ZEB1","url":"https://www.omim.org/entry/189909"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GPX4"},"hgnc":{"alias_symbol":["PHGPx","MCSP"],"prev_symbol":[]},"alphafold":{"accession":"P36969","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P36969","model_url":"","pae_url":"","plddt_mean":null},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GPX4","jax_strain_url":"https://www.jax.org/strain/search?query=GPX4"},"sequence":{"accession":"P36969","fasta_url":"https://rest.uniprot.org/uniprotkb/P36969.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P36969/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P36969"}},"corpus_meta":[{"pmid":"24439385","id":"PMC_24439385","title":"Regulation of ferroptotic cancer cell death by GPX4.","date":"2014","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/24439385","citation_count":6113,"is_preprint":false},{"pmid":"31634900","id":"PMC_31634900","title":"The CoQ oxidoreductase FSP1 acts parallel to GPX4 to 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identified via chemoproteomics; GPX4 overexpression suppresses and knockdown promotes ferroptotic cell death induced by 12 different ferroptosis inducers, establishing GPX4 as an essential regulator of ferroptosis.\",\n      \"method\": \"Chemoproteomics (direct target identification), GPX4 overexpression/knockdown with cell viability readouts, targeted metabolomic profiling\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — chemoproteomics-based direct target identification combined with genetic gain- and loss-of-function, replicated across multiple compounds and cell lines\",\n      \"pmids\": [\"24439385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GPX4 functions as the glutathione-dependent lipid hydroperoxidase that converts lipid hydroperoxides into non-toxic lipid alcohols, thereby suppressing ferroptosis; FSP1 acts in a parallel non-mitochondrial CoQ antioxidant pathway independent of GPX4.\",\n      \"method\": \"Synthetic lethal CRISPR-Cas9 screen, biochemical oxidoreductase assays, xenograft tumor models\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — CRISPR screen with biochemical validation, multiple orthogonal methods, replicated in vivo\",\n      \"pmids\": [\"31634900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation; cells expressing a cysteine variant of GPX4 (Sec→Cys) are highly sensitive to peroxide-induced ferroptosis, demonstrating that selenolate-based catalysis is specifically required for neuronal survival and prevention of fatal epileptic seizures in vivo.\",\n      \"method\": \"Knock-in mouse models (Gpx4cys/cys), in vitro peroxide sensitivity assays, neuronal cell-specific conditional knockouts\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — genetic reconstitution in mouse and cell models with multiple orthogonal functional readouts, mechanistic mutagenesis of active-site residue\",\n      \"pmids\": [\"29290465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Drug-tolerant persister cancer cells in a high mesenchymal therapy-resistant state acquire dependency on GPX4 for survival; loss of GPX4 function causes selective ferroptotic death of persister cells in vitro and prevents tumor relapse in mice.\",\n      \"method\": \"GPX4 genetic knockdown/inhibition, ferroptosis cell death assays, in vivo mouse tumor relapse models\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined cellular ferroptosis phenotype confirmed in vivo, replicated across multiple cancer types\",\n      \"pmids\": [\"29088702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GPX4 deficiency enhances cellular lipid peroxidation, which leads to carbonylation of STING at C88 and inhibits STING trafficking from the ER to the Golgi complex, thereby specifically attenuating the cGAS-STING innate immune pathway; GPX4-maintained redox homeostasis is required for STING activation.\",\n      \"method\": \"GPX4 knockdown/knockout, carbonylation assays, subcellular fractionation and trafficking assays (ER-to-Golgi), viral infection models in vivo\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with biochemical mechanism (carbonylation at specific residue, trafficking defect), in vivo validation\",\n      \"pmids\": [\"32541831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PHGPx (GPX4) acts as a protein thiol peroxidase in sperm, using its selenocysteine to oxidize specific capsule protein thiols via a peroxide-dependent mechanism, leading to cross-linking of mitochondrial capsule proteins and accounting for the selenium dependency of spermatogenesis.\",\n      \"method\": \"In vitro biochemical assays with H2O2, monobromobimane labeling, SDS-PAGE, mercaptoethanol release of active enzyme\",\n      \"journal\": \"BioFactors (Oxford, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with defined substrates and stoichiometry, mechanistic characterization of catalytic cross-linking activity\",\n      \"pmids\": [\"11568459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GPX4 is the only enzyme capable of reducing esterified phospholipid hydroperoxides within a cellular context; a patient-derived R152H mutation destabilizes a critical loop adjacent to the active site, causing substantial loss of enzymatic function while paradoxically reducing GPX4 susceptibility to degradation.\",\n      \"method\": \"Structure-based analysis, cell models including patient fibroblasts, enzymatic activity assays, protein stability assays\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structure-based mechanistic characterization combined with cell-based functional validation and patient-derived fibroblasts\",\n      \"pmids\": [\"34931062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"An allosteric site on GPX4 (distinct from the active site) is involved in native regeneration of GPX4 under low glutathione conditions; covalent binding of inhibitors to this site causes conformational change, inhibition of activity, and subsequent cellular GPX4 protein degradation. Co-crystal structures of six inhibitors bound in this site were determined.\",\n      \"method\": \"Co-crystal structure determination (X-ray), biophysical binding assays, enzymatic activity assays, compound library screen\",\n      \"journal\": \"Cell chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple co-crystal structures with functional validation of allosteric mechanism, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"36423641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NMR characterization of GPX4 reveals an electrostatic (cationic patch) mechanism for membrane binding; mutagenesis identifies specific cationic patch residues required for membrane and cardiolipin headgroup binding, and a novel lipid binding site adjacent to the catalytic site. The cationic patch also mediates DNA binding, explaining the nuclear isoform's ability to target DNA-bound protamines.\",\n      \"method\": \"NMR spectroscopy (first NMR assignments of GPX4), site-directed mutagenesis, lipid/DNA binding assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with mutagenesis validation, multiple binding partners characterized, single lab\",\n      \"pmids\": [\"34492183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Testis-specific nuclear form of GPX4 (PHGPx/snGPx) is generated by transcription initiation from an alternative promoter (not by alternative splicing) located in intron 1, producing an arginine-rich N-terminus responsible for nuclear localization and chromatin binding, with expression restricted to late stages of spermatogenesis.\",\n      \"method\": \"5'-RACE, RT-PCR, S1 nuclease analysis, in situ hybridization\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple complementary molecular methods establishing alternative promoter usage and isoform-specific localization\",\n      \"pmids\": [\"12751792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Copper promotes GPX4 autophagic degradation and ferroptosis by directly binding to GPX4 cysteines C107 and C148, inducing GPX4 ubiquitination and aggregation; TAX1BP1 then acts as the autophagic receptor mediating GPX4 degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, autophagic flux assays, copper chelation experiments, mouse pancreatic cancer models\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP identifying specific cysteine binding sites and autophagic receptor, validated in vivo, single lab\",\n      \"pmids\": [\"36622894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Creatine kinase B (CKB), when phosphorylated at T133 by AKT downstream of IGF1R signaling, acts as a non-canonical protein kinase to phosphorylate GPX4 at S104; this phosphorylation prevents HSC70 binding to GPX4 and blocks chaperone-mediated autophagy (CMA)-dependent GPX4 degradation, thereby suppressing ferroptosis.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, CMA degradation assays, mutagenesis, mouse tumor models, human HCC specimen correlation\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay establishing CKB as protein kinase, combined with Co-IP, mutagenesis, and in vivo validation\",\n      \"pmids\": [\"37156912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Legumain facilitates chaperone-mediated autophagy (CMA) of GPX4 in acute kidney injury: legumain directly interacts with HSC70, HSP90, and GPX4 (shown by immunoprecipitation), and legumain deficiency prevents CMA-dependent GPX4 degradation and tubular ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation, lysosomal fractionation, proteasome/autophagy inhibitor experiments, legumain KO mice with IRI model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP identifying protein complex, genetic KO with defined ferroptosis phenotype, in vivo model, single lab\",\n      \"pmids\": [\"33431801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CST1 interacts with GPX4 (confirmed by Co-IP and mass spectrometry) and recruits the deubiquitinase OTUB1 to relieve GPX4 ubiquitination, thereby stabilizing GPX4 protein and inhibiting ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation combined with mass spectrometry, ubiquitination assays, siRNA knockdown, in vivo mouse metastasis models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS identifying the complex and ubiquitination readout, validated in vivo, single lab\",\n      \"pmids\": [\"36369321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LUBAC (via its catalytic subunit HOIP) binds GPX4 and stabilizes it through linear (M1-linked) ubiquitination both under basal conditions and oxidative stress; LUBAC deficiency promotes GPX4 degradation and sensitizes cells to ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation, linear ubiquitination assays, LUBAC genetic deficiency models, lipid peroxidation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with ubiquitination linkage-type characterization, genetic deficiency model, single lab\",\n      \"pmids\": [\"36279464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP8 (ubiquitin-specific protease 8) directly interacts with and deubiquitinates GPX4, leading to GPX4 protein stabilization; USP8 inhibition destabilizes GPX4 and sensitizes cancer cells to ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assays, intestinal epithelial cell-specific Usp8 knockout mice, in vivo tumor models\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with deubiquitination biochemical assay, conditional KO mice, single lab\",\n      \"pmids\": [\"38598341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD5 acts as a deubiquitinase that binds GPX4, stabilizes it, and confers ferroptosis resistance; during ischemia-reperfusion, mTORC1-mediated autophagy degrades OTUD5, causing GPX4 decay and subsequent ferroptosis in renal tubular cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, spatial transcriptomics, AAV-mediated OTUD5 delivery, mouse IRI model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP establishing GPX4-OTUD5 interaction and deubiquitination, in vivo rescue experiment, single lab\",\n      \"pmids\": [\"38110369\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM26 directly interacts with GPX4 via its RING domain and catalyzes K63-linked ubiquitination of GPX4 at K107 and K117, switching polyubiquitination from K48 to K63 linkage and thereby enhancing GPX4 protein stability; PLK1-mediated S127 phosphorylation of TRIM26 enhances this interaction.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin linkage-specific assays (K48 vs K63), mutagenesis of ubiquitination sites, TRIM26 knockdown in glioma\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with ubiquitin linkage characterization and site mutagenesis, single lab\",\n      \"pmids\": [\"37872147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GPX4 is S-palmitoylated on cysteine 66 by the acyltransferase ZDHHC20, which increases GPX4 protein stability; APT2 acts as the depalmitoylase of GPX4. Disrupting GPX4 palmitoylation sensitizes cancer cells to ferroptosis in vitro and in vivo.\",\n      \"method\": \"Palmitoylation assays (acyl-RAC), mutagenesis (C66), ZDHHC20/APT2 knockdown/inhibition, xenograft and orthotopic tumor models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — site-specific PTM identification with mutagenesis, writer/eraser identified biochemically, validated in multiple in vivo models\",\n      \"pmids\": [\"39833225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZDHHC8 palmitoylates GPX4 at Cys75, stabilizing GPX4 and suppressing ferroptosis; pharmacological inhibition of ZDHHC8 with PF-670462 promotes ZDHHC8 degradation, reduces GPX4 palmitoylation, and enhances ferroptosis sensitivity in tumor cells.\",\n      \"method\": \"Palmitoylation assays, mutagenesis (C75), small-molecule drug screening, xenograft tumor models, CD8+ T cell co-culture assays\",\n      \"journal\": \"Nature cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific palmitoylation with mutagenesis and pharmacological validation, single lab\",\n      \"pmids\": [\"40108413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PRMT5 catalyzes symmetric dimethylation of GPX4 at arginine 152 (R152), which prevents Cullin1-FBW7 E3 ligase binding to GPX4 and blocks ubiquitination-mediated GPX4 degradation, thereby prolonging GPX4 half-life and suppressing ferroptosis.\",\n      \"method\": \"In vitro methylation assays, Co-immunoprecipitation, ubiquitination assays, PRMT5 inhibition, mutagenesis (R152), mouse tumor models\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro methylation assay establishing writer, Co-IP identifying E3 ligase exclusion mechanism, mutagenesis, in vivo validation\",\n      \"pmids\": [\"40033101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NEDD4L directly interacts with GPX4 (confirmed by Co-IP) and promotes GPX4 ubiquitination and degradation, facilitating granulosa cell ferroptosis and contributing to PCOS pathology.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, CCK-8 cell viability, ferroptosis marker assays\",\n      \"journal\": \"Endocrine connections\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with ubiquitination assay, single lab, no in vivo validation\",\n      \"pmids\": [\"36662677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"STUB1 is identified as an E3 ubiquitin ligase of GPX4, promoting GPX4 ubiquitination at site K191 and its degradation, thereby inducing ferroptosis in gastrointestinal stromal tumors.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays with site mutagenesis (K191), STUB1 knockdown and GPX4 overexpression, in vivo tumor models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination site mutagenesis with genetic rescue experiments, in vivo validation, single lab\",\n      \"pmids\": [\"38110356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD5 deubiquitylates and stabilizes GPX4; p53 suppresses OTUD5 transcription, thereby promoting GPX4 ubiquitination and degradation and inducing ferroptosis in gastric cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay, CRISPR-Cas9 Otud5 KO, luciferase reporter for p53-OTUD5 transcription, mouse tumor models\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with biochemical deubiquitination, genetic KO in vivo, transcriptional regulation validated, single lab\",\n      \"pmids\": [\"40070026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"FUNDC1 interacts with GPX4 via its 96–133 amino acid domain (shown by Co-IP), facilitating GPX4 recruitment from cytoplasm into mitochondria via the TOM/TIM import complex; within mitochondria, GPX4 is degraded by mitophagy, triggering hepatocyte ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation (domain mapping), immunofluorescence colocalization, FUNDC1 knockout mouse (CCl4 model), mitochondrial fractionation\",\n      \"journal\": \"Journal of advanced research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-mapped Co-IP with subcellular fractionation and genetic KO in vivo, single lab\",\n      \"pmids\": [\"36828120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"STAT3 binds to the GPX4 promoter region and promotes its transcription (confirmed by ChIP and dual-luciferase reporter assay); thiostrepton blocks GPX4 expression by inhibiting STAT3, thereby inducing ferroptosis in pancreatic cancer cells.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), dual-luciferase reporter assay, STAT3 inhibition, in vivo xenograft\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase assays directly establishing STAT3-GPX4 promoter interaction, in vivo validation, single lab\",\n      \"pmids\": [\"35859150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Itaconate (produced by Irg1/aconitate decarboxylase 1) directly alkylates GPX4 at cysteine 66, allosterically enhancing GPX4 enzymatic activity, thereby protecting neurons from ferroptosis after intracerebral hemorrhage.\",\n      \"method\": \"Bioorthogonal itaconate-alkyne (ITalk) probe, GPX4 activity assay using phosphatidylcholine hydroperoxide, Irg1 transgenic/KO mouse models, in vitro cortical neuron ferroptosis assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — bioorthogonal probe-based direct covalent modification identification with enzymatic activity assay, genetic mouse model validation, mechanistically linked to C66 of GPX4\",\n      \"pmids\": [\"38719928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"PHGPx (GPX4) shares an active site catalytic triad (selenocysteine, glutamine, tryptophan) with tetrameric GPXs; its unique ability to accommodate large lipophilic substrates (phospholipid hydroperoxides) is defined by structural and kinetic data. PHGPx also exhibits broad donor-substrate specificity (not restricted to GSH) and binds specific mitochondrial proteins via protein-protein interaction, suggesting a role in oxidizing specific protein thiols.\",\n      \"method\": \"Structural analysis, kinetic assays, protein-protein interaction (protein binding to mitochondrial proteins released by ionic strength and thiols)\",\n      \"journal\": \"Biomedical and environmental sciences : BES\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural and kinetic characterization of catalytic mechanism, single review-based summary but citing original structural/kinetic data\",\n      \"pmids\": [\"9315326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Overexpression of mitochondrial PHGPx (mPHGPx) in neonatal rat cardiac myocytes reduces lipid peroxidation (malondialdehyde/HNE), decreases cytochrome c release from mitochondria, and preserves electron transport chain complex IV function following simulated ischemia/reoxygenation, demonstrating a direct mitochondrial protective role.\",\n      \"method\": \"Adenoviral overexpression of mPHGPx and cPHGPx, MDA/HNE quantification, cytochrome c release assay, complex IV activity assay, simulated I/R in neonatal cardiomyocytes\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct overexpression with multiple biochemical readouts including mitochondrial function, single lab\",\n      \"pmids\": [\"14583338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GPX4 is required for follicular helper T (TFH) cell survival; selective deletion of GPX4 in T cells abrogates TFH cells and germinal center responses in immunized mice, establishing the selenium-GPX4-ferroptosis axis as a critical regulator of TFH homeostasis.\",\n      \"method\": \"T cell-specific GPX4 conditional knockout mice, immunization experiments, flow cytometry for TFH and germinal center B cells, selenium supplementation experiments\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific conditional KO with defined cellular phenotype (TFH depletion, germinal center loss), in vivo immunization model, replicated with selenium intervention\",\n      \"pmids\": [\"34413521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"GPX4 deficiency in macrophages and lung tissue results in ferroptotic cell death during Mycobacterium tuberculosis infection; Gpx4-deficient mice show increased lung necrosis and bacterial burden while Gpx4-overexpressing transgenic mice show decreased necrosis, establishing the GPX4/GSH axis as a host-resistance determinant in TB.\",\n      \"method\": \"Gpx4-deficient and Gpx4-transgenic mouse models, Mtb infection, ferrostatin-1 rescue in macrophages, lipid peroxidation assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — both loss- and gain-of-function mouse genetic models with defined infectious disease phenotypes, pharmacological rescue with lipid peroxidation inhibitor\",\n      \"pmids\": [\"36069923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Adipocyte-specific GPX4 knockout spontaneously causes adipocyte hypertrophy, lipid peroxidation, inflammatory cytokine expression (TNF-α, IL-1β, IL-6, CXCL1), macrophage infiltration, glucose intolerance, and hepatic insulin resistance independent of ferroptosis-associated cell death.\",\n      \"method\": \"Conditional adipocyte-specific Gpx4 knockout (Adipoq-Cre and Fabp4-Cre), metabolic phenotyping (GTT, ITT), lipid peroxidation assays, cytokine measurement, TUNEL staining\",\n      \"journal\": \"International journal of obesity (2005)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent conditional KO mouse models with metabolic phenotyping and orthogonal biochemical readouts, single lab\",\n      \"pmids\": [\"35031697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PKA phosphorylates and promotes degradation of ALKBH5 (an m6A demethylase), thereby increasing m6A modification and stability of GPX4 mRNA; ALKBH5 deletion maintains GPX4 m6A modification and suppresses ferroptotic cell death, establishing a PKA-ALKBH5-GPX4 m6A regulatory axis.\",\n      \"method\": \"PKA kinase assays, ALKBH5 phosphorylation and degradation assays, m6A quantification of GPX4 mRNA, ALKBH5 knockout, ferroptosis cell death assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase assay with downstream m6A mRNA modification readout and genetic validation, single lab\",\n      \"pmids\": [\"39901038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RSL3 and ML162 (commonly used GPX4 inhibitors) completely lack capacity to inhibit recombinant selenoprotein GPX4 enzymatic activity in vitro; instead, they are efficient inhibitors of TXNRD1. This negative result suggests prior mechanistic studies attributing ferroptosis induction by these compounds solely to GPX4 inhibition need re-evaluation.\",\n      \"method\": \"In vitro enzymatic activity assays with recombinant GPX4 and TXNRD1, cell death assays with ferrostatin-1 rescue\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct in vitro enzymatic assay with recombinant proteins, single lab; important negative result regarding direct GPX4 inhibition by RSL3/ML162\",\n      \"pmids\": [\"37087975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRAF6 ubiquitinates GPX4, promoting its recognition by the autophagic receptor p62 and selective autophagic degradation; a GPX4-targeted AUTAC exploiting this pathway induces GPX4 degradation and ferroptosis in breast cancer.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, selective autophagy flux assays, AUTAC compound design, patient-derived organoids, in vivo tumor models\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with ubiquitination and autophagic receptor identification, validated with TPD approach in organoids and in vivo, single lab\",\n      \"pmids\": [\"40394165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PHGPx (GPX4) was cloned as an anti-apoptotic and growth-promoting gene in Burkitt lymphoma cells, where cystine availability limiting glutathione biosynthesis determines cell susceptibility to oxidative stress-induced apoptosis, and PHGPx overexpression provides protection.\",\n      \"method\": \"Expression cloning in BL cells, overexpression rescue of low-density/low-serum-induced apoptosis\",\n      \"journal\": \"BioFactors (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional cloning with defined cellular phenotypic rescue, single lab\",\n      \"pmids\": [\"11568455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Co-transfection of both PHGPx cDNA and selenophosphate synthase (SelD) cDNA into endothelial cells produces significantly higher PHGPx activity than either alone, especially under selenium-limiting conditions, demonstrating that selenocysteine incorporation machinery (SelD) is rate-limiting for GPX4 expression and function.\",\n      \"method\": \"Stable transfection of endothelial cells (ECV304), PHGPx activity assays, hydroperoxide reduction assays, H2O2 cytotoxicity assays\",\n      \"journal\": \"Biomedical and environmental sciences : BES\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional co-transfection experiment with biochemical and cytotoxicity readouts, single lab\",\n      \"pmids\": [\"9315307\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GPX4 is a selenocysteine-containing lipid hydroperoxidase that reduces phospholipid hydroperoxides to non-toxic lipid alcohols using glutathione as the reducing substrate, thereby preventing iron-dependent lipid peroxidation and ferroptosis; its activity, stability, and subcellular localization are regulated at multiple levels including selenocysteine-based catalysis (superior to Cys for resisting overoxidation), alternative promoter-driven isoform expression (cytosolic, mitochondrial, nuclear), and an extensive post-translational modification network encompassing K48/K63/M1-linked ubiquitination by E3 ligases (STUB1, NEDD4L, CUL1-FBW7) and stabilization by deubiquitinases (USP8, OTUD5, OTUB1), S-palmitoylation at C66/C75 by ZDHHC20/ZDHHC8 (reversed by APT2), arginine-152 methylation by PRMT5, and serine-104 phosphorylation by CKB; direct covalent modification at C66 by itaconate allosterically activates GPX4; copper-induced ubiquitination at C107/C148 and chaperone-mediated autophagy (legumain/HSC70) or selective autophagy (TAX1BP1, TRAF6-p62) drive its degradation; transcriptional control by STAT3, KLF11, and epigenetic mechanisms (m6A via METTL3/ALKBH5/FTO, DNMT-mediated promoter methylation) modulates GPX4 levels; functionally, GPX4 is essential for TFH cell survival, innate immune STING activation (by preventing lipid peroxidation-induced STING carbonylation at C88), and host resistance to Mycobacterium tuberculosis, and in adipocytes it suppresses metabolic inflammation independently of overt ferroptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GPX4 is a selenocysteine-dependent glutathione peroxidase that constitutes the central cellular defense against iron-dependent lipid peroxidation, and its loss precipitates ferroptotic cell death [#0, #1]. It is uniquely capable of reducing esterified phospholipid hydroperoxides within membranes to non-toxic lipid alcohols, a reaction no other enzyme performs in a cellular context [#1, #6]; an electrostatic cationic patch mediates membrane and cardiolipin binding adjacent to the catalytic site [#8], and the selenolate active-site catalysis is specifically required to resist irreversible overoxidation, distinguishing GPX4 from a cysteine variant and being essential for neuronal survival in vivo [#2]. This antioxidant function makes GPX4 a survival dependency in therapy-resistant high-mesenchymal persister cancer cells [#3], and a patient-derived R152H active-site loop mutation that abolishes activity links GPX4 dysfunction to human disease [#6]. Beyond its canonical hydroperoxidase role, GPX4 acts as a protein-thiol peroxidase that cross-links mitochondrial capsule proteins during spermatogenesis [#5], and alternative-promoter usage generates cytosolic, mitochondrial, and arginine-rich nuclear isoforms with distinct localizations [#9, #28]. GPX4 abundance is set by a dense post-translational network: stabilizing palmitoylation at C66/C75 (ZDHHC20/ZDHHC8, reversed by APT2) [#18, #19], R152 symmetric dimethylation by PRMT5 that excludes the Cullin1-FBW7 ligase [#20], S104 phosphorylation by CKB that blocks chaperone-mediated autophagy [#11], stabilizing deubiquitination (USP8, OTUD5, OTUB1 recruited by CST1) and linear ubiquitination by LUBAC [#13, #14, #15, #16], opposed by degradative ubiquitination (STUB1 at K191, NEDD4L, copper-induced modification of C107/C148) and selective/chaperone-mediated autophagy via TAX1BP1, p62/TRAF6, legumain-HSC70, and FUNDC1-mediated mitochondrial import [#10, #22, #12, #24, #34], with transcriptional and m6A control by STAT3 and the PKA-ALKBH5 axis [#25, #32]. Functionally, GPX4-maintained redox homeostasis sustains follicular helper T cell and germinal center responses [#29], enables cGAS-STING innate immunity by preventing lipid-peroxidation-driven STING carbonylation at C88 [#4], confers host resistance to Mycobacterium tuberculosis [#30], and in adipocytes suppresses metabolic inflammation independently of overt ferroptotic death [#31]. Direct covalent modification of C66 by itaconate allosterically activates the enzyme to protect neurons [#26].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established the catalytic basis for GPX4's distinctive substrate range, answering how a glutathione peroxidase could act on bulky lipid hydroperoxides and protein thiols.\",\n      \"evidence\": \"Structural and kinetic analysis with protein-binding assays defining a Sec/Gln/Trp catalytic triad and broad donor specificity\",\n      \"pmids\": [\"9315326\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not resolve the membrane-binding mode\", \"Protein-thiol substrates not identified at residue level\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Showed that selenocysteine incorporation machinery, not the GPX4 coding sequence alone, limits functional enzyme production, framing selenium availability as a control point.\",\n      \"evidence\": \"Co-transfection of PHGPx with selenophosphate synthase (SelD) in endothelial cells with activity and cytotoxicity readouts\",\n      \"pmids\": [\"9315307\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cell type\", \"Did not address endogenous regulation of Sec machinery\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrated a protein-thiol peroxidase moonlighting activity, explaining the selenium dependency of spermatogenesis beyond simple antioxidant function.\",\n      \"evidence\": \"In vitro biochemistry with H2O2, thiol labeling and SDS-PAGE of mitochondrial capsule protein cross-linking\",\n      \"pmids\": [\"11568459\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro only\", \"Identity of all physiological thiol substrates incomplete\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Connected GPX4 expression to cell survival under cystine/glutathione limitation, an early anti-apoptotic/growth-promoting role.\",\n      \"evidence\": \"Expression cloning in Burkitt lymphoma cells with overexpression rescue of oxidative apoptosis\",\n      \"pmids\": [\"11568455\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Predates ferroptosis framework\", \"Mechanism of protection not at molecular resolution\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the molecular origin of the nuclear GPX4 isoform, answering how subcellular targeting diversity arises without alternative splicing.\",\n      \"evidence\": \"5'-RACE, RT-PCR, S1 nuclease and in situ hybridization identifying an intron-1 alternative promoter generating an Arg-rich N-terminus\",\n      \"pmids\": [\"12751792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation of alternative promoter not defined\", \"Function of nuclear isoform beyond chromatin binding unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Provided direct evidence that mitochondrial GPX4 protects organelle function, linking isoform localization to physiological cardioprotection.\",\n      \"evidence\": \"Adenoviral mPHGPx/cPHGPx overexpression in cardiomyocytes with MDA/HNE, cytochrome c release and complex IV assays in I/R\",\n      \"pmids\": [\"14583338\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Overexpression only\", \"Endogenous mitochondrial GPX4 contribution not isolated\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified GPX4 as the essential regulator of ferroptosis, defining the cell death modality it suppresses.\",\n      \"evidence\": \"Chemoproteomic target identification of RSL3 plus GPX4 gain/loss-of-function across multiple inducers and cell lines\",\n      \"pmids\": [\"24439385\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymatic inhibition by RSL3 later questioned\", \"Endogenous lipid substrate spectrum not fully mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Explained why GPX4 uses selenocysteine rather than cysteine, establishing resistance to overoxidation as the catalytic rationale for an essential enzyme.\",\n      \"evidence\": \"Gpx4 Sec-to-Cys knock-in mice and conditional neuronal knockouts with peroxide sensitivity assays\",\n      \"pmids\": [\"29290465\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of selenolate regeneration in cells not fully defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Revealed GPX4 as a selective vulnerability of drug-tolerant persister cancer cells, giving therapeutic relevance to the ferroptosis dependency.\",\n      \"evidence\": \"GPX4 inhibition/knockdown in mesenchymal persister cells with ferroptosis assays and in vivo relapse models\",\n      \"pmids\": [\"29088702\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why the mesenchymal state imposes GPX4 dependency not fully mechanistic\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined GPX4 as the glutathione-dependent lipid hydroperoxidase and positioned FSP1 as a parallel, GPX4-independent antioxidant arm of ferroptosis defense.\",\n      \"evidence\": \"Synthetic-lethal CRISPR screen, biochemical oxidoreductase assays and xenografts\",\n      \"pmids\": [\"31634900\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosstalk thresholds between GPX4 and FSP1 pathways not quantified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked GPX4 redox control to innate immunity, showing lipid peroxidation carbonylates STING and blocks its trafficking.\",\n      \"evidence\": \"GPX4 knockdown/knockout with STING C88 carbonylation, ER-to-Golgi trafficking assays and viral infection models\",\n      \"pmids\": [\"32541831\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct lipid species mediating STING carbonylation not pinned down\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the structural basis of membrane and DNA engagement, explaining how GPX4 reaches membrane lipid substrates and how the nuclear isoform targets chromatin.\",\n      \"evidence\": \"First NMR assignments of GPX4 with mutagenesis of a cationic patch and lipid/DNA binding assays\",\n      \"pmids\": [\"34492183\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Membrane orientation during catalysis not directly visualized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established GPX4 as required for follicular helper T cell survival, extending the selenium-GPX4-ferroptosis axis to adaptive immunity.\",\n      \"evidence\": \"T cell-specific Gpx4 conditional knockout with immunization, flow cytometry and selenium supplementation\",\n      \"pmids\": [\"34413521\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why TFH cells are selectively vulnerable not fully explained\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Initiated the degradation arm of GPX4 regulation, showing chaperone-mediated autophagy controls GPX4 turnover in injury.\",\n      \"evidence\": \"Co-IP of legumain with HSC70/HSP90/GPX4, lysosomal fractionation and legumain-KO IRI model\",\n      \"pmids\": [\"33431801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab Co-IP\", \"Direct CMA targeting motif on GPX4 not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established GPX4 as the only enzyme reducing esterified phospholipid hydroperoxides in cells and connected an active-site-loop mutation to human disease.\",\n      \"evidence\": \"Structure-based analysis, patient fibroblasts and enzymatic/stability assays of R152H\",\n      \"pmids\": [\"34931062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full clinical spectrum of GPX4 mutations not enumerated here\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified an allosteric regeneration site distinct from the active site, defining a druggable conformational control point.\",\n      \"evidence\": \"Six co-crystal structures of inhibitors with biophysical and activity assays\",\n      \"pmids\": [\"36423641\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous ligand of the allosteric site unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Began assembling the ubiquitin-stabilization network by showing LUBAC adds protective linear ubiquitin chains to GPX4.\",\n      \"evidence\": \"Co-IP, M1-linkage ubiquitination assays and LUBAC-deficient ferroptosis sensitivity\",\n      \"pmids\": [\"36279464\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Site of M1 attachment not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed deubiquitinase recruitment stabilizes GPX4, identifying CST1-OTUB1 as a protective module.\",\n      \"evidence\": \"Co-IP/MS, ubiquitination assays and in vivo metastasis models\",\n      \"pmids\": [\"36369321\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Generality across tissues untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended GPX4's protective role to infection, defining the GPX4/GSH axis as a tuberculosis host-resistance determinant.\",\n      \"evidence\": \"Gpx4-deficient and -transgenic mice in Mtb infection with ferrostatin-1 rescue\",\n      \"pmids\": [\"36069923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-intrinsic vs tissue-level contributions not fully separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a ferroptosis-independent metabolic function of GPX4 in adipocytes restraining inflammation.\",\n      \"evidence\": \"Two adipocyte-specific Gpx4 KO lines with metabolic phenotyping, cytokine and TUNEL assays\",\n      \"pmids\": [\"35031697\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lipid mediators driving inflammation not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined copper as a direct trigger of GPX4 autophagic degradation, coupling metal stress to ferroptosis.\",\n      \"evidence\": \"Co-IP, ubiquitination/aggregation assays mapping Cu binding to C107/C148 and TAX1BP1 receptor in pancreatic cancer models\",\n      \"pmids\": [\"36622894\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Stoichiometry of copper binding undefined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified CKB as a non-canonical kinase that phosphorylates GPX4 to block its CMA degradation, linking growth-factor signaling to ferroptosis resistance.\",\n      \"evidence\": \"In vitro kinase assay, Co-IP, S104 mutagenesis, CMA assays and HCC tumor models\",\n      \"pmids\": [\"37156912\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase reversing S104 not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed USP8 directly deubiquitinates and stabilizes GPX4, adding a DUB to the protective network.\",\n      \"evidence\": \"Co-IP, deubiquitination assays and intestinal-epithelial Usp8 conditional KO mice\",\n      \"pmids\": [\"38598341\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Linkage type removed not specified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified OTUD5 as a stabilizing DUB whose autophagic loss drives GPX4 decay during ischemia-reperfusion.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, spatial transcriptomics and AAV-OTUD5 rescue in IRI\",\n      \"pmids\": [\"38110369\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Ubiquitin site on GPX4 not mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a ubiquitin-linkage switch in which TRIM26 builds stabilizing K63 chains, illustrating linkage-type control of GPX4 fate.\",\n      \"evidence\": \"Co-IP, K48/K63 linkage assays and K107/K117 mutagenesis in glioma\",\n      \"pmids\": [\"37872147\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"PLK1-TRIM26 axis generality untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified STUB1 as a degradative E3 ligase ubiquitinating GPX4 at K191, defining a pro-ferroptotic destabilizer.\",\n      \"evidence\": \"Co-IP, K191 mutagenesis, genetic rescue and in vivo GIST tumor models\",\n      \"pmids\": [\"38110356\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Conditions selecting STUB1 over stabilizers unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established direct transcriptional control of GPX4 by STAT3, defining a targetable input to GPX4 abundance.\",\n      \"evidence\": \"ChIP, dual-luciferase reporter, STAT3 inhibition and xenografts in pancreatic cancer\",\n      \"pmids\": [\"35859150\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Other transcription factors at the locus not co-assessed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Added NEDD4L as a degradative E3 ligase of GPX4 in a granulosa-cell ferroptosis context.\",\n      \"evidence\": \"Co-IP and ubiquitination assays with viability and ferroptosis markers\",\n      \"pmids\": [\"36662677\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without in vivo validation\", \"Ubiquitination site not mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed itaconate directly alkylates C66 to allosterically activate GPX4, defining an endogenous metabolite as a positive regulator.\",\n      \"evidence\": \"Bioorthogonal ITalk probe, phosphatidylcholine-hydroperoxide activity assay and Irg1 mouse models in ICH\",\n      \"pmids\": [\"38719928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of allosteric activation not solved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined an m6A layer of control, with PKA-driven ALKBH5 degradation increasing GPX4 mRNA stability.\",\n      \"evidence\": \"PKA kinase assays, ALKBH5 degradation, GPX4 mRNA m6A quantification and ALKBH5 KO\",\n      \"pmids\": [\"39901038\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"m6A reader for GPX4 transcript not identified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked tumor suppressor p53 to GPX4 turnover via transcriptional repression of the DUB OTUD5.\",\n      \"evidence\": \"Co-IP, deubiquitination assay, Otud5 CRISPR KO, p53-OTUD5 luciferase reporter and tumor models\",\n      \"pmids\": [\"40070026\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Relation to other p53-ferroptosis routes unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified S-palmitoylation at C66 by ZDHHC20 (reversed by APT2) as a stability switch for GPX4.\",\n      \"evidence\": \"Acyl-RAC, C66 mutagenesis, ZDHHC20/APT2 perturbation and multiple in vivo tumor models\",\n      \"pmids\": [\"39833225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interplay between C66 palmitoylation and C66 itaconate alkylation not reconciled\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Added ZDHHC8-mediated C75 palmitoylation as a second stabilizing acylation and a pharmacological target.\",\n      \"evidence\": \"Palmitoylation assays, C75 mutagenesis, PF-670462 and xenograft/CD8 T cell co-culture\",\n      \"pmids\": [\"40108413\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Relative contribution of C66 vs C75 palmitoylation unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed PRMT5-mediated R152 methylation stabilizes GPX4 by excluding the Cullin1-FBW7 ligase, adding arginine methylation to the PTM network.\",\n      \"evidence\": \"In vitro methylation, Co-IP, R152 mutagenesis, ubiquitination assays and tumor models\",\n      \"pmids\": [\"40033101\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Demethylase for R152 not identified\", \"Relationship of R152 methylation to the R152H disease mutation not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined TRAF6-p62 selective autophagy as a route for GPX4 degradation and validated it with a degrader strategy.\",\n      \"evidence\": \"Co-IP, ubiquitination/autophagy flux assays, AUTAC design, organoids and tumor models\",\n      \"pmids\": [\"40394165\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Ubiquitin site engaged by p62 not mapped\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed FUNDC1 recruits GPX4 into mitochondria for mitophagic degradation, coupling subcellular import to GPX4 turnover.\",\n      \"evidence\": \"Domain-mapped Co-IP, colocalization, mitochondrial fractionation and FUNDC1-KO liver injury model\",\n      \"pmids\": [\"36828120\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"How import competes with cytosolic GPX4 function unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether widely used inhibitors directly inhibit GPX4 enzymatic activity remains unsettled, complicating interpretation of decades of pharmacological ferroptosis data.\",\n      \"evidence\": \"In vitro assays showing RSL3 and ML162 fail to inhibit recombinant selenoprotein GPX4 and instead inhibit TXNRD1\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Need reconciliation of cellular target engagement with recombinant-enzyme data\", \"Identity of true cellular targets of these compounds incompletely resolved\", \"Whether allosteric/covalent inhibition occurs only on membrane-bound or modified GPX4 untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 1, 5, 6, 27]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [1, 6, 26]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"GO:0016209\", \"supporting_discovery_ids\": [0, 1, 28]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [9, 24]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [24, 28]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4, 29, 30]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [11, 18, 20, 22, 34]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [10, 12, 34, 24]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"STING\", \"CKB\", \"PRMT5\", \"ZDHHC20\", \"USP8\", \"OTUD5\", \"STUB1\", \"TRAF6\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}