{"gene":"AKR1C4","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2000,"finding":"AKR1C4 (type 1 3α-HSD) functions as a highly catalytically efficient NAD(P)(H)-dependent ketosteroid reductase and hydroxysteroid oxidase, acting on 3-, 17-, and 20-positions of steroids. It is the most catalytically efficient isoform (k_cat/K_m 10–30-fold higher than AKR1C1–1C3), inactivates 5α-DHT to 3α-androstanediol, oxidizes testosterone to androstene-3,17-dione, reduces oestrone to 17β-oestradiol, and forms 5α/5β-tetrahydrosteroids. It is virtually liver-specific in tissue distribution.","method":"Recombinant protein expression, kinetic parameter determination, product identification by biochemical assay, isoform-specific RT-PCR for tissue distribution","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic characterization with purified recombinant protein, comprehensive kinetics and product identification, replicated across multiple substrates and compared across four isoforms","pmids":["10998348"],"is_preprint":false},{"year":2011,"finding":"AKR1C4 is the major enzyme responsible for the hepatic formation of the 5β-tetrahydrosteroid of testosterone via the 5β-pathway: it reduces 5β-dihydrotestosterone predominantly to the 3α-hydroxy configuration, with the stereochemistry explained by molecular docking. AKR1C4 has the highest kinetic efficiency for this substrate among AKR1C1–1C4.","method":"In vitro enzymatic assay with purified recombinant AKR1C1–1C4, product characterization by liquid chromatography-MS, kinetic parameter determination, molecular docking","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with LC-MS product identification, kinetics, and structural docking; orthogonal methods in a single rigorous study","pmids":["21521174"],"is_preprint":false},{"year":2000,"finding":"His-216 in AKR1C4 (unique compared to the conserved Tyr-216 in AKR1C1–1C3) plays a key role in orienting the nicotinamide ring of the coenzyme and shaping the substrate-binding cavity. Replacement of His-216 with Tyr or Phe decreased K_m for NADP+ ~3-fold, differentially altered K_m and k_cat for substrates depending on structure (bile acids with 12α-OH vs. others), changed inhibitor sensitivity, and reduced stimulatory effects of non-essential activators.","method":"Site-directed mutagenesis of AKR1C4 (H216Y, H216F), kinetic analysis with purified mutant enzymes, inhibitor and activator binding assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — active-site mutagenesis with quantitative kinetics, multiple substrates and inhibitors tested, mechanistic interpretation grounded in crystallographic context of family members","pmids":["11104674"],"is_preprint":false},{"year":2001,"finding":"Transcription of the human AKR1C4 (DD4) gene in hepatic cells is cooperatively regulated by HNF-4α and HNF-4γ binding to a cis-element at −701 to −684, and HNF-1α binding to a cis-element at −682 to −666. Mutation of either element reduces luciferase reporter activity to ~10% and ~8% of wild-type, respectively.","method":"Luciferase reporter assay in HepG2 cells, 1,10-phenanthroline-copper footprinting, gel-shift (EMSA) assay, supershift assay with antibodies to HNF-4α, HNF-4γ, HNF-1α, promoter deletion/mutation analysis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (footprinting, EMSA, supershift, reporter mutagenesis) in a single study establishing transcriptional regulation mechanism","pmids":["11284743"],"is_preprint":false},{"year":2002,"finding":"Cell-type-specific expression of AKR1C4 (DD4) is determined by differential occupancy of the HNF-4/HNF-1 promoter elements: in hepatic HepG2 cells, HNF-4α, HNF-4γ, and HNF-1α activate transcription, whereas in non-expressing ACHN renal cells, vHNF-1-C (a truncated isoform lacking transcriptional activation domain) occupies the same elements without activating transcription.","method":"Reporter gene (luciferase) assay, supershift EMSA with isoform-specific antibodies, semi-quantitative RT-PCR, transfection with vHNF-1-C expression plasmid","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (reporter, EMSA, RT-PCR, transfection) in a single lab extending the prior study","pmids":["12220531"],"is_preprint":false},{"year":2007,"finding":"LXRα (NR1H3) binds to a response element ~1.5 kb upstream of the AKR1C4 transcription start site and mediates transcriptional activation of AKR1C4, leading to increased AKR1C4 protein expression. This was identified by hidden Markov model prediction combined with chromatin immunoprecipitation/microarray analysis.","method":"Hidden Markov model prediction of nuclear receptor response elements, chromatin immunoprecipitation (ChIP)/microarray, demonstration of LXRα binding to the LXRE and transcriptional activation of AKR1C4","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP combined with functional protein expression data, single lab, two complementary methods","pmids":["18024509"],"is_preprint":false},{"year":1995,"finding":"The AKR1C4 gene (as CHDR/chlordecone reductase) and closely related dihydrodiol dehydrogenase genes (DDH1, DDH2) are all located on human chromosome 10p14–p15.","method":"PCR with gene-specific primers on human/hamster hybrid DNA panel, fluorescence in situ hybridization (FISH)","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal mapping methods (PCR on hybrid panel + FISH), single study","pmids":["7789999"],"is_preprint":false},{"year":2024,"finding":"AKR1C4 enzymatic activity is required to suppress ferroptosis and confer chemotherapy resistance in colorectal cancer cells: CRISPR/Cas9 knockout of AKR1C4 enhances sensitivity to 5-FU, irinotecan, and oxaliplatin by inducing ferroptosis (increased total iron and lipid peroxidation). A Y55A catalytic mutant of AKR1C4 fails to rescue chemoresistance, demonstrating that enzymatic activity is necessary.","method":"CRISPR/Cas9 knockout, catalytic point mutant (Y55A) rescue assay, SRB cell viability assay, total iron content measurement, lipid peroxidation measurement, acquisition of chemoresistant cell lines by long-term drug induction","journal":"Cancer chemotherapy and pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with defined phenotype plus catalytic-dead mutant rescue, multiple orthogonal ferroptosis readouts, single lab","pmids":["38890190"],"is_preprint":false}],"current_model":"AKR1C4 is a liver-specific, NAD(P)(H)-dependent aldo-keto reductase that catalyzes the stereospecific reduction of ketosteroids (including 5α-DHT and 5β-dihydrosteroids) and oxidation of hydroxysteroids to regulate active sex hormone levels and bile acid metabolism; its catalytic efficiency depends critically on His-216 in the substrate-binding cavity, its hepatic expression is cooperatively activated by HNF-4α/γ and HNF-1α binding to the promoter and further induced by LXRα, and its enzymatic activity suppresses ferroptosis to promote chemotherapy resistance in colorectal cancer cells."},"narrative":{"mechanistic_narrative":"AKR1C4 is a virtually liver-specific, NAD(P)(H)-dependent aldo-keto reductase that catalyzes the stereospecific interconversion of ketosteroids and hydroxysteroids at the 3-, 17-, and 20-positions, thereby governing the levels of active sex hormones and dihydrosteroid metabolites [PMID:10998348]. It is the most catalytically efficient AKR1C isoform, inactivating 5α-DHT to 3α-androstanediol, oxidizing testosterone, reducing oestrone to 17β-oestradiol, and forming 5α/5β-tetrahydrosteroids [PMID:10998348]; it is the major hepatic enzyme of the 5β-pathway, reducing 5β-dihydrotestosterone preferentially to the 3α-hydroxy configuration [PMID:21521174]. Its distinctive catalytic behavior derives in part from His-216 (in place of the Tyr conserved in AKR1C1–1C3), which orients the nicotinamide ring of the coenzyme and shapes the substrate-binding cavity, modulating cofactor affinity, substrate-dependent kinetics, and inhibitor sensitivity [PMID:11104674]. Hepatic, cell-type-restricted expression is established by cooperative transactivation through HNF-4α/HNF-4γ and HNF-1α binding to adjacent promoter elements—with non-activating vHNF-1-C occupancy explaining silencing in non-hepatic cells—and is further induced by LXRα binding to an upstream response element [PMID:11284743, PMID:12220531, PMID:18024509]. Beyond steroid metabolism, AKR1C4 enzymatic activity suppresses ferroptosis to confer chemotherapy resistance in colorectal cancer cells, an effect lost in a catalytically dead mutant [PMID:38890190].","teleology":[{"year":2000,"claim":"Established that AKR1C4 is a bona fide, highly efficient steroid-metabolizing oxidoreductase, defining its molecular activity and the liver-restricted context in which it operates.","evidence":"Recombinant protein kinetics, product identification, and isoform-specific RT-PCR across AKR1C1–1C4","pmids":["10998348"],"confidence":"High","gaps":["In vitro substrate preferences do not establish the dominant physiological reaction direction in hepatocytes","Did not resolve structural basis of catalytic superiority over other isoforms"]},{"year":2000,"claim":"Pinpointed His-216 as a determinant of AKR1C4's cofactor orientation and substrate cavity, explaining its kinetic distinctiveness from the Tyr-216 paralogs.","evidence":"Site-directed mutagenesis (H216Y, H216F) with kinetic, inhibitor, and activator binding analysis on purified enzymes","pmids":["11104674"],"confidence":"High","gaps":["No crystal structure of AKR1C4 itself; interpretation rests on family member structures","Effect of His-216 on the full physiological substrate panel not exhaustively mapped"]},{"year":2001,"claim":"Resolved how hepatic AKR1C4 transcription is driven, showing cooperative control by adjacent HNF-4α/γ and HNF-1α cis-elements.","evidence":"Luciferase reporters, footprinting, EMSA/supershift, and promoter mutagenesis in HepG2 cells","pmids":["11284743"],"confidence":"High","gaps":["Did not address signals upstream that modulate HNF factor activity","Promoter analysis in a single hepatic cell line"]},{"year":2002,"claim":"Explained tissue-restricted expression as differential transcription-factor occupancy, with non-activating vHNF-1-C occupying the same elements in non-hepatic cells.","evidence":"Reporter assays, supershift EMSA, RT-PCR, and vHNF-1-C transfection in HepG2 vs ACHN cells","pmids":["12220531"],"confidence":"Medium","gaps":["Single-lab extension of the prior promoter study","Does not establish in vivo relevance of vHNF-1-C silencing across tissues"]},{"year":2007,"claim":"Added a nuclear-receptor input by showing LXRα directly activates AKR1C4 transcription, linking the gene to lipid/sterol-sensing regulation.","evidence":"HMM prediction of response elements plus ChIP/microarray and protein expression analysis","pmids":["18024509"],"confidence":"Medium","gaps":["Physiological ligand/condition triggering LXRα-driven induction not defined","Two complementary methods from a single lab"]},{"year":2011,"claim":"Identified AKR1C4 as the principal hepatic enzyme of the 5β-pathway, defining its stereochemical product preference for 5β-dihydrotestosterone.","evidence":"In vitro reconstitution with LC-MS product characterization, kinetics, and molecular docking across AKR1C1–1C4","pmids":["21521174"],"confidence":"High","gaps":["In vivo flux through the 5β-pathway not directly quantified","Docking model not validated by experimental structure"]},{"year":2024,"claim":"Extended AKR1C4 function beyond steroid metabolism, demonstrating that its catalytic activity suppresses ferroptosis and drives chemoresistance in colorectal cancer.","evidence":"CRISPR/Cas9 knockout, Y55A catalytic-dead rescue, viability/iron/lipid-peroxidation readouts in chemoresistant cell lines","pmids":["38890190"],"confidence":"Medium","gaps":["The metabolic substrate/product responsible for ferroptosis suppression is not identified","Single-lab cell-line study without in vivo confirmation","Mechanism linking enzymatic output to lipid peroxidation unresolved"]},{"year":null,"claim":"It remains unknown how AKR1C4's steroid-reductase chemistry mechanistically connects to lipid-peroxidation/ferroptosis control, and whether its in vivo physiological role is dominated by steroid clearance or by the cancer-associated activity.","evidence":"","pmids":[],"confidence":"Low","gaps":["No identified endogenous substrate bridging steroid metabolism and ferroptosis","No experimentally determined AKR1C4 structure","In vivo physiological consequences of expression changes not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,1,2,7]}],"localization":[],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,1]}],"complexes":[],"partners":[],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P17516","full_name":"Aldo-keto reductase family 1 member C4","aliases":["3-alpha-hydroxysteroid 3-dehydrogenase type I","3-alpha-HSD1","Chlordecone reductase","CDR","Dihydrodiol dehydrogenase 4","DD-4","DD4","HAKRA"],"length_aa":323,"mass_kda":37.1,"function":"Cytosolic aldo-keto reductase that catalyzes NADPH-dependent reduction of ketosteroids to hydroxysteroids. Displays broad substrate specificity with distinct positional and stereochemistry, primarily generating 3alpha/beta-, 17beta- and 20alpha-hydroxysteroids (PubMed:10634139, PubMed:10998348, PubMed:11158055, PubMed:14672942, PubMed:1530633, PubMed:12604236, PubMed:19218247, PubMed:21802064, PubMed:7650035). Required for male sex determination as a component of the 'backdoor' androgen biosynthesis pathway that generates 5alpha-dihydrotestosterone (5alpha-DHT) via pregnanes. Acts together with AKR1C2 to convert 5alpha-dihydroprogesterone (5alpha-DHP) to 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP/allopregnanolone), leading to 5alpha-DHT secretion necessary for embryonic gonad differentiation into testis (PubMed:21802064). May regulate the concentrations of circulating neurosteroids. Reduces 5alpha-dihydroprogesterone (5-alpha-DHP) and 5alpha-dihydrodeoxycorticosterone (5-alpha-DHDOC) precursors to 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP/allopregnanolone) and 3alpha,21-dihydroxy-5alpha-pregnane-20-one (3alpha,5alpha-THDOC) neuroactive steroids known to alter neural excitability via allosteric activation of gamma-aminobutyric acid type A receptors (GABAAR) (PubMed:12604236). Regulates ligand availability for steroid hormone receptors. Catalyzes the inactivation of 5alpha-DHT and progesterone converting them into 3alpha/beta-androstanediols and (20S)-hydroxypregn-4-en-3-one, respectively (PubMed:10998348, PubMed:11158055, PubMed:14672942). May contribute to the metabolism of adrenal-derived androgens via reduction of 11-keto-5alpha-androstane-3,17-dione (11K-Adione) into 11-ketoandrosterone (11KAST) and of 11-ketodihydrotestosterone (11KDHT) into 11-keto-5alpha-androstane-3alpha/beta,17beta-diol (11K-A3diol) (PubMed:31926269). Catalyzes the reduction of estrone into 17beta-estradiol but with low efficiency (PubMed:14672942). In androgen catabolism, may predominantly act as a phase I enzyme by introducing a hydroxyl group prior to conjugation. It can nevertheless participate in the alternative phase II pathway by directly reducing sulfate- or glucuronide-conjugated androgens (PubMed:19218247). Catalyzes the biotransformation of the pesticide chlordecone (kepone) to its corresponding alcohol, leading to increased biliary excretion of the pesticide and concomitant reduction of its neurotoxicity since bile is the major excretory route (PubMed:2427522). In vitro can efficiently catalyze bidirectional conversion between ketosteroids and hydroxysteroids using NADPH/NADP(+) or NADH/NAD(+) as cofactors. In vivo however, the reductase activity prevails since the major reducing cofactor NADPH inhibits NAD(+)-dependent oxidase activity (PubMed:14672942)","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/P17516/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AKR1C4","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/AKR1C4","total_profiled":1310},"omim":[{"mim_id":"614279","title":"46,XY SEX REVERSAL 8; SRXY8","url":"https://www.omim.org/entry/614279"},{"mim_id":"603966","title":"ALDO-KETO REDUCTASE FAMILY 1, MEMBER C3; AKR1C3","url":"https://www.omim.org/entry/603966"},{"mim_id":"600451","title":"ALDO-KETO REDUCTASE FAMILY 1, MEMBER C4; AKR1C4","url":"https://www.omim.org/entry/600451"},{"mim_id":"600450","title":"ALDO-KETO REDUCTASE FAMILY 1, MEMBER C2; AKR1C2","url":"https://www.omim.org/entry/600450"},{"mim_id":"600449","title":"ALDO-KETO REDUCTASE FAMILY 1, MEMBER C1; AKR1C1","url":"https://www.omim.org/entry/600449"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Nucleoli fibrillar center","reliability":"Additional"},{"location":"Endoplasmic reticulum","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"liver","ntpm":670.3}],"url":"https://www.proteinatlas.org/search/AKR1C4"},"hgnc":{"alias_symbol":["DD4","HAKRA","C11","3-alpha-HSD","CDR","MGC22581"],"prev_symbol":["CHDR"]},"alphafold":{"accession":"P17516","domains":[{"cath_id":"3.20.20.100","chopping":"4-226_266-285","consensus_level":"high","plddt":97.1743,"start":4,"end":285}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P17516","model_url":"https://alphafold.ebi.ac.uk/files/AF-P17516-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P17516-F1-predicted_aligned_error_v6.png","plddt_mean":96.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AKR1C4","jax_strain_url":"https://www.jax.org/strain/search?query=AKR1C4"},"sequence":{"accession":"P17516","fasta_url":"https://rest.uniprot.org/uniprotkb/P17516.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P17516/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P17516"}},"corpus_meta":[{"pmid":"10998348","id":"PMC_10998348","title":"Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones.","date":"2000","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/10998348","citation_count":516,"is_preprint":false},{"pmid":"28352233","id":"PMC_28352233","title":"Aldo-Keto Reductase AKR1C1-AKR1C4: Functions, Regulation, and Intervention for Anti-cancer Therapy.","date":"2017","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/28352233","citation_count":105,"is_preprint":false},{"pmid":"21521174","id":"PMC_21521174","title":"Stereospecific reduction of 5β-reduced steroids by human ketosteroid reductases of the AKR (aldo-keto reductase) superfamily: role of AKR1C1-AKR1C4 in the metabolism of testosterone and progesterone via the 5β-reductase pathway.","date":"2011","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/21521174","citation_count":38,"is_preprint":false},{"pmid":"12571290","id":"PMC_12571290","title":"Drosophila dd4 mutants reveal that gammaTuRC is required to maintain juxtaposed half spindles in spermatocytes.","date":"2003","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/12571290","citation_count":27,"is_preprint":false},{"pmid":"21570127","id":"PMC_21570127","title":"AKR1C4 gene variant associated with low euthymic serum progesterone and a history of mood irritability in males with bipolar disorder.","date":"2011","source":"Journal of affective disorders","url":"https://pubmed.ncbi.nlm.nih.gov/21570127","citation_count":26,"is_preprint":false},{"pmid":"18024509","id":"PMC_18024509","title":"Regulation of human 3 alpha-hydroxysteroid dehydrogenase (AKR1C4) expression by the liver X receptor alpha.","date":"2007","source":"Molecular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/18024509","citation_count":25,"is_preprint":false},{"pmid":"32591384","id":"PMC_32591384","title":"Discovery of an Inducible Toluene Monooxygenase That Cooxidizes 1,4-Dioxane and 1,1-Dichloroethylene in Propanotrophic Azoarcus sp. Strain DD4.","date":"2020","source":"Applied and environmental microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/32591384","citation_count":23,"is_preprint":false},{"pmid":"11006119","id":"PMC_11006119","title":"Molecular cloning of the crustacean DD4 cDNA encoding a Ca(2+)-binding protein.","date":"2000","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/11006119","citation_count":23,"is_preprint":false},{"pmid":"7789999","id":"PMC_7789999","title":"Localization of multiple human dihydrodiol dehydrogenase (DDH1 and DDH2) and chlordecone reductase (CHDR) genes in chromosome 10 by the polymerase chain reaction and fluorescence in situ hybridization.","date":"1995","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/7789999","citation_count":23,"is_preprint":false},{"pmid":"22356824","id":"PMC_22356824","title":"Polymorphisms in AKR1C4 and HSD3B2 and differences in serum DHEAS and progesterone are associated with paranoid ideation during mania or hypomania in bipolar disorder.","date":"2012","source":"European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/22356824","citation_count":22,"is_preprint":false},{"pmid":"11284743","id":"PMC_11284743","title":"Co-operative regulation of the transcription of human dihydrodiol dehydrogenase (DD)4/aldo-keto reductase (AKR)1C4 gene by hepatocyte nuclear factor (HNF)-4alpha/gamma and HNF-1alpha.","date":"2001","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/11284743","citation_count":17,"is_preprint":false},{"pmid":"34021356","id":"PMC_34021356","title":"Large trans-ethnic meta-analysis identifies AKR1C4 as a novel gene associated with age at menarche.","date":"2021","source":"Human reproduction (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/34021356","citation_count":10,"is_preprint":false},{"pmid":"12175496","id":"PMC_12175496","title":"Expression pattern of dd4, a sole member of the d4 family of transcription factors in Drosophila melanogaster.","date":"2002","source":"Mechanisms of development","url":"https://pubmed.ncbi.nlm.nih.gov/12175496","citation_count":7,"is_preprint":false},{"pmid":"11104674","id":"PMC_11104674","title":"Kinetic alteration of a human dihydrodiol/3alpha-hydroxysteroid dehydrogenase isoenzyme, AKR1C4, by replacement of histidine-216 with tyrosine or phenylalanine.","date":"2000","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/11104674","citation_count":6,"is_preprint":false},{"pmid":"12220531","id":"PMC_12220531","title":"Hepatocyte nuclear factor (HNF)-4 alpha/gamma, HNF-1 alpha, and vHNF-1 regulate the cell-specific expression of the human dihydrodiol dehydrogenase (DD)4/AKR1C4 gene.","date":"2002","source":"Archives of biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/12220531","citation_count":5,"is_preprint":false},{"pmid":"38890190","id":"PMC_38890190","title":"AKR1C4 regulates the sensitivity of colorectal cancer cells to chemotherapy through ferroptosis modulation.","date":"2024","source":"Cancer chemotherapy and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/38890190","citation_count":1,"is_preprint":false},{"pmid":"40721470","id":"PMC_40721470","title":"Genetic and computational analysis of AKR1C4 gene rs17134592 polymorphism in breast cancer among the Bangladeshi population.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40721470","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.06.25327120","title":"Protocol for Bayesian combined multi-genotype and concentration informed tacrolimus dosing in paediatric solid organ transplantation (BRUNO-PIC)","date":"2025-05-07","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.06.25327120","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13434,"output_tokens":2409,"usd":0.038219,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9620,"output_tokens":2646,"usd":0.057125,"stage2_stop_reason":"end_turn"},"total_usd":0.095344,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"AKR1C4 (type 1 3α-HSD) functions as a highly catalytically efficient NAD(P)(H)-dependent ketosteroid reductase and hydroxysteroid oxidase, acting on 3-, 17-, and 20-positions of steroids. It is the most catalytically efficient isoform (k_cat/K_m 10–30-fold higher than AKR1C1–1C3), inactivates 5α-DHT to 3α-androstanediol, oxidizes testosterone to androstene-3,17-dione, reduces oestrone to 17β-oestradiol, and forms 5α/5β-tetrahydrosteroids. It is virtually liver-specific in tissue distribution.\",\n      \"method\": \"Recombinant protein expression, kinetic parameter determination, product identification by biochemical assay, isoform-specific RT-PCR for tissue distribution\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic characterization with purified recombinant protein, comprehensive kinetics and product identification, replicated across multiple substrates and compared across four isoforms\",\n      \"pmids\": [\"10998348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AKR1C4 is the major enzyme responsible for the hepatic formation of the 5β-tetrahydrosteroid of testosterone via the 5β-pathway: it reduces 5β-dihydrotestosterone predominantly to the 3α-hydroxy configuration, with the stereochemistry explained by molecular docking. AKR1C4 has the highest kinetic efficiency for this substrate among AKR1C1–1C4.\",\n      \"method\": \"In vitro enzymatic assay with purified recombinant AKR1C1–1C4, product characterization by liquid chromatography-MS, kinetic parameter determination, molecular docking\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with LC-MS product identification, kinetics, and structural docking; orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"21521174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"His-216 in AKR1C4 (unique compared to the conserved Tyr-216 in AKR1C1–1C3) plays a key role in orienting the nicotinamide ring of the coenzyme and shaping the substrate-binding cavity. Replacement of His-216 with Tyr or Phe decreased K_m for NADP+ ~3-fold, differentially altered K_m and k_cat for substrates depending on structure (bile acids with 12α-OH vs. others), changed inhibitor sensitivity, and reduced stimulatory effects of non-essential activators.\",\n      \"method\": \"Site-directed mutagenesis of AKR1C4 (H216Y, H216F), kinetic analysis with purified mutant enzymes, inhibitor and activator binding assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — active-site mutagenesis with quantitative kinetics, multiple substrates and inhibitors tested, mechanistic interpretation grounded in crystallographic context of family members\",\n      \"pmids\": [\"11104674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Transcription of the human AKR1C4 (DD4) gene in hepatic cells is cooperatively regulated by HNF-4α and HNF-4γ binding to a cis-element at −701 to −684, and HNF-1α binding to a cis-element at −682 to −666. Mutation of either element reduces luciferase reporter activity to ~10% and ~8% of wild-type, respectively.\",\n      \"method\": \"Luciferase reporter assay in HepG2 cells, 1,10-phenanthroline-copper footprinting, gel-shift (EMSA) assay, supershift assay with antibodies to HNF-4α, HNF-4γ, HNF-1α, promoter deletion/mutation analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (footprinting, EMSA, supershift, reporter mutagenesis) in a single study establishing transcriptional regulation mechanism\",\n      \"pmids\": [\"11284743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Cell-type-specific expression of AKR1C4 (DD4) is determined by differential occupancy of the HNF-4/HNF-1 promoter elements: in hepatic HepG2 cells, HNF-4α, HNF-4γ, and HNF-1α activate transcription, whereas in non-expressing ACHN renal cells, vHNF-1-C (a truncated isoform lacking transcriptional activation domain) occupies the same elements without activating transcription.\",\n      \"method\": \"Reporter gene (luciferase) assay, supershift EMSA with isoform-specific antibodies, semi-quantitative RT-PCR, transfection with vHNF-1-C expression plasmid\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (reporter, EMSA, RT-PCR, transfection) in a single lab extending the prior study\",\n      \"pmids\": [\"12220531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"LXRα (NR1H3) binds to a response element ~1.5 kb upstream of the AKR1C4 transcription start site and mediates transcriptional activation of AKR1C4, leading to increased AKR1C4 protein expression. This was identified by hidden Markov model prediction combined with chromatin immunoprecipitation/microarray analysis.\",\n      \"method\": \"Hidden Markov model prediction of nuclear receptor response elements, chromatin immunoprecipitation (ChIP)/microarray, demonstration of LXRα binding to the LXRE and transcriptional activation of AKR1C4\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP combined with functional protein expression data, single lab, two complementary methods\",\n      \"pmids\": [\"18024509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The AKR1C4 gene (as CHDR/chlordecone reductase) and closely related dihydrodiol dehydrogenase genes (DDH1, DDH2) are all located on human chromosome 10p14–p15.\",\n      \"method\": \"PCR with gene-specific primers on human/hamster hybrid DNA panel, fluorescence in situ hybridization (FISH)\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal mapping methods (PCR on hybrid panel + FISH), single study\",\n      \"pmids\": [\"7789999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AKR1C4 enzymatic activity is required to suppress ferroptosis and confer chemotherapy resistance in colorectal cancer cells: CRISPR/Cas9 knockout of AKR1C4 enhances sensitivity to 5-FU, irinotecan, and oxaliplatin by inducing ferroptosis (increased total iron and lipid peroxidation). A Y55A catalytic mutant of AKR1C4 fails to rescue chemoresistance, demonstrating that enzymatic activity is necessary.\",\n      \"method\": \"CRISPR/Cas9 knockout, catalytic point mutant (Y55A) rescue assay, SRB cell viability assay, total iron content measurement, lipid peroxidation measurement, acquisition of chemoresistant cell lines by long-term drug induction\",\n      \"journal\": \"Cancer chemotherapy and pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with defined phenotype plus catalytic-dead mutant rescue, multiple orthogonal ferroptosis readouts, single lab\",\n      \"pmids\": [\"38890190\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AKR1C4 is a liver-specific, NAD(P)(H)-dependent aldo-keto reductase that catalyzes the stereospecific reduction of ketosteroids (including 5α-DHT and 5β-dihydrosteroids) and oxidation of hydroxysteroids to regulate active sex hormone levels and bile acid metabolism; its catalytic efficiency depends critically on His-216 in the substrate-binding cavity, its hepatic expression is cooperatively activated by HNF-4α/γ and HNF-1α binding to the promoter and further induced by LXRα, and its enzymatic activity suppresses ferroptosis to promote chemotherapy resistance in colorectal cancer cells.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AKR1C4 is a virtually liver-specific, NAD(P)(H)-dependent aldo-keto reductase that catalyzes the stereospecific interconversion of ketosteroids and hydroxysteroids at the 3-, 17-, and 20-positions, thereby governing the levels of active sex hormones and dihydrosteroid metabolites [#0]. It is the most catalytically efficient AKR1C isoform, inactivating 5\\u03b1-DHT to 3\\u03b1-androstanediol, oxidizing testosterone, reducing oestrone to 17\\u03b2-oestradiol, and forming 5\\u03b1/5\\u03b2-tetrahydrosteroids [#0]; it is the major hepatic enzyme of the 5\\u03b2-pathway, reducing 5\\u03b2-dihydrotestosterone preferentially to the 3\\u03b1-hydroxy configuration [#1]. Its distinctive catalytic behavior derives in part from His-216 (in place of the Tyr conserved in AKR1C1\\u20131C3), which orients the nicotinamide ring of the coenzyme and shapes the substrate-binding cavity, modulating cofactor affinity, substrate-dependent kinetics, and inhibitor sensitivity [#2]. Hepatic, cell-type-restricted expression is established by cooperative transactivation through HNF-4\\u03b1/HNF-4\\u03b3 and HNF-1\\u03b1 binding to adjacent promoter elements\\u2014with non-activating vHNF-1-C occupancy explaining silencing in non-hepatic cells\\u2014and is further induced by LXR\\u03b1 binding to an upstream response element [#3, #4, #5]. Beyond steroid metabolism, AKR1C4 enzymatic activity suppresses ferroptosis to confer chemotherapy resistance in colorectal cancer cells, an effect lost in a catalytically dead mutant [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that AKR1C4 is a bona fide, highly efficient steroid-metabolizing oxidoreductase, defining its molecular activity and the liver-restricted context in which it operates.\",\n      \"evidence\": \"Recombinant protein kinetics, product identification, and isoform-specific RT-PCR across AKR1C1\\u20131C4\",\n      \"pmids\": [\"10998348\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro substrate preferences do not establish the dominant physiological reaction direction in hepatocytes\", \"Did not resolve structural basis of catalytic superiority over other isoforms\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Pinpointed His-216 as a determinant of AKR1C4's cofactor orientation and substrate cavity, explaining its kinetic distinctiveness from the Tyr-216 paralogs.\",\n      \"evidence\": \"Site-directed mutagenesis (H216Y, H216F) with kinetic, inhibitor, and activator binding analysis on purified enzymes\",\n      \"pmids\": [\"11104674\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal structure of AKR1C4 itself; interpretation rests on family member structures\", \"Effect of His-216 on the full physiological substrate panel not exhaustively mapped\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Resolved how hepatic AKR1C4 transcription is driven, showing cooperative control by adjacent HNF-4\\u03b1/\\u03b3 and HNF-1\\u03b1 cis-elements.\",\n      \"evidence\": \"Luciferase reporters, footprinting, EMSA/supershift, and promoter mutagenesis in HepG2 cells\",\n      \"pmids\": [\"11284743\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address signals upstream that modulate HNF factor activity\", \"Promoter analysis in a single hepatic cell line\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Explained tissue-restricted expression as differential transcription-factor occupancy, with non-activating vHNF-1-C occupying the same elements in non-hepatic cells.\",\n      \"evidence\": \"Reporter assays, supershift EMSA, RT-PCR, and vHNF-1-C transfection in HepG2 vs ACHN cells\",\n      \"pmids\": [\"12220531\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab extension of the prior promoter study\", \"Does not establish in vivo relevance of vHNF-1-C silencing across tissues\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Added a nuclear-receptor input by showing LXR\\u03b1 directly activates AKR1C4 transcription, linking the gene to lipid/sterol-sensing regulation.\",\n      \"evidence\": \"HMM prediction of response elements plus ChIP/microarray and protein expression analysis\",\n      \"pmids\": [\"18024509\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological ligand/condition triggering LXR\\u03b1-driven induction not defined\", \"Two complementary methods from a single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified AKR1C4 as the principal hepatic enzyme of the 5\\u03b2-pathway, defining its stereochemical product preference for 5\\u03b2-dihydrotestosterone.\",\n      \"evidence\": \"In vitro reconstitution with LC-MS product characterization, kinetics, and molecular docking across AKR1C1\\u20131C4\",\n      \"pmids\": [\"21521174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo flux through the 5\\u03b2-pathway not directly quantified\", \"Docking model not validated by experimental structure\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended AKR1C4 function beyond steroid metabolism, demonstrating that its catalytic activity suppresses ferroptosis and drives chemoresistance in colorectal cancer.\",\n      \"evidence\": \"CRISPR/Cas9 knockout, Y55A catalytic-dead rescue, viability/iron/lipid-peroxidation readouts in chemoresistant cell lines\",\n      \"pmids\": [\"38890190\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The metabolic substrate/product responsible for ferroptosis suppression is not identified\", \"Single-lab cell-line study without in vivo confirmation\", \"Mechanism linking enzymatic output to lipid peroxidation unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how AKR1C4's steroid-reductase chemistry mechanistically connects to lipid-peroxidation/ferroptosis control, and whether its in vivo physiological role is dominated by steroid clearance or by the cancer-associated activity.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No identified endogenous substrate bridging steroid metabolism and ferroptosis\", \"No experimentally determined AKR1C4 structure\", \"In vivo physiological consequences of expression changes not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 1, 2, 7]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}