{"gene":"ALOX5","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2009,"finding":"Loss of Alox5 gene prevents BCR-ABL-induced chronic myeloid leukemia in mice by impairing leukemia stem cell (LSC) function—affecting differentiation, cell division, and survival of long-term LSCs—without significantly affecting normal hematopoietic stem cells.","method":"Genetic knockout (Alox5-/- mice), BCR-ABL retroviral transduction, bone marrow transplantation, LSC functional assays","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined cellular phenotype replicated with pharmacological inhibitor (zileuton), multiple orthogonal readouts","pmids":["19503090"],"is_preprint":false},{"year":2023,"finding":"ALOX5 mediates ACSL4-independent ferroptosis in Huntington's disease by stabilizing FLAP (its essential cofactor), thereby enhancing ALOX5-mediated lipoxygenase activity and lipid peroxidation in neurons; mHTT (HTTQ94) activates this pathway by stabilizing FLAP.","method":"RNAi screening, CRISPR/Cas9 knockout, overexpression, in vivo HD mouse model with lifespan analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (RNAi screen, KO, in vivo rescue), mechanistic dissection of FLAP stabilization","pmids":["36921996"],"is_preprint":false},{"year":2013,"finding":"Cannabinoid receptor 2 (Cnr2) suppresses leukocyte inflammatory migration by downregulating Alox5 expression via inhibition of the JNK/c-Jun signaling pathway; alox5 is a transcriptional target of c-Jun, and inactivation of Alox5 blocks leukocyte migration.","method":"Chemical genetic screen in zebrafish, zinc finger nuclease-mediated mutagenesis of Cnr2, zebrafish leukocyte migration assay, human myeloid cell migration assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic inactivation in two model systems, pathway epistasis established","pmids":["23539630"],"is_preprint":false},{"year":2022,"finding":"ERK1-dependent phosphorylation of Alox5 is critical for targeting Alox5 to the nuclear membrane to mediate lipid peroxidation, resulting in nuclear translocation of cytolytic molecules and caspase-9-independent cell death; double knockout of caspase-9 and Alox5 in mice leads to significant T cell expansion, demonstrating these pathways act in parallel.","method":"Genome-wide siRNA library screening, double-knockout mice (caspase-9/Alox5), subcellular localization studies, lipid peroxidation assays","journal":"Cells","confidence":"High","confidence_rationale":"Tier 2 / Strong — unbiased whole-genome screen, confirmed by double-KO in vivo, mechanistic localization established","pmids":["36231015"],"is_preprint":false},{"year":2019,"finding":"Mutagenesis of 'triad determinant' residues (F359W/A424I/N425M/A603I) in human ALOX5 shifts reaction specificity from dominant 5(S)-HpETE production to 15(S)- and 8(S)-HpETE, abolishes leukotriene synthase activity, broadens substrate acceptance to C18 fatty acids, and alters substrate orientation at the active site as confirmed by QM/MM calculations.","method":"Site-directed mutagenesis, Sf9 insect cell expression, product analysis by chiral HPLC, kinetic studies with deutero-labeled substrates, molecular dynamics and QM/MM simulations","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro enzyme with mutagenesis, structural modeling, and stereospecific product analysis","pmids":["31664810"],"is_preprint":false},{"year":2015,"finding":"Zebrafish ALOX2 (functional ortholog of human ALOX5) is an arachidonic acid 5S-lipoxygenating enzyme with leukotriene synthase activity; mutagenesis of its triad determinants (F359W/A424I/N425M) shifts specificity from 5S- to dominant 15S-lipoxygenation, analogous to human ALOX5.","method":"Cloning and expression in pro- and eukaryotic systems, product characterization, site-directed mutagenesis","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted enzyme activity, mutagenesis validation, single lab","pmids":["26456699"],"is_preprint":false},{"year":2009,"finding":"Oxygenation of the 5-lipoxygenase product 5S-HETE by COX-2 yields a bicyclic di-endoperoxide; heme-catalyzed cleavage of this di-endoperoxide produces 4S-HNE, 8-oxo-5S-hydroxy-6E-octenoic acid, and malondialdehyde, establishing a convergent 5-LOX/COX-2 pathway to classic lipid peroxidation products.","method":"In vitro enzymatic reactions, hematin/ferrous iron treatment, chiral HPLC analysis of products","journal":"Journal of lipid research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with structural product characterization, chiral analysis","pmids":["19553698"],"is_preprint":false},{"year":2022,"finding":"LysOX (lysyl oxidase) promotes ALOX5-mediated ferroptotic lipid peroxidation in neurons via activation of ERK signaling; adeno-associated virus-mediated LysOX overexpression enhances ferroptosis sensitivity and aggravates seizure-induced hippocampal damage, while LysOX inhibition blocks Alox5 activation and neuronal death.","method":"AAV-based gene transfer (overexpression), pharmacological LysOX inhibition (BAPN), seizure mouse model, lipid peroxidation assays","journal":"Acta pharmaceutica Sinica. B","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic overexpression and pharmacological inhibition, single lab, two orthogonal approaches","pmids":["36176900"],"is_preprint":false},{"year":2024,"finding":"ALOX5 increases LTB4 production in RA CD4+ T cells, which stimulates Ca2+ influx through ORAI3 channels, leading to NLRP3 inflammasome activation and pyroptosis; knockdown or pharmacological inhibition of ALOX5 suppresses pyroptosis and improves symptoms in rodent RA models.","method":"siRNA knockdown, pharmacological inhibition, Ca2+ flux assays, NLRP3 inflammasome activation assays, rodent RA models","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — mechanistic pathway (ALOX5→LTB4→ORAI3→NLRP3→pyroptosis) established by multiple orthogonal methods including in vivo validation","pmids":["38412254"],"is_preprint":false},{"year":2021,"finding":"PKC-β upregulates Alox5 expression via ERK1/2 signaling in CML cells, and Alox5 promotes TKI resistance by inactivating PTEN; this PKC-β/ERK1/2/Alox5/PTEN axis mediates BCR-Abl-independent TKI resistance.","method":"shRNA knockdown, gene expression comparison of 84 leukemia-related genes, PTEN activity assays, in vivo CML-PDX mouse model","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway epistasis and in vivo validation, single lab","pmids":["33561320"],"is_preprint":false},{"year":2024,"finding":"PARP-1 interacts with and PARylates (post-translationally modifies) ALOX5 to stabilize it in cancer-educated neutrophils, leading to increased MMP-9 expression; blocking PARP-1 decreases ALOX5 expression and MMP-9 production, and eliminates neutrophil-mediated lung cancer cell invasion.","method":"Immunoprecipitation coupled to mass spectrometry (IP/MS), co-immunoprecipitation (Co-IP), gene knockdown, in vitro invasion assays, in vivo tumor model","journal":"Cancer biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus MS identified PARP-1/ALOX5 interaction, functional rescue with gene knockdown, single lab","pmids":["38172525"],"is_preprint":false},{"year":2020,"finding":"ALOX5 expression and activity are upregulated by HER2 in breast cancer cells; HER2 inhibition decreases ALOX5 expression and activity but not ALOX5AP expression; ALOX5 mediates breast cancer growth and migration through RhoA, focal adhesion, and PI3K/Akt/mTOR signaling.","method":"HER2 inhibitor treatment, siRNA knockdown, proliferation and migration assays, signaling pathway analysis","journal":"BioMed research international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — HER2 inhibition plus KD with pathway readouts, single lab","pmids":["33224971"],"is_preprint":false},{"year":2017,"finding":"ALOX5 is transcriptionally repressed in MLL-rearranged AML via Polycomb repressive complex 2 (PRC2); restoration of Alox5 expression sensitizes leukemic cells to doxorubicin and cytarabine, with this drug-sensitizing effect associated with negative correlation of Stat and K-Ras signaling pathways.","method":"Affymetrix microarray profiling, ChIP assays (PRC2), colony-forming/replating assays, bone marrow transplantation, in vivo AML model with chemotherapy","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP established PRC2-mediated repression, in vivo rescue with defined signaling readouts, single lab","pmids":["28500307"],"is_preprint":false},{"year":2008,"finding":"Alox5 deficiency in mice impairs glucose-stimulated insulin secretion and reduces expression of insulin and Pdx1 genes in islets; siRNA knockdown of ALOX5 in isolated human islets similarly decreases insulin and PDX1 gene expression by ~50% and insulin secretion threefold, demonstrating a direct role of 5-LO in pancreatic beta cell function.","method":"Alox5-/- mouse characterization, siRNA knockdown in human islets, glucose tolerance tests, insulin secretion assays","journal":"Diabetologia","confidence":"High","confidence_rationale":"Tier 2 / Strong — mouse KO and human siRNA knockdown with orthogonal readouts, replicated across species","pmids":["18421434"],"is_preprint":false},{"year":2000,"finding":"In aging rat brain, 5-LOX mRNA and protein increase in hippocampus and cerebellum, and the membrane/cytosol 5-LOX ratio is larger in older rats, indicating increased 5-LOX protein translocation/activation during aging.","method":"Quantitative RT-PCR with internal standards, quantitative Western immunoblotting, subcellular fractionation (membrane vs. cytosol)","journal":"Neurobiology of aging","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct subcellular fractionation showing translocation, replicated in two brain regions, single lab","pmids":["11016533"],"is_preprint":false},{"year":2007,"finding":"5-LOX protein is expressed in peripheral blood T lymphocytes and is present primarily in the cytoplasm with some nuclear localization; it translocates to the nuclear periphery after mitogenic stimulation. Purified resting T lymphocytes cannot synthesize leukotrienes without exogenous arachidonic acid, whereas Jurkat cells produce LTC4 and LTB4 upon CD3-CD28 cross-linking, which is abolished by 5-LOX inhibitors.","method":"Western blot, immunofluorescence, FACS, in situ RT-PCR, leukotriene synthesis assays with inhibitors","journal":"Immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by immunofluorescence and fractionation, functional leukotriene synthesis linked to localization/activation state","pmids":["17484769"],"is_preprint":false},{"year":2012,"finding":"5-LOX and its metabolite LTB4 activate NF-κB in hepatoma cells: inhibition or knockdown of 5-LOX reduces NF-κB p65 mRNA expression, decreases IκBα phosphorylation in the cytoplasm, and reduces nuclear NF-κB p65 levels; exogenous LTB4 activates NF-κB in a dose-dependent manner.","method":"siRNA knockdown, MK886 pharmacological inhibition, Western blot, immunofluorescence, transcriptional activity reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA plus pharmacological inhibition with multiple readouts, single lab","pmids":["22293202"],"is_preprint":false},{"year":2021,"finding":"ALOX5-derived 5-HETE in gastric cancer cells activates MEK/ERK signaling to promote tumor growth and reduce chemotherapy sensitivity; ALOX5 inhibition suppresses ERK-mediated signaling and enhances chemotherapy efficacy.","method":"ALOX5 overexpression and genetic/pharmacological inhibition, ERK pathway analysis by Western blot, growth/survival assays, exogenous 5-HETE addition","journal":"Cancer medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with defined signaling readout, exogenous metabolite rescue, single lab","pmids":["34121352"],"is_preprint":false},{"year":2024,"finding":"In glioma, ALOX5-derived 5-HETE promotes M2 polarization, PD-L1 expression, and migration of glioma-associated microglia/macrophages by facilitating nuclear translocation of NRF2; an ALOX5-targeted nanobody suppresses 5-HETE efflux and attenuates M2 polarization.","method":"UHPLC-MS/MS oxylipin profiling, orthotopic glioma mouse model, immunofluorescence, flow cytometry, nanobody development, in vivo bioluminescent imaging","journal":"Journal for immunotherapy of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic pathway (ALOX5→5-HETE→NRF2 nuclear translocation→M2/PD-L1) shown with multiple methods, single lab","pmids":["39142719"],"is_preprint":false},{"year":2023,"finding":"In intrahepatic cholangiocarcinoma, LTB4 (metabolite of ALOX5) binds BLT1/BLT2 receptors on tumor-associated macrophages (TAMs) to activate the PI3K pathway, promoting M2 macrophage migration toward tumor cells and ICC progression.","method":"In vitro co-culture model, bulk RNA-seq after co-culture, scRNA-seq analysis, xenograft tumor model with ALOX5 inhibitor (zileuton) plus CSF1R inhibitor","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-culture plus in vivo validation, pathway defined as LTB4-BLT1/2-PI3K, single lab","pmids":["38124204"],"is_preprint":false},{"year":2017,"finding":"ALOX5 expression in bladder cancer is transcriptionally regulated by EGR1; ALOX5 deficiency (via CRISPR/Cas9) confers resistance to RSL3-induced ferroptosis in high-pathological-stage bladder cancer cells, identifying ALOX5 as a pro-ferroptotic factor downstream of EGR1-mediated transcription.","method":"RNA-seq, RNAi loss-of-function, CRISPR/Cas9 knockout, EGR1 transcriptional regulation assays, in vitro and in vivo ferroptosis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO plus transcription factor upstream mechanism, single lab","pmids":["38062004"],"is_preprint":false},{"year":2020,"finding":"Mass spectrometric DNA pulldown identified 66 specific ALOX5 promoter-binding proteins, including known regulators Sp1 and Sp3, novel zinc finger proteins (KLF13, KLF16, MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281, ZNF579), two helicases (BLM, DHX36), and hnRNPD/hnRNPK; the GC-rich ALOX5 promoter was confirmed to form DNA G-quadruplex structures by spectroscopic and antibody-based methods.","method":"DNA pulldown, label-free quantitative mass spectrometry, G-quadruplex spectroscopy, antibody-based detection","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic proteomics of promoter interactome with G-quadruplex confirmation by orthogonal methods, single lab","pmids":["32096311"],"is_preprint":false},{"year":2020,"finding":"LPS activates the ALOX5 promoter and synergizes with TGF-β to increase 5-LO mRNA (up to 54-fold in MM1 cells) and protein expression in human monocytic cells, dramatically increasing leukotriene biosynthesis; this represents a receptor-mediated transcriptional control mechanism for 5-LO expression.","method":"ALOX5 promoter-luciferase reporter assays, RT-PCR, Western blot, leukotriene product quantification","journal":"Prostaglandins, leukotrienes, and essential fatty acids","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter reporter plus protein/product assays, two cell lines tested, single lab","pmids":["32120263"],"is_preprint":false},{"year":2011,"finding":"In Alox5-/- mice on a high-fat diet, enhanced osteoclastogenesis and bone loss occurs with increased RANKL, PPARγ, and COX-2 gene expression in femur; treatment with celecoxib (COX-2 inhibitor) suppresses these increases, placing 5-LO upstream of COX-2-mediated osteoclast activity in a gene-by-environment interaction.","method":"Alox5-/- mice on high-fat diet, microarchitectural analysis, histomorphometry, in vitro osteoclastogenesis assay, gene expression, celecoxib rescue","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with pharmacological rescue establishing epistasis, multiple skeletal readouts, single lab","pmids":["22128029"],"is_preprint":false},{"year":2014,"finding":"In 3xTg Alzheimer's disease mice, ALOX5 gene deficiency prevents stress-induced worsening of tau phosphorylation, impaired tau solubility, increased GSK3β activity, compromised long-term potentiation, and fear-conditioned memory deficits caused by restraint/isolation stress.","method":"Triple-transgenic 3xTg mice crossed with 5-LO-/- mice, restraint/isolation stress paradigm, tau phosphorylation/solubility assays, GSK3β activity measurement, electrophysiology (LTP), behavioral fear conditioning","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in vivo with multiple orthogonal phenotypic readouts, single lab","pmids":["25122659"],"is_preprint":false},{"year":2016,"finding":"PEDF-R (PNPLA2 gene product) physically binds human recombinant 5-LOX via ionic interactions through a specific peptide region (E5b/P1 spanning Leu159–Met325); this binding inhibits 5-LOX lipoxygenase activity and reduces LTB4 levels and RPE cell death during oxidative stress.","method":"Peptide-affinity chromatography, pulldown assays with recombinant 5-LOX, lipoxygenase activity assay, LTB4 ELISA, cell viability assays","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding pulldown with recombinant protein, functional enzyme inhibition assay, single lab","pmids":["27635633"],"is_preprint":false},{"year":2009,"finding":"ALOX5 silencing by RNAi in isolated rat cardiomyocytes reduces ALOX5 expression 4.7-fold, decreases LTC4 levels, and reduces necrotic cell death during anoxia-reoxygenation; in vivo ALOX5 knockdown in rat heart reduces infarct size 3.8-fold during ischemia-reperfusion.","method":"siRNA knockdown in cardiomyocytes, real-time PCR, LTC4 measurement, necrosis/viability assays, in vivo rat I/R model","journal":"Acta biochimica Polonica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo KD with mechanistic readout (LTC4), single lab","pmids":["20011686"],"is_preprint":false},{"year":2023,"finding":"ALOX5 regulates tumor-associated macrophage M2 polarization via the JAK/STAT pathway in pancreatic cancer; ALOX5 overexpression promotes chemotaxis of macrophages toward cancer cells, while zileuton inhibits these effects.","method":"Lentiviral ALOX5 overexpression in PANC-1 and macrophages, JAK/STAT pathway analysis, nude mouse orthotopic transplantation model","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function with defined pathway (JAK/STAT), in vivo confirmation, single lab","pmids":["37348233"],"is_preprint":false},{"year":2023,"finding":"ALOX5 promotes autophagy-dependent ferroptosis in melanoma by activating the AMPK/mTOR pathway and inhibiting GPX4 expression; inhibition of autophagy reduces ALOX5-enhanced ferroptosis, demonstrating synergy between autophagy and ALOX5 in this cell death pathway.","method":"qRT-PCR, Western blot, IHC, iron/GSH/MDA assays, autophagy inhibition, xenograft model with recombinant ALOX5 protein","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo with mechanistic AMPK/mTOR/GPX4 pathway readouts, single lab","pmids":["37080437"],"is_preprint":false},{"year":2020,"finding":"CaMKIIγ knockdown in AML stem-like cell lines downregulates Alox5 levels and decreases c-Myc and p-IκBα (with unchanged total IκBα), placing CaMKIIγ upstream of Alox5 in regulating the Alox5/NF-κB pathway controlling AML stem-like cell viability and self-renewal.","method":"shRNA lentiviral knockdown of CaMKIIγ, qRT-PCR, Western blot for NF-κB pathway components, CD34+ cell proportion analysis","journal":"International journal of laboratory hematology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single KD with pathway readouts, no reciprocal rescue, single lab","pmids":["33369192"],"is_preprint":false}],"current_model":"ALOX5 encodes arachidonate 5-lipoxygenase, a key enzyme that oxygenates arachidonic acid to produce 5(S)-HpETE and subsequently leukotrienes (including LTA4, LTB4, and cysteinyl leukotrienes); its catalytic specificity is determined by 'triad determinant' residues in the active site, its activity requires FLAP as an essential cofactor, it is transcriptionally regulated by Sp1-family zinc finger proteins and the ALOX5 promoter G-quadruplex, it translocates from cytoplasm to the nuclear membrane (via ERK1-dependent phosphorylation) to execute lipid peroxidation-driven ferroptosis, it activates NF-κB via LTB4, it drives CD4+ T cell pyroptosis via LTB4→ORAI3→NLRP3 inflammasome signaling, it is stabilized by PARP-1 PARylation and by mHTT-mediated FLAP stabilization, and it functions as a critical regulator of leukemia stem cell survival, Alzheimer's-related tau pathology, and multiple forms of regulated cell death including ferroptosis."},"narrative":{"mechanistic_narrative":"ALOX5 encodes arachidonate 5-lipoxygenase, the enzyme that oxygenates arachidonic acid to 5(S)-HpETE and downstream 5-HETE and leukotrienes (LTB4, LTC4), and through these lipid mediators it acts as a central regulator of inflammatory cell behavior, regulated cell death, and tumor biology [PMID:31664810, PMID:17484769]. Catalytic specificity for 5(S)-lipoxygenation and leukotriene synthase activity is set by a small set of 'triad determinant' active-site residues (F359/A424/N425/A603); mutating these shifts the enzyme toward 15(S)- and 8(S)-HpETE production, abolishes leukotriene synthesis, and broadens substrate acceptance, a specificity logic conserved in the zebrafish ortholog [PMID:31664810, PMID:26456699]. Enzymatic output bifurcates into two functional arms: a leukotriene arm in which LTB4 drives downstream signaling — activating NF-κB in hepatoma cells, driving Ca2+ influx through ORAI3 to license NLRP3 inflammasome-mediated pyroptosis in CD4+ T cells, and recruiting BLT-receptor-bearing M2 macrophages — and a lipid-peroxidation arm in which ALOX5 executes ferroptosis [PMID:22293202, PMID:38412254, PMID:38124204, PMID:38062004]. ALOX5-driven ferroptosis depends on its essential cofactor FLAP, whose stabilization (including by mutant huntingtin) enhances lipoxygenase activity and lipid peroxidation, and on ERK-dependent phosphorylation that targets ALOX5 to the nuclear membrane, where it can drive caspase-independent death; this peroxidation activity also engages autophagy/AMPK-mTOR signaling and GPX4 suppression [PMID:36921996, PMID:36231015, PMID:37080437]. Activity is gated by translocation from cytoplasm to nuclear membrane upon cell activation [PMID:17484769, PMID:11016533], and the enzyme is controlled both transcriptionally — its GC-rich, G-quadruplex-forming promoter is bound by Sp1/Sp3-family and other zinc-finger factors, by EGR1, and by PRC2-mediated repression — and post-translationally by FLAP stabilization, PARP-1 PARylation, and inhibitory binding by PEDF-R/PNPLA2 [PMID:32096311, PMID:38062004, PMID:28500307, PMID:38172525, PMID:27635633]. Functionally, ALOX5 is required for BCR-ABL leukemia stem cell maintenance, contributes to TKI resistance and chemoresistance across leukemia and solid tumors, supports tumor-associated macrophage polarization and invasion, and modulates beta-cell insulin secretion, osteoclastogenesis, ischemic injury, and stress-induced tau pathology [PMID:19503090, PMID:33561320, PMID:18421434, PMID:22128029, PMID:25122659, PMID:20011686, PMID:37348233].","teleology":[{"year":2000,"claim":"Established that 5-LOX is dynamically regulated at the level of subcellular distribution, with activation reflected by increased membrane-associated enzyme — framing translocation as a control point for activity.","evidence":"RT-PCR, Western blot, and membrane/cytosol fractionation in aging rat brain","pmids":["11016533"],"confidence":"Medium","gaps":["Does not define the molecular trigger for translocation","Correlative with aging, not causal for any phenotype"]},{"year":2008,"claim":"Showed ALOX5 has a physiological role beyond inflammation, supporting glucose-stimulated insulin secretion and beta-cell gene expression, indicating broad tissue functions.","evidence":"Alox5-/- mice and siRNA knockdown in human islets with insulin secretion and PDX1/insulin expression readouts","pmids":["18421434"],"confidence":"High","gaps":["Lipid-mediator effector linking ALOX5 to insulin transcription not identified","Mechanism connecting enzyme activity to PDX1 regulation unknown"]},{"year":2009,"claim":"Defined ALOX5 as a non-redundant requirement for leukemia stem cell function, establishing it as a selective therapeutic vulnerability in BCR-ABL CML.","evidence":"Alox5-/- mice with BCR-ABL transduction, bone marrow transplantation, and LSC functional assays plus zileuton","pmids":["19503090"],"confidence":"High","gaps":["The downstream lipid mediator driving LSC maintenance not pinpointed","Why normal HSCs are spared mechanistically unresolved"]},{"year":2009,"claim":"Demonstrated chemically how 5-LOX products converge with COX-2 to generate classic lipid peroxidation aldehydes, linking the enzyme to oxidative damage chemistry.","evidence":"In vitro enzymatic reactions with heme/iron treatment and chiral HPLC product characterization","pmids":["19553698"],"confidence":"High","gaps":["Cellular relevance of the di-endoperoxide pathway not demonstrated","Does not test contribution to disease phenotypes"]},{"year":2007,"claim":"Linked ALOX5 localization state to functional leukotriene output in T cells, showing activation-dependent nuclear translocation and stimulus-gated leukotriene synthesis.","evidence":"Western blot, immunofluorescence, and leukotriene synthesis assays with inhibitors in T lymphocytes and Jurkat cells","pmids":["17484769"],"confidence":"Medium","gaps":["Signal driving translocation not defined","Requirement for exogenous arachidonate leaves endogenous substrate supply open"]},{"year":2012,"claim":"Connected ALOX5 enzymatic output to transcriptional signaling by showing LTB4 activates NF-κB, providing a route from lipid mediator to gene expression.","evidence":"siRNA knockdown, MK886 inhibition, and NF-κB reporter/Western assays with exogenous LTB4 in hepatoma cells","pmids":["22293202"],"confidence":"Medium","gaps":["LTB4 receptor mediating NF-κB activation not identified here","Single cell-type context"]},{"year":2013,"claim":"Placed ALOX5 within an upstream signaling cascade for inflammatory migration, identifying it as a c-Jun transcriptional target repressed by CB2 signaling.","evidence":"Chemical genetic screen and zinc-finger nuclease mutagenesis in zebrafish plus human myeloid migration assays","pmids":["23539630"],"confidence":"High","gaps":["Direct binding of c-Jun to the ALOX5 promoter not shown","Lipid mediator executing migration not specified"]},{"year":2017,"claim":"Identified opposing transcriptional control of ALOX5 in leukemia, with PRC2 silencing the gene and its restoration sensitizing cells to chemotherapy.","evidence":"Microarray, PRC2 ChIP, colony assays, and in vivo AML chemotherapy model","pmids":["28500307"],"confidence":"Medium","gaps":["Mechanism by which restored ALOX5 sensitizes to drugs not established","Relationship to its LSC-promoting role left unreconciled"]},{"year":2019,"claim":"Mapped the structural basis of 5-LOX reaction specificity to discrete triad-determinant residues, defining how positional and stereo selectivity and leukotriene synthase activity are encoded.","evidence":"Site-directed mutagenesis, Sf9 expression, chiral HPLC, kinetics, and QM/MM simulations on human ALOX5; corroborated in the zebrafish ortholog","pmids":["31664810","26456699"],"confidence":"High","gaps":["In vitro specificity not tied to a cellular phenotype","FLAP dependence not addressed in the reconstituted system"]},{"year":2020,"claim":"Resolved the cis- and trans-elements controlling ALOX5 transcription, cataloguing promoter-binding zinc-finger factors and confirming a regulatory G-quadruplex; also defined receptor-driven (LPS/TGF-β) promoter activation.","evidence":"DNA pulldown mass spectrometry, G-quadruplex spectroscopy/antibody detection, and promoter-luciferase/product assays in monocytic cells","pmids":["32096311","32120263"],"confidence":"Medium","gaps":["Functional contribution of individual zinc-finger factors not dissected","Role of the G-quadruplex in cellular ALOX5 regulation not tested"]},{"year":2022,"claim":"Defined the activation logic of ALOX5-driven ferroptosis, showing ERK-dependent phosphorylation targets the enzyme to the nuclear membrane to execute caspase-independent death, with LysOX/ERK as an upstream driver.","evidence":"Genome-wide siRNA screen, caspase-9/Alox5 double-KO mice, localization and lipid peroxidation assays; AAV LysOX overexpression and BAPN inhibition in a seizure model","pmids":["36231015","36176900"],"confidence":"High","gaps":["Identity of the ERK isoform phosphosite on ALOX5 not fully mapped","How nuclear-membrane localization generates cytolytic lipid species unresolved"]},{"year":2023,"claim":"Established FLAP stabilization as the rate-limiting determinant of ALOX5-mediated, ACSL4-independent ferroptosis and showed mutant huntingtin hijacks this axis in neurodegeneration.","evidence":"RNAi screen, CRISPR knockout, overexpression, and in vivo HD mouse lifespan analysis","pmids":["36921996"],"confidence":"High","gaps":["Molecular mechanism of FLAP stabilization by mHTT not defined","Generality across other ferroptosis settings untested"]},{"year":2023,"claim":"Extended ALOX5 ferroptosis control to additional effector branches, linking it to autophagy-dependent ferroptosis via AMPK/mTOR and GPX4 suppression in melanoma.","evidence":"qRT-PCR, Western blot, iron/GSH/MDA assays, autophagy inhibition, and xenograft with recombinant ALOX5","pmids":["37080437"],"confidence":"Medium","gaps":["Direct mechanism connecting ALOX5 to AMPK/mTOR not established","Whether GPX4 suppression is cause or consequence unclear"]},{"year":2024,"claim":"Delineated a complete LTB4 effector cascade in autoimmune inflammation: ALOX5→LTB4→ORAI3 Ca2+ influx→NLRP3→pyroptosis in CD4+ T cells, providing a defined disease-relevant signaling axis.","evidence":"siRNA knockdown, pharmacological inhibition, Ca2+ flux and NLRP3 assays, and rodent RA models","pmids":["38412254"],"confidence":"High","gaps":["LTB4 receptor coupling to ORAI3 not molecularly resolved","Contribution relative to other inflammatory pathways in RA not quantified"]},{"year":2024,"claim":"Identified post-translational stabilization of ALOX5 by PARP-1 PARylation, defining a non-transcriptional route to elevated enzyme levels in pro-metastatic neutrophils.","evidence":"IP/MS, Co-IP, knockdown, in vitro invasion assays, and an in vivo tumor model","pmids":["38172525"],"confidence":"Medium","gaps":["PARylation sites on ALOX5 not mapped","Reciprocal validation of the interaction limited to single lab"]},{"year":2024,"claim":"Showed ALOX5-derived 5-HETE reprograms tumor-associated myeloid cells toward immunosuppressive M2/PD-L1 states via NRF2 nuclear translocation, broadening its tumor-microenvironment role.","evidence":"UHPLC-MS/MS oxylipin profiling, orthotopic glioma model, flow cytometry, and an ALOX5-targeting nanobody","pmids":["39142719"],"confidence":"Medium","gaps":["How 5-HETE triggers NRF2 translocation mechanistically unknown","Receptor mediating 5-HETE efflux/uptake not identified"]},{"year":null,"claim":"It remains unresolved how a single enzyme is partitioned between its pro-survival leukotriene-signaling outputs and its pro-death lipid-peroxidation/ferroptosis outputs within and across cell types, and what governs that switch.","evidence":"No single study in the corpus reconciles the opposing leukotriene-signaling versus ferroptosis-executing roles","pmids":[],"confidence":"Low","gaps":["No unified model linking localization, FLAP availability, and product identity to outcome","Determinants selecting LTB4 versus lipid-peroxidation fate uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[4,5,6]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[4,5]},{"term_id":"GO:0016829","term_label":"lyase activity","supporting_discovery_ids":[4]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[4,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[14,15]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[3,14,15]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[15]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[4,5,6]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,8,20,28]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,8,15,16]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,1,24]}],"complexes":[],"partners":["ALOX5AP","PARP1","PNPLA2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P09917","full_name":"Polyunsaturated fatty acid 5-lipoxygenase","aliases":["Arachidonate 5-lipoxygenase","5-LO","5-lipoxygenase"],"length_aa":674,"mass_kda":78.0,"function":"Catalyzes the oxygenation of arachidonate ((5Z,8Z,11Z,14Z)-eicosatetraenoate) to 5-hydroperoxyeicosatetraenoate (5-HPETE) followed by the dehydration to 5,6- epoxyeicosatetraenoate (Leukotriene A4/LTA4), the first two steps in the biosynthesis of leukotrienes, which are potent mediators of inflammation (PubMed:19022417, PubMed:21233389, PubMed:22516296, PubMed:23246375, PubMed:24282679, PubMed:24893149, PubMed:31664810, PubMed:8615788, PubMed:8631361). Also catalyzes the oxygenation of arachidonate into 8-hydroperoxyicosatetraenoate (8-HPETE) and 12-hydroperoxyicosatetraenoate (12-HPETE) (PubMed:23246375). Displays lipoxin synthase activity being able to convert (15S)-HETE into a conjugate tetraene (PubMed:31664810). Although arachidonate is the preferred substrate, this enzyme can also metabolize oxidized fatty acids derived from arachidonate such as (15S)-HETE, eicosapentaenoate (EPA) such as (18R)- and (18S)-HEPE or docosahexaenoate (DHA) which lead to the formation of specialized pro-resolving mediators (SPM) lipoxin and resolvins E and D respectively, therefore it participates in anti-inflammatory responses (PubMed:17114001, PubMed:21206090, PubMed:31664810, PubMed:32404334, PubMed:32841762, PubMed:8615788). Oxidation of DHA directly inhibits endothelial cell proliferation and sprouting angiogenesis via peroxisome proliferator-activated receptor gamma (PPARgamma) (By similarity). It does not catalyze the oxygenation of linoleic acid and does not convert (5S)-HETE to lipoxin isomers (PubMed:31664810). In addition to inflammatory processes, it participates in dendritic cell migration, wound healing through an antioxidant mechanism based on heme oxygenase-1 (HO-1) regulation expression, monocyte adhesion to the endothelium via ITGAM expression on monocytes (By similarity). Moreover, it helps establish an adaptive humoral immunity by regulating primary resting B cells and follicular helper T cells and participates in the CD40-induced production of reactive oxygen species (ROS) after CD40 ligation in B cells through interaction with PIK3R1 that bridges ALOX5 with CD40 (PubMed:21200133). May also play a role in glucose homeostasis, regulation of insulin secretion and palmitic acid-induced insulin resistance via AMPK (By similarity). Can regulate bone mineralization and fat cell differentiation increases in induced pluripotent stem cells (By similarity)","subcellular_location":"Cytoplasm; Nucleus matrix; Nucleus membrane; Cytoplasm, perinuclear region; Cytoplasm, cytosol; Nucleus envelope; Nucleus intermembrane space","url":"https://www.uniprot.org/uniprotkb/P09917/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ALOX5","classification":"Not Classified","n_dependent_lines":11,"n_total_lines":1208,"dependency_fraction":0.009105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ALOX5","total_profiled":1310},"omim":[{"mim_id":"621173","title":"G PROTEIN-COUPLED RECEPTOR 146; GPR146","url":"https://www.omim.org/entry/621173"},{"mim_id":"608557","title":"MYOCARDIAL INFARCTION, SUSCEPTIBILITY TO, 2","url":"https://www.omim.org/entry/608557"},{"mim_id":"608232","title":"LEUKEMIA, CHRONIC MYELOID; CML","url":"https://www.omim.org/entry/608232"},{"mim_id":"606748","title":"COACTOSIN-LIKE PROTEIN 1; COTL1","url":"https://www.omim.org/entry/606748"},{"mim_id":"606241","title":"DICER 1, RIBONUCLEASE III; DICER1","url":"https://www.omim.org/entry/606241"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lung","ntpm":69.4},{"tissue":"lymphoid tissue","ntpm":90.4}],"url":"https://www.proteinatlas.org/search/ALOX5"},"hgnc":{"alias_symbol":["5-LOX"],"prev_symbol":[]},"alphafold":{"accession":"P09917","domains":[{"cath_id":"2.60.60.20","chopping":"4-115","consensus_level":"high","plddt":94.9491,"start":4,"end":115},{"cath_id":"1.20.245.10","chopping":"149-176_367-558_606-668","consensus_level":"medium","plddt":97.5775,"start":149,"end":668},{"cath_id":"3.10.450.60","chopping":"180-359_562-594","consensus_level":"medium","plddt":97.9318,"start":180,"end":594}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P09917","model_url":"https://alphafold.ebi.ac.uk/files/AF-P09917-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P09917-F1-predicted_aligned_error_v6.png","plddt_mean":97.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ALOX5","jax_strain_url":"https://www.jax.org/strain/search?query=ALOX5"},"sequence":{"accession":"P09917","fasta_url":"https://rest.uniprot.org/uniprotkb/P09917.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P09917/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P09917"}},"corpus_meta":[{"pmid":"10369259","id":"PMC_10369259","title":"Pharmacogenetic 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gene.","date":"2011","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/22128029","citation_count":18,"is_preprint":false},{"pmid":"36605525","id":"PMC_36605525","title":"Interference of ALOX5 alleviates inflammation and fibrosis in high glucose‑induced renal mesangial cells.","date":"2022","source":"Experimental and therapeutic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/36605525","citation_count":17,"is_preprint":false},{"pmid":"38340032","id":"PMC_38340032","title":"Magnolin alleviated DSS-induced colitis by inhibiting ALOX5-mediated ferroptosis.","date":"2024","source":"The Kaohsiung journal of medical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38340032","citation_count":17,"is_preprint":false},{"pmid":"26959713","id":"PMC_26959713","title":"Copy number variation in ALOX5 and PTGER1 is associated with NSAIDs-induced urticaria and/or angioedema.","date":"2016","source":"Pharmacogenetics and genomics","url":"https://pubmed.ncbi.nlm.nih.gov/26959713","citation_count":17,"is_preprint":false},{"pmid":"34303896","id":"PMC_34303896","title":"Discovery of small molecule acting as multitarget inhibitor of colorectal cancer by simultaneous blocking of the key COX-2, 5-LOX and PIM-1 kinase enzymes.","date":"2021","source":"Bioorganic chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/34303896","citation_count":17,"is_preprint":false},{"pmid":"32120263","id":"PMC_32120263","title":"LPS induces ALOX5 promoter activation and 5-lipoxygenase expression in human monocytic cells.","date":"2020","source":"Prostaglandins, leukotrienes, and essential fatty acids","url":"https://pubmed.ncbi.nlm.nih.gov/32120263","citation_count":16,"is_preprint":false},{"pmid":"38172525","id":"PMC_38172525","title":"Cancer-educated neutrophils promote lung cancer progression via PARP-1-ALOX5-mediated MMP-9 expression.","date":"2024","source":"Cancer biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38172525","citation_count":16,"is_preprint":false},{"pmid":"32096311","id":"PMC_32096311","title":"Analysis of proximal ALOX5 promoter binding proteins by quantitative proteomics.","date":"2020","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/32096311","citation_count":16,"is_preprint":false},{"pmid":"20011686","id":"PMC_20011686","title":"Cardioprotective effect of 5-lipoxygenase gene (ALOX5) silencing in ischemia-reperfusion.","date":"2009","source":"Acta biochimica Polonica","url":"https://pubmed.ncbi.nlm.nih.gov/20011686","citation_count":16,"is_preprint":false},{"pmid":"27293987","id":"PMC_27293987","title":"Simultaneous targeting of 5-LOX-COX and ODC block NNK-induced lung adenoma progression to adenocarcinoma in A/J mice.","date":"2016","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/27293987","citation_count":16,"is_preprint":false},{"pmid":"39278904","id":"PMC_39278904","title":"Multiomics integrated analysis and experimental validation identify TLR4 and ALOX5 as oxidative stress-related biomarkers in intracranial aneurysms.","date":"2024","source":"Journal of neuroinflammation","url":"https://pubmed.ncbi.nlm.nih.gov/39278904","citation_count":15,"is_preprint":false},{"pmid":"17241301","id":"PMC_17241301","title":"5-LOX inhibitor modulates the inflammatory responses provoked by Helicobacter pylori infection.","date":"2007","source":"Helicobacter","url":"https://pubmed.ncbi.nlm.nih.gov/17241301","citation_count":15,"is_preprint":false},{"pmid":"33369192","id":"PMC_33369192","title":"CaMKIIγ regulates the viability and self-renewal of acute myeloid leukaemia stem-like cells by the Alox5/NF-κB pathway.","date":"2020","source":"International journal of laboratory 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archives of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/37955689","citation_count":13,"is_preprint":false},{"pmid":"31918059","id":"PMC_31918059","title":"Effects of ALOX5, IL6R and SFTPD gene polymorphisms on the risk of lung cancer: A case-control study in China.","date":"2020","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/31918059","citation_count":13,"is_preprint":false},{"pmid":"28623130","id":"PMC_28623130","title":"Leukotriene signaling via ALOX5 and cysteinyl leukotriene receptor 1 is dispensable for in vitro growth of CD34+CD38- stem and progenitor cells in chronic myeloid leukemia.","date":"2017","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/28623130","citation_count":13,"is_preprint":false},{"pmid":"33408499","id":"PMC_33408499","title":"Discovery and Development of a Novel mPGES-1/5-LOX Dual Inhibitor LFA-9 for Prevention and Treatment of Chronic Inflammatory Diseases.","date":"2020","source":"Journal of inflammation research","url":"https://pubmed.ncbi.nlm.nih.gov/33408499","citation_count":13,"is_preprint":false},{"pmid":"36231015","id":"PMC_36231015","title":"Mitochondrion-Mediated Cell Death through Erk1-Alox5 Independent of Caspase-9 Signaling.","date":"2022","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/36231015","citation_count":12,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":57064,"output_tokens":7422,"usd":0.141261,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16756,"output_tokens":5428,"usd":0.10974,"stage2_stop_reason":"end_turn"},"total_usd":0.251001,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Loss of Alox5 gene prevents BCR-ABL-induced chronic myeloid leukemia in mice by impairing leukemia stem cell (LSC) function—affecting differentiation, cell division, and survival of long-term LSCs—without significantly affecting normal hematopoietic stem cells.\",\n      \"method\": \"Genetic knockout (Alox5-/- mice), BCR-ABL retroviral transduction, bone marrow transplantation, LSC functional assays\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined cellular phenotype replicated with pharmacological inhibitor (zileuton), multiple orthogonal readouts\",\n      \"pmids\": [\"19503090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALOX5 mediates ACSL4-independent ferroptosis in Huntington's disease by stabilizing FLAP (its essential cofactor), thereby enhancing ALOX5-mediated lipoxygenase activity and lipid peroxidation in neurons; mHTT (HTTQ94) activates this pathway by stabilizing FLAP.\",\n      \"method\": \"RNAi screening, CRISPR/Cas9 knockout, overexpression, in vivo HD mouse model with lifespan analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (RNAi screen, KO, in vivo rescue), mechanistic dissection of FLAP stabilization\",\n      \"pmids\": [\"36921996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Cannabinoid receptor 2 (Cnr2) suppresses leukocyte inflammatory migration by downregulating Alox5 expression via inhibition of the JNK/c-Jun signaling pathway; alox5 is a transcriptional target of c-Jun, and inactivation of Alox5 blocks leukocyte migration.\",\n      \"method\": \"Chemical genetic screen in zebrafish, zinc finger nuclease-mediated mutagenesis of Cnr2, zebrafish leukocyte migration assay, human myeloid cell migration assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic inactivation in two model systems, pathway epistasis established\",\n      \"pmids\": [\"23539630\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ERK1-dependent phosphorylation of Alox5 is critical for targeting Alox5 to the nuclear membrane to mediate lipid peroxidation, resulting in nuclear translocation of cytolytic molecules and caspase-9-independent cell death; double knockout of caspase-9 and Alox5 in mice leads to significant T cell expansion, demonstrating these pathways act in parallel.\",\n      \"method\": \"Genome-wide siRNA library screening, double-knockout mice (caspase-9/Alox5), subcellular localization studies, lipid peroxidation assays\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — unbiased whole-genome screen, confirmed by double-KO in vivo, mechanistic localization established\",\n      \"pmids\": [\"36231015\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Mutagenesis of 'triad determinant' residues (F359W/A424I/N425M/A603I) in human ALOX5 shifts reaction specificity from dominant 5(S)-HpETE production to 15(S)- and 8(S)-HpETE, abolishes leukotriene synthase activity, broadens substrate acceptance to C18 fatty acids, and alters substrate orientation at the active site as confirmed by QM/MM calculations.\",\n      \"method\": \"Site-directed mutagenesis, Sf9 insect cell expression, product analysis by chiral HPLC, kinetic studies with deutero-labeled substrates, molecular dynamics and QM/MM simulations\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro enzyme with mutagenesis, structural modeling, and stereospecific product analysis\",\n      \"pmids\": [\"31664810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Zebrafish ALOX2 (functional ortholog of human ALOX5) is an arachidonic acid 5S-lipoxygenating enzyme with leukotriene synthase activity; mutagenesis of its triad determinants (F359W/A424I/N425M) shifts specificity from 5S- to dominant 15S-lipoxygenation, analogous to human ALOX5.\",\n      \"method\": \"Cloning and expression in pro- and eukaryotic systems, product characterization, site-directed mutagenesis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted enzyme activity, mutagenesis validation, single lab\",\n      \"pmids\": [\"26456699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Oxygenation of the 5-lipoxygenase product 5S-HETE by COX-2 yields a bicyclic di-endoperoxide; heme-catalyzed cleavage of this di-endoperoxide produces 4S-HNE, 8-oxo-5S-hydroxy-6E-octenoic acid, and malondialdehyde, establishing a convergent 5-LOX/COX-2 pathway to classic lipid peroxidation products.\",\n      \"method\": \"In vitro enzymatic reactions, hematin/ferrous iron treatment, chiral HPLC analysis of products\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with structural product characterization, chiral analysis\",\n      \"pmids\": [\"19553698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LysOX (lysyl oxidase) promotes ALOX5-mediated ferroptotic lipid peroxidation in neurons via activation of ERK signaling; adeno-associated virus-mediated LysOX overexpression enhances ferroptosis sensitivity and aggravates seizure-induced hippocampal damage, while LysOX inhibition blocks Alox5 activation and neuronal death.\",\n      \"method\": \"AAV-based gene transfer (overexpression), pharmacological LysOX inhibition (BAPN), seizure mouse model, lipid peroxidation assays\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic overexpression and pharmacological inhibition, single lab, two orthogonal approaches\",\n      \"pmids\": [\"36176900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALOX5 increases LTB4 production in RA CD4+ T cells, which stimulates Ca2+ influx through ORAI3 channels, leading to NLRP3 inflammasome activation and pyroptosis; knockdown or pharmacological inhibition of ALOX5 suppresses pyroptosis and improves symptoms in rodent RA models.\",\n      \"method\": \"siRNA knockdown, pharmacological inhibition, Ca2+ flux assays, NLRP3 inflammasome activation assays, rodent RA models\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mechanistic pathway (ALOX5→LTB4→ORAI3→NLRP3→pyroptosis) established by multiple orthogonal methods including in vivo validation\",\n      \"pmids\": [\"38412254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PKC-β upregulates Alox5 expression via ERK1/2 signaling in CML cells, and Alox5 promotes TKI resistance by inactivating PTEN; this PKC-β/ERK1/2/Alox5/PTEN axis mediates BCR-Abl-independent TKI resistance.\",\n      \"method\": \"shRNA knockdown, gene expression comparison of 84 leukemia-related genes, PTEN activity assays, in vivo CML-PDX mouse model\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway epistasis and in vivo validation, single lab\",\n      \"pmids\": [\"33561320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PARP-1 interacts with and PARylates (post-translationally modifies) ALOX5 to stabilize it in cancer-educated neutrophils, leading to increased MMP-9 expression; blocking PARP-1 decreases ALOX5 expression and MMP-9 production, and eliminates neutrophil-mediated lung cancer cell invasion.\",\n      \"method\": \"Immunoprecipitation coupled to mass spectrometry (IP/MS), co-immunoprecipitation (Co-IP), gene knockdown, in vitro invasion assays, in vivo tumor model\",\n      \"journal\": \"Cancer biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus MS identified PARP-1/ALOX5 interaction, functional rescue with gene knockdown, single lab\",\n      \"pmids\": [\"38172525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALOX5 expression and activity are upregulated by HER2 in breast cancer cells; HER2 inhibition decreases ALOX5 expression and activity but not ALOX5AP expression; ALOX5 mediates breast cancer growth and migration through RhoA, focal adhesion, and PI3K/Akt/mTOR signaling.\",\n      \"method\": \"HER2 inhibitor treatment, siRNA knockdown, proliferation and migration assays, signaling pathway analysis\",\n      \"journal\": \"BioMed research international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — HER2 inhibition plus KD with pathway readouts, single lab\",\n      \"pmids\": [\"33224971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALOX5 is transcriptionally repressed in MLL-rearranged AML via Polycomb repressive complex 2 (PRC2); restoration of Alox5 expression sensitizes leukemic cells to doxorubicin and cytarabine, with this drug-sensitizing effect associated with negative correlation of Stat and K-Ras signaling pathways.\",\n      \"method\": \"Affymetrix microarray profiling, ChIP assays (PRC2), colony-forming/replating assays, bone marrow transplantation, in vivo AML model with chemotherapy\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP established PRC2-mediated repression, in vivo rescue with defined signaling readouts, single lab\",\n      \"pmids\": [\"28500307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Alox5 deficiency in mice impairs glucose-stimulated insulin secretion and reduces expression of insulin and Pdx1 genes in islets; siRNA knockdown of ALOX5 in isolated human islets similarly decreases insulin and PDX1 gene expression by ~50% and insulin secretion threefold, demonstrating a direct role of 5-LO in pancreatic beta cell function.\",\n      \"method\": \"Alox5-/- mouse characterization, siRNA knockdown in human islets, glucose tolerance tests, insulin secretion assays\",\n      \"journal\": \"Diabetologia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mouse KO and human siRNA knockdown with orthogonal readouts, replicated across species\",\n      \"pmids\": [\"18421434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"In aging rat brain, 5-LOX mRNA and protein increase in hippocampus and cerebellum, and the membrane/cytosol 5-LOX ratio is larger in older rats, indicating increased 5-LOX protein translocation/activation during aging.\",\n      \"method\": \"Quantitative RT-PCR with internal standards, quantitative Western immunoblotting, subcellular fractionation (membrane vs. cytosol)\",\n      \"journal\": \"Neurobiology of aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct subcellular fractionation showing translocation, replicated in two brain regions, single lab\",\n      \"pmids\": [\"11016533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"5-LOX protein is expressed in peripheral blood T lymphocytes and is present primarily in the cytoplasm with some nuclear localization; it translocates to the nuclear periphery after mitogenic stimulation. Purified resting T lymphocytes cannot synthesize leukotrienes without exogenous arachidonic acid, whereas Jurkat cells produce LTC4 and LTB4 upon CD3-CD28 cross-linking, which is abolished by 5-LOX inhibitors.\",\n      \"method\": \"Western blot, immunofluorescence, FACS, in situ RT-PCR, leukotriene synthesis assays with inhibitors\",\n      \"journal\": \"Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by immunofluorescence and fractionation, functional leukotriene synthesis linked to localization/activation state\",\n      \"pmids\": [\"17484769\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"5-LOX and its metabolite LTB4 activate NF-κB in hepatoma cells: inhibition or knockdown of 5-LOX reduces NF-κB p65 mRNA expression, decreases IκBα phosphorylation in the cytoplasm, and reduces nuclear NF-κB p65 levels; exogenous LTB4 activates NF-κB in a dose-dependent manner.\",\n      \"method\": \"siRNA knockdown, MK886 pharmacological inhibition, Western blot, immunofluorescence, transcriptional activity reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus pharmacological inhibition with multiple readouts, single lab\",\n      \"pmids\": [\"22293202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALOX5-derived 5-HETE in gastric cancer cells activates MEK/ERK signaling to promote tumor growth and reduce chemotherapy sensitivity; ALOX5 inhibition suppresses ERK-mediated signaling and enhances chemotherapy efficacy.\",\n      \"method\": \"ALOX5 overexpression and genetic/pharmacological inhibition, ERK pathway analysis by Western blot, growth/survival assays, exogenous 5-HETE addition\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with defined signaling readout, exogenous metabolite rescue, single lab\",\n      \"pmids\": [\"34121352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In glioma, ALOX5-derived 5-HETE promotes M2 polarization, PD-L1 expression, and migration of glioma-associated microglia/macrophages by facilitating nuclear translocation of NRF2; an ALOX5-targeted nanobody suppresses 5-HETE efflux and attenuates M2 polarization.\",\n      \"method\": \"UHPLC-MS/MS oxylipin profiling, orthotopic glioma mouse model, immunofluorescence, flow cytometry, nanobody development, in vivo bioluminescent imaging\",\n      \"journal\": \"Journal for immunotherapy of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic pathway (ALOX5→5-HETE→NRF2 nuclear translocation→M2/PD-L1) shown with multiple methods, single lab\",\n      \"pmids\": [\"39142719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In intrahepatic cholangiocarcinoma, LTB4 (metabolite of ALOX5) binds BLT1/BLT2 receptors on tumor-associated macrophages (TAMs) to activate the PI3K pathway, promoting M2 macrophage migration toward tumor cells and ICC progression.\",\n      \"method\": \"In vitro co-culture model, bulk RNA-seq after co-culture, scRNA-seq analysis, xenograft tumor model with ALOX5 inhibitor (zileuton) plus CSF1R inhibitor\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-culture plus in vivo validation, pathway defined as LTB4-BLT1/2-PI3K, single lab\",\n      \"pmids\": [\"38124204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALOX5 expression in bladder cancer is transcriptionally regulated by EGR1; ALOX5 deficiency (via CRISPR/Cas9) confers resistance to RSL3-induced ferroptosis in high-pathological-stage bladder cancer cells, identifying ALOX5 as a pro-ferroptotic factor downstream of EGR1-mediated transcription.\",\n      \"method\": \"RNA-seq, RNAi loss-of-function, CRISPR/Cas9 knockout, EGR1 transcriptional regulation assays, in vitro and in vivo ferroptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO plus transcription factor upstream mechanism, single lab\",\n      \"pmids\": [\"38062004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Mass spectrometric DNA pulldown identified 66 specific ALOX5 promoter-binding proteins, including known regulators Sp1 and Sp3, novel zinc finger proteins (KLF13, KLF16, MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281, ZNF579), two helicases (BLM, DHX36), and hnRNPD/hnRNPK; the GC-rich ALOX5 promoter was confirmed to form DNA G-quadruplex structures by spectroscopic and antibody-based methods.\",\n      \"method\": \"DNA pulldown, label-free quantitative mass spectrometry, G-quadruplex spectroscopy, antibody-based detection\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic proteomics of promoter interactome with G-quadruplex confirmation by orthogonal methods, single lab\",\n      \"pmids\": [\"32096311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LPS activates the ALOX5 promoter and synergizes with TGF-β to increase 5-LO mRNA (up to 54-fold in MM1 cells) and protein expression in human monocytic cells, dramatically increasing leukotriene biosynthesis; this represents a receptor-mediated transcriptional control mechanism for 5-LO expression.\",\n      \"method\": \"ALOX5 promoter-luciferase reporter assays, RT-PCR, Western blot, leukotriene product quantification\",\n      \"journal\": \"Prostaglandins, leukotrienes, and essential fatty acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter reporter plus protein/product assays, two cell lines tested, single lab\",\n      \"pmids\": [\"32120263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In Alox5-/- mice on a high-fat diet, enhanced osteoclastogenesis and bone loss occurs with increased RANKL, PPARγ, and COX-2 gene expression in femur; treatment with celecoxib (COX-2 inhibitor) suppresses these increases, placing 5-LO upstream of COX-2-mediated osteoclast activity in a gene-by-environment interaction.\",\n      \"method\": \"Alox5-/- mice on high-fat diet, microarchitectural analysis, histomorphometry, in vitro osteoclastogenesis assay, gene expression, celecoxib rescue\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with pharmacological rescue establishing epistasis, multiple skeletal readouts, single lab\",\n      \"pmids\": [\"22128029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In 3xTg Alzheimer's disease mice, ALOX5 gene deficiency prevents stress-induced worsening of tau phosphorylation, impaired tau solubility, increased GSK3β activity, compromised long-term potentiation, and fear-conditioned memory deficits caused by restraint/isolation stress.\",\n      \"method\": \"Triple-transgenic 3xTg mice crossed with 5-LO-/- mice, restraint/isolation stress paradigm, tau phosphorylation/solubility assays, GSK3β activity measurement, electrophysiology (LTP), behavioral fear conditioning\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in vivo with multiple orthogonal phenotypic readouts, single lab\",\n      \"pmids\": [\"25122659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PEDF-R (PNPLA2 gene product) physically binds human recombinant 5-LOX via ionic interactions through a specific peptide region (E5b/P1 spanning Leu159–Met325); this binding inhibits 5-LOX lipoxygenase activity and reduces LTB4 levels and RPE cell death during oxidative stress.\",\n      \"method\": \"Peptide-affinity chromatography, pulldown assays with recombinant 5-LOX, lipoxygenase activity assay, LTB4 ELISA, cell viability assays\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding pulldown with recombinant protein, functional enzyme inhibition assay, single lab\",\n      \"pmids\": [\"27635633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALOX5 silencing by RNAi in isolated rat cardiomyocytes reduces ALOX5 expression 4.7-fold, decreases LTC4 levels, and reduces necrotic cell death during anoxia-reoxygenation; in vivo ALOX5 knockdown in rat heart reduces infarct size 3.8-fold during ischemia-reperfusion.\",\n      \"method\": \"siRNA knockdown in cardiomyocytes, real-time PCR, LTC4 measurement, necrosis/viability assays, in vivo rat I/R model\",\n      \"journal\": \"Acta biochimica Polonica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo KD with mechanistic readout (LTC4), single lab\",\n      \"pmids\": [\"20011686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALOX5 regulates tumor-associated macrophage M2 polarization via the JAK/STAT pathway in pancreatic cancer; ALOX5 overexpression promotes chemotaxis of macrophages toward cancer cells, while zileuton inhibits these effects.\",\n      \"method\": \"Lentiviral ALOX5 overexpression in PANC-1 and macrophages, JAK/STAT pathway analysis, nude mouse orthotopic transplantation model\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function with defined pathway (JAK/STAT), in vivo confirmation, single lab\",\n      \"pmids\": [\"37348233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALOX5 promotes autophagy-dependent ferroptosis in melanoma by activating the AMPK/mTOR pathway and inhibiting GPX4 expression; inhibition of autophagy reduces ALOX5-enhanced ferroptosis, demonstrating synergy between autophagy and ALOX5 in this cell death pathway.\",\n      \"method\": \"qRT-PCR, Western blot, IHC, iron/GSH/MDA assays, autophagy inhibition, xenograft model with recombinant ALOX5 protein\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo with mechanistic AMPK/mTOR/GPX4 pathway readouts, single lab\",\n      \"pmids\": [\"37080437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CaMKIIγ knockdown in AML stem-like cell lines downregulates Alox5 levels and decreases c-Myc and p-IκBα (with unchanged total IκBα), placing CaMKIIγ upstream of Alox5 in regulating the Alox5/NF-κB pathway controlling AML stem-like cell viability and self-renewal.\",\n      \"method\": \"shRNA lentiviral knockdown of CaMKIIγ, qRT-PCR, Western blot for NF-κB pathway components, CD34+ cell proportion analysis\",\n      \"journal\": \"International journal of laboratory hematology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single KD with pathway readouts, no reciprocal rescue, single lab\",\n      \"pmids\": [\"33369192\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ALOX5 encodes arachidonate 5-lipoxygenase, a key enzyme that oxygenates arachidonic acid to produce 5(S)-HpETE and subsequently leukotrienes (including LTA4, LTB4, and cysteinyl leukotrienes); its catalytic specificity is determined by 'triad determinant' residues in the active site, its activity requires FLAP as an essential cofactor, it is transcriptionally regulated by Sp1-family zinc finger proteins and the ALOX5 promoter G-quadruplex, it translocates from cytoplasm to the nuclear membrane (via ERK1-dependent phosphorylation) to execute lipid peroxidation-driven ferroptosis, it activates NF-κB via LTB4, it drives CD4+ T cell pyroptosis via LTB4→ORAI3→NLRP3 inflammasome signaling, it is stabilized by PARP-1 PARylation and by mHTT-mediated FLAP stabilization, and it functions as a critical regulator of leukemia stem cell survival, Alzheimer's-related tau pathology, and multiple forms of regulated cell death including ferroptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ALOX5 encodes arachidonate 5-lipoxygenase, the enzyme that oxygenates arachidonic acid to 5(S)-HpETE and downstream 5-HETE and leukotrienes (LTB4, LTC4), and through these lipid mediators it acts as a central regulator of inflammatory cell behavior, regulated cell death, and tumor biology [#4, #15]. Catalytic specificity for 5(S)-lipoxygenation and leukotriene synthase activity is set by a small set of 'triad determinant' active-site residues (F359/A424/N425/A603); mutating these shifts the enzyme toward 15(S)- and 8(S)-HpETE production, abolishes leukotriene synthesis, and broadens substrate acceptance, a specificity logic conserved in the zebrafish ortholog [#4, #5]. Enzymatic output bifurcates into two functional arms: a leukotriene arm in which LTB4 drives downstream signaling — activating NF-\\u03baB in hepatoma cells, driving Ca2+ influx through ORAI3 to license NLRP3 inflammasome-mediated pyroptosis in CD4+ T cells, and recruiting BLT-receptor-bearing M2 macrophages — and a lipid-peroxidation arm in which ALOX5 executes ferroptosis [#16, #8, #19, #20]. ALOX5-driven ferroptosis depends on its essential cofactor FLAP, whose stabilization (including by mutant huntingtin) enhances lipoxygenase activity and lipid peroxidation, and on ERK-dependent phosphorylation that targets ALOX5 to the nuclear membrane, where it can drive caspase-independent death; this peroxidation activity also engages autophagy/AMPK-mTOR signaling and GPX4 suppression [#1, #3, #28]. Activity is gated by translocation from cytoplasm to nuclear membrane upon cell activation [#15, #14], and the enzyme is controlled both transcriptionally — its GC-rich, G-quadruplex-forming promoter is bound by Sp1/Sp3-family and other zinc-finger factors, by EGR1, and by PRC2-mediated repression — and post-translationally by FLAP stabilization, PARP-1 PARylation, and inhibitory binding by PEDF-R/PNPLA2 [#21, #20, #12, #10, #25]. Functionally, ALOX5 is required for BCR-ABL leukemia stem cell maintenance, contributes to TKI resistance and chemoresistance across leukemia and solid tumors, supports tumor-associated macrophage polarization and invasion, and modulates beta-cell insulin secretion, osteoclastogenesis, ischemic injury, and stress-induced tau pathology [#0, #9, #13, #23, #24, #26, #27].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that 5-LOX is dynamically regulated at the level of subcellular distribution, with activation reflected by increased membrane-associated enzyme — framing translocation as a control point for activity.\",\n      \"evidence\": \"RT-PCR, Western blot, and membrane/cytosol fractionation in aging rat brain\",\n      \"pmids\": [\"11016533\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not define the molecular trigger for translocation\", \"Correlative with aging, not causal for any phenotype\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showed ALOX5 has a physiological role beyond inflammation, supporting glucose-stimulated insulin secretion and beta-cell gene expression, indicating broad tissue functions.\",\n      \"evidence\": \"Alox5-/- mice and siRNA knockdown in human islets with insulin secretion and PDX1/insulin expression readouts\",\n      \"pmids\": [\"18421434\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lipid-mediator effector linking ALOX5 to insulin transcription not identified\", \"Mechanism connecting enzyme activity to PDX1 regulation unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined ALOX5 as a non-redundant requirement for leukemia stem cell function, establishing it as a selective therapeutic vulnerability in BCR-ABL CML.\",\n      \"evidence\": \"Alox5-/- mice with BCR-ABL transduction, bone marrow transplantation, and LSC functional assays plus zileuton\",\n      \"pmids\": [\"19503090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The downstream lipid mediator driving LSC maintenance not pinpointed\", \"Why normal HSCs are spared mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrated chemically how 5-LOX products converge with COX-2 to generate classic lipid peroxidation aldehydes, linking the enzyme to oxidative damage chemistry.\",\n      \"evidence\": \"In vitro enzymatic reactions with heme/iron treatment and chiral HPLC product characterization\",\n      \"pmids\": [\"19553698\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular relevance of the di-endoperoxide pathway not demonstrated\", \"Does not test contribution to disease phenotypes\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Linked ALOX5 localization state to functional leukotriene output in T cells, showing activation-dependent nuclear translocation and stimulus-gated leukotriene synthesis.\",\n      \"evidence\": \"Western blot, immunofluorescence, and leukotriene synthesis assays with inhibitors in T lymphocytes and Jurkat cells\",\n      \"pmids\": [\"17484769\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Signal driving translocation not defined\", \"Requirement for exogenous arachidonate leaves endogenous substrate supply open\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected ALOX5 enzymatic output to transcriptional signaling by showing LTB4 activates NF-\\u03baB, providing a route from lipid mediator to gene expression.\",\n      \"evidence\": \"siRNA knockdown, MK886 inhibition, and NF-\\u03baB reporter/Western assays with exogenous LTB4 in hepatoma cells\",\n      \"pmids\": [\"22293202\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"LTB4 receptor mediating NF-\\u03baB activation not identified here\", \"Single cell-type context\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed ALOX5 within an upstream signaling cascade for inflammatory migration, identifying it as a c-Jun transcriptional target repressed by CB2 signaling.\",\n      \"evidence\": \"Chemical genetic screen and zinc-finger nuclease mutagenesis in zebrafish plus human myeloid migration assays\",\n      \"pmids\": [\"23539630\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding of c-Jun to the ALOX5 promoter not shown\", \"Lipid mediator executing migration not specified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified opposing transcriptional control of ALOX5 in leukemia, with PRC2 silencing the gene and its restoration sensitizing cells to chemotherapy.\",\n      \"evidence\": \"Microarray, PRC2 ChIP, colony assays, and in vivo AML chemotherapy model\",\n      \"pmids\": [\"28500307\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which restored ALOX5 sensitizes to drugs not established\", \"Relationship to its LSC-promoting role left unreconciled\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Mapped the structural basis of 5-LOX reaction specificity to discrete triad-determinant residues, defining how positional and stereo selectivity and leukotriene synthase activity are encoded.\",\n      \"evidence\": \"Site-directed mutagenesis, Sf9 expression, chiral HPLC, kinetics, and QM/MM simulations on human ALOX5; corroborated in the zebrafish ortholog\",\n      \"pmids\": [\"31664810\", \"26456699\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro specificity not tied to a cellular phenotype\", \"FLAP dependence not addressed in the reconstituted system\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved the cis- and trans-elements controlling ALOX5 transcription, cataloguing promoter-binding zinc-finger factors and confirming a regulatory G-quadruplex; also defined receptor-driven (LPS/TGF-\\u03b2) promoter activation.\",\n      \"evidence\": \"DNA pulldown mass spectrometry, G-quadruplex spectroscopy/antibody detection, and promoter-luciferase/product assays in monocytic cells\",\n      \"pmids\": [\"32096311\", \"32120263\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional contribution of individual zinc-finger factors not dissected\", \"Role of the G-quadruplex in cellular ALOX5 regulation not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined the activation logic of ALOX5-driven ferroptosis, showing ERK-dependent phosphorylation targets the enzyme to the nuclear membrane to execute caspase-independent death, with LysOX/ERK as an upstream driver.\",\n      \"evidence\": \"Genome-wide siRNA screen, caspase-9/Alox5 double-KO mice, localization and lipid peroxidation assays; AAV LysOX overexpression and BAPN inhibition in a seizure model\",\n      \"pmids\": [\"36231015\", \"36176900\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the ERK isoform phosphosite on ALOX5 not fully mapped\", \"How nuclear-membrane localization generates cytolytic lipid species unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established FLAP stabilization as the rate-limiting determinant of ALOX5-mediated, ACSL4-independent ferroptosis and showed mutant huntingtin hijacks this axis in neurodegeneration.\",\n      \"evidence\": \"RNAi screen, CRISPR knockout, overexpression, and in vivo HD mouse lifespan analysis\",\n      \"pmids\": [\"36921996\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of FLAP stabilization by mHTT not defined\", \"Generality across other ferroptosis settings untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended ALOX5 ferroptosis control to additional effector branches, linking it to autophagy-dependent ferroptosis via AMPK/mTOR and GPX4 suppression in melanoma.\",\n      \"evidence\": \"qRT-PCR, Western blot, iron/GSH/MDA assays, autophagy inhibition, and xenograft with recombinant ALOX5\",\n      \"pmids\": [\"37080437\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanism connecting ALOX5 to AMPK/mTOR not established\", \"Whether GPX4 suppression is cause or consequence unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Delineated a complete LTB4 effector cascade in autoimmune inflammation: ALOX5\\u2192LTB4\\u2192ORAI3 Ca2+ influx\\u2192NLRP3\\u2192pyroptosis in CD4+ T cells, providing a defined disease-relevant signaling axis.\",\n      \"evidence\": \"siRNA knockdown, pharmacological inhibition, Ca2+ flux and NLRP3 assays, and rodent RA models\",\n      \"pmids\": [\"38412254\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"LTB4 receptor coupling to ORAI3 not molecularly resolved\", \"Contribution relative to other inflammatory pathways in RA not quantified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified post-translational stabilization of ALOX5 by PARP-1 PARylation, defining a non-transcriptional route to elevated enzyme levels in pro-metastatic neutrophils.\",\n      \"evidence\": \"IP/MS, Co-IP, knockdown, in vitro invasion assays, and an in vivo tumor model\",\n      \"pmids\": [\"38172525\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PARylation sites on ALOX5 not mapped\", \"Reciprocal validation of the interaction limited to single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed ALOX5-derived 5-HETE reprograms tumor-associated myeloid cells toward immunosuppressive M2/PD-L1 states via NRF2 nuclear translocation, broadening its tumor-microenvironment role.\",\n      \"evidence\": \"UHPLC-MS/MS oxylipin profiling, orthotopic glioma model, flow cytometry, and an ALOX5-targeting nanobody\",\n      \"pmids\": [\"39142719\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How 5-HETE triggers NRF2 translocation mechanistically unknown\", \"Receptor mediating 5-HETE efflux/uptake not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how a single enzyme is partitioned between its pro-survival leukotriene-signaling outputs and its pro-death lipid-peroxidation/ferroptosis outputs within and across cell types, and what governs that switch.\",\n      \"evidence\": \"No single study in the corpus reconciles the opposing leukotriene-signaling versus ferroptosis-executing roles\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model linking localization, FLAP availability, and product identity to outcome\", \"Determinants selecting LTB4 versus lipid-peroxidation fate uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [4, 5, 6]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0016829\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [4, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [14, 15]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [3, 14, 15]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [4, 5, 6]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 8, 20, 28]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 8, 15, 16]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 1, 24]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ALOX5AP\", \"PARP1\", \"PNPLA2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}