{"gene":"ACSL4","run_date":"2026-06-09T22:02:39","timeline":{"discoveries":[{"year":2016,"finding":"ACSL4 is an essential component for ferroptosis execution; Gpx4-Acsl4 double-knockout cells show marked resistance to ferroptosis, placing ACSL4 downstream of or parallel to GPX4. Mechanistically, ACSL4 enriches cellular membranes with long polyunsaturated ω6 fatty acids, providing peroxidation substrates required for ferroptosis.","method":"Genome-wide CRISPR-based genetic screen, microarray analysis of resistant cell lines, Gpx4/Acsl4 double-knockout mouse genetics, lipidomics","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — two independent discovery approaches (CRISPR screen + transcriptomics), genetic epistasis with double-KO, lipidomic mechanistic validation, replicated across studies","pmids":["27842070"],"is_preprint":false},{"year":2016,"finding":"ACSL4-mediated production of 5-hydroxyeicosatetraenoic acid (5-HETE) contributes to ferroptosis; knockdown of ACSL4 inhibits erastin-induced ferroptosis in sensitive cells, while overexpression restores ferroptosis sensitivity in resistant cells.","method":"shRNA knockdown, gene transfection overexpression, pharmacological inhibition of 5-HETE production (zileuton), cell death assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function in multiple cell lines, single lab, pharmacological corroboration","pmids":["27565726"],"is_preprint":false},{"year":1998,"finding":"ACSL4 (FACL4) encodes a functional long-chain fatty acid-CoA ligase with substrate preference for arachidonic acid; the gene is located on chromosome Xq23 and is highly expressed in brain, placenta, testis, ovary, spleen, and adrenal cortex.","method":"cDNA cloning, enzymatic activity assay, Northern hybridization, FISH chromosomal localization","journal":"Genomics","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct enzymatic characterization with substrate specificity, cloning and expression, replicated in multiple studies","pmids":["9598324"],"is_preprint":false},{"year":1998,"finding":"FACL4 (ACSL4) encodes a long-chain acyl-CoA synthetase of 670 amino acids (711 in brain isoform); the gene is deleted in patients with Alport syndrome with mental retardation, establishing loss of ACSL4 enzymatic function as contributing to the MR phenotype.","method":"Genomic deletion mapping, RACE cloning, Northern blot, sequence conservation analysis","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct mapping of deletion to clinical phenotype, structural characterization of protein, single study","pmids":["9480748"],"is_preprint":false},{"year":2002,"finding":"Point mutations in FACL4 (ACSL4) causing reduced enzymatic activity lead to nonspecific X-linked mental retardation; analysis of lymphoblastoid cell lines from affected individuals showed low levels of fatty acid-CoA ligase enzymatic activity, confirming loss-of-function.","method":"Mutation identification (missense and splice-site), enzymatic activity assay in lymphoblastoid cell lines, X-inactivation analysis","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct enzymatic activity measurement in patient-derived cells, two independent families, functional null mutations confirmed","pmids":["11889465"],"is_preprint":false},{"year":2003,"finding":"A missense mutation (P375L) in the first luciferase domain of FACL4 markedly reduces enzymatic activity and co-segregates with MRX68; a rapid enzymatic assay on peripheral blood is sufficient for diagnostic screening.","method":"Mutation screening, enzymatic activity assay on peripheral blood, co-segregation analysis","journal":"Journal of medical genetics","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — enzymatic assay in patient-derived cells, single lab, confirms functional domain importance","pmids":["12525535"],"is_preprint":false},{"year":2022,"finding":"PKCβII phosphorylates and activates ACSL4, amplifying lipid peroxidation and ferroptosis; PKCβII acts as a sensor of initial lipid peroxides and the lipid peroxidation-PKCβII-ACSL4 positive-feedback axis drives ferroptosis. Activated ACSL4 catalyzes PUFA-containing lipid biosynthesis, leading to accumulation of lipid peroxidation products.","method":"Genome-wide CRISPR-Cas9 screen, kinase inhibitor library screen, phosphorylation assays, lipidomics, in vitro and in vivo functional studies","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — two independent screening approaches, biochemical phosphorylation demonstration, lipidomic validation, in vivo corroboration","pmids":["35027735"],"is_preprint":false},{"year":2022,"finding":"CDK1 directly binds to and phosphorylates ACSL4 at S447, triggering recruitment of E3 ubiquitin ligase UBR5 and polyubiquitination of ACSL4 at K388, K498, and K690, leading to ACSL4 protein degradation and consequent suppression of ferroptosis.","method":"CRISPR/Cas9 screening, direct binding assays, phosphorylation site mutagenesis, ubiquitination assays, mass spectrometry, xenograft models","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — phosphorylation site identified and mutated, ubiquitination sites mapped by MS, in vivo xenograft validation","pmids":["37428466"],"is_preprint":false},{"year":2022,"finding":"IFNγ stimulates ACSL4 and alters tumor cell lipid patterns, increasing incorporation of arachidonic acid into C16 and C18 acyl chain-containing phospholipids, thereby enabling CD8+ T cell-mediated immunogenic tumor ferroptosis. Palmitoleic acid and oleic acid promote ACSL4-dependent tumor ferroptosis induced by IFNγ plus arachidonic acid.","method":"Lipidomics, genetic ACSL4 deletion in tumors, in vivo tumor models, combination IFNγ + fatty acid treatment assays","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — lipidomic mechanistic validation, genetic deletion in vivo, multiple orthogonal approaches","pmids":["35216678"],"is_preprint":false},{"year":2019,"finding":"Sp1 is a transcription factor that increases ACSL4 transcription by binding to the ACSL4 promoter region during ischemia-induced ferroptosis in intestinal ischemia/reperfusion injury.","method":"Promoter binding assay (Sp1 binding to ACSL4 promoter), siRNA knockdown, in vivo and in vitro ischemia/hypoxia models, pharmacological inhibition with rosiglitazone","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter binding demonstrated, siRNA and pharmacological corroboration, single lab","pmids":["30737476"],"is_preprint":false},{"year":2021,"finding":"HIF-1α negatively regulates ACSL4 expression; ACSL4 expression is suppressed in the early phase of ischemic stroke by HIF-1α induction, and overexpression of ACSL4 exacerbates ischemic brain injury via enhanced lipid peroxidation/ferroptosis.","method":"ACSL4 knockdown/overexpression in vivo, HIF-1α manipulation, lipid peroxidation assays, in vivo ischemia models","journal":"Brain, behavior, and immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic gain- and loss-of-function in vivo, HIF-1α regulatory link shown, single lab","pmids":["33444733"],"is_preprint":false},{"year":2022,"finding":"Thrombin promotes arachidonic acid mobilization and subsequent esterification by ACSL4, generating pro-ferroptotic phosphatidylethanolamine lipid products; multi-omics identified thrombin and ACSL4 as prominently altered in middle cerebral artery occlusion. Genetic or pharmacological inhibition of this pathway attenuated ischemic outcomes.","method":"Unbiased multi-omics (proteomics, lipidomics), genetic and pharmacological inhibition of thrombin-ACSL4 pathway, in vitro and in vivo ischemia models","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multi-omics identification, mechanistic pathway validation with inhibitors, single lab","pmids":["35197442"],"is_preprint":false},{"year":2024,"finding":"ACSL4 compound AS-252424 directly binds to glutamine 464 of ACSL4 to inhibit its enzymatic activity, suppressing lipid peroxidation and ferroptosis; identified via kinase inhibitor library screening and validated with binding and enzymatic assays.","method":"Kinase inhibitor library screening, direct binding assay, enzymatic activity assay with site-specific mutation (Q464), nanoparticle delivery in vivo","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct enzymatic inhibition demonstrated, specific binding site identified (Q464), in vivo validation, single lab","pmids":["38552012"],"is_preprint":false},{"year":2021,"finding":"ACSL4-mediated lipid peroxidation/ferroptosis pathway is regulated by the transcription factor SP1, which promotes ACSL4 expression; inhibition of SP1 or ACSL4 rescues Aβ-induced cardiomyocyte lipid peroxidation defects in the context of ALDH2-mediated protection.","method":"SP1 inhibitor (tolfenamic acid), ACSL4 inhibitor (triacsin C), cardiomyocyte in vitro models, ALDH2 transgenic mice","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic corroboration of SP1-ACSL4 axis, multiple inhibitors used, single lab","pmids":["33767380"],"is_preprint":false},{"year":2022,"finding":"GLIA maturation factor-β (GMFB) impairs chaperone-mediated autophagy (CMA) degradation of ACSL4: GMFB translocates ATP6V1A from lysosomes preventing assembly and alkalinizing lysosomes, blocking HSC70-mediated autophagic digestion of ACSL4, leading to ACSL4 accumulation and ferroptosis in retinal pigment epithelial cells.","method":"Protein interaction/co-localization, lysosomal pH assays, CMA pathway perturbation, HSC70 recognition assay, in vitro and in vivo diabetic retinopathy models","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic dissection of CMA-ACSL4 axis with multiple pathway interventions, single lab","pmids":["35325805"],"is_preprint":false},{"year":2023,"finding":"CARM1 methylates ACSL4 at arginine 339 (R339); this methylation promotes binding of E3 ubiquitin ligase RNF25 to ACSL4, leading to ACSL4 ubiquitylation and degradation. Inhibition of CARM1 thus stabilizes ACSL4 and increases ferroptosis sensitivity.","method":"Methylation site identification (R339), RNF25 co-IP and binding assays, ubiquitination assays, in vitro and in vivo ferroptosis assays","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — specific modification site identified, writer (CARM1) and reader/effector (RNF25) characterized, ubiquitination assay, in vivo validation","pmids":["37946697"],"is_preprint":false},{"year":2023,"finding":"CYP1B1-derived 20-HETE activates the protein kinase C pathway to increase FBXO10 expression, which promotes ubiquitination and degradation of ACSL4, rendering tumor cells resistant to ferroptosis.","method":"CYP1B1 manipulation, 20-HETE treatment, FBXO10 expression analysis, ubiquitination assay for ACSL4, ferroptosis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway dissection from CYP1B1 to FBXO10 to ACSL4 ubiquitination, single lab, multiple approaches","pmids":["37059712"],"is_preprint":false},{"year":2023,"finding":"STING directly interacts with ACSL4 at D53 and K412 amino acids of ACSL4 (identified by co-immunoprecipitation and LC-MS/MS); STING induces renal inflammatory response and fibrosis through ACSL4-dependent ferroptosis.","method":"Co-immunoprecipitation, liquid chromatography-tandem mass spectrometry (protein interaction mapping), ACSL4 siRNA, pharmacological inhibition, in vivo kidney injury models","journal":"Molecular therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein interaction identified with interaction sites by MS, functional validation by siRNA and inhibitor, single lab","pmids":["37533255"],"is_preprint":false},{"year":2023,"finding":"ACSL4 regulates lipid metabolism and reduces VGLL4 expression to promote NF-κB signal transduction, driving LPS-induced proinflammatory responses in microglia; knockdown of ACSL4 decreases proinflammatory cytokine production.","method":"ACSL4 knockdown in microglia, NF-κB pathway analysis, VGLL4 expression assays, LPS-induced neuroinflammation models in vitro and in vivo","journal":"Brain, behavior, and immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function in vitro and in vivo, pathway analysis connecting ACSL4 to VGLL4/NF-κB, single lab","pmids":["36791893"],"is_preprint":false},{"year":2023,"finding":"RB1 loss activates E2F transcription factors which directly upregulate ACSL4 expression (ACSL4 is a direct E2F target gene), enriching arachidonic acid-containing phospholipids and sensitizing cells to ferroptosis via an RB/E2F/ACSL4 molecular axis.","method":"RB1 loss/E2F activation genetic models, ACSL4 promoter analysis (E2F target), lipidomics, xenograft tumor models, GPX4 inhibitor treatment in vivo","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct E2F target gene identification, lipidomic validation, in vivo xenograft and genetically engineered models, multiple orthogonal methods","pmids":["36928314"],"is_preprint":false},{"year":2023,"finding":"TRIM28 binds to ACSL4 and promotes SUMO3 modification of ACSL4 at lysine 532, inhibiting K63-linked ACSL4 ubiquitination and thereby suppressing OPTN-dependent autophagic degradation of ACSL4, leading to ACSL4 accumulation and neuronal ferroptosis. SENP3 was identified as the deSUMOylation enzyme that reverses this process.","method":"Co-IP (TRIM28-ACSL4 binding), SUMOylation site mapping (K532), ubiquitination assays, autophagy-receptor (OPTN) analysis, Trim28 genetic deletion in mice, SCI model","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — specific SUMOylation site identified, writer (TRIM28) and eraser (SENP3) defined, ubiquitination cross-talk mapped, in vivo genetic validation","pmids":["39875520"],"is_preprint":false},{"year":2023,"finding":"AIM2 promotes FOXO3a phosphorylation and proteasome degradation, reducing FOXO3a transcriptional activation of ACSL4 and inhibiting ferroptosis in renal cell carcinoma; this pathway drives sunitinib resistance.","method":"AIM2 overexpression/knockdown, FOXO3a phosphorylation and degradation assays, ACSL4 transcriptional regulation assays, ferroptosis assays","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptional regulation pathway dissected, phosphorylation of FOXO3a linked to ACSL4, single lab","pmids":["36923928"],"is_preprint":false},{"year":2023,"finding":"Radiation-induced ACSL4 transcription is regulated by the STAT1/IRF1 axis; STAT1 and IRF1 drive ACSL4 upregulation in irradiated intestinal epithelial cells, promoting ferroptosis-mediated radiation intestinal injury.","method":"RNA sequencing, ACSL4 promoter/transcription analysis (STAT1/IRF1), ACSL4 knockdown, AMPK activation assays, in vivo radiation injury model","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcription factor axis identified, ACSL4 knockdown functional validation, single lab","pmids":["37611494"],"is_preprint":false},{"year":2024,"finding":"Lactate induces ACSL4 expression via histone H3K18 lactylation at the ACSL4 promoter, and also directly lactylates ACSL4 at K412 (post-translational lactylation); decreased SIRT3 expression elevates ACSL4 lactylation, driving ferroptosis in nucleus pulposus cells during intervertebral disc degeneration.","method":"Single-cell RNA sequencing, H3K18 lactylation assay at ACSL4 promoter, ACSL4 K412 lactylation site identification, SIRT3 expression analysis, AAV9-siLdha in vivo, 2-DG treatment","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — specific lactylation site on ACSL4 (K412) identified, histone lactylation at promoter shown, in vivo validation, single lab","pmids":["40171826"],"is_preprint":false},{"year":2024,"finding":"METTL3-mediated m6A modification is enriched in ACSL4 mRNA and its stability is regulated through a YTHDC1-dependent pathway; lactate promotes p300-mediated H3K18la binding to the METTL3 promoter, upregulating METTL3 which then stabilizes ACSL4 mRNA, promoting ferroptosis in alveolar epithelial cells.","method":"m6A modification assays, METTL3 knockdown/inhibition, YTHDC1 pathway analysis, H3K18la ChIP, GPR81 signaling assays, in vitro and in vivo sepsis models","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m6A modification on ACSL4 mRNA demonstrated, YTHDC1 reader identified, histone lactylation-driven transcription upstream characterized, single lab","pmids":["38852200"],"is_preprint":false},{"year":2024,"finding":"FTO demethylase activity targets ACSL4 and TFRC mRNA stability in an m6A-dependent manner; FTO downregulation in older livers increases ACSL4 and TFRC expression, exacerbating ferroptosis during ischemia/reperfusion injury.","method":"Mass spectrometry (FTO identification), FTO overexpression in vivo, m6A modification assays for ACSL4 mRNA, mRNA stability assays, older vs. young liver comparison","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — m6A eraser (FTO) mechanistically linked to ACSL4 mRNA stability, in vivo gain-of-function, mass spectrometry identification, multiple orthogonal methods","pmids":["38834654"],"is_preprint":false},{"year":2023,"finding":"MMD physically interacts with both ACSL4 and MBOAT7 (two enzymes catalyzing sequential steps of arachidonic acid incorporation into phosphatidylinositol), increasing flux of AA into PI and other AA-containing phospholipid species, thereby promoting ferroptosis susceptibility.","method":"Co-IP (MMD-ACSL4 and MMD-MBOAT7 interactions), lipidomics, genetic loss-of-function in ovarian and renal carcinoma cells","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct protein-protein interaction demonstrated by Co-IP, lipidomic consequences quantified, functional ferroptosis outcomes measured, single lab","pmids":["37691145"],"is_preprint":false},{"year":2024,"finding":"ACSL4 promotes metastatic extravasation by enhancing membrane fluidity and cellular invasiveness through PUFA-lipid incorporation; ACSL4 is a pro-hematogenous metastasis factor identified by metabolism-focused in vivo CRISPR screens in ovarian cancer.","method":"Two rounds of in vivo CRISPR screen in mouse ovarian cancer metastasis model, membrane fluidity assays, invasion assays, ACSL4 genetic deletion","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo CRISPR screen replicated with two rounds of selection, membrane fluidity mechanistic assays, multiple cancer models","pmids":["39591965"],"is_preprint":false},{"year":2024,"finding":"ACSL4-mediated phospholipid remodeling of cell membranes induces lipid-raft localization and activation of integrin β1 in a CD47-dependent manner, leading to downstream focal adhesion kinase phosphorylation that promotes TNBC metastasis.","method":"Lipidomics of metastatic vs. primary TNBC, lipid raft fractionation, integrin β1 activation assays, CD47 dependence assays, FAK phosphorylation assays, in vivo pharmacological ACSL4 inhibition","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — lipidomic mechanism, specific integrin activation pathway, CD47 dependence established, in vivo validation, multiple orthogonal methods","pmids":["38471082"],"is_preprint":false},{"year":2024,"finding":"ACSL4-mediated lipid peroxidation promotes lipid raft formation in melanoma cell membranes, which inhibits immunogenic ferroptosis and pyroptosis by reducing cell membrane pore formation; disruption of ACSL4-mediated lipid rafts (by cholesterol removal) promotes immunogenic cell death.","method":"Lipid raft isolation, ferroptosis/pyroptosis assays, cholesterol depletion experiments, ACSL4 manipulation, immune cell co-culture","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — lipid raft mechanistic link to membrane pore formation established, pharmacological and genetic approaches, single lab","pmids":["39343834"],"is_preprint":false},{"year":2024,"finding":"ACSL4 modulates fatty acid oxidation (FAO) and intracellular acetyl-CoA levels, leading to hyperacetylation of H3K9ac and H3K27ac marks and overexpression of SNAIL, driving TNBC metastasis via an epigenetic mechanism.","method":"Global transcriptome analysis, acetyl-CoA metabolic assays, histone acetylation (H3K9ac, H3K27ac) quantification, ACSL4 genetic ablation/pharmacological inhibition, in vivo metastasis models","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic link from ACSL4-FAO to acetyl-CoA to histone acetylation to SNAIL established, multiple methods, single lab","pmids":["39700137"],"is_preprint":false},{"year":2023,"finding":"ACSL4 upregulates the lipogenesis master regulator SREBP1 and its downstream lipogenic enzymes via c-Myc in HCC cells, promoting de novo lipogenesis and accumulation of triglycerides, cholesterols, and lipid droplets; SREBP1 is required for ACSL4-mediated lipogenesis and oncogenic capabilities.","method":"ACSL4 knockdown/overexpression, SREBP1 rescue experiments, c-Myc pathway analysis, lipid droplet quantification, in vitro and in vivo HCC models","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway dissected via rescue experiments, multiple lipid readouts, in vivo corroboration, single lab","pmids":["33340617"],"is_preprint":false},{"year":2022,"finding":"ACSL4 and LPCAT2 overexpression together sensitize cells to RSL3-induced ferroptosis; mitochondrial ROS formation and mitochondrial membrane potential deterioration are essential downstream events in ACSL4/LPCAT2-driven ferroptosis, and mitoquinone (MitoQ) protection confirms mitochondrial ROS as a key mediator.","method":"ACSL4 and LPCAT2 co-overexpression in HEK293T cells, ferroptosis assays, mitochondrial ROS measurement, mitochondrial membrane potential assay, mitochondrial respiration assay, MitoQ treatment","journal":"Antioxidants (Basel, Switzerland)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct overexpression reconstitution, multiple mitochondrial parameter readouts, pharmacological confirmation, single lab","pmids":["37627584"],"is_preprint":false},{"year":2022,"finding":"FUNDC1 (mitophagy receptor) co-immunoprecipitates with ACSL4, indicating a direct protein-protein interaction; FUNDC1 deficiency leads to upregulation of ACSL4 and enhanced ferroptosis in cardiomyocytes under high-fat diet challenge.","method":"Co-immunoprecipitation (FUNDC1-ACSL4), FUNDC1 knockout mice, ACSL4 expression analysis, ferroptosis assays, in vitro arachidonic acid treatment","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct Co-IP interaction, genetic knockout model, functional ferroptosis readout, single lab","pmids":["39326685"],"is_preprint":false},{"year":2023,"finding":"HIV-1 Tat protein downregulates miR-204 in microglia, releasing its suppression of ACSL4, leading to increased ACSL4 expression, oxidized phosphatidylethanolamine generation, lipid peroxidation, and ferroptosis-driven microglial activation with proinflammatory cytokine release.","method":"miR-204 mimic transfection, ACSL4 gene silencing, lipid peroxidation assays, co-immunoprecipitation, HIV-1 transgenic rat validation, human HIV+ brain sample analysis","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — miR-204 upstream regulation validated, ACSL4 silencing confirmation, validated in animal model and human samples, single lab","pmids":["37023693"],"is_preprint":false},{"year":2025,"finding":"HAT1 directly promotes acetylation of ACSL4 at lysine 383, enhancing its protein stability; SIRT3 mediates deacetylation of ACSL4, while HDAC2 enhances ACSL4 acetylation by inhibiting SIRT3 transcription. Acetylation at K383 inhibits FBXO10-mediated K48-linked ubiquitination of ACSL4, stabilizing the protein.","method":"Acetylation site mapping (K383), HAT1/SIRT3/HDAC2 manipulation, ubiquitination assays, FBXO10 interaction assay, in vitro and in vivo NPC models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — specific acetylation site identified, writer (HAT1) and eraser (SIRT3) defined, ubiquitination cross-talk demonstrated, single lab","pmids":["40050614"],"is_preprint":false},{"year":2025,"finding":"Parkin E3 ubiquitin ligase promotes ubiquitination of ACSL4, inhibiting iron overload-induced ferroptosis in cardiomyocytes; p53 transcriptionally suppresses Parkin expression in iron-overloaded cardiomyocytes, establishing a p53-Parkin-ACSL4 regulatory pathway in cardiac ferroptosis.","method":"Parkin-ACSL4 ubiquitination assays, cardiac-specific Parkin knockout mice (Myh6-CreER/Parkin), p53 transcriptional regulation of Parkin, iron overload and I/R models, ferroptosis inhibitor (Fer-1) rescue","journal":"Acta pharmaceutica Sinica. B","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assay, cardiac-specific KO in vivo, transcriptional regulation by p53 demonstrated, single lab","pmids":["40370554"],"is_preprint":false},{"year":2024,"finding":"TRIM21 (E3 ligase) and USP15 (deubiquitinase) together control ACSL4 protein stability: TRIM21 promotes ACSL4 degradation while USP15 stabilizes it; reduced ACSL4 expression due to excessive TRIM21-mediated degradation underlies imatinib resistance in GISTs.","method":"Co-immunoprecipitation (TRIM21-ACSL4, USP15-ACSL4), shRNA interference, Western blot, xenograft model, GIST patient sample analysis","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP binding, E3 ligase and deubiquitinase both characterized for same substrate, clinical correlation, single lab","pmids":["38182686"],"is_preprint":false},{"year":2022,"finding":"The silibinin (SIL) natural compound directly binds to ACSL4 at the K536-proximal region and inhibits ACSL4 enzymatic activity, mitigating ACSL4-mediated ferroptosis; living cell-target responsive accessibility profiling (LC-TRAP) identified ACSL4 as a SIL target.","method":"LC-TRAP proteomics, biophysical binding assays, SIL-derivatized chemical probe, enzymatic activity assay, site-specific binding region identification (K536-proximal)","journal":"Analytical chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct enzymatic inhibition demonstrated, binding region mapped by LC-TRAP, biophysical corroboration, single lab","pmids":["36260072"],"is_preprint":false},{"year":2023,"finding":"MFN2 overexpression suppresses the mitochondrial translocation of ACSL4, inhibiting mitochondria-associated ferroptosis; PRDX2-mediated suppression of oxidative stress operates upstream via MFN2-dependent mitochondrial dynamics to prevent ACSL4 mitochondrial localization.","method":"MFN2 overexpression, ACSL4 mitochondrial localization by immunofluorescence, ferroptosis assays in db/db mice and cardiac microvascular endothelial cells, PRDX2 overexpression","journal":"Diabetes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mitochondrial localization of ACSL4 directly shown by immunofluorescence, MFN2 gain-of-function effect on ACSL4 localization, in vivo corroboration, single lab","pmids":["36367849"],"is_preprint":false},{"year":2024,"finding":"Mitochondria-localized AMPKα1 phosphorylation promotes Pink1/Parkin-dependent mitophagy, which inhibits the mitochondrial translocation of ACSL4, suppressing mitochondria-associated ferroptosis in cardiac microvascular endothelial cells during diabetic cardiomyopathy.","method":"AMPKα1 mitochondrial localization assay, mitophagy assay (mt-Keima, TEM), ACSL4 mitochondrial translocation by immunofluorescence, mitoAα1 overexpression, in vivo diabetic model","journal":"Pharmacological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ACSL4 mitochondrial localization directly assayed, AMPKα1-Parkin-ACSL4 pathway dissected with gain-of-function, single lab","pmids":["38218357"],"is_preprint":false},{"year":2023,"finding":"NFIL3 (a circadian rhythm gene) directly regulates ACSL4 expression; NFIL3 knockdown attenuates ferroptosis and inflammation in renal tubular epithelial cells by downregulating ACSL4 in sepsis-associated AKI.","method":"NFIL3 loss-of-function in vitro and in vivo, ACSL4 expression analysis, ferroptosis markers, SA-AKI model","journal":"Cell death discovery","confidence":"Low","confidence_rationale":"Tier 3 / Weak — NFIL3-ACSL4 regulatory link shown by correlation and knockdown, mechanism of direct regulation not biochemically established, single lab","pmids":["39097582"],"is_preprint":false},{"year":2024,"finding":"STAT3 signaling drives ACSL4 expression in tubular epithelial cells; however, in the context of ER stress-induced AKI, ACSL4 activity directs PUFA metabolism toward triglycerides rather than phospholipids, and ACSL4 alone is insufficient to sensitize cells to ferroptosis, highlighting context-dependence.","method":"STAT3 inhibition, lipidomics, ACSL4 expression analysis, mouse ER stress AKI model","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — STAT3-ACSL4 transcriptional link and lipidomic consequences demonstrated, important negative/contextual finding, single lab","pmids":["38799564"],"is_preprint":false},{"year":2025,"finding":"In IBD, ACSL4 is overexpressed in intestinal fibroblasts where it reprograms lipid metabolism and mediates sensitivity of adjacent intestinal epithelial cells to ferroptosis through heterocellular crosstalk; fibroblast-specific ACSL4 overexpression increases epithelial ferroptosis and worsens colitis, while fibroblast ACSL4 deletion ameliorates colitis.","method":"Fibroblast-specific ACSL4 overexpression and deletion in mouse models of colitis, IBD tissue analysis, lipid metabolism assays, ferroptosis markers","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific genetic manipulation (both gain- and loss-of-function), multiple colitis models, heterocellular crosstalk mechanism established","pmids":["40571769"],"is_preprint":false},{"year":2024,"finding":"EGR1 transcriptionally upregulates ACSL4 expression; YAP transcriptionally upregulates EGR1, forming a YAP/EGR1/ACSL4 axis that promotes ferroptosis in ischemic stroke; chromatin immunoprecipitation and dual luciferase assays verified these molecular interactions.","method":"Chromatin immunoprecipitation, dual luciferase assay (EGR1-ACSL4 promoter, YAP-EGR1 promoter), OGD/R neuronal models, MCAO mouse model, YAP/EGR1 overexpression/silencing","journal":"Experimental neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct promoter binding established by ChIP and luciferase for both EGR1-ACSL4 and YAP-EGR1, single lab","pmids":["39889877"],"is_preprint":false}],"current_model":"ACSL4 is a long-chain acyl-CoA synthetase with substrate preference for polyunsaturated fatty acids (especially arachidonic and adrenic acids) that acts as a critical pro-ferroptotic enzyme by esterifying PUFAs into membrane phospholipids, providing the peroxidation substrates required for ferroptosis execution; its activity and stability are tightly regulated by multiple post-translational modifications—including phosphorylation by PKCβII (activating) and CDK1 (triggering UBR5-mediated ubiquitin degradation), SUMOylation by TRIM28 at K532 (stabilizing), acetylation by HAT1 at K383 (stabilizing, blocking FBXO10-mediated degradation), lactylation at K412, m6A-dependent mRNA stabilization via METTL3/YTHDC1, and autophagic degradation via HSC70/CMA or OPTN-selective autophagy—while its transcription is driven by SP1, Sp1, STAT1/IRF1, STAT3, E2F (downstream of RB1), EGR1 (downstream of YAP), and NFIL3, and inhibited by HIF-1α; beyond ferroptosis, ACSL4 promotes tumor metastasis by remodeling membrane phospholipid composition to activate integrin β1 signaling and enhance membrane fluidity, reprograms lipid metabolism through the c-Myc/SREBP1 axis and FAO-dependent histone acetylation, regulates neuroinflammation via VGLL4/NF-κB, and interacts directly with partner proteins including STING, FUNDC1, and MMD/MBOAT7 to modulate disease-specific lipid peroxidation pathways."},"narrative":{"mechanistic_narrative":"ACSL4 (FACL4) is a long-chain acyl-CoA synthetase that activates polyunsaturated fatty acids—with marked preference for arachidonic acid—and esterifies them into membrane phospholipids, thereby supplying the peroxidation substrates that license ferroptotic cell death [PMID:9598324, PMID:27842070]. Loss-of-function point mutations and genomic deletions that abolish its enzymatic activity cause nonspecific X-linked mental retardation and the Alport-syndrome-with-mental-retardation contiguous gene deletion phenotype [PMID:9480748, PMID:11889465, PMID:12525535]. In ferroptosis, ACSL4 acts downstream of or parallel to GPX4: combined Gpx4/Acsl4 ablation confers resistance, and ACSL4 enriches membranes with long ω6 PUFAs that become peroxidation substrates [PMID:27842070], with its partner enzymes MMD/MBOAT7 channeling arachidonate into specific phospholipid species [PMID:37691145]. Its pro-ferroptotic output is set by a dense post-translational and transcriptional control network: PKCβII phosphorylation activates ACSL4 in a lipid-peroxidation feedback loop [PMID:35027735], whereas CDK1 phosphorylation, CARM1 methylation, and several E3 ligases (UBR5, RNF25, FBXO10, TRIM21, Parkin) drive its ubiquitin-dependent degradation, opposed by stabilizing SUMOylation (TRIM28), acetylation (HAT1 at K383), and deubiquitination (USP15) [PMID:37428466, PMID:37946697, PMID:37059712, PMID:39875520, PMID:40050614, PMID:38182686, PMID:40370554]. Transcription is driven by Sp1, STAT1/IRF1, STAT3, E2F downstream of RB1 loss, and a YAP/EGR1 axis, and repressed by HIF-1α, while m6A writers/erasers (METTL3/YTHDC1, FTO) tune ACSL4 mRNA stability [PMID:30737476, PMID:37611494, PMID:36928314, PMID:39889877, PMID:33444733, PMID:38852200, PMID:38834654]. Beyond ferroptosis, ACSL4-driven phospholipid remodeling enhances membrane fluidity and lipid-raft–dependent integrin β1/FAK signaling to promote metastatic extravasation, and reprograms lipid metabolism through c-Myc/SREBP1 lipogenesis and FAO-fueled histone acetylation [PMID:39591965, PMID:38471082, PMID:33340617, PMID:39700137]. ACSL4 mitochondrial translocation is a regulated step that sensitizes cells to mitochondria-associated ferroptosis [PMID:36367849, PMID:38218357, PMID:37627584].","teleology":[{"year":1998,"claim":"Establishing that ACSL4 is an enzyme—a long-chain acyl-CoA synthetase preferring arachidonic acid—defined its core biochemical activity and tissue distribution before any disease or ferroptosis role was known.","evidence":"cDNA cloning, enzymatic activity assays, Northern blot, and FISH localization to Xq23","pmids":["9598324","9480748"],"confidence":"High","gaps":["Structural basis of arachidonate selectivity not resolved","Subcellular distribution of activity not defined"]},{"year":2002,"claim":"Patient missense/splice mutations that reduce enzymatic activity causally linked ACSL4 loss-of-function to X-linked mental retardation, proving the enzyme's physiological importance in brain.","evidence":"Mutation identification with enzymatic activity measurement in patient lymphoblastoid lines and co-segregation/X-inactivation analysis","pmids":["11889465","12525535"],"confidence":"High","gaps":["Neuronal substrate/pathway downstream of ACSL4 in cognition not defined","Mechanism connecting reduced acyl-CoA synthesis to MR unknown"]},{"year":2016,"claim":"Unbiased genetic screens placed ACSL4 as an essential, substrate-supplying enzyme for ferroptosis, reframing it from a generic lipid enzyme to a determinant of a regulated cell death pathway.","evidence":"Genome-wide CRISPR screen, Gpx4/Acsl4 double-knockout genetics, lipidomics, plus shRNA/overexpression with 5-HETE pharmacology","pmids":["27842070","27565726"],"confidence":"High","gaps":["Whether ACSL4 acts upstream, parallel, or downstream of GPX4 not fully resolved","Identity of the critical peroxidized phospholipid species not pinned down in 2016"]},{"year":2022,"claim":"Discovery of PKCβII activating phosphorylation and CDK1/UBR5-mediated degradative phosphorylation showed ACSL4 ferroptotic output is gated by opposing post-translational switches.","evidence":"CRISPR and kinase-inhibitor screens, phosphosite mutagenesis (S447), ubiquitination site mapping (K388/K498/K690), lipidomics, and xenografts","pmids":["35027735","37428466"],"confidence":"High","gaps":["Stoichiometry and interplay between activating and degradative phosphorylation unresolved","Phosphatases reversing these marks not identified"]},{"year":2022,"claim":"Immune and ischemic contexts defined transcriptional and substrate-channeling control of ACSL4: IFNγ/T-cell-driven arachidonate incorporation enables immunogenic ferroptosis, while Sp1 and HIF-1α set opposing transcriptional tone.","evidence":"Lipidomics with genetic ACSL4 deletion in tumors, IFNγ + fatty-acid assays, promoter-binding/siRNA and HIF-1α manipulation in ischemia models","pmids":["35216678","30737476","33444733"],"confidence":"High","gaps":["Direct transcription-factor occupancy at promoter not biochemically resolved for all factors","How substrate availability and transcription are coordinated in vivo unclear"]},{"year":2023,"claim":"A broad regulatory network was mapped: direct partners (STING, FUNDC1, MMD/MBOAT7), additional transcriptional inputs (RB1/E2F, STAT1/IRF1, FOXO3a, c-Myc/SREBP1), and a methylation-coupled degradation axis (CARM1/RNF25, FBXO10) that together tune ACSL4 level and ferroptosis or lipogenesis output.","evidence":"Co-IP/LC-MS interaction mapping, methylation/ubiquitination site assays, lipidomics, promoter analyses, and in vivo disease models across kidney, brain, and cancer","pmids":["37533255","39326685","37691145","36928314","37611494","36923928","33340617","37946697","37059712"],"confidence":"High","gaps":["Whether these partner interactions are direct vs. complex-mediated not uniformly established","Tissue-specific dominance among competing regulators unclear"]},{"year":2024,"claim":"ACSL4 was shown to drive cancer metastasis independent of cell death—remodeling membrane phospholipids to raise fluidity and activate lipid-raft integrin β1/FAK signaling, and rerouting metabolism into lipogenesis and FAO-fueled histone acetylation.","evidence":"In vivo CRISPR metastasis screens, membrane-fluidity and lipid-raft fractionation assays, CD47/integrin β1/FAK readouts, and acetyl-CoA/histone-acetylation profiling","pmids":["39591965","38471082","39700137"],"confidence":"High","gaps":["How a single enzyme partitions between pro-death and pro-metastatic phospholipid pools is unresolved","Direct phospholipid species responsible for raft/integrin activation not fully defined"]},{"year":2024,"claim":"RNA-level (m6A via METTL3/YTHDC1 and FTO) and metabolite-level (lactylation, acetylation) control further refined how ACSL4 abundance and activity respond to the metabolic environment.","evidence":"m6A modification and mRNA-stability assays, lactylation site mapping (K412), acetylation site mapping (K383) with writer/eraser manipulation in disease models","pmids":["38852200","38834654","40171826","40050614"],"confidence":"Medium","gaps":["Cross-talk hierarchy among mRNA, lactylation, and acetylation controls not integrated","Direct catalytic consequence of each PTM on enzyme kinetics not measured"]},{"year":2025,"claim":"Cell-type-resolved genetics revealed ACSL4 acts non-cell-autonomously, with fibroblast ACSL4 dictating ferroptosis sensitivity of neighboring epithelium, expanding its role to heterocellular tissue crosstalk in inflammation.","evidence":"Fibroblast-specific gain- and loss-of-function in multiple colitis models with lipid metabolism and ferroptosis readouts","pmids":["40571769"],"confidence":"High","gaps":["Identity of the transferred lipid mediator between fibroblasts and epithelium unknown","Generalizability beyond intestinal tissue untested"]},{"year":null,"claim":"It remains unresolved how ACSL4 substrate flux is partitioned between pro-ferroptotic phospholipids, neutral lipids/triglycerides, and pro-metastatic raft lipids, and what structural features dictate this context-dependent channeling.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model linking active-site chemistry to lipid product fate","Context-dependence (e.g., ER-stress AKI directing PUFA to triglyceride rather than phospholipid) mechanistically unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[2,3,4,5]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,2,26]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,8,28]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[39,40,32]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[28,29]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,6,19]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[2,31,30]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,43,27]}],"complexes":[],"partners":["MBOAT7","MMD","STING","FUNDC1","UBR5","RNF25","TRIM28","FBXO10"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O60488","full_name":"Long-chain-fatty-acid--CoA ligase 4","aliases":["Arachidonate--CoA ligase","Long-chain acyl-CoA synthetase 4","LACS 4"],"length_aa":711,"mass_kda":79.2,"function":"Catalyzes the conversion of long-chain fatty acids to their active form acyl-CoA for both synthesis of cellular lipids, and degradation via beta-oxidation (PubMed:21242590, PubMed:22633490, PubMed:24269233, PubMed:31061331, PubMed:38720107). Preferentially activates arachidonate and eicosapentaenoate as substrates (PubMed:21242590). Preferentially activates 8,9-EET > 14,15-EET > 5,6-EET > 11,12-EET (PubMed:21242590). Modulates glucose-stimulated insulin secretion by regulating the levels of unesterified EETs (By similarity). Modulates prostaglandin E2 secretion (PubMed:21242590). Acts as an activator of ferroptosis by activating polyunsaturated fatty acids, especially arachidonate and adrenate, to their active form, generating the primary lipid-peroxidation substrates that contribute to ferroptosis (PubMed:27842070, PubMed:35027735, PubMed:38720107)","subcellular_location":"Mitochondrion outer membrane; Endoplasmic reticulum membrane; Cell membrane","url":"https://www.uniprot.org/uniprotkb/O60488/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ACSL4","classification":"Not Classified","n_dependent_lines":93,"n_total_lines":1208,"dependency_fraction":0.07698675496688742},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ACSL4","total_profiled":1310},"omim":[{"mim_id":"612024","title":"OTU DOMAIN-CONTAINING PROTEIN 7A; OTUD7A","url":"https://www.omim.org/entry/612024"},{"mim_id":"607379","title":"NF2, MOESIN-EZRIN-RADIXIN-LIKE (MERLIN) TUMOR SUPPRESSOR; NF2","url":"https://www.omim.org/entry/607379"},{"mim_id":"606608","title":"YES1-ASSOCIATED TRANSCRIPTIONAL REGULATOR; YAP1","url":"https://www.omim.org/entry/606608"},{"mim_id":"300387","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, X-LINKED 63; XLID63","url":"https://www.omim.org/entry/300387"},{"mim_id":"300157","title":"ACYL-CoA SYNTHETASE LONG CHAIN FAMILY, MEMBER 4; ACSL4","url":"https://www.omim.org/entry/300157"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"},{"location":"Mitochondria","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ACSL4"},"hgnc":{"alias_symbol":["ACS4","LACS4"],"prev_symbol":["FACL4","MRX63","MRX68"]},"alphafold":{"accession":"O60488","domains":[{"cath_id":"2.30.38.10","chopping":"50-68_375-571","consensus_level":"medium","plddt":92.3716,"start":50,"end":571},{"cath_id":"3.40.50.12780","chopping":"80-293","consensus_level":"high","plddt":93.28,"start":80,"end":293},{"cath_id":"3.30.300.30","chopping":"575-709","consensus_level":"high","plddt":88.9263,"start":575,"end":709}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O60488","model_url":"https://alphafold.ebi.ac.uk/files/AF-O60488-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O60488-F1-predicted_aligned_error_v6.png","plddt_mean":90.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ACSL4","jax_strain_url":"https://www.jax.org/strain/search?query=ACSL4"},"sequence":{"accession":"O60488","fasta_url":"https://rest.uniprot.org/uniprotkb/O60488.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O60488/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O60488"}},"corpus_meta":[{"pmid":"27842070","id":"PMC_27842070","title":"ACSL4 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Mechanistically, ACSL4 enriches cellular membranes with long polyunsaturated ω6 fatty acids, providing peroxidation substrates required for ferroptosis.\",\n      \"method\": \"Genome-wide CRISPR-based genetic screen, microarray analysis of resistant cell lines, Gpx4/Acsl4 double-knockout mouse genetics, lipidomics\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — two independent discovery approaches (CRISPR screen + transcriptomics), genetic epistasis with double-KO, lipidomic mechanistic validation, replicated across studies\",\n      \"pmids\": [\"27842070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ACSL4-mediated production of 5-hydroxyeicosatetraenoic acid (5-HETE) contributes to ferroptosis; knockdown of ACSL4 inhibits erastin-induced ferroptosis in sensitive cells, while overexpression restores ferroptosis sensitivity in resistant cells.\",\n      \"method\": \"shRNA knockdown, gene transfection overexpression, pharmacological inhibition of 5-HETE production (zileuton), cell death assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function in multiple cell lines, single lab, pharmacological corroboration\",\n      \"pmids\": [\"27565726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"ACSL4 (FACL4) encodes a functional long-chain fatty acid-CoA ligase with substrate preference for arachidonic acid; the gene is located on chromosome Xq23 and is highly expressed in brain, placenta, testis, ovary, spleen, and adrenal cortex.\",\n      \"method\": \"cDNA cloning, enzymatic activity assay, Northern hybridization, FISH chromosomal localization\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct enzymatic characterization with substrate specificity, cloning and expression, replicated in multiple studies\",\n      \"pmids\": [\"9598324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"FACL4 (ACSL4) encodes a long-chain acyl-CoA synthetase of 670 amino acids (711 in brain isoform); the gene is deleted in patients with Alport syndrome with mental retardation, establishing loss of ACSL4 enzymatic function as contributing to the MR phenotype.\",\n      \"method\": \"Genomic deletion mapping, RACE cloning, Northern blot, sequence conservation analysis\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct mapping of deletion to clinical phenotype, structural characterization of protein, single study\",\n      \"pmids\": [\"9480748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Point mutations in FACL4 (ACSL4) causing reduced enzymatic activity lead to nonspecific X-linked mental retardation; analysis of lymphoblastoid cell lines from affected individuals showed low levels of fatty acid-CoA ligase enzymatic activity, confirming loss-of-function.\",\n      \"method\": \"Mutation identification (missense and splice-site), enzymatic activity assay in lymphoblastoid cell lines, X-inactivation analysis\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct enzymatic activity measurement in patient-derived cells, two independent families, functional null mutations confirmed\",\n      \"pmids\": [\"11889465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"A missense mutation (P375L) in the first luciferase domain of FACL4 markedly reduces enzymatic activity and co-segregates with MRX68; a rapid enzymatic assay on peripheral blood is sufficient for diagnostic screening.\",\n      \"method\": \"Mutation screening, enzymatic activity assay on peripheral blood, co-segregation analysis\",\n      \"journal\": \"Journal of medical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — enzymatic assay in patient-derived cells, single lab, confirms functional domain importance\",\n      \"pmids\": [\"12525535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PKCβII phosphorylates and activates ACSL4, amplifying lipid peroxidation and ferroptosis; PKCβII acts as a sensor of initial lipid peroxides and the lipid peroxidation-PKCβII-ACSL4 positive-feedback axis drives ferroptosis. Activated ACSL4 catalyzes PUFA-containing lipid biosynthesis, leading to accumulation of lipid peroxidation products.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 screen, kinase inhibitor library screen, phosphorylation assays, lipidomics, in vitro and in vivo functional studies\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — two independent screening approaches, biochemical phosphorylation demonstration, lipidomic validation, in vivo corroboration\",\n      \"pmids\": [\"35027735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CDK1 directly binds to and phosphorylates ACSL4 at S447, triggering recruitment of E3 ubiquitin ligase UBR5 and polyubiquitination of ACSL4 at K388, K498, and K690, leading to ACSL4 protein degradation and consequent suppression of ferroptosis.\",\n      \"method\": \"CRISPR/Cas9 screening, direct binding assays, phosphorylation site mutagenesis, ubiquitination assays, mass spectrometry, xenograft models\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — phosphorylation site identified and mutated, ubiquitination sites mapped by MS, in vivo xenograft validation\",\n      \"pmids\": [\"37428466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"IFNγ stimulates ACSL4 and alters tumor cell lipid patterns, increasing incorporation of arachidonic acid into C16 and C18 acyl chain-containing phospholipids, thereby enabling CD8+ T cell-mediated immunogenic tumor ferroptosis. Palmitoleic acid and oleic acid promote ACSL4-dependent tumor ferroptosis induced by IFNγ plus arachidonic acid.\",\n      \"method\": \"Lipidomics, genetic ACSL4 deletion in tumors, in vivo tumor models, combination IFNγ + fatty acid treatment assays\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — lipidomic mechanistic validation, genetic deletion in vivo, multiple orthogonal approaches\",\n      \"pmids\": [\"35216678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Sp1 is a transcription factor that increases ACSL4 transcription by binding to the ACSL4 promoter region during ischemia-induced ferroptosis in intestinal ischemia/reperfusion injury.\",\n      \"method\": \"Promoter binding assay (Sp1 binding to ACSL4 promoter), siRNA knockdown, in vivo and in vitro ischemia/hypoxia models, pharmacological inhibition with rosiglitazone\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter binding demonstrated, siRNA and pharmacological corroboration, single lab\",\n      \"pmids\": [\"30737476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HIF-1α negatively regulates ACSL4 expression; ACSL4 expression is suppressed in the early phase of ischemic stroke by HIF-1α induction, and overexpression of ACSL4 exacerbates ischemic brain injury via enhanced lipid peroxidation/ferroptosis.\",\n      \"method\": \"ACSL4 knockdown/overexpression in vivo, HIF-1α manipulation, lipid peroxidation assays, in vivo ischemia models\",\n      \"journal\": \"Brain, behavior, and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic gain- and loss-of-function in vivo, HIF-1α regulatory link shown, single lab\",\n      \"pmids\": [\"33444733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Thrombin promotes arachidonic acid mobilization and subsequent esterification by ACSL4, generating pro-ferroptotic phosphatidylethanolamine lipid products; multi-omics identified thrombin and ACSL4 as prominently altered in middle cerebral artery occlusion. Genetic or pharmacological inhibition of this pathway attenuated ischemic outcomes.\",\n      \"method\": \"Unbiased multi-omics (proteomics, lipidomics), genetic and pharmacological inhibition of thrombin-ACSL4 pathway, in vitro and in vivo ischemia models\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multi-omics identification, mechanistic pathway validation with inhibitors, single lab\",\n      \"pmids\": [\"35197442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ACSL4 compound AS-252424 directly binds to glutamine 464 of ACSL4 to inhibit its enzymatic activity, suppressing lipid peroxidation and ferroptosis; identified via kinase inhibitor library screening and validated with binding and enzymatic assays.\",\n      \"method\": \"Kinase inhibitor library screening, direct binding assay, enzymatic activity assay with site-specific mutation (Q464), nanoparticle delivery in vivo\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct enzymatic inhibition demonstrated, specific binding site identified (Q464), in vivo validation, single lab\",\n      \"pmids\": [\"38552012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ACSL4-mediated lipid peroxidation/ferroptosis pathway is regulated by the transcription factor SP1, which promotes ACSL4 expression; inhibition of SP1 or ACSL4 rescues Aβ-induced cardiomyocyte lipid peroxidation defects in the context of ALDH2-mediated protection.\",\n      \"method\": \"SP1 inhibitor (tolfenamic acid), ACSL4 inhibitor (triacsin C), cardiomyocyte in vitro models, ALDH2 transgenic mice\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic corroboration of SP1-ACSL4 axis, multiple inhibitors used, single lab\",\n      \"pmids\": [\"33767380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"GLIA maturation factor-β (GMFB) impairs chaperone-mediated autophagy (CMA) degradation of ACSL4: GMFB translocates ATP6V1A from lysosomes preventing assembly and alkalinizing lysosomes, blocking HSC70-mediated autophagic digestion of ACSL4, leading to ACSL4 accumulation and ferroptosis in retinal pigment epithelial cells.\",\n      \"method\": \"Protein interaction/co-localization, lysosomal pH assays, CMA pathway perturbation, HSC70 recognition assay, in vitro and in vivo diabetic retinopathy models\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic dissection of CMA-ACSL4 axis with multiple pathway interventions, single lab\",\n      \"pmids\": [\"35325805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CARM1 methylates ACSL4 at arginine 339 (R339); this methylation promotes binding of E3 ubiquitin ligase RNF25 to ACSL4, leading to ACSL4 ubiquitylation and degradation. Inhibition of CARM1 thus stabilizes ACSL4 and increases ferroptosis sensitivity.\",\n      \"method\": \"Methylation site identification (R339), RNF25 co-IP and binding assays, ubiquitination assays, in vitro and in vivo ferroptosis assays\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — specific modification site identified, writer (CARM1) and reader/effector (RNF25) characterized, ubiquitination assay, in vivo validation\",\n      \"pmids\": [\"37946697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CYP1B1-derived 20-HETE activates the protein kinase C pathway to increase FBXO10 expression, which promotes ubiquitination and degradation of ACSL4, rendering tumor cells resistant to ferroptosis.\",\n      \"method\": \"CYP1B1 manipulation, 20-HETE treatment, FBXO10 expression analysis, ubiquitination assay for ACSL4, ferroptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway dissection from CYP1B1 to FBXO10 to ACSL4 ubiquitination, single lab, multiple approaches\",\n      \"pmids\": [\"37059712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"STING directly interacts with ACSL4 at D53 and K412 amino acids of ACSL4 (identified by co-immunoprecipitation and LC-MS/MS); STING induces renal inflammatory response and fibrosis through ACSL4-dependent ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation, liquid chromatography-tandem mass spectrometry (protein interaction mapping), ACSL4 siRNA, pharmacological inhibition, in vivo kidney injury models\",\n      \"journal\": \"Molecular therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein interaction identified with interaction sites by MS, functional validation by siRNA and inhibitor, single lab\",\n      \"pmids\": [\"37533255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ACSL4 regulates lipid metabolism and reduces VGLL4 expression to promote NF-κB signal transduction, driving LPS-induced proinflammatory responses in microglia; knockdown of ACSL4 decreases proinflammatory cytokine production.\",\n      \"method\": \"ACSL4 knockdown in microglia, NF-κB pathway analysis, VGLL4 expression assays, LPS-induced neuroinflammation models in vitro and in vivo\",\n      \"journal\": \"Brain, behavior, and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in vitro and in vivo, pathway analysis connecting ACSL4 to VGLL4/NF-κB, single lab\",\n      \"pmids\": [\"36791893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RB1 loss activates E2F transcription factors which directly upregulate ACSL4 expression (ACSL4 is a direct E2F target gene), enriching arachidonic acid-containing phospholipids and sensitizing cells to ferroptosis via an RB/E2F/ACSL4 molecular axis.\",\n      \"method\": \"RB1 loss/E2F activation genetic models, ACSL4 promoter analysis (E2F target), lipidomics, xenograft tumor models, GPX4 inhibitor treatment in vivo\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct E2F target gene identification, lipidomic validation, in vivo xenograft and genetically engineered models, multiple orthogonal methods\",\n      \"pmids\": [\"36928314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM28 binds to ACSL4 and promotes SUMO3 modification of ACSL4 at lysine 532, inhibiting K63-linked ACSL4 ubiquitination and thereby suppressing OPTN-dependent autophagic degradation of ACSL4, leading to ACSL4 accumulation and neuronal ferroptosis. SENP3 was identified as the deSUMOylation enzyme that reverses this process.\",\n      \"method\": \"Co-IP (TRIM28-ACSL4 binding), SUMOylation site mapping (K532), ubiquitination assays, autophagy-receptor (OPTN) analysis, Trim28 genetic deletion in mice, SCI model\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — specific SUMOylation site identified, writer (TRIM28) and eraser (SENP3) defined, ubiquitination cross-talk mapped, in vivo genetic validation\",\n      \"pmids\": [\"39875520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AIM2 promotes FOXO3a phosphorylation and proteasome degradation, reducing FOXO3a transcriptional activation of ACSL4 and inhibiting ferroptosis in renal cell carcinoma; this pathway drives sunitinib resistance.\",\n      \"method\": \"AIM2 overexpression/knockdown, FOXO3a phosphorylation and degradation assays, ACSL4 transcriptional regulation assays, ferroptosis assays\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptional regulation pathway dissected, phosphorylation of FOXO3a linked to ACSL4, single lab\",\n      \"pmids\": [\"36923928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Radiation-induced ACSL4 transcription is regulated by the STAT1/IRF1 axis; STAT1 and IRF1 drive ACSL4 upregulation in irradiated intestinal epithelial cells, promoting ferroptosis-mediated radiation intestinal injury.\",\n      \"method\": \"RNA sequencing, ACSL4 promoter/transcription analysis (STAT1/IRF1), ACSL4 knockdown, AMPK activation assays, in vivo radiation injury model\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcription factor axis identified, ACSL4 knockdown functional validation, single lab\",\n      \"pmids\": [\"37611494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Lactate induces ACSL4 expression via histone H3K18 lactylation at the ACSL4 promoter, and also directly lactylates ACSL4 at K412 (post-translational lactylation); decreased SIRT3 expression elevates ACSL4 lactylation, driving ferroptosis in nucleus pulposus cells during intervertebral disc degeneration.\",\n      \"method\": \"Single-cell RNA sequencing, H3K18 lactylation assay at ACSL4 promoter, ACSL4 K412 lactylation site identification, SIRT3 expression analysis, AAV9-siLdha in vivo, 2-DG treatment\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — specific lactylation site on ACSL4 (K412) identified, histone lactylation at promoter shown, in vivo validation, single lab\",\n      \"pmids\": [\"40171826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"METTL3-mediated m6A modification is enriched in ACSL4 mRNA and its stability is regulated through a YTHDC1-dependent pathway; lactate promotes p300-mediated H3K18la binding to the METTL3 promoter, upregulating METTL3 which then stabilizes ACSL4 mRNA, promoting ferroptosis in alveolar epithelial cells.\",\n      \"method\": \"m6A modification assays, METTL3 knockdown/inhibition, YTHDC1 pathway analysis, H3K18la ChIP, GPR81 signaling assays, in vitro and in vivo sepsis models\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m6A modification on ACSL4 mRNA demonstrated, YTHDC1 reader identified, histone lactylation-driven transcription upstream characterized, single lab\",\n      \"pmids\": [\"38852200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FTO demethylase activity targets ACSL4 and TFRC mRNA stability in an m6A-dependent manner; FTO downregulation in older livers increases ACSL4 and TFRC expression, exacerbating ferroptosis during ischemia/reperfusion injury.\",\n      \"method\": \"Mass spectrometry (FTO identification), FTO overexpression in vivo, m6A modification assays for ACSL4 mRNA, mRNA stability assays, older vs. young liver comparison\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — m6A eraser (FTO) mechanistically linked to ACSL4 mRNA stability, in vivo gain-of-function, mass spectrometry identification, multiple orthogonal methods\",\n      \"pmids\": [\"38834654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MMD physically interacts with both ACSL4 and MBOAT7 (two enzymes catalyzing sequential steps of arachidonic acid incorporation into phosphatidylinositol), increasing flux of AA into PI and other AA-containing phospholipid species, thereby promoting ferroptosis susceptibility.\",\n      \"method\": \"Co-IP (MMD-ACSL4 and MMD-MBOAT7 interactions), lipidomics, genetic loss-of-function in ovarian and renal carcinoma cells\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein-protein interaction demonstrated by Co-IP, lipidomic consequences quantified, functional ferroptosis outcomes measured, single lab\",\n      \"pmids\": [\"37691145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ACSL4 promotes metastatic extravasation by enhancing membrane fluidity and cellular invasiveness through PUFA-lipid incorporation; ACSL4 is a pro-hematogenous metastasis factor identified by metabolism-focused in vivo CRISPR screens in ovarian cancer.\",\n      \"method\": \"Two rounds of in vivo CRISPR screen in mouse ovarian cancer metastasis model, membrane fluidity assays, invasion assays, ACSL4 genetic deletion\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo CRISPR screen replicated with two rounds of selection, membrane fluidity mechanistic assays, multiple cancer models\",\n      \"pmids\": [\"39591965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ACSL4-mediated phospholipid remodeling of cell membranes induces lipid-raft localization and activation of integrin β1 in a CD47-dependent manner, leading to downstream focal adhesion kinase phosphorylation that promotes TNBC metastasis.\",\n      \"method\": \"Lipidomics of metastatic vs. primary TNBC, lipid raft fractionation, integrin β1 activation assays, CD47 dependence assays, FAK phosphorylation assays, in vivo pharmacological ACSL4 inhibition\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — lipidomic mechanism, specific integrin activation pathway, CD47 dependence established, in vivo validation, multiple orthogonal methods\",\n      \"pmids\": [\"38471082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ACSL4-mediated lipid peroxidation promotes lipid raft formation in melanoma cell membranes, which inhibits immunogenic ferroptosis and pyroptosis by reducing cell membrane pore formation; disruption of ACSL4-mediated lipid rafts (by cholesterol removal) promotes immunogenic cell death.\",\n      \"method\": \"Lipid raft isolation, ferroptosis/pyroptosis assays, cholesterol depletion experiments, ACSL4 manipulation, immune cell co-culture\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lipid raft mechanistic link to membrane pore formation established, pharmacological and genetic approaches, single lab\",\n      \"pmids\": [\"39343834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ACSL4 modulates fatty acid oxidation (FAO) and intracellular acetyl-CoA levels, leading to hyperacetylation of H3K9ac and H3K27ac marks and overexpression of SNAIL, driving TNBC metastasis via an epigenetic mechanism.\",\n      \"method\": \"Global transcriptome analysis, acetyl-CoA metabolic assays, histone acetylation (H3K9ac, H3K27ac) quantification, ACSL4 genetic ablation/pharmacological inhibition, in vivo metastasis 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 — mechanistic link from ACSL4-FAO to acetyl-CoA to histone acetylation to SNAIL established, multiple methods, single lab\",\n      \"pmids\": [\"39700137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ACSL4 upregulates the lipogenesis master regulator SREBP1 and its downstream lipogenic enzymes via c-Myc in HCC cells, promoting de novo lipogenesis and accumulation of triglycerides, cholesterols, and lipid droplets; SREBP1 is required for ACSL4-mediated lipogenesis and oncogenic capabilities.\",\n      \"method\": \"ACSL4 knockdown/overexpression, SREBP1 rescue experiments, c-Myc pathway analysis, lipid droplet quantification, in vitro and in vivo HCC models\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway dissected via rescue experiments, multiple lipid readouts, in vivo corroboration, single lab\",\n      \"pmids\": [\"33340617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ACSL4 and LPCAT2 overexpression together sensitize cells to RSL3-induced ferroptosis; mitochondrial ROS formation and mitochondrial membrane potential deterioration are essential downstream events in ACSL4/LPCAT2-driven ferroptosis, and mitoquinone (MitoQ) protection confirms mitochondrial ROS as a key mediator.\",\n      \"method\": \"ACSL4 and LPCAT2 co-overexpression in HEK293T cells, ferroptosis assays, mitochondrial ROS measurement, mitochondrial membrane potential assay, mitochondrial respiration assay, MitoQ treatment\",\n      \"journal\": \"Antioxidants (Basel, Switzerland)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct overexpression reconstitution, multiple mitochondrial parameter readouts, pharmacological confirmation, single lab\",\n      \"pmids\": [\"37627584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FUNDC1 (mitophagy receptor) co-immunoprecipitates with ACSL4, indicating a direct protein-protein interaction; FUNDC1 deficiency leads to upregulation of ACSL4 and enhanced ferroptosis in cardiomyocytes under high-fat diet challenge.\",\n      \"method\": \"Co-immunoprecipitation (FUNDC1-ACSL4), FUNDC1 knockout mice, ACSL4 expression analysis, ferroptosis assays, in vitro arachidonic acid treatment\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct Co-IP interaction, genetic knockout model, functional ferroptosis readout, single lab\",\n      \"pmids\": [\"39326685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HIV-1 Tat protein downregulates miR-204 in microglia, releasing its suppression of ACSL4, leading to increased ACSL4 expression, oxidized phosphatidylethanolamine generation, lipid peroxidation, and ferroptosis-driven microglial activation with proinflammatory cytokine release.\",\n      \"method\": \"miR-204 mimic transfection, ACSL4 gene silencing, lipid peroxidation assays, co-immunoprecipitation, HIV-1 transgenic rat validation, human HIV+ brain sample analysis\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — miR-204 upstream regulation validated, ACSL4 silencing confirmation, validated in animal model and human samples, single lab\",\n      \"pmids\": [\"37023693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HAT1 directly promotes acetylation of ACSL4 at lysine 383, enhancing its protein stability; SIRT3 mediates deacetylation of ACSL4, while HDAC2 enhances ACSL4 acetylation by inhibiting SIRT3 transcription. Acetylation at K383 inhibits FBXO10-mediated K48-linked ubiquitination of ACSL4, stabilizing the protein.\",\n      \"method\": \"Acetylation site mapping (K383), HAT1/SIRT3/HDAC2 manipulation, ubiquitination assays, FBXO10 interaction assay, in vitro and in vivo NPC models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — specific acetylation site identified, writer (HAT1) and eraser (SIRT3) defined, ubiquitination cross-talk demonstrated, single lab\",\n      \"pmids\": [\"40050614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Parkin E3 ubiquitin ligase promotes ubiquitination of ACSL4, inhibiting iron overload-induced ferroptosis in cardiomyocytes; p53 transcriptionally suppresses Parkin expression in iron-overloaded cardiomyocytes, establishing a p53-Parkin-ACSL4 regulatory pathway in cardiac ferroptosis.\",\n      \"method\": \"Parkin-ACSL4 ubiquitination assays, cardiac-specific Parkin knockout mice (Myh6-CreER/Parkin), p53 transcriptional regulation of Parkin, iron overload and I/R models, ferroptosis inhibitor (Fer-1) rescue\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assay, cardiac-specific KO in vivo, transcriptional regulation by p53 demonstrated, single lab\",\n      \"pmids\": [\"40370554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRIM21 (E3 ligase) and USP15 (deubiquitinase) together control ACSL4 protein stability: TRIM21 promotes ACSL4 degradation while USP15 stabilizes it; reduced ACSL4 expression due to excessive TRIM21-mediated degradation underlies imatinib resistance in GISTs.\",\n      \"method\": \"Co-immunoprecipitation (TRIM21-ACSL4, USP15-ACSL4), shRNA interference, Western blot, xenograft model, GIST patient sample analysis\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP binding, E3 ligase and deubiquitinase both characterized for same substrate, clinical correlation, single lab\",\n      \"pmids\": [\"38182686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The silibinin (SIL) natural compound directly binds to ACSL4 at the K536-proximal region and inhibits ACSL4 enzymatic activity, mitigating ACSL4-mediated ferroptosis; living cell-target responsive accessibility profiling (LC-TRAP) identified ACSL4 as a SIL target.\",\n      \"method\": \"LC-TRAP proteomics, biophysical binding assays, SIL-derivatized chemical probe, enzymatic activity assay, site-specific binding region identification (K536-proximal)\",\n      \"journal\": \"Analytical chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct enzymatic inhibition demonstrated, binding region mapped by LC-TRAP, biophysical corroboration, single lab\",\n      \"pmids\": [\"36260072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MFN2 overexpression suppresses the mitochondrial translocation of ACSL4, inhibiting mitochondria-associated ferroptosis; PRDX2-mediated suppression of oxidative stress operates upstream via MFN2-dependent mitochondrial dynamics to prevent ACSL4 mitochondrial localization.\",\n      \"method\": \"MFN2 overexpression, ACSL4 mitochondrial localization by immunofluorescence, ferroptosis assays in db/db mice and cardiac microvascular endothelial cells, PRDX2 overexpression\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mitochondrial localization of ACSL4 directly shown by immunofluorescence, MFN2 gain-of-function effect on ACSL4 localization, in vivo corroboration, single lab\",\n      \"pmids\": [\"36367849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mitochondria-localized AMPKα1 phosphorylation promotes Pink1/Parkin-dependent mitophagy, which inhibits the mitochondrial translocation of ACSL4, suppressing mitochondria-associated ferroptosis in cardiac microvascular endothelial cells during diabetic cardiomyopathy.\",\n      \"method\": \"AMPKα1 mitochondrial localization assay, mitophagy assay (mt-Keima, TEM), ACSL4 mitochondrial translocation by immunofluorescence, mitoAα1 overexpression, in vivo diabetic model\",\n      \"journal\": \"Pharmacological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ACSL4 mitochondrial localization directly assayed, AMPKα1-Parkin-ACSL4 pathway dissected with gain-of-function, single lab\",\n      \"pmids\": [\"38218357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NFIL3 (a circadian rhythm gene) directly regulates ACSL4 expression; NFIL3 knockdown attenuates ferroptosis and inflammation in renal tubular epithelial cells by downregulating ACSL4 in sepsis-associated AKI.\",\n      \"method\": \"NFIL3 loss-of-function in vitro and in vivo, ACSL4 expression analysis, ferroptosis markers, SA-AKI model\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — NFIL3-ACSL4 regulatory link shown by correlation and knockdown, mechanism of direct regulation not biochemically established, single lab\",\n      \"pmids\": [\"39097582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"STAT3 signaling drives ACSL4 expression in tubular epithelial cells; however, in the context of ER stress-induced AKI, ACSL4 activity directs PUFA metabolism toward triglycerides rather than phospholipids, and ACSL4 alone is insufficient to sensitize cells to ferroptosis, highlighting context-dependence.\",\n      \"method\": \"STAT3 inhibition, lipidomics, ACSL4 expression analysis, mouse ER stress AKI model\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — STAT3-ACSL4 transcriptional link and lipidomic consequences demonstrated, important negative/contextual finding, single lab\",\n      \"pmids\": [\"38799564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In IBD, ACSL4 is overexpressed in intestinal fibroblasts where it reprograms lipid metabolism and mediates sensitivity of adjacent intestinal epithelial cells to ferroptosis through heterocellular crosstalk; fibroblast-specific ACSL4 overexpression increases epithelial ferroptosis and worsens colitis, while fibroblast ACSL4 deletion ameliorates colitis.\",\n      \"method\": \"Fibroblast-specific ACSL4 overexpression and deletion in mouse models of colitis, IBD tissue analysis, lipid metabolism assays, ferroptosis markers\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific genetic manipulation (both gain- and loss-of-function), multiple colitis models, heterocellular crosstalk mechanism established\",\n      \"pmids\": [\"40571769\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EGR1 transcriptionally upregulates ACSL4 expression; YAP transcriptionally upregulates EGR1, forming a YAP/EGR1/ACSL4 axis that promotes ferroptosis in ischemic stroke; chromatin immunoprecipitation and dual luciferase assays verified these molecular interactions.\",\n      \"method\": \"Chromatin immunoprecipitation, dual luciferase assay (EGR1-ACSL4 promoter, YAP-EGR1 promoter), OGD/R neuronal models, MCAO mouse model, YAP/EGR1 overexpression/silencing\",\n      \"journal\": \"Experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct promoter binding established by ChIP and luciferase for both EGR1-ACSL4 and YAP-EGR1, single lab\",\n      \"pmids\": [\"39889877\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ACSL4 is a long-chain acyl-CoA synthetase with substrate preference for polyunsaturated fatty acids (especially arachidonic and adrenic acids) that acts as a critical pro-ferroptotic enzyme by esterifying PUFAs into membrane phospholipids, providing the peroxidation substrates required for ferroptosis execution; its activity and stability are tightly regulated by multiple post-translational modifications—including phosphorylation by PKCβII (activating) and CDK1 (triggering UBR5-mediated ubiquitin degradation), SUMOylation by TRIM28 at K532 (stabilizing), acetylation by HAT1 at K383 (stabilizing, blocking FBXO10-mediated degradation), lactylation at K412, m6A-dependent mRNA stabilization via METTL3/YTHDC1, and autophagic degradation via HSC70/CMA or OPTN-selective autophagy—while its transcription is driven by SP1, Sp1, STAT1/IRF1, STAT3, E2F (downstream of RB1), EGR1 (downstream of YAP), and NFIL3, and inhibited by HIF-1α; beyond ferroptosis, ACSL4 promotes tumor metastasis by remodeling membrane phospholipid composition to activate integrin β1 signaling and enhance membrane fluidity, reprograms lipid metabolism through the c-Myc/SREBP1 axis and FAO-dependent histone acetylation, regulates neuroinflammation via VGLL4/NF-κB, and interacts directly with partner proteins including STING, FUNDC1, and MMD/MBOAT7 to modulate disease-specific lipid peroxidation pathways.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ACSL4 (FACL4) is a long-chain acyl-CoA synthetase that activates polyunsaturated fatty acids—with marked preference for arachidonic acid—and esterifies them into membrane phospholipids, thereby supplying the peroxidation substrates that license ferroptotic cell death [#2, #0]. Loss-of-function point mutations and genomic deletions that abolish its enzymatic activity cause nonspecific X-linked mental retardation and the Alport-syndrome-with-mental-retardation contiguous gene deletion phenotype [#3, #4, #5]. In ferroptosis, ACSL4 acts downstream of or parallel to GPX4: combined Gpx4/Acsl4 ablation confers resistance, and ACSL4 enriches membranes with long ω6 PUFAs that become peroxidation substrates [#0], with its partner enzymes MMD/MBOAT7 channeling arachidonate into specific phospholipid species [#26]. Its pro-ferroptotic output is set by a dense post-translational and transcriptional control network: PKCβII phosphorylation activates ACSL4 in a lipid-peroxidation feedback loop [#6], whereas CDK1 phosphorylation, CARM1 methylation, and several E3 ligases (UBR5, RNF25, FBXO10, TRIM21, Parkin) drive its ubiquitin-dependent degradation, opposed by stabilizing SUMOylation (TRIM28), acetylation (HAT1 at K383), and deubiquitination (USP15) [#7, #15, #16, #20, #35, #37, #36]. Transcription is driven by Sp1, STAT1/IRF1, STAT3, E2F downstream of RB1 loss, and a YAP/EGR1 axis, and repressed by HIF-1α, while m6A writers/erasers (METTL3/YTHDC1, FTO) tune ACSL4 mRNA stability [#9, #22, #19, #44, #10, #24, #25]. Beyond ferroptosis, ACSL4-driven phospholipid remodeling enhances membrane fluidity and lipid-raft–dependent integrin β1/FAK signaling to promote metastatic extravasation, and reprograms lipid metabolism through c-Myc/SREBP1 lipogenesis and FAO-fueled histone acetylation [#27, #28, #31, #30]. ACSL4 mitochondrial translocation is a regulated step that sensitizes cells to mitochondria-associated ferroptosis [#39, #40, #32].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing that ACSL4 is an enzyme—a long-chain acyl-CoA synthetase preferring arachidonic acid—defined its core biochemical activity and tissue distribution before any disease or ferroptosis role was known.\",\n      \"evidence\": \"cDNA cloning, enzymatic activity assays, Northern blot, and FISH localization to Xq23\",\n      \"pmids\": [\"9598324\", \"9480748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of arachidonate selectivity not resolved\", \"Subcellular distribution of activity not defined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Patient missense/splice mutations that reduce enzymatic activity causally linked ACSL4 loss-of-function to X-linked mental retardation, proving the enzyme's physiological importance in brain.\",\n      \"evidence\": \"Mutation identification with enzymatic activity measurement in patient lymphoblastoid lines and co-segregation/X-inactivation analysis\",\n      \"pmids\": [\"11889465\", \"12525535\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Neuronal substrate/pathway downstream of ACSL4 in cognition not defined\", \"Mechanism connecting reduced acyl-CoA synthesis to MR unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Unbiased genetic screens placed ACSL4 as an essential, substrate-supplying enzyme for ferroptosis, reframing it from a generic lipid enzyme to a determinant of a regulated cell death pathway.\",\n      \"evidence\": \"Genome-wide CRISPR screen, Gpx4/Acsl4 double-knockout genetics, lipidomics, plus shRNA/overexpression with 5-HETE pharmacology\",\n      \"pmids\": [\"27842070\", \"27565726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ACSL4 acts upstream, parallel, or downstream of GPX4 not fully resolved\", \"Identity of the critical peroxidized phospholipid species not pinned down in 2016\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovery of PKCβII activating phosphorylation and CDK1/UBR5-mediated degradative phosphorylation showed ACSL4 ferroptotic output is gated by opposing post-translational switches.\",\n      \"evidence\": \"CRISPR and kinase-inhibitor screens, phosphosite mutagenesis (S447), ubiquitination site mapping (K388/K498/K690), lipidomics, and xenografts\",\n      \"pmids\": [\"35027735\", \"37428466\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and interplay between activating and degradative phosphorylation unresolved\", \"Phosphatases reversing these marks not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Immune and ischemic contexts defined transcriptional and substrate-channeling control of ACSL4: IFNγ/T-cell-driven arachidonate incorporation enables immunogenic ferroptosis, while Sp1 and HIF-1α set opposing transcriptional tone.\",\n      \"evidence\": \"Lipidomics with genetic ACSL4 deletion in tumors, IFNγ + fatty-acid assays, promoter-binding/siRNA and HIF-1α manipulation in ischemia models\",\n      \"pmids\": [\"35216678\", \"30737476\", \"33444733\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct transcription-factor occupancy at promoter not biochemically resolved for all factors\", \"How substrate availability and transcription are coordinated in vivo unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A broad regulatory network was mapped: direct partners (STING, FUNDC1, MMD/MBOAT7), additional transcriptional inputs (RB1/E2F, STAT1/IRF1, FOXO3a, c-Myc/SREBP1), and a methylation-coupled degradation axis (CARM1/RNF25, FBXO10) that together tune ACSL4 level and ferroptosis or lipogenesis output.\",\n      \"evidence\": \"Co-IP/LC-MS interaction mapping, methylation/ubiquitination site assays, lipidomics, promoter analyses, and in vivo disease models across kidney, brain, and cancer\",\n      \"pmids\": [\"37533255\", \"39326685\", \"37691145\", \"36928314\", \"37611494\", \"36923928\", \"33340617\", \"37946697\", \"37059712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether these partner interactions are direct vs. complex-mediated not uniformly established\", \"Tissue-specific dominance among competing regulators unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"ACSL4 was shown to drive cancer metastasis independent of cell death—remodeling membrane phospholipids to raise fluidity and activate lipid-raft integrin β1/FAK signaling, and rerouting metabolism into lipogenesis and FAO-fueled histone acetylation.\",\n      \"evidence\": \"In vivo CRISPR metastasis screens, membrane-fluidity and lipid-raft fractionation assays, CD47/integrin β1/FAK readouts, and acetyl-CoA/histone-acetylation profiling\",\n      \"pmids\": [\"39591965\", \"38471082\", \"39700137\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single enzyme partitions between pro-death and pro-metastatic phospholipid pools is unresolved\", \"Direct phospholipid species responsible for raft/integrin activation not fully defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"RNA-level (m6A via METTL3/YTHDC1 and FTO) and metabolite-level (lactylation, acetylation) control further refined how ACSL4 abundance and activity respond to the metabolic environment.\",\n      \"evidence\": \"m6A modification and mRNA-stability assays, lactylation site mapping (K412), acetylation site mapping (K383) with writer/eraser manipulation in disease models\",\n      \"pmids\": [\"38852200\", \"38834654\", \"40171826\", \"40050614\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cross-talk hierarchy among mRNA, lactylation, and acetylation controls not integrated\", \"Direct catalytic consequence of each PTM on enzyme kinetics not measured\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cell-type-resolved genetics revealed ACSL4 acts non-cell-autonomously, with fibroblast ACSL4 dictating ferroptosis sensitivity of neighboring epithelium, expanding its role to heterocellular tissue crosstalk in inflammation.\",\n      \"evidence\": \"Fibroblast-specific gain- and loss-of-function in multiple colitis models with lipid metabolism and ferroptosis readouts\",\n      \"pmids\": [\"40571769\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the transferred lipid mediator between fibroblasts and epithelium unknown\", \"Generalizability beyond intestinal tissue untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how ACSL4 substrate flux is partitioned between pro-ferroptotic phospholipids, neutral lipids/triglycerides, and pro-metastatic raft lipids, and what structural features dictate this context-dependent channeling.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model linking active-site chemistry to lipid product fate\", \"Context-dependence (e.g., ER-stress AKI directing PUFA to triglyceride rather than phospholipid) mechanistically unexplained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [2, 3, 4, 5]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 2, 26]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 8, 28]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [39, 40, 32]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [28, 29]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 6, 19]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 31, 30]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 43, 27]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MBOAT7\", \"MMD\", \"STING\", \"FUNDC1\", \"UBR5\", \"RNF25\", \"TRIM28\", \"FBXO10\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}