{"gene":"FDX1","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2010,"finding":"Human FDX1 (adrenodoxin/ferredoxin 1) is specifically required for steroidogenesis via electron donation to mitochondrial cytochrome P450 enzymes (e.g., CYP11A1 converting cholesterol to pregnenolone), but is NOT required for Fe/S cluster or heme A biosynthesis in human cells; that role belongs exclusively to FDX2.","method":"RNAi-mediated depletion of FDX1 or FDX2 in human cells; functional assays for steroid conversion, heme A synthesis, and Fe/S protein maturation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean RNAi KD with multiple orthogonal functional readouts (steroidogenesis, heme A, Fe/S), replicated concept across FDX1 and FDX2 in a single rigorous study","pmids":["20547883"],"is_preprint":false},{"year":2022,"finding":"FDX1 is specifically required for steroidogenesis, heme a biosynthesis, and lipoyl cofactor biosynthesis. In the lipoylation pathway FDX1 provides electrons to kickstart the radical chain reaction catalyzed by lipoyl synthase (LIAS). Target specificity of FDX1 versus FDX2 was assigned to small conserved sequence motifs; swapping these motifs exchanged their substrate specificities.","method":"Cell-based functional assays with FDX1/FDX2 KO or swapped motif mutants; in vitro reconstitution of lipoyl synthase radical reaction; structural analysis","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including in vitro reconstitution, mutagenesis of specificity motifs, and cell-based KO assays in one rigorous study","pmids":["36280795"],"is_preprint":false},{"year":2023,"finding":"FDX1 directly binds the lipoyl synthase enzyme LIAS, promoting LIAS's functional interaction with the lipoyl carrier protein GCSH and thereby driving mitochondrial protein lipoylation; this regulation is independent of Fe/S cluster biosynthesis. Loss of FDX1 impairs all four lipoylation-dependent TCA cycle enzymes, abolishes cellular respiration, and is conditionally lethal under low glucose.","method":"Co-immunoprecipitation/pulldown (direct FDX1–LIAS binding); metabolite profiling; transcriptional profiling; CRISPR/RNAi loss-of-function with defined metabolic phenotypes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding demonstrated by pulldown, mechanism separated from Fe/S by epistasis, replicated metabolomics and transcriptomics, confirmed in peer-reviewed and preprint versions (37453661, 36778498)","pmids":["37453661","36778498"],"is_preprint":false},{"year":2023,"finding":"FDX1 is dispensable under 1% O2 in cultured human cells. FDX1 is essential for lipoic acid cofactor production via the ISC-containing enzyme lipoyl synthase; hypoxia rescues the growth defect of FDX1 KO cells but does not rescue lipoylation, arguing against an alternative biosynthetic route for lipoate under hypoxia.","method":"CRISPR KO of FDX1 or lipoyl synthase; growth assays at normoxia vs. 1% O2; biochemical measurement of lipoylation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean CRISPR KO, two orthogonal conditions (normoxia/hypoxia), direct measurement of lipoylation as molecular readout","pmids":["37481209"],"is_preprint":false},{"year":2023,"finding":"FDX1 acts as the mitochondrial matrix reductase that catalyzes reduction of ES-Cu(II) to Cu(I), releasing copper inside mitochondria where it is bioavailable for metalation of cytochrome c oxidase. In FDX1-null cells, ES fails to rescue CcO abundance/activity under copper deficiency, while ES-mediated copper delivery to non-mitochondrial cuproproteins continues via FDX1-independent mechanisms.","method":"Genetic KO of FDX1 combined with elesclomol-copper treatment; biochemical measurement of CcO activity and abundance; copper quantification; cell-biological assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean CRISPR KO, multiple orthogonal readouts (CcO activity, copper levels, cell survival), positive and negative controls included","pmids":["36848556"],"is_preprint":false},{"year":2023,"finding":"FDX1 is required for the biogenesis of cytochrome c oxidase (CcO) in mammalian cardiomyocytes; FDX1 KO reduces CcO abundance, assembly, copper levels, and heme a/a3 levels. Copper supplementation does not rescue CcO, but overexpression of heme a synthase (COX15) partially rescues COX1 abundance, placing FDX1 upstream of COX15 in CcO biogenesis.","method":"CRISPR-Cas9 KO in rat cardiomyocyte cell line; measurement of CcO complex assembly, copper, and heme a; rescue experiments with COX15 overexpression","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean CRISPR KO with multiple biochemical readouts and epistasis rescue experiment placing FDX1 upstream of COX15","pmids":["37858707"],"is_preprint":false},{"year":1998,"finding":"Crystal structure of truncated bovine adrenodoxin (Adx/FDX1 ortholog) Adx(4-108) determined at 1.85 Å resolution. The protein has a core domain and an interaction domain (35 residues) containing all residues involved in binding adrenodoxin reductase (AR) and cytochrome P450. A domain motion coupled to the redox state of the [2Fe-2S] cluster was identified, and a strikingly asymmetric charge distribution implicated in electrostatic steering of AR and P450 interactions.","method":"X-ray crystallography at 1.85 Å (MAD phasing using Fe atoms); functional validation from prior biochemical studies","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional domain annotation supported by extensive prior biochemical data","pmids":["9551550"],"is_preprint":false},{"year":2017,"finding":"Both human FDX1 and FDX2 bind the cysteine desulfurase complex (NFS1/ISD11/ACP) with residues near the Fe/S cluster involved in interaction; FDX2 binds more tightly than FDX1. Both reduced ferredoxins become oxidized in the presence of the complex + L-cysteine, and both can support in vitro Fe/S cluster assembly on ISCU, though FDX2 is faster.","method":"NMR spectroscopy (binding interface mapping); isothermal titration calorimetry; in vitro Fe/S cluster assembly assay","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution (in vitro cluster assembly) + NMR + ITC, multiple orthogonal methods in one study","pmids":["28001042"],"is_preprint":false},{"year":2025,"finding":"Deep mutational scanning of FDX1 identified two conserved solvent-exposed residues on alpha helix 3, D136 and D139, as essential for both cuproptosis induction and protein lipoylation in cells; charge-reversal mutations abolish both activities despite retaining full in vitro enzymatic (electron transfer) activity. Dihydrolipoamide dehydrogenase (DLD, the E3 subunit) was identified as an alternative FDX1 reductase both in cells and in vitro.","method":"Deep mutational scanning; site-directed mutagenesis; cell-based cuproptosis and lipoylation assays; in vitro enzymatic assays; structural and genomic analyses","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — deep mutational scanning + site-directed mutagenesis + in vitro enzymatic assay + cell functional assays, multiple orthogonal methods in one study","pmids":["41423452"],"is_preprint":false},{"year":2025,"finding":"AKT1 phosphorylates FDX1, abrogating FDX1-induced cuproptosis and aerobic respiration while promoting glycolysis in triple-negative breast cancer cells. Copper activates AKT signaling, which in turn inhibits FDX1 via phosphorylation.","method":"Kinase-substrate phosphorylation assays; AKT1 overexpression/knockdown; cell-based cuproptosis and metabolic readouts; in vitro and in vivo rescue experiments with AKT1 inhibitors + copper ionophores","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphorylation identified with functional rescue, but single lab; abstract does not detail in vitro kinase assay explicitly","pmids":["39976173"],"is_preprint":false},{"year":2025,"finding":"PUMA directly binds FDX1 at its R155 site, enhancing FDX1 function and promoting DLAT/DLST oligomerization and cuproptosis. PUMA binding also raises ubiquitination at FDX1 K182, leading to FDX1 degradation during cuproptosis.","method":"Co-IP; immunofluorescence; dual-luciferase reporter assay (for upstream miR-144-3p/PUMA axis); Western blot for DLAT oligomerization and FDX1 ubiquitination","journal":"Cellular oncology (Dordrecht, Netherlands)","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — Co-IP with defined residues (R155, K182), single lab, limited orthogonal validation of direct interaction","pmids":["41212484"],"is_preprint":false},{"year":2025,"finding":"EPR spectroscopy provided direct spectroscopic evidence that reduced FDX1 transfers electrons to the ES-Cu2+ complex in vitro and in cells; ES-Cu2+ accepts electrons from FDX1 more readily than free Cu2+. EPR and molecular docking showed FDX1 has higher binding affinity for ES-Cu2+ than its homolog FDX2, explaining functional specificity.","method":"Low-temperature and power saturation EPR spectroscopy; molecular docking; in vitro and in vivo electron transfer assays","journal":"Chemistry (Weinheim an der Bergstrasse, Germany)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct spectroscopic (EPR) evidence of electron transfer in vitro and in vivo, combined with molecular docking, rigorous single study","pmids":["40484707"],"is_preprint":false},{"year":2020,"finding":"Substrate binding and adrenodoxin (Adx/FDX1) recognition in CYP24A1 are allosterically coupled: Adx binding induces conformational changes in the F and G helices of CYP24A1 that are required for substrate binding, as shown by differential chemical cross-linking and NMR perturbations. A semiconserved nonpolar interaction at the CYP24A1–Adx interface influences CYP24A1 regioselectivity.","method":"Chemical cross-linking coupled to mass spectrometry; NMR spectroscopy; CYP24A1 functional assays","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — cross-linking MS + NMR + functional assay in one study, single lab","pmids":["32259445"],"is_preprint":false},{"year":2005,"finding":"Overexpression of adrenodoxin (Adx/FDX1) in human cell lines causes ROS production in mitochondria, disruption of mitochondrial transmembrane potential, cytochrome c release, and caspase activation, leading to apoptosis independent of p53.","method":"Transient overexpression; ROS assays; mitochondrial membrane potential measurement; cytochrome c release assay; caspase activation assay; cell viability across 11 cell lines","journal":"Biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal cellular assays with defined mechanistic readouts, single lab","pmids":["15927889"],"is_preprint":false},{"year":2017,"finding":"Transcription factors SF1 and cJUN cooperate to activate the mouse Fdx1 promoter in Leydig cells. SF1 is recruited to a region between -124 and -306 bp of the Fdx1 promoter and is required (by RNA interference) for Fdx1 transcription, while cJUN is dispensable but cooperates with SF1 to upregulate expression.","method":"Luciferase reporter assays; chromatin immunoprecipitation (ChIP); RNA interference; promoter deletion analysis in MA-10 and TM3 Leydig cells","journal":"The Journal of steroid biochemistry and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP + reporter assays + RNAi in two cell lines, single lab","pmids":["28274746"],"is_preprint":false},{"year":2023,"finding":"METTL16 lactylation at K229 (inhibited by SIRT2) promotes m6A modification on FDX1 mRNA, increasing FDX1 expression and sensitizing gastric cancer cells to cuproptosis. Copper stress promotes METTL16 lactylation, linking metabolic state to FDX1-dependent cell death.","method":"m6A-seq/MeRIP; METTL16 lactylation site mapping; SIRT2 inhibition (AGK2); cell-based cuproptosis assays in vitro and in vivo","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epitranscriptomic mapping with defined modification site and functional rescue, single lab","pmids":["37863889"],"is_preprint":false},{"year":2025,"finding":"p53 enhances elesclomol-Cu-induced cuproptosis in HCC via FDXR-mediated FDX1 upregulation: p53 activation increases FDXR expression, which promotes FDX1 upregulation and subsequent DLAT oligomerization; FDXR knockdown reverses p53-mediated sensitization to cuproptosis.","method":"p53 overexpression/knockdown; siRNA-mediated FDXR/FDX1 knockdown; Western blot for DLAT oligomerization; cell viability assay; in vivo xenograft","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis (FDXR KD reverses p53 effect) with downstream molecular readout, single lab","pmids":["40630211"],"is_preprint":false},{"year":2023,"finding":"FDX1 interacts with G6PD and reduces G6PD protein stability, decreasing NADPH and GSH levels and thereby enhancing cuproptosis in endometriosis cells.","method":"Co-immunoprecipitation (FDX1–G6PD interaction); G6PD stability assays; NADPH/GSH measurement; cell-based cuproptosis assays; mouse ectopic endometrial tissue model","journal":"Apoptosis : an international journal on programmed cell death","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with functional consequence assays, single lab, limited mechanistic depth on how stability is reduced","pmids":["37119432"],"is_preprint":false},{"year":2025,"finding":"FDX1 overexpression triggers mitochondrial permeability transition pore opening, leading to cytosolic release of mitochondrial DNA and mt-dsRNA, activating cGAS and RIG-I/MDA5 sensors, TBK1 phosphorylation, and a type I interferon response that precedes ferroptosis and reshapes the tumor microenvironment.","method":"FDX1 overexpression; mitochondrial permeability transition assays; cytosolic fractionation for mtDNA/mt-dsRNA; cGAS/RIG-I pathway activation assays; orthotopic syngeneic models","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal molecular readouts (mtDNA/dsRNA release, sensor activation, IFN response) with in vivo validation, single lab","pmids":["41199656"],"is_preprint":false},{"year":2025,"finding":"FDX1 bound directly to FMR1 protein and upregulated FMR1 expression, subsequently restraining Bcl-2 and N-cadherin while enhancing ALCAM, cleaved caspase-3, and E-cadherin; FMR1 knockdown reversed FDX1-mediated suppression of ccRCC growth and metastasis.","method":"Co-IP; TMT proteomic sequencing; RNA sequencing; cell phenotype and in vivo mouse orthotopic tumor experiments","journal":"Cell death discovery","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP for FDX1–FMR1 interaction, epistasis by FMR1 KD rescue, single lab","pmids":["40118855"],"is_preprint":false},{"year":2025,"finding":"PVT1 lncRNA directly binds the FDX1 promoter (35/98 nt of PVT1 binding -104/-41 bp region), increases H3K27ac deposition, and activates FDX1 transcription; PVT1 also recruits SF1 to the FDX1 promoter, further enhancing FDX1 expression and cuproptosis in colorectal cancer.","method":"ChIRP; ChIP; luciferase reporter assay; bioinformatics; molecular docking; xenograft model","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct promoter binding confirmed by ChIRP and ChIP with defined nucleotide coordinates, multiple orthogonal methods, single lab","pmids":["40505346"],"is_preprint":false},{"year":2025,"finding":"Trilobatin (TLB) directly binds FDX1 (confirmed by surface plasmon resonance) and suppresses FDX1-mediated cuproptosis after doxorubicin treatment, reducing mitochondrial oxidative stress and cuproptosis-related proteins in a dose-dependent manner.","method":"Surface plasmon resonance binding assay; molecular dynamics simulation; siRNA knockdown of FDX1; in vivo and in vitro cardiotoxicity models; Western blot for cuproptosis markers","journal":"British journal of pharmacology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — SPR confirms direct binding, functional consequence shown by siRNA, single lab","pmids":["39933533"],"is_preprint":false},{"year":2025,"finding":"Icaritin (ICT) directly binds FDX1 with high affinity (confirmed by surface plasmon resonance and molecular dynamics simulation), downregulates FDX1 protein expression, and inhibits FDX1-mediated cuproptosis in hippocampal neurons, reducing DLAT oligomerization and restoring copper homeostasis.","method":"Surface plasmon resonance; computational molecular docking and trajectory simulations; FDX1 knockdown; Western blot for DLAT and lipoylation markers; mitochondrial functional assays","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — SPR with MD simulation + FDX1 KD epistasis, single lab","pmids":["40998065"],"is_preprint":false}],"current_model":"FDX1 is a mitochondrial [2Fe-2S] ferredoxin that functions as an electron shuttle with at least three distinct biochemical roles: (1) it donates electrons to mitochondrial cytochrome P450 enzymes (e.g., CYP11A1, CYP24A1) to drive steroid hormone and vitamin D metabolism; (2) it directly binds lipoyl synthase (LIAS), facilitating electron donation to kickstart the radical SAM reaction that installs lipoyl cofactors on TCA cycle enzymes, with its acidic alpha-helix 3 (D136/D139) being the critical protein–protein interaction interface; and (3) it catalyzes the reduction of Cu(II) (as the elesclomol-Cu complex) to Cu(I) inside the mitochondrial matrix, releasing bioavailable copper for CcO metalation and, when copper overloads, triggering cuproptosis through accumulation of lipoylated protein aggregates; FDX2, not FDX1, handles Fe/S cluster and heme A biosynthesis in human cells."},"narrative":{"mechanistic_narrative":"FDX1 (adrenodoxin/ferredoxin 1) is a mitochondrial [2Fe-2S] ferredoxin that acts as an electron shuttle dedicated to a defined set of biosynthetic and metabolic reactions, functionally distinct from its homolog FDX2, which carries out Fe/S cluster and heme A biosynthesis [PMID:20547883, PMID:36280795]. Its founding role is to donate electrons to mitochondrial cytochrome P450 enzymes to drive steroidogenesis (e.g., CYP11A1) and vitamin D metabolism (CYP24A1), where Adx binding allosterically remodels the P450 active site to enable substrate binding and tune regioselectivity [PMID:20547883, PMID:32259445]. A second, central role is in mitochondrial protein lipoylation: FDX1 directly binds lipoyl synthase (LIAS), supplies the reducing equivalents that initiate its radical-SAM chemistry, and promotes LIAS engagement with the lipoyl carrier GCSH, thereby lipoylating all four lipoylation-dependent TCA cycle enzymes; loss of FDX1 abolishes respiration and is conditionally lethal under low glucose, a defect rescued for growth but not lipoylation by hypoxia [PMID:36280795, PMID:37453661, PMID:36778498, PMID:37481209]. FDX1 is also required upstream of COX15 for cytochrome c oxidase biogenesis, controlling CcO assembly, copper, and heme a/a3 content [PMID:37858707]. As a matrix reductase, FDX1 reduces the elesclomol-Cu(II) complex to Cu(I) to release bioavailable copper for CcO metalation, and excess copper handling through this activity drives cuproptosis via accumulation of lipoylated protein aggregates [PMID:36848556, PMID:40484707]. The acidic solvent-exposed residues D136 and D139 on alpha-helix 3 form the critical protein-protein interface required for both lipoylation and cuproptosis, independent of intrinsic electron-transfer activity [PMID:41423452]. FDX1 activity is constrained by AKT1-mediated phosphorylation, which suppresses cuproptosis and respiration in favor of glycolysis [PMID:39976173].","teleology":[{"year":1998,"claim":"Established the structural basis for how the ferredoxin engages its electron-acceptor partners, revealing a redox-coupled domain motion and an asymmetric charge surface that electrostatically steers interactions with adrenodoxin reductase and P450.","evidence":"X-ray crystallography of truncated bovine adrenodoxin at 1.85 Å with functional domain annotation","pmids":["9551550"],"confidence":"High","gaps":["Resolved an ortholog fragment rather than full-length human FDX1","Did not address the lipoylation or copper-reductase interfaces later defined on alpha-helix 3"]},{"year":2010,"claim":"Resolved the long-standing ambiguity between the two human ferredoxins, demonstrating FDX1 is specifically required for steroidogenic P450 electron donation and not for Fe/S cluster or heme A biosynthesis, which belong to FDX2.","evidence":"RNAi depletion of FDX1 vs FDX2 in human cells with orthogonal readouts for steroidogenesis, heme A, and Fe/S maturation","pmids":["20547883"],"confidence":"High","gaps":["Did not yet identify lipoylation or copper roles for FDX1","Mechanism of substrate discrimination between FDX1 and FDX2 not defined"]},{"year":2017,"claim":"Tested whether FDX1 can participate in Fe/S assembly biochemically, showing both ferredoxins bind the NFS1/ISD11/ACP desulfurase and can support in vitro cluster assembly, but FDX2 binds tighter and is faster — clarifying the in vivo division of labor.","evidence":"NMR interface mapping, ITC, and in vitro Fe/S cluster assembly on ISCU","pmids":["28001042"],"confidence":"High","gaps":["In vitro competence does not establish in vivo Fe/S role for FDX1","Structural determinants of differential affinity not defined"]},{"year":2020,"claim":"Defined the mechanism of FDX1-P450 functional coupling, showing adrenodoxin binding allosterically reorganizes the CYP24A1 F/G helices to permit substrate binding and influence regioselectivity.","evidence":"Chemical cross-linking mass spectrometry, NMR perturbation, and CYP24A1 functional assays","pmids":["32259445"],"confidence":"Medium","gaps":["Single P450 partner examined","Allosteric model not extended to other mitochondrial P450s"]},{"year":2022,"claim":"Assigned FDX1 a third biosynthetic role in lipoyl cofactor synthesis and pinpointed the sequence motifs that distinguish FDX1 from FDX2 substrate specificity.","evidence":"Cell-based KO and motif-swap mutants, in vitro reconstitution of the lipoyl synthase radical reaction, and structural analysis","pmids":["36280795"],"confidence":"High","gaps":["Did not resolve the physical FDX1–LIAS interface at residue level","Did not separate lipoylation from Fe/S epistatically"]},{"year":2023,"claim":"Demonstrated FDX1 directly binds LIAS to promote its interaction with GCSH and drive lipoylation independent of Fe/S biosynthesis, with loss abolishing respiration and causing conditional lethality.","evidence":"Co-IP/pulldown, metabolite and transcriptional profiling, and CRISPR/RNAi loss-of-function with metabolic phenotyping","pmids":["37453661","36778498"],"confidence":"High","gaps":["Stoichiometry and structure of the FDX1–LIAS–GCSH assembly unresolved","Reductant source for the reaction not defined in this study"]},{"year":2023,"claim":"Tested whether an alternative lipoate route exists under hypoxia, showing hypoxia rescues FDX1-KO growth but not lipoylation, confirming FDX1/LIAS as the sole biosynthetic path.","evidence":"CRISPR KO of FDX1 or LIAS, growth at normoxia vs 1% O2, and direct lipoylation measurement","pmids":["37481209"],"confidence":"High","gaps":["Mechanism by which hypoxia bypasses the growth requirement not fully defined"]},{"year":2023,"claim":"Identified FDX1 as the matrix reductase that converts elesclomol-Cu(II) to bioavailable Cu(I) for cytochrome c oxidase metalation, distinguishing mitochondrial from non-mitochondrial copper delivery.","evidence":"FDX1 KO with elesclomol-copper treatment, CcO activity and abundance assays, and copper quantification","pmids":["36848556"],"confidence":"High","gaps":["FDX1-independent copper delivery routes to non-mitochondrial cuproproteins not molecularly defined"]},{"year":2023,"claim":"Placed FDX1 within the CcO biogenesis pathway upstream of heme a synthase COX15, showing copper supplementation cannot rescue but COX15 overexpression partially restores COX1.","evidence":"CRISPR KO in rat cardiomyocytes with CcO assembly, copper, and heme a measurements plus COX15 rescue","pmids":["37858707"],"confidence":"High","gaps":["Direct mechanism linking FDX1 to heme a synthesis not defined","Whether FDX1 acts via lipoylation or independently in this context unclear"]},{"year":2023,"claim":"Began connecting FDX1 to upstream regulation and tumor cell death by linking epitranscriptomic (METTL16 m6A) and proteostatic control to FDX1 expression and cuproptosis sensitivity.","evidence":"m6A-seq/MeRIP, METTL16 lactylation site mapping, and cuproptosis assays in gastric cancer","pmids":["37863889"],"confidence":"Medium","gaps":["Single cancer context","Direct effect of m6A on FDX1 protein output vs mRNA stability not fully separated"]},{"year":2025,"claim":"Used unbiased mutational scanning to define the essential functional interface, showing D136/D139 on alpha-helix 3 are required for both lipoylation and cuproptosis despite preserved electron-transfer activity, and identified DLD as an alternative FDX1 reductase.","evidence":"Deep mutational scanning, site-directed mutagenesis, in vitro enzymatic assays, and cell-based cuproptosis/lipoylation readouts","pmids":["41423452"],"confidence":"High","gaps":["Structural detail of how D136/D139 engages partners not solved","Relative in vivo contributions of DLD vs canonical reductase unquantified"]},{"year":2025,"claim":"Provided direct spectroscopic confirmation of FDX1-driven electron transfer to elesclomol-Cu and explained FDX1's specificity over FDX2 by higher binding affinity for the ES-Cu complex.","evidence":"Low-temperature/power-saturation EPR, molecular docking, and in vitro/in vivo electron-transfer assays","pmids":["40484707"],"confidence":"High","gaps":["Behavior toward free copper vs chelated copper in physiological settings not fully resolved"]},{"year":2025,"claim":"Revealed post-translational and protein-level regulation of FDX1 activity through AKT1 phosphorylation that toggles cells from FDX1-dependent respiration/cuproptosis toward glycolysis.","evidence":"Kinase-substrate assays, AKT1 gain/loss-of-function, metabolic and cuproptosis readouts, and AKT inhibitor + ionophore rescue in vivo","pmids":["39976173"],"confidence":"Medium","gaps":["Phosphosite(s) on FDX1 not explicitly mapped in abstract","Single lab; mechanism of how phosphorylation impairs electron transfer unclear"]},{"year":2025,"claim":"Extended the regulatory network with transcriptional, partner-binding, and immunogenic consequences of FDX1, including PVT1/SF1-driven transcription, FDXR/p53-mediated upregulation, PUMA binding, and induction of an mtDNA/dsRNA-driven interferon response.","evidence":"ChIRP/ChIP, Co-IP, dual-luciferase, fractionation for cytosolic mtDNA/dsRNA, and in vivo tumor models across multiple cancers","pmids":["40505346","40630211","41212484","41199656"],"confidence":"Medium","gaps":["Most interactions rest on single Co-IP/single-lab evidence","Direct vs indirect nature of several partner interactions not reciprocally validated"]},{"year":null,"claim":"How FDX1's three biochemical activities (P450 electron donation, lipoylation, copper reduction) are partitioned, regulated, and prioritized within a single mitochondrion remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of full-length human FDX1 bound to LIAS or to the copper complex","Physiological reductant choice (canonical reductase vs DLD) under different metabolic states unclear","Whether cuproptosis is a physiological program or a pharmacological liability not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[4,8,11]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[0,6,7]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[4,13,18]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,2,3]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,8,13]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[5]}],"complexes":[],"partners":["LIAS","GCSH","NFS1","CYP11A1","CYP24A1","G6PD","PUMA","FMR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P10109","full_name":"Adrenodoxin, mitochondrial","aliases":["Adrenal ferredoxin","Ferredoxin-1","Hepatoredoxin"],"length_aa":184,"mass_kda":19.4,"function":"Essential for the synthesis of various steroid hormones (PubMed:20547883, PubMed:21636783). Participates in the reduction of mitochondrial cytochrome P450 for steroidogenesis (PubMed:20547883, PubMed:21636783). Transfers electrons from adrenodoxin reductase to CYP11A1, a cytochrome P450 that catalyzes cholesterol side-chain cleavage (PubMed:20547883, PubMed:21636783). Does not form a ternary complex with adrenodoxin reductase and CYP11A1 but shuttles between the two enzymes to transfer electrons (By similarity)","subcellular_location":"Mitochondrion matrix","url":"https://www.uniprot.org/uniprotkb/P10109/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FDX1","classification":"Not Classified","n_dependent_lines":279,"n_total_lines":1208,"dependency_fraction":0.23096026490066227},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FDX1","total_profiled":1310},"omim":[{"mim_id":"614585","title":"FERREDOXIN 2; FDX2","url":"https://www.omim.org/entry/614585"},{"mim_id":"103260","title":"FERREDOXIN 1; FDX1","url":"https://www.omim.org/entry/103260"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Mitochondria","reliability":"Enhanced"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"adrenal gland","ntpm":300.4}],"url":"https://www.proteinatlas.org/search/FDX1"},"hgnc":{"alias_symbol":["ADX"],"prev_symbol":["FDX"]},"alphafold":{"accession":"P10109","domains":[{"cath_id":"3.10.20.30","chopping":"67-167","consensus_level":"high","plddt":96.2034,"start":67,"end":167}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P10109","model_url":"https://alphafold.ebi.ac.uk/files/AF-P10109-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P10109-F1-predicted_aligned_error_v6.png","plddt_mean":76.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FDX1","jax_strain_url":"https://www.jax.org/strain/search?query=FDX1"},"sequence":{"accession":"P10109","fasta_url":"https://rest.uniprot.org/uniprotkb/P10109.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P10109/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P10109"}},"corpus_meta":[{"pmid":"9582371","id":"PMC_9582371","title":"Assembly of iron-sulfur clusters. 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cosponsored by the American Society of Preventive Oncology","url":"https://pubmed.ncbi.nlm.nih.gov/39699293","citation_count":5,"is_preprint":false},{"pmid":"40484707","id":"PMC_40484707","title":"Electron Paramagnetic Resonance Insights into Direct Electron Transfer Between FDX1 and Elesclomol-Cu2+ Complex in Cuproptosis.","date":"2025","source":"Chemistry (Weinheim an der Bergstrasse, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/40484707","citation_count":5,"is_preprint":false},{"pmid":"41355968","id":"PMC_41355968","title":"Precision targeting of FDX1-mediated cuproptosis by a ROS-responsive hydrogel for myocardial ischemia-reperfusion injury treatment.","date":"2026","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/41355968","citation_count":4,"is_preprint":false},{"pmid":"40118855","id":"PMC_40118855","title":"FDX1 overexpression inhibits the growth and metastasis of clear cell renal cell carcinoma by upregulating FMR1 expression.","date":"2025","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/40118855","citation_count":4,"is_preprint":false},{"pmid":"40843273","id":"PMC_40843273","title":"Tanshinone IIA promotes METTL3/METTL14-mediated FDX1 m6A modification to induce cuproptosis in bladder cancer.","date":"2025","source":"Toxicology research","url":"https://pubmed.ncbi.nlm.nih.gov/40843273","citation_count":4,"is_preprint":false},{"pmid":"40998065","id":"PMC_40998065","title":"Targeting FDX1 with Icaritin attenuates neuronal cuproptosis by reconciling mitochondrial fission-fusion dynamics and bioenergetic homeostasis.","date":"2025","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40998065","citation_count":4,"is_preprint":false},{"pmid":"40850113","id":"PMC_40850113","title":"Polystyrene nanoplastics carrying copper ion induce FDX1-mediated cuproptosis.","date":"2025","source":"Ecotoxicology and environmental safety","url":"https://pubmed.ncbi.nlm.nih.gov/40850113","citation_count":4,"is_preprint":false},{"pmid":"40244269","id":"PMC_40244269","title":"FDX1 Regulates the Phosphorylation of ATM, DNA-PKcs Akt, and EGFR and Affects Radioresistance Under Severe Hypoxia in the Glioblastoma Cell Line T98G.","date":"2025","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40244269","citation_count":3,"is_preprint":false},{"pmid":"41212484","id":"PMC_41212484","title":"Unraveling the miR-144-3p/PUMA pathway: a novel regulator of FDX1-mediated cuproptosis in colorectal cancer.","date":"2025","source":"Cellular oncology (Dordrecht, Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/41212484","citation_count":3,"is_preprint":false},{"pmid":"37516708","id":"PMC_37516708","title":"Effect of the mGlu4 positive allosteric modulator ADX-88178 on parkinsonism, psychosis-like behaviours and dyskinesia in the MPTP-lesioned marmoset.","date":"2023","source":"Psychopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/37516708","citation_count":3,"is_preprint":false},{"pmid":"41199656","id":"PMC_41199656","title":"Fe-S Protein FDX1 Triggers Tumor-Intrinsic Innate Immunity via Mitochondrial Nucleic Acids Release to Orchestrate Ferroptosis in CCRCC.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/41199656","citation_count":2,"is_preprint":false},{"pmid":"40911146","id":"PMC_40911146","title":"Cuproptosis-Related Gene FDX1 Induces Malignant Progression and Immune Suppression in Triple-Negative Breast Cancer.","date":"2025","source":"Biochemical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/40911146","citation_count":2,"is_preprint":false},{"pmid":"41501140","id":"PMC_41501140","title":"METTL3-mediated m6A modification of FDX1 confers resistance to cuproptosis and promotes hepatocellular carcinoma progression.","date":"2026","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/41501140","citation_count":2,"is_preprint":false},{"pmid":"40354319","id":"PMC_40354319","title":"Modulation of Cuproptosis Pathway Genes (DLAT, FDX1) and Antioxidant Enzyme Activities in Obese Mice in Response to Quercetin and Calorie Restriction.","date":"2025","source":"DNA and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/40354319","citation_count":2,"is_preprint":false},{"pmid":"41513631","id":"PMC_41513631","title":"FDX1-mediated cuproptosis promotes cholestatic liver injury exacerbated by taurocholic acid-enhanced copper accumulation.","date":"2026","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/41513631","citation_count":2,"is_preprint":false},{"pmid":"41407052","id":"PMC_41407052","title":"Mitochondrial uncoupling sensitizes gastric cancer cells to elesclomol-induced cuproptosis via FDX1/DLAT upregulation.","date":"2025","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41407052","citation_count":2,"is_preprint":false},{"pmid":"40691025","id":"PMC_40691025","title":"[Acupuncture inhibits cuproptosis to prolong the time window of thrombolysis by down-regulating cerebral FDX1 and DLAT in rats with cerebral infarction].","date":"2025","source":"Zhen ci yan jiu = Acupuncture research","url":"https://pubmed.ncbi.nlm.nih.gov/40691025","citation_count":2,"is_preprint":false},{"pmid":"40629130","id":"PMC_40629130","title":"Modulating ovarian cancer progression through FDX1-driven autophagy.","date":"2025","source":"NPJ precision oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40629130","citation_count":1,"is_preprint":false},{"pmid":"40769372","id":"PMC_40769372","title":"METTL14 promotes hippocampal neuronal cuproptosis via m6A modification on FDX1 mRNA in cerebral ischemia-reperfusion injury.","date":"2025","source":"Brain research bulletin","url":"https://pubmed.ncbi.nlm.nih.gov/40769372","citation_count":1,"is_preprint":false},{"pmid":"40449270","id":"PMC_40449270","title":"Mycobacterium tuberculosis stimulates cuproptosis by regulating Lnc-Gm5532 to target FDX1 for bacteria intracellular survival.","date":"2025","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40449270","citation_count":1,"is_preprint":false},{"pmid":"41232822","id":"PMC_41232822","title":"Mettl1 mitigates sepsis-induced cardiomyopathy via inhibition of FDX1-dependent cuproptosis.","date":"2025","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/41232822","citation_count":1,"is_preprint":false},{"pmid":"41100003","id":"PMC_41100003","title":"Integrated Multi-omics and Experimental Validation Reveal FDX1/LIAS-Mediated Cuproptosis as a Potential Driver of Diabetic Kidney Disease.","date":"2025","source":"Biological trace element research","url":"https://pubmed.ncbi.nlm.nih.gov/41100003","citation_count":1,"is_preprint":false},{"pmid":"41648530","id":"PMC_41648530","title":"Client distribution between Chlamydomonas FDX1 and FDX2 in carbon, nitrogen and sulfur assimilation.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41648530","citation_count":1,"is_preprint":false},{"pmid":"41469708","id":"PMC_41469708","title":"Atractylenolide III mitigates polycystic ovary syndrome by activating FDX1-mediated proliferation of ovarian granulocyte cells via PI3K/AKT/mTOR.","date":"2025","source":"Journal of ovarian research","url":"https://pubmed.ncbi.nlm.nih.gov/41469708","citation_count":1,"is_preprint":false},{"pmid":"40987411","id":"PMC_40987411","title":"Targeting cuproptosis by FDX1 in acetaminophen-induced liver injury.","date":"2025","source":"Chemico-biological interactions","url":"https://pubmed.ncbi.nlm.nih.gov/40987411","citation_count":0,"is_preprint":false},{"pmid":"41673093","id":"PMC_41673093","title":"PRR15 suppresses renal cell carcinoma progression via the NF-κB/FDX1 axis to induce cuproptosis and mitochondrial dysfunction.","date":"2026","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/41673093","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":55832,"output_tokens":6099,"usd":0.129491,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14794,"output_tokens":4722,"usd":0.09601,"stage2_stop_reason":"end_turn"},"total_usd":0.225501,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"Human FDX1 (adrenodoxin/ferredoxin 1) is specifically required for steroidogenesis via electron donation to mitochondrial cytochrome P450 enzymes (e.g., CYP11A1 converting cholesterol to pregnenolone), but is NOT required for Fe/S cluster or heme A biosynthesis in human cells; that role belongs exclusively to FDX2.\",\n      \"method\": \"RNAi-mediated depletion of FDX1 or FDX2 in human cells; functional assays for steroid conversion, heme A synthesis, and Fe/S protein maturation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean RNAi KD with multiple orthogonal functional readouts (steroidogenesis, heme A, Fe/S), replicated concept across FDX1 and FDX2 in a single rigorous study\",\n      \"pmids\": [\"20547883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FDX1 is specifically required for steroidogenesis, heme a biosynthesis, and lipoyl cofactor biosynthesis. In the lipoylation pathway FDX1 provides electrons to kickstart the radical chain reaction catalyzed by lipoyl synthase (LIAS). Target specificity of FDX1 versus FDX2 was assigned to small conserved sequence motifs; swapping these motifs exchanged their substrate specificities.\",\n      \"method\": \"Cell-based functional assays with FDX1/FDX2 KO or swapped motif mutants; in vitro reconstitution of lipoyl synthase radical reaction; structural analysis\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including in vitro reconstitution, mutagenesis of specificity motifs, and cell-based KO assays in one rigorous study\",\n      \"pmids\": [\"36280795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FDX1 directly binds the lipoyl synthase enzyme LIAS, promoting LIAS's functional interaction with the lipoyl carrier protein GCSH and thereby driving mitochondrial protein lipoylation; this regulation is independent of Fe/S cluster biosynthesis. Loss of FDX1 impairs all four lipoylation-dependent TCA cycle enzymes, abolishes cellular respiration, and is conditionally lethal under low glucose.\",\n      \"method\": \"Co-immunoprecipitation/pulldown (direct FDX1–LIAS binding); metabolite profiling; transcriptional profiling; CRISPR/RNAi loss-of-function with defined metabolic phenotypes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding demonstrated by pulldown, mechanism separated from Fe/S by epistasis, replicated metabolomics and transcriptomics, confirmed in peer-reviewed and preprint versions (37453661, 36778498)\",\n      \"pmids\": [\"37453661\", \"36778498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FDX1 is dispensable under 1% O2 in cultured human cells. FDX1 is essential for lipoic acid cofactor production via the ISC-containing enzyme lipoyl synthase; hypoxia rescues the growth defect of FDX1 KO cells but does not rescue lipoylation, arguing against an alternative biosynthetic route for lipoate under hypoxia.\",\n      \"method\": \"CRISPR KO of FDX1 or lipoyl synthase; growth assays at normoxia vs. 1% O2; biochemical measurement of lipoylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean CRISPR KO, two orthogonal conditions (normoxia/hypoxia), direct measurement of lipoylation as molecular readout\",\n      \"pmids\": [\"37481209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FDX1 acts as the mitochondrial matrix reductase that catalyzes reduction of ES-Cu(II) to Cu(I), releasing copper inside mitochondria where it is bioavailable for metalation of cytochrome c oxidase. In FDX1-null cells, ES fails to rescue CcO abundance/activity under copper deficiency, while ES-mediated copper delivery to non-mitochondrial cuproproteins continues via FDX1-independent mechanisms.\",\n      \"method\": \"Genetic KO of FDX1 combined with elesclomol-copper treatment; biochemical measurement of CcO activity and abundance; copper quantification; cell-biological assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean CRISPR KO, multiple orthogonal readouts (CcO activity, copper levels, cell survival), positive and negative controls included\",\n      \"pmids\": [\"36848556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FDX1 is required for the biogenesis of cytochrome c oxidase (CcO) in mammalian cardiomyocytes; FDX1 KO reduces CcO abundance, assembly, copper levels, and heme a/a3 levels. Copper supplementation does not rescue CcO, but overexpression of heme a synthase (COX15) partially rescues COX1 abundance, placing FDX1 upstream of COX15 in CcO biogenesis.\",\n      \"method\": \"CRISPR-Cas9 KO in rat cardiomyocyte cell line; measurement of CcO complex assembly, copper, and heme a; rescue experiments with COX15 overexpression\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean CRISPR KO with multiple biochemical readouts and epistasis rescue experiment placing FDX1 upstream of COX15\",\n      \"pmids\": [\"37858707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Crystal structure of truncated bovine adrenodoxin (Adx/FDX1 ortholog) Adx(4-108) determined at 1.85 Å resolution. The protein has a core domain and an interaction domain (35 residues) containing all residues involved in binding adrenodoxin reductase (AR) and cytochrome P450. A domain motion coupled to the redox state of the [2Fe-2S] cluster was identified, and a strikingly asymmetric charge distribution implicated in electrostatic steering of AR and P450 interactions.\",\n      \"method\": \"X-ray crystallography at 1.85 Å (MAD phasing using Fe atoms); functional validation from prior biochemical studies\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional domain annotation supported by extensive prior biochemical data\",\n      \"pmids\": [\"9551550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Both human FDX1 and FDX2 bind the cysteine desulfurase complex (NFS1/ISD11/ACP) with residues near the Fe/S cluster involved in interaction; FDX2 binds more tightly than FDX1. Both reduced ferredoxins become oxidized in the presence of the complex + L-cysteine, and both can support in vitro Fe/S cluster assembly on ISCU, though FDX2 is faster.\",\n      \"method\": \"NMR spectroscopy (binding interface mapping); isothermal titration calorimetry; in vitro Fe/S cluster assembly assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution (in vitro cluster assembly) + NMR + ITC, multiple orthogonal methods in one study\",\n      \"pmids\": [\"28001042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Deep mutational scanning of FDX1 identified two conserved solvent-exposed residues on alpha helix 3, D136 and D139, as essential for both cuproptosis induction and protein lipoylation in cells; charge-reversal mutations abolish both activities despite retaining full in vitro enzymatic (electron transfer) activity. Dihydrolipoamide dehydrogenase (DLD, the E3 subunit) was identified as an alternative FDX1 reductase both in cells and in vitro.\",\n      \"method\": \"Deep mutational scanning; site-directed mutagenesis; cell-based cuproptosis and lipoylation assays; in vitro enzymatic assays; structural and genomic analyses\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — deep mutational scanning + site-directed mutagenesis + in vitro enzymatic assay + cell functional assays, multiple orthogonal methods in one study\",\n      \"pmids\": [\"41423452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AKT1 phosphorylates FDX1, abrogating FDX1-induced cuproptosis and aerobic respiration while promoting glycolysis in triple-negative breast cancer cells. Copper activates AKT signaling, which in turn inhibits FDX1 via phosphorylation.\",\n      \"method\": \"Kinase-substrate phosphorylation assays; AKT1 overexpression/knockdown; cell-based cuproptosis and metabolic readouts; in vitro and in vivo rescue experiments with AKT1 inhibitors + copper ionophores\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphorylation identified with functional rescue, but single lab; abstract does not detail in vitro kinase assay explicitly\",\n      \"pmids\": [\"39976173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PUMA directly binds FDX1 at its R155 site, enhancing FDX1 function and promoting DLAT/DLST oligomerization and cuproptosis. PUMA binding also raises ubiquitination at FDX1 K182, leading to FDX1 degradation during cuproptosis.\",\n      \"method\": \"Co-IP; immunofluorescence; dual-luciferase reporter assay (for upstream miR-144-3p/PUMA axis); Western blot for DLAT oligomerization and FDX1 ubiquitination\",\n      \"journal\": \"Cellular oncology (Dordrecht, Netherlands)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — Co-IP with defined residues (R155, K182), single lab, limited orthogonal validation of direct interaction\",\n      \"pmids\": [\"41212484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EPR spectroscopy provided direct spectroscopic evidence that reduced FDX1 transfers electrons to the ES-Cu2+ complex in vitro and in cells; ES-Cu2+ accepts electrons from FDX1 more readily than free Cu2+. EPR and molecular docking showed FDX1 has higher binding affinity for ES-Cu2+ than its homolog FDX2, explaining functional specificity.\",\n      \"method\": \"Low-temperature and power saturation EPR spectroscopy; molecular docking; in vitro and in vivo electron transfer assays\",\n      \"journal\": \"Chemistry (Weinheim an der Bergstrasse, Germany)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct spectroscopic (EPR) evidence of electron transfer in vitro and in vivo, combined with molecular docking, rigorous single study\",\n      \"pmids\": [\"40484707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Substrate binding and adrenodoxin (Adx/FDX1) recognition in CYP24A1 are allosterically coupled: Adx binding induces conformational changes in the F and G helices of CYP24A1 that are required for substrate binding, as shown by differential chemical cross-linking and NMR perturbations. A semiconserved nonpolar interaction at the CYP24A1–Adx interface influences CYP24A1 regioselectivity.\",\n      \"method\": \"Chemical cross-linking coupled to mass spectrometry; NMR spectroscopy; CYP24A1 functional assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — cross-linking MS + NMR + functional assay in one study, single lab\",\n      \"pmids\": [\"32259445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Overexpression of adrenodoxin (Adx/FDX1) in human cell lines causes ROS production in mitochondria, disruption of mitochondrial transmembrane potential, cytochrome c release, and caspase activation, leading to apoptosis independent of p53.\",\n      \"method\": \"Transient overexpression; ROS assays; mitochondrial membrane potential measurement; cytochrome c release assay; caspase activation assay; cell viability across 11 cell lines\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal cellular assays with defined mechanistic readouts, single lab\",\n      \"pmids\": [\"15927889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Transcription factors SF1 and cJUN cooperate to activate the mouse Fdx1 promoter in Leydig cells. SF1 is recruited to a region between -124 and -306 bp of the Fdx1 promoter and is required (by RNA interference) for Fdx1 transcription, while cJUN is dispensable but cooperates with SF1 to upregulate expression.\",\n      \"method\": \"Luciferase reporter assays; chromatin immunoprecipitation (ChIP); RNA interference; promoter deletion analysis in MA-10 and TM3 Leydig cells\",\n      \"journal\": \"The Journal of steroid biochemistry and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP + reporter assays + RNAi in two cell lines, single lab\",\n      \"pmids\": [\"28274746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"METTL16 lactylation at K229 (inhibited by SIRT2) promotes m6A modification on FDX1 mRNA, increasing FDX1 expression and sensitizing gastric cancer cells to cuproptosis. Copper stress promotes METTL16 lactylation, linking metabolic state to FDX1-dependent cell death.\",\n      \"method\": \"m6A-seq/MeRIP; METTL16 lactylation site mapping; SIRT2 inhibition (AGK2); cell-based cuproptosis assays in vitro and in vivo\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epitranscriptomic mapping with defined modification site and functional rescue, single lab\",\n      \"pmids\": [\"37863889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"p53 enhances elesclomol-Cu-induced cuproptosis in HCC via FDXR-mediated FDX1 upregulation: p53 activation increases FDXR expression, which promotes FDX1 upregulation and subsequent DLAT oligomerization; FDXR knockdown reverses p53-mediated sensitization to cuproptosis.\",\n      \"method\": \"p53 overexpression/knockdown; siRNA-mediated FDXR/FDX1 knockdown; Western blot for DLAT oligomerization; cell viability assay; in vivo xenograft\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis (FDXR KD reverses p53 effect) with downstream molecular readout, single lab\",\n      \"pmids\": [\"40630211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FDX1 interacts with G6PD and reduces G6PD protein stability, decreasing NADPH and GSH levels and thereby enhancing cuproptosis in endometriosis cells.\",\n      \"method\": \"Co-immunoprecipitation (FDX1–G6PD interaction); G6PD stability assays; NADPH/GSH measurement; cell-based cuproptosis assays; mouse ectopic endometrial tissue model\",\n      \"journal\": \"Apoptosis : an international journal on programmed cell death\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with functional consequence assays, single lab, limited mechanistic depth on how stability is reduced\",\n      \"pmids\": [\"37119432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FDX1 overexpression triggers mitochondrial permeability transition pore opening, leading to cytosolic release of mitochondrial DNA and mt-dsRNA, activating cGAS and RIG-I/MDA5 sensors, TBK1 phosphorylation, and a type I interferon response that precedes ferroptosis and reshapes the tumor microenvironment.\",\n      \"method\": \"FDX1 overexpression; mitochondrial permeability transition assays; cytosolic fractionation for mtDNA/mt-dsRNA; cGAS/RIG-I pathway activation assays; orthotopic syngeneic models\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal molecular readouts (mtDNA/dsRNA release, sensor activation, IFN response) with in vivo validation, single lab\",\n      \"pmids\": [\"41199656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FDX1 bound directly to FMR1 protein and upregulated FMR1 expression, subsequently restraining Bcl-2 and N-cadherin while enhancing ALCAM, cleaved caspase-3, and E-cadherin; FMR1 knockdown reversed FDX1-mediated suppression of ccRCC growth and metastasis.\",\n      \"method\": \"Co-IP; TMT proteomic sequencing; RNA sequencing; cell phenotype and in vivo mouse orthotopic tumor experiments\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP for FDX1–FMR1 interaction, epistasis by FMR1 KD rescue, single lab\",\n      \"pmids\": [\"40118855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PVT1 lncRNA directly binds the FDX1 promoter (35/98 nt of PVT1 binding -104/-41 bp region), increases H3K27ac deposition, and activates FDX1 transcription; PVT1 also recruits SF1 to the FDX1 promoter, further enhancing FDX1 expression and cuproptosis in colorectal cancer.\",\n      \"method\": \"ChIRP; ChIP; luciferase reporter assay; bioinformatics; molecular docking; xenograft model\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct promoter binding confirmed by ChIRP and ChIP with defined nucleotide coordinates, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"40505346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Trilobatin (TLB) directly binds FDX1 (confirmed by surface plasmon resonance) and suppresses FDX1-mediated cuproptosis after doxorubicin treatment, reducing mitochondrial oxidative stress and cuproptosis-related proteins in a dose-dependent manner.\",\n      \"method\": \"Surface plasmon resonance binding assay; molecular dynamics simulation; siRNA knockdown of FDX1; in vivo and in vitro cardiotoxicity models; Western blot for cuproptosis markers\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — SPR confirms direct binding, functional consequence shown by siRNA, single lab\",\n      \"pmids\": [\"39933533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Icaritin (ICT) directly binds FDX1 with high affinity (confirmed by surface plasmon resonance and molecular dynamics simulation), downregulates FDX1 protein expression, and inhibits FDX1-mediated cuproptosis in hippocampal neurons, reducing DLAT oligomerization and restoring copper homeostasis.\",\n      \"method\": \"Surface plasmon resonance; computational molecular docking and trajectory simulations; FDX1 knockdown; Western blot for DLAT and lipoylation markers; mitochondrial functional assays\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — SPR with MD simulation + FDX1 KD epistasis, single lab\",\n      \"pmids\": [\"40998065\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FDX1 is a mitochondrial [2Fe-2S] ferredoxin that functions as an electron shuttle with at least three distinct biochemical roles: (1) it donates electrons to mitochondrial cytochrome P450 enzymes (e.g., CYP11A1, CYP24A1) to drive steroid hormone and vitamin D metabolism; (2) it directly binds lipoyl synthase (LIAS), facilitating electron donation to kickstart the radical SAM reaction that installs lipoyl cofactors on TCA cycle enzymes, with its acidic alpha-helix 3 (D136/D139) being the critical protein–protein interaction interface; and (3) it catalyzes the reduction of Cu(II) (as the elesclomol-Cu complex) to Cu(I) inside the mitochondrial matrix, releasing bioavailable copper for CcO metalation and, when copper overloads, triggering cuproptosis through accumulation of lipoylated protein aggregates; FDX2, not FDX1, handles Fe/S cluster and heme A biosynthesis in human cells.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FDX1 (adrenodoxin/ferredoxin 1) is a mitochondrial [2Fe-2S] ferredoxin that acts as an electron shuttle dedicated to a defined set of biosynthetic and metabolic reactions, functionally distinct from its homolog FDX2, which carries out Fe/S cluster and heme A biosynthesis [#0, #1]. Its founding role is to donate electrons to mitochondrial cytochrome P450 enzymes to drive steroidogenesis (e.g., CYP11A1) and vitamin D metabolism (CYP24A1), where Adx binding allosterically remodels the P450 active site to enable substrate binding and tune regioselectivity [#0, #12]. A second, central role is in mitochondrial protein lipoylation: FDX1 directly binds lipoyl synthase (LIAS), supplies the reducing equivalents that initiate its radical-SAM chemistry, and promotes LIAS engagement with the lipoyl carrier GCSH, thereby lipoylating all four lipoylation-dependent TCA cycle enzymes; loss of FDX1 abolishes respiration and is conditionally lethal under low glucose, a defect rescued for growth but not lipoylation by hypoxia [#1, #2, #3]. FDX1 is also required upstream of COX15 for cytochrome c oxidase biogenesis, controlling CcO assembly, copper, and heme a/a3 content [#5]. As a matrix reductase, FDX1 reduces the elesclomol-Cu(II) complex to Cu(I) to release bioavailable copper for CcO metalation, and excess copper handling through this activity drives cuproptosis via accumulation of lipoylated protein aggregates [#4, #11]. The acidic solvent-exposed residues D136 and D139 on alpha-helix 3 form the critical protein-protein interface required for both lipoylation and cuproptosis, independent of intrinsic electron-transfer activity [#8]. FDX1 activity is constrained by AKT1-mediated phosphorylation, which suppresses cuproptosis and respiration in favor of glycolysis [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established the structural basis for how the ferredoxin engages its electron-acceptor partners, revealing a redox-coupled domain motion and an asymmetric charge surface that electrostatically steers interactions with adrenodoxin reductase and P450.\",\n      \"evidence\": \"X-ray crystallography of truncated bovine adrenodoxin at 1.85 Å with functional domain annotation\",\n      \"pmids\": [\"9551550\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Resolved an ortholog fragment rather than full-length human FDX1\", \"Did not address the lipoylation or copper-reductase interfaces later defined on alpha-helix 3\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved the long-standing ambiguity between the two human ferredoxins, demonstrating FDX1 is specifically required for steroidogenic P450 electron donation and not for Fe/S cluster or heme A biosynthesis, which belong to FDX2.\",\n      \"evidence\": \"RNAi depletion of FDX1 vs FDX2 in human cells with orthogonal readouts for steroidogenesis, heme A, and Fe/S maturation\",\n      \"pmids\": [\"20547883\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not yet identify lipoylation or copper roles for FDX1\", \"Mechanism of substrate discrimination between FDX1 and FDX2 not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Tested whether FDX1 can participate in Fe/S assembly biochemically, showing both ferredoxins bind the NFS1/ISD11/ACP desulfurase and can support in vitro cluster assembly, but FDX2 binds tighter and is faster — clarifying the in vivo division of labor.\",\n      \"evidence\": \"NMR interface mapping, ITC, and in vitro Fe/S cluster assembly on ISCU\",\n      \"pmids\": [\"28001042\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro competence does not establish in vivo Fe/S role for FDX1\", \"Structural determinants of differential affinity not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined the mechanism of FDX1-P450 functional coupling, showing adrenodoxin binding allosterically reorganizes the CYP24A1 F/G helices to permit substrate binding and influence regioselectivity.\",\n      \"evidence\": \"Chemical cross-linking mass spectrometry, NMR perturbation, and CYP24A1 functional assays\",\n      \"pmids\": [\"32259445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single P450 partner examined\", \"Allosteric model not extended to other mitochondrial P450s\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Assigned FDX1 a third biosynthetic role in lipoyl cofactor synthesis and pinpointed the sequence motifs that distinguish FDX1 from FDX2 substrate specificity.\",\n      \"evidence\": \"Cell-based KO and motif-swap mutants, in vitro reconstitution of the lipoyl synthase radical reaction, and structural analysis\",\n      \"pmids\": [\"36280795\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the physical FDX1–LIAS interface at residue level\", \"Did not separate lipoylation from Fe/S epistatically\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated FDX1 directly binds LIAS to promote its interaction with GCSH and drive lipoylation independent of Fe/S biosynthesis, with loss abolishing respiration and causing conditional lethality.\",\n      \"evidence\": \"Co-IP/pulldown, metabolite and transcriptional profiling, and CRISPR/RNAi loss-of-function with metabolic phenotyping\",\n      \"pmids\": [\"37453661\", \"36778498\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and structure of the FDX1–LIAS–GCSH assembly unresolved\", \"Reductant source for the reaction not defined in this study\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Tested whether an alternative lipoate route exists under hypoxia, showing hypoxia rescues FDX1-KO growth but not lipoylation, confirming FDX1/LIAS as the sole biosynthetic path.\",\n      \"evidence\": \"CRISPR KO of FDX1 or LIAS, growth at normoxia vs 1% O2, and direct lipoylation measurement\",\n      \"pmids\": [\"37481209\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which hypoxia bypasses the growth requirement not fully defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified FDX1 as the matrix reductase that converts elesclomol-Cu(II) to bioavailable Cu(I) for cytochrome c oxidase metalation, distinguishing mitochondrial from non-mitochondrial copper delivery.\",\n      \"evidence\": \"FDX1 KO with elesclomol-copper treatment, CcO activity and abundance assays, and copper quantification\",\n      \"pmids\": [\"36848556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"FDX1-independent copper delivery routes to non-mitochondrial cuproproteins not molecularly defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placed FDX1 within the CcO biogenesis pathway upstream of heme a synthase COX15, showing copper supplementation cannot rescue but COX15 overexpression partially restores COX1.\",\n      \"evidence\": \"CRISPR KO in rat cardiomyocytes with CcO assembly, copper, and heme a measurements plus COX15 rescue\",\n      \"pmids\": [\"37858707\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mechanism linking FDX1 to heme a synthesis not defined\", \"Whether FDX1 acts via lipoylation or independently in this context unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Began connecting FDX1 to upstream regulation and tumor cell death by linking epitranscriptomic (METTL16 m6A) and proteostatic control to FDX1 expression and cuproptosis sensitivity.\",\n      \"evidence\": \"m6A-seq/MeRIP, METTL16 lactylation site mapping, and cuproptosis assays in gastric cancer\",\n      \"pmids\": [\"37863889\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cancer context\", \"Direct effect of m6A on FDX1 protein output vs mRNA stability not fully separated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Used unbiased mutational scanning to define the essential functional interface, showing D136/D139 on alpha-helix 3 are required for both lipoylation and cuproptosis despite preserved electron-transfer activity, and identified DLD as an alternative FDX1 reductase.\",\n      \"evidence\": \"Deep mutational scanning, site-directed mutagenesis, in vitro enzymatic assays, and cell-based cuproptosis/lipoylation readouts\",\n      \"pmids\": [\"41423452\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural detail of how D136/D139 engages partners not solved\", \"Relative in vivo contributions of DLD vs canonical reductase unquantified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided direct spectroscopic confirmation of FDX1-driven electron transfer to elesclomol-Cu and explained FDX1's specificity over FDX2 by higher binding affinity for the ES-Cu complex.\",\n      \"evidence\": \"Low-temperature/power-saturation EPR, molecular docking, and in vitro/in vivo electron-transfer assays\",\n      \"pmids\": [\"40484707\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Behavior toward free copper vs chelated copper in physiological settings not fully resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed post-translational and protein-level regulation of FDX1 activity through AKT1 phosphorylation that toggles cells from FDX1-dependent respiration/cuproptosis toward glycolysis.\",\n      \"evidence\": \"Kinase-substrate assays, AKT1 gain/loss-of-function, metabolic and cuproptosis readouts, and AKT inhibitor + ionophore rescue in vivo\",\n      \"pmids\": [\"39976173\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphosite(s) on FDX1 not explicitly mapped in abstract\", \"Single lab; mechanism of how phosphorylation impairs electron transfer unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the regulatory network with transcriptional, partner-binding, and immunogenic consequences of FDX1, including PVT1/SF1-driven transcription, FDXR/p53-mediated upregulation, PUMA binding, and induction of an mtDNA/dsRNA-driven interferon response.\",\n      \"evidence\": \"ChIRP/ChIP, Co-IP, dual-luciferase, fractionation for cytosolic mtDNA/dsRNA, and in vivo tumor models across multiple cancers\",\n      \"pmids\": [\"40505346\", \"40630211\", \"41212484\", \"41199656\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Most interactions rest on single Co-IP/single-lab evidence\", \"Direct vs indirect nature of several partner interactions not reciprocally validated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How FDX1's three biochemical activities (P450 electron donation, lipoylation, copper reduction) are partitioned, regulated, and prioritized within a single mitochondrion remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of full-length human FDX1 bound to LIAS or to the copper complex\", \"Physiological reductant choice (canonical reductase vs DLD) under different metabolic states unclear\", \"Whether cuproptosis is a physiological program or a pharmacological liability not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [4, 8, 11]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [0, 6, 7]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [4, 13, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 8, 13]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"LIAS\", \"GCSH\", \"NFS1\", \"CYP11A1\", \"CYP24A1\", \"G6PD\", \"PUMA\", \"FMR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}