{"gene":"ALDH1L1","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2022,"finding":"Cryo-EM structures of tetrameric rat ALDH1L1 revealed that it is a natural fusion of three unrelated domains: N-terminal domains remove formyl from 10-formyltetrahydrofolate, intermediate domains (homologs of acyl/peptidyl carrier proteins with covalently attached 4'-phosphopantetheine arm) transfer the formyl group between catalytic domains of different protomers, and C-terminal aldehyde dehydrogenase domains convert formyl to CO2. The tetrameric state is indispensable for catalysis because the intermediate domain transfers the formyl group between protomers across the tetramer interface.","method":"Cryo-EM structure determination of tetrameric rat ALDH1L1","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with functional domain interaction validation in a single rigorous structural study","pmids":["35013550"],"is_preprint":false},{"year":2011,"finding":"Crystal structures of C-terminal domain (Ct-FDH) mutants showed that Glu-673 restricts coenzyme affinity (E673A causes irreversible NADP+ binding) and Cys-707 acts as a sensor of the coenzyme redox state (C707A mutant cannot differentiate between NADP+ and NADPH). These two conserved catalytic residues adjacent to the nicotinamide ring control binding and discharge of the NADP+ coenzyme.","method":"Crystal structures of Cys707 and Glu673 point mutants combined with coenzyme binding experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of multiple active-site mutants combined with biochemical coenzyme binding assays in one rigorous study","pmids":["21540484"],"is_preprint":false},{"year":2013,"finding":"Crystal structures of Ct-FDH with thio-NADP+ and of the C707S mutant with NADP+ and NADPH revealed that Cys-707 forms a covalent bond with the C4N atom of the nicotinamide ring during catalysis, trapping the coenzyme in a contracted conformation during the transition from oxidized to reduced form. This mechanism allows the enzyme to discriminate between oxidized and reduced coenzyme.","method":"Crystal structures of C707S mutant and thio-NADP+ complexes","journal":"Chemico-biological interactions","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures with mechanistic mutagenesis, directly extending prior structural study","pmids":["23295222"],"is_preprint":false},{"year":2017,"finding":"Computational modeling using available domain structures showed that ALDH1L1 is a tetramer of identical subunits, each with three functional domains; the intermediate acyl carrier protein domain possesses a covalently attached 4'-phosphopantetheine prosthetic group that functions as a swinging arm to transfer the formyl reaction intermediate between the N-terminal and C-terminal catalytic domains. Models defined positions of the 4'-phosphopantetheine arm within both catalytic domains and predicted inter-domain interfaces.","method":"Computer modeling of domain interactions using available crystal structures","journal":"Chemico-biological interactions","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational modeling only, no experimental validation in this paper","pmids":["28414156"],"is_preprint":false},{"year":2010,"finding":"ALDH1L1 (FDH) inhibits cell motility by stabilizing F-actin and promoting actin stress fibers through PP1- and PP2A-mediated dephosphorylation of cofilin at Ser-3. The PP1/PP2A inhibitor calyculin A prevented cofilin dephosphorylation and restored motility. This effect is independent of FDH-induced apoptosis (JNK inhibitor or pan-caspase inhibitor did not restore motility), is folate-dependent (increased folate prevented cofilin dephosphorylation), and is mediated through cofilin (siRNA knockdown of cofilin or expression of phosphorylation-deficient S3A mutant mimicked FDH effects, while S3D phosphomimetic mutant blocked them).","method":"FRAP of GFP-actin, pyrene-actin polymerization/depolymerization assays, pharmacological inhibitors, siRNA knockdown, cofilin phosphomutant expression in A549 cells","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (FRAP, biochemical actin assays, pharmacological rescue, genetic rescue with mutants) in a single rigorous study establishing the mechanism","pmids":["20729910"],"is_preprint":false},{"year":2014,"finding":"In response to ALDH1L1 expression, JNK1 and JNK2 phosphorylate Bid at Thr59 within its caspase-8 cleavage site. Thr59 phosphorylation protects Bid from caspase-8 cleavage, causing accumulation of full-length Bid and its translocation to mitochondria. In vitro, all three JNK isoforms (JNK1-3) phosphorylated Thr59, with JNK1 being least active. siRNA silencing of JNK1/2 or Bid prevented Bid phosphorylation and accumulation, and rescued ALDH1L1-expressing cells from apoptosis. A T59D phosphomimetic mutant promoted cleavage of Bid to jBid.","method":"In vitro kinase assay, siRNA knockdown, expression of Bid phosphomutants, co-immunoprecipitation, subcellular fractionation in PC-3 prostate cancer cells","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay combined with multiple genetic rescue experiments (siRNA, phosphomutants) and subcellular localization in one rigorous study","pmids":["25077544"],"is_preprint":false},{"year":2018,"finding":"ALDH1L1 is degraded during S-phase via proteasomal degradation mediated by the chaperone-dependent E3 ubiquitin ligase CHIP. CHIP co-localizes with ALDH1L1 and interacts with it (demonstrated by co-immunoprecipitation); siRNA silencing of CHIP halts ALDH1L1 loss, while transient CHIP overexpression promotes ALDH1L1 degradation. Proteasome inhibitor MG-132 prevents ALDH1L1 loss in proliferating cells. Downregulation of ALDH1L1 leads to accumulation of its substrate 10-formyltetrahydrofolate, required for de novo purine biosynthesis during S-phase.","method":"Co-immunoprecipitation, confocal microscopy co-localization, MG-132 proteasome inhibition, siRNA knockdown of CHIP, thymidine block cell-cycle arrest in NIH3T3 cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and multiple orthogonal approaches (pharmacological, genetic), single lab","pmids":["29979702"],"is_preprint":false},{"year":2011,"finding":"ALDH1L1 promoter methylation is the major mechanism controlling FDH levels in human cancers. An extensive CpG island spanning -525 to +918 bp (96 CpG pairs, covering the promoter, exon 1, and part of intron 1) is extensively methylated (76-95% of CpGs) in cancer cell lines, while unmethylated in normal tissues. Treatment of FDH-deficient A549 cells with the methyltransferase inhibitor 5-aza-2'-deoxycytidine restored FDH expression. Exon 1 significantly increases ALDH1L1 transcriptional activity in a luciferase reporter assay.","method":"Bisulfite sequencing, 5-aza-2'-deoxycytidine treatment, luciferase reporter assay, patient tumor/normal tissue pairs","journal":"Genes & cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (bisulfite sequencing of patient samples, pharmacological demethylation rescue, luciferase reporter) establishing the epigenetic silencing mechanism","pmids":["21779486"],"is_preprint":false},{"year":2021,"finding":"DNMT3A suppresses ALDH1L1 transcription in skeletal muscle by binding to its promoter region and altering its epigenetic profile. Muscle-specific Dnmt3a knockout mice show elevated ALDH1L1 expression. Forced expression of ALDH1L1 elevates NADPH levels, which causes overproduction of ROS via the NADPH oxidase complex, resulting in mitochondrial dysfunction. In vivo knockdown of Aldh1l1 largely rescues exercise intolerance in Dnmt3a-deficient mice, placing ALDH1L1 downstream of DNMT3A in this pathway.","method":"Muscle-specific Dnmt3a knockout mice, ALDH1L1 forced expression in myotubes, in vivo Aldh1l1 shRNA knockdown, chromatin binding assay (DNMT3A binding to Aldh1l1 promoter), NADPH measurement, ROS measurement, mitochondrial respiration assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis (KO plus rescue knockdown), promoter binding, and multiple metabolic/functional readouts in a single rigorous study","pmids":["33847380"],"is_preprint":false},{"year":2022,"finding":"Loss of ALDH1L1 in RT4 bladder cancer cells (via shRNA or CRISPR knockout) leads to decreased glycine (8-fold) and decreased metabolites from S-adenosylmethionine-utilizing (methylation) pathways, establishing ALDH1L1 as a direct regulator of glycine biosynthesis and methyl group flux in living cells. Additional changes include decreased amino acids, Krebs cycle intermediates, and ribose-5-phosphate, and increased nicotinic acid.","method":"shRNA knockdown and CRISPR knockout of ALDH1L1 in RT4 cells, untargeted UHPLC-HR-MS metabolomics, supervised and unsupervised multivariate analysis","journal":"Molecules (Basel, Switzerland)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent genetic perturbations (shRNA and CRISPR KO) with metabolomics, single lab","pmids":["36500483"],"is_preprint":false},{"year":2023,"finding":"ALDH1L1 expression in HuH-7 hepatocellular carcinoma cells consumes 10-formyltetrahydrofolate, causing ZMP (5-aminoimidazole-4-carboxamide ribonucleotide) accumulation by blocking the ZMP formylation step of de novo purine synthesis. This results in serine depletion and glycine increase intracellularly. The ZMP accumulation inhibits mitochondrial activity through a serine catabolism mechanism, and ALDH1L1-expressing cells show reduced ZMP sensitivity and higher mitochondrial activity.","method":"Metabolome analysis of ALDH1L1-expressing HuH-7 cells, intracellular metabolite measurement, mitochondrial activity assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based metabolomics with ALDH1L1 gain-of-function, single lab, multiple metabolic readouts","pmids":["37596270"],"is_preprint":false},{"year":2024,"finding":"Gossypol binds to an allosteric site on the C-terminal aldehyde dehydrogenase domain of human ALDH1L1 and disrupts folate metabolism by preventing NADP+ binding, causing a shift in structural conformation to a closed-form NADP+-binding site. Cryo-EM structures of tetrameric C-terminal ALDH1L1 in complex with gossypol confirmed this allosteric inhibition mechanism.","method":"Cryo-EM structure of human ALDH1L1 C-terminal domain in complex with gossypol, ALDH1L1 inhibition activity assay in NSCLC cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — cryo-EM structure with inhibitor, single lab, limited functional validation beyond structural observation","pmids":["38917634"],"is_preprint":false},{"year":2025,"finding":"Cytoplasmic ALDH1L1 translocates into mitochondria in a ROS-dependent feedback manner in cancer cells. ROS-mediated oxidative modification of ALDH1L1 is necessary for its interaction with HSP90β, which enables translocation into mitochondria via the TOM70 import channel. Once inside, mitochondrial ALDH1L1 produces NADPH to maintain mitochondrial redox homeostasis and binds TFAM to prevent its degradation by the protease LONP1. This was identified by co-immunoprecipitation followed by quantitative mass spectrometry.","method":"Co-immunoprecipitation followed by quantitative mass spectrometry, subcellular fractionation, co-localization with mitochondrial markers, HSP90β and TOM70 interaction assays, LONP1 degradation assay, ALDH1L1 knockout studies","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP/MS identification plus multiple follow-up interaction and functional assays, single lab","pmids":["41184641"],"is_preprint":false},{"year":2025,"finding":"ALDH1L1 directly interacts with the E3 ubiquitin ligase STUB1 and the autophagy cargo receptor TOLLIP to mediate degradation of porcine epidemic diarrhea virus nucleocapsid (N) and envelope (E) proteins via the autophagosome-lysosomal pathway. This ALDH1L1-STUB1-TOLLIP axis constitutes a novel antiviral restriction mechanism.","method":"Co-immunoprecipitation, knockdown and overexpression experiments, autophagy pathway inhibitor assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP establishing the interaction complex plus functional knockdown/overexpression, single lab, novel finding","pmids":["41467838"],"is_preprint":false},{"year":2025,"finding":"ALDH1L1 directly interacts with the glutamine synthetase enzyme GLUL (demonstrated by molecular docking and co-immunoprecipitation), leading to L-glutamate accumulation in the tumor microenvironment. This accumulated L-glutamate suppresses the PI3K/Akt/FoxO1 signaling axis in CD8+ T cells, impairing mitochondrial function and inhibiting oxidative phosphorylation, thereby driving CD8+ T-cell exhaustion.","method":"Co-immunoprecipitation, molecular docking, transcriptomic sequencing of CD8+ T cells, mitochondrial functional assays, in vivo mouse models","journal":"Journal of translational medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP for GLUL interaction, single lab, mechanistic pathway supported by transcriptomics and functional assays but multi-step inference","pmids":["41654886"],"is_preprint":false},{"year":2025,"finding":"In a Xenopus pax3-knockdown model of folic acid-rescueable neural tube defects, ALDH1L1 (FTHFD) was shown to be required for folic acid protection: FA upregulates aldh1l1 expression, and CRISPR/Cas9 knockdown of ALDH1L1 abolishes the FA protective effect. Human ALDH1L1 enzyme was shown in vitro to convert retinaldehyde to retinoic acid (RA), establishing ALDH1L1 as an enzymatic bridge between folate metabolism and RA biosynthesis. Overexpression of ALDH1L1 restored neural tube closure in aldh1l1-knockdown embryos when retinaldehyde was provided.","method":"Xenopus pax3-knockdown NTD model, CRISPR/Cas9 knockdown of aldh1l1, in vitro enzymatic assay of human ALDH1L1 with retinaldehyde substrate, rescue experiments with RA/retinaldehyde and ALDH1L1 overexpression","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro enzymatic assay establishing retinaldehyde-to-RA activity, combined with genetic rescue in Xenopus model; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.10.14.681787"],"is_preprint":true}],"current_model":"ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase) is a tetrameric enzyme whose cryo-EM structure reveals three functional domains per subunit—an N-terminal formyl-transfer domain, an intermediate acyl-carrier-protein domain with a 4'-phosphopantetheine swinging arm that transfers formyl groups between protomers, and a C-terminal ALDH domain that oxidizes formyl to CO2 in an NADP+-dependent reaction—with Cys-707 acting as a redox sensor and Glu-673 controlling coenzyme affinity; beyond its metabolic role in folate one-carbon metabolism (regulating glycine biosynthesis, methylation reactions, and de novo purine synthesis by consuming 10-formylTHF), ALDH1L1 inhibits cell motility through PP1/PP2A-mediated cofilin dephosphorylation, activates JNK1/2-dependent Bid phosphorylation at Thr59 as a pro-apoptotic signal, is targeted for proteasomal degradation during S-phase via the CHIP E3 ubiquitin ligase, is transcriptionally silenced in cancers by CpG island promoter methylation, is suppressed in muscle by DNMT3A binding to its promoter, can translocate to mitochondria under ROS stress via HSP90β/TOM70 to produce NADPH and protect TFAM, and has been shown in vitro to catalyze retinaldehyde-to-retinoic acid conversion, suggesting a role as a molecular link between folate and retinoic acid signaling in neural tube development."},"narrative":{"mechanistic_narrative":"ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase) is a tetrameric enzyme central to folate one-carbon metabolism that consumes 10-formyltetrahydrofolate and thereby controls glycine biosynthesis, methylation flux, and de novo purine synthesis [PMID:35013550, PMID:36500483, PMID:37596270]. Each identical subunit is a natural fusion of three functional domains: an N-terminal formyl-transfer domain, an intermediate acyl-carrier-protein domain bearing a covalently attached 4'-phosphopantetheine swinging arm, and a C-terminal NADP+-dependent aldehyde dehydrogenase domain; the swinging arm shuttles the formyl intermediate between protomers across the tetramer interface, making the tetrameric state obligatory for catalysis [PMID:35013550]. In the C-terminal domain, Glu-673 restricts coenzyme affinity and Cys-707 senses coenzyme redox state by forming a transient covalent bond with the nicotinamide ring, allowing the enzyme to discriminate oxidized from reduced NADP [PMID:21540484, PMID:23295222]. By depleting 10-formylTHF, ALDH1L1 expression elevates glycine while depleting serine, blocks the ZMP formylation step of purine synthesis, and reprograms downstream mitochondrial and methylation metabolism [PMID:36500483, PMID:37596270]. ALDH1L1 also exerts non-metabolic regulatory functions: it inhibits cell motility through PP1/PP2A-mediated dephosphorylation of cofilin at Ser-3 to stabilize F-actin [PMID:20729910], and it drives a pro-apoptotic program in which JNK1/2 phosphorylate Bid at Thr59 to block its caspase-8 cleavage [PMID:25077544]. Its abundance is tightly controlled: CHIP-mediated proteasomal degradation removes ALDH1L1 during S-phase to permit 10-formylTHF accumulation for purine synthesis [PMID:29979702], while in tumors and skeletal muscle the gene is epigenetically silenced by CpG-island promoter methylation and by DNMT3A promoter binding, respectively [PMID:21779486, PMID:33847380]. Under oxidative stress, oxidatively modified ALDH1L1 binds HSP90β and translocates into mitochondria via TOM70, where it produces NADPH and protects TFAM from LONP1-mediated degradation [PMID:41184641].","teleology":[{"year":2011,"claim":"Defining how the C-terminal ALDH domain handles its NADP coenzyme established the active-site logic of formyl oxidation, answering how the enzyme binds and discharges cofactor.","evidence":"Crystal structures of Glu-673 and Cys-707 point mutants with coenzyme binding assays","pmids":["21540484"],"confidence":"High","gaps":["Did not resolve the full tetrameric architecture or inter-domain formyl transfer","Studied an isolated C-terminal domain rather than the intact enzyme"]},{"year":2013,"claim":"Capturing a covalent Cys-707-nicotinamide adduct explained the chemical basis by which the enzyme discriminates oxidized from reduced coenzyme.","evidence":"Crystal structures of C707S mutant with NADP+/NADPH and thio-NADP+ complexes","pmids":["23295222"],"confidence":"High","gaps":["Catalytic relevance of the contracted conformation in the full-length tetramer not directly tested"]},{"year":2010,"claim":"Identifying ALDH1L1 as an inhibitor of cell motility via cofilin dephosphorylation revealed a non-metabolic cytoskeletal regulatory role distinct from its apoptotic function.","evidence":"FRAP, pyrene-actin assays, pharmacological PP1/PP2A inhibition, cofilin siRNA and phosphomutants in A549 cells","pmids":["20729910"],"confidence":"High","gaps":["How ALDH1L1 connects mechanistically to PP1/PP2A activation is not defined","Whether the catalytic activity is required for cofilin effects beyond folate-dependence is unresolved"]},{"year":2014,"claim":"Linking ALDH1L1 to JNK1/2-mediated Bid Thr59 phosphorylation defined a pro-apoptotic signaling axis downstream of the enzyme.","evidence":"In vitro kinase assay, siRNA of JNK1/2 and Bid, Bid phosphomutants, fractionation in PC-3 cells","pmids":["25077544"],"confidence":"High","gaps":["The signal connecting ALDH1L1 expression to JNK activation is not identified","Whether folate metabolism or the protein itself triggers JNK is unclear"]},{"year":2011,"claim":"Establishing CpG-island promoter methylation as the dominant mechanism silencing ALDH1L1 in cancers explained its frequent loss in tumors.","evidence":"Bisulfite sequencing of tumor/normal pairs, 5-aza-2'-deoxycytidine rescue, luciferase reporter","pmids":["21779486"],"confidence":"High","gaps":["Does not identify the methyltransferase responsible in tumors","Functional consequence of restored expression on tumor phenotype not tested here"]},{"year":2018,"claim":"Showing CHIP-mediated proteasomal degradation of ALDH1L1 during S-phase explained how cells transiently relieve 10-formylTHF consumption for purine synthesis.","evidence":"Reciprocal co-IP, confocal co-localization, MG-132, CHIP siRNA/overexpression, thymidine block in NIH3T3 cells","pmids":["29979702"],"confidence":"Medium","gaps":["Ubiquitination sites on ALDH1L1 not mapped","Single lab; cell-cycle dependence not validated in additional systems"]},{"year":2021,"claim":"Placing ALDH1L1 downstream of DNMT3A in skeletal muscle revealed an epigenetic repression circuit whose loss causes NADPH-driven ROS and mitochondrial dysfunction.","evidence":"Muscle-specific Dnmt3a KO mice, ALDH1L1 forced expression in myotubes, in vivo Aldh1l1 knockdown rescue, promoter chromatin binding, NADPH/ROS/respiration assays","pmids":["33847380"],"confidence":"High","gaps":["Mechanistic link between ALDH1L1-driven NADPH and NADPH oxidase activation not fully detailed","Tissue specificity of this circuit outside muscle unknown"]},{"year":2022,"claim":"Cryo-EM of the intact tetramer resolved the obligate inter-protomer formyl transfer via the 4'-phosphopantetheine swinging arm, explaining why the tetramer is indispensable for catalysis.","evidence":"Cryo-EM structure determination of tetrameric rat ALDH1L1 with functional domain interaction analysis","pmids":["35013550"],"confidence":"High","gaps":["Dynamics of arm movement during turnover not directly visualized","Structure is of rat enzyme; human-specific features inferred"]},{"year":2022,"claim":"Genetic loss-of-function metabolomics confirmed in living cells that ALDH1L1 controls glycine biosynthesis and methyl-group flux, validating its predicted metabolic role.","evidence":"shRNA and CRISPR knockout in RT4 bladder cancer cells with untargeted UHPLC-HR-MS metabolomics","pmids":["36500483"],"confidence":"Medium","gaps":["Untargeted metabolomics does not establish direct flux through ALDH1L1","Single cell-line context"]},{"year":2023,"claim":"Gain-of-function metabolomics showed ALDH1L1 consumption of 10-formylTHF blocks the ZMP formylation step of purine synthesis, linking the enzyme to ZMP-dependent mitochondrial control.","evidence":"Metabolome analysis and mitochondrial activity assays in ALDH1L1-expressing HuH-7 cells","pmids":["37596270"],"confidence":"Medium","gaps":["Causal mechanism of ZMP-mediated mitochondrial inhibition only partially defined","Single cell-line, single lab"]},{"year":2024,"claim":"Structural identification of an allosteric gossypol site on the C-terminal ALDH domain provided a pharmacological route to inhibit ALDH1L1 folate metabolism.","evidence":"Cryo-EM of human ALDH1L1 C-terminal domain with gossypol plus inhibition assay in NSCLC cells","pmids":["38917634"],"confidence":"Medium","gaps":["Functional validation beyond structural observation is limited","Selectivity of gossypol for ALDH1L1 over related enzymes not established"]},{"year":2025,"claim":"Discovery of ROS-induced mitochondrial translocation via HSP90β/TOM70 revealed a moonlighting role in maintaining mitochondrial redox and TFAM stability.","evidence":"Co-IP/quantitative MS, fractionation, mitochondrial co-localization, HSP90β/TOM70 interaction, LONP1 degradation and knockout assays in cancer cells","pmids":["41184641"],"confidence":"Medium","gaps":["The oxidative modification on ALDH1L1 required for HSP90β binding is not chemically defined","Generality across cell types not established"]},{"year":2025,"claim":"Identification of an ALDH1L1-STUB1-TOLLIP axis degrading viral N and E proteins extended ALDH1L1 function into autophagy-mediated antiviral restriction.","evidence":"Co-IP, knockdown/overexpression, autophagy inhibitor assays for porcine epidemic diarrhea virus","pmids":["41467838"],"confidence":"Medium","gaps":["Direct vs. indirect nature of the complex interactions not fully resolved","Relevance to mammalian/human viruses untested"]},{"year":2025,"claim":"A reported ALDH1L1-GLUL interaction driving glutamate accumulation and CD8+ T-cell exhaustion proposed a tumor-immune-microenvironment role.","evidence":"Co-IP, molecular docking, CD8+ T-cell transcriptomics, mitochondrial assays, in vivo mouse models","pmids":["41654886"],"confidence":"Low","gaps":["Single co-IP for GLUL interaction without reciprocal validation","Multi-step causal chain from interaction to T-cell exhaustion relies on inference","Whether enzymatic activity is required is unknown"]},{"year":2025,"claim":"Demonstrating in vitro retinaldehyde-to-retinoic-acid conversion and a requirement for ALDH1L1 in folic-acid rescue of neural tube defects proposed the enzyme as a metabolic bridge between folate and retinoic acid signaling.","evidence":"Xenopus pax3-knockdown NTD model, CRISPR/Cas9 aldh1l1 knockdown, in vitro enzymatic assay, retinaldehyde/RA rescue (preprint)","pmids":["bio_10.1101_2025.10.14.681787"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Physiological relevance of retinaldehyde oxidation versus its canonical formyl-THF activity in vivo unclear","Catalytic efficiency on retinaldehyde not benchmarked"]},{"year":null,"claim":"How ALDH1L1's catalytic folate activity is mechanistically coupled to its diverse moonlighting roles (cytoskeletal, apoptotic, mitochondrial, antiviral, immunomodulatory) remains unresolved.","evidence":"No single study integrates the metabolic and non-metabolic functions","pmids":[],"confidence":"Low","gaps":["Whether enzymatic activity is required for each moonlighting function is mostly untested","Tissue- and context-specificity of these roles is not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,1,2,15]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,9,10]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,12]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[12]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,9,10]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[6]}],"complexes":[],"partners":["STUB1","TOLLIP","HSP90B1","TOMM70","TFAM","GLUL"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75891","full_name":"Cytosolic 10-formyltetrahydrofolate dehydrogenase","aliases":["Aldehyde dehydrogenase family 1 member L1"],"length_aa":902,"mass_kda":98.8,"function":"Cytosolic 10-formyltetrahydrofolate dehydrogenase that catalyzes the NADP(+)-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and carbon dioxide (PubMed:19933275, PubMed:21238436). May also have an NADP(+)-dependent aldehyde dehydrogenase activity towards formaldehyde, acetaldehyde, propionaldehyde, and benzaldehyde (By similarity)","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/O75891/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ALDH1L1","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ALDH1L1","total_profiled":1310},"omim":[{"mim_id":"613584","title":"ALDEHYDE DEHYDROGENASE 1 FAMILY, MEMBER L2; ALDH1L2","url":"https://www.omim.org/entry/613584"},{"mim_id":"600249","title":"ALDEHYDE DEHYDROGENASE 1 FAMILY, MEMBER L1; ALDH1L1","url":"https://www.omim.org/entry/600249"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in 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The tetrameric state is indispensable for catalysis because the intermediate domain transfers the formyl group between protomers across the tetramer interface.\",\n      \"method\": \"Cryo-EM structure determination of tetrameric rat ALDH1L1\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with functional domain interaction validation in a single rigorous structural study\",\n      \"pmids\": [\"35013550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structures of C-terminal domain (Ct-FDH) mutants showed that Glu-673 restricts coenzyme affinity (E673A causes irreversible NADP+ binding) and Cys-707 acts as a sensor of the coenzyme redox state (C707A mutant cannot differentiate between NADP+ and NADPH). These two conserved catalytic residues adjacent to the nicotinamide ring control binding and discharge of the NADP+ coenzyme.\",\n      \"method\": \"Crystal structures of Cys707 and Glu673 point mutants combined with coenzyme binding experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of multiple active-site mutants combined with biochemical coenzyme binding assays in one rigorous study\",\n      \"pmids\": [\"21540484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structures of Ct-FDH with thio-NADP+ and of the C707S mutant with NADP+ and NADPH revealed that Cys-707 forms a covalent bond with the C4N atom of the nicotinamide ring during catalysis, trapping the coenzyme in a contracted conformation during the transition from oxidized to reduced form. This mechanism allows the enzyme to discriminate between oxidized and reduced coenzyme.\",\n      \"method\": \"Crystal structures of C707S mutant and thio-NADP+ complexes\",\n      \"journal\": \"Chemico-biological interactions\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures with mechanistic mutagenesis, directly extending prior structural study\",\n      \"pmids\": [\"23295222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Computational modeling using available domain structures showed that ALDH1L1 is a tetramer of identical subunits, each with three functional domains; the intermediate acyl carrier protein domain possesses a covalently attached 4'-phosphopantetheine prosthetic group that functions as a swinging arm to transfer the formyl reaction intermediate between the N-terminal and C-terminal catalytic domains. Models defined positions of the 4'-phosphopantetheine arm within both catalytic domains and predicted inter-domain interfaces.\",\n      \"method\": \"Computer modeling of domain interactions using available crystal structures\",\n      \"journal\": \"Chemico-biological interactions\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational modeling only, no experimental validation in this paper\",\n      \"pmids\": [\"28414156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ALDH1L1 (FDH) inhibits cell motility by stabilizing F-actin and promoting actin stress fibers through PP1- and PP2A-mediated dephosphorylation of cofilin at Ser-3. The PP1/PP2A inhibitor calyculin A prevented cofilin dephosphorylation and restored motility. This effect is independent of FDH-induced apoptosis (JNK inhibitor or pan-caspase inhibitor did not restore motility), is folate-dependent (increased folate prevented cofilin dephosphorylation), and is mediated through cofilin (siRNA knockdown of cofilin or expression of phosphorylation-deficient S3A mutant mimicked FDH effects, while S3D phosphomimetic mutant blocked them).\",\n      \"method\": \"FRAP of GFP-actin, pyrene-actin polymerization/depolymerization assays, pharmacological inhibitors, siRNA knockdown, cofilin phosphomutant expression in A549 cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (FRAP, biochemical actin assays, pharmacological rescue, genetic rescue with mutants) in a single rigorous study establishing the mechanism\",\n      \"pmids\": [\"20729910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In response to ALDH1L1 expression, JNK1 and JNK2 phosphorylate Bid at Thr59 within its caspase-8 cleavage site. Thr59 phosphorylation protects Bid from caspase-8 cleavage, causing accumulation of full-length Bid and its translocation to mitochondria. In vitro, all three JNK isoforms (JNK1-3) phosphorylated Thr59, with JNK1 being least active. siRNA silencing of JNK1/2 or Bid prevented Bid phosphorylation and accumulation, and rescued ALDH1L1-expressing cells from apoptosis. A T59D phosphomimetic mutant promoted cleavage of Bid to jBid.\",\n      \"method\": \"In vitro kinase assay, siRNA knockdown, expression of Bid phosphomutants, co-immunoprecipitation, subcellular fractionation in PC-3 prostate cancer cells\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay combined with multiple genetic rescue experiments (siRNA, phosphomutants) and subcellular localization in one rigorous study\",\n      \"pmids\": [\"25077544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ALDH1L1 is degraded during S-phase via proteasomal degradation mediated by the chaperone-dependent E3 ubiquitin ligase CHIP. CHIP co-localizes with ALDH1L1 and interacts with it (demonstrated by co-immunoprecipitation); siRNA silencing of CHIP halts ALDH1L1 loss, while transient CHIP overexpression promotes ALDH1L1 degradation. Proteasome inhibitor MG-132 prevents ALDH1L1 loss in proliferating cells. Downregulation of ALDH1L1 leads to accumulation of its substrate 10-formyltetrahydrofolate, required for de novo purine biosynthesis during S-phase.\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy co-localization, MG-132 proteasome inhibition, siRNA knockdown of CHIP, thymidine block cell-cycle arrest in NIH3T3 cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and multiple orthogonal approaches (pharmacological, genetic), single lab\",\n      \"pmids\": [\"29979702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ALDH1L1 promoter methylation is the major mechanism controlling FDH levels in human cancers. An extensive CpG island spanning -525 to +918 bp (96 CpG pairs, covering the promoter, exon 1, and part of intron 1) is extensively methylated (76-95% of CpGs) in cancer cell lines, while unmethylated in normal tissues. Treatment of FDH-deficient A549 cells with the methyltransferase inhibitor 5-aza-2'-deoxycytidine restored FDH expression. Exon 1 significantly increases ALDH1L1 transcriptional activity in a luciferase reporter assay.\",\n      \"method\": \"Bisulfite sequencing, 5-aza-2'-deoxycytidine treatment, luciferase reporter assay, patient tumor/normal tissue pairs\",\n      \"journal\": \"Genes & cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (bisulfite sequencing of patient samples, pharmacological demethylation rescue, luciferase reporter) establishing the epigenetic silencing mechanism\",\n      \"pmids\": [\"21779486\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DNMT3A suppresses ALDH1L1 transcription in skeletal muscle by binding to its promoter region and altering its epigenetic profile. Muscle-specific Dnmt3a knockout mice show elevated ALDH1L1 expression. Forced expression of ALDH1L1 elevates NADPH levels, which causes overproduction of ROS via the NADPH oxidase complex, resulting in mitochondrial dysfunction. In vivo knockdown of Aldh1l1 largely rescues exercise intolerance in Dnmt3a-deficient mice, placing ALDH1L1 downstream of DNMT3A in this pathway.\",\n      \"method\": \"Muscle-specific Dnmt3a knockout mice, ALDH1L1 forced expression in myotubes, in vivo Aldh1l1 shRNA knockdown, chromatin binding assay (DNMT3A binding to Aldh1l1 promoter), NADPH measurement, ROS measurement, mitochondrial respiration assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis (KO plus rescue knockdown), promoter binding, and multiple metabolic/functional readouts in a single rigorous study\",\n      \"pmids\": [\"33847380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Loss of ALDH1L1 in RT4 bladder cancer cells (via shRNA or CRISPR knockout) leads to decreased glycine (8-fold) and decreased metabolites from S-adenosylmethionine-utilizing (methylation) pathways, establishing ALDH1L1 as a direct regulator of glycine biosynthesis and methyl group flux in living cells. Additional changes include decreased amino acids, Krebs cycle intermediates, and ribose-5-phosphate, and increased nicotinic acid.\",\n      \"method\": \"shRNA knockdown and CRISPR knockout of ALDH1L1 in RT4 cells, untargeted UHPLC-HR-MS metabolomics, supervised and unsupervised multivariate analysis\",\n      \"journal\": \"Molecules (Basel, Switzerland)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent genetic perturbations (shRNA and CRISPR KO) with metabolomics, single lab\",\n      \"pmids\": [\"36500483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALDH1L1 expression in HuH-7 hepatocellular carcinoma cells consumes 10-formyltetrahydrofolate, causing ZMP (5-aminoimidazole-4-carboxamide ribonucleotide) accumulation by blocking the ZMP formylation step of de novo purine synthesis. This results in serine depletion and glycine increase intracellularly. The ZMP accumulation inhibits mitochondrial activity through a serine catabolism mechanism, and ALDH1L1-expressing cells show reduced ZMP sensitivity and higher mitochondrial activity.\",\n      \"method\": \"Metabolome analysis of ALDH1L1-expressing HuH-7 cells, intracellular metabolite measurement, mitochondrial activity assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based metabolomics with ALDH1L1 gain-of-function, single lab, multiple metabolic readouts\",\n      \"pmids\": [\"37596270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Gossypol binds to an allosteric site on the C-terminal aldehyde dehydrogenase domain of human ALDH1L1 and disrupts folate metabolism by preventing NADP+ binding, causing a shift in structural conformation to a closed-form NADP+-binding site. Cryo-EM structures of tetrameric C-terminal ALDH1L1 in complex with gossypol confirmed this allosteric inhibition mechanism.\",\n      \"method\": \"Cryo-EM structure of human ALDH1L1 C-terminal domain in complex with gossypol, ALDH1L1 inhibition activity assay in NSCLC cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — cryo-EM structure with inhibitor, single lab, limited functional validation beyond structural observation\",\n      \"pmids\": [\"38917634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cytoplasmic ALDH1L1 translocates into mitochondria in a ROS-dependent feedback manner in cancer cells. ROS-mediated oxidative modification of ALDH1L1 is necessary for its interaction with HSP90β, which enables translocation into mitochondria via the TOM70 import channel. Once inside, mitochondrial ALDH1L1 produces NADPH to maintain mitochondrial redox homeostasis and binds TFAM to prevent its degradation by the protease LONP1. This was identified by co-immunoprecipitation followed by quantitative mass spectrometry.\",\n      \"method\": \"Co-immunoprecipitation followed by quantitative mass spectrometry, subcellular fractionation, co-localization with mitochondrial markers, HSP90β and TOM70 interaction assays, LONP1 degradation assay, ALDH1L1 knockout studies\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP/MS identification plus multiple follow-up interaction and functional assays, single lab\",\n      \"pmids\": [\"41184641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALDH1L1 directly interacts with the E3 ubiquitin ligase STUB1 and the autophagy cargo receptor TOLLIP to mediate degradation of porcine epidemic diarrhea virus nucleocapsid (N) and envelope (E) proteins via the autophagosome-lysosomal pathway. This ALDH1L1-STUB1-TOLLIP axis constitutes a novel antiviral restriction mechanism.\",\n      \"method\": \"Co-immunoprecipitation, knockdown and overexpression experiments, autophagy pathway inhibitor assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP establishing the interaction complex plus functional knockdown/overexpression, single lab, novel finding\",\n      \"pmids\": [\"41467838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALDH1L1 directly interacts with the glutamine synthetase enzyme GLUL (demonstrated by molecular docking and co-immunoprecipitation), leading to L-glutamate accumulation in the tumor microenvironment. This accumulated L-glutamate suppresses the PI3K/Akt/FoxO1 signaling axis in CD8+ T cells, impairing mitochondrial function and inhibiting oxidative phosphorylation, thereby driving CD8+ T-cell exhaustion.\",\n      \"method\": \"Co-immunoprecipitation, molecular docking, transcriptomic sequencing of CD8+ T cells, mitochondrial functional assays, in vivo mouse models\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP for GLUL interaction, single lab, mechanistic pathway supported by transcriptomics and functional assays but multi-step inference\",\n      \"pmids\": [\"41654886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In a Xenopus pax3-knockdown model of folic acid-rescueable neural tube defects, ALDH1L1 (FTHFD) was shown to be required for folic acid protection: FA upregulates aldh1l1 expression, and CRISPR/Cas9 knockdown of ALDH1L1 abolishes the FA protective effect. Human ALDH1L1 enzyme was shown in vitro to convert retinaldehyde to retinoic acid (RA), establishing ALDH1L1 as an enzymatic bridge between folate metabolism and RA biosynthesis. Overexpression of ALDH1L1 restored neural tube closure in aldh1l1-knockdown embryos when retinaldehyde was provided.\",\n      \"method\": \"Xenopus pax3-knockdown NTD model, CRISPR/Cas9 knockdown of aldh1l1, in vitro enzymatic assay of human ALDH1L1 with retinaldehyde substrate, rescue experiments with RA/retinaldehyde and ALDH1L1 overexpression\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro enzymatic assay establishing retinaldehyde-to-RA activity, combined with genetic rescue in Xenopus model; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.10.14.681787\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase) is a tetrameric enzyme whose cryo-EM structure reveals three functional domains per subunit—an N-terminal formyl-transfer domain, an intermediate acyl-carrier-protein domain with a 4'-phosphopantetheine swinging arm that transfers formyl groups between protomers, and a C-terminal ALDH domain that oxidizes formyl to CO2 in an NADP+-dependent reaction—with Cys-707 acting as a redox sensor and Glu-673 controlling coenzyme affinity; beyond its metabolic role in folate one-carbon metabolism (regulating glycine biosynthesis, methylation reactions, and de novo purine synthesis by consuming 10-formylTHF), ALDH1L1 inhibits cell motility through PP1/PP2A-mediated cofilin dephosphorylation, activates JNK1/2-dependent Bid phosphorylation at Thr59 as a pro-apoptotic signal, is targeted for proteasomal degradation during S-phase via the CHIP E3 ubiquitin ligase, is transcriptionally silenced in cancers by CpG island promoter methylation, is suppressed in muscle by DNMT3A binding to its promoter, can translocate to mitochondria under ROS stress via HSP90β/TOM70 to produce NADPH and protect TFAM, and has been shown in vitro to catalyze retinaldehyde-to-retinoic acid conversion, suggesting a role as a molecular link between folate and retinoic acid signaling in neural tube development.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase) is a tetrameric enzyme central to folate one-carbon metabolism that consumes 10-formyltetrahydrofolate and thereby controls glycine biosynthesis, methylation flux, and de novo purine synthesis [#0, #9, #10]. Each identical subunit is a natural fusion of three functional domains: an N-terminal formyl-transfer domain, an intermediate acyl-carrier-protein domain bearing a covalently attached 4'-phosphopantetheine swinging arm, and a C-terminal NADP+-dependent aldehyde dehydrogenase domain; the swinging arm shuttles the formyl intermediate between protomers across the tetramer interface, making the tetrameric state obligatory for catalysis [#0]. In the C-terminal domain, Glu-673 restricts coenzyme affinity and Cys-707 senses coenzyme redox state by forming a transient covalent bond with the nicotinamide ring, allowing the enzyme to discriminate oxidized from reduced NADP [#1, #2]. By depleting 10-formylTHF, ALDH1L1 expression elevates glycine while depleting serine, blocks the ZMP formylation step of purine synthesis, and reprograms downstream mitochondrial and methylation metabolism [#9, #10]. ALDH1L1 also exerts non-metabolic regulatory functions: it inhibits cell motility through PP1/PP2A-mediated dephosphorylation of cofilin at Ser-3 to stabilize F-actin [#4], and it drives a pro-apoptotic program in which JNK1/2 phosphorylate Bid at Thr59 to block its caspase-8 cleavage [#5]. Its abundance is tightly controlled: CHIP-mediated proteasomal degradation removes ALDH1L1 during S-phase to permit 10-formylTHF accumulation for purine synthesis [#6], while in tumors and skeletal muscle the gene is epigenetically silenced by CpG-island promoter methylation and by DNMT3A promoter binding, respectively [#7, #8]. Under oxidative stress, oxidatively modified ALDH1L1 binds HSP90\\u03b2 and translocates into mitochondria via TOM70, where it produces NADPH and protects TFAM from LONP1-mediated degradation [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Defining how the C-terminal ALDH domain handles its NADP coenzyme established the active-site logic of formyl oxidation, answering how the enzyme binds and discharges cofactor.\",\n      \"evidence\": \"Crystal structures of Glu-673 and Cys-707 point mutants with coenzyme binding assays\",\n      \"pmids\": [\"21540484\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the full tetrameric architecture or inter-domain formyl transfer\", \"Studied an isolated C-terminal domain rather than the intact enzyme\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Capturing a covalent Cys-707-nicotinamide adduct explained the chemical basis by which the enzyme discriminates oxidized from reduced coenzyme.\",\n      \"evidence\": \"Crystal structures of C707S mutant with NADP+/NADPH and thio-NADP+ complexes\",\n      \"pmids\": [\"23295222\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic relevance of the contracted conformation in the full-length tetramer not directly tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identifying ALDH1L1 as an inhibitor of cell motility via cofilin dephosphorylation revealed a non-metabolic cytoskeletal regulatory role distinct from its apoptotic function.\",\n      \"evidence\": \"FRAP, pyrene-actin assays, pharmacological PP1/PP2A inhibition, cofilin siRNA and phosphomutants in A549 cells\",\n      \"pmids\": [\"20729910\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ALDH1L1 connects mechanistically to PP1/PP2A activation is not defined\", \"Whether the catalytic activity is required for cofilin effects beyond folate-dependence is unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Linking ALDH1L1 to JNK1/2-mediated Bid Thr59 phosphorylation defined a pro-apoptotic signaling axis downstream of the enzyme.\",\n      \"evidence\": \"In vitro kinase assay, siRNA of JNK1/2 and Bid, Bid phosphomutants, fractionation in PC-3 cells\",\n      \"pmids\": [\"25077544\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The signal connecting ALDH1L1 expression to JNK activation is not identified\", \"Whether folate metabolism or the protein itself triggers JNK is unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Establishing CpG-island promoter methylation as the dominant mechanism silencing ALDH1L1 in cancers explained its frequent loss in tumors.\",\n      \"evidence\": \"Bisulfite sequencing of tumor/normal pairs, 5-aza-2'-deoxycytidine rescue, luciferase reporter\",\n      \"pmids\": [\"21779486\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify the methyltransferase responsible in tumors\", \"Functional consequence of restored expression on tumor phenotype not tested here\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showing CHIP-mediated proteasomal degradation of ALDH1L1 during S-phase explained how cells transiently relieve 10-formylTHF consumption for purine synthesis.\",\n      \"evidence\": \"Reciprocal co-IP, confocal co-localization, MG-132, CHIP siRNA/overexpression, thymidine block in NIH3T3 cells\",\n      \"pmids\": [\"29979702\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination sites on ALDH1L1 not mapped\", \"Single lab; cell-cycle dependence not validated in additional systems\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placing ALDH1L1 downstream of DNMT3A in skeletal muscle revealed an epigenetic repression circuit whose loss causes NADPH-driven ROS and mitochondrial dysfunction.\",\n      \"evidence\": \"Muscle-specific Dnmt3a KO mice, ALDH1L1 forced expression in myotubes, in vivo Aldh1l1 knockdown rescue, promoter chromatin binding, NADPH/ROS/respiration assays\",\n      \"pmids\": [\"33847380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic link between ALDH1L1-driven NADPH and NADPH oxidase activation not fully detailed\", \"Tissue specificity of this circuit outside muscle unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Cryo-EM of the intact tetramer resolved the obligate inter-protomer formyl transfer via the 4'-phosphopantetheine swinging arm, explaining why the tetramer is indispensable for catalysis.\",\n      \"evidence\": \"Cryo-EM structure determination of tetrameric rat ALDH1L1 with functional domain interaction analysis\",\n      \"pmids\": [\"35013550\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of arm movement during turnover not directly visualized\", \"Structure is of rat enzyme; human-specific features inferred\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Genetic loss-of-function metabolomics confirmed in living cells that ALDH1L1 controls glycine biosynthesis and methyl-group flux, validating its predicted metabolic role.\",\n      \"evidence\": \"shRNA and CRISPR knockout in RT4 bladder cancer cells with untargeted UHPLC-HR-MS metabolomics\",\n      \"pmids\": [\"36500483\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Untargeted metabolomics does not establish direct flux through ALDH1L1\", \"Single cell-line context\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Gain-of-function metabolomics showed ALDH1L1 consumption of 10-formylTHF blocks the ZMP formylation step of purine synthesis, linking the enzyme to ZMP-dependent mitochondrial control.\",\n      \"evidence\": \"Metabolome analysis and mitochondrial activity assays in ALDH1L1-expressing HuH-7 cells\",\n      \"pmids\": [\"37596270\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal mechanism of ZMP-mediated mitochondrial inhibition only partially defined\", \"Single cell-line, single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Structural identification of an allosteric gossypol site on the C-terminal ALDH domain provided a pharmacological route to inhibit ALDH1L1 folate metabolism.\",\n      \"evidence\": \"Cryo-EM of human ALDH1L1 C-terminal domain with gossypol plus inhibition assay in NSCLC cells\",\n      \"pmids\": [\"38917634\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional validation beyond structural observation is limited\", \"Selectivity of gossypol for ALDH1L1 over related enzymes not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Discovery of ROS-induced mitochondrial translocation via HSP90\\u03b2/TOM70 revealed a moonlighting role in maintaining mitochondrial redox and TFAM stability.\",\n      \"evidence\": \"Co-IP/quantitative MS, fractionation, mitochondrial co-localization, HSP90\\u03b2/TOM70 interaction, LONP1 degradation and knockout assays in cancer cells\",\n      \"pmids\": [\"41184641\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The oxidative modification on ALDH1L1 required for HSP90\\u03b2 binding is not chemically defined\", \"Generality across cell types not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of an ALDH1L1-STUB1-TOLLIP axis degrading viral N and E proteins extended ALDH1L1 function into autophagy-mediated antiviral restriction.\",\n      \"evidence\": \"Co-IP, knockdown/overexpression, autophagy inhibitor assays for porcine epidemic diarrhea virus\",\n      \"pmids\": [\"41467838\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect nature of the complex interactions not fully resolved\", \"Relevance to mammalian/human viruses untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A reported ALDH1L1-GLUL interaction driving glutamate accumulation and CD8+ T-cell exhaustion proposed a tumor-immune-microenvironment role.\",\n      \"evidence\": \"Co-IP, molecular docking, CD8+ T-cell transcriptomics, mitochondrial assays, in vivo mouse models\",\n      \"pmids\": [\"41654886\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single co-IP for GLUL interaction without reciprocal validation\", \"Multi-step causal chain from interaction to T-cell exhaustion relies on inference\", \"Whether enzymatic activity is required is unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating in vitro retinaldehyde-to-retinoic-acid conversion and a requirement for ALDH1L1 in folic-acid rescue of neural tube defects proposed the enzyme as a metabolic bridge between folate and retinoic acid signaling.\",\n      \"evidence\": \"Xenopus pax3-knockdown NTD model, CRISPR/Cas9 aldh1l1 knockdown, in vitro enzymatic assay, retinaldehyde/RA rescue (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.10.14.681787\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Physiological relevance of retinaldehyde oxidation versus its canonical formyl-THF activity in vivo unclear\", \"Catalytic efficiency on retinaldehyde not benchmarked\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ALDH1L1's catalytic folate activity is mechanistically coupled to its diverse moonlighting roles (cytoskeletal, apoptotic, mitochondrial, antiviral, immunomodulatory) remains unresolved.\",\n      \"evidence\": \"No single study integrates the metabolic and non-metabolic functions\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Whether enzymatic activity is required for each moonlighting function is mostly untested\", \"Tissue- and context-specificity of these roles is not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 1, 2, 15]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 9, 10]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 12]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 9, 10]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"STUB1\", \"TOLLIP\", \"HSP90B1\", \"TOMM70\", \"TFAM\", \"GLUL\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}