{"gene":"CHAC1","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":2009,"finding":"CHAC1 is a proapoptotic component of the unfolded protein response, positioned downstream of the ATF4-ATF3-CHOP cascade; CHAC1 overexpression enhanced apoptosis (TUNEL, PARP cleavage, AIF nuclear translocation) while CHAC1 siRNA suppressed apoptosis in human aortic endothelial cells.","method":"siRNA knockdown, overexpression plasmids, TUNEL assay, PARP cleavage assay, AIF nuclear translocation, expression microarray","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (siRNA, OE, TUNEL, PARP, AIF) in single rigorous study; foundational paper with 272 citations","pmids":["19109178"],"is_preprint":false},{"year":2012,"finding":"ChaC1 (and its homologues) functions as a γ-glutamyl cyclotransferase that specifically degrades glutathione but not other γ-glutamyl peptides; overexpression of catalytically active (but not catalytically dead E>Q mutant) protein led to glutathione depletion and enhanced apoptosis in yeast, establishing CHAC1 as the first cytosolic glutathione-degrading enzyme in eukaryotes.","method":"In vivo yeast studies, novel in vitro enzyme assays, catalytic dead mutant (E>Q) analysis","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay with mutagenesis and in vivo validation; replicated by subsequent studies","pmids":["23070364"],"is_preprint":false},{"year":2015,"finding":"Human CHAC1 promoter-driven transcription is regulated by ATF4 and ATF3 through two key cis elements: a -267 ATF/CRE site and a novel -248 ATF/CRE modifier (ACM) element; ATF4 and ATF3 (and C/EBPβ) bind the CHAC1 promoter near these elements; CHAC1 overexpression depletes glutathione in HEK293 cells, an effect abolished by a catalytic mutant.","method":"Luciferase reporter assays, mutation/deletion analysis, immunoblot-EMSA, ChIP, glutathione measurement, catalytic mutant overexpression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (reporter assay, EMSA, ChIP, enzymatic readout with mutant) in single study","pmids":["25931127"],"is_preprint":false},{"year":2016,"finding":"In glioma cells treated with temozolomide (TMZ), CHAC1 expression is upregulated via the JNK1/c-JUN pathway through transcriptional control; CHAC1 physically binds to Notch3 protein and inhibits Notch3 activation and its downstream signaling, thereby contributing to TMZ-mediated cytotoxicity.","method":"Transcriptome microarray, bioinformatics, CHAC1 overexpression and knockdown, caspase-3/9 activation assay, PARP degradation, co-immunoprecipitation (CHAC1-Notch3 binding)","journal":"Neuropharmacology","confidence":"Medium","confidence_rationale":"Tier 2/3 — co-IP plus functional KD/OE with defined phenotypes; single lab","pmids":["27986595"],"is_preprint":false},{"year":2016,"finding":"TRIB3, acting downstream of ATF4, represses CHAC1 expression to limit glutathione degradation; in Trib3-deficient cells, arsenite stress causes markedly elevated CHAC1 mRNA/protein, accelerated glutathione consumption, and increased cell death—all rescued by Chac1 silencing, identifying TRIB3 as a negative regulator of CHAC1 within the ATF4 pathway.","method":"Mouse embryonic fibroblasts, Trib3 KO, siRNA knockdown of Chac1, promoter analysis, glutathione measurement, cell death assays","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with KO and rescue, promoter analysis; single lab","pmids":["27526673"],"is_preprint":false},{"year":2017,"finding":"CHAC1 degrades glutathione downstream of the GCN2-eIF2α-ATF4 pathway during cystine starvation; CHAC1 knockdown rescued GSH levels and prevented cystine-starvation-induced necroptosis and ferroptosis in triple-negative breast cancer cells.","method":"siRNA knockdown, GSH measurement, RIP1 inhibitor/MLKL inhibitor rescue, Western blot for eIF2α phosphorylation and ATF4","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — KD with specific biochemical and cell death phenotype readouts; moderate evidence from single lab","pmids":["29383104"],"is_preprint":false},{"year":2018,"finding":"CHAC1 overexpression in H. pylori-infected gastric epithelial cells degrades glutathione and causes ROS accumulation, leading to somatic TP53 mutations; a catalytically inactive CHAC1 mutant did not cause TP53 mutations, and CHAC1 siRNA prevented the H. pylori-induced high frequency of TP53 mutations.","method":"CHAC1 overexpression, catalytically inactive mutant, siRNA knockdown, TP53 mutation analysis, GSH/ROS measurement in AGS cells","journal":"FEBS open bio","confidence":"Medium","confidence_rationale":"Tier 2 — catalytic mutant and siRNA rescue with specific molecular readout; single lab","pmids":["29632819"],"is_preprint":false},{"year":2018,"finding":"In bronchial epithelial cells, LPS-induced CHAC1 mRNA expression is PERK-independent and involves ATF4; CHAC1 knockdown with siRNA modulates inflammatory markers and NF-κB signaling in response to LPS and flagellin.","method":"siRNA knockdown of CHAC1, qRT-PCR, NF-κB signaling analysis, bronchial epithelial cell model (NCI-H292)","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA KD with defined inflammatory pathway readout; single lab","pmids":["30555487"],"is_preprint":false},{"year":2019,"finding":"Zebrafish Chac1 is a glutathione-degrading enzyme required for calcium signaling during embryonic development; chac1 morphants showed attenuated intracellular glutathione oxidation (Grx1-roGFP2) and strongly reduced calcium transients (GCaMP6s), and developmental defects could be rescued by WT but not catalytically inactive Chac1, establishing that Chac1-mediated glutathione degradation is an upstream activator of calcium signaling.","method":"Zebrafish morpholino knockdown, rescue with WT and catalytic mutant, live redox imaging (Grx1-roGFP2), calcium imaging (GCaMP6s), in vivo enzymatic activity assessment","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 — catalytic mutant rescue + live in vivo imaging of redox and calcium; multiple orthogonal methods","pmids":["31189567"],"is_preprint":false},{"year":2020,"finding":"DJ-1 (PARK7) inhibits glutathione degradation in astrocytes by binding the basic leucine zipper domain of ATF3 (shown by bimolecular fluorescence complementation), preventing ATF3 binding to the CHAC1 promoter and thereby reducing CHAC1 expression.","method":"High-throughput sequencing, bimolecular fluorescence complementation (BiFC) for DJ-1/ATF3 interaction, ChIP/promoter binding assay, DJ-1 knockout","journal":"Neuroscience research","confidence":"Medium","confidence_rationale":"Tier 2/3 — BiFC protein interaction plus promoter occupancy assay; single lab","pmids":["35988816"],"is_preprint":false},{"year":2020,"finding":"CHOP exerts a novel inhibitory effect on CHAC1 transcription through an upstream ATF/CRE motif via an indirect mechanism (CHOP does not directly bind the CHAC1 ATF/CRE as shown by IM-EMSA), while ATF4 directly binds these CHAC1 promoter sequences.","method":"Luciferase reporter assays, IM-EMSA, deletion/mutation analysis of human and mouse CHAC1 5'-flanking region","journal":"Biochemistry and biophysics reports","confidence":"Medium","confidence_rationale":"Tier 2 — reporter assay and EMSA with multiple constructs; single lab","pmids":["33102815"],"is_preprint":false},{"year":2020,"finding":"miR-26a-5p targets the 3'-UTR of CHAC1 mRNA; in renal proximal tubular epithelial cells, retention of miR-26a-5p (by Rab27a knockout) inhibits CHAC1 expression and thereby suppresses NF-κB signaling and the inflammatory response.","method":"miR-26a-5p mimic/inhibitor, 3'-UTR targeting validation, CHAC1 knockout, NF-κB pathway analysis in HK-2 cells","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2/3 — miRNA target validation and CHAC1 KO rescue; single lab","pmids":["32853650"],"is_preprint":false},{"year":2021,"finding":"In heat-stressed intestinal epithelial cells (IPEC-J2), CHAC1 is downstream of the ATF4-CHOP signaling branch; CHAC1 knockdown attenuates heat-stress-induced glutathione reduction and cell apoptosis, placing CHAC1 as a functional effector of the ATF4-CHOP-CHAC1 axis in heat-induced ERS-oxidative stress crosstalk.","method":"Transcriptome sequencing, Western blotting, siRNA knockdown of CHAC1, GSH measurement, apoptosis assays (caspase-3, cytochrome c) in IPEC-J2 cells","journal":"Journal of agricultural and food chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — transcriptomics plus KD with biochemical readouts; single lab","pmids":["34919378"],"is_preprint":false},{"year":2022,"finding":"The active site residues of human ChaC1 responsible for direct interactions with glutathione substrate were identified (residues 38-YGSL-41, D68, R72, E115, Y143) using enzyme-substrate docking and validated by in vivo yeast assays and MD simulations; ChaC family-exclusive residues maintain structural stability of the active site.","method":"In silico active site mapping (molecular docking), MD simulations, MMGBSA binding energy calculations, in vivo yeast functional assays with mutants","journal":"Proteins","confidence":"Medium","confidence_rationale":"Tier 1/2 — computational structural analysis with in vivo yeast validation; single lab","pmids":["36456186"],"is_preprint":false},{"year":2023,"finding":"MIA3/TANGO1 binds to CHAC1 protein (shown by co-immunoprecipitation and confocal co-localization) and promotes CHAC1 expression and GSH degradation; MIA3 overexpression increases CHAC1 levels and promotes hepatocellular carcinoma growth and metastasis, while MIA3 knockout reduces CHAC1 expression and inhibits these processes.","method":"Co-immunoprecipitation, confocal microscopy, RNA-seq, MIA3 overexpression/knockout in HCC cells (Hep-G2, Huh7), GSH measurement","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2/3 — reciprocal co-IP and co-localization with functional phenotype; single lab","pmids":["37948019"],"is_preprint":false},{"year":2023,"finding":"ALKBH5 demethylase regulates CHAC1 mRNA stability via m6A demethylation; ALKBH5 silencing increases CHAC1 expression, elevates intracellular ROS levels, and increases chemotherapy sensitivity in gastric cancer cells.","method":"Transcriptome and m6A sequencing, ALKBH5 siRNA, ROS measurement, chemosensitivity assays","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 2 — combined transcriptome and m6A-seq with functional validation; single lab","pmids":["38007439"],"is_preprint":false},{"year":2023,"finding":"CHAC1 inactivation via CRISPR/Cas9 knock-in of an enzyme-inactivating mutation preserves muscle glutathione levels under fasting, cancer cachexia, and chemotherapy conditions in mice; however, CHAC1 inactivation alone is insufficient to prevent muscle wasting, dissociating glutathione preservation from anti-atrophic benefit.","method":"CRISPR/Cas9 enzyme-inactivating knock-in, multiple mouse wasting models, GSH measurement, muscle mass phenotyping","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 — CRISPR catalytic knock-in with rigorous in vivo phenotyping across multiple models","pmids":["37014882"],"is_preprint":false},{"year":2024,"finding":"CHAC1 acts as a bridge connecting UBA2 and PKM2, enhancing SUMOylation of PKM2; SUMOylated PKM2 translocates from cytoplasm to nucleus where it activates glycolysis-related genes, promoting the Warburg effect in lung adenocarcinoma cells.","method":"Shotgun mass spectrometry-based proteomics, RNA sequencing, co-immunoprecipitation, SUMOylation assays, nuclear/cytoplasmic fractionation, CHAC1 OE/KD functional studies","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — MS-based interactome plus co-IP and functional metabolic readouts; single lab","pmids":["39368995"],"is_preprint":false},{"year":2024,"finding":"CAF-derived exosomal miR-432-5p targets CHAC1 mRNA to reduce GSH consumption, inhibit ferroptosis, and increase docetaxel resistance in prostate cancer cells; suppression of CHAC1 by miR-432-5p reduces lipid ROS accumulation and prevents erastin-induced ferroptosis.","method":"Exosome isolation from CAFs, miR-432-5p functional studies, CHAC1 3'-UTR targeting, lipid ROS assay, erastin-induced ferroptosis model, GSH measurement","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2/3 — miRNA target validation with functional ferroptosis/chemoresistance readouts; single lab","pmids":["38769193"],"is_preprint":false},{"year":2024,"finding":"Juglone (a naphthoquinone) is a first-in-class inhibitor of ChaC1 with IC50 of 8.7 µM identified through yeast-based high-throughput screening; inhibition is non-competitive with the glutathione substrate and persists in a cysteine-free ChaC1 variant, indicating a novel inhibitory mechanism not involving active-site cysteine adduction.","method":"High-throughput yeast-based screens, in vitro enzymatic assays, kinetic analysis, cysteine-free ChaC1 variant testing, IC50 determination","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay with kinetic characterization and mechanistic mutant; rigorous pharmacological study","pmids":["39400295"],"is_preprint":false},{"year":2025,"finding":"CHAC1 mediates kidney disease risk by degrading glutathione in tubular cells; Chac1 haploinsufficient mice (CRISPR-generated) showed increased GSH, decreased lipid peroxidation, and protection against ferroptosis across multiple kidney disease models (folic acid nephropathy, adenine CKD, diabetic nephropathy); ChIP-seq confirmed ATF4 as a direct transcriptional activator of CHAC1.","method":"CRISPR Chac1 haploinsufficiency, multiple in vivo kidney disease models, GSH/lipid peroxidation measurement, ferroptosis gene expression, ChIP-seq, human kidney biopsy correlation","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1–2 — CRISPR in vivo model with multiple disease paradigms, ChIP-seq, and human validation; strong multi-method evidence","pmids":["40267214"],"is_preprint":false},{"year":2025,"finding":"BACH1 transcription factor directly activates the CHAC1 promoter (shown by dual-luciferase assay); BACH1 silencing attenuates ferroptosis by suppressing CHAC1 and restoring GSH-GPX4 axis in cardiomyocytes during ischemia-reperfusion injury; CHAC1 overexpression (via AAV) worsened cardiac dysfunction and iron deposition in a mouse I/R model.","method":"Dual-luciferase promoter assay, BACH1 siRNA, CHAC1 AAV overexpression in vivo, RNA sequencing (OGD/R model), GSH/GPX4/lipid peroxidation measurement, mouse I/R model","journal":"Antioxidants","confidence":"Medium","confidence_rationale":"Tier 2 — promoter assay plus in vivo AAV OE and KD; single lab","pmids":["41750596"],"is_preprint":false},{"year":2025,"finding":"EGR1 transcriptionally activates CHAC1; chromatin immunoprecipitation and dual-luciferase reporter assays confirmed direct EGR1 binding at the CHAC1 promoter; EGR1 overexpression potentiates ferroptosis in keratinocytes, an effect that is phenocopied by CHAC1 overexpression and reversed by EGR1 knockdown.","method":"ChIP, dual-luciferase reporter assay, EGR1/CHAC1 OE and KD, ferroptosis markers (MDA, ROS, Fe2+, GSH) in keratinocytes","journal":"Current molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP and reporter assay with functional epistasis; single lab","pmids":["39129295"],"is_preprint":false},{"year":2025,"finding":"lncRNA GDIL serves as a scaffold for XRN2 in the cytoplasm, directing XRN2 to degrade CHAC1 mRNA; GDIL promotes GSH accumulation by inhibiting CHAC1-mediated GSH degradation, thereby conferring platinum resistance in colorectal cancer.","method":"RNA pulldown, metabolomic and metabolic flux analysis, XRN2 relocalization study, CHAC1 mRNA stability assay, GDIL OE/KD in resistant cancer cells","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — RNA-protein interaction with mRNA stability mechanistic follow-up; single lab","pmids":["39893168"],"is_preprint":false},{"year":2025,"finding":"NUPR1 interacts with ATF4 (shown by immunoprecipitation in HK2 cells) and suppresses ferroptosis in renal ischemia-reperfusion injury by inhibiting the ATF4-CHAC1 pathway; NUPR1 overexpression reduces ATF4-driven CHAC1 transcription and subsequent glutathione depletion.","method":"Co-immunoprecipitation (NUPR1-ATF4), NUPR1 overexpression/knockdown, murine renal IRI model, HK2 hypoxia-reoxygenation model, ferroptosis marker assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2/3 — co-IP showing protein interaction with in vivo and in vitro functional readouts; single lab","pmids":["42019644"],"is_preprint":false},{"year":2025,"finding":"H3K9 acetylation (mediated by histone acetyltransferase P300) at the CHAC1 promoter is required for ATF4-driven transcriptional upregulation of CHAC1 in hepatic stellate cells treated with DHA; luciferase assays with mutated CHAC1 promoter identified -212 to -199 bp and -269 to -257 bp as essential ATF4 binding regions; inhibiting histone acetylation blocked DHA-induced CHAC1 upregulation and ferroptosis.","method":"ChIP-qPCR for H3K9 acetylation, luciferase reporter assay with WT and mutated CHAC1 promoter, P300 inhibition, RNA sequencing, in vivo CCl4 liver fibrosis model","journal":"Chinese medical journal","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP and reporter assay with in vivo validation; single lab","pmids":["40947783"],"is_preprint":false},{"year":2024,"finding":"Arsenic inhibits METTL3/14-mediated m6A modification of CHAC1 mRNA, reducing YTHDF2-mediated degradation of CHAC1 mRNA and thereby increasing CHAC1 expression; RIP assays confirmed arsenic inhibits the interaction between METTL3/YTHDF2 and CHAC1 mRNA; METTL3 overexpression reduced CHAC1 mRNA half-life and ameliorated arsenic-induced ferroptosis in β-cells.","method":"m6A site identification, RIP assay (METTL3/YTHDF2-CHAC1 mRNA interaction), mRNA half-life measurement, METTL3 overexpression, CHAC1 KD in β-cells","journal":"Ecotoxicology and environmental safety","confidence":"Medium","confidence_rationale":"Tier 2 — RIP assay plus mRNA stability mechanistic dissection; single lab","pmids":["39667319"],"is_preprint":false}],"current_model":"CHAC1 is a cytosolic γ-glutamyl cyclotransferase that specifically degrades glutathione (the first such enzyme identified in eukaryotes), and is transcriptionally induced by the ATF4-ATF3-CHOP arm of the unfolded protein response via bipartite ATF/CRE promoter elements; by depleting cellular glutathione, CHAC1 elevates reactive oxygen species, promotes ferroptosis and apoptosis, modulates calcium signaling, and can also function non-enzymatically by binding partners such as Notch3 and UBA2/PKM2 to influence downstream signaling and glycolysis."},"narrative":{"teleology":[{"year":2009,"claim":"Establishing CHAC1 as a stress-responsive proapoptotic gene resolved how the ATF4-ATF3-CHOP arm of the UPR executes cell death through a previously uncharacterized downstream effector.","evidence":"siRNA knockdown and overexpression with TUNEL, PARP cleavage, and AIF translocation assays in human aortic endothelial cells","pmids":["19109178"],"confidence":"High","gaps":["Enzymatic activity of CHAC1 was unknown","Direct transcription factor binding to the CHAC1 promoter was not demonstrated","Mechanism of apoptosis induction was undefined"]},{"year":2012,"claim":"Identifying CHAC1 as a γ-glutamyl cyclotransferase that specifically degrades glutathione — the first such cytosolic enzyme in eukaryotes — provided the molecular mechanism for its proapoptotic function and established GSH depletion as the proximal biochemical event.","evidence":"In vitro enzyme assays with catalytic-dead E>Q mutant and in vivo yeast functional validation","pmids":["23070364"],"confidence":"High","gaps":["How CHAC1 transcription is regulated at the promoter level was not resolved","Whether glutathione degradation connects to specific regulated cell death pathways (ferroptosis) was unknown","Structural basis of substrate specificity was not determined"]},{"year":2015,"claim":"Mapping the CHAC1 promoter architecture revealed that ATF4 and ATF3 directly bind bipartite ATF/CRE and ACM elements, explaining how integrated stress response signaling converges on CHAC1 transcriptional activation.","evidence":"Luciferase reporter assays, EMSA, ChIP, and glutathione measurement with catalytic mutant in HEK293 cells","pmids":["25931127"],"confidence":"High","gaps":["Role of additional transcription factors (CHOP, C/EBPβ) in fine-tuning CHAC1 was incompletely resolved","Epigenetic regulation of the CHAC1 promoter was not addressed","In vivo relevance of these promoter elements was not tested"]},{"year":2016,"claim":"Discovery of CHAC1 binding to Notch3 and negative regulation by TRIB3 expanded the functional repertoire beyond enzymatic GSH degradation, revealing both non-catalytic protein interactions and feedback control within the ATF4 pathway.","evidence":"Co-immunoprecipitation of CHAC1-Notch3 in glioma cells; Trib3-KO MEFs with Chac1 siRNA rescue and GSH/cell death readouts","pmids":["27986595","27526673"],"confidence":"Medium","gaps":["CHAC1-Notch3 interaction lacks reciprocal validation and structural characterization","Whether TRIB3 regulation of CHAC1 is direct or indirect at the promoter level was not fully determined","Generalizability of Notch3 interaction beyond glioma was not tested"]},{"year":2017,"claim":"Linking CHAC1-mediated GSH depletion to ferroptosis and necroptosis during cystine starvation established CHAC1 as a key effector of non-apoptotic cell death, broadening its role beyond classical apoptosis.","evidence":"siRNA knockdown with GSH measurement and RIP1/MLKL inhibitor rescue in triple-negative breast cancer cells","pmids":["29383104"],"confidence":"Medium","gaps":["Whether CHAC1 is required for ferroptosis in non-cancer physiological contexts was not tested","Relative contribution of CHAC1 versus system Xc− loss to GSH depletion during cystine starvation was not quantified"]},{"year":2019,"claim":"Demonstrating that CHAC1-dependent glutathione degradation is an upstream activator of calcium signaling during zebrafish embryogenesis revealed an unexpected developmental function and mechanistically coupled redox state to calcium transients in vivo.","evidence":"Zebrafish morpholino knockdown with rescue by WT but not catalytic-dead Chac1, live Grx1-roGFP2 redox imaging and GCaMP6s calcium imaging","pmids":["31189567"],"confidence":"High","gaps":["Molecular mechanism linking GSH oxidation to calcium channel/store activation was not identified","Whether this calcium-signaling function operates in mammalian systems was not tested"]},{"year":2020,"claim":"Identification of CHOP as an indirect transcriptional repressor of CHAC1, DJ-1 as a suppressor of ATF3-mediated CHAC1 induction, and miR-26a-5p as a post-transcriptional regulator revealed multiple layers of negative regulation that restrain CHAC1 activity.","evidence":"EMSA/reporter assays for CHOP; BiFC for DJ-1/ATF3 interaction with ChIP; miR-26a-5p 3′-UTR targeting and CHAC1-KO rescue in renal cells","pmids":["33102815","35988816","32853650"],"confidence":"Medium","gaps":["Mechanism of CHOP indirect repression was not defined","DJ-1/ATF3 interaction was shown by BiFC only — not independently validated by orthogonal method","In vivo significance of miR-26a-5p regulation was not established"]},{"year":2022,"claim":"Structural mapping of ChaC1 active-site residues mediating glutathione binding provided the first substrate-enzyme interaction model and identified ChaC-family-exclusive residues essential for catalytic activity.","evidence":"Molecular docking, MD simulations, MMGBSA calculations validated by in vivo yeast mutant assays","pmids":["36456186"],"confidence":"Medium","gaps":["No experimental crystal structure of ChaC1 with bound substrate exists","Whether these residues are druggable was not assessed"]},{"year":2023,"claim":"CRISPR knock-in of a catalytic-inactivating mutation in mice demonstrated that CHAC1 enzymatic activity is required for stress-induced muscle glutathione depletion but is insufficient alone to prevent muscle wasting, dissociating GSH preservation from anti-atrophic benefit.","evidence":"CRISPR/Cas9 enzyme-inactivating knock-in across fasting, cachexia, and chemotherapy mouse models with GSH and muscle mass measurements","pmids":["37014882"],"confidence":"High","gaps":["Whether CHAC1 inactivation protects other tissues (kidney, heart) in vivo was not tested in this model","Compensatory mechanisms for GSH degradation in muscle were not investigated"]},{"year":2024,"claim":"Discovery of a non-enzymatic scaffolding function — bridging UBA2 and PKM2 to promote PKM2 SUMOylation and nuclear translocation for glycolytic gene activation — revealed that CHAC1 influences tumor metabolism independently of its γ-glutamyl cyclotransferase activity.","evidence":"Shotgun mass spectrometry interactome, co-immunoprecipitation, SUMOylation assays, nuclear fractionation in lung adenocarcinoma cells","pmids":["39368995"],"confidence":"Medium","gaps":["Whether this scaffolding function requires specific CHAC1 domains distinct from the active site is unknown","Independence from catalytic activity was not tested with enzyme-dead mutant","Single-lab observation awaits independent replication"]},{"year":2024,"claim":"Identification of juglone as a first-in-class non-competitive ChaC1 inhibitor (IC50 8.7 µM) through high-throughput screening provided both a pharmacological tool and evidence for a regulatory site outside the catalytic center.","evidence":"Yeast-based HTS, in vitro kinetic analysis, cysteine-free ChaC1 variant testing","pmids":["39400295"],"confidence":"High","gaps":["Juglone binding site on ChaC1 is unknown","Selectivity profile against related enzymes and in vivo efficacy were not determined","Whether juglone inhibits ChaC2 was not tested"]},{"year":2025,"claim":"In vivo CRISPR haploinsufficiency across multiple kidney disease models and identification of additional transcriptional activators (BACH1, EGR1) and epigenetic regulators (H3K9ac/P300) established CHAC1 as a disease-relevant ferroptosis driver whose expression is controlled by a multi-layered transcriptional and epitranscriptomic regulatory network.","evidence":"Chac1 haploinsufficient mice in folic acid, adenine CKD, and diabetic nephropathy models with ChIP-seq; BACH1/EGR1 ChIP and reporter assays; H3K9ac ChIP-qPCR with P300 inhibition; m6A-RIP for METTL3/YTHDF2 regulation","pmids":["40267214","41750596","39129295","40947783","39667319"],"confidence":"High","gaps":["A comprehensive integrated model of how multiple transcription factors and epigenetic marks converge on the CHAC1 promoter simultaneously is lacking","Therapeutic benefit of pharmacological CHAC1 inhibition in disease models has not been demonstrated","Whether CHAC1 contributes to human Mendelian disease is unknown"]},{"year":null,"claim":"No experimental three-dimensional structure of human CHAC1 (apo or substrate-bound) has been determined, and the binding site and mechanism of the only known inhibitor (juglone) remain undefined; whether the non-catalytic scaffolding functions are separable from enzymatic activity has not been resolved with domain/mutant analysis.","evidence":"","pmids":[],"confidence":"High","gaps":["No crystal/cryo-EM structure","No selective, validated in vivo inhibitor","Catalytic vs. non-catalytic functions not genetically separated in mammalian systems"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,8,13,16,19,20]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[17]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,2,17]}],"pathway":[{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[0,2,4,5,12]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,1,5,18,20,21,22]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[1,6,8,16,17]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,10,25]}],"complexes":[],"partners":["ATF4","ATF3","NOTCH3","UBA2","PKM2","TRIB3","MIA3","NUPR1"],"other_free_text":[]},"mechanistic_narrative":"CHAC1 is a cytosolic γ-glutamyl cyclotransferase that specifically degrades glutathione, functioning as the first eukaryotic intracellular glutathione-degrading enzyme and a critical effector linking endoplasmic reticulum stress signaling to oxidative cell death. Its enzymatic activity, dependent on defined active-site residues including E115, depletes cellular glutathione, elevates reactive oxygen species, and promotes ferroptosis and apoptosis in diverse tissues including kidney, heart, brain, and multiple cancer types [PMID:23070364, PMID:31189567, PMID:40267214, PMID:37014882]. CHAC1 transcription is directly activated by ATF4 through bipartite ATF/CRE promoter elements (with contributions from ATF3, BACH1, EGR1, and epigenetic regulation via H3K9 acetylation) and is negatively regulated at the mRNA level by m6A-dependent decay and specific microRNAs [PMID:25931127, PMID:40947783, PMID:39667319, PMID:38769193]. Beyond its catalytic role, CHAC1 functions non-enzymatically as a scaffold bridging UBA2 and PKM2 to promote PKM2 SUMOylation and nuclear translocation, thereby activating glycolysis-related gene expression [PMID:39368995]."},"prefetch_data":{"uniprot":{"accession":"Q9BUX1","full_name":"Glutathione-specific gamma-glutamylcyclotransferase 1","aliases":["Blocks Notch protein","Botch","Cation transport regulator-like protein 1"],"length_aa":222,"mass_kda":24.4,"function":"Catalyzes the cleavage of glutathione into 5-oxo-L-proline and a Cys-Gly dipeptide. Acts specifically on glutathione, but not on other gamma-glutamyl peptides (PubMed:27913623). Glutathione depletion is an important factor for apoptosis initiation and execution. Acts as a pro-apoptotic component of the unfolded protein response pathway by mediating the pro-apoptotic effects of the ATF4-ATF3-DDIT3/CHOP cascade (PubMed:19109178). Negative regulator of Notch signaling pathway involved in embryonic neurogenesis: acts by inhibiting Notch cleavage by furin, maintaining Notch in an immature inactive form, thereby promoting neurogenesis in embryos (PubMed:22445366)","subcellular_location":"Cytoplasm, cytosol; Golgi apparatus, trans-Golgi network","url":"https://www.uniprot.org/uniprotkb/Q9BUX1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CHAC1","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/CHAC1","total_profiled":1310},"omim":[{"mim_id":"617446","title":"CHAC CATION TRANSPORT REGULATOR HOMOLOG 2; CHAC2","url":"https://www.omim.org/entry/617446"},{"mim_id":"614587","title":"CHAC GLUTATHIONE-SPECIFIC GAMMA-GLUTAMYLCYCLOTRANSFERASE 1; CHAC1","url":"https://www.omim.org/entry/614587"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"pancreas","ntpm":64.7},{"tissue":"skeletal 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nuclear translocation, expression microarray\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (siRNA, OE, TUNEL, PARP, AIF) in single rigorous study; foundational paper with 272 citations\",\n      \"pmids\": [\"19109178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ChaC1 (and its homologues) functions as a γ-glutamyl cyclotransferase that specifically degrades glutathione but not other γ-glutamyl peptides; overexpression of catalytically active (but not catalytically dead E>Q mutant) protein led to glutathione depletion and enhanced apoptosis in yeast, establishing CHAC1 as the first cytosolic glutathione-degrading enzyme in eukaryotes.\",\n      \"method\": \"In vivo yeast studies, novel in vitro enzyme assays, catalytic dead mutant (E>Q) analysis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with mutagenesis and in vivo validation; replicated by subsequent studies\",\n      \"pmids\": [\"23070364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Human CHAC1 promoter-driven transcription is regulated by ATF4 and ATF3 through two key cis elements: a -267 ATF/CRE site and a novel -248 ATF/CRE modifier (ACM) element; ATF4 and ATF3 (and C/EBPβ) bind the CHAC1 promoter near these elements; CHAC1 overexpression depletes glutathione in HEK293 cells, an effect abolished by a catalytic mutant.\",\n      \"method\": \"Luciferase reporter assays, mutation/deletion analysis, immunoblot-EMSA, ChIP, glutathione measurement, catalytic mutant overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (reporter assay, EMSA, ChIP, enzymatic readout with mutant) in single study\",\n      \"pmids\": [\"25931127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In glioma cells treated with temozolomide (TMZ), CHAC1 expression is upregulated via the JNK1/c-JUN pathway through transcriptional control; CHAC1 physically binds to Notch3 protein and inhibits Notch3 activation and its downstream signaling, thereby contributing to TMZ-mediated cytotoxicity.\",\n      \"method\": \"Transcriptome microarray, bioinformatics, CHAC1 overexpression and knockdown, caspase-3/9 activation assay, PARP degradation, co-immunoprecipitation (CHAC1-Notch3 binding)\",\n      \"journal\": \"Neuropharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — co-IP plus functional KD/OE with defined phenotypes; single lab\",\n      \"pmids\": [\"27986595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TRIB3, acting downstream of ATF4, represses CHAC1 expression to limit glutathione degradation; in Trib3-deficient cells, arsenite stress causes markedly elevated CHAC1 mRNA/protein, accelerated glutathione consumption, and increased cell death—all rescued by Chac1 silencing, identifying TRIB3 as a negative regulator of CHAC1 within the ATF4 pathway.\",\n      \"method\": \"Mouse embryonic fibroblasts, Trib3 KO, siRNA knockdown of Chac1, promoter analysis, glutathione measurement, cell death assays\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with KO and rescue, promoter analysis; single lab\",\n      \"pmids\": [\"27526673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CHAC1 degrades glutathione downstream of the GCN2-eIF2α-ATF4 pathway during cystine starvation; CHAC1 knockdown rescued GSH levels and prevented cystine-starvation-induced necroptosis and ferroptosis in triple-negative breast cancer cells.\",\n      \"method\": \"siRNA knockdown, GSH measurement, RIP1 inhibitor/MLKL inhibitor rescue, Western blot for eIF2α phosphorylation and ATF4\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with specific biochemical and cell death phenotype readouts; moderate evidence from single lab\",\n      \"pmids\": [\"29383104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CHAC1 overexpression in H. pylori-infected gastric epithelial cells degrades glutathione and causes ROS accumulation, leading to somatic TP53 mutations; a catalytically inactive CHAC1 mutant did not cause TP53 mutations, and CHAC1 siRNA prevented the H. pylori-induced high frequency of TP53 mutations.\",\n      \"method\": \"CHAC1 overexpression, catalytically inactive mutant, siRNA knockdown, TP53 mutation analysis, GSH/ROS measurement in AGS cells\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — catalytic mutant and siRNA rescue with specific molecular readout; single lab\",\n      \"pmids\": [\"29632819\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In bronchial epithelial cells, LPS-induced CHAC1 mRNA expression is PERK-independent and involves ATF4; CHAC1 knockdown with siRNA modulates inflammatory markers and NF-κB signaling in response to LPS and flagellin.\",\n      \"method\": \"siRNA knockdown of CHAC1, qRT-PCR, NF-κB signaling analysis, bronchial epithelial cell model (NCI-H292)\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA KD with defined inflammatory pathway readout; single lab\",\n      \"pmids\": [\"30555487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Zebrafish Chac1 is a glutathione-degrading enzyme required for calcium signaling during embryonic development; chac1 morphants showed attenuated intracellular glutathione oxidation (Grx1-roGFP2) and strongly reduced calcium transients (GCaMP6s), and developmental defects could be rescued by WT but not catalytically inactive Chac1, establishing that Chac1-mediated glutathione degradation is an upstream activator of calcium signaling.\",\n      \"method\": \"Zebrafish morpholino knockdown, rescue with WT and catalytic mutant, live redox imaging (Grx1-roGFP2), calcium imaging (GCaMP6s), in vivo enzymatic activity assessment\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — catalytic mutant rescue + live in vivo imaging of redox and calcium; multiple orthogonal methods\",\n      \"pmids\": [\"31189567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DJ-1 (PARK7) inhibits glutathione degradation in astrocytes by binding the basic leucine zipper domain of ATF3 (shown by bimolecular fluorescence complementation), preventing ATF3 binding to the CHAC1 promoter and thereby reducing CHAC1 expression.\",\n      \"method\": \"High-throughput sequencing, bimolecular fluorescence complementation (BiFC) for DJ-1/ATF3 interaction, ChIP/promoter binding assay, DJ-1 knockout\",\n      \"journal\": \"Neuroscience research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — BiFC protein interaction plus promoter occupancy assay; single lab\",\n      \"pmids\": [\"35988816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CHOP exerts a novel inhibitory effect on CHAC1 transcription through an upstream ATF/CRE motif via an indirect mechanism (CHOP does not directly bind the CHAC1 ATF/CRE as shown by IM-EMSA), while ATF4 directly binds these CHAC1 promoter sequences.\",\n      \"method\": \"Luciferase reporter assays, IM-EMSA, deletion/mutation analysis of human and mouse CHAC1 5'-flanking region\",\n      \"journal\": \"Biochemistry and biophysics reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reporter assay and EMSA with multiple constructs; single lab\",\n      \"pmids\": [\"33102815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"miR-26a-5p targets the 3'-UTR of CHAC1 mRNA; in renal proximal tubular epithelial cells, retention of miR-26a-5p (by Rab27a knockout) inhibits CHAC1 expression and thereby suppresses NF-κB signaling and the inflammatory response.\",\n      \"method\": \"miR-26a-5p mimic/inhibitor, 3'-UTR targeting validation, CHAC1 knockout, NF-κB pathway analysis in HK-2 cells\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — miRNA target validation and CHAC1 KO rescue; single lab\",\n      \"pmids\": [\"32853650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In heat-stressed intestinal epithelial cells (IPEC-J2), CHAC1 is downstream of the ATF4-CHOP signaling branch; CHAC1 knockdown attenuates heat-stress-induced glutathione reduction and cell apoptosis, placing CHAC1 as a functional effector of the ATF4-CHOP-CHAC1 axis in heat-induced ERS-oxidative stress crosstalk.\",\n      \"method\": \"Transcriptome sequencing, Western blotting, siRNA knockdown of CHAC1, GSH measurement, apoptosis assays (caspase-3, cytochrome c) in IPEC-J2 cells\",\n      \"journal\": \"Journal of agricultural and food chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — transcriptomics plus KD with biochemical readouts; single lab\",\n      \"pmids\": [\"34919378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The active site residues of human ChaC1 responsible for direct interactions with glutathione substrate were identified (residues 38-YGSL-41, D68, R72, E115, Y143) using enzyme-substrate docking and validated by in vivo yeast assays and MD simulations; ChaC family-exclusive residues maintain structural stability of the active site.\",\n      \"method\": \"In silico active site mapping (molecular docking), MD simulations, MMGBSA binding energy calculations, in vivo yeast functional assays with mutants\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1/2 — computational structural analysis with in vivo yeast validation; single lab\",\n      \"pmids\": [\"36456186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MIA3/TANGO1 binds to CHAC1 protein (shown by co-immunoprecipitation and confocal co-localization) and promotes CHAC1 expression and GSH degradation; MIA3 overexpression increases CHAC1 levels and promotes hepatocellular carcinoma growth and metastasis, while MIA3 knockout reduces CHAC1 expression and inhibits these processes.\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy, RNA-seq, MIA3 overexpression/knockout in HCC cells (Hep-G2, Huh7), GSH measurement\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — reciprocal co-IP and co-localization with functional phenotype; single lab\",\n      \"pmids\": [\"37948019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALKBH5 demethylase regulates CHAC1 mRNA stability via m6A demethylation; ALKBH5 silencing increases CHAC1 expression, elevates intracellular ROS levels, and increases chemotherapy sensitivity in gastric cancer cells.\",\n      \"method\": \"Transcriptome and m6A sequencing, ALKBH5 siRNA, ROS measurement, chemosensitivity assays\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — combined transcriptome and m6A-seq with functional validation; single lab\",\n      \"pmids\": [\"38007439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CHAC1 inactivation via CRISPR/Cas9 knock-in of an enzyme-inactivating mutation preserves muscle glutathione levels under fasting, cancer cachexia, and chemotherapy conditions in mice; however, CHAC1 inactivation alone is insufficient to prevent muscle wasting, dissociating glutathione preservation from anti-atrophic benefit.\",\n      \"method\": \"CRISPR/Cas9 enzyme-inactivating knock-in, multiple mouse wasting models, GSH measurement, muscle mass phenotyping\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — CRISPR catalytic knock-in with rigorous in vivo phenotyping across multiple models\",\n      \"pmids\": [\"37014882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CHAC1 acts as a bridge connecting UBA2 and PKM2, enhancing SUMOylation of PKM2; SUMOylated PKM2 translocates from cytoplasm to nucleus where it activates glycolysis-related genes, promoting the Warburg effect in lung adenocarcinoma cells.\",\n      \"method\": \"Shotgun mass spectrometry-based proteomics, RNA sequencing, co-immunoprecipitation, SUMOylation assays, nuclear/cytoplasmic fractionation, CHAC1 OE/KD functional studies\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS-based interactome plus co-IP and functional metabolic readouts; single lab\",\n      \"pmids\": [\"39368995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CAF-derived exosomal miR-432-5p targets CHAC1 mRNA to reduce GSH consumption, inhibit ferroptosis, and increase docetaxel resistance in prostate cancer cells; suppression of CHAC1 by miR-432-5p reduces lipid ROS accumulation and prevents erastin-induced ferroptosis.\",\n      \"method\": \"Exosome isolation from CAFs, miR-432-5p functional studies, CHAC1 3'-UTR targeting, lipid ROS assay, erastin-induced ferroptosis model, GSH measurement\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — miRNA target validation with functional ferroptosis/chemoresistance readouts; single lab\",\n      \"pmids\": [\"38769193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Juglone (a naphthoquinone) is a first-in-class inhibitor of ChaC1 with IC50 of 8.7 µM identified through yeast-based high-throughput screening; inhibition is non-competitive with the glutathione substrate and persists in a cysteine-free ChaC1 variant, indicating a novel inhibitory mechanism not involving active-site cysteine adduction.\",\n      \"method\": \"High-throughput yeast-based screens, in vitro enzymatic assays, kinetic analysis, cysteine-free ChaC1 variant testing, IC50 determination\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with kinetic characterization and mechanistic mutant; rigorous pharmacological study\",\n      \"pmids\": [\"39400295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CHAC1 mediates kidney disease risk by degrading glutathione in tubular cells; Chac1 haploinsufficient mice (CRISPR-generated) showed increased GSH, decreased lipid peroxidation, and protection against ferroptosis across multiple kidney disease models (folic acid nephropathy, adenine CKD, diabetic nephropathy); ChIP-seq confirmed ATF4 as a direct transcriptional activator of CHAC1.\",\n      \"method\": \"CRISPR Chac1 haploinsufficiency, multiple in vivo kidney disease models, GSH/lipid peroxidation measurement, ferroptosis gene expression, ChIP-seq, human kidney biopsy correlation\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — CRISPR in vivo model with multiple disease paradigms, ChIP-seq, and human validation; strong multi-method evidence\",\n      \"pmids\": [\"40267214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"BACH1 transcription factor directly activates the CHAC1 promoter (shown by dual-luciferase assay); BACH1 silencing attenuates ferroptosis by suppressing CHAC1 and restoring GSH-GPX4 axis in cardiomyocytes during ischemia-reperfusion injury; CHAC1 overexpression (via AAV) worsened cardiac dysfunction and iron deposition in a mouse I/R model.\",\n      \"method\": \"Dual-luciferase promoter assay, BACH1 siRNA, CHAC1 AAV overexpression in vivo, RNA sequencing (OGD/R model), GSH/GPX4/lipid peroxidation measurement, mouse I/R model\",\n      \"journal\": \"Antioxidants\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — promoter assay plus in vivo AAV OE and KD; single lab\",\n      \"pmids\": [\"41750596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EGR1 transcriptionally activates CHAC1; chromatin immunoprecipitation and dual-luciferase reporter assays confirmed direct EGR1 binding at the CHAC1 promoter; EGR1 overexpression potentiates ferroptosis in keratinocytes, an effect that is phenocopied by CHAC1 overexpression and reversed by EGR1 knockdown.\",\n      \"method\": \"ChIP, dual-luciferase reporter assay, EGR1/CHAC1 OE and KD, ferroptosis markers (MDA, ROS, Fe2+, GSH) in keratinocytes\",\n      \"journal\": \"Current molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and reporter assay with functional epistasis; single lab\",\n      \"pmids\": [\"39129295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"lncRNA GDIL serves as a scaffold for XRN2 in the cytoplasm, directing XRN2 to degrade CHAC1 mRNA; GDIL promotes GSH accumulation by inhibiting CHAC1-mediated GSH degradation, thereby conferring platinum resistance in colorectal cancer.\",\n      \"method\": \"RNA pulldown, metabolomic and metabolic flux analysis, XRN2 relocalization study, CHAC1 mRNA stability assay, GDIL OE/KD in resistant cancer cells\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNA-protein interaction with mRNA stability mechanistic follow-up; single lab\",\n      \"pmids\": [\"39893168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NUPR1 interacts with ATF4 (shown by immunoprecipitation in HK2 cells) and suppresses ferroptosis in renal ischemia-reperfusion injury by inhibiting the ATF4-CHAC1 pathway; NUPR1 overexpression reduces ATF4-driven CHAC1 transcription and subsequent glutathione depletion.\",\n      \"method\": \"Co-immunoprecipitation (NUPR1-ATF4), NUPR1 overexpression/knockdown, murine renal IRI model, HK2 hypoxia-reoxygenation model, ferroptosis marker assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — co-IP showing protein interaction with in vivo and in vitro functional readouts; single lab\",\n      \"pmids\": [\"42019644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"H3K9 acetylation (mediated by histone acetyltransferase P300) at the CHAC1 promoter is required for ATF4-driven transcriptional upregulation of CHAC1 in hepatic stellate cells treated with DHA; luciferase assays with mutated CHAC1 promoter identified -212 to -199 bp and -269 to -257 bp as essential ATF4 binding regions; inhibiting histone acetylation blocked DHA-induced CHAC1 upregulation and ferroptosis.\",\n      \"method\": \"ChIP-qPCR for H3K9 acetylation, luciferase reporter assay with WT and mutated CHAC1 promoter, P300 inhibition, RNA sequencing, in vivo CCl4 liver fibrosis model\",\n      \"journal\": \"Chinese medical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and reporter assay with in vivo validation; single lab\",\n      \"pmids\": [\"40947783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Arsenic inhibits METTL3/14-mediated m6A modification of CHAC1 mRNA, reducing YTHDF2-mediated degradation of CHAC1 mRNA and thereby increasing CHAC1 expression; RIP assays confirmed arsenic inhibits the interaction between METTL3/YTHDF2 and CHAC1 mRNA; METTL3 overexpression reduced CHAC1 mRNA half-life and ameliorated arsenic-induced ferroptosis in β-cells.\",\n      \"method\": \"m6A site identification, RIP assay (METTL3/YTHDF2-CHAC1 mRNA interaction), mRNA half-life measurement, METTL3 overexpression, CHAC1 KD in β-cells\",\n      \"journal\": \"Ecotoxicology and environmental safety\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RIP assay plus mRNA stability mechanistic dissection; single lab\",\n      \"pmids\": [\"39667319\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CHAC1 is a cytosolic γ-glutamyl cyclotransferase that specifically degrades glutathione (the first such enzyme identified in eukaryotes), and is transcriptionally induced by the ATF4-ATF3-CHOP arm of the unfolded protein response via bipartite ATF/CRE promoter elements; by depleting cellular glutathione, CHAC1 elevates reactive oxygen species, promotes ferroptosis and apoptosis, modulates calcium signaling, and can also function non-enzymatically by binding partners such as Notch3 and UBA2/PKM2 to influence downstream signaling and glycolysis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CHAC1 is a cytosolic γ-glutamyl cyclotransferase that specifically degrades glutathione, functioning as the first eukaryotic intracellular glutathione-degrading enzyme and a critical effector linking endoplasmic reticulum stress signaling to oxidative cell death. Its enzymatic activity, dependent on defined active-site residues including E115, depletes cellular glutathione, elevates reactive oxygen species, and promotes ferroptosis and apoptosis in diverse tissues including kidney, heart, brain, and multiple cancer types [PMID:23070364, PMID:31189567, PMID:40267214, PMID:37014882]. CHAC1 transcription is directly activated by ATF4 through bipartite ATF/CRE promoter elements (with contributions from ATF3, BACH1, EGR1, and epigenetic regulation via H3K9 acetylation) and is negatively regulated at the mRNA level by m6A-dependent decay and specific microRNAs [PMID:25931127, PMID:40947783, PMID:39667319, PMID:38769193]. Beyond its catalytic role, CHAC1 functions non-enzymatically as a scaffold bridging UBA2 and PKM2 to promote PKM2 SUMOylation and nuclear translocation, thereby activating glycolysis-related gene expression [PMID:39368995].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Establishing CHAC1 as a stress-responsive proapoptotic gene resolved how the ATF4-ATF3-CHOP arm of the UPR executes cell death through a previously uncharacterized downstream effector.\",\n      \"evidence\": \"siRNA knockdown and overexpression with TUNEL, PARP cleavage, and AIF translocation assays in human aortic endothelial cells\",\n      \"pmids\": [\"19109178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Enzymatic activity of CHAC1 was unknown\", \"Direct transcription factor binding to the CHAC1 promoter was not demonstrated\", \"Mechanism of apoptosis induction was undefined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identifying CHAC1 as a γ-glutamyl cyclotransferase that specifically degrades glutathione — the first such cytosolic enzyme in eukaryotes — provided the molecular mechanism for its proapoptotic function and established GSH depletion as the proximal biochemical event.\",\n      \"evidence\": \"In vitro enzyme assays with catalytic-dead E>Q mutant and in vivo yeast functional validation\",\n      \"pmids\": [\"23070364\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CHAC1 transcription is regulated at the promoter level was not resolved\", \"Whether glutathione degradation connects to specific regulated cell death pathways (ferroptosis) was unknown\", \"Structural basis of substrate specificity was not determined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapping the CHAC1 promoter architecture revealed that ATF4 and ATF3 directly bind bipartite ATF/CRE and ACM elements, explaining how integrated stress response signaling converges on CHAC1 transcriptional activation.\",\n      \"evidence\": \"Luciferase reporter assays, EMSA, ChIP, and glutathione measurement with catalytic mutant in HEK293 cells\",\n      \"pmids\": [\"25931127\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of additional transcription factors (CHOP, C/EBPβ) in fine-tuning CHAC1 was incompletely resolved\", \"Epigenetic regulation of the CHAC1 promoter was not addressed\", \"In vivo relevance of these promoter elements was not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Discovery of CHAC1 binding to Notch3 and negative regulation by TRIB3 expanded the functional repertoire beyond enzymatic GSH degradation, revealing both non-catalytic protein interactions and feedback control within the ATF4 pathway.\",\n      \"evidence\": \"Co-immunoprecipitation of CHAC1-Notch3 in glioma cells; Trib3-KO MEFs with Chac1 siRNA rescue and GSH/cell death readouts\",\n      \"pmids\": [\"27986595\", \"27526673\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CHAC1-Notch3 interaction lacks reciprocal validation and structural characterization\", \"Whether TRIB3 regulation of CHAC1 is direct or indirect at the promoter level was not fully determined\", \"Generalizability of Notch3 interaction beyond glioma was not tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linking CHAC1-mediated GSH depletion to ferroptosis and necroptosis during cystine starvation established CHAC1 as a key effector of non-apoptotic cell death, broadening its role beyond classical apoptosis.\",\n      \"evidence\": \"siRNA knockdown with GSH measurement and RIP1/MLKL inhibitor rescue in triple-negative breast cancer cells\",\n      \"pmids\": [\"29383104\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CHAC1 is required for ferroptosis in non-cancer physiological contexts was not tested\", \"Relative contribution of CHAC1 versus system Xc− loss to GSH depletion during cystine starvation was not quantified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating that CHAC1-dependent glutathione degradation is an upstream activator of calcium signaling during zebrafish embryogenesis revealed an unexpected developmental function and mechanistically coupled redox state to calcium transients in vivo.\",\n      \"evidence\": \"Zebrafish morpholino knockdown with rescue by WT but not catalytic-dead Chac1, live Grx1-roGFP2 redox imaging and GCaMP6s calcium imaging\",\n      \"pmids\": [\"31189567\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism linking GSH oxidation to calcium channel/store activation was not identified\", \"Whether this calcium-signaling function operates in mammalian systems was not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of CHOP as an indirect transcriptional repressor of CHAC1, DJ-1 as a suppressor of ATF3-mediated CHAC1 induction, and miR-26a-5p as a post-transcriptional regulator revealed multiple layers of negative regulation that restrain CHAC1 activity.\",\n      \"evidence\": \"EMSA/reporter assays for CHOP; BiFC for DJ-1/ATF3 interaction with ChIP; miR-26a-5p 3′-UTR targeting and CHAC1-KO rescue in renal cells\",\n      \"pmids\": [\"33102815\", \"35988816\", \"32853650\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of CHOP indirect repression was not defined\", \"DJ-1/ATF3 interaction was shown by BiFC only — not independently validated by orthogonal method\", \"In vivo significance of miR-26a-5p regulation was not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Structural mapping of ChaC1 active-site residues mediating glutathione binding provided the first substrate-enzyme interaction model and identified ChaC-family-exclusive residues essential for catalytic activity.\",\n      \"evidence\": \"Molecular docking, MD simulations, MMGBSA calculations validated by in vivo yeast mutant assays\",\n      \"pmids\": [\"36456186\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental crystal structure of ChaC1 with bound substrate exists\", \"Whether these residues are druggable was not assessed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"CRISPR knock-in of a catalytic-inactivating mutation in mice demonstrated that CHAC1 enzymatic activity is required for stress-induced muscle glutathione depletion but is insufficient alone to prevent muscle wasting, dissociating GSH preservation from anti-atrophic benefit.\",\n      \"evidence\": \"CRISPR/Cas9 enzyme-inactivating knock-in across fasting, cachexia, and chemotherapy mouse models with GSH and muscle mass measurements\",\n      \"pmids\": [\"37014882\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CHAC1 inactivation protects other tissues (kidney, heart) in vivo was not tested in this model\", \"Compensatory mechanisms for GSH degradation in muscle were not investigated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery of a non-enzymatic scaffolding function — bridging UBA2 and PKM2 to promote PKM2 SUMOylation and nuclear translocation for glycolytic gene activation — revealed that CHAC1 influences tumor metabolism independently of its γ-glutamyl cyclotransferase activity.\",\n      \"evidence\": \"Shotgun mass spectrometry interactome, co-immunoprecipitation, SUMOylation assays, nuclear fractionation in lung adenocarcinoma cells\",\n      \"pmids\": [\"39368995\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether this scaffolding function requires specific CHAC1 domains distinct from the active site is unknown\", \"Independence from catalytic activity was not tested with enzyme-dead mutant\", \"Single-lab observation awaits independent replication\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of juglone as a first-in-class non-competitive ChaC1 inhibitor (IC50 8.7 µM) through high-throughput screening provided both a pharmacological tool and evidence for a regulatory site outside the catalytic center.\",\n      \"evidence\": \"Yeast-based HTS, in vitro kinetic analysis, cysteine-free ChaC1 variant testing\",\n      \"pmids\": [\"39400295\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Juglone binding site on ChaC1 is unknown\", \"Selectivity profile against related enzymes and in vivo efficacy were not determined\", \"Whether juglone inhibits ChaC2 was not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"In vivo CRISPR haploinsufficiency across multiple kidney disease models and identification of additional transcriptional activators (BACH1, EGR1) and epigenetic regulators (H3K9ac/P300) established CHAC1 as a disease-relevant ferroptosis driver whose expression is controlled by a multi-layered transcriptional and epitranscriptomic regulatory network.\",\n      \"evidence\": \"Chac1 haploinsufficient mice in folic acid, adenine CKD, and diabetic nephropathy models with ChIP-seq; BACH1/EGR1 ChIP and reporter assays; H3K9ac ChIP-qPCR with P300 inhibition; m6A-RIP for METTL3/YTHDF2 regulation\",\n      \"pmids\": [\"40267214\", \"41750596\", \"39129295\", \"40947783\", \"39667319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"A comprehensive integrated model of how multiple transcription factors and epigenetic marks converge on the CHAC1 promoter simultaneously is lacking\", \"Therapeutic benefit of pharmacological CHAC1 inhibition in disease models has not been demonstrated\", \"Whether CHAC1 contributes to human Mendelian disease is unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"No experimental three-dimensional structure of human CHAC1 (apo or substrate-bound) has been determined, and the binding site and mechanism of the only known inhibitor (juglone) remain undefined; whether the non-catalytic scaffolding functions are separable from enzymatic activity has not been resolved with domain/mutant analysis.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal/cryo-EM structure\", \"No selective, validated in vivo inhibitor\", \"Catalytic vs. non-catalytic functions not genetically separated in mammalian systems\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 8, 13, 16, 19, 20]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 2, 17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [0, 2, 4, 5, 12]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 1, 5, 18, 20, 21, 22]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [1, 6, 8, 16, 17]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 10, 25]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"ATF4\",\n      \"ATF3\",\n      \"NOTCH3\",\n      \"UBA2\",\n      \"PKM2\",\n      \"TRIB3\",\n      \"MIA3\",\n      \"NUPR1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}